[Dextrose and vigilance: the effect of dextrose on the spectro-analytic parameters of pharmaco-EEG]. In a randomized single-blind cross-over-design the effect of 15 g dextrose was investigated against placebo with 48 healthy subjects at two different days each. The quantitative pharmaco-EEG was recorded occipitocentral at the left side (O1-Cz) under vigilance controlled conditions and then under resting conditions (three minutes each). Having measured its baseline it was repeated six times every 20 minutes starting 10 minutes after the application of the substance. After the administration of dextrose a stabilization of vigilance could be seen in the EEG by the following differences of statistical significance (p < = 0.05) between dextrose and placebo under resting recording conditions: In the first control (14.-16. minute post) there was a significant decrease of the relative power in the delta and in the theta range combined with an increase of alpha 2. In the second control (34.-36. minute post) there was a significant decrease of relative power in the theta range and significant increase in the alpha 1 range. In the third control (54.-56. minute post) and later on no effects of dextrose were left in the pharmaco-EEG. The significant differences in the first and second control have to be interpreted as follows: After the administration of dextrose the level of vigilance was increased compared to the placebo group.


Current and future trends in wound healing. New adjuncts to outpatient wound care focus on diminishing infection rates and improving the speed and effectiveness of laceration repair. This article provides an overview of growth factors, proteolytic enzymes, topical antimicrobial agents, and new materials that facilitate wound closure. Also, the pathophysiology of surface wound repair is summarized.


Wound evaluation and cleansing.

Wound evaluation and cleansing. The approach to wound management in the Emergency Department does not differ from that used in evaluating other complaints. This consists of a problem-directed history and physical examination. The history should probe for host and wound factors that increase the risk of a poor outcome. The examination should emphasize a search for the involvement of underlying structures. Appropriate cleansing remains the foundation for good wound management. Until a rapid inexpensive test is available to identify wounds with low bacterial counts, the clinician should assume that all wounds have bacterial loads capable of causing infection. Therefore, most wounds should be cleansed by irrigation with at least 200 mL of normal saline. Larger quantities or 1% povidone-iodine solution may be used in wounds that have high wound or host risk factors. Wound exploration should occur in a well-lit environment and combine good hemostasis with appropriate analgesia and anesthesia.


Suture materials and mechanical after care. Proper wound closure necessitates a familiarity with the various types of materials and the indications and contraindications of the many techniques available. Proper wound dressing and after-care instruction ensure optimal results and help minimize complications.


Sedation and analgesia.

Sedation and analgesia. The control of pain and the alleviation of anxiety are integral components in the management of soft tissue injuries in the Emergency Department. Pharmacologic agents utilized for analgesia and sedation must possess properties favorable for emergency department use. This article reviews the pharmacologic approach to patients who require sedation and/or analgesia for the management of a soft tissue injury in the Emergency Department.


Local anesthetics.

Local anesthetics. Emergency physicians often rely on the use of local anesthetic agents to relieve patient discomfort, and research continues in an effort to develop new agents with improved anesthetic qualities. Eventually, a nontoxic, rapidly acting agent may become available that could provide profound anesthesia of long duration when applied topically to intact skin or wounds. Until the "perfect" agent is developed, physicians can help the patient by making knowledgeable choices regarding local anesthetic techniques. By choosing topical agents when appropriate and buffering agents to be infiltrated, using courteous techniques of injection, and being cognizant of potential adverse reactions, the physician can turn a potentially unpleasant and frightening situation for the patient into a positive experience that promotes satisfaction and cooperation.


Emergency management of dog and cat bite wounds. Most dog and cat bite injuries are minor wounds that require only local wound care as well a patient evaluation for tetanus and rabies postexposure immunoprophylaxis. For larger wounds, primary or delayed primary closure can be performed safely where indicated. When antimicrobial agents are necessary, inexpensive penicillins or cephalosporins are adequate for initial therapy. Large-scale, prospective, controlled clinical studies are needed to define further the role of prophylactic antimicrobial therapy for uninfected wounds.


Soft tissue infections.

Soft tissue infections. Infections in soft tissues present with one or more of the cardinal signs of infections; however, presence of these signs may indicate manifestation of a deep systemic infection or of a noninfectious process. One of the earliest determinations to be made is whether the problem is likely to require patient admission or referral to another specialty. Only then is the primary management clear.


The rational use of antimicrobial agents in simple wounds. The wounds in most patients presenting to the Emergency Department will heal uneventfully and do not require the use of antimicrobial agents. Specific indications for antimicrobial use include simple wounds in patients with lymphedema, orthopedic prostheses, or in those patients prone to bacterial endocarditis. Relative indications include high-risk wounds in compromised hosts, wounds contaminated with feces, saliva, or vaginal secretions, or wounds clinically infected at the time of presentation. More important than antimicrobial administration is the commitment to aggressive, timely wound care. Pitfalls in therapy center around the injudicious use of antimicrobial agents. Such therapy does not give the physician permission to close wounds that should be left open or to give less than optimal wound care. Physicians should be keenly aware of the fact that antimicrobial use is not innocuous. The economic burden and potential for adverse side effects must be weighed against the fact that the wounds in 90% to 95% of patients presenting to the Emergency Department will heal without the use of antimicrobial agents.


Detection and management of foreign bodies in soft tissue. The discovery and management of foreign bodies in soft tissue are challenging problems for emergency physicians. History, physical examination, and thorough wound exploration serve as the screening test for the presence of foreign bodies. Various radiographic studies can be performed to confirm the diagnosis, to identify the object's composition and shape, and to determine its approximate location in the tissues. Once the object is discovered, the clinician must weigh the potential harm of the foreign body in its current location against the risks of attempting removal.


The mangled extremity. Compartment syndrome and amputations. Injuries that amputate, crush, or otherwise mangle an extremity are devastating. The possibilities of medical and psychologic disability, multiple operations, and protracted rehabilitation loom before these patients. Fortunately, expert care given by the emergency department physician maximizes the opportunity for success should the limb be replanted or revascularized. The first priority for the emergency department staff is the efficient and aggressive resuscitation of the patient. Only after this has been carried out and other life-threatening injuries addressed can attention be directed toward the injured limb. It is in the patient's best interest if the emergency department staff assumes that a mangled extremity will be replanted or revascularized. Advances in microsurgical technique and equipment have enabled surgeons to achieve salvage rates greater than 90%. Continued improvements in technique, equipment, rehabilitation, and patient selection will enable more patients to benefit from limb salvage procedures.


Chronic skin ulcers.

Chronic skin ulcers. Chronic skin ulceration is a common complication of diabetes, peripheral vascular disease, and disorders that decrease mobility. Local ulcer care will be successful only if the underlying cause is correctly identified and steps are taken to reverse it. This article reviews the emergency department assessment and management of the patient with chronic skin ulceration.


Hyperbaric medicine for outpatient wound care. After proper cleansing, debridement, and appropriate antibiotic and closure decisions, some wounds will fail to heal properly or may develop serious complications. Hyperbaric oxygen therapy offers a relatively safe noninvasive method of improving wound healing by enhancing tissue oxygenation and decreasing edema formation. Recommendation for the use of hyperbaric oxygen therapy in outpatient wound care is reviewed and the rationale behind the benefits is discussed.


FOS expression in gonadotropin-releasing hormone neurons: enhancement by steroid treatment and mating. Expression of the protooncoprotein FOS is now widely believed to be a marker for neuronal activation. In female rats, a steroid-induced LH surge is accompanied by an increase in FOS-positive GnRH neurons, especially in the region of the organum vasculosum of the lamina terminalis. The present study, conducted in mice, has examined the effects of both steroid hormone treatment and sexual behavior on the expression of FOS in GnRH neurons and their distribution in the central nervous system. Thirty-three ovariectomized mice, each bearing a sc priming capsule of 17 beta-estradiol, were divided into five groups, four of which were treated sequentially with estradiol benzoate (1 microgram) and progesterone (500 micrograms). In females maintained on 17 beta-estradiol only and killed between 1400-1530 h, only 1.3 +/- 0.7% of GnRH neurons contained FOS, while treatment with estradiol benzoate/progesterone increased FOS expression significantly to 31.7 +/- 8.5% in the same time period. In animals killed at 1530-1700 h, FOS expression declined in the absence of a male (13.8 +/- 2.2%) or when the male present in the cage displayed some sexual behavior but did not ejaculate (13.0 +/- 8.6%). Interestingly, the expression of FOS was maintained at a high level (42.3 +/- 11.4%) into the late afternoon in females paired with a reproductively successful (ejaculating) male. There was a positive correlation (r2 = 0.65; P < 0.01) between the level of LH and the number of FOS-positive GnRH neurons. Hence, the expression of FOS in GnRH neurons was enhanced by both a steroid regimen leading to a LH surge and an intense level of mating behavior. Mapping of the GnRH neurons indicates that in animals with the highest level of FOS expression, FOS-positive GnRH neurons were not confined to the region of the organum vasculosum of the lamina terminalis, but were found more widely distributed along the entire rostro-caudal axis of these cells.


Role of luteinizing hormone in the expression of cholesterol side-chain cleavage cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase, delta 5-4 isomerase messenger ribonucleic acids in the primate corpus luteum. It is well established that LH has an obligatory role in the acute production of progesterone by the primate corpus luteum in vivo because interruption of LH support to the corpus luteum at any time during the luteal phase is accompanied by an immediate and sustained fall in serum progesterone concentrations. However, recent studies have demonstrated that maximal steroidogenic capacity of cultured human luteal cells and maximal levels of messenger RNAs (mRNAs) for cholesterol side chain cleavage cytochrome P450 (P450scc) and 3 beta-hydroxysteroid dehydrogenase, delta 5-4 isomerase (3 beta-HSD) in luteal tissue are observed shortly after luteinization and decline thereafter throughout the remainder of the luteal phase. These findings would suggest that the role of LH in the acute regulation of progesterone production may differ from its role in the expression of mRNAs for steroidogenic enzymes. We initiated the current studies to define the role of LH upon the expression of mRNAs for P450scc and 3 beta-HSD by the primate corpus luteum. For this purpose, we treated cynomolgus monkeys with a potent GnRH antagonist for 1, 2, and 3 days during the luteal phase of the menstrual cycle and measured levels of mRNAs for P450scc and 3 beta-HSD in corpora lutea. Treatment of monkeys with the GnRH antagonist reduced bioactive LH concentrations to less than 5 ng/ml by 48 h of treatment, and LH concentrations remained less than 5 ng/ml thereafter. Serum progesterone concentrations were reduced by 74% after 1 day of antagonist treatment, 88% after 2 days of antagonist treatment, and by more than 95% after 3 days of GnRH antagonist treatment. Although progesterone secretion was markedly diminished after 24 h of antagonist treatment, there were no differences in mRNAs for P450scc and 3 beta-HSD between antagonist-treated and control animals. However, mRNAs for P450scc and 3 beta-HSD were significantly (P < 0.05) reduced after 2 days of antagonist treatment and were nearly nondetectable after 3 days of antagonist treatment. These results demonstrate a temporal dissociation of the effects of LH on the acute regulation of progesterone secretion and the maintenance of specific mRNAs involved in progesterone production. Nonetheless, the results clearly show that LH is required for the continued expression of mRNAs for P450scc and 3 beta-HSD by the primate corpus luteum.


Pituitary adenomas in mice transgenic for growth hormone-releasing hormone. It has been shown that mice transgenic for human GH-releasing hormone (GRH) develop hyperplasia of pituitary somatotrophs, lactotrophs, and mammosomatotrophs, cells capable of producing both GH and PRL, by 8 months of age. We now report that GRH transgenic mice 10-24 months of age develop pituitary adenomas, which we characterized by histology, immunohistochemistry, in situ hybridization, and electron microscopy. Of 13 animals examined, all developed GH-immunoreactive neoplasms that had diffuse positivity for GH mRNA by in situ hybridization. Eleven also contained PRL immunoreactivity; in situ hybridization demonstrated focal PRL mRNA in 3 of 5 immunohistochemically positive tumors. Alpha-Subunit was positive by immunohistochemistry in 8 adenomas, and TSH beta was localized in tumor cells of 5 adenomas. The adenomas had variable ultrastructural appearances, ranging from cells that resembled somatotrophs or mammosomatotrophs to cells with features of the glycoprotein hormone cell line. These findings provide conclusive evidence that protracted GRH stimulation of secretory activity can result in proliferation, hyperplasia, and adenoma of adenohypophysial cells.


Cloning of an ovine 11 beta-hydroxysteroid dehydrogenase complementary deoxyribonucleic acid: tissue and temporal distribution of its messenger ribonucleic acid during fetal and neonatal development. Glucocorticoids promote the development of many organ systems vital for extrauterine survival, and fetal cortisol provides the trigger for birth in sheep. The activity of glucocorticoids may be influenced at a cellular level by 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which is responsible for the interconversion of cortisol and cortisone. To examine 11 beta-HSD gene expression during fetal development, two overlapping clones which yield a 1.4 kilobase (kb) complementary DNA encoding sheep 11 beta-HSD from a liver library were isolated by using a rat 11 beta-HSD cDNA as the probe. This cDNA contains a 879 base pair open reading frame for a protein of 292 amino acids that has more than 70% sequence identity to rat and human 11 beta-HSDs. To define the tissue distribution of 11 beta-HSD messenger RNA in sheep, selected tissues were collected from one fetus at day 130 and term (approximately 145 days), and from a nonpregnant ewe. Cellular RNA was extracted and subjected to Northern blot analysis, and a single 1.8 kb transcript was detected in the fetal and adult liver, lung, hypothalamus, anterior pituitary, and placenta. This was undetectable in adrenals and kidneys, but a smaller (1.5 kb) transcript was present in fetal and adult kidney RNA. The relative abundance of 11 beta-HSD mRNA was greatest in fetal and adult livers, and it was much higher in adult liver, lung, and kidney than in the corresponding fetal tissues. To examine whether 11 beta-HSD gene expression is developmentally regulated in the fetal sheep, liver, lung, and kidney tissues were taken from fetuses at day 60-70, day 100-110, day 125-130, at term, and from newborn lambs (24-48 h old). In the lung and kidney, the relative abundance of 11 beta-HSD mRNA did not change from day 60 to term but increased in the lungs of newborn lambs. In contrast, 11 beta-HSD mRNA levels in the liver increased between day 125 and term and rose further in the newborn. Collectively, these results demonstrate that 11 beta-HSD gene expression in sheep is regulated in a tissue-specific and developmentally programmed manner.


Progesterone control of fibronectin secretion in guinea pig endometrium. Immunohistochemistry with a polyclonal antibody raised against human plasma fibronectin (Fn) was used to determine the localization of Fn in endometrial sections of guinea pig uteri isolated at the first, fourth, sixth, or tenth day of the estrous cycle. Immunoreactive Fn was constantly visualized in the endometrial stroma but absent from the epithelial layer. Fn was detected in the uterine lumen on the first or fourth day of the estrous cycle and was absent from the other sections. To determine the origin of this luminal Fn the ability of subcultured endometrial cells to produce Fn was tested, and the hormonal regulation of Fn secretion was studied. Cells were treated by estradiol alone or in association with progesterone, progesterone alone, or untreated. Whatever the hormonal treatment, stromal cells constantly secreted immunoreactive Fn into the culture medium. In the same way, the amount of Fn synthesized and basally secreted by epithelial cells was not affected by any hormonal treatments. However, Fn was found in the apical secretions of the untreated or estradiol-treated epithelial cells but was undetectable in the apical compartment when the epithelial cells were treated by progesterone alone or in association with estradiol. These results indicate that Fn is constitutively secreted by stromal cells and that subcultured epithelial cells of guinea pig endometrium secrete Fn from both their basal and apical membrane domains. However, the apical secretion of Fn is specifically suppressed by progesterone.


Continuous infusion of interleukin-1 beta induces a nonthyroidal illness syndrome in the rat. We studied the effects of continuous administration of recombinant human interleukin-1 beta (IL-1) on pituitary-thyroid function. Rats were equipped with minipumps loaded with either IL-1 (delivery rate, 0.5, 2.0, or 4.0 micrograms/day, ip, for 1 week) or saline. Infusion of 2.0 and 4.0 micrograms IL-1/day caused a significant decrease in plasma free T4 levels during the first 2-4 days, whereas plasma total T4 levels and T4 binding were significantly lowered throughout the week of the study. The infusion of 0.5 micrograms IL-1/day did not significantly change plasma TSH or total and free T4 levels. During the infusion of 2.0 micrograms IL-1/day, the decrease in plasma free T4 levels was paralleled by a significant decline in plasma TSH values and an impaired TSH responsiveness to TRH administration on the second day of infusion. IL-1 (2.0 micrograms/day) treatment significantly lowered plasma levels of T4-binding prealbumin, whereas it did not influence the plasma T3/T4 ratio or hepatic 5'-deiodinase activity. Plasma rT3 levels remained undetectable in both control and IL-1-treated rats. Chronic infusion of rats with 4.0 micrograms IL-1/day induced prolonged fever, whereas at the lower doses of IL-1, temperatures were elevated only on the first 2 days. IL-1 at doses of 2.0 and 4.0 micrograms/day induced a transient decrease in food intake and a suppression of body weight gain. Restriction of food consumption to the level observed in the 2.0 micrograms IL-1 experiment caused small decreases in T3, total and free T4, and TSH levels compared to those in ad libitum fed rats, but had no effects on T4 binding. We conclude that 1) continuous infusion of rats with 2.0 and 4.0 micrograms IL-1/day induces changes in thyroid economy commonly seen during infectious diseases and other systemic illnesses in rats [decreased plasma levels of TSH, T3, and (free) T4; diminished T4 binding; and decreased plasma T4-binding prealbumin levels], 2) the decrease in food intake during IL-1 treatment cannot completely explain the observed changes in thyroid hormone and TSH levels; and 3) it is highly unlikely that the decrease in thyroid hormone binding during chronic IL-1 infusion is caused by decreased food intake. Further studies are needed to clarify whether the observed alterations in thyroid economy during IL-1 infusion reflect direct effects of IL-1 per se or indirect effects caused by the mild illness induced by the cytokine.


Rat somatotroph insulin-like growth factor-II (IGF-II) signaling: role of the IGF-I receptor. The anterior pituitary contains insulin-like growth factor-II (IGF-II) and expresses abundant IGF-II-binding sites, but the IGF-II signaling pathway in the pituitary has not been defined. We, therefore, tested the effects of recombinant human IGF-II on pituitary function by assessing the GH responsiveness of the primary rat somatotroph to IGF-II. IGF-II (3.3 nM) suppressed GH secretion by 50%, similar to the effect elicited by equimolar doses of IGF-I. In contrast, a low concentration of IGF-II (0.2 nM) did not attenuate GH secretion, while a similar IGF-I dose was sufficient to produce 50% inhibition of basal GH secretion. Fifty percent competition for [125I]IGF-I binding by IGF-I and IGF-II in GC rat pituitary cells demonstrated a 14-fold lesser affinity of the IGF-II ligand for the IGF-I receptor compared to IGF-I; therefore, the binding affinity of IGF-II for the IGF-I receptor correlates with the concentration of IGF-II required for 50% GH inhibition. Transfected GH-secreting cell lines derived from GC cells overexpressing intact human IGF-I receptors exhibited enhanced responsiveness to IGF-II. In contrast, cells transfected with a truncated IGF-I receptor cDNA lacking the cytoplasmic receptor beta-subunit (952STOP) failed to transduce the IGF-II signal, indicating that functional IGF-I receptors are required for IGF-II signaling. In addition, a mutant IGF ligand, [Leu27]IGF-II, which selectively exhibits high affinity for the type II receptor, but only minimal binding to the IGF-I receptor, did not attenuate GH secretion. However, the analog [Arg54,Arg55]IGF-II, which exhibits high affinity to the IGF-I receptor, but no binding to the type II receptor, appropriately suppressed GH secretion. This unique model of somatotroph signaling provides evidence for IGF-II regulation of polypeptide hormone secretion mediated by the IGF-I receptor.


Estradiol increases prolactin synthesis and prolactin messenger ribonucleic acid in selected brain regions in the hypophysectomized female rat. Immunoreactive PRL which is not of pituitary origin, has been identified in many regions of the rat brain. We have previously demonstrated that estradiol increases hypothalamic immunoreactive PRL content in hypophysectomized female rats. To determine if estradiol stimulates PRL synthesis, we examined the effect of estradiol on the in vivo production of PRL, and on the expression of PRL messenger RNA (mRNA) in the hypothalamus, pons, and cerebral cortex. To examine the effect of estradiol on the in vivo production of PRL, [35S] methionine was injected into the lateral ventricle and its incorporation into immunoprecipitable PRL was determined by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In estradiol, but not vehicle-treated hypophysectomized rats, a 24,000 M(r) immunoprecipitable PRL protein was detected in the hypothalamus and pons-medulla, 2 and 4 h after methionine administration. No immunoprecipitable PRL proteins were detected in the amygdala, hippocampus, cortex, or serum at either time point. In addition, in the hypothalamus, but not the pons-medulla, a second PRL band was detected with an apparent mol wt of 16,000K. To determine if estradiol increased the expression of PRL mRNA, copy DNA was obtained by reverse transcription of poly(A+) mRNA prepared from intact and vehicle or estradiol-treated hypophysectomized rats and analyzed by polymerase chain reaction amplification. In tissues from hypophysectomized rats, there was little, or no, detectable levels of PRL mRNA. In contrast, in estradiol-treated hypophysectomized rats PRL mRNA was easily detected in the hypothalamus and pons-medulla by polymerase chain reaction amplification. These data suggest that estradiol increases the PRL content in the hypothalamus and pons-medulla by increasing PRL gene expression, in a manner similar to that reported in the pituitary.


Interleukin-1 inhibits Leydig cell steroidogenesis primarily by decreasing 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 expression. Macrophage-secreted cytokines, such as interleukin-1 (IL-1), have been shown to modulate Leydig cell function. The present study examined the effect of recombinant murine IL-1 alpha on Leydig cell steroidogenesis and its mechanism of action. Addition of IL-1 to macrophage-depleted primary cultures of mouse Leydig cells caused a dose-dependent decrease in cAMP-dependent testosterone production and 17 alpha-hydroxylase/C17-20 lyase (P450c17) mRNA levels. Chronic treatment (48 h) of Leydig cells in culture with 50 microM 8-bromo-cAMP (8-Br-cAMP) resulted in a 60-fold increase in testosterone production. Treatment with 8-Br-cAMP plus 0.2 and 2 U/ml IL-1 decreased testosterone production to 63 +/- 14% and 41 +/- 19%, respectively, while 5, 10, and 20 U/ml IL-1 decreased testosterone production to less than 5% that of cells treated with 8-Br-cAMP alone. Chronic treatment with IL-1 plus 8-Br-cAMP caused a shift in steroid production from testosterone to progesterone, but total steroid production (the sum of testosterone plus progesterone) was unaffected by IL-1 treatment. Treatment with 8-Br-cAMP alone caused a marked increase in P450c17 mRNA levels compared to that in control cultures, where P450c17 mRNA was undetectable. IL-1 caused a dose-dependent decrease in 8-Br-cAMP-stimulated P450c17 levels (0.2 U/ml by 31 +/- 9%, 2 U/ml by 82 +/- 12%, and 10 or 20 U/ml by 100% compared to that in cells treated with 8-Br-cAMP alone). In contrast to the effect on P450c17 mRNA, only the highest concentrations of IL-1 (10 and 20 U/ml) had any effect on cholesterol side-chain cleavage enzyme (P450scc) mRNA levels (53 +/- 16% and 38 +/- 20% decreases, respectively). The inhibitory effect of IL-1 on 8-Br-cAMP-stimulated P450c17 expression was reversible. Within 12 h after the removal of IL-1, P450c17 mRNA was restored to 24%; after 24 h, to 36%; after 36 h, to 65%; and after 48 h, to 84% of that with 8-Br-cAMP alone. P450c17 expression was more sensitive to IL-1-mediated inhibition than P450scc; therefore, inhibition of P450c17 is most likely primarily responsible for the observed inhibitory effects of IL-1 on Leydig cell testosterone production.


Differential capacity for cholesterol transport and processing in large and small rat luteal cells. The aim of this investigation was to determine whether a specific luteal subpopulation is responsible for the hypertrophic development of the corpus luteum at midpregnancy in the rat and to determine whether there was an underlying cellular basis for the differential production of steroids by the luteal cell subtypes. To examine this, we have dispersed and separated rat luteal steroidogenic cell populations into small (< 20 microns) and large (> 30 microns) cell types by elutriation. Luteal cells were examined at early (day 3) and midpregnancy (day 14) for differences in protein content and for differential expression of proteins required for steroid production. Specific proteins examined include the P450side chain cleavage enzyme (P450scc), adrenodoxin and adrenodoxin reductase, proteins required for cholesterol conversion to progestagens in the corpus luteum, and sterol carrier protein-2 (SCP2), a protein thought to be involved in intracellular cholesterol transport. The cytochrome P450(17)alpha hydroxylase (P450(17)alpha), a key enzyme responsible for androgen biosynthesis was also examined in the isolated luteal cells. The large luteal cell population displayed an increase in total cellular protein content while the small cell type did not change with luteal development. In addition, the large luteal cells expressed proteins unique to or elevated in that cell type. Analysis by two-dimensional polyacrylamide gel electrophoresis revealed that the large cell-specific proteins had molecular masses of 23 K and 32 K and that a 14 kilodalton (kDa) protein was elevated in the large cell type relative to the small cells. The small luteal cell on day 3 of pregnancy expressed a 36 kDa protein which was barely detectable in the large cell. Immunocytochemical and Western analysis indicated that the large luteal cells contain 5.3-fold more SCP2 (P < 0.05) and 5.6-fold more P450scc (P < 0.001) relative to the small cell type. Immunocytochemical staining of adrenodoxin and adrenodoxin reductase indicate these proteins were elevated in the large cell as well. Human CG administration stimulated P450(17)alpha expression mainly in the large luteal cell population. The results of this investigation indicate, for the first time, that the large luteal cell of the rat, in contrast to the small cell type, undergoes a dramatic increase in protein content with luteal development, and that with this increase in cell size there is a concomitant increase in the large cell capacity to produce steroids. This occurs as a direct result of the enhanced expression of SCP2, P450scc, adrenodoxin and adrenodoxin reductase, proteins specifically required to transport and process cholesterol for steroid production in the large luteal cell.


Hormonal and immunological characterization of the 32 kilodalton ovarian-specific protein. Recent studies from this laboratory have shown that the large luteal cell of the pregnant rat contains an abundant 32 kilodalton (32K) phosphoprotein which is up-regulated by estradiol. In order to assess the potential importance of this protein and to more fully understand its function, a specific polyclonal antibody was produced against the 32K and was used to examine its intraovarian localization, its tissue specificity, and its developmental regulation. Immunocytochemical localization of the 32K in the ovary of the pregnant rat found this protein to be selectively and abundantly expressed in the corpus luteum. Immunofluorescence study of small and large luteal cell populations clearly revealed an extensive localization of the 32K in the large luteal cells. Western blot analysis revealed that the 32K was absent from all steroidogenic and nonsteroidogenic tissues. Whereas this protein was absent from all other tissues examined in the rat, it was clearly expressed in corpora lutea of different animal species, including the mouse, hamster, cow, human, and pig. Although undetectable by immunohistochemistry, Western blot analysis showed this protein to be present in the follicle but at levels markedly lower than in the corpus luteum. Analysis of theca and granulosa cells revealed the presence of the 32K in both cell types. To further examine the developmental expression of this protein throughout gestation, Western blot analysis of microsomal fractions isolated from rat corpora lutea on days 3-21 of pregnancy was performed. The 32K was detected at low levels in early pregnancy, increased markedly on day 11, reached a peak on days 14-15, and remained elevated through day 21. Treatment with estradiol and PRL increased the content of the 32K in the corpus luteum. Human CG, known to cause follicular development to the preovulatory stage and to enhance luteal estradiol synthesis, also increased levels of the 32K in the corpus luteum, while it concomitantly decreased this protein in the follicle. In summary, the presence of a unique ovarian-specific 32 kilodalton protein has been established. This protein, which is present in low abundance in theca and granulosa cells, is localized to the large luteal cell and becomes abundantly expressed during midpregnancy, a time when there is a remarkable increase in luteal cell size and activity. Results of this study also demonstrate a multihormonal regulation of the 32K by tropic hormones. Thus, because of its apparent uniqueness and its timely and highly regionalized expression, the 32K may play a central role in the regulation of corpus luteum growth and function.


Expression of Fos-like proteins in gonadotropin-releasing hormone neurons of Syrian hamsters: effects of estrous cycles and metabolic fuels. In female mammals, reproduction is sensitive to the availability of metabolic fuels, and food deprivation has been shown to suppress pulsatile LH secretion, attenuate the preovulatory LH surge, and prevent ovulation. It has been suggested that food deprivation impairs fertility by reducing the secretion of GnRH by GnRH-producing neurons in the forebrain. A series of experiments tested this hypothesis by examining the effects of estrous cycles and manipulations of metabolic fuel availability on the expression of Fos-like proteins (Fos-IR) in GnRH-immunoreactive (GnRH-IR) neurons in the forebrain of Syrian hamsters. GnRH-IR neurons were detected in several areas, including the diagonal band of Broca (DBB), medial septum (MS), rostral medial preoptic area (mPOA), and caudal POA. In the more rostral regions (DBB and MS/mPOA), GnRH-IR neurons expressed Fos-IR almost exclusively on day 4 of the cycle, just after the preovulatory LH surge. However, in the caudal POA, GnRH-IR neurons expressed Fos-IR across the entire cycle, including days 1-3, when LH secretion is pulsatile. Food deprivation on days 1 and 2 of the cycle, which attenuates the LH surge and blocks ovulation in hamsters, significantly reduced the proportion of GnRH-IR neurons that expressed Fos-IR on days 2 and 4 (caudal POA) or only on day 4 (DBB and MS/mPOA). Suppression of fuel availability with insulin or 2-deoxy-D-glucose on day 1 of the cycle mimicked the effects of food deprivation and reduced the proportion of caudal POA GnRH-IR neurons that expressed Fos-IR. The results of these experiments suggest that in Syrian hamsters, there are separate populations of GnRH-IR neurons associated with pulsatile and surge modes of LH secretion. In addition, the fact that manipulations of metabolic fuel availability cause changes in the expression of Fos-IR in both populations of GnRH-IR neurons provides strong support for the hypothesis that nutritional infertility is due in part to decreased GnRH secretion.


Evidence for an autocrine/paracrine role for interleukin-6 in bone resorption by giant cells from giant cell tumors of bone. Interleukin-6 (IL-6) is a multifunctional cytokine whose role in osteoclastic bone resorption has not been clearly defined. Therefore, we have used giant cells, which express many features of osteoclasts, from giant cell tumors of bone as a model to examine the role that IL-6 may play in human osteoclastic bone resorption. We found that conditioned medium from 24-h cultures of highly purified giant cells (10(6)/ml) contained large amounts of IL-6 (37.9 +/- 8.8 ng/ml), similar to the amount of IL-6 produced by tumor stromal cells (29.8 +/- 11.5 ng/ml). Giant cells and stromal cells from giant cell tumors expressed IL-6 mRNA, as indicated by polymerase chain reaction analysis and in situ hybridization studies, and immunohistochemical techniques demonstrated that the giant cells expressed IL-6 receptors. The addition of a neutralizing antibody to IL-6 significantly decreased the area of dentine resorbed by purified giant cells in a dose-dependent manner, and the addition of IL-6 to cultures of purified giant cells pretreated with anti-IL-6 restored the resorbing capacity of the giant cells. These data suggest that IL-6 may act as both an autocrine and a paracrine factor for human osteoclasts and play an important role in the bone-resorbing capacity of these cells.


Ca(2+)-independent induction of acrosome reaction by protein kinase C in human sperm. We report that activated protein kinase C (PKC) can induce acrosome reaction independently of elevated Ca2+. Addition of 12-O-tetradecanoyl phorbol-13-acetate or the membrane-permeable diacylglycerol analog 1-oleoyl-2-acetylglycerol to ejaculated human sperm resulted in stimulation of acrosomal reaction (2- to 3-fold), provided the sperm underwent capacitation. Induction of acrosome reaction by 12-O-tetradecanoyl phorbol-13-acetate was blocked by the PKC inhibitor staurosporine or by down-regulation of endogenous PKC, but not by removal of extracellular Ca2+. Acrosome reaction was also enhanced by the Ca2+ ionophore ionomycin in a Ca(2+)-dependent, PKC-independent fashion. Immunohistochemical analysis with type-specific PKC antibodies revealed the presence of PKC alpha and PKC beta II in the equatorial segment, whereas PKC beta I and PKC epsilon staining was found in the principal piece of the tail. Acrosome reaction, thus far believed to be induced only by elevated Ca2+, can therefore be triggered by activated PKC in a Ca(2+)-independent fashion. The PKC subtypes potentially involved in acrosome reaction are most likely alpha and beta II, whereas the beta I- and epsilon-subspecies might be involved in regulation of flagellar motility of human sperm.


Expression of type I insulin-like growth factor receptors on human peripheral blood mononuclear cells. Insulin-like growth factor-I and -II (IGF-I and IGF-II), both of which bind to type I IGF receptors, can modulate certain functions of the immune system. We, therefore, studied the expression of type I IGF receptors on purified subpopulations of peripheral blood mononuclear cells. Using two-color flow cytometry, specific binding of a monoclonal antibody directed against the type I IGF receptor (alpha IR3) was found in every subpopulation. Relatively high numbers of receptors were detected on monocytes, natural killer cells, and CD4+ T-helper cells, an intermediate number of receptors on CD8+ suppressor/cytotoxic T-cells, and a relatively low number of receptors on B-cells. The presence of these receptors was confirmed by specific binding of [125I] IGF-I to purified subpopulations. alpha IR3 inhibited the binding of [125I] IGF-I. The specific binding of [125I]IGF-I to monocytes could be completely inhibited by IGF-II and insulin, but higher doses of these peptides were needed than of IGF-I. Scatchard analysis revealed the presence of 734 +/- 426 receptors/monocyte, with a Kd of 0.23 +/- 0.05 nM. A lower number of receptors (230 +/- 52), but with a higher affinity (Kd = 0.05 +/- 0.02 nM), was found on purified T-cells. The positive effect of IGF-I on natural killer cell cytotoxicity indicates that the type I IGF receptors on this cell type are functional. The possibility that IGF-I mediates hormonal effects on the immune system is discussed.


Set-point for parathyroid hormone release increases with postmaturational aging in the rat. The plasma concentration of immunoreactive PTH (iPTH) increases with postmaturational aging in both humans and animals. In the present study we determined the basal, maximally stimulated, and maximally suppressed levels of iPTH and the concentration of whole blood ionized calcium sufficient to produce half-maximal suppression of the plasma concentration of iPTH (set-point for PTH release) in male Fischer 344 rats aged 3, 6, 12, 18, 24, and 28 months. Basal iPTH increased 2.3-fold from 3 to 28 months of age, whereas basal blood ionized calcium remained unchanged. The set-point for PTH release increased steadily and significantly (P < 0.001) from 1.19 +/- 0.09 mM at 3 months to 1.37 +/- 0.13 mM at 24 months and then declined slightly to 1.32 +/- 0.11 mM at 28 months of age. Basal iPTH correlated significantly with set-point. Neither maximally stimulated nor maximally suppressed iPTH levels showed any significant change with advancing age. These results suggest that the age-related increase in basal plasma iPTH in the rat may be in part a consequence of an increase in the set-point for PTH release.


Progesterone regulation of a pregnancy-specific transcription repressor to beta-casein gene promoter in mouse mammary gland. Progesterone inhibits casein gene expression in the mammary gland during pregnancy. A pregnancy-specific mammary nuclear factor (PMF) is a transcriptional repressor that binds to the two separate sites of the beta-casein promoter region and mediates the inhibition of mouse beta-casein gene expression by progesterone. PMF has been detected in mouse mammary tissue during pregnancy, but not during the virginal or lactational periods. Southwestern hybridization showed that the PMF-binding sequence (TGAT/ATCA) bound a protein with a mol wt of 65 kilodaltons from mammary nuclear extract from pregnant mice, but not that from lactating animals. In this study the hormonal regulation of binding activity of PMF and its relationship to casein gene expression were also examined in vivo using ovariectomy of pregnant mice. Ovariectomy rapidly reduces the circulating progesterone and estrogen levels and induces lactogenesis in the mammary tissue of pregnant mice. After ovariectomy, the binding activity of PMF in the mammary gland was reduced to a very low level (< 5% of the sham-operated level) at 24 h, whereas the binding activity of another nuclear protein, octamer-like binding factor, was not altered 24 h after the operation. Progesterone injection at the time of ovariectomy inhibited the loss of PMF-binding activity, and the level of its activity was maintained at about 66% the level of sham-operated pregnant animals 24 h after steroid administration. The ability of progesterone to prevent the ovariectomy-mediated decrease in PMF-binding activity was specific, since other steroid hormones, such as estrogen, hydrocortisone, or testosterone, were incapable of doing so. In contrast to PMF, ovariectomy increased the level of beta-casein mRNA in mammary tissue, and this increase was inhibited by concomitant administration of progesterone, suggesting that the extent of beta-casein gene expression was inversely related to PMF-binding activity in mammary tissue. These results are consistent with the view that PMF plays a role in the regulation of casein gene expression by mediating the inhibitory action of progesterone.


Suramin inhibits bone resorption and reduces osteoblast number in a neonatal mouse calvarial bone resorption assay. The antineoplastic properties of suramin, a polyanionic agent with demonstrated antigrowth factor activity, are under evaluation in vitro, in vivo, and in clinical trials. Suramin has been shown to have antitumor activity in patients with advanced, hormone refractory prostate cancer. During these trials, significant resolution of osseous pain was observed in nearly three quarters of the patients treated with suramin. To evaluate the effect of suramin on bone cells, we studied the effect of suramin on bone resorption in a neonatal mouse calvarial assay. Suramin inhibited bone-resorbing activity in a dose-related fashion and had an additive effect with calcitonin. Calvaria pretreated with suramin had less bone-resorbing activity, fewer attached osteoblasts, and less medium alkaline phosphatase activity than control calvaria. Suramin also inhibited osteoclastic release of tritiated proline from labeled bone in a dose-dependent fashion. The effect of metastatic prostate carcinoma on bone is incompletely understood, but may be moderated by tumor-produced factors and/or cytokines. The effects of several such agents, therefore, were examined in combination with suramin. Bone resorption induced by PTH, epidermal growth factor, tumor necrosis factor, and a tumor-produced factor, PTH related-protein, was blocked by suramin. The ability of suramin to inhibit the bone-resorbing effects of several cytokines suggests that its mechanism may involve direct action on bone metabolism. Autoradiography performed on calvaria treated with labeled suramin demonstrated heavy deposition of suramin on the outer surface of the matrix, adjacent to osteoblasts and osteoclasts lining the outer table, suggesting that bone cells may be subject to high local concentrations of the drug, in keeping with this hypothesis.


Posttranslational processing of proenkephalin in AtT-20 cells: evidence for cleavage at a Lys-Lys site. Proteolytic processing of proenkephalin was examined in several subclones of AtT-20 cells stably transfected with rat proenkephalin cDNA (AT/PE cells). Proenkephalin is synthesized in both N-glycosylated and unglycosylated forms, as demonstrated by treatment with tunicamycin. RIAs and Western blot studies showed that AT/PE clones process proenkephalin at some, but not all, Lys-Arg sequences in a limited processing profile reminiscent of bovine adrenal chromaffin cells. Pulse-chase studies using Met5-enkephalin-Arg-Gly-Leu antiserum demonstrated that 50% of the precursor is processed within 1 h, and processing is complete after 2.5 h with the production of the 5.3-kilodalton (kDa) peptide. Further cleavage to the octapeptide Met5-enkephalin-Arg-Gly-Leu is minimal. Radiosequencing results verified the efficient cleavage of a Lys-Lys site within proenkephalin that resulted in the production of the 5.3-kDa peptide. Proenkephalin cleavage products stored within cells, which included the 5.3-kDa peptide, could be released upon stimulation of cells with BaCl2 (2-fold above basal levels), 8-bromo-cAMP or CRF (7- and 8-fold above basal levels, respectively), and a mixture of BaCl2 and 8-bromo-cAMP (20-fold above basal levels). An important difference between the processing of proenkephalin and the ACTH/endorphin precursor (POMC) in AtT-20 cells is efficient cleavage of a Lys-Lys site in proenkephalin and not in POMC. The ability of AT/PE to process proenkephalin in a natural manner makes it a suitable model system to investigate elements involved in the processing of proenkephalin at Lys-Lys sites.


Interleukin-1 stimulates release of insulin-like growth factor-I from neonatal mouse calvaria by a prostaglandin synthesis-dependent mechanism. Interleukin-1 alpha (IL-1 alpha) and IL-1 beta are cytokines produced by cells of the immune system and nonimmune cells, such as osteoblasts. IL-1 alpha and IL-1 beta have potent stimulatory effects on bone resorption and also have mixed effects on bone formation, suggesting that they play an important role in local regulation of bone turnover. The present study examined paracrine mechanisms underlying effects of IL-1 on bone formation. Bone formation is regulated at the local level by polypeptide growth factors such as insulin-like growth factor-I (IGF-I). IGF-I is a relatively abundant growth factor in bone matrix, is produced by osteoblasts, and stimulates osteoblastic cell proliferation and bone matrix synthesis. Recent evidence suggests that IGF-I also stimulates osteoclast formation and bone resorption. To determine whether increased or decreased IGF-I production in bone might mediate some of the effects of IL-1 on bone turnover, the effects of IL-1 alpha and IL-1 beta on IGF-I release from neonatal mouse calvaria in organ culture were examined. IL-1 alpha and IL-1 beta stimulated resorption and increased the release of IGF-I into the medium during 6 days of culture. Maximal stimulation of resorption occurred at lower concentrations of IL-1 alpha and IL-1 beta (1 pM) than were required for maximum stimulation of IGF-I release (10 pM). IL-1 beta also increased steady state levels of 7.5- and 0.9-kilobase IGF-I mRNA transcripts in total RNA extracted from cultured calvaria at 24 h. The cyclooxygenase inhibitor indomethacin (1 microM) inhibited IL-1 beta-induced prostaglandin E2 (PGE2) production and inhibited resorption and release of IGF-I from calvaria. Indomethacin had a smaller effect on PTH-induced IGF-I release and had no significant effect on PTH-induced resorption. These results suggest that the effects of IL-1 on resorption and release of IGF-I may be mediated in part by a prostaglandin-dependent mechanism. Consistent with this hypothesis, PGE1 and PGE2 stimulated resorption and release of IGF-I. The dose responses for PGE2 stimulation of resorption and stimulation of IGF-I release were equivalent. The results are consistent with the hypotheses that 1) IL-1 stimulates IGF-I production by bone cells, in part by PG-dependent mechanisms; 2) the effects of IL-1 and PGs on bone formation and resorption may be mediated by locally induced secretion of IGF-I and other growth factors in bone.


Ontogeny of aromatase messenger ribonucleic acid in mouse brain: fluorometrical quantitation by polymerase chain reaction. A sensitive method was developed for the determination of trace amounts of aromatase mRNA in various tissues. Aromatase mRNA was quantitated by subjecting it to reverse transcription in the presence of an internal standard RNA and then amplifying the resulting cDNA by polymerase chain reaction with a fluorescent primer. The tissue distribution of aromatase mRNA in mice was examined by this polymerase chain reaction method. Results showed that aromatase mRNA is expressed in the brain, testis, and ovary, but scarcely at all in other tissues, including the placenta. In mouse brain, aromatase is mainly located in the forebrain, especially the diencephalon. In adult male and female diencephala, aromatase mRNA levels were 0.022 +/- 0.004 and 0.014 +/- 0.003 attomoles/micrograms total RNA, respectively. Aromatase in the diencephalon is known to participate in brain differentiation and sexual behavior, so changes in its mRNA levels in the brain during development were examined. Aromatase mRNA was first detected in the 12-day-old fetus and was found to increase rapidly during the fetal and neonatal periods. Its mRNA levels in male and female brains reached maxima of 0.068 +/- 0.008 and 0.059 +/- 0.006 attomoles/micrograms total RNA, respectively, 3-4 days after birth and then gradually decreased to adult levels. These observations are consistent with previous data indicating that the marginal period of neonatal imprinting of sexual differences is within a week after birth and suggest that brain aromatase may be important in sexual differentiation and behavior.


Potential involvement of the carboxy-terminus of the Glut 1 transporter in glucose transport. The role of the carboxy-terminal domain of the Glut 1 glucose transporter was investigated using an antipeptide antibody to the C-terminal part of the molecule. The study was performed in fibroblasts transfected with the cDNA coding for the human insulin receptor. These cells acutely respond to insulin for glucose transport. Using antipeptide antibodies to Glut 1 and Glut 4, we first established that these cells expressed only Glut 1. Then, to define the role of the C-terminal part of Glut 1 in glucose transport, the antibodies were loaded into the cells by electroporation. When anti-Glut 1 immunoglobulins were introduced into the cells, a 60% increase in basal deoxyglucose and 3-O-methylglucose transport was observed compared to that in cells electroporated with nonimmune immunoglobulins. The stimulatory action of the antipeptide was not due to an increase in the total amount of transporters. It was found only at low glucose concentrations, suggesting that the affinity of the transporter, rather than its maximal capacity, was changed. Finally, the effect of antibody was additive to that of insulin. The interaction between the anti-Glut 1 antibody and the carboxy-tail of the transporter seems to lead to an increase in the intrinsic activity of the transporter, suggesting that this part of the molecule could be implicated in the regulation of glucose uptake.


The carboxy-terminus of parathyroid hormone is essential for hormone processing and secretion. The biological function of the carboxy-terminal portion of the PTH molecule is unknown. We hypothesized that the carboxy-terminus of PTH may be essential for hormone processing and secretion. To ascertain the potential role of the carboxy-terminus in transport through the secretory pathway, we constructed a series of carboxy-terminally deleted mutants of human preproPTH. PreproPTH and these truncated peptides were expressed in cell-free extracts and in acutely transfected COS 7 cells. Intact preproPTH(1-84) (115 residues) was processed to proPTH(1-84); the PTH was secreted very efficiently. In contrast, translocation across the membrane of the endoplasmic reticulum and signal sequence cleavage were mildly impaired for PTH(1-52) (83 residues), moderately impaired for PTH(1-40) (71 residues), and dramatically impaired for PTH(1-34) (65 residues). Subsequent cleavage of the prosequence and secretion from intact cells were also dramatically impaired for PTH(1-52) and undetectable for PTH(1-40) and PTH(1-34). We conclude that more than 65 residues are needed to cross the endoplasmic reticulum and that more than 83 residues are needed for efficient prosequence cleavage and secretion. Perhaps similar requirements for full transport through the secretory pathway explains why most secreted peptides are synthesized as long propeptides of at least 80 residues.


Selective removal of glycosylation sites from sex hormone-binding globulin by site-directed mutagenesis. Sex hormone-binding globulin (SHBG) is a homodimeric plasma glycoprotein with high affinity for sex steroid hormones. It contains two N-linked carbohydrate chains and one O-linked oligosaccharide per subunit, but their functional significance is not known. Site-directed mutagenesis of a human SHBG cDNA has enabled us to selectively disrupt the known glycosylation sites individually and in various combinations. The mutant cDNAs were expressed in Chinese hamster ovary (CHO) cells, and it was found that the presence of carbohydrates is not an absolute requirement for the secretion of SHBG from these cells, but the absence of both N-linked oligosaccharides reduced the amount of SHBG in the culture medium. In addition, the affinity and specificity of SHBG for steroid ligands was unaffected by the lack of one or more carbohydrate chains. Proportionally greater amounts (26-31%) of the mutants lacking a single N-linked carbohydrate chain failed to interact with Concanavalin-A. (Con-A) compared to normal SHBG produced by CHO cells (15%). Western analysis demonstrated that both consensus sites for N-glycosylation are used and that the typical heavy [mol wt (M(r)), approximately 51,000] and light (M(r), approximately 47,000) subunit size-heterogeneity was maintained regardless of the absence of an O-linked carbohydrate at residue 7. Furthermore, the SHBG mutants containing only one N-linked oligosaccharide comprise only a single subunit with a M(r) of approximately 47,000. This suggests that the heavy subunit contains two N-linked oligosaccharides, while only one of these sites is used on the light subunit. The M(r) of the various SHBG mutants were also examined by gel filtration, and this indicated that they are all produced as homodimers and that carbohydrates are not involved in subunit association.


Diminished insulinotropic effects of gastrin-releasing peptide in pregnant sheep are reproduced by progesterone treatment of nonpregnant animals. Gastrin-releasing peptide (GRP) is insulinotropic in several species, but possible alterations in this action during pregnancy have not been explored. Therefore, changes in plasma insulin and glucagon concentrations were examined in response to exogenous GRP in nonpregnant and pregnant sheep that were feed restricted, fed ad libitum, or infused with glucose. Administered GRP provoked insulin and glucagon release in pregnant and nonpregnant fed animals. This effect was reduced with feed restriction and potentiated in the presence of glucose. The responses were less in pregnant than in nonpregnant animals. Interpretation of this result, however, was confounded by lower plasma immunoreactive concentrations of GRP achieved in pregnant than in nonpregnant sheep in response to the same infusion rate (picomoles per kg BW) of exogenous GRP. Therefore, nonpregnant ovariectomized sheep were treated with estradiol (E2) or E2 plus progesterone (P4), given to reproduce circulating steroid levels in pregnancy, in order to examine insulinotropic responses to GRP in the absence or presence of concurrent glucose administration. Similar plasma GRP and glucose concentrations were achieved by exogenous infusions in the different groups of nonpregnant animals treated with steroids and in control animals not receiving steroids. E2 alone did not alter the insulin response to GRP compared to control, but E2 plus P4 treatment attenuated these responses to values similar to those in pregnant animals. We conclude that GRP is insulinotropic in sheep, and this action is modulated by the circulating glucose concentration. The response to GRP is less in pregnant than in nonpregnant animals, and this attenuation is mimicked in nonpregnant animals treated with E2 plus P4. We suggest that in species such as sheep, GRP is a potentiator of glucose-stimulated insulin release. This action is diminished in response to the altered endocrine environment of pregnancy and may contribute to the metabolic changes that occur at this time.


Insulin-like growth factors selectively stimulate spermatogonial, but not meiotic, deoxyribonucleic acid synthesis during rat spermatogenesis. The in vitro effects of insulin-like growth factor-I (IGF-I), insulin-like growth factor-II (IGF-II), truncated IGF-I, insulin, and human GH (hGH) on premitotic and premeiotic DNA synthesis of adult rat germ cells in vitro were investigated. Two-millimeter segments of seminiferous tubules from four different stages containing type A4-spermatogonia (stage I), type B spermatogonia (stage V), resting preleptotene spermatocytes (stage VIIa), and preleptotene spermatocytes in the S-phase (stage VIII-IX), respectively, were isolated by transillumination-assisted microdissection. They were cultured in serum-free medium at 34 or 37 C with and without growth factors, labeled for 4 h with tritiated thymidine, and harvested at 24, 48, and 72 h. Spontaneous progression of spermatogenesis was noted at both incubation temperatures, with a more rapid rate at 37 C. IGF-I significantly stimulated [3H]thymidine uptake in originally stage I and stage V tubule segments (type A4 and B spermatogonia, respectively) after 48 h of culture at 37 C. Improved maintenance of the DNA synthesis of stage VIII-IX tubules was found after 48 h at 37 C and 72 h at 34 C. Truncated IGF-I produced a similar response, but was more potent. IGF-II showed slight stimulation of stage V tubules after 72 h at both 34 and 37 C and maintenance of stage VIII-IX tubules after 48 h at 37 C and 72 h at 34 C. hGH was effective only at 34 C, showing slight stimulation of stage I tubule segments after 48 and 72 h of incubation. Insulin at high concentrations was effective only at 37 C and stimulated DNA synthesis in stages I, V, and VIIa after 48 h and stages V and VIIa after 72 h of incubation. It is concluded that IGFs stimulate premitotic DNA synthesis of rat germ cells in vitro and may also maintain premeiotic DNA synthesis. Whether the slight response to hGH is mediated via local production of IGF-I by the tissue cultures remains to be investigated. As IGF-I and IGF-II are locally produced in the testis, the present results suggest that these factors have a selective paracrine or autocrine role in the regulation of spermatogonial proliferation during spermatogenesis.


Interaction between activin and follicle-stimulating hormone-suppressing protein/follistatin in the regulation of basal inhibin production by cultured rat granulosa cells. There is evidence that FSH-suppressing protein (FSP) antagonizes the action of activin on the differentiation of rat granulosa cells by binding activin in vitro. We tested the interaction of activin and FSP in this in vitro system by examining the effects of FSP on activin dose-related stimulation of immunoreactive inhibin release by rat granulosa cells. Granulosa cells (2 x 10(5) viable cells/well) from diethylstilbestrol-treated immature rats were cultured for 48 h in McCoy's 5a serum-free medium with additives and increasing doses of bovine FSP (0-30 nM) and human recombinant activin (0-20 nM). Inhibin was measured in the medium by RIA. Activin caused a dose-related increase in basal inhibin production, which was maximal between 4-10 nM activin (ED50, 0.6 nM). With the addition of FSP, an apparent increase in the ED50 of the activin dose-response curves was observed, but there were no changes in the maximum response. This pattern closely resembled that of chemical antagonism of an agonist by an agent that binds with relatively high affinity to form a biologically inactive complex. Based on this premise, apparent high affinity activin binding to FSP was determined by Scatchard analysis to have a Kd of 0.13 +/- 0.07 nM (mean +/- SD) and to occur in a 2:1 or greater FSP/activin molar ratio. These data support the proposition that the antagonistic effect of FSP on activin is due to the formation of an inactive complex.


The stimulation of liver angiotensinogen by glucocorticoids depends on the type of steroid and its mode of administration. Glucocorticoids are well known inducers of transcription of the liver angiotensinogen (AOG) gene. However, the doses and the conditions under which they exert this effect in vivo are not known. To investigate this question, we have implanted rats with wax pellets containing 80% corticosterone (B). These pellets increased plasma B and induced clear signs of hypercorticism. However, they did not stimulate plasma AOG, whereas acute injections of dexamethasone (DEX) had a robust effect. In additional experiments, we have determined that: 1) chronic exposure to DEX was less effective than acute DEX in stimulating the production of liver AOG in rats and AOG secretion by rat hepatoma cells; 2) at maximally effective doses, B stimulated the production of AOG by hepatoma cells less effectively than DEX; and 3) DEX had less effect on AOG secretion than on AOG messenger RNA concentration, both in vivo and in vitro. All three mechanisms may have contributed concomitantly to the absence of response of plasma AOG to mild and chronic elevations of plasma B. These results suggest that glucocorticoids are unlikely to be primary regulators of liver AOG.


Opposite effects of insulin-like growth factor-I on the formation of trabecular and cortical bone in adult female rats. We have previously found that in intact adult female rats, 17 beta-estradiol (E2) stimulates trabecular bone formation, which can be detected by triple fluorochrome labeling within a few days of commencing E2 treatment. We have also observed that E2 stimulates trabecular bone formation in ovariectomised (Ovx) rats given 3-amino-1-hydroxypropylidene bisphosphonate (AHPrBP). This anabolic action of E2 is presumably mediated through the local release of growth factors, of which insulin-like growth factor-I (IGF-I) is a strong contender. To assess the role of IGF-I as mediator of the anabolic action of E2 on rat trabecular bone, we examined the effect of daily sc administration of IGF-I (200 micrograms/kg) for 17 days on trabecular bone formation at the proximal tibial metaphysis in experiments using the above two models. Intact animals received fluorochrome labels on days 1, 8, and 15 of IGF-I treatment, and Ovx animals received AHPrBP (0.3 mg/kg) on days 1, 8, and 15 and fluorochrome labels on days 8 and 15. Trabecular surfaces covered by first and second labels only (termed arrested surfaces), second and third labels only (induction surfaces), and all three labels (persisting surfaces) were assessed in intact animals; double labeled surfaces were assessed in Ovx animals. We found that IGF-I increased the longitudinal growth rate in both intact rats and Ovx animals given AHPrBP. However, to our surprise, we found that IGF-I inhibited trabecular bone formation in intact animals, reducing both arrested and persisting trabecular surfaces by approximately 50%. This was associated with a doubling of periosteal bone formation, as measured at the tibial diaphysis. In Ovx animals given AHPrBP, the AHPrBP markedly inhibited trabecular bone formation compared with that in Ovx controls, and IGF-I was without effect. We conclude that, in contrast to E2, IGF-I acts to increase the length and circumference of long bones and, perhaps through reactive mechanisms such as mechanical compensation, reduces rather than stimulates trabecular bone formation.


Insulin-like growth factor-I reverses the impairment of wound healing induced by corticosteroids in rats. Corticosteroids are widely used therapeutic agents that have as a major side-effect the impairment of wound healing. Two hypotheses were tested: 1) that antiinflammatory corticosteroids decrease the local insulin-like growth factor-I (IGF-I) response to injury; and 2) that locally administered IGF-I would overcome methylprednisolone-mediated suppression of healing. The IGF-I concentration was measured in fluid from wire mesh cylinder wounds in rats given saline or methylprednisolone im. Rats receiving 8 and 16 mg had decreased wound IGF-I levels of 32% and 56%, respectively, compared to the saline-injected controls. IGF-I was infused (15 micrograms/day) by osmotic minipumps into wire mesh cylinder wound chambers of methylprednisolone (8 mg)-treated rats for 7 and 14 days. Methylprednisolone decreased wound DNA to 21%, hydroxyproline to 30%, and total protein to 5% of the values found in saline-infused controls. A 14-day treatment with IGF-I completely reversed the effect of methylprednisolone and increased DNA, hydroxyproline, and total protein to 216%, 109%, and 96% of saline control values, respectively. In conclusion, corticosteroids depressed wound IGF-I concentrations in rats, and an infusion of IGF-I into wound chambers reversed the corticosteroid-induced impairment of wound healing, as determined by the DNA, hydroxyproline, and total protein contents in wound chambers.


Thyroid hormone-dependent redistribution of the 55-kilodalton monomer of protein disulfide isomerase in cultured glial cells. In addition to the effects of thyroid hormone that are mediated through interaction with chromatin-associated receptors, T4 modulates the activity of the cellular content of the membrane-associated protein type II iodothyronine 5'-deiodinase (5'D-II) by regulating its degradation through an actin-dependent extranuclear mechanism. Under the influence of thyroid hormone, the substrate-binding subunit of 5'D-II is translocated from the plasma membrane to an intracellular microfilament-associated pool. In glial cells, a 55-kilodalton (kDa) protein (glial-p55), which was shown to be identical to the 55-kDa monomer of protein disulfide isomerase (PDI) also demonstrates a similar T4-dependent association to the F-actin microfilaments. To explore the role of glial-p55 in the extranuclear effect of thyroid hormone in glial cells, the effects of thyroid hormone on the subcellular localization of glial-p55 were further examined. The current study demonstrates the presence of two pools of glial-p55. While the majority of glial-p55 is associated with endoplasmic reticulum and represents PDI, approximately 25% of glial-p55 is cytosolic in the absence of thyroid hormone. Cytosolic glial-p55 is lost from the cells after mild permeabilization with saponin, and treatment of cells with T4 causes the shift of glial-p55 from the cytosolic pool to the subcellular fractions that contain the actin cytoskeleton. Crude microsomal preparations were prepared which contain membranes, microfilaments, and other particulate cell structures. In the absence of thyroid hormone, glial cells lack an intact actin cytoskeleton, and glial-p55 is easily removed from these preparations by conditions that remove extrinsic membrane proteins like PDI, such as alkaline pH and detergent extraction. In contrast, glial-p55 is not removed from the crude microsomes prepared from thyroid hormone-replete glial cells that contain an intact actin cytoskeleton. Since previous work in our laboratory indicated that glial-p55 becomes actin associated in a thyroid-dependent manner along with the substrate-binding subunit of 5'D-II, this study suggests that the 55-kDa monomer of PDI may play a role in the thyroid hormone-dependent regulation of actin polymerization and the degradation of 5'D-II.


Antipeptide polyclonal antibodies specifically recognize each human thyroid hormone receptor isoform. We characterized four antipeptide polyclonal antibodies (abs) able to specifically recognize each thyroid hormone receptor (TR) isoform. The abs immunoprecipitated both the in vitro synthesized receptor and the receptor expressed in E. coli and their specificity was confirmed by competition studies and immunohistochemistry. Ab activity measured by enzyme-linked immunosorbent assay decreased after preabsorption of each ab with the immunizing peptide or the specific receptor protein expressed in E. coli. No specific activity was detectable in enzyme-linked immunosorbent assay, no nuclear staining was observed after affinity column immunoabsorption, and the specific bands obtained in Western blot analysis disappeared after preabsorption with the specific TR isoform expressed in E. coli. By immunohistochemical studies we detected coordinate expression of each receptor isoform in most tissues examined. However, in heart and muscle, the beta-isoform is expressed at a very low level compared to the alpha-isoform in spite of the significant TR beta mRNA levels previously demonstrated by Northern blot analysis. We also demonstrated a different pattern of distribution of alpha- and beta-isoforms in rat testis. In this tissue the TR alpha is significantly expressed in spermatogonia nuclei, but in spermatids the beta-isoform is predominant, and only the TR beta is detectable in mature spermatozoa.


Carbohydrate moiety of follitropin receptor is not required for high affinity hormone-binding or for functional coupling between receptor and guanine nucleotide-binding protein in bovine calf testis membranes. Previously, we have extensively studied FSH-receptor interactions using bovine calf testis membranes, and demonstrated that the high-affinity FSH binding to receptors and coupling of FSH receptors with guanine nucleotide-binding protein (Gs protein) in a GTP-sensitive state are important initial events in FSH action. In this study, using the same plasma membrane system, we examined the glycoprotein nature of the FSH receptor and determined the contribution of carbohydrate moieties to these functions of the FSH receptor. Our approach involved enzymic deglycosylation of FSH receptors present in calf testis plasma membranes and then removal of incompletely deglycosylated FSH receptors by lectin affinity chromatography. Following treatment of testis membranes with peptide N-glycosidase, the receptor, as identified by ligand-blot analysis, had a higher electrophoretic mobility indicating a decrease in M(r) from 240-200K. Treatment of testis membranes with neuraminidase caused a reduction (to approximately 225K) in the size of the receptor consistent with desialylation. However, digestion with O-glycosidase (endo-alpha-N-acetylgalactosaminidase) did not affect the mobility of the FSH receptor. These results suggest that bovine testis FSH receptor contains predominantly N-linked oligosaccharide chains, a finding which is consistent with recent predictions that N-linked glycosylation, but not O-linked glycosylation sites are present in cloned FSH receptor from rat testis. Moreover, calf testis membranes after treatment with peptide N-glycosidase F, were solubilized with Triton X-100 under optimum conditions that preserve the physical and functional coupling of FSH receptors with guanine nucleotide-binding protein, and then subjected to lectin affinity chromatography. Scatchard analysis indicated that intact and deglycosylated FSH receptors bound 125I-human FSH with similar affinities. In the presence of GTP, the binding of 125I-human FSH to intact and deglycosylated receptors decreased similarly and in a noncompetitive manner. Treatment of testis membranes with NAD plus cholera toxin, but not NAD plus pertussis toxin, eliminated the GTP effect on FSH binding to enzymic deglycosylated as well as intact receptors, suggesting that the guanine nucleotide binding protein mediating GTP regulation of FSH binding in these membranes is probably Gs protein. Our results suggest that the bovine testis FSH receptor contains predominantly N-linked oligosaccharide chains consistent with recently predicted N-linked glycosylation sites of cloned FSH receptor of rat testis. The bovine testis FSH receptor does not require N-linked carbohydrate for high-affinity hormone binding.(ABSTRACT TRUNCATED AT 400 WORDS)


Luteal control of endometrial receptivity and its modification by progesterone antagonists. The objective of this study was to determine preimplantational effects of progesterone antagonists (PA) on the cell biology of the endometrium, on corpus luteum (CL) function and on the complex interactions between these two organs. The PA onapristone (ZK 98.299) or lilopristone (ZK 98.734) was given to pseudopregnant rabbits at days 5, 6, and 7 p.hCG. Three treatment protocols were investigated: Exp I, onapristone or lilopristone treatment only; Exp II, onapristone treatment after hysterectomy at day 1 p.hCG; Exp III, onapristone treatment together with 17 beta-estradiol, which represents the ultimate luteotropic hormone in the rabbit. In Exp I, onapristone and lilopristone gave rise to endometrial regression (inhibition of epithelial proliferation and differentiation, increase of apoptosis). Simultaneous addition of 17 beta-estradiol in Exp III did not alter these findings. A rapid luteolysis was found in Exp I. In Exp II and III, however, onapristone was unable to impair luteal development and function. Due to the unaffected CL in Exp III and due to the 17 beta-estradiol substitution, the endometrium was capable of starting a new transformation, which met all requirements for receptivity at day 12 p.hCG. Transfers of day 4 p.c. blastocysts from untreated donors into such delayed secretion recipient rabbits at days 12 p.hCG resulted in normal implantations and normal embryonic development. Contrary to Exp III, the missing of any luteotropic substitutions in Exp I resulted in a complete inhibition of further uterine transformation. The present findings suggest that PA can exert a direct inhibitory effect on the endometrium, which is followed by an indirect luteolytic effect via endometrial mediators. The simultaneous addition of a proper luteotropic signal to the PA protocol results in survival of CL. Furthermore, this prolongation of the CL life span can be interpreted as a functional dissociation of the endometrium from the CL.


Neuropeptide-Y innervation of beta-endorphin-containing cells in the rat mediobasal hypothalamus: a light and electron microscopic double immunostaining analysis. Central administration of neuropeptide-Y (NPY) inhibits pituitary LH release in ovariectomized rats and stimulates LH release in intact and ovariectomized rats pretreated with ovarian steroids. Although the precise neural mechanism of this dual effect of NPY is not known, experimental evidence suggests an underlying interaction between hypothalamic NPY and the inhibitory beta-endorphin (beta END) systems in the neuroendocrine regulation of pituitary LH release in the rat. The present study was undertaken to examine the morphological basis of the interaction between these two peptidergic systems in the hypothalamus. Sections of the mediobasal hypothalamus of colchicine-pretreated female rats were double immunostained for NPY and beta END and examined by light and electron microscopy. The light brown diaminobenzidine reaction was used to visualize beta END cells, while NPY neurons were labeled with a dark blue nickel ammonium sulfate-intensified diaminobenzidine reaction. Under the light microscope, a dense network of NPY-immunoreactive axons and axon terminals was observed in close apposition with beta END-immunoreactive neurons throughout the medial basal hypothalamus. Electron microscopic examination revealed that NPY-immunoreactive boutons formed axosomatic and axo-dendritic synaptic connections with beta END cells. A majority of these synaptic membrane specializations appeared asymmetrical [corrected]. In light of the previous evidence of excitatory and inhibitory effects on LH release and the existence of direct synaptic connections between NPY and LHRH neurons in the hypothalamus, the current results imply that the dual effects of NPY on LH secretion may involve modulation of LHRH secretion, both by the direct route and indirectly through the hypothalamic beta END system.


1 alpha,25-dihydroxyvitamin D3 regulates in vivo production of the third component of complement (C3) in bone. We previously reported that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] specifically stimulates production of the third component of complement (C3) by murine osteoblastic cells and marrow-derived stromal cells (ST2) in vitro. In the present study we examined tissue-specific production of C3 in vivo in vitamin D-deficient mice, some of which received supplemental 1 alpha,25-(OH)2D3. Western blot analysis indicated that the C3 protein band in bone was undetectable in vitamin D-deficient mice, but became distinct 48 h after 1 alpha,25-(OH)2D3 administration. The mRNA expression of C3 in bone was also undetectable in vitamin D-deficient mice and appeared as early as 24 h after 1 alpha,25-(OH)2D3 administration. mRNA expression apparently preceded the appearance of C3 protein. In contrast, there was no significant difference in the expression of hepatic C3 mRNA among normal mice fed laboratory chow and vitamin D-deficient mice with and without 1 alpha,25-(OH)2D3 administration. The serum concentration of C3 in vitamin D-deficient mice was almost identical to that in normal mice and was unchanged after 1 alpha,25-(OH)2D3 administration. 1 alpha,25-(OH)2D3 receptor (VDR) mRNAs were detected in the kidney and intestine, whereas no appreciable mRNA expression of VDR occurred in the liver. Osteopontin mRNA was expressed in response to 1 alpha,25-(OH)2D3 in the kidney, but not in the intestine. Immunohistochemical studies showed that in normal mice, the C3 protein was located mainly in the periosteal regions of calvaria and on the surfaces of bone trabeculae in the tibial metaphyses. These results demonstrate that 1 alpha,25-(OH)2D3 tissue-specifically regulates in vivo production of C3 in bone. The production of bone C3 cannot be attributed to the presence of VDR alone, and we speculate that other tissue-specific factors are required.


Endothelin-1 as a luteinization inhibitor: inhibition of rat granulosa cell progesterone accumulation via selective modulation of key steroidogenic steps affecting both progesterone formation and degradation. Endothelin (ET)-1, is a 21 amino acid vasoactive peptide subject to regulation by cellular oxygen tension. However, an increasing body of information now suggests that ET-1 is a multifunctional peptidergic regulator the actions of which are not limited to the vascular system. Although ET-1 has been shown to inhibit the gonadotropin-supported accumulation of progesterone by cultured granulosa cells, the precise cellular mechanism(s) involved remain unknown. It was therefore the objective of this study to examine in greater detail the effects of ET-1 on progestin economy in cultured granulosa cells from immature rats. Treatment with ET-1 was inhibitory to the FSH-supported accumulation of progesterone in a dose-dependent manner, an action characterized by a median inhibitory dose of 2 x 10(-11) M and a maximal inhibitory effect of 90%. This inhibitory action of ET-1 was reversible following extensive washing and could not be accounted for by a decrease in the viable cell mass. Evaluation of the activities of progesterone-forming enzymes revealed ET-1 to be a potent (P < 0.01) inhibitor of cholesterol side-chain cleavage and 3 beta-hydroxysteroid dehydrogenase (HSD)/isomerase (76.1 +/- 1.2% and 47.3 +/- 8.6% inhibition, respectively). Cellular radiolabeling with [3H]pregnenolone confirmed an ET-1-induced inhibition of the FSH-supported accumulation of radiolabeled progesterone. However, this effect was concomitant with enhancement of the accumulation of more distal metabolites, i.e. 20 alpha-dihydroprogesterone, 5 alpha-pregnane-3 alpha, 20 alpha-diol, and 5 alpha-pregnane-3 alpha-ol-20-one. Analysis of the FSH-supported activities of the progesterone-degrading enzymes revealed ET-1 as a potent (P < 0.05) stimulator of 20 alpha-HSD and 5 alpha-reductase (3.6 +/- 1.0 and 1.7 +/- 0.3-fold, stimulation respectively). In contrast, no significant changes were observed in 3 alpha-HSD activity. Taken together, our findings demonstrate that the ET-1 induced inhibition of gonadotropin-supported progesterone accumulation constitutes a complex phenomenon wherein ET-1 inhibits the activities of steroidogenic enzymes concerned with progesterone formation while enhancing the activities of enzymes concerned with progesterone degradation. We speculate that ET-1, possibly of intraovarian origin, acts as a luteinization-inhibitor to suppress premature luteinization at a time when continued preovulatory expression of ET-1 (in the intact but not ruptured follicle) may be contingent upon relative intrafollicular hypoxia.


Vitamin E protects hypothalamic beta-endorphin neurons from estradiol neurotoxicity. Estradiol valerate (EV) treatment has been shown to result in the destruction of 60% of beta-endorphin neurons in the hypothalamic arcuate nucleus. Evidence suggests that the mechanism of EV-induced neurotoxicity involves the conversion of estradiol to catechol estrogen and subsequent oxidation to free radicals in local peroxidase-positive astrocytes. In this study, we examined whether treatment with the antioxidant, vitamin E, protects beta-endorphin neurons from the neurotoxic action of estradiol. Our results demonstrate that chronic vitamin E treatment prevents the decrement in hypothalamic beta-endorphin concentrations resulting from arcuate beta-endorphin cell loss, suggesting that the latter is mediated by free radicals. Vitamin E treatment also prevented the onset of persistent vaginal cornification and polycystic ovarian condition which have been shown to result from the EV-induced hypothalamic pathology.


Reversion of thyrotoxic atrial fibrillation in hypothyroid state after radioiodine treatment. Twenty patients with thyrotoxic Basedow's disease complicated by atrial fibrillation lasting more than one month despite treatment with antithyroidal drugs were treated with radioiodine supplemented with an antithyroidal drug or inorganic iodine. We classified the 20 patients on the basis of atrial fibrillation reversion into two groups, one with reversion (group I) and the other without reversion (group II). In all 12 patients in group I, T4 and T3 decreased to hypothyroid levels in 3.2 +/- 1.3 months, and one month later all patients had their sinus rhythm restored while T4 and T3 remained below normal (2.6 +/- 1.1 micrograms/dl and 77.9 +/- 34.4 ng/dl, respectively). Although T4 and T3 also decreased within 3.5 +/- 1.8 months in all 8 patients in group II, one month later, atrial fibrillation persisted while T4 and T3 (10.4 +/- 5.3 micrograms/dl and 157.7 +/- 67.5 ng/dl, respectively) rose significantly compared to those in group I (P less than 0.001 and P less than 0.01, respectively). For reversion of atrial fibrillation it is important that the onset of hypothyroidism is rapidly induced by radioiodine and that hypothyroidism continues for at least one month.


Possible role of cingulate cortex in regulating sexual behavior in male rats: effects of lesions and cuts. The role of the cingulate cortex in regulating male sexual behavior was studied in testosterone propionate-treated castrated male rats. Males with lesions in the anterior part of the cingulate cortex showed lower levels of mount, intromission and ejaculation activities than sham-operated control males and males with lesions in the posterior part of the cingulate cortex or the frontal cortex. In male rats in which lateral connections of the anterior cingulate cortex were bilaterally interrupted by sagittal cuts, the sexual activity was much lower than in the control rats, being comparable to that of the anterior cingulate cortex lesion group, but transection of the anterior connections by a transverse cut made in the anterior part of the anterior cingulate had no effect. These results suggest that the anterior cingulate cortex and its lateral connections are critical in regulating male sexual behavior in male rats.


Regulation of androgen receptor by androgen and epidermal growth factor in a human prostatic cancer cell line, LNCaP. The effects of androgen and epidermal growth factor (EGF) on cell proliferation and the expression of mRNA and protein of androgen receptor (AR) were examined in an androgen-sensitive human prostatic cancer cell line, LNCaP, by Northern and Western blot analyses. The addition of 1 nM dihydrotestosterone (DHT), at which the proliferation of the cells was most stimulated, did not change the level of AR mRNA but increased the level of AR protein by reducing the turnover rate of the AR protein. EGF also stimulated the proliferation of the cells but repressed the expression of AR mRNA and protein. This repression was found to be exerted primarily at the level of transcription. When DHT and EGF were added simultaneously to the cells, the level of AR mRNA was reduced to the same degree as was accomplished by the addition of EGF alone. On the other hand, the level of AR protein increased but this increase was about 70% of that attained following the addition of DHT alone. The stimulatory effects of EGF and DHT on cell proliferation were found to be additive. These results indicate that EGF down-regulates the level of AR mRNA and thereby also that of AR protein irrespective of the presence of DHT, and that EGF stimulates the proliferation of LNCaP cells through a different pathway from that of DHT.


A case of hypothyroidism with simultaneous presence of stimulating type anti-thyrotropin (TSH) receptor antibodies and anti-thyroxine (T4) autoantibodies. We have examined a hypothyroid patient with stimulating type anti-thyrotropin (TSH) receptor antibodies and without blocking type anti-TSH receptor antibodies. Although she had high serum TSH (240 microU/ml) and low free triiodothyronine (FT3, 0.49 pg/ml) concentrations, which agree with physical findings of hypothyroidism, she had an unusually high free thyroxine (FT4) concentration (3.56 ng/dl). Incubation of her serum with 125I-T4, followed by precipitation with 12.5% polyethylene glycol (PEG) disclosed a higher binding of 125I-T4 (34.4%) than in normal controls, being 5-7%. In addition, binding of 125I-T4 to her serum gamma-globulin was completely displaced by the addition of unlabelled T4. From these results it was concluded that her serum contained anti-T4 autoantibodies. Treatment with synthetic T4 was begun and her thyroid function was monitored by sensitive TSH radioimmunoassay (RIA) and RIA of FT4 after PEG treatment. Since both sensitive TSH RIA and FT4 RIA results after PEG treatment give results concordant with the physical findings, it was concluded that both of the RIA results are useful for the evaluation of thyroid function in patients with thyroid hormone autoantibodies.


A child with pituitary gigantism and precocious adrenarche: does GH and/or PRL advance the onset of adrenarche? We describe a female child with pituitary gigantism and precocious adrenarche. From two years of age she showed unusual overgrowth, and at 5 years old she was 133.5 cm (+ 5.5 SD) tall and weighed 40.5 kg. Her precocious manifestations were public hair, acne vulgaris, hirsutism, and advanced bone age. Endocrinological examination revealed markedly increased serum growth hormone (GH) and prolactin (PRL), which responded paradoxically to a TRH test. In addition, the concentrations of serum dehydroepiandrosterone (DHA) and its sulfate (DHAS) were increased to adult levels, moving in accordance with changes in ACTH, which suggested that these androgens were secreted from the adrenal glands functionally. These androgens seemed to be responsible for her partial precocity. Prior reports have suggested that GH and/or PRL overproduction might have played a role in the induction of adrenarche. Also, in previous reports of 9 gigantism patients under 10 years old, the manifestation of precocious adrenarche was suggested in 8. Further investigation of the influence of GH and PRL on adrenal androgen production in children with pituitary gigantism is required. On the other hand, in short children with normal GH secretion, attention should be paid to whether or not the GH therapy in early childhood induces precocious adrenarche.


Synergistic actions of cytokines and growth factors in enhancing porcine granulosa cell growth. In our studies of the growth-promoting effect of a cytokine, interleukin-1 (IL-1), on cultured porcine granulosa cells, we found that the potency of IL-1 action correlated with the serum concentration in the culture medium and that IL-1 acted synergistically with insulin to increase the number of cells in the presence of low serum concentrations (0.1-1%). With granulosa cells maintained in a quiescent state under serum-free conditions, we therefore examined the effects of combined treatment with IL-1 and peptide growth factors, including insulin, on [3H]thymidine incorporation by these cells. IL-1 by itself enhanced [3H]thymidine incorporation in a concentration-dependent manner. Moreover, IL-1 acted synergistically with insulin, epidermal growth factor (EGF), or fibroblast growth factor (FGF) to enhance [3H]thymidine incorporation. Combinations of maximally effective concentrations of insulin (1 micrograms/ml), EGF (1 ng/ml), or FGF (50 ng/ml) with the maximally effective concentration of IL-1 (10 ng/ml) increased the levels of [3H]thymidine incorporation to 10-, 22-, and 20-fold, respectively, over the control values. Whereas IL-2 (0.1-100 ng/ml) did not affect [3H]thymidine incorporation, tumor necrosis factor alpha (TNF alpha) stimulated [3H]thymidine incorporation by itself and reproduced the actions of IL-1 to act synergistically with insulin, EGF, or FGF. When IL-1 and TNF alpha were added together in relatively low concentrations (1 ng/ml each), the combination had synergistic effects in enhancing [3H]thymidine incorporation. The present study demonstrates that cytokines and peptide growth factors act synergistically to markedly enhance porcine granulosa cell growth in vitro.


Paracrine effect of folliculo-stellate cells on the growth factor-like action of activin A in anterior pituitary cultures. Several studies have shown that pituitary folliculo-stellate (FS) cells exhibit local functions within the pituitary gland. On the other hand, we have shown previously that activin A increases the number of FSH-producing gonadotropes in cultured rat anterior pituitary cells. In this study, we investigated whether FS cells exert an influence on the action of activin A. FS cells were prepared by culturing the dispersed rat anterior pituitary cells in media containing 15% fetal calf serum and 6 mM glutamine for 15 days. Most cells had the morphological characteristics of FS cells and S-100 protein immunoreactivity, a specific marker of FS cells. The number of FSH cells, which was higher in activin A-treated than in control cultures, was reduced to the control level by incubation with activin A plus conditioned media from FS cell-enriched cultures (FSCM). This inhibitory effect of FSCM was neutralized by a follistatin antibody, but not by anti-S-100 protein or anti-basic fibroblast growth factor. Furthermore, follistatin suppressed activin A stimulated increases in the number of FSH cells in a similar inhibitory pattern to that of FSCM. Meanwhile, the number of FSH cells was not affected by FSCM or follistatin in the absence of activin A. These results suggest that FS cells are involved in the regulation of the function and/or the morphogenesis of the FSH cell-lineage by affecting the action of activin A, and that this paracrine effect of FS cells is mediated by follistatin.


Inhibition by epidermal growth factor (EGF) of epidermal DNA synthesis in cultured chick embryonic skin pretreated with retinol and/or hydrocortisone: specific increment in EGF binding activity in both retinol- and hydrocortisone-pretreated epidermis without correlation to EGF-mediated inhibition of cell growth. When tarsometatarsal skin of 13-day-old chick embryos that had been cultured in medium containing 5% delipidized FCS with or without retinol (20 microM) and/or hydrocortisone (20 nM) for 1 day was cultured in a chemically defined medium without either the hormone or retinol for 1 day, epidermal DNA synthesis of hydrocortisone- and/or retinol-pretreated skin was inhibited when compared to that of control skin. The addition of epidermal growth factor (EGF, 10 ng/ml) to retinol- or hydrocortisone-pretreated skin further inhibited the epidermal DNA synthesis. Epidermal DNA synthesis in retinol- and hydrocortisone-pretreated skin was more strongly inhibited than in retinol- or hydrocortisone-pretreated skin, but was not further inhibited by EGF. In epidermis which was induced to differentiation toward keratinization by hydrocortisone or mucous metaplasia by retinol, EGF inhibited DNA synthesis. The extent of [125I]-EGF binding to the epidermis of retinol- and hydrocortisone-pretreated skin was 160-180% that in control skin, with no change in affinity. Hence there is no correlation between EGF-binding and the mitogenic activity of EGF.


A case of autoimmune insulin antibody syndrome associated with polymyositis, empty sella and apparent high urinary output of immunoreactive insulin. Patients with autoimmune insulin antibody are characterized by hypoglycemic attacks and antibodies to insulin in serum without prior insulin administration. In the present report, a patient with hypoglycemia due to autoimmune insulin antibody associated with primary empty sella syndrome and polymyositis appeared to have high urinary immunoreactive insulin (IRI) in the face of normal urinary C peptide. Consequently, the urinary IRI/C peptide ratio was apparently high. The amelioration of hypoglycemic attacks and polymyositis by prednisolone treatment was accompanied by the disappearance of the antibodies and complete normalization of the urinary IRI and IRI/C peptide ratio. No comparable rise in the urinary IRI and IRI/C peptide ratio was observed in the patients with other disorders studied. Glucose clamp and glucose tolerance study showed decreased sensitivity to exogenous or newly secreted insulin, prolonged half disappearance time of serum insulin, and normal disappearance of blood glucose. These results were consistent with the idea that autoantibodies buffered the effect of exogenous or newly secreted insulin and maintained a relatively constant level of serum free insulin which was not high enough when a large amount of glucose was loaded, but was too high after prolonged fasting, which eventually caused hypoglycemic attacks.


Comparison of the incidence of association of periodic paralysis and hyperthyroidism in Japan in 1957 and 1991. Periodic paralysis has been known to be associated with thyrotoxicosis in Japan. The incidence was 8.6% among male and 0.4% among female thyrotoxic patients according to a survey performed in the three major thyroid clinics in Japan in 1957. To determine the changes in the incidence during the intervening 34 years, the same type of survey was carried out again in 1991 at the same three major thyroid clinics previously involved. The incidence of paralysis in 1991 was 4.3% among male and 0.04% among female thyrotoxic patients, indicating more than a 40% decrease in the incidence. The possible cause of the decrease is related to the changes in food consumption, namely, to the fact that less carbohydrate and more potassium were taken in 1991 than in 1957.


A clinical decision analysis to assess the value of laparoscopic cholecystectomy in the treatment of gallstone disease. Laparoscopic cholecystectomy has been introduced for gallstone therapy and its use appears currently to be spreading very rapidly. A clinical decision analysis was performed to assess this new technique by quantitative comparison with other therapeutic modalities. A decision tree-based state transition model implemented to simulate a five-year period of follow-up was constructed. Non-surgical treatments were superior to surgical treatments in enhancing both life expectancy and quality-adjusted life expectancy on the basis of the optimal indications; one radiolucent stone < 2 cm for extracorporeal shock-wave lithotripsy and multiple floating stones for oral dissolution therapy respectively, especially in the elderly. Laparoscopic cholecystectomy was superior to open cholecystectomy in enhancing the quality of life for all eligible patients. Sensitivity analysis revealed that a higher incidence of the bile duct injury resulting from laparoscopic cholecystectomy would result in a poorer quality-adjusted life expectancy. The results of the analysis suggest the following. Nonsurgical treatments are acceptable alternatives to surgery on the basis of their optimal indications. Laparoscopic cholecystectomy has an advantage over open cholecystectomy in enhancing the patient's utility, and appears to be an attractive therapeutic modality. A proper training program is necessary to ensure the quality of this procedure in order to maintain its advantage.