Immunocytochemical study of ballooned neurons in cortical degeneration with neuronal achromasia. We studied the immunocytochemical characteristics of the ballooned neurons (BN) in three patients with cortical degeneration with neuronal achromasia (CDNA) using antibodies to phosphorylated neurofilaments (PNF), tau, Alz-50, ubiquitin, beta (A4) amyloid, and glial fibrillary acidic protein. All BN exhibited intense perikaryal staining for PNF protein. Most BN and some normal-appearing neurons also stained for ubiquitin and Alz-50. The BN did not immunostain for tau protein, and none of the cases had tau-reactive neocortical neurofibrillary tangles or Pick bodies. One case had occasional senile plaques that stained for beta amyloid; no case had amyloid angiopathy. Our findings suggest that the pathophysiologic basis of the cortical degeneration in CDNA involves an alteration of neuronal cytoskeletal metabolism affecting neurofilament and possibly microtubular proteins in conjunction with activation of the ubiquitin proteolytic system.


Involvement of the brain stem reticular formation in familial amyotrophic lateral sclerosis. The reticular formation of the brain stem of three cases of familial amyotrophic lateral sclerosis (ALS) with posterior column and spinocerebellar tract involvement showed a paucity of large neurons, chromatolytic neurons and Lewy body-like intracytoplasmic inclusions. Some of the remaining neurons were stained focally by antibodies to phosphorylated neurofilaments and ubiquitin; the staining patterns were similar to those seen in anterior horn cells. Within the reticular formation, the medial part of the medulla oblongata was most pronouncedly affected. The reticular formation should be among the systems other than motor neurons that may be involved in this type of familial ALS.


Plasma levels of endothelin in chronic renal failure and after renal transplantation: impact on hypertension and cyclosporin A-associated nephrotoxicity. 1. Plasma levels of endothelin were measured in 30 patients with chronic renal failure, 32 patients on chronic haemodialysis treatment and 25 renal graft recipients with stable renal graft function. 2. In patients with chronic renal failure as well as in patients on regular haemodialysis treatment, mean plasma levels of endothelin were significantly increased (4.59 +/- 2.09 pg/ml, 10.08 +/- 3.12 pg/ml, respectively) when compared with normal subjects (1.88 +/- 0.6 pg/ml, P less than 0.01, P less than 0.001, respectively). 3. In the group with chronic renal failure a positive correlation between the plasma level of endothelin and the plasma concentration of creatinine was observed (P less than 0.003). 4. Renal graft recipients on cyclosporin A with stable renal graft function had a normal plasma level of endothelin suggesting that cyclosporin A nephrotoxicity is not mediated by endothelin. 5. Hypertensive patients with chronic renal failure or on regular haemodialysis and hypertensive renal graft recipients did not differ from the corresponding normotensive population with regard to the plasma level of endothelin, demonstrating that an increased plasma level of endothelin does not play a major role in the pathogenesis of renal hypertension.


Peripheral motor-sensory neuropathy in membranous lipodystrophy (Nasu's disease): a case report. A case of membranous lipodystrophy (ML) with peripheral polyneuropathy (PN) is described. A 40-year-old woman presented with dementia, multiple lytic bone lesions with typical membranous material in adipose tissues and neurophysiological and pathological findings of peripheral neuropathy. We describe the first case of ML with PN. It is interesting since nerve involvement might be an additional cause of symptoms usually attributed to bone lesions.


Interactions between hypoxic and almitrine-induced vasoconstriction in the rat lung. 1. To test whether almitrine might improve the arterial partial pressure of O2 in patients with chronic obstructive airways disease by improvement of ventilation-perfusion matching, we looked at the interaction between hypoxic and almitrine-induced vasoconstriction in isolated rat lungs perfused with blood at constant flow. Increases in pressure represented increases in resistance. 2. Almitrine, given in increasing doses between challenges with 2% O2, enhanced hypoxic vasoconstriction at low doses but attenuated it at high doses. 3. Stimulus-response curves to hypoxia of increasing severity gave a sigmoid curve. 4. Almitrine solvent caused small changes in pulmonary artery pressure and shifted the stimulus-response curve slightly in a parallel fashion. 5. Small doses of almitrine enhanced the action of mild to moderate hypoxia, medium doses attenuated moderately severe hypoxia, whereas high doses depressed vasoconstriction due to all degrees of hypoxia. 6. These effects of almitrine on hypoxic vasoconstriction were compared with the effect of solvent by analysis of variance; the results substantiated significant enhancement of hypoxia by small doses and attenuation by large doses. 7. In patients, if similar effects apply, small doses of almitrine would assist ventilation-perfusion matching, but large doses might worsen it. 8. Almitrine-induced vasoconstriction was attenuated by a fall in perfusate temperature in a similar manner to hypoxic vasoconstriction. It was also attenuated by three drugs, chlorpheniramine, propanolol and diethylcarbamazine, all of which also decrease hypoxic vasoconstriction. The similarity between hypoxic and almitrine-induced pulmonary vasoconstriction is further confirmed.


Enhanced synthesis of neutrophil-activating peptide-1/interleukin-8 in active ulcerative colitis. 1. We studied neutrophil-activating peptide-1/interleukin-8 in inflammatory bowel disease. 2. Mucosal levels of neutrophil-activating peptide-1/interleukin-8 were significantly higher in patients with active ulcerative colitis [median 74.5 (range 17.7-450.8) pg/mg] than in patients with active Crohn's disease [10.4 (4-46.9) pg/mg; P less than 0.002] or in normal control subjects [10.4 (4-16.6) pg/mg; P less than 0.002]. 3. Circulating neutrophil-activating peptide-1/interleukin-8 was generally undetectable but there were higher levels of anti-neutrophil-activating peptide-1/interleukin-8 antibodies in patients with active ulcerative colitis [62.9 (3.4-239) ng/ml] than in patients with active Crohn's disease [5.9 (2.1-18.10) ng/ml; P less than 0.001] or in control subjects [6.1 (3.2-15.8) ng/ml; P less than 0.001]. 4. Neutrophil-activating peptide-1/interleukin-8 may be of specific functional importance in mediating inflammation in ulcerative colitis.


Exposure of rat colonic mucosa to human semen in vivo induces mucosal cytolysis, abolishes fluid absorption and raises paracellular permeability. 1. The effects of human semen on rat descending colon fluid absorption, permeability to 3H-labelled polyethylene glycol 4000 and the histological appearance of the mucosa were examined. Also, the semen was fractioned by centrifugation into plasma and sperm fractions and the effects of these fractions on rat colonic function were examined. The effects of trypsin and bacterial collagenase, mimetics of acrosin and seminal collagenase activity, were examined in order to investigate which component of human semen alters colonic permeability. 2. Contact between human semen and rat descending colonic mucosa for 3 h decreased fluid absorption from 52.0 +/- 2.9 microliters h-1 cm-2 (control) to 10.7 +/- 3.4 microliters h-1 cm-2 (P less than or equal to 0.001), increased the permeability to polyethylene glycol 4000 from 0.099 +/- 0.006 cm/h (control) to 0.31 +/- 0.04 cm/h (P less than or equal to 0.001) and caused cytolysis of the surface mucosa. 3. Spermatozoa inside the colonic lumen were destroyed within 1 h with release of acrosomal contents; this raised the activity of the acrosomal proteolytic enzyme acrosin by 40-fold (P less than or equal to 0.005) and of seminal plasma metalloproteinase (collagenase) by about twofold (mean activity 1623 +/- 240 units/ml of luminal fluid). 4. The changes in colonic permeability induced by seminal plasma were similar to those induced by similar activities of clostridial collagenase. 5. We conclude that seminal collagenase is present in sufficient amounts to cause acute damage to the colonic mucosa, and that this could be a factor in facilitating viral transmission across the colonic wall.


Nitrogen absorption from isonitrogenous solutions of L-leucyl-L-leucine and L-leucine: a study in the isolated perfused rat small intestine. 1. We assessed the efficacy of nitrogen absorption from luminal L-leucine (1.2 and 12 mmol/l) and from isonitrogenous L-leucyl-L-leucine (0.6 and 6.0 mmol/l) in a preparation of vascularly and luminally perfused rat small intestine by measuring luminal leucyl-leucine disappearance and venous leucine appearance. 2. No intact dipeptide was found in the vascular perfusate. Leucine-nitrogen absorption, as judged by venous leucine appearance, was as efficient from free leucine (29 +/- 3 and 245 +/- 19 ng-atom min-1 g-1) as from leucyl-leucine (27 +/- 4 and 211 +/- 58 ng-atom min-1 g-1). It was not reduced in the presence of glycyl-L-proline or of the brush-border dipeptidase inhibitors alanine-beta-naphthylamide and cilastatin. 3. After one passage of the whole small intestine, only trace amounts of dipeptide, but large amounts of free leucine, were detected in the luminal effluent. Peptidase activity in the luminal effluent was demonstrated in 100,000 g supernatant and was inhibited by p-hydroxy-mercuribenzoate, but not by brush-border dipeptidase inhibitors. 4. We propose that nitrogen absorption from luminal leucyl-leucine may proceed predominantly via intraluminal peptide hydrolysis and subsequent transport of free leucine. Nevertheless, our findings and conclusions are at variance with previous observations and current opinion on intestinal handling of dipeptides, which may be due, in part, to the different methodological approach.


Kinetics of sodium-lithium countertransport activity in patients with uncomplicated type 1 diabetes. 1. Increased erythrocyte sodium-lithium countertransport activity has been reported to be associated with nephropathy in type 1 diabetes and linked to a family history of essential hypertension. 2. This study aimed to determine the mechanism of increased sodium-lithium countertransport activity. Sodium-lithium countertransport kinetics were measured in uncomplicated and hyperlipidaemic type 1 diabetic patients. 3. In the nine out of 31 uncomplicated type 1 diabetic patients who had high sodium-lithium countertransport activity, the sodium affinity (Km) was normal but the maximum velocity (Vmax) was increased. 4. Hyperlipidaemia, when present in diabetic patients, was associated with increased sodium-lithium countertransport activity, but could not explain the high activity in uncomplicated type 1 diabetic patients in whom plasma lipid concentrations were normal. 5. Sodium-lithium countertransport activity is increased in type 1 diabetes by a mechanism different to that in essential hypertension, where the mechanism is a low Km (increased sodium affinity). Hence familial hypertension cannot explain the raised sodium-lithium countertransport activity in type 1 diabetes.


Changes in erythrocyte sodium-lithium countertransport kinetics in diabetic nephropathy. 1. It has been proposed that raised erythrocyte sodium-lithium countertransport activity in type 1 diabetic patients is associated with an increased risk of developing diabetic nephropathy. Diabetic patients with established nephropathy would therefore be expected to have high activity. 2. Standard sodium-lithium countertransport activity, sodium affinity (Km) and maximum velocity (Vmax) were measured in type 1 diabetic patients at different stages of diabetic nephropathy and in appropriately matched uncomplicated diabetic patients and normal control subjects. 3. A small proportion (15%) of patients with nephropathy had standard sodium-lithium countertransport activity higher than the control range. However, mean standard sodium-lithium countertransport activity in the diabetic patients with nephropathy [mean +/- SEM, 0.26 +/- 0.12 mmol of Li+ h-1 (l of cells)-1] was not significantly higher than in the uncomplicated diabetic patients [0.27 +/- 0.03 mmol of Li+ h-1 (l of cells)-1] or in the normal control subjects [0.25 +/- 0.02 mmol of Li+ h-1 (l of cells)-1]. 4. There were marked changes in the kinetic characteristics of the sodium-lithium countertransport in the diabetic patients with nephropathy so that there were decreases in both Km and Vmax. 5. These kinetic changes could not be attributed to an effect of either renal failure per se or the duration of diabetes. 6. The characteristic kinetic changes in sodium-lithium countertransport may indicate underlying alterations in membrane function with the onset of nephropathy in type 1 diabetes.


Decrease in erythrocyte glycophorin sialic acid content is associated with increased erythrocyte aggregation in human diabetes. 1. Sialic acid moieties of erythrocyte membrane glycoproteins are the principal determinants of the negative charge on the cell surface. The resultant electrostatic repulsion between the cells reduces erythrocyte aggregation and hence the low shear rate viscosity and yield stress of blood. 2. Using g.c.-m.s., a decrease in sialic acid content has been observed in the major erythrocyte membrane glycoprotein, glycophorin A, obtained from nine diabetic patients compared with that from seven normal control subjects [median (range): 3.30 (0.01-11.90) versus 18.60 (3.20-32.60) micrograms/100 micrograms of protein, P less than 0.02]. 3. Erythrocyte aggregation, measured by viscometry as the ratio of suspension viscosity to supernatant viscosity (LS/S) in fibrinogen solution, was increased in ten diabetic patients compared with ten normal control subjects (mean +/- SEM, 37.6 +/- 1.3 versus 33.8 +/- 0.6, P less than 0.02). 4. In the patients in whom both viscometry and carbohydrate analysis were performed, the decrease in erythrocyte glycophorin sialylation and the increase in erythrocyte aggregation in fibrinogen solution were related statistically (LS/S correlated negatively with glycophorin sialic acid content, r = 0.73, P less than 0.05). 5. Decreased glycophorin sialylation provides an explanation at the molecular level for increased erythrocyte aggregation and it may be important in the pathogenesis of vascular disease in diabetes.


Leg extensor power and functional performance in very old men and women. 1. Residents of a chronic care hospital (13 men of mean age 88.5 +/- 6 SD years and 13 women of mean age 86.5 +/- 6 SD years) who had multiple pathologies were assessed for leg extensor capability in several ways. 2. A custom-built rig was used to assess leg extensor power, that is, maximal power output over less than 1 s in a single extension of one leg. Performance measures were obtained by timing chair rises (from a standard chair 0.43 m high), stair climbing (four risers, total height 0.635 m) and a walk (6.1 m). For each measurement the best of several trials were recorded as definitive. 3. Leg extensor power was significantly correlated with all performance measures, but the performance measures were not related to each other except for chair rising and walking speed. 4. Women had significantly less extensor power than men, but their power explained more of the variance in performance, e.g. power accounted for 86% of the variance in walking speed. 5. There was no relation within the group between age and any of the variables measured. 6. Measurement of leg extensor power in frail elderly people may prove useful in focusing effective rehabilitation programmes.


Hysteresis of the venous pressure-volume relationship in the forearm of borderline hypertensive subjects. 1. The forearm venous pressure-volume relationship was studied in 14 young men with borderline hypertension and in 16 control subjects of the same age and sex. Strain-gauge plethysmography was used to evaluate volume changes after slow increases and decreases in distention, in order to estimate the amplitude of the hysteresis curve. 2. Compared with normotensive control subjects, subjects with borderline hypertension had significantly higher values of blood pressure, heart rate and forearm blood flow. 3. Baseline forearm venous tone was slightly, but not significantly, increased in borderline hypertensive subjects (21.35 +/- 6.53 versus 18.75 +/- 5.95 mmHg ml-1 100 ml-1) and was significantly enhanced after a cold pressor test. The increase was no higher in the borderline hypertensive subjects than in the normotensive control subjects. 4. The area of the hysteresis curve was significantly decreased (7.58 +/- 3.58 versus 10.34 +/- 5.67 arbitrary units; P = 0.0092) as was the extent of isotonic relaxation (creep) (0.28 +/- 0.11 versus 0.39 +/- 0.22 ml/100 ml; P = 0.0098) in borderline hypertensive subjects compared with control subjects. Both parameters were unaffected by the cold pressor test. 5. The study suggests that the viscous component of the venous wall is altered in young patients with borderline hypertension, indicating intrinsic changes in vascular segments which are not exposed to increased intraluminal pressure.


Extent of ischaemia caused by hepatic vascular exclusion as evaluated in a canine model. 1. The difference in the extent of liver ischaemia between a hepatic vascular exclusion model and an inflow occlusion model were investigated by determining Indocyanine Green retention and hepatic mitochondrial redox state during 2 h of ischaemia in 10 mongrel dogs. The splanchnic venous bed and/or the infra-hepatic inferior vena cava were decompressed by pump-driven veno-venous bypass. 2. The Indocyanine Green retention test revealed that there was no hepatic blood flow in the hepatic vascular exclusion model during ischaemia (96.8 +/- 0.73% retention of the dye after 20 min), whereas hepatic blood perfusion was still present significantly in the inflow occlusion model (78.1 +/- 1.19% retention of the dye after 20 min) (P less than 0.01). 3. The mitochondrial redox potential of the liver in the dogs with hepatic vascular exclusion decreased immediately after the induction of ischaemia and remained fixed at extremely low levels. By contrast, in the dogs with inflow occlusion the redox potential decreased gradually after induction and was maintained significantly higher than that in dogs with hepatic vascular exclusion during 2 h of ischaemia (P less than 0.01). 4. It is concluded that the extent of liver ischaemia in the hepatic vascular exclusion model with pump-driven shunt is significantly different from that in the inflow occlusion model with shunt.


Infection of bovine fetuses at 120 days' gestation with virulent and avirulent strains of bluetongue virus serotype 11. Bluetongue virus infection in sheep and cattle during fetal development causes neuropathology. Two strains of bluetongue virus serotype 11 designated as UC-2 and UC-8 have different virulence patterns in newborn mice. These viruses have distinctly different electropherotype patterns on polyacrylamide gel electrophoresis indicating a genetic difference in these two viruses of the same serotype. Four bovine fetuses each were inoculated intramuscularly with either UC-2 or UC-8, and one fetus was inoculated with placebo. The inoculation was made intramuscularly through the uterine wall at 120 days' gestation, and the bovine fetuses were recovered by cesarean section 12 or 20 days after inoculation. Fetal blood was collected for virus isolation and serology. Virus was reisolated from brain, blood, lung and liver. Both strains, UC-2 and UC-8, cause severe lesions in the 120 day fetuses. The encephalomalacic lesions occurred earlier and were more severe in fetuses inoculated with UC-8 as compared to those inoculated with UC-2. The subtle differences observed in the fetuses inoculated with the two different strains suggest that there is a difference in pathogenic potential of the two viruses. These differences do not appear to be completely dependent upon the host species.


Investigations of environmental conditions during cluster indicate probable vectors of unknown exogenous agent(s) of multiple sclerosis. During the tail-end of an active cluster several environmental investigations indicated that wildbirds were very probably the vectors of the unknown exogenous agent of MS. Canine distemper and genetic-autoimmune theories were very definitely eliminated because of the unusual pattern of the cluster. Studies of several avian pathogens unveiled Marek's (MDV) and/or IBD (Gumboro) as the most likely candidates for exogenous agent of MS.


Cholecystokinin-octapeptide stimulates hypothalamic-pituitary-adrenal function in rats: role of corticotropin-releasing hormone. Peripherally-administered cholecystokinin (CCK) is a profound suppressor of food intake, can promote anxiety, and causes the acute release of ACTH into plasma. Centrally administered corticotropin-releasing hormone (CRH), on the other hand, not only represents the principal stimulus to the pituitary corticotroph cell, but also has been shown to suppress appetite and to be profoundly anxiogenic. Because of the overlap in the effects of peripherally administered CCK and of centrally administered CRH, we report here a study to determine whether sulphated CCK octapeptide (CCK-8) could induce the release of CRH within the central nervous system. To accomplish this task, we first assessed the dose-related effects of CCK-8 on ACTH release. Graded doses of CCK-8 (0.1-10 micrograms/kg BW) given in an i.v. bolus to freely moving male rats, resulted in a dose-dependent increase of plasma immunoreactive (IR)-ACTH (ED50: 1-10 micrograms/kg BW). The lowest maximal stimulatory dose of CCK-8 (5 micrograms/kg BW) was used in all subsequent experiments. To evaluate whether CCK-induced ACTH secretion was mediated by a peripheral CCK receptor, an i.v. bolus injection of vehicle or L-364,718 (1 mg/kg BW), a specific, highly potent peripheral CCK receptor antagonist, was given before the i.v. administration of CCK-8 or vehicle. Plasma IR-ACTH response to CCK-8 was significantly attenuated by L-364,718. A role for the vagal afferents that contain CCK receptors in peripherally administered CCK-mediated hypothalamic-pituitary-adrenal (HPA) axis activation was examined in animals that had been pretreated with capsaicin, a potent neurotoxin that destroys vagal afferents. Plasma IR-ACTH and IR-corticosterone responses in capsaicin-treated animals were significantly lower than those in vehicle treated rats. In subsequent in vivo experiments, pituitary stalk-transected and sham-operated animals were used to evaluate whether CCK-8 stimulates the HPA axis via a centrally mediated mechanism. IR-ACTH and IR-corticosterone responses to i.v. CCK-8 were significantly reduced in the pituitary stalk-transected compared to sham-operated animals. In further effort to determine whether the central nervous system was involved in the plasma IR-ACTH response to the peripheral administration of i.v. CCK-8, we compared the effects of the i.v. administration of CRH antisera vs. normal rabbit serum on this parameter. IR-ACTH and IR-corticosterone responses to i.v. CCK-8 were significantly reduced in the context of pretreatment with CRH antisera compared to the administration of normal rabbit serum.(ABSTRACT TRUNCATED AT 400 WORDS)


Chronic activation of the hypothalamo-pituitary-adrenal axis and loss of circadian rhythm during adjuvant-induced arthritis in the rat. Adjuvant-induced arthritis (AA) in Sprague-Dawley rats resulted in a chronic increase in plasma levels of ACTH and corticosterone (B). Joint inflammation became clinically apparent between days 12-15 after injection of adjuvant and reached a peak on day 21, after which time it subsided. In AA animals, plasma ACTH and B levels in the morning (0800-0900 h) on days 7, 8, 9, and 21 were significantly higher than those in control animals (day 0). The corresponding evening ACTH and B levels in AA animals were not significantly different from evening levels in the control animals. Adrenal weight in AA animals was increased on day 21, while thymic weight diminished gradually from days 7-21 postinjection. Development of AA was associated with activation of the hypothalamo-pituitary-adrenal axis, with increased morning ACTH and B levels and abolition of normal diurnal ACTH and B rhythms. This model of chronic inflammatory stress clearly activates the ACTH drive despite increased corticosteroid feedback in the morning, resulting overall in chronically increased B secretion.


Regulation of corticotropin-releasing factor secretion from Leydig cells by serotonin. CRF is produced in the Leydig cells and acts as a negative autocrine regulator of Leydig cell function. To clarify the hormonal control of CRF secretion by Leydig cells, we evaluated the participation of serotonin (5HT) and serotonin agonists in the release of CRF from Leydig cells and their effects on hCG-induced cAMP generation and steroidogenesis. Serotonin stimulated CRF secretion up to 4-fold above basal levels and inhibited basal and hCG-stimulated cAMP generation and testosterone production (ID50, 1 nM). The inhibitory action of 5HT was prevented by a CRF antibody and the alpha-helical CRF-(9-41) antagonist. The selective 5HT2 receptor agonist (+-)1-[2,5-dimethoxy-4-iodophyryl]2-amino propane hydrochloride (DOI) also stimulated CRF secretion and inhibited hCG-stimulated cAMP generation and testosterone production to control levels (ID50, 7 microM). Serotonergic 5HT1A, 5HT1B/1C, 5HT1D, and 5HT3/5HT2 agonists were less effective inhibitors of hCG-stimulated cAMP and testosterone production, while agonists for the 5HT3 receptor had no effect. [125I]DOI binding studies in Leydig cells demonstrated two sets of receptors with Kd values in the nanomolar and micromolar range, with low and high capacities, respectively. The low affinity site differed from that of brain receptors (Kd, 4.2 nM) and displayed higher binding capacity (50-fold). The selective 5HT2 receptor antagonist ketanserin prevented CRF stimulation and blocked the inhibitory actions of 5HT and DOI, while the alpha 1-adrenergic antagonist prazosin had no effect. Also, treatment of cells with ketanserin increased sensitivity to hCG and raised maximal cAMP and testosterone production. 5HT was a more effective stimulus than hCG in stimulating CRF secretion, and gonadotropin-induced CRF release was inhibited by ketanserin. Inhibitory effects of exogenous CRF were demonstrable after blockade of 5HT action by ketanserin. The inhibitory actions of 5HT were unaffected by pertussis and cholera toxins and were reversed by the addition of 8-bromo-cAMP. These results demonstrate that 5HT acts on 5HT2 receptors in Leydig cells that are distinct from those in the brain to stimulate CRF secretion through a pertussis toxin-insensitive G-protein. This action of 5HT is predominantly mediated by the low affinity 5HT2-binding site and requires full occupancy for maximal CRF stimulation, indicating the absence of spare receptors. 5HT-stimulated CRF inhibits basal and hCG-induced cAMP generation and steroidogenesis. Furthermore, 5HT mediates the stimulatory action of LH/hCG on CRF secretion from Leydig cells and, thus, participates in a negative autoregulatory loop to limit the testosterone response to the gonadotropic stimulus.


Morphology of adenohypophysial tumors in mice transgenic for vasopressin-SV40 hybrid oncogene. Transgenic mice for the promoter sequence of bovine arginine vasopressin (AVP) gene fused to large SV40 T-antigen coding sequence develop pituitary tumors and insulin-producing pancreatic tumors. In order to establish the cellular composition of the pituitary tumors, histological, immunocytochemical, in situ hybridization, and electron microscopic technics were applied. Pituitary anterior lobe tumors were identified in 10 out of 14 glands examined. In 2 of these cases, intermediate lobe tumors were also found. The anterior lobe tumors contained a variable number of GH immunoreactive cells. In situ hybridization performed in 7 cases revealed a diffuse distribution of GH messenger RNA over all tumor cells. Ultrastructurally, the tumors contained undifferentiated cells with very small secretory granules and rare cells showing some resemblance to somatotrophs. The results indicate that these pituitary tumors are composed of undifferentiated somatotrophs. The presence of a few PRL immunoreactive cells in four tumors and scattered TSH immunoreactive cells in two tumors supports the view that somatotrophs have the potential to produce PRL and TSH. The intermediate lobe tumors were immunoreactive for ACTH and intensely positive for POMC mRNA. In the nontumorous adenohypophyses, no hyperplasia of any cell type was noted. Several GH immunoreactive cells exhibited pleomorphic, giant nuclei and mitoses. In conclusion, the majority of transgenic mice for AVP/large T-antigen develop pituitary tumors originating in and composed of somatotrophs. Less frequently, intermediary lobe tumors were present as well. AVP/SV40 transgenic mice provide a unique experimental model for somatotroph tumors that are neither preceded by, nor associated with somatotroph hyperplasia.


Growth of the endometrial adenocarcinoma cell line AN3 CA is modulated by tumor necrosis factor and its receptor is up-regulated by estrogen in vitro. We demonstrate that tumor necrosis factor (TNF) has a biphasic effect on the growth of the human endometrial adenocarcinoma cell line AN3 CA in vitro. Low levels (0.2-5 pg/ml) of TNF were moderately growth stimulatory (up to 20% enhancement), while levels over 100 pg were growth inhibitory (up to 45% inhibition). Northern blot analysis showed expression of the 75-kilodalton (kDa) TNF receptor mRNA, but no expression of the 55-kDa TNF receptor mRNA or TNF mRNA. The growth of these cells was not directly affected by physiological concentrations (10(-7)-10(-9) M) of 17 beta-estradiol (E2). However, [125I]TNF binding studies and Scatchard analysis showed that 18-h coculture with 10(-8) M E2 increased the number of TNF receptors expressed on these cells 3-fold. Quantitative mRNA analysis confirmed that 75-kDa TNF receptor mRNA expression increased within 4 h of incubation with E2. These observations suggest an interaction between the endocrine and the immune systems, with an important implication for the homeostasis of endometrial tissues.


Mechanism of regulation of thyrotropin-releasing hormone receptor messenger ribonucleic acid in stably transfected rat pituitary cells. We showed previously that the level of TRH receptor (TRH-R) mRNA in rat pituitary GH3 cells is down-regulated by TRH. Here, we study the mechanism of regulation of TRH-R mRNA in a line of GH3 cells that are stably transfected with mouse pituitary TRH-R cDNA (GH-mTRHR-1 cells). GH-mTRHR-1 cells were found to have 2.4 times the number of TRH-Rs and to stimulate a 2.5-fold greater increase in inositol phosphates in response to TRH than the parent cell line and to show TRH-induced down-regulation of TRH-R number. GH-mTRHR-1 cells contained 26 +/- 1.6 molecules of mouse TRH-R mRNA/cell and 230 +/- 31 molecules of mRNA for the neomycin resistance gene (NEO) with which it was cotransfected. In GH-mTRHR-1 cells, TRH caused a dose-dependent transient decrease in mouse TRH-R mRNA, with a nadir to 20% of control levels after 6 h. In contrast, TRH did not affect NEO mRNA or glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA, an endogenous gene product. TRH stimulated the rate of transcription of mouse TRH-R DNA by approximately 2-fold, but did not affect total poly(A) RNA synthesis. Most importantly, TRH caused a 4-fold increase in the rate of degradation of mouse TRH-R mRNA, but did not affect degradation of GAPDH mRNA. The half-lives of mouse TRH-R and GAPDH mRNAs were 3 and more than 20 h in control cells and 0.75 and more than 20 h in cells treated with 1 microM TRH for 1.5 h, respectively. These data show that the predominant effect of TRH on mouse TRH-R mRNA in GH-mTRHR-1 cells is to enhance the rate of its degradation. We suggest, therefore, that down-regulation of TRH-R mRNA caused by TRH in the parent GH3 cell line is secondary to increased TRH-R mRNA degradation.


Distinct tissue distribution and cellular localization of two messenger ribonucleic acids encoding different subtypes of rat endothelin receptors. Endothelins (ETs) are very potent vasoconstrictive peptides and have diverse functions in both vascular and nonvascular tissues. This investigation concerns the tissue distribution and cellular localization of rat mRNAs encoding two different subtypes of ET receptors (ETA and ETB). We isolated 46 cDNA clones from a rat lung cDNA library by hybridization with the bovine ETA cDNA. The characterization of these cDNA clones indicated that they represent either the ETA or ETB cDNA. In situ and blot hybridization analyses revealed that the rat ETA mRNA is predominantly expressed in vascular smooth muscle cells of a variety of tissues, bronchial smooth muscle cells, myocardium, and the pituitary gland. There is no significant expression of ETB mRNA in vascular smooth muscle cells, and ETA, thus, plays a primary role in ET-induced vascular contraction. ETB mRNA is more widely distributed in various cell types of many tissues. Its prominent expression is seen in glial cells throughout the brain regions, epithelial cells of the choroid plexus, ependymal cells lining the ventricle, myocardium, endothelial cells of glomeruli, and epithelial cells of the thin segments of Henle's loops. Our investigation demonstrates that the mRNAs for the two subtypes of rat ET receptors show specialized expression patterns of cell types in both brain and peripheral tissues.


Acute intravenous corticotropin-releasing factor administration: effects on insulin secretion in lean and genetically obese fa/fa rats. The effect of an i.v. administration of different doses (250, 500, and 1000 pmol) of ovine CRF (oCRF) on plasma glucose and insulin levels in lean and genetically obese fa/fa rats was investigated. In both phenotypes, i.v. CRF promoted a rapid (peak at 1 min) transient doubling of basal insulin levels without a concomitant change in glycemia. The dose-dependency of this early insulin response was bell-shaped in both lean and obese animals, with a maximal response at 500 pmol oCRF. After this early rise in insulinemia, glycemia increased in a dose-dependent manner in both lean and obese rats. It was accompanied by a bell-shaped insulin response in lean rats, while such a response was linear in obese rats. The early transient stimulatory effect of CRF on plasma insulin levels could not be prevented by the prior administration of an anti-CRF serum or the alpha-helical CRF-(9-41) antagonist, although administration of either one of these compounds was effective in preventing the CRF-induced changes in the pituitary-adrenal axis. The effect of CRF on the early insulin response was, however, completely suppressed by an acute cholinergic blockade (i.v. injection of atropine). It is suggested that i.v. CRF administration mimics the reflex, cephalic phase insulin secretion. Such cephalic phase insulin output is known to play a role in oral glucose tolerance and may be of physiopathological importance in the Zucker rat strain.


Hypothalamic-pituitary-adrenal axis function in the Zucker obese rat. The development of many endocrine, metabolic, and behavioral abnormalities characteristic of genetically obese Zucker rats is dependent upon the presence of glucocorticoids, the secretion of which is regulated by a neuroendocrine cascade initiated by hypothalamic release of CRF. Recent reports have inferred alterations in central CRF tone as a putative factor contributing to dysregulation of the pituitary-adrenal axis and of metabolic processes in this phenotype. In the current study the hypothalamic CRF system in Zucker lean (FA/?) and obese (fa/fa) phenotypes was functionally evaluated. Neither the stalk median eminence content of CRF or arginine vasopressin (AVP) nor hypothalamic levels of CRF or AVP mRNA differed in the lean and obese phenotypes. No phenotypic differences were observed in either basal or stimulated CRF release from hypothalamic tissue obtained from lean and obese rats. Furthermore, in intact rats the magnitude of pituitary-adrenal responses to various stressors was also similar between phenotypes. However, secretion of CRF and AVP into the hypophysial-portal circulation of obese rats was, respectively, 73% and 35% lower than that of the lean rats. Adrenalectomy was associated with a 3-fold elevation of hypophysial-portal CRF levels in obese rats compared to intact controls. Corticosterone infusion was more effective in suppressing portal CRF levels in adrenalectomized obese compared to adrenalectomized lean rats. Finally, neither CRF receptor number and affinity nor the magnitude of pituitary-adrenal responses to rat CRF challenge (1 micrograms, iv) differed between Zucker phenotypes. These observations lead us to infer that rats of the obese phenotype exhibit reduced hypothalamic CRF tone due to dysregulation of the HPA axis at a site proximal to the hypophysiotropic CRF system that mediates glucocorticoid feedback regulation.


Ectopic corticotropin-releasing hormone produced by a transfected cell line chronically activates the pituitary-adrenal axis in transkaryotic rats. Hypothalamic CRH is the primary positive regulatory factor of the pituitary-adrenal axis. The purpose of our study was to analyze the chronic effects of CRH on the production and secretion of POMC peptides from both the anterior lobe (AL) and neurointermediate lobe (NIL) of the pituitary by mimicking the syndrome of ectopic CRH secretion from neuroendocrine tumors. We first generated stably transfected W2 medullary thyroid carcinoma cell lines with a rat CRH expression vector under the transcriptional control of a cytomegalovirus gene promoter. These cell lines constitutively expressed the foreign gene, accurately processed the encoded prepro-CRH, and secreted biologically active CRH with an estimated potency equivalent to that of synthetic CRH-(1-41)NH2. The cell line designated W2CRH-7 was implanted sc in the syngeneic rat strain WAG/Rij and produced tumors that abundantly secreted CRH into the peripheral circulation. Four weeks postimplantation, W2CRH-7, but not wild-type W2, cells caused significant increases in the AL content of beta-endorphin-like immunoreactivity comparable to that caused by adrenalectomy (ADX). Plasma ACTH and serum beta-endorphin-like immunoreactivity were increased to a greater extent by ADX than by W2CRH-7 cell implantation. The NIL of both male and female rats showed either no change or a tendency to decreased beta-endorphin concentrations with no change in the acetylation or carboxy-shortening profiles judged by cation exchange chromatography in response to the ectopic CRH treatment. Rats of both sexes maintained a profound activation of the pituitary adrenal axis up to 16 weeks postimplantation, with normalized adrenal gland weights 5 times that of controls. The chronic secretion of CRH by W2CRH-7 cells resulted in a complete cessation of body growth in all rats up to the maximum time tested of 16 weeks. The lack of growth was partly ameliorated by concomitant ADX, suggesting an important role for adrenal glucocorticoids in these effects. We conclude that 1) the transplantable W2CRH-7 cell line provides a highly effective and reproducible means of sustained CRH treatment that mimics the syndrome of ectopic CRH expression by neuroendocrine tumors; 2) AL corticotrophs respond to chronic CRH by a sustained production and secretion of POMC peptides, leading to a marked adrenal cortical hyperplasia, with no evidence of biologically significant desensitization; 3) chronic CRH tends to decrease the NIL content of beta-endorphin,with remarkably little effect on posttranslational processing; and 4) the syndrome of chronic ectopic CRH in WAG/Rij rats includes a cessation of body growth at least partly due to products of the adrenal glands.


Endogenous excitatory amino acids and glutamate receptor subtypes involved in the control of hypothalamic luteinizing hormone-releasing hormone secretion. These studies were designed to evaluate the actions and relative potencies of different endogenous and excitatory amino acid (EAA) selective analogs on EAA-induced neuropeptide secretion as well as to analyze the receptor subtypes involved. For this purpose, different glutamate agonists were tested for their ability to evoke release of the hypothalamic neuropeptide LHRH from arcuate nucleus-median eminence (AN-ME) fragments incubated in vitro. Different glutamate agonists, i.e. 3-amino-3-hydroxy-5-methyl-isoxazole-4-propionic (AMPA), kainic, quisqualic, homocysteic (HCA), quinolinic (QUIN), N-methyl-D-aspartic (NMDA), and pyroglutamic (PYR) acids, elicited LHRH release from AN-ME fragments in vitro. Further evaluation of the range of activity of several of these compounds, both in terms of the dose inducing a half-maximal response and the LHRH-releasing effect at that particular dose, indicated that AMPA greater than HCA greater than QUIN greater than PYR, suggesting that non-NMDA receptors are primarily involved in EAA-induced LHRH release at the level of the AN-ME. Evaluation of the receptor types involved using two specific antagonists for NMDA and non-NMDA receptors, D,L-2-amino-7-phosphoheptanoic acid and 6,7-cyanoquinoxaline-2,3-dione, respectively, showed that the effects of AMPA and HCA on LHRH release can be completely blocked by 6,7-cyanoquinoxaline-2,3-dione, whereas QUIN activity was blocked by D,L-2-amino-7-phosphoheptanoic acid. The effects of PYR on LHRH release were abolished by both receptor blockers. The metabotropic receptor agonist trans-1-amino-cyclopentyl-1,1,3-dicarboxylic acid was not active in eliciting LHRH secretion. The data indicate that endogenous substances active at EAA receptor sites, such as HCA, QUIN, and PYR, can significantly increase the secretion of the neuropeptide LHRH and, thus, may participate in the physiological regulation of the activity of this important neuroendocrine neuronal system. In addition, the results suggest that non-NMDA receptor sites may be preferentially activated at lower ligand concentrations, although NMDA receptors may also be involved in the response to certain endogenous agonists.


Effects of parathyroid hormone-related peptide on adenosine 3',5'-monophosphate and ornithine decarboxylase in a human colonic cell line. PTH-related peptide (PTHrP) is widely distributed in normal tissues, including the gut, and is considered a potential autocrine or paracrine regulator of cellular growth and differentiation. With this in mind, a human colonic cell line (LoVo) was used to study the effect of PTHrP on ornithine decarboxylase (ODC), because ODC is known to have profound effects on the growth and differentiation of many cell types via stimulation of synthesis of polyamines. cAMP also was measured, because this second messenger has been implicated in the regulation of ODC activity. Nearly confluent LoVo cells, grown in F-12 medium and 10% fetal bovine serum (FBS), were preincubated in 1% FBS for at least 5 h, and then PTHrP-(1-34) was added, and the incubation was continued for up to 6 h. Cell extracts were analyzed for ODC activity by measuring 14CO2 liberated from 14C-labeled ornithine, for cAMP by RIA, and for ODC mRNA by Northern analysis. PTHrP produced dose-related increases in both cAMP (2- to 3-fold) and ODC (3- to 5-fold), with a maximal effect at 0.1-1 microM and an ED50 of 1-10 nM. Comparison of the cAMP and ODC responses to PTHrP showed a strong correlation (r = 0.96; P less than 0.001). The effects of 1 microM PTHrP-(1-34) to increase cAMP and ODC were completely inhibited by 10-20 microM of the specific antagonist [Asn10,Leu11]PTHrP-(7-34). PTHrP-(1-34) did not stimulate ODC activity when cells were incubated without FBS. The stimulation of ODC activity by PTHrP-(1-34) was maximal at 2 h, a time at which an increase in ODC mRNA also was evident. PTH-(1-34) and forskolin also stimulated ODC activity, but PTHrP-(67-86) amide was ineffective. The results indicate that the N-terminal portion of the PTHrP molecule can stimulate ODC activity in a human colon cell line and that the effect is probably mediated by cAMP. The results are consistent with the idea that PTHrP may influence cell growth and differentiation in the gut via an effect on polyamine biosynthesis. Since LoVo cells also express PTHrP mRNA, this gastrointestinal cell line may serve as a useful model for studying autocrine regulation of gut cell growth and differentiation by PTHrP.


Time course of the in vivo effects of thyroid hormone on cardiac gene expression. The rate of response to thyroid hormone on cardiac growth, heart rate, and the relative changes in messenger RNA (mRNA) coding for alpha- and beta-myosin heavy chain (MHC), slow sarcoplasmic reticulum calcium-adenosine triphosphatase, and thyroid hormone receptors in ventricular tissue of hypothyroid rats was investigated. Hypothyroid rats had significantly smaller hearts, with slower heart rates and expressed no alpha-MHC mRNA as analyzed by an S1 nuclease protection assay when compared to euthyroid animals that expressed 79% alpha-MHC. Twelve hours after treating hypothyroid rats with 20 micrograms of L-T4, detectable levels of alpha-MHC mRNA were present and the shift to alpha-MHC mRNA was complete by 72 h of treatment. Northern blot analysis showed that hypothyroidism resulted in a 60% decrease in the level of sarcoplasmic reticulum calcium-adenosine triphosphatase mRNA which increased after 12 h of T4 administration and was 2.5-fold (P less than 0.05) greater than euthyroid levels after 72 h. In contrast, thyroid hormone receptor mRNA levels measured in poly(A)+ RNA were elevated in hypothyroid rats and decreased to euthyroid levels within 24 h after thyroid hormone treatment. These changes in cardiac gene expression occurred simultaneously with changes in both cardiac size and heart rate. The current studies characterize the coordinated changes and the time course for gene expression that occur in the hypothyroid heart after acute T4 administration.


Expression of testicular 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase: regulation by luteinizing hormone and forskolin in Leydig cells of adult rats. LH is required to maintain the activity of 3 beta-hydroxysteriod dehydrogenase/delta 5----4-isomerase (3 beta HSD) in testicular Leydig cells. The objective of the present study was to determine whether LH and effectors such as forskolin, which act via the intracellular cAMP signal transduction pathway, can regulate the expression of 3 beta HSD in rat Leydig cells in vitro. Primary cultures of Leydig cells were prepared from testes of adult rats and treated with oLH, forskolin, (Bu)2cAMP, or cholera toxin. The effects of treatment on 3 beta HSD activity were measured using [3 alpha-3H]dehydroepiandrosterone as substrate. Immunoreactive 3 beta HSD was quantified by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a polyclonal antiserum against 3 beta HSD. The synthesis of 3 beta HSD was quantified after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated cellular lysates of Leydig cells radiolabeled with L-[35S]methionine. The levels of 3 beta HSD mRNA were quantified by Northern analysis and hybridization with a cDNA encoding testicular 3 beta HSD (rat type I). A cell-free protein-synthesizing system was used to test the ability of 3 beta HSD mRNA to be translated into immunoreactive 3 beta HSD. 3 beta HSD activity increased 3.5- and 5.0-fold in Leydig cell cultures treated with forskolin (1 microM) and (Bu)2cAMP (1 mM), respectively, compared with control cultures. Maximal activity was attained after 48-72 h and maintained through 120 h of treatment. The increase in 3 beta HSD activity could be accounted for quantitatively by increases in the steady state levels and the rates of synthesis of 3 beta HSD. The cellular levels of immunoreactive 3 beta HSD increased 4.0- and 7.6-fold in Leydig cells treated with forskolin and (Bu)2cAMP, respectively. Moreover, both of these effectors increased by 6- to 8-fold the levels of newly synthesized 3 beta HSD after 24-72 h of treatment. Ovine LH, forskolin, cholera toxin, and (Bu)2cAMP increased the cellular levels of 3 beta HSD mRNA in a dose-dependent manner. The magnitude of the increases ranged from 2- to 42-fold, compared with that in control cultures, after 12 h of treatment. Maximal responses were effected by 1 ng/ml ovine LH, 1 microM forskolin, 1 ng/ml cholera toxin, and 1 mM (Bu)2cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)


Endothelin-1 suppression of rat adipocyte precursor cell differentiation in serum-free culture. The effect of endothelin-1 (ET-1) on differentiation of rat adipocyte precursor cells in serum-free culture and of the adipogenic fibroblast cell line TA1 was studied. ET-1 inhibited differentiation of rat adipocyte precursor cells into adipocytes in a dose-dependent fashion, while the peptide exerted no effect on TA1 cells. Rat adipose precursor cells possessed a single class of high affinity ET-1 receptor with a Kd of 0.71 nM and a binding capacity of 47,000 sites/cell. Affinity cross-linking of [125I]ET-1 showed two bands with molecular masses of 86 and 50 kilodaltons in rat adipose precursor cells and a single broad band with a molecular mass of 55-60 kilodaltons in TA1 cells. Pertussis toxin and the protein kinase-C inhibitors, H-7 and staurosporine, all of which enhanced adipocyte differentiation of rat adipose precursor cells, partially reversed ET-1 inhibition. These results showed the divergent effect of ET-1 on adipocyte conversion and indicated the possible involvement of a pertussis toxin-sensitive pathway and protein kinase-C at least in part in the inhibitory action of ET-1 on adipocyte differentiation.


Cardiovascular responsiveness to parathyroid hormone (PTH) and PTH-related protein in genetic hypertension. Hypertension is often accompanied by abnormalities of calcium homeostasis, including hyperparathyroidism with reduced target organ responses to PTH in kidney and bone. Due to this association between PTH and hypertension and since PTH and the paracrine factor PTH-related protein (PTHrp) have both been shown to exert marked changes in cardiovascular activity, these actions of PTH and PTHrp were examined in spontaneously hypertensive rats (SHR) and in control normotensive Wistar-Kyoto rats (WKY). Fourteen-week-old SHR [systolic blood pressure (SBP), 201 +/- 4.4 mm Hg] and WKY (SBP, 141 +/- 2.5 mm Hg) were studied. Renal cortical membranes were prepared and assayed for radioligand binding with [125I]PTH-(1-34) and [125I]PTHrp-(1-34). There was no apparent alteration in the affinity of the binding sites to either peptide in the SHR, but specific binding in SHR renal tissue was only 60% of that observed in WKY tissue for both peptides. Serum immunoreactive PTH levels were 4-fold higher in SHR than WKY, while serum total calcium and 1,25-dihydroxyvitamin D3 levels were not different. The iv administration of both PTH and PTHrp produced dose-dependent reductions in SBP and increases in heart rate in conscious unrestrained SHR and WKY. Both peptides caused greater absolute reductions in blood pressure in SHR than in WKY. However, when the hypotensive response was normalized for the higher baseline pressure in the SHR, the blood pressure reductions caused by PTH and PTHrp were not different in SHR and WKY. Conversely, the chronotropic responses to PTH and PTHrp were lower in SHR compared to WKY. These findings indicate that the SHR exhibits elevated PTH levels, with a reduced number of renal PTH/PTHrp receptors and a depressed chronotropic response to either PTH or PTHrp. In contrast, the hypotensive response to PTH or PTHrp was not altered, indicating possible tissue-specific receptor subclasses or tissue-specific regulation of PTH and PTHrp receptors.


Differential effects of platelet-derived growth factor isoforms on plasminogen activator activity in fetal rat osteoblasts due to isoform-specific receptor functions. We examined receptor binding and metabolic effects of the platelet-derived growth factor (PDGF) isoforms AA, AB, and BB in cultures of osteoblastic cells from fetal rat calvaria. Saturation binding experiments demonstrated the presence of 6,000 binding sites for PDGF-AA, 42,000 for PDGF-AB, and 60,000 for PDGF-BB. Binding competition experiments were compatible with the recently postulated model of three PDGF receptor subtypes with differential affinity for the PDGF isoforms. The effects of the PDGF isoforms on DNA synthesis, collagen synthesis, and alkaline phosphatase activity in osteoblasts strictly correlated with the number of available binding sites. Accordingly, PDGF-BB was the most potent isoform, PDGF-AB was slightly less potent, and PDGF-AA was the least potent. In contrast, we found that PDGF-BB was less potent than PDGF-AB in increasing plasminogen activator activity in the osteoblast-conditioned medium. Our data strongly suggest that the PDGF receptor subtypes in fetal rat osteoblasts not only selectively bind one or more PDGF isoforms, but are also capable of responding differently to these isoforms.


Bradykinin stimulates aldosterone release from cultured bovine adrenocortical cells through bradykinin B2 receptors. The adrenal cortex contains a kallikrein-like enzyme that may lead to bradykinin (BK) formation. This study was designed to determine whether BK acts on adrenocortical cells to stimulate steroid secretion. BK, Lys-BK, a specific BK 2 (B2) receptor agonist, and desArg9-BK, a specific BK 1 (B1) receptor agonist, all stimulated aldosterone secretion from cultured bovine adrenal zona glomerulosa cells. BK and Lys-BK were equipotent (EC50 = 2 x 10(-9) M), whereas desArg9-BK was 1000-fold less potent. The maximal effects of BK and BK analogs were comparable to the maximal effects of adrenocorticotropin or angiotensin II. A B2, but not a B1, receptor antagonist inhibited BK-stimulated aldosterone release. Verapamil and N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate, which reduce intracellular calcium concentrations, reduced BK-stimulated aldosterone secretion. Although BK stimulated both prostacyclin and aldosterone production, indomethacin abolished prostacyclin production without affecting aldosterone secretion. In cultured adrenal fasciculata cells, high concentrations of BK stimulated cortisol release, but B1 or B2 receptor agonists were not effective. BK-stimulated cortisol secretion was reduced by N,N-diethylamino-octyl-3,4,5-trimethoxybenzoate but not by indomethacin. In summary, BK stimulates aldosterone release from cultured adrenal glomerulosa cells via high affinity B2 receptors. The effect is calcium-dependent and independent of prostaglandins. BK also increases cortisol release; however, this stimulation requires high concentrations of BK and may be mediated by an unknown receptor or by a receptor-independent mechanism.


Beta-adrenergic and peptide N-terminal histidine and C-terminal isoleucine stimulation of N-acetyl-transferase activity and melatonin production in the cultured rat pineal gland. The purpose of this investigation was to compare the effect of peptide N-terminal histidine and C-terminal isoleucine (PHI) with that of the beta-adrenergic agonist isoproterenol (ISO) on N-acetyltransferase (NAT) activity and melatonin production in the cultured rat pineal gland. Pineal glands were removed and placed in organ culture containing PHI, ISO, or PHI plus ISO at different dosages, and subsequent changes in NAT activity and melatonin levels were measured. PHI stimulated these parameters in both a time- and dose-dependent manner. Combined treatment with PHI (10(-6) M) and high doses of ISO (either 10(-7) or 10(-8) M) did not potentiate the effect of the peptide in terms of either NAT activity or melatonin levels in the pineal gland. However, at a lower concentration, ISO (10(-9) M) had additive effects to those of PHI in both cultured pineals and medium. The results suggest that PHI modulates melatonin synthesis in the rat pineal gland. Furthermore, stimulation of the pineal with both PHI and ISO demonstrates an additive effect rather than a synergistic action of these compounds. It is presumed that ISO and PHI stimulate pineal melatonin production via separate receptors, but they probably use the same intracellular second messenger, cAMP, to do so. This is the first study showing an effect of the peptide PHI on pineal melatonin production in any vertebrate.


Activin-A stimulates the synthesis of gonadotropin-releasing hormone receptors. The number of GnRH receptors on gonadotropes is regulated by GnRH as well as by heterologous modulators. We have used the density shift technique to measure the synthetic rate of GnRH receptors in pituitary cell cultures and found it to be stimulated by GnRH, an action that is antagonized by inhibin. In the present study, we evaluated the effects of activin-A on the GnRH receptor synthesis rate as well as effects of activin on stimulation of GnRH receptor synthesis by the homologous hormone. Recombinant human activin-A (50 ng/ml) was incubated with pituitary cell cultures from female weanling rats and the incorporation of densely labeled amino acids into receptors for GnRH was measured. The rate of GnRH receptor synthesis of cells treated with activin (50 ng/ml) together with either GnRH (0.1 ng/ml) or inhibin (12 ng/ml) was also quantified. Activin significantly stimulated the synthetic rate of GnRH receptors similarly to that observed after GnRH treatment (time for synthesis of half the population of GnRH receptors was 12.6 +/- 1.1, 16.1 +/- 1.3 vs. 28.3 +/- 1.2 h for GnRH, activin, and control, respectively), although the time course for stimulation by GnRH and activin appeared to differ. Inclusion of activin in cultures did not affect homologous stimulation of GnRH receptor synthesis. The stimulatory effects of activin were unaffected by combined treatment with inhibin (t1/2 of synthesis 17.2 +/- 2.0 h). Together, these data indicate that activin stimulates GnRH receptor synthesis in cell culture through a distinct mechanism from GnRH. Additionally, inhibin did not antagonize the stimulatory effects of activin on synthesis of GnRH receptors. This is, to our knowledge, the first demonstration of an action of activin-A on GnRH receptor synthesis.


Lymphocyte-derived adrenocorticotropin is insufficient to stimulate adrenal steroidogenesis in hypophysectomized rats. Cells of the immune system can produce and respond to peptide hormones associated with the endocrine system. However, the physiological significance of these endocrine-immune interactions is not known. It has been postulated that cells of the immune system, when stimulated with viruses that induce interferon-alpha, produce sufficient levels of ACTH to stimulate adrenal steroidogenesis and, thus, function as an auxiliary source of ACTH that may have a role in the response to stress. However, we have confirmed that levels of ACTH-related peptides produced by immunocompetent cells are far lower than those produced by the pituitary, raising questions about the ability of lymphocyte-derived ACTH to stimulate adrenal function. Furthermore, we have rigorously examined this issue using intact and hypophysectomized rats treated with Newcastle disease virus. Although high levels of interferon-alpha were produced by both intact and hypophysectomized rats, and the plasma corticosterone concentration increased dramatically in intact animals, corticosterone remained undetectable in hypophysectomized rats. The lack of a corticosterone response in these animals was not due to adrenal insensitivity to ACTH, as shown by a normal rise in corticosterone following Cosyntropin injection 8 h after hypophysectomy. The findings demonstrate that levels of ACTH produced by nonpituitary sources in response to viral infection are not sufficient to stimulate adrenal steroidogenesis.


Solubilization of high affinity peptide-YY receptors from porcine brain. We have previously characterized peptide-YY (PYY) receptors in porcine hippocampal membranes. We demonstrate here that brain PYY receptors can be extracted in the active state using digitonin. Among several detergents tested for their suitability to extract active PYY receptors, digitonin gave the most favorable results, as judged by specific binding of [125I]PYY to the solubilized receptors. The binding of [125I]PYY to digitonin extract was dependent on incubation time, temperature, and protein and magnesium ion concentrations and had a pH optimum of 6-7. Solubilized PYY receptors maintained the rank order of potencies for various related peptides and PYY fragments characteristic of the membrane PYY receptor: PYY greater than neuropeptide Y (NPY) much much greater than avian and porcine pancreatic polypeptide, and PYY greater than PYY-(22-36) much much greater than PYY-(1-22) and PYY-(22-28), respectively. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract; the high affinity component had affinities and binding capacities similar to those of the membrane PYY receptor. Solubilized PYY receptors also retained their sensitivity to guanine nucleotides. PYY was cross-linked to its receptors with disuccinimidyl suberate, solubilized with digitonin, and cross-linked to digitonin-solubilized receptors. The resulting complexes were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography. Using these procedures, we identified a PYY receptor species with a molecular size of 50,000, which was the same size as the labeled protein in native membrane homogenates. Solubilized NPY receptors were also the same size. The solubilized cross-linked PYY receptor was adsorbed by wheat germ agglutinin-agarose and Concanavalin-A, suggesting its glycoprotein nature. These data suggest that the specific binding properties of the PYY receptor are inherent in the solubilized glycoprotein molecules. The solubilization in digitonin of PYY receptors from membranes should allow a more complete molecular and functional characterization of PYY-mediated events and purification of the receptor.


Regulation of the adrenal renin angiotensin system in cultured bovine zona glomerulosa cells: effect of catecholamines. The renin-angiotensin system consists of two main enzymes, renin and angiotensin-converting enzyme, which lead to the formation of angiotensin-II. Angiotensin-II is a potent vasoconstrictor and stimulates the production of aldosterone. In this study we examined the effect of ACTH, potassium, (Bu)2cAMP (dbcAMP), and catecholamines on the adrenal renin-angiotensin system. To study the production of renin and aldosterone in vitro, we developed a monolayer culture of bovine zona glomerulosa cells in serum-free medium. Collagenase-dispersed zona glomerulosa cells were incubated in Pasadena Foundation for Medical Research-4 medium containing 10% fetal calf serum for 72 h, and the medium was replaced with serum-free medium for the next 24 h of the experimental period. The cells during this 24 h were exposed to various doses of ACTH, potassium, dbcAMP, and sympathomimetic agents. ACTH and dbcAMP stimulated aldosterone secretion, and this secretion was associated with an increase in renin activity in cells and medium. Aldosterone was also stimulated by high doses of potassium, and potassium had a stimulatory effect on the secretion of renin in medium. Catecholamines had a weak stimulating effect on aldosterone secretion and were potent stimulators of adrenal renin activity in cells and medium. Dopamine had no significant effect on basal aldosterone secretion or renin activity in cells and medium. In conclusion, these data indicate that adrenal renin is synthesized in bovine zona glomerulosa cells in vitro, and that ACTH and dbcAMP stimulate adrenal renin and aldosterone production. Furthermore, adrenal renin, like renal renin, may be under the control of the sympathetic nervous system.


Differential regulation of the rat melatonin receptors: selective age-associated decline and lack of melatonin-induced changes. To gain some understanding of the factors regulating high affinity melatonin (MT) receptors in the rat, we conducted a series of studies using quantitative autoradiography of [2-125I]iodo-MT binding in vitro with validated assay conditions. MT receptor status and the relative protein content of the autoradiographic sections were assessed in the anterior pituitary gland, the area postrema, the caudal (tail) artery (CA), the anterior cerebral artery (ACA), and the suprachiasmatic nuclei (SCN) of 9-, 96-, and 306-day-old Wistar rats. When age-associated changes in protein content were taken into account, MT receptor expression in the area postrema and the SCN remained relatively constant between 9-306 days of age. On the contrary, a dramatic loss of MT receptors was observed in the arteries of 306-day-old rats (98% and 89% loss compared to the 9-day-old rats in the ACA and CA, respectively). In the anterior pituitary gland, MT receptors were expressed only in the 9-day-old rats. The above changes reflected major changes in binding capacity and minor changes in binding affinity. Neither removal of endogenous circulating MT (acute light exposure for 24 h, pinealectomy, or superior cervical ganglionectomy) nor MT injections (1 mg/kg for 10 days 6 h after lights on) affected MT receptor status in the ACA, CA, area postrema, or SCN. Our data suggest that MT receptor expression is differentially regulated during development and that permanent alterations in MT levels do not affect rat MT receptor status.


Inhibition of 25-hydroxyvitamin D3-24-hydroxylase by forskolin: evidence for a 3',5'-cyclic adenosine monophosphate-independent mechanism. Forskolin has long been used to demonstrate the involvement of cAMP in the regulation of cellular function, by virtue of its ability to stimulate adenylate cyclase directly. Recently, however, forskolin has been shown to affect plasma membrane transporter and channel function in a manner unrelated to cAMP. The present study examines whether forskolin-mediated inhibition of a mitochondrial membrane-associated enzyme, 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase), also occurs by a cAMP-independent mechanism. Both forskolin and PTH stimulated cAMP accumulation and inhibited 24-hydroxylase activity in a dose-dependent manner in fresh mouse renal tubules. However, the level of inhibition of 24-hydroxylase achieved with forskolin was consistently greater than that obtained with PTH, at comparable levels of cAMP. 1',9'-Dideoxyforskolin, a cyclase-inactive analog of forskolin, also inhibited 24-hydroxylase activity, without stimulating cAMP production. Moreover, both forskolin and 1',9'-dideoxyforskolin directly inhibited 24-hydroxylase in isolated renal mitochondria. Kinetic analysis revealed a competitive mode of inhibition for both agents; however, 1',9'-dideoxyforskolin proved to be a more potent inhibitor of 24-hydroxylase than forskolin (inhibitory constant, 0.25 vs. 22 microM, respectively). Finally, both forskolin and 1',9'-dideoxyforskolin also inhibited inducible 24-hydroxylase in renal tubules prepared from 1,25-(OH)2D3-treated mice. However, inducible 24-hydroxylase activity was less susceptible to inhibition by the diterpenes than the basal enzyme activity. The present study provides evidence for cAMP-independent inhibition of 24-hydroxylase by forskolin and represents the first demonstration of a cAMP-independent effect of forskolin on a protein that is not a plasma membrane-associated transporter or channel. Our data advocate caution in the interpretation of studies using forskolin to assess the role of cAMP in cellular processes.


Melatonin signal transduction and mechanism of action in the central nervous system: using the rabbit cortex as a model. The cortex of the rabbit (Oryctolagus cuniculus) is rich in melatonin binding sites, and particularly abundant is the parietal cortex. Consequently, we characterized the putative melatonin receptor in the parietal cortex by a series of in vitro ligand-receptor binding experiments and biochemical and electrophysiological studies. The in vitro saturation and competition experiments demonstrated that the binding in the crude cortical membrane preparations was of high affinity and specificity. Guanine nucleotides (GDP, GTP, and GTP gamma S) inhibited the specific 2-[125I]iodomelatonin binding in a dose-dependent manner. Coincubation with a nonhydrolyzable GTP analog provoked a shift in the binding affinity; the numerical values of the Kd increased from 20-30 to 200-600 pM. Melatonin, in nanomolar concentrations, was able to inhibit the forskolin-stimulated accumulation of cAMP in parietal cortex explants, and preincubation with pertussis toxin counteracted this effect of melatonin. Apparently, the melatonin binding site in the rabbit parietal cortex is linked to its second messenger via a pertussis toxin-sensitive G-protein, probably of the inhibitory Gi class, similar to what has been described for different parts of the brain of other vertebrates. The experiments on the spontaneous firing activity of single neurons in the third to fourth layer of the parietal cortex in anesthetized animals showed that melatonin and its potent agonist 2-iodomelatonin exhibited gamma-aminobutyric acid (GABA)-like effects and were able alone, in nanomolar concentrations, to significantly slow the neuronal firing activity. Moreover, both melatonin and 2-iodomelatonin potentiated the effect of GABA on the neuronal activity, leading to powerful inhibition of the tested neurons. Undoubtedly, the binding site in the rabbit parietal cortex possesses all of the characteristics of a functional receptor. We suggest that melatonin is involved in the control of fundamental cortical functions and that it acts in concert with GABA, one of the two major inhibitory neurotransmitters in the central nervous system.


Oxytocin at physiological concentrations evokes adrenocorticotropin (ACTH) release from corticotrophs by increasing intracellular free calcium mobilized mainly from intracellular stores. Oxytocin displays synergistic or additive effects on ACTH-releasing factor or arginine vasopressin-induced ACTH secretion, respectively. The potency of oxytocin (OT) in evoking ACTH secretion by isolated, superfused rat adenohypophyseal corticotrophs and its enhancement by CRF and arginine vasopressin (AVP) were analyzed. Each secretagogue effectively released ACTH from adenohypophyseal cells when added separately in pulsatile fashion in physiological concentrations based on hypophyseal portal blood (OT, 10 nM; AVP, 0.5 nM; CRF, 0.1 nM). OT released ACTH at concentrations as low as 1 nM. Moreover, a dose-response relationship up to 10 microM was revealed. Combinations of a constant amount of CRF (0.1 nM) with increasing concentrations of OT exerted a synergistic effect on ACTH release. In contrast, OT given in various concentrations in combination with AVP (0.5 nM) produced an additive effect on ACTH release. To study the mechanism of action of OT on ACTH secretion, cytosolic free calcium levels in single pituitary cells exposed to OT or AVP were measured using the calcium-sensitive fluorescent indicator Fura-2. Corticotrophs among mixed adenohypophyseal cell types in the primary cultures were identified by immunocytochemistry. More than 500 cells were individually stimulated with OT or AVP. Basal cytosolic free calcium levels ranged between 80-130 nM free calcium. The addition of 100 nM OT or 1 microM AVP increased the cytosolic free calcium concentration within 3 sec to values ranging from 500-800 nM. An increase in intracellular calcium ranging from 200-500 nM due to OT could still be observed after extracellular calcium depletion. Taken together, our data demonstrate that physiological concentrations of OT stimulate ACTH secretion, independent of the other ACTH secretagogues, by mobilizing calcium mainly from intracellular stores.


Inactivation of early glucocorticoid feedback by corticotropin-releasing factor in vitro. We have investigated the interaction between hypothalamic ACTH secretagogues and adrenocortical glucocorticoids in rat anterior pituitary tissue using an in vitro perifusion system. Repeated 5 min pulses of 41-residue CRF (CRF-41) or arginine vasopressin (AVP) were applied at 1 h intervals for up to 7 h. Administration of 0.1 microM corticosterone 30 min before and during the 5 min 0.1 nM CRF-41 stimulus at 5 h resulted in a significant inhibition of CRF-41 stimulated ACTH release within 30 min. Inhibition of ACTH release also developed if no CRF-41 stimulus was applied in conjunction with steroid at 5 h. In contrast, if the exposure to corticosterone (0.1 microM, 35 min total duration) was started simultaneously with the application of CRF-41 at 5 h, no inhibition of ACTH release ensued. Similarly, no inhibition of CRF-41-stimulated ACTH release was observed when corticosterone was started simultaneously with a 5 min pulse of cyclic 8-(4-Chlorophenylthio) AMP (8-CPT-cAMP), a cell membrane permeant analog of cAMP. In contrast to CRF-41 and 8-CPT-cAMP, AVP failed to modify glucocorticoid-induced inhibition of AVP- or CRF-41-stimulated ACTH release. Moreover, CRF-41 did not prevent the glucocorticoid-induced inhibition of AVP-stimulated ACTH release. In summary: 1) CRF-41 inactivates early glucocorticoid inhibition of CRF-41-stimulated ACTH secretion, and this is mimicked by a cell membrane permeant analog of cAMP; 2) AVP does not inactivate glucocorticoid-induced inhibition of stimulated ACTH release; 3) the data point to an acute interaction between the cAMP/protein kinase A and glucocorticoid-responsive intracellular pathways. Such differential modulation of feedback inhibition by CRFs may be of functional importance in vivo.


Rat Leydig cells bind platelet-derived growth factor through specific receptors and produce platelet-derived growth factor-like molecules. The platelet-derived growth factor (PDGF) is a major mitogen for cells of mesenchymal origin. Because Leydig cells arise from mesenchymal precursors, we tested the hypothesis that these cells might be a target for PDGF. We also investigated a possible production of a PDGF-like substance by Leydig cells in culture and the distribution of PDGF-like material in the rat testis using immunohistochemistry. PDGF was found to bind specifically to high affinity receptors on the surface of purified adult rat Leydig cells. Conditioned medium from cultured Leydig cells competed with 125I-labeled PDGF for binding to the Leydig cells. The secretion of PDGF receptor-competing activity was stimulated in a dose-dependent manner by the trophic hormone hCG. Immunohistochemical studies revealed specific staining for PDGF in the Leydig cells of adult rat testis. Taken together these observations suggest that PDGF may play a role in the local control mechanisms of testicular function.


Bidirectional regulation of the 1,25-dihydroxyvitamin D3 receptor by phorbol ester-activated protein kinase-C in osteoblast-like cells: interaction with adenosine 3',5'-monophosphate-induced up-regulation of the 1,25-dihydroxyvitamin D3 receptor. In the present study the involvement of protein kinase-C (PKC) in the regulation of the vitamin D receptor (VDR) and interaction of PKC with cAMP-induced up-regulation of VDR in osteoblast-like cells were examined. Activation of PKC by incubation for 4 h with the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a comparable dose-dependent decrease in 1,25-dihydroxyvitamin D3 binding in the osteoblast-like cell lines UMR 106 and ROS 17/2.8, with a maximum inhibition at 100 nM and an IC50 at 5 nM PMA. Time-course studies revealed that in both UMR 106 and ROS 17/2.8 cells, 24-h incubation with PMA caused an increase in 1,25-dihydroxyvitamin D3 binding. This can be related to down-regulation of PKC. Scatchard analysis demonstrated that activation of PKC resulted not in a change in receptor affinity, but, rather, in an increase in VDR number. This is supported by Northern blot analysis, which shows at 2 h a decrease and at 24 h an increase in VDR mRNA. At 4 h, when activation of the cAMP pathway results in an increase in VDR, activation of PKC results in a decrease in VDR. Coincubation for 4 h with PMA caused a decrease in PTH- and forskolin-induced up-regulation of VDR. This inhibition is not due to a reduction in cAMP production, as PTH-stimulated cAMP production was potentiated by PMA. The effect of activation of PKC on VDR is not a general effect, as PMA does not affect basal ornithine decarboxylase activity and potentiates PTH-induced ornithine decarboxylase activity. The present study demonstrates that PKC is involved in the regulation of VDR in UMR 106 and ROS 17/2.8 and that PKC interacts with cAMP in the regulation of VDR. The current data point to a negative controlling role for PKC in the regulation of VDR. Moreover, two different cAMP-regulated actions in UMR 106 cells (VDR up-regulation and ornithine decarboxylase activity) are differently modulated by PKC. Although the precise mechanism by which PKC represses and stimulates gene expression is not yet clear, this study demonstrates the important regulatory role for PKC in two osteoblast-like sarcoma cell lines.


Effect of chronic active immunization anti-corticotropin-releasing factor on the pituitary-adrenal function in the sheep. ACTH and cortisol diurnal variations and responses to two types of stress (insulin-induced hypoglycemia and isolation-restraint stress) or to an acute injection of lysine-vasopressin were studied in intact and anti-corticotropin-releasing factor (CRF) actively immunized rams. Immunization was obtained by the injection of synthetic ovine CRF coupled to BSA with carbodiimide. All animals developed antibodies anti-CRF and displayed an alteration of their general condition and a body weight reduction. The mean basal ACTH and cortisol secretion as well as the number and mean amplitude of diurnal pulses of these hormones was significantly reduced in the group of anti-CRF immunized rams. However, the reduction in all three parameters was much more pronounced for cortisol than for ACTH. No ACTH and cortisol response to insulin-induced hypoglycemia and isolation-restraint stress was observed. The stimulating action of lysine-vasopressin on ACTH release was significantly reduced as compared to controls. These results indicate that CRF is a major physiological component of the ovine hypothalamo-hypophysial-adrenal axis and participates in the events that regulate ACTH and cortisol diurnal variations and response to stress.


Basic fibroblast growth factor: an autocrine mitogen of rat thyroid follicular cells? Basic fibroblast growth factor (FGF) is a mitogen for the rat thyroid cell line FRTL-5. A possible autocrine role for this growth factor has been investigated in rat thyroid follicular cells both in vitro and in vivo. We report here the synthesis and localisation of basic FGF and one of its high affinity receptors (flg) in FRTL-5 cells, shown by Northern hybridization analysis, Western blotting, and immunohistochemistry. Two major species of basic FGF mRNA of approximately 2.2 and 7.0 kilobases and one major species of flg mRNA of approximately 4.2 kilobases were identified in FRTL-5 cells. The basic FGF immunoreactivity observed histologically was attributed to a heparin-binding protein of approximately 20 kilodaltons mol wt. The physiological relevance of basic FGF to the thyroid is underlined by the demonstration of significant stores of immunoreactive protein, predominantly in the basement membrane of thyroid follicular cells, in paraffin sections of the normal rat thyroid, although basic FGF mRNA was not detected by in situ or Northern hybridization analysis. The mitogenic response of FRTL-5 cells to human recombinant basic FGF has been further characterized, and the factor shown to stimulate with an ED50 of 4 ng/ml. The mitogenic effects of exogenously supplied and endogenously produced basic FGF were shown to be potentiated by heparin. Examination of the mitogenic activity of both exogenous and endogenous basic FGF and its immunoneutralization in vitro suggests that locally produced basic FGF may be an important autocrine regulator of thyroid follicular cell growth.


Platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and EGF and PDGF beta-receptors in human endometrial tissue: localization and in vitro action. Human endometrial tissue and primary stromal cell culture contain immunoreactive epidermal growth factor (EGF), platelet-derived growth factor (PDGF)-AB as well as EGF and PDGF-beta receptors. The immunostaining for EGF, EGF receptor, and PDGF beta-receptor were associated with endometrial luminal and glandular epithelial and stromal cells, whereas only the stromal cells contain immunoreactive PDGF-AB. The immunostaining intensity of EGF, EGF receptor, and PDGF-AB was similar in both phases of the menstrual cycle, whereas, PDGF-beta receptor immunostaining was highest in proliferative phase and considerably reduced, particularly in luminal and glandular epithelial cells in the secretory phase. In addition primary stromal cell cultures express EGF, PDGF-AB, and contain EGF and PDGF-beta receptors, and very low levels of PDGF-alpha receptor. 3H-Thymidine incorporation indicate that after 48 h of incubation in serum-free medium approximately 75-80% of stromal cells are quiescent. Incubation of quiescent stromal cells with 10% fetal bovine serum stimulate 3H-thymidine incorporation in a time-dependent manner reaching maximal after 30-48 h, with a doubling time of 38.2 h. EGF (1.5-15 ng/ml) stimulates 3H-thymidine incorporation by quiescent stromal cells (P less than 0.001). This effect was significantly reduced at concentrations above 15 ng/ml (P less than 0.005). PDGF-AB (3-10 ng/ml) and PDGF-BB (0.5-10 ng/ml) also stimulate 3H-thymidine incorporation in quiescent stromal cells compared to controls (P less than 0.005). The action of EGF (15 ng/ml) and PDGF-AB (10 ng/ml) was time dependent, reaching maximal after 36 and 48 h of incubation (P less than 0.002). Addition of PDGF-AB (10 ng/ml) to EGF (15 ng/ml) significantly enhanced the action of EGF or PDGF-AB used individually (P less than 0.001). 17 beta-estradiol or progesterone at 1 microM did not stimulate 3H-thymidine incorporation, although they were stimulatory in combination (P less than 0.001), they did not alter the action of EGF or PDGF when added in combination. These observations provide further evidence that human endometrial tissue contains specific immunoreactive EGF receptors. It also demonstrates the presence of immunoreactive EGF, PDGF-AB, and PDGF-beta receptors in endometrial tissue as well as stromal cells in primary culture. Both EGF and PDGF are mitogenic for endometrial stromal cells, suggesting an autocrine/paracrine role in modulation of endometrial cell growth and differentiation.


Heterogeneous intracellular free calcium responses to parathyroid hormone correlate with morphology and receptor distribution in osteogenic sarcoma cells. The osteogenic sarcoma cell line UMR 106-01 exhibits heterogeneous morphology and hormone response in subconfluent monolayer cultures. In these studies we have explored the correlation between morphological profiles and patterns of cytosolic calcium [Ca2+]i response to PTH and other agonists in single UMR 106-01 cells loaded with the Ca(2+)-sensitive fluorescent indicator, fura-2. Realtime recording of [Ca2+]i revealed that PTH (10(-7) M) produced a transient [Ca2+]i rise in 19% of the cells studied. [Ca2+]i transients were also induced by prostaglandins E2 and F2 alpha, and fetal bovine serum, but with different response frequencies (20%, 12%, and 58%, respectively). Spatial resolution of changes in [Ca2+]i by video image analysis revealed that the response to PTH was more frequent in large polygonal cells with long cytoplasmic processes and less common in smaller cells growing in clusters, whereas there was no clear subtype specificity for the effects of epidermal growth factor and fetal bovine serum on [Ca2+]i. Autoradiographic analysis of cell monolayers demonstrated a higher density of PTH-binding sites on cells with cytoplasmic extensions, whereas epidermal growth factor-binding sites were largely on colony-forming cells. Thus, the [Ca2+]i response to hormonal stimulation is heterogeneous within UMR 106-01 cell populations and within single cells, and it correlates with receptor density. This suggests that osteoblastic cells respond to PTH by activation of changes in [Ca2+]i only at certain specific steps during osteoblast development or stages of the cell cycle.


Norepinephrine stimulates immunoreactive (ir) atrial natriuretic peptide (ANP) secretion and pro-ANP mRNA expression from rat hypothalamic neurons in culture: effects of alpha 2-adrenoceptors. Although ANP or its smaller congeners are produced and secreted from rat hypothalami, the role or roles of neurotransmitter(s) in regulating their release and production from the neurons remains unclear. We report here that norepinephrine or epinephrine (NE/EPI) facilitates irANP secretion and pro-ANP mRNA expression in long term cultures of rat hypothalamic neurons through their effects on alpha 2-adrenoceptors. Hypothalami of 3 day-old Sprague-Dawley rats were removed and digested with collagenase. The dispersed cells were plated on poly-D-lysine coated culture dishes (10(6) cells/well) in Hepes buffered Dulbecco's Modified Eagle Medium supplemented with 8% fetal calf serum. Six days after plating, media were replenished with serum free media and the cultures incubated for 4 more days with vehicle or various doses of NE, EPI, alpha- or beta-adrenoceptor agonists in the presence of absence of antagonists. Culture media were then extracted with C18 Sep-pak and the levels of irANP determined by a well characterised RIA for ANP. NE or EPI treatment significantly increased irANP secretion from the cultures in a dose related manner with ED50 and Emax of approximately 0.2 microM and 1 microM respectively. The stimulation effect of NE was blocked by yohimbine (alpha 2-antagonist), but not prazosin (alpha 1-antagonist) or propranolol (beta-antagonist). Clonidine (alpha 2-agonist), but not phenylephrine (alpha 1-agonist) or isoprenaline (beta-agonist) mimicked the effects of NE or EPI. At the concentration of 0.1 microM, clonidine increased irANP release approximately 3 fold above that of control values (34.7 +/- 3.3; mean +/- SE, n = 4). These changes were accompanied by corresponding increments in the abundance of pro-ANP mRNA in the cultures as examined by a colorimetric Northern blot analysis. Our results indicate that NE or EPI, acting through its alpha 2-adrenoceptors, may modulate the function of ANP neurons in rat hypothalami by regulating the secretion and production of the neuropeptide at the genomic level.


Excitatory amino acids modulate phosphoinositide signal transduction in human epileptic neocortex. Stimulation of phosphoinositide (PI) hydrolysis by norepinephrine (NE), carbachol (Carb), and excitatory amino acids (EAAs) was measured in slices prepared from neocortex excised during epilepsy surgery. NE and Carb markedly enhanced PI turnover (EC50: NE, 12 microM; Carb, 661 microM) as reflected by [3H]inositol monophosphate (IP1) accumulation in tissue slices prelabeled with [3H]myoinositol. These effects were dose-dependent, saturable, and five to six times higher than basal IP1 accumulation. A weaker stimulation (twofold) was observed with quisqualate (QUIS; EC50, 1.1 microM) and glutamate (GLU; EC50, greater than 1 mM), while minimal or no stimulation was seen with kainate (KA) and N-methyl-D-aspartate (NMDA). Agonist-stimulated PI turnover was significantly reduced in samples from actively spiking epileptic neocortex versus nonspiking areas as defined by electrocorticography (NE, -23%, p less than 0.05; Carb, -44%, p less than 0.01). Preincubation of slices with various EEAs inhibited Carb-induced IP1 formation. The maximal extent of inhibition (1 mM) was both amino acid-dependent (IC50: NMDA, 5 microM; KA, 3.3 microM; QUIS, 47 microM; GLU, greater than 1 mM). These data suggest that epileptic activity modulates PI metabolism and alters receptor-effector coupling. As important mediators of epileptogenesis, EAAs may interfere++ with the efficiency of this second messenger system.


Evolution of the nuclear receptor gene superfamily. Nuclear receptor genes represent a large family of genes encoding receptors for various hydrophobic ligands such as steroids, vitamin D, retinoic acid and thyroid hormones. This family also contains genes encoding putative receptors for unknown ligands. Nuclear receptor gene products are composed of several domains important for transcriptional activation, DNA binding (C domain), hormone binding and dimerization (E domain). It is not known whether these genes have evolved through gene duplication from a common ancestor or if their different domains came from different independent sources. To test these possibilities we have constructed and compared the phylogenetic trees derived from two different domains of 30 nuclear receptor genes. The tree built from the DNA binding C domain clearly shows a common progeny of all nuclear receptors, which can be grouped into three subfamilies: (i) thyroid hormone and retinoic acid receptors, (ii) orphan receptors and (iii) steroid hormone receptors. The tree constructed from the central part of the E domain which is implicated in transcriptional regulation and dimerization shows the same distribution in three subfamilies but two groups of receptors are in a different position from that in the C domain tree: (i) the Drosophila knirps family genes have acquired very different E domains during evolution, and (ii) the vitamin D and ecdysone receptors, as well as the FTZ-F1 and the NGF1B genes, seem to have DNA binding and hormone binding domains belonging to different classes. These data suggest a complex evolutionary history for nuclear receptor genes in which gene duplication events and swapping between domains of different origins took place.


Demonstration of retrotransposition of the Tf1 element in fission yeast. Tf1, a retrotransposon from fission yeast, has LTRs and coding sequences resembling the protease, reverse transcriptase and integrase domains of retroviral pol genes. A unique aspect of Tf1 is that it contains a single open reading frame whereas other retroviruses and retrotransposons usually possess two or more open reading frames. To determine whether Tf1 can transpose, we overproduced Tf1 transcripts encoded by a plasmid copy of the element marked with a neo gene. Approximately 0.1-4.0% of the cell population acquired chromosomally inherited resistance to G418. DNA blot analysis demonstrated that such strains had acquired both Tf1 and neo specific sequences within a restriction fragment of the same size; the size of this restriction fragment varied between different isolates. Structural analysis of the cloned DNA flanking the Tf1-neo element of two transposition candidates with the same regions in the parent strain showed that the ability to grow on G418 was due to transposition of Tf1-neo and not other types of recombination events.


Magnetic resonance imaging in infantile spasms: effects of hormonal therapy. Magnetic resonance imaging (MRI) was performed on five patients with infantile spasms who were treated with relatively low doses of adrenocorticotrophic hormone (ACTH) to study the extent of brain shrinkage induced by ACTH therapy. MRI prior to ACTH therapy revealed periventricular hyperintensity (PVH) areas and poor myelination in four patients. In one case, MRI performed 2 days after initiation of ACTH therapy also showed PVH and poor myelination. Brain shrinkage was observed 2 weeks after initiation of ACTH therapy. The most impressive follow-up finding upon MRI was the decrease in PVH found in four patients. The differentiation between myelinated white matter and surrounding cortex became poorer in three cases. Cortical atrophy progressed in all patients but ventricular dilation progressed in only one patient. At the end of ACTH therapy, ventricular dilation progressed in all cases. These findings suggest that loss of water not only from periventricular white matter but also from cortex is the main etiological factor of brain shrinkage induced by ACTH.


Symmetry, flexibility and permeability in the structure of yeast retrotransposon virus-like particles. The virus-like particles (VLPs) of the yeast retrotransposon Ty are genetically, structurally and functionally analogous to retroviral nucleocapsids or cores. Like retroviral cores Ty-VLPs package and possibly promote the enzyme activities for reverse transcription and integration, as well as encapsulating the RNA that is the intermediate in retrotransposition. Here we show that Ty-VLPs assemble into symmetrical structures across a broad distribution of particle sizes. This spread of sizes violates the principle of quasi-equivalent packing. In addition, RNase accessibility experiments suggest that these particles form an open structure that does not protect the encapsulated RNA. These features distinguish Ty-VLPs from typical spherical viral capsids in both structure and function.


A nuclear matrix attachment region organizes the Epstein-Barr viral plasmid in Raji cells into a single DNA domain. The extrachromosomal Epstein-Barr virus (EBV) plasmid in the Burkitt lymphoma cell line, Raji, is stably associated with the nuclear matrix. This association is effected by a nuclear matrix attachment region (MAR) located in the BamHI C fragment of the viral genome; no other region of EBV DNA was found to be attached to the nuclear matrix with high affinity. The MAR was mapped to 5.2 kbp of DNA, greater than 80% of which is found on the nuclear matrix in unsynchronized cells expressing only viral latent cycle products. Thus the majority of viral plasmids in Raji cells use the same MAR. The MAR of EBV DNA contains the origin of latent viral DNA replication (oriP), the genes for the small viral RNAs (EBERs) and a 500 bp region immediately upstream of the EBER-1 gene. The clustering of the latent viral replication origin and the nearby enhancer and promoters for latent viral transcription on the nuclear matrix is likely to be crucial for regulation of the latent viral genome.


Precursor protein translocation by the Escherichia coli translocase is directed by the protonmotive force. The SecY/E protein of Escherichia coli was coreconstituted with the proton pump bacteriorhodopsin and cytochrome c oxidase yielding proteoliposomes capable of sustaining a protonmotive force (delta p) of defined polarity and composition. Proteoliposomes support the ATP- and SecA-dependent translocation of proOmpA which is stimulated by a delta p, inside acid and positive. delta p of opposite polarity, inside alkaline and negative, suppresses translocation while SecA-mediated ATP hydrolysis continues unabated. delta psi and delta pH are equally effective in promoting or inhibiting translocation. Membrane-spanning translocation intermediates move backwards in the presence of a reversed delta p. These results support a model [Schiebel, E., Driessen, A.J.M., Hartl, F.-U. and Wickner, W. (1991) Cell, 64, 927-939] in which the delta p defines the direction of translocation after ATP hydrolysis has released proOmpA from its association with SecA. The polarity effect of the delta p challenges models involving delta p-dependent membrane destabilization and provides further evidence for a role of the delta p as driving force in precursor protein translocation.


Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain. Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L-type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.


Cytoplasmic truncation of the p55 tumour necrosis factor (TNF) receptor abolishes signalling, but not induced shedding of the receptor. The mechanistic relationship between the signalling for the TNF effects by the human p55 TNF receptor (hu-p55-TNF-R) and the formation of a soluble form of the receptor, which is inhibitory to these effects, was explored by examining the function of C-terminally truncated mutants of the receptor, expressed in rodent cells. The 'wild-type' receptor signalled for a cytocidal effect when cross-linked with specific antibodies and exhibited spontaneous shedding. Shedding of the receptor was not affected by TNF but was markedly enhanced by 4 beta-phorbol-12-myristate-13-acetate (PMA). Receptor mutants with 53%, 83% and 96% C-terminal deletions could not signal for the cytocidal effect. Furthermore, they were found to associate with the endogenous rodent receptors, interfering with their signalling. Yet even the deletion of 96% of the intracellular domain did not abolish shedding of the receptor in response to PMA. These findings suggest that signalling and shedding of the p55 TNF-R are mechanistically distinct.