Characterization of endothelin-converting enzyme from endothelial cells and rat brain: detection of the formation of biologically active endothelin-1 by rapid bioassay. Using the endothelin-1 (ET-1)-stimulated elevation in cGMP in LLC-PK1 cells as a biological detector system for the conversion of big ET-1 (bET-1) to ET-1, we detected bET-1-converting activities in subcellular fractions from bovine aortic cultured endothelial cells (BAE) and rat brain. Within the particulate fraction of BAE, we detected two activities, at pH 3.4 and pH 5.4-7.4. The latter but not the former activity was inhibited in a concentration-dependent manner by phosphoramidon (approximate IC50, 1 microM) and converted bET-1 to ET-1 at a rate of 0.6 nmol/hr/mg of protein. It could be solubilized from the particulate fraction by detergent treatment. Phosphoramidon-inhibitable converting activity was also detected in the cytosolic fraction of BAE. Within the rat brain, phosphoramidon-inhibitable conversion of bET-1 to ET-1 was detected principally in the cytoskeletal fraction, i.e., that fraction from the membrane that was not solubilized by detergent treatment. These results show the presence of at least two different endothelin-converting enzyme activities in endothelial cells and a third within the rat brain. They also demonstrate the use of LLC-PK1 cells as a rapid assay that permits the sensitive detection and measurement of the formation of biologically active ET-1 from its precursor bET-1.


Lactosylated high density lipoprotein: a potential carrier for the site-specific delivery of drugs to parenchymal liver cells. Mammalian liver contains two types of galactose receptors, specific for Kupffer or parenchymal cells. Because galactose-specific receptors are largely confined to the liver, galactose-bearing carriers are promising vehicles for the specific delivery of drugs to liver cells. In the present study, high density lipoprotein (HDL), a spherical particle with a diameter of 10 nm, in which a variety of lipophilic drugs can be incorporated, was provided with galactose residues by reductive lactosamination. After injection into rats, lactosylated 125I-HDL was rapidly cleared from the plasma (half-life, less than 1 min). Ten minutes after injection, the liver contained about 95% of the dose, whereas only small amounts of radioactivity were found in other tissues. The hepatic uptake was inhibited by preinjection with N-acetylgalactosamine, which indicates that the hepatic recognition sites are galactose specific. Subcellular fractionation of the liver indicated that recognition of lactosylated HDL is followed by internalization and degradation of the apoprotein in the lysosomes. Liver cells were isolated at 10 min after injection of lactosylated 125I-HDL, and it was found that uptake occurs almost exclusively by parenchymal cells. These cells contained about 98% of the hepatic radioactivity. The liver uptake of the lipid moiety of lactosylated HDL, labeled with [3H]cholesteryl oleate, was identical to that of the 125I-labeled apoproteins, which indicates that the particle is taken up as a unit. Thus, lactosylated HDL is taken up rapidly and selectively by parenchymal liver cells, and it appears that it might be a very effective vehicle for the specific delivery of lipophilic drugs to these cells.


Inside polyomavirus at 25-A resolution.

Inside polyomavirus at 25-A resolution. Empty capsids and complete virions of polyomavirus crystallize isomorphously. Here we use difference Fourier analysis of X-ray diffraction data at 25-A resolution from these crystals to obtain an electron-density map of the inside of the virion. The polyomavirus capsid is built from 72 pentamers of VP1 that form three different types of connections in the T = 7d icosahedral surface lattice. Self-assembly of purified recombinant VP1 into capsid-like aggregates has shown that switching of the bonding specificity to form the unanticipated non-equivalent connections is an inherent property of the VP1 pentamers. Our map of the inside of the virion displays 72 prongs of electron density extending from the core into the axial cavities of the VP1 pentamers. We identify these prongs with the VP2 and VP3 molecules, which may function to guide the assembly of the highly ordered capsid on the nucleohistone core. The atomic structure of the closely related simian virus-40 capsid has been determined from the high-resolution diffraction data. Our polyomavirus map, calculated using all the low-resolution diffraction data, shows no indication of regular order inside the spherical core.


Nature of biological electron transfer.

Nature of biological electron transfer. Powerful first-order analysis of intraprotein electron transfer is developed from electron-transfer measurements both in biological and in chemical systems. A variation of 20 A in the distance between donors and acceptors in protein changes the electron-transfer rate by 10(12)-fold. Protein presents a uniform electronic barrier to electron tunnelling and a uniform nuclear characteristic frequency, properties similar to an organic glass. Selection of distance, free energy and reorganization energy are sufficient to define rate and directional specificity of biological electron transfer, meeting physiological requirements in diverse systems.


P-type calcium channels blocked by the spider toxin omega-Aga-IVA. Voltage-dependent calcium channels mediate calcium entry into neurons, which is crucial for many processes in the brain including synaptic transmission, dendritic spiking, gene expression and cell death. Many types of calcium channels exist in mammalian brains, but high-affinity blockers are available for only two types, L-type channels (targeted by nimodipine and other dihydropyridine channel blockers) and N-type channels (targeted by omega-conotoxin). In a search for new channel blockers, we have identified a peptide toxin from funnel web spider venom, omega-Aga-IVA, which is a potent inhibitor of both calcium entry into rat brain synaptosomes and of 'P-type' calcium channels in rat Purkinje neurons. omega-Aga-IVA will facilitate characterization of brain calcium channels resistant to existing channel blockers and may assist in the design of neuroprotective drugs.


Clonal expansion of p53 mutant cells is associated with brain tumour progression. Tumour progression is a fundamental feature of the biology of cancer. Cancers do not arise de novo in their final form, but begin as small, indolent growths, which gradually acquire characteristics associated with malignancy. In the brain, for example, low-grade tumours (astrocytomas) evolve into faster growing, more dysplastic and invasive high-grade tumours (glioblastomas). To define the genetic events underlying brain tumour progression, we analysed the p53 gene in ten primary brain tumour pairs. Seven pairs consisted of tumours that were high grade both at presentation and recurrence (group A) and three pairs consisted of low-grade tumours that had progressed to higher grade tumours (group B). In group A pairs, four of the recurrent tumours contained a p53 gene mutation; in three of them, the same mutation was found in the primary tumour. In group B pairs, progression to high grade was associated with a p53 gene mutation. A subpopulation of cells were present in the low-grade tumours that contained the same p53 gene mutation predominant in the cells of the recurrent tumours that had progressed to glioblastoma. Thus, the histological progression of brain tumours was associated with a clonal expansion of cells that had previously acquired a mutation in the p53 gene, endowing them with a selective growth advantage. These experimental observations strongly support Nowell's clonal evolution model of tumour progression.


A chloride channel widely expressed in epithelial and non-epithelial cells. Chloride channels have several functions, including the regulation of cell volume, stabilizing membrane potential, signal transduction and transepithelial transport. The plasma membrane Cl- channels already cloned belong to different structural classes: ligand-gated channels, voltage-gated channels, and possibly transporters of the ATP-binding-cassette type (if the cystic fibrosis transmembrane regulator is a Cl- channel). The importance of chloride channels is illustrated by the phenotypes that can result from their malfunction: cystic fibrosis, in which transepithelial transport is impaired, and myotonia, in which ClC-1, the principal skeletal muscle Cl- channel, is defective. Here we report the properties of ClC-2, a new member of the voltage-gated Cl- channel family. Its sequence is approximately 50% identical to either the Torpedo electroplax Cl- channel, ClC-0 (ref. 8), or the rat muscle Cl- channel, ClC-1 (ref. 9). Isolated initially from rat heart and brain, it is also expressed in pancreas, lung and liver, for example, and in pure cell lines of fibroblastic, neuronal, and epithelial origin, including tissues and cells affected by cystic fibrosis. Expression in Xenopus oocytes induces Cl- currents that activate slowly upon hyperpolarization and display a linear instantaneous current-voltage relationship. The conductivity sequence is Cl- greater than or equal to Br- greater than I-. The presence of ClC-2 in such different cell types contrasts with the highly specialized expression of ClC-1 (ref. 9) and also with the cloned cation channels, and suggests that its function is important for most cells.


Product of vav proto-oncogene defines a new class of tyrosine protein kinase substrates. Several proteins implicated in the regulation of cellular responses to mitogenic stimuli contain a common non-catalytic domain, SH2 (for src-homologous domain 2), that mediates their interaction with activated tyrosine protein kinases. Here we report that p95vav, a proto-oncogene product specifically expressed in cells of the haematopoietic system, contains an SH2 domain and is a substrate for tyrosine protein kinases. Exposure of quiescent NIH3T3 cells ectopically expressing p95vav to either epidermal or platelet-derived growth factors induces the rapid phosphorylation of this protein on tyrosine residues. Activation of the receptors for these growth factors by their cognate ligand results in their association with p95vav, a process mediated by its SH2 domain. In T cells, co-activation of the T-cell receptor and the accessory CD4 cell-surface protein also results in the phosphorylation of the endogenous p95vav protein in tyrosine residues. Phosphorylation of p95vav is rapid, transient and precedes the appearance of most other phosphotyrosine-containing proteins. In addition to the SH2 domain, p95vav contains structural motifs not found in other tyrosine kinase substrates. One such motif is a helix-loop-helix/leucine zipper-like domain which shares some sequence similarity with these motifs in the Myc and Max proteins. Deletion of the helix-loop-helix-like motif causes oncogenic activation of p95vav. These results indicate that p95vav is a new type of signal transduction molecule and suggest a possible role for this protein in the transduction of tyrosine phosphorylation signalling into transcriptional events.


The kinetics of the inhibition by dihydroouabain of the sodium pump current in single rabbit cardiac Purkinje cells. The kinetics of the inhibition by dihydroouabain (DHO) of the Na pump current were studied in isolated rabbit cardiac Purkinje cells by means of whole-cell recording. A fast exchange of the extracellular solution at the membrane of the cell studied was performed via two multi-barrelled pipettes nearby. Judging from the steady-state inhibition of the Na pump current at various DHO concentrations half maximal inhibition occurred at 1 x 10(-5) mol/l DHO in a medium containing 2 mmol/l K+ and at 3.5 x 10(-5) mol/l DHO in a solution containing 10.8 mmol/l K+. Assuming a reversible one-to-one binding reaction, the time course of DHO binding and unbinding was analysed under a variety of conditions. It was shown that the antagonistic effect of K+ on the inhibition of the pump current was entirely due to a decrease of the association rate constant of DHO binding to the sodium pump at higher K+ concentrations. The association rate constant amounted to 1 x 10(4) (mol/l)-1.s-1 at 2 mmol/l K+ and to 2.8 x 10(3) (mol/l)-1.s-1 at 10.8 mmol/l K+. The dissociation rate constant of DHO unbinding remained unchanged (approximately 0.06 s-1). The equilibrium dissociation constant KD for the inhibition of the pump current by DHO decreased by a factor 3 to 5 if the Na+ concentration of the patch pipette solution was augmented from 5 to 50 mmol/l. Increasing DHO concentrations inhibited the pump current increasingly faster and to a larger extent. The KD values derived showed little variation with the concentration of DHO applied.(ABSTRACT TRUNCATED AT 250 WORDS)


Functional role of sodium pump in human placental arteries. Ouabain (10(-7) to 10(-4) M) elicited concentration-dependent contractile responses in human placental arteries. The contractions were reduced by 10(-4) M amiloride and Ca(2+)-free medium, but not affected by 10(-6) M nifedipine or 10(-6) M Bay-K-8644, which markedly reduced or potentiated 75 nM K(+)-induced contractions, respectively. After contracting the vessels with 10(-6) M prostaglandin F2 alpha in a K(+)-free medium, the subsequent addition of 7.5 mM K+ induced a marked relaxation, which was blocked by 10(-6) M ouabain. This glycoside (10(-8) to 10(-4) M) also produced a concentration-dependent reduction of 86Rb+ uptake. Scatchard analysis of the [3H]-ouabain binding to membrane fractions from human placental arteries suggests a single class of binding sites with a KD of 88.3 nM and a Bmax of 345 fmol/mg. 5-Hydroxytryptamine (5-HT; 10(-9) to 10(-5) M) caused concentration-dependent contractions. Single concentrations produced transient responses composed of an initial contraction, followed by a slow fall in tension. Ouabain (10(-8) to 10(-6) M), K(+)-free medium or the reduction of bath temperature (28 degrees C) did not modify contractions but inhibited the relaxant phase of the response. 5-HT (10(-8) to 10(-6) M) increased both total and ouabain-insensitive 86Rb+ uptake, but the difference between them was not modified. These data indicate that: (1) human placental arteries possess an important sodium pump activity, inhibition or stimulation of which markedly alters vascular tone, (2) ouabain-evoked contractions are produced by Ca2+ entry mainly through Na(+)-Ca2+ exchange, secondary to intracellular Na+ accumulation, (3) the relaxant component of 5-HT response is dependent on the activity of the sodium pump, (4) the activation of Na+,K(+)-ATPase activity by this amine is not apparently due to direct effect, and (5) the inhibition of the sodium pump can cause long lasting increases of placental vascular resistance in the presence of physiological concentrations of 5-HT.


Isoprenaline-induced increase in the 40/41 kDa pertussis toxin substrates and functional consequences on contractile response in rat heart. Chronic beta-adrenoceptor stimulation leads to desensitization of the myocardial adenylyl cyclase signalling pathway which includes beta-adrenoceptor downregulation and upregulation of Gi-protein alpha-subunits. However, these investigations have mainly been done in cellular preparations. In this study we report that isoprenaline infusion in vivo leads to an increase in myocardial Gi alpha and present evidence for functional consequences of this increase. Rats were treated by a 4-day subcutaneous infusion with isoprenaline (2.4 mg/kg.d), propranolol (9.9 mg/kg.d) and triiodothyronine (T3, 0.5 mg/kg.d) for comparison. Isoprenaline treatment increased the pertussis toxin-sensitive amount of Gi alpha by 22 +/- 6% and decreased beta 1- and beta 2-adrenoceptor density from 35 +/- 4 to 23 +/- 6 fmol/mg protein and 24 +/- 4 to 8 +/- 6 fmol/mg protein, respectively. Contraction experiments on electrically driven papillary muscles revealed that the negative inotropic potency of the M-cholinoceptor agonist carbachol in the presence of isoprenaline was increased as compared to control (mean EC50-values: 0.04 mumol/l vs. 0.28 mumol/l). All isoprenaline-induced effects were antagonized by simultaneously administered propranolol. T3 treatment had no influence on the parameters investigated. The results suggest that chronic beta-adrenoceptor stimulation desensitizes myocardial adenylyl cyclase by at least two mechanisms: beta-adrenoceptor downregulation leading to diminished signal transduction in the stimulatory pathway and Gi alpha upregulation leading to sensitization of the inhibitory pathway. Such adaptation might protect the heart from chronic exposure to catecholamines in heart diseases with elevated plasma catecholamine levels.


Comparison of the effects of neuropeptide K and neuropeptide gamma with neurokinin A at NK2 receptors in the hamster urinary bladder. Neuropeptide K (NPK) and neuropeptide gamma (NP gamma) are two endogenous N-terminally extended forms of neurokinin A (NKA). Here, we compared their effects with those of NKA on 125I-NKA binding, phosphatidylinositol (PI) turnover and smooth muscle contraction in the hamster urinary bladder. NPK, NP gamma and NKA were equipotent in competing 125I-NKA from NK2 receptors in crude hamster bladder membranes. All three peptides stimulated PI turnover by approximately 750% with similar potency. In a third series of experiments, these peptides had similar efficacy in inducing a dose-dependent contraction of bladder smooth muscle. The NK2 receptor selective antagonist L-659,877 (cyclo[Leu-Met-Gln-Trp-Phe-Gly]) inhibited the stimulation of PI turnover and bladder contractions induced by all three tachykinins. The present results show that NKA, NPK and NP gamma display a similar biological profile. The N-terminal extensions of NPK and NP gamma appear not to influence binding of these peptides to NK2 receptors, NK2 receptor mediated stimulation of PI turnover, or smooth muscle contraction in hamster urinary bladder.


Phenytoin partially antagonized L-type Ca2+ current in glucagon-secreting tumor cells (ITC-1). Transmembrane Ca2+ currents were investigated by means of a whole-cell clamp technique in a hamster glucagon-secreting tumor cell line (ITC-1). Two types of Ca2+ current were identified in ITC-1 cells. The low-threshold and transient (T-type) current became detectable above the potential level around -60 mV and decayed rapidly with an inactivation time constant of 95 ms (at -40 mV and 23 degrees C), while the high-threshold and long-lasting (L-type) one was activated by depolarization more positive to -30 mV with non-inactivating kinetics. The voltage dependence and kinetics of these currents were identical to those reported in guinea-pig pancreatic alpha 2 cells. Both currents were augmented by equimolar substitution of Ca2+ with Ba2+ and completely abolished by adding 1 microM La3+. Phenytoin, a well known anti-epileptic drug and a postulated T-type specific Ca2+ current antagonist, surprisingly blocked the L-type current without affecting the T-type current in ITC-1 cells. While phenytoin antagonized the L-type Ba2+ current selectively, 60% of the current remained even in supramaximal concentration range over 500 microM. The residual component of the L-type current was completely abolished by adding nifedipine.


Effect of 3-morpholinosydnonimine (SIN-1) and NG-nitro-L-arginine (NNA) on isolated perfused anaphylactic guinea-pig hearts. The modulating effects of exogenous and endogenous nitric oxide (NO) on the cardiac anaphylactic reaction and eicosanoid release were investigated in isolated perfused sensitized guinea-pig hearts using 3-morpholinosydnonimine (SIN-1), the active metabolite of molsidomine, as NO-donor and NG-nitro-L-arginine (NNA) as an inhibitor of NO biosynthesis. Infusion of SIN-1 (final concentrations in the perfusates 0.3 or 1.0 mmol/l) elevated coronary flow under basal conditions as well as during cardiac anaphylaxis, while NNA (0.1 mmol/l) decreased basal coronary flow and aggravated the anaphylactic coronary constriction. Both drugs did not modify the characteristic biphasic profile of the coronary constriction after antigen challenge with an initial more severe phase followed by a less pronounced long-lasting flow reduction. Neither SIN-1 nor NNA affected spontaneous heart rate. However, while NNA tended to prolong the duration of antigen-induced arrhythmias, SIN-1 (1 mmol/l) had an inhibitory effect. This protection might be related to the increased coronary flow in the presence of SIN-1. SIN-1 inhibited anaphylactic release of cysteinyl-leukotrienes (LT) and 6-keto-prostaglandin (PG) F1 alpha, but did not influence thromboxane (TX) B2 release. On the other hand, NNA (0.1 mmol/l) inhibited anaphylactic release of TXB2, but had only marginal effects on the release of cysteinyl-LT and 6-keto-PGF1 alpha. The results suggest that exogenous and endogenous NO functionally antagonize the effects of vasoconstrictor mediators released after antigen challenge. Additional effects of high concentrations of SIN-1 and NNA on antigen-induced eicosanoid release could modulate the vascular actions of these drugs during cardiac anaphylaxis.


Autoradiographic localization of gamma-aminobutyric acidA receptors within the ventral tegmental area. Destruction of intrinsic neurons in the ventral tegmental area (VTA) with the excitotoxin, quinolinic acid produced a significant decrease (80%) in [3H]muscimol binding to GABAA receptors within the parabrachial pigmented and paranigral nuclei of the VTA. Selective destruction of the dopaminergic neurons with 6-hydroxydopamine (6-OHDA) did not reduce [3H]muscimol binding within the VTA. However, the destruction of dopaminergic neurons did produce an increase (20%) in [3H]muscimol binding contralateral to the lesion, suggesting a reduction in the GABAergic innervation to this region. Additionally, destruction of the VTA afferents with quinolinic acid injections in the medial accumbens failed to produce alterations in [3H]muscimol binding within the VTA. These results are consistent with the predominant localization of GABAA receptors to non-dopaminergic neurons intrinsic to the VTA.


Trophic regulation of acetylcholinesterase isoenzymes in adult mammalian skeletal muscles. This work addresses the physiological regulation of skeletal muscle acetylcholinesterase (AChE) isoforms by examining endplate-enriched samples from adult rat gracilis muscles 48 h after: low-intensity treadmill exercise; obturator nerve transection; nerve impulse conduction blockade by tetrodotoxin; acetylcholine (ACh) receptor (AChR) inactivation by alpha-bungarotoxin; and, addition of obturator nerve extracts to muscles in organ culture. Results document the important role(s) of functional AChRs and ACh-AChR interactions in the differential control of individual AChE isoenzymes. A theoretical model based on these and other findings considers that: AChR activation by spontaneously released ACh is the only neural factor required for the maintenance of G1 + G2 AChE; the amount of A12 AChE is determined by the combined effects of ACh and another neurogenic substance; although mechanisms intrinsic to myofibers control normal levels of G4 AChE, enhanced production of this isoform is initiated through increasing the frequency of ACh-AChR interactions.


[3H]D-aspartic acid release in brain slices of adult and aged Fischer 344 rates. Alterations in glutamate content and uptake have been reported to occur in aged animals. The present studies used [3H]D-Aspartic acid [( 3H]-D-ASP) release as a marker for glutamate neurotransmission. Frequency dependent [3H]-D-ASP release was measured in adult (8 month) and aged (28-30 month) Fischer 344 rats. Relatively high stimulation frequencies (greater than 10 Hz) were required to induce [3H]-D-ASP release in both adult and aged F344 rats in temporal cortex and hippocampus. In both brain areas aged animals showed significantly more [3H]-D-ASP release than adult animals. Kainic acid 1 mM failed to induce the release of [3H]-D-ASP in either temporal cortex or hippocampus. Omega conotoxin GVIA (5 x 10(-9) M) a N and L type voltage sensitive calcium channel antagonist failed to inhibit [3H]-D-ASP stimulated release. These results demonstrate an increase in [3H]-D-ASP release in aged compared to adult F344 rats. The data also suggest a novel calcium channel may be involved in [3H]-D-ASP release.


gamma-Hydroxybutyric acid binding sites: interaction with the GABA-benzodiazepine-picrotoxin receptor complex. The effect of three compounds known to allosterically modulate binding to the GABA/benzodiazepine/picrotoxin receptor complex on 4-hydroxy-2,3 [3H]butyric acid (GHB) binding was investigated. Pentobarbital, pentylenetetrazole, and picrotoxin enhanced [3H]GHB binding in a dose dependent fashion. Pentobarbital enhanced 4-hydroxy-2,3 [3H]butyric acid binding was associated with an increase in Bmax while pentylenetetrazole and picrotoxin altered the affinity of GHB for its binding site producing a decrease in Kd. These findings suggest that the GHB and GABA receptor complex may share certain moieties in common.


Effects of selected vasoactive substances on adenylate cyclase activity in brain, isolated brain microvessels, and primary cultures of brain microvessel endothelial cells. The specific activity of adenylate cyclase was assayed in homogenates of gray matter, freshly isolated and primary cultured microvessel endothelial cells from bovine cerebral cortex. Specific activities for the tissues were 14.6 +/- 2.1, 15.6 +/- 2.7, and 8.4 +/- 1.5 pmol cAMP/mg protein/min +/- SD for gray matter, cultured microvessels, and freshly isolated microvessels, respectively. Adenylate cyclase associated with gray matter and cultured microvessels was sensitive to histamine and selected catecholamines. Perhaps due to metabolic deficiencies, adenylate cyclase of freshly isolated microvessels exhibited little or no response to either the catecholamines or histamine. Angiotensin II stimulated adenylate cyclase of both freshly isolated and cultured microvessels but had no effect on gray matter. Bradykinin did not stimulate cAMP generation in any of the tissues. Overall results support the role of cAMP in regulating brain microvessel functions and suggest that primary cultures of brain microvessels may be useful in examining cAMP-mediated biochemical pathways at the blood-brain barrier.


Efficacy of hydrated sodium calcium aluminosilicate to reduce the individual and combined toxicity of aflatoxin and ochratoxin A. A 2 x 2 x 2 factorial arrangement of treatments consisting of dietary aflatoxin (3.5 micrograms/g), ochratoxin A (2.0 micrograms/g), and hydrated sodium calcium aluminosilicate (HSCAS, .5%) was used to evaluate the individual and combined effects of these treatments. There were six replicate pens of 10 broilers per pen for each of the eight treatments. The broilers were maintained on these treatments from 1 day to 3 wk of age with feed and water available for ad libitum intake. Aflatoxin and ochratoxin A each significantly decreased body weight, serum protein, albumin, and cholesterol and increased the relative weight of the liver, kidney, and proventriculus. Aflatoxin increased the relative weight of the heart and decreased serum aspartate aminotransferase activity and ochratoxin A increased serum uric acid. The toxicity resulting from the combination of aflatoxin and ochratoxin A was more severe than when either of these mycotoxins were present alone. Addition of HSCAS alone did not alter any of the parameters evaluated. The HSCAS reduced the toxicity of aflatoxin, but had little effect on either the toxicity of ochratoxin A alone or the toxicity resulting from the combination of aflatoxin and ochratoxin A.


Regulation of steroid hormone action in target cells by specific hormone-inactivating enzymes. The target cell sensitivity of steroid hormones is determined by the concerted action of specific hormone receptors and steroid-inactivating enzymes. In recent years, a considerable amount of knowledge has been obtained on hormone receptor concentration-based target cell sensitivity. However, an equal understanding of the role of specific steroid-inactivating enzymes in hormone action is absent. This review highlights the importance of specific steroid-inactivating enzymes in the control of target cell sensitivity of mineralocorticoids, glucocorticoids, androgens, and estrogens. Two classes of enzymes that are actively involved in this process are hydroxysteroid dehydrogenases and hydroxysteroid sulfotransferases. Some of the target cells in which the critical roles of these enzymes have been extensively characterized are those of the kidney, endometrium, and liver. cDNA for many of these enzymes have already been cloned, and rapid progress in the elucidation of this component of steroid hormone action is anticipated.


Effects of dietary fermentable fiber on fatty acid synthesis and triglyceride secretion in rats fed fructose-based diet: studies with sugar-beet fiber. In an attempt to elucidate the role of the dietary fermentable fiber in reduction of hyperlipidemia, we substituted 30% wheat starch with 30% sugar-beet fiber in rats fed a fructose-based (41% fructose), low-fat (2% corn oil) diet. Male Wistar rats ate the test diets for 3 weeks. Feeding the sugar-beet fiber (SBF) diet resulted in a significant enlargement of the cecum; it also increased the concentration of volatile fatty acids compared with rats fed a fiber-free (FF) diet. Feeding SBF decreased plasma triglyceride and cholesterol concentrations in the postprandial as well as the postabsorptive period. In the liver, triglyceride levels were depressed in concert with the decreased liver lipogenesis and the post-Triton triglyceride secretion. Liver cholesterol levels were unaffected by SBF diet feeding. SBF-fed animals were markedly less fat compared with fiber-free-diet-fed rats. Adipose tissue lipogenesis was depressed in the postprandial period in SBF-fed animals. In short, this study suggests that substitution of easily digested carbohydrates by certain fermentable fibers may play an interesting role in the reduction of hyperlipidemia and obesity.


Diazepam impairs retention of spatial information without affecting retrieval or cue learning. We further investigated the effect of diazepam on the processing of spatial information in a water maze task. Diazepam significantly impaired the retention of spatial information in a group of rats trained to locate a hidden platform. In a free swim trial carried out after training, diazepam-treated rats showed no bias to the target quadrant. There was no effect of diazepam on retrieval of spatial information in well-trained rats, and diazepam was devoid of any effect on cue learning in the water maze. However, diazepam blocked latent place learning during cue training in the water maze. Our results indicate that the GABA-BZD receptor modulates spatial information processing and that diazepam specifically impairs the retention of spatial information without affecting retrieval or cue learning.


Effects of [D-Ala2, D-Leu5]enkephalin and [D-Pen2, L-Pen5]enkephalin on apomorphine-induced motor activity in the mouse. The effects of intracerebroventricular injections of opioid peptides such as DADL [( D-Ala2, D-Leu5]enkephalin) and DPLPE [( D-Pen2, L-Pen5]enkephalin) with different degrees of selectivity for delta- over mu-receptor on apomorphine (0.1, 0.3, 1.0 and/or 3.0 mg/kg)-induced motor activity were investigated in the mouse using multi-dimensional behavioral analyses. Lower doses (0.1 and 0.3 mg/kg) of apomorphine failed to affect significantly motor activity, whilst higher doses (1.0 and 3.0 mg/kg) of the drug produced a marked increase in linear locomotion, circling, rearing, and/or grooming behaviors. DADL (0.03, 0.1 or 0.3 microgram) by itself did not influence behaviors, while the peptide (0.1 and 0.3 microgram) produced a marked inhibition on apomorphine (1.0 but not 3.0 mg/kg)-induced increase in rearing behaviors. Furthermore, the inhibitory effect of DADL (0.3 micrograms) on the apomorphine (1.0 mg/kg)-induced increase in rearing was reversed by treatment with the alkylating agent beta-FNA (beta-funaltrexamine) (5.0 micrograms). In contrast to the effects of DADL, the much more delta-selective opioid agonist DPLPE (0.3, 1.0 or 1.75 micrograms) had no marked effects on apomorphine (1.0 mg/kg)-induced behaviors. These results suggest that delta opioid receptors do not play a principal role in the apomorphine-induced increase in circling, rearing or grooming behaviors.


Cocaine binding sites in mouse striatum, dopamine autoreceptors, and cocaine-induced locomotion. BALB/cByJ mice received cocaine (25 mg/kg IP) once a day for 3 days, resulting in a greater locomotor response to cocaine on day 3 than on day 1. On day 4, a dose (0.03 mg/kg SC) of apomorphine, targeted at dopamine autoreceptors, caused the same degree of locomotor depression in cocaine- as in saline-pretreated mice. In addition, no change was found in either the affinity or density of cocaine binding sites in their striatum as measured by the binding of [3H]CFT. C57BL/6ByJ, mice displayed a greater locomotor response to cocaine than BALB/cByJ mice, but had the same number of striatal [3H]CFT binding sites with the same affinity. Factors other than striatal cocaine binding sites, or dopamine autoreceptors as measured by apomorphine-induced depression of locomotion, should be considered for the explanation of the enhancement of the locomotor response upon daily cocaine administration in BALB/cByJ mice, or for the different locomotor response to cocaine of this strain compared with the C57BL/6ByJ strain of mice.


Effects of acute and chronic ketocyclazocine and its modulation by oxytocin or vasopressin on food intake in rats. The effects of acute and chronic ketocyclazocine (KCZ, a kappa receptor agonist) and its interactions with oxytocin (OXY) or vasopressin (AVP) were investigated on food intake in free-fed rats. Acute treatment with KCZ (1 mg/kg) produced a generalized hyperphagia during the light phase (0-6 h) without influencing dark phase (6-24 h) food intake. On chronic administration, tolerance developed to hyperphagic effect during light phase, whereas an enhancement in the food intake was seen during dark phase. OXY or AVP (both at 10 micrograms/kg) per se, did not affect the food intake response during either the light or the dark phase, after acute as well as chronic treatment. In the interaction studies, acute AVP or OXY attenuated the hyperphagia of KCZ during the light phase. On chronic treatment, both AVP and OXY blocked (a) the tolerance, and (b) the "reverse tolerance" to the food intake response to KCZ during light and dark phases, respectively. These results are discussed in light of complex opioid-OXY/AVP interactions during food intake in rats.


The regulation of connective tissue metabolism by vasoactive intestinal polypeptide. Immunohistochemical studies have confirmed the innervation of bone with neuropeptidergic neurons containing vasoactive intestinal polypeptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP). In this study, we report effects of VIP on connective tissue cell metabolism. VIP stimulated PGE2 production in human articular chondrocytes, human osteoblast-like cells and human synovial cells, however, stromelysin production was unaffected. VIP also stimulated cAMP production in human osteoblast-like cells, but not in human articular chondrocytes or synovial cells. These findings are suggestive of a role of VIP in connective tissue cell metabolism which may contribute to the inflammatory processes of arthritis.


Heterogeneity of NK-2 tachykinin receptors in hamster and rabbit smooth muscles. The possible existence of NK-2 receptor subtypes in peripheral smooth muscle preparations from rabbit and hamster was investigated by studying the effect of neurokinin A, the selective NK-2 receptor agonist [beta Ala8] neurokinin A (4-10), the selective NK-2 tachykinin receptor antagonists, MEN 10,376, L 659,877 and R 396, and the pseudopeptide derivative of neurokinin A (4-10), MDL 28,564. All experiments were performed in the presence of peptidase inhibitors (captopril, bestatin and thiorphan, 1 microM each). Both neurokinin A and [beta Ala8] neurokinin A (4-10) produced concentration-dependent contractions of the rabbit isolated bronchus and hamster isolated stomach and colon, as well as enhancement of the nerve-mediated twitches of rabbit isolated vas deferens (pars prostatica). MEN 10,376, L 659,877 and R 396 antagonized the effect of the NK-2 receptor selective agonist in all four tissues under study, although marked differences in antagonist potency were evident for the three antagonists. Thus MEN 10,376 was distinctly more potent (about 100 times) in rabbit than in hamster preparations while L 659,877 and R 396 were more potent in hamster than rabbit preparations. MDL 28,564 showed a distinct agonist character in rabbit preparations while it was virtually inactive in hamster preparations, where it antagonized the effect of the NK-2 receptor selective agonist.(ABSTRACT TRUNCATED AT 250 WORDS)


[Oat bran as lipid-lowering agent?].

[Oat bran as lipid-lowering agent?]. Hypercholesterolemia is one of the major risk factors for ischemic heart disease and for the development of atherosclerotic plaques. Water soluble plant fibers do exert a binding of intestinal cholesterol and are not digested or metabolized. We have investigated the effect of a highly water soluble fiber containing oat brain on serum cholesterol concentrations in 14 volunteers (nine males, five females, age 39 to 67 years, serum cholesterol concentration: 7.14 +/- 0.80 mmol/l; x +/- SD) during an intervention with 50 g oat brain per day for nine weeks. We have documented in the whole study population a modest, but significant reduction of serum cholesterol levels of 4.2% (p less than 0.05) at six weeks; however at nine weeks the efficacy of this regimen was less pronounced (3.2%, p = n.s.) speaking for a decreased compliance although this regimen was well tolerated and accepted. In subsets of high risk volunteers for coronary heart disease of male gender (n = 9), with hypercholesterolemia (n = 8) or essential hypertension (n = 8) the reduction of serum cholesterol levels was more consistent (5.6%, p less than 0.05) with no change in normolipemic volunteers.


The practice of cardiothoracic surgeons in the perioperative staging of non-small cell lung cancer. The treatment and prognosis of non-small cell lung cancer, and assessment of the results of treatment, depend on accurate perioperative staging. The extent to which this is carried out in the United Kingdom is unknown. A postal questionnaire survey was undertaken in 1990 to determine the perioperative staging practices of cardiothoracic surgeons in the United Kingdom. Replies from 77 surgeons, who between them performed about 4833 pulmonary resections a year for lung cancer, were analysed. Forty four per cent of surgeons, operating on 43% of the patients, do not perform computed tomography of the thorax or mediastinal exploration before surgery. They may therefore embark on a thoracotomy for stage III disease. At thoracotomy 45% of surgeons, operating on 40% of patients, do not sample macroscopically normal lymph nodes. They may therefore understage cases as N0/N1 when there is at least microscopic disease in mediastinal lymph nodes. The staging of lung cancer in the United Kingdom in 1990 appears in many instances to be inadequate. There should be a more organised approach to perioperative staging so that prognosis may be assessed and comparisons between groups of patients can be made.


Cisplatin-induced loss of kidney copper and nephrotoxicity is ameliorated by single dose diethyldithiocarbamate, but not mesna. Platinum, copper, and zinc concentrations in kidney and liver were monitored following administration of cis-diamminedichloroplatinum (Cisplatin, CDDP) alone or in combination with diethyldithiocarbamate (DDC) or mercaptoethanesulfonate (mesna). Compounds were administered in saline to F344 female rats as single bolus ip doses: 7.5 mg CDDP/kg body wt; 500 mg DDC/kg body wt 1 hr after CDDP; and 100 mg mesna/kg body wt 1 hr before CDDP, at the same time as CDDP, or 1 hr after CDDP. Tissues were collected at 4 hr, 1 day, 4 days, and 7 days post-CDDP dosing. CDDP alone produced significant increases in blood urea nitrogen (fourfold) and plasma creatinine (threefold) concentrations by Day 4. Concurrent with the toxicity, CDDP lowered kidney copper (-71%) by Day 4, but had little effect on liver copper except in copper-pretreated rats. Copper-pretreated rats initially had a twofold higher kidney copper concentration and a fourfold higher liver copper concentration, but by Day 4, CDDP lowered copper concentrations in both organs to near the noncopper-treated levels. Platinum in kidney and liver rose 72-100% of peak levels within 4 hr post-CDDP and was relatively stable throughout the 7-day test period. Kidney zinc rose significantly by day 4 only in CDDP-treated rats. DDC protected against the kidney toxicity of CDDP and markedly changed kidney copper loss. Within 4 hr, DDC reduced kidney copper 60% while increasing kidney platinum to the highest concentration of any of the treatments. By Day 4, DDC-treated rats had approximately 50% lower kidney platinum while copper returned toward control levels. A single dose of mesna did not significantly protect against CDDP nephrotoxicity and had little effect on kidney platinum, copper, or zinc. The patterns of copper loss and toxicity from CDDP alone or with DDC suggest that copper be further evaluated for its role in the mechanism of CDDP cytotoxicity.


Comparison of oxidant-generation and BP-diol activation by bone marrow cells from C57Bl/6 and DBA/2 mice: implications for risk of bone marrow toxicity induced by polycyclic hydrocarbons. Neutrophil-derived oxidants have been implicated in both the damage to biomolecules and the metabolic activation of xenobiotics. Bone marrow, a relatively neutrophil-rich tissue with low cytochrome P450 activity, is subject to toxicity from orally administered benzo[a]pyrene (BP) in mice with noninducible P450 monooxygenase systems. Thus, we have compared the oxidant generation and chemical activation by neutrophilic cells isolated from femurs of male DBA/2 and C57Bl/6 mice, strains that are susceptible and nonsusceptible, respectively, to bone marrow toxicity from BP. Oxidant generation of neutrophilic preparations was assayed by superoxide anion generation and oxidant-dependent chemiluminescence (CL) from luminol or lucigenin. In all assays, cells from DBA/2 mice demonstrated increased oxidant generation. CL from BP-7,8-dihydrodiol (BP-diol) has previously been shown to correlate with its ability to elicit genotoxic effects. A twofold enhancement of oxidant-dependent CL from BP-diol was observed with TPA-stimulated neutrophilic cells from DBA/2 mice as compared to cells from C57Bl/6 mice. DBA/2-derived bone marrow cells also activated more BP-diol to a DNA covalent binding species than did bone marrow cells derived from the less BP-sensitive C57Bl/6 mouse. Tetraol analysis of BP-diol metabolism by activated bone marrow cells confirmed this greater bioactivation of the diol by DBA/2-derived cells. These results suggest that the increased risk of DBA/2 mice for BP-induced bone marrow toxicity may be related to their greater ability to bioactivate xenobiotics through oxidant-dependent mechanisms.


Meso-2,3-dimercaptosuccinic acid (DMSA) affects maternal and fetal copper metabolism in Swiss mice. Meso-2,3-dimercaptosuccinic acid (DMSA) is a chelating agent used to treat heavy metal intoxication. DMSA has been reported to be teratogenic in the mouse, and it has been suggested that this teratogenicity may be secondary to DMSA-induced alterations in Zn metabolism. In the present study, 0, 400 or 800 mg DMSA/kg body weight were administered on gestation days 6-15 to pregnant Swiss mice by gavage (PO) or subcutaneous injection (SC). Mice were fed a diet containing 14 micrograms Zn, 10 micrograms Cu, 120 micrograms Fe, 1175 micrograms Mg and 6.8 mg Ca/g diet. A sub-group of mice in the 800 mg DMSA/kg SC group was fed a diet containing 250 micrograms Zn/g. DMSA administration did not result in overt maternal toxicity. There was no effect of the drug on fetal or placental weight, or on crown-rump length. However, some fetuses from DMSA-treated dams were characterized by skeletal abnormalities including supernumerary ribs, unossified anterior phalanges and malformed sternebrae. Drug exposure was not associated with consistent changes in tissue Zn, Fe, Ca or Mg levels. Supplemental Zn had no marked effects on the fetus. Fetal liver Cu concentrations exhibited dose-dependent decreases with increasing DMSA dose. This finding suggests that the developmental toxicity of DMSA may be mediated through disturbed maternal/fetal copper metabolism.


Trichothecene mycotoxins inhibit phosphoinositide hydrolysis in bovine platelets stimulated with platelet activating factor. The effects of the trichothecene mycotoxins, acetyl T-2 toxin, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON) and T-2 tetraol on phospholipid turnover were determined in bovine platelets prelabelled with [1-14C]arachidonic acid (AA). In resting, non-stimulated platelets exposed to acetyl T-2 toxin, a marked decrease in [1-14C]phosphatidylinositol (PI) along with a marked increase in [1-14C]phosphatidic acid (PA) were observed, whereas T-2 toxin, and HT-2 toxin only induced a significant increase in [1-14C]PA. In contrast, in platelet activating factor (PAF)-stimulated platelets, the mycotoxins were found to suppress both the agonist-induced loss of [1-14C]PI and the appearance of [1-14C]PA with acetyl T-2 toxin being the most effective and T-2 toxin, HT-2 toxin, and DAS essentially equally effective. T-2 tetraol and DON did not affect phospholipid metabolism either in unstimulated or PAF stimulated platelets. The alterations in [1-14C]PI and [1-14C]PA suggest that the inhibitory toxins may activate a specific phospholipase C (PLC) in the unstimulated platelets and then impede further PLC activation in PAF-stimulated platelets.


Glutathione release by pulmonary alveolar macrophages in response to particles in vitro. We studied whether cultured rat pulmonary alveolar macrophages (PAM) could release glutathione (GSH) in response to latex beads, quartz, and crocidolite asbestos. PAM were exposed for 2 h to 0-100 micrograms of particles per 1 x 10(6) cells. Both quartz and asbestos produced concentration-dependent increases (up to 8-10-fold) in the amount of GSH recovered in the medium and decreases in the cellular GSH levels. In contrast, latex beads did not produce any changes in GSH levels. We also measured lactate dehydrogenase (LDH) levels as an index of toxicity. Only quartz and asbestos were able to produce increases in LDH levels in the medium. The release of GSH occurred at particle concentrations that did not cause release of LDH. Our results indicated that, in vitro, PAM release GSH in response to toxic particles.


Primary oat cell (neuroendocrine) carcinoma of the breast. Report of four cases. Four cases of oat cell (neuroendocrine) carcinoma of the breast are reported. Three patients died within 15 months of the diagnosis and the fourth patient is alive after 44 months. Histochemical, ultrastructural and mRNA markers of endocrine differentiation were present in three cases. These tumours show histological similarities to breast metastases of bronchial oat cell carcinoma, but a distinguishing feature is the presence of in situ ductal lesions. It appears that the breast is a further site which has to be added to the long list of extrapulmonary oat cell carcinomas.


Smooth muscle contraction bands in intestinal infarction. We studied microscopic sections of 24 cases of intestinal infarction looking for contraction bands (CB) in the muscularis propria of the bowel wall. Controls were 11 surgical and 11 autopsy cases from patients who did not suffer any form of ischaemic disease. The difference of CB frequency was significant (P less than 0.001) in infarction versus the surgical control group. Moreover, within the study group the CB frequency was also related to the severity of the ischaemic lesion (P less than 0.01). With immunostaining, CB were not reactive with antibodies against vimentin, desmin, actin or myosin. We propose that CB genesis in intestinal smooth muscle is related to hypoxia, possibly through altered homeostasis of calcium and catecholamine metabolism.


Immunohistochemical study of skin lesions in herpes zoster. Thirty-seven biopsy skin tissues of herpes zoster taken from 27 patients were analysed immunohistochemically using two monoclonal antibodies detecting either nucleocapsid or glycoproteins of varicella-zoster virus (VZV) on paraffin sections of formalin fixed tissues. Skin lesions of herpes zoster were divided clinically into four stages: erythematous, vesicular, pustular and ulcerative. In the erythematous stage, VZV antigens, if detected, were found only within ballooning cells in the lower epidermis or follicular epithelium. In the vesicular stage, antigens were detected in the cells around and within the intraepidermal vesicles and in histiocytes or fibrocytes of the dermis in all cases and in the endothelial or perineural cells in 10 of 14 cases. In the pustular stage, the antigens were observed in degenerated or necrotic keratinocytes and multinucleated giant cells within pustules and some necrotic cells in the dermis. In the ulcerative stage, the viral antigens were detected only at the ulcer margin and around the hair shaft in 2 of 7 cases. These results suggest that VZV initially involves the epidermis in the erythematous stage, subsequently invades the dermis in the vesicular stage, and disappears in the early ulcerative stage.


[Cushing's syndrome and pregnancy: apropos of a case and review of the literature]. The occurrence of Cushing's syndrome during pregnancy is rare. Sixty-eight cases have been reported in the literature of which 42.7% were secondary to an adrenal adenoma, 10.2% to an adrenal carcinoma, 26.5% to a bilateral adrenal hyperplasia and 11.8% to Cushing's disease. We report the case of a patient in whom the diagnostic of Cushing's syndrome with an adrenal adenoma was established during the postpartum period.


Protective efficacy of virus-like particles for bluetongue disease. Bluetongue virus-like particles (VLPs) derived from multiple baculovirus expression vectors have been administered in the presence of various adjuvants to sheep, a vertebrate host susceptible to the virus, and the neutralizing antibody responses are measured. Vaccinated sheep are challenged after 4 months of inoculation, and clinical reaction indices and viraemia determined. The results indicate that these multiprotein virus-like particles lacking the genetic material are highly immunogenic and as little as 10 micrograms of VLPs in conjunction with appropriate adjuvant elicit an immune response which protects against infectious virus challenge. The formation of virus-like particles using this new technology offers a novel approach in vaccinology.


Construction of a genetically defined Salmonella typhi Ty2 aroA, aroC mutant for the engineering of a candidate oral typhoid-tetanus vaccine. The construction of a Salmonella typhi Ty2 strain harbouring defined deletions in both the aroA and aroC genes is described. These deletions have been fully defined at the molecular level by DNA sequencing and have been introduced in such a way that no foreign DNA remains in the S. typhi genome. This strain is attenuated in mice when given by the intraperitoneal route suspended in hog gastric mucin and is attenuated to a similar level to strains harbouring deletions in aroA or aroC alone indicating that both lesions are capable of attenuating independently. We have used this defined S. typhi aroA aroC strain to express stably a non-toxic 50 kDa fragment of tetanus toxin (fragment C) from a gene incorporated into the chromosome. This strain has the advantage of harbouring no antibiotic-resistance markers and we consider it to be a candidate bivalent oral typhoid-tetanus vaccine.


Potential use of non-replicating vectors as recombinant vaccines. Avipoxviruses, members of the Poxvirus family, are naturally restricted in that productive replication takes place only in avian species. Recent work has described the construction of Avipox recombinants using fowlpox and canarypox viruses. Preparation of recombinant fowlpox viruses which express immunogens from avian pathogens and successful vaccination of poultry have been reported. Recombinant fowlpox and canarypox viruses which express immunogens from mammalian pathogens have also been described and have been demonstrated to provide protective immunity on inoculation in non-avian species. This is a surprising result. Such non-replicating expression vectors provide the possibility of developing safe, effective vaccines which combine the advantages of killed and live vaccines.


Induction of anti-viral immune responses by immunization with recombinant-DNA encoded avian coronavirus nucleocapsid protein. Immune responses to the infectious bronchitis virus (IBV) nucleocapsid protein were studied using a recombinant-DNA expression product. In mice, a lymphocyte proliferative response and a delayed-type hypersensitivity reaction to IBV were induced upon immunization with this nucleocapsid protein. Next, we studied the role of the expressed nucleocapsid protein in induction of a protective immune response to IBV in chickens. Chickens were primed with nucleocapsid protein and subsequently boosted with inactivated IBV, strain M41. Proliferative responses of blood mononuclear cells corresponded with increased mean haemagglutination inhibition and virus neutralization titres. Finally, an increased tracheal protection against challenge with live IBV was observed. These results indicate that infectious bronchitis virus nucleocapsid protein is a relevant target for immune recognition in both the mouse and the chicken.


Current progress towards vaccines for arenavirus-caused diseases. The arenaviruses are primarily viruses of rodents, but some members of the group cause severe disease (Argentine and Bolivian haemorrhagic fevers and Lassa fever) when transmitted to humans in the specific areas of the world where they are enzootic. Current research of relevance to the provision of vaccines against these diseases, which highlights many of the problems encountered generally in the development of vaccines, is reviewed here. Although one of the classical approaches to vaccine production, the use of inactivated preparations of virus of varying degrees of purity, has produced no results of promise, attenuation of a virulent strain of Junin virus by passage in cultured cells has yielded a vaccine strain currently being tested for efficacy in protecting against Argentine haemorrhagic fever in the human population at risk. The experimental evidence for protection in animal model systems by related, apparently non-pathogenic, viruses and by recombinant vaccinia viruses expressing arenavirus proteins is discussed, together with some of the potential difficulties of these approaches.


In vivo potentiation of atracurium neuromuscular blockade by nimodipine in rabbits. The interaction between nimodipine, a calcium channel blocker, used for the treatment of subarachnoid haemorrhage, and atracurium, was studied in rabbits. An intravenous dose of 0.1 mg.kg-1 of nimodipine given over 3 min caused a potentiation of the neuromuscular blocking action of atracurium (administered at an infusion rate of 7.5 micrograms.kg-1.min-1), measured on the indirectly stimulated tibialis-anterior muscle of the animal. (ED50)inf and (ED95)inf were significantly reduced (30.7% and 23.3%) in nimodipine-treated animals (P less than 0.05 and P less than 0.02, respectively). No changes were observed in recovery rate.


Effect of guar, pectin, psyllium, soy polysaccharide, and cellulose on breath hydrogen and methane in healthy subjects. Different types of dietary fiber are fermented to various extents in vitro, but little is known about the effects of fiber on breath hydrogen and methane levels in vivo. Therefore, we studied the effects on breath hydrogen and methane of 15 g of guar, pectin, psyllium, soy polysaccharide, or cellulose in eight healthy subjects over a 12-h period. None of the fibers had a significant effect on breath hydrogen or methane concentrations, compared with the control (fasting). The four methane producers had lower breath hydrogen levels than the nonproducers 1 h after 15 g of lactulose (3 +/- 1 vs. 42 +/- 9, p less than 0.005) and 5-12 h after the different fibers (3.3 vs. 4.8 ppm; pooled SEM = 0.8; p less than 0.025). When the methane responses of the methane producers were expressed as increments relative to the control, there were small differences between treatments, with guar producing a larger response, 8.2 +/- 3.3 ppm, than cellulose, -2.9 +/- 2.3 ppm (p less than 0.05). The incremental methane responses of the different fibers in vivo were related to the previously reported production of propionic acid (r = 0.94, n = 5, p less than 0.02) and methane (r = 0.93, n = 4, NS) from in vitro fermentation of the same fibers. We conclude that methane producers have lower breath hydrogen levels than nonproducers. Purified fermentable and nonfermentable dietary fibers have no effect on breath hydrogen levels over 12 h in subjects previously consuming a normal diet. However, fermentable fibers may produce small increases in breath methane in methane-producing subjects.


Assignment of the urokinase-type plasminogen activator receptor gene (PLAUR) to chromosome 19q13.1-q13.2. The urokinase-type plasminogen activator receptor (uPAR) is a key molecule in the regulation of cell-surface plasminogen activation and, as such, plays an important role in many normal as well as pathological processes. We applied a cDNA probe from the corresponding gene (PLAUR) in a location analysis using a panel of human/rodent cell hybrids and in a multipoint linkage analysis of 40 CEPH families. These two independent studies both found PLAUR to be located on chromosome 19. The cell hybrid study suggested that PLAUR is located at chromosome 19q13-qter, and the multipoint analysis indicated that PLAUR is located at chromosome 19q13.1-q13.2 and surrounded by DNA markers in the following way (with distances given in recombination fractions): D19S27-.11-CYP2A-.06-PLAUR-.03-D19S8-.04-APOC 2-.24-PRKCG. Further, a ligand-binding study performed on cell hybrids verified the species specificity of the uPAR and confirmed the chromosome assignment.


The osteoclast as a unicellular proton-transporting epithelium. Osteoclasts dissolve bone mineral by the vectorial secretion of hydrogen ion at their osseous attachment site. To accomplish this, the osteoclast employs a vacuolar H+ ATPase that is polarized to the specialized proton-secreting plasma membrane domain, the ruffled border. Physiologically and biochemically, they resemble the specialized proton-secreting intercalated cells of the renal collecting duct, which also use a polarized vacuolar H+ ATPase to effect transepithelial hydrogen ion transport. Studies on the mechanism of hydrogen ion transport by the kidney may therefore provide insights into the control of acid secretion by the osteoclast.


Focal ventricular thinning caused by indomethacin in the late phase of coxsackievirus B4 murine myocarditis. Indomethacin has been shown to increase virus titers and to worsen cardiac injury in the acute phase of coxsackievirus B4 murine myocarditis. The authors evaluated the effects of indomethacin on the histopathologic changes in a later phase of this disease after virus clearance. Two-day old CD1 mice were infected with coxsackievirus B4. Ten days later, surviving animals were randomized to receive indomethacin or saline intraperitoneally for 10 days. They were then euthanatized, and their hearts were examined for the presence of inflammation, necrosis, scarring, and focal thinning. Mortality was slightly higher among treated animals (7/15 versus 2/12, p = 0.3). The index of inflammation (0.6 +/- 0.5 versus 0.7 +/- 0.5) necrosis and scarring (0.4 +/- 0.5 versus 0.3 +/- 0.5) among treated and control animals, respectively, was not significantly different, but the size of involved myocardium (149742 +/- 201982 versus 35300 +/- 45413 microns2) was remarkably larger (p less than 0.05), and focal ventricular thinning (5/12 versus 0/10, p = 0.03) was encountered among indomethacin recipients exclusively. These findings indicate that indomethacin treatment in the late phase of coxsackievirus B4 myocarditis enhances myocardial damage and increases the incidence of focal ventricular thinning.


Hyperoxia induces alkalinization and dome formation in MDCK epithelial cells. We observed that confluent Madin-Darby canine kidney (MDCK) epithelial cells exposed to 95% O2 showed intensive dome formation, a manifestation of cell differentiation and transepithelial fluid transport, whereas cells exposed to 40% O2 or to normoxia did not. Hyperoxia-induced dome formation (HIDF) was preceded and accompanied by a significant rise in intracellular pH (pHi) on days 2 (7.53 vs. 7.42) and 3 (7.62 vs. 7.45), as compared with controls. Inhibition of the Na(+)-H+ exchanger by 0.1 or 1.0 mM amiloride caused 29 or 69% reduction of HIDF and completely abolished hyperoxia-induced alkalinization of the cells. HIDF was altered by modification of extracellular pH (pHo); there was a decrease by 84% with pHo 6.8, while pHo 7.8 led to earlier and more intensive dome formation (day 2, +472%; day 3, +27%). We also found that adenosine 3',5'-cyclic monophosphate (cAMP) was increased in hyperoxic cells, a change that was independent from the rise of pHi. We conclude that high-level hyperoxia induces dome formation in MDCK epithelial monolayers by a process involving activation of the Na(+)-H+ exchanger, together with increased intracellular cAMP.


Direct evidence for a basolateral membrane Cl- conductance in toad retinal pigment epithelium. There is now evidence that a Cl- conductance on the basal membrane of the retinal pigment epithelium (RPE) is involved in the generation of both the fast oscillation and the light peak of the direct-current electroretinogram as well as being critical for transepithelial fluid and salt movement. In the present study, we characterized the basolateral membrane Cl- conductance of an in vitro preparation of toad RPE-choroid using conventional and Cl(-)-selective microelectrodes. Under control conditions, the potential across the apical (Vap) and basal (Vba) membranes averaged -60 +/- 2 and -45 +/- 2 mV, respectively (n = 40). Intracellular Cl- activity (aiCl = 20 +/- 1 mM) was distributed above equilibrium across both membranes, consistent with active accumulation of Cl-. A sixfold decrease in Cl- in the basal bath depolarized Vba by 12 +/- 1 mV (n = 17) and increased the apparent basal membrane resistance. By sequential measurement of aiCl and subepithelial Cl- activity during a step decrease in basal Cl-, we constructed the change in Cl- equilibrium potential (ECl) across the basal membrane. Estimation of the change in basal membrane electromotive force during the change in ECl gave an average value for the Cl- transference number (TCl) of 0.45. Further evidence for a Cl- conductance was obtained by measuring changes in aiCl induced by transepithelial current. Depolarizing Vba elevated aiCl, whereas hyperpolarizing Vba had the opposite effect, consistent with conductive Cl- movement across the basal membrane. Both the amplitude of the Cl- diffusion potential and the current-induced changes in aiCl were reduced by basal perfusion with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (250-500 microM), a blocker of Cl- channels in some epithelia.


GTP gamma S-dependent regulation of smooth muscle contractile elements. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) increases the sensitivity of the contractile response to activation by Ca2+ in permeabilized tracheal smooth muscle. Increased tension was associated with a proportional increase in myosin light chain phosphorylation. The site of phosphorylation was determined to be serine-19, which corresponds to the site rapidly phosphorylated by myosin light chain kinase. GTP gamma S did not affect the contraction induced by the protein phosphatase inhibitor okadaic acid but did enhance contraction produced by Ca(2+)-independent myosin light chain kinase. In tracheal homogenates Ca(2+)-dependent myosin light chain kinase activity was not affected by GTP gamma S; however, dephosphorylation of 32P-labeled heavy meromyosin by phosphatase was inhibited. Thus GTP gamma S may increase the Ca2+ sensitivity of contractile elements in tracheal smooth muscle by inhibition of protein phosphatase activity toward myosin light chain.


Actions of thiocyanate and N-phenylmaleimide on volume-responsive Na and K transport in dog red cells. Two sets of observations suggest a linkage between volume-responsive Na and K transport systems in dog red blood cells. 1) The lyotropic anion thiocyanate inhibits shrinkage-induced Na-H exchange and stimulates swelling-induced K-Cl cotransport. 2) The effect of a brief incubation with N-phenylmaleimide (NPM) on Na and K transport depends on the volume of the cells at the time of exposure to the sulfhydryl reagent. Cells shrunken during the NPM incubation and then brought back to normal volume behave as though they were still shrunken, i.e., they show an increased Na flux and a decreased K flux. Cells incubated with NPM in a swollen state retain fluxes characteristic of swollen cells when returned to a normal volume. The electrophoretic mobility of the membrane-associated enzyme glyceraldehyde-3-phosphate dehydrogenase is influenced by the cell volume at the time of NPM exposure. These findings point to the existence of a system in cells that perceives volume changes and coordinates the responses of membrane transporters.


Synthesis and translocation of Na(+)-K(+)-ATPase alpha- and beta-subunits to plasma membrane in MDCK cells. Synthesis and translocation of Na(+)-K(+)-ATPase alpha-catalytic and beta-glycoprotein subunits from intracellular membranes to the plasma membrane were studied in Madin-Darby canine kidney cells (MDCK-T) by combining the methods of pulse-chase labeling, subcellular fractionation on sorbitol gradients, and immunoprecipitation. Immunoprecipitation from homogenates revealed that radioactive methionine incorporated into beta-subunit was equal to that incorporated into alpha-subunit after 15 min of labeling. Because the ratio of total methionines in alpha- vs. beta-subunit is approximately 5:1, these results suggest that beta-subunit is synthesized in molar excess over alpha-subunit. Half of the newly synthesized beta-subunit, likely unassembled units, were degraded by 60 min after labeling, while alpha-subunits were stable through 120 min after synthesis, suggesting alpha may be limiting for alpha beta-assembly. By 120 min the ratio of counts incorporated into alpha vs. beta approached 5, which is predicted by a 1:1 ratio of alpha to beta. The sorbitol gradient resolved two major membrane samples: a mixture of endoplasmic reticulum and Golgi populations and a plasma membrane-enriched sample. Immature beta (beta i) could not be detected in the plasma membrane-enriched samples at levels greater than could be attributed to cross-contamination by intracellular membranes. Mature beta (beta m) became detectable after 30 min, and conversion of beta i to beta m was 90% complete at 120 min. A peak of labeled alpha-subunit appeared in the plasma membrane-enriched sample at 60 min, coincident with the appearance of labeled beta m-subunit in this sample, suggesting movement as alpha beta-heterodimers.


Thyroid hormone induction of rat myocardial Na(+)-K(+)-ATPase: alpha 1-, alpha 2-, and beta 1-mRNA and -protein levels at steady state. In this study, we measured the time courses of change in Na(+)-K(+)-ATPase alpha 1-, alpha 2-, and beta 1-subunit mRNA and protein abundance in cardiac myocytes isolated from euthyroid, hypothyroid, and hyperthyroid (hypothyroids injected daily with 1 microgram T3/g body wt) rats. In hypothyroids, alpha 1-, alpha 2-, and beta 1-protein levels were decreased to 0.55, 0.42, and 0.57 of euthyroids, predicting the decrease in Na(+)-K(+)-ATPase activity to 0.53 of control. There was no change in these subunits' mRNA levels, indicating that the decreases in protein levels were not due to decreased subunit transcription rates. In hyperthyroids, the alpha 1-mRNA increased to a steady state of threefold over hypothyroid by 1 day of T3 treatment, and the alpha 1-protein levels increased to twofold over hypothyroid by 4 days of T3. alpha 2-mRNA increased to 5-fold over hypothyroid by 2 days, whereas the alpha 2-protein levels increased to 14-fold above hypothyroid by 4 days of T3. Beta 1-mRNA increased to 12-fold above hypothyroid by 1 day of T3 treatment, whereas beta 1-protein increased to only 2.5-fold over hypothyroid by 4 days of T3. The discoordinate changes in alpha 2- and beta 1-mRNA vs. protein can be reconciled with the hypothesis that beta 1 is rate limiting for assembly in eu- and hypothyroids, and favors assembly with alpha 1, while excess unassembled alpha 2 is degraded. In the hyperthyroids we predict beta 1 is not rate limiting and there is increased alpha 2 beta 1-assembly. We calculate that T3 decreases the alpha 1-to-alpha 2 ratio from 24:1 in hypothyroid to 3.4:1 in hyperthyroid cardiomyocytes.


Role of G proteins in stimulation of Na-H exchange by cell shrinkage. Many cells respond to shrinkage by stimulating specific ion transport processes (e.g., Na-H exchange). However, it is not known how the cell senses this volume change, nor how this signal is transduced to an ion transporter. We have studied the activation of Na-H exchange in internally dialyzed barnacle muscle fibers, measuring intracellular pH (pHi) with glass microelectrodes. When cells are dialyzed to a pHi of approximately 7.2, Na-H exchange is active only in shrunken cells. We found that the shrinkage-induced stimulation of Na-H exchange, elicited by increasing medium osmolality from 975 to 1,600 mosmol/kgH2O, is inhibited approximately 72% by including in the dialysis fluid 1 mM guanosine 5'-O-(2-thiodiphosphate). The latter is an antagonist of G protein activation. Even in unshrunken cells, Na-H exchange is activated by dialyzing the cell with 1 mM guanosine 5'-O-(3-thiotriphosphate), which causes the prolonged activation of G proteins. Activation of Na-H exchange is also elicited in unshrunken cells by injecting cholera toxin, which activates certain G proteins. Neither exposing cells to 100 nM phorbol 12-myristate 13-acetate nor dialyzing them with a solution containing 20 microM adenosine 3',5'-cyclic monophosphate (cAMP) (or 50 microM dibutyryl cAMP) plus 0.5 mM 3-isobutyl-1-methylxanthine substantially stimulates the exchanger. Thus our data suggest that a G protein plays a key role in the transduction of the shrinkage signal to the Na-H exchanger via a pathway that involves neither protein kinase C nor cAMP.


Role of cAMP in mediating effects of fasting on dephosphorylation of insulin receptor. We studied the effect of fasting on phosphotyrosine phosphatase (PTPase) activities in particulate (PF) and cytosolic (CF) fractions of rat adipocytes and liver. PTPase activity was assessed using [32P]tyrosine insulin receptor (IR). In adipocytes, 48 h fasting significantly inhibited PTPase activity. Dephosphorylation of IR by PF and CF PTPases was reduced by 80 and 65%, respectively. Similar reductions of lesser magnitude were observed in fasted rat livers. The effect of fasting was completely reversed by either refeeding or by incubating "fasted" adipocytes for 2 h in tissue culture medium containing 5 mM glucose. Neither 20 mM glucose nor the presence of insulin influenced phosphatase activity. Because fasting is accompanied by elevated protein kinase C (PKC) and adenosine 3',5'-cyclic monophosphate (cAMP) levels, we examined their influence on adipocyte PTPases. Neither activation (1 microM 12-O-tetradecanoylphorbol-13-acetate) nor inhibition (20 microM sphingosine) of PKC affected PTPase activity. In contrast, cAMP (2 mM) significantly inhibited PTPase activity (80% inhibition at 2 h), and its effect was prevented by a cAMP antagonist RpcAMP. Fasting- and cAMP-induced inhibition of PTPase activity was restored by incubating PF with trypsin (4 micrograms/ml for 5 min), which separated the putative inhibitors from the phosphatases. We conclude that fasting-induced inhibition of PTPases is mediated by elevated cAMP levels, most likely by activating phosphatase inhibitors.


Insulin receptor kinase activity in muscles and white adipose tissue during course of VMH obesity. Early after lesion of the ventromedial hypothalamus nuclei (VMH), insulin-induced glucose utilization is increased in white adipose tissue (WAT), whereas oxidative and glycolytic muscles are, respectively, normoresponsive or resistant to insulin. Five weeks later, all of the muscles are resistant, whereas WAT returns to normal responsiveness. The aim of this study was to characterize the insulin receptor kinase activity in WAT and muscles 1 and 6 wk after lesion. The number and affinity of insulin receptors were not modified in any of the tissues studied. Autophosphorylation and phosphorylation of an exogenous substrate were similar in oxidative and glycolytic muscles of VMH and control rats both 1 and 6 wk after the lesion. Insulin receptors from WAT of 1-wk VMH rats exhibited a 2.5-fold increase in insulin-stimulated autophosphorylation and phosphorylation. Six weeks after the lesion, both autophosphorylation and phosphorylation returned to normal values. This suggests that insulin receptor tyrosine kinase activity does not play a significant role in the insulin resistance of skeletal muscles but has a crucial role in mediating the variations of insulin action on WAT observed during the development of VMH obesity.


Xanthine oxidase and dehydrogenase activities in rat ovarian tissues. The production of reactive oxygen species in the ovary is rapidly inducible, but the nature of the generator is unknown. One possibility is xanthine oxidase (XO), an enzyme that produces superoxide in the presence of hypoxanthine (or xanthine) and oxygen. The objective of the present studies was to measure levels of XO in follicular and luteal tissue to determine whether XO may be a source of reactive oxygen species in the rat ovary. Ovarian levels of XO were about one-fifth of that seen in the liver and adrenal, and XO levels were about one-third of xanthine dehydrogenase (XDH). Preovulatory ovarian levels of XO activity were unchanged after induction of ovulation with gonadotropin and in follicles incubated with gonadotropin. Luteal XO activity was not changed during natural or prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis. Allopurinol, an inhibitor of XO, did not inhibit ovulation or PGF2 alpha-induced luteal regression. Finally, neither catalase and superoxide dismutase nor oxypurinol altered luteal cell function in the presence of hypoxanthine. Thus, while XO is present in the ovary, it does not appear that it is a major source of reactive oxygen species in this organ.


Termination of the peripubertal FSH increase in male rats. To determine if the pubertal testosterone rise plays a role in the termination of the peripubertal follicle-stimulating hormone (FSH) increase, male rats were injected with ethylene dimethanesulfonate (EDS) at 40 days of age to eradicate the Leydig cells just before the onset of the testosterone rise. Rats were decapitated at weekly intervals from age 26 to 96 days. Compared with vehicle-injected controls, EDS treatment resulted in a delay in the peripubertal increase in the relative weights of prostates and seminal vesicles of approximately 2 wk. Serum testosterone remained at pretreatment levels for 1 wk postinjection. Testicular interstitial fluid testosterone remained at pretreatment concentrations for considerably longer and was significantly lower than controls for 2 wk postinjection. EDS treatment resulted in serum FSH levels that were elevated by 1 wk postinjection. They remained significantly higher than controls until 96 days of age. Compared with controls, serum alpha-inhibin was elevated after EDS as was serum luteinizing hormone. These results suggest that the pubertal testosterone increase plays an important role in terminating the peripubertal FSH rise.


Intrinsic factor receptor activity and cobalamin transport in bile duct-ligated rats. The intrinsic factor (IF)-cobalamin (Cbl) receptor activity in the mucosal homogenates progressively decreased after bile duct ligation in the rat, and 80% of the receptor activity was decreased in 96 h after ligation. The activity was restored to normal values of 5.5-6 pmol of IF-[57Co]Cbl bound/g mucosa when the assays were performed with both conjugated and unconjugated bile acids. When [57Co]Cbl bound to intrinsic factor was orally administered, the tissue levels of [57Co]Cbl were decreased by 75-80% in bile duct-ligated rats. The apical membrane receptor activity was also decreased after bile duct ligation; however, the activity was stimulated twofold by the addition of ileal cytosol and threefold with the addition of both ileal cytosol and taurocholate (1 mM). Enhanced binding of IF-[57Co]Cbl to the apical ileal brush-border membrane occurred with the use of dialyzed ileal cytosol but not with cytosol isolated from duodenal or proximal jejunal mucosa. The enhanced binding obtained with ileal cytosol was abolished after its treatment with trypsin. These results suggest that luminal bile acids optimize the binding of IF-Cbl by the ileal membrane receptor via interactions with a cytosolic factor and thus influence the gastrointestinal absorption of cobalamin.


Phorbol esters stimulate and inhibit Cl- secretion by different mechanisms in a colonic cell line. Phorbol 12-myristate 13-acetate (PMA) was found to increase both the short-circuit current (Isc) and the efflux of 125I- or 36Cl- in the colonic epithelial cell line HT-29.cl19A. Neither the PMA-provoked rise in Isc nor the stimulation of 125I- efflux was affected by the cyclooxygenase inhibitor indomethacin. The PMA-induced increase in Cl- efflux was not accompanied by a rise in adenosine 3',5'-cyclic monophosphate (cAMP) levels. A prolonged incubation with PMA (3 h), however, inhibited the PMA- and the cAMP-stimulated Isc by greater than 90%, whereas the cAMP-provoked 125I- and 36Cl- efflux was not inhibited. The long-term PMA treatment was found to inhibit the basal and cAMP-provoked 86Rb+ efflux by 65 +/- 9 and 86 +/- 7%, respectively. A 3-h incubation with PMA also strongly inhibited the Ca2+ ionophore A23187-induced increase in 86Rb+ efflux, whereas the A23187-stimulated 125I- efflux was only marginally inhibited. These data suggest that phorbol esters, presumably by activation of protein kinase C, can provoke Cl- secretion in HT-29.cl19A colonocytes independently of a prostaglandin- or cAMP-mediated pathway. Prolonged exposure to PMA, however, causes an inhibition of net electrogenic Cl- secretion by downregulation of the activity of K+ transporters.


EndothelinB receptor on guinea pig small intestinal smooth muscle cells. We investigated the binding characteristics of the endothelin (ET) receptor and the mechanism by which ET induces contraction of longitudinal smooth muscle cells of the guinea pig small intestine by using vasoactive intestinal contractor (VIC), a mouse variant of ET-2. A functional receptor for VIC was found to exist on longitudinal smooth muscle cells. These cells showed a similar binding of and contractile response to ET-1, ET-2, and ET-3. Inhibitors of both intracellular and extracellular Ca2+ movement attenuated the VIC-induced contraction of longitudinal smooth muscle cells. These results suggest that smooth muscle cells of the guinea pig small intestine express the ETB receptor that primarily mediates the contractile effect on smooth muscle cells. In addition, ET-induced contraction depends on intracellular as well as extracellular Ca2+.


Assessment of signal transduction mechanisms regulating airway smooth muscle contractility. Agonist-receptor interactions regulate airway smooth muscle tone through activation of guanine nucleotide binding proteins (G proteins) which are coupled to second-messenger pathways that mediate changes in the tissue's contractile state. Various methods have been applied to identify the structure/function characteristics of G proteins and their role in signal transduction in airway smooth muscle, including the use of exotoxins, nonhydrolyzable analogs of guanosine-triphosphate (GTP), antibodies to purified G proteins, and membrane reconstitution studies. In elucidating mechanisms of airway smooth muscle relaxation, considerable progress has been made in identifying the molecular basis for receptor/G protein coupling and other regulatory processes leading to both the activation and down-regulation of the adenylate cyclase/adenosine 3',5'-cyclic monophosphate system. Further, with respect to airway smooth muscle contraction, various approaches have been used to evaluate the role of membrane phosphoinositide turnover and the mechanisms of action of the bifurcating signal transduction pathways associated with the production and metabolism of inositol 1,4,5-trisphosphate and 1,2-diacylglycerol, and activation of protein kinase C. This review identifies much of the information gained to date on the above signal transduction pathways, with an emphasis placed on various methodological approaches used to determine membrane and transmembrane signaling processes in airway smooth muscle.


Platelet-derived growth factor and growth-related genes in rat lung. II. Effect of exposure to 85% O2. The expression of platelet-derived growth factor (PDGF), its receptor, and related genes was studied in the lung tissue of rats exposed to air or 85% O2. PDGF-B chain mRNA was increased by 6 days and PDGF B-type receptor mRNA was increased by 4 and 6 days of exposure to 85% O2. Despite a continued increase of cell division, both PDGF-B chain and B-type receptor mRNAs had returned to control values by 14 days of exposure to 85% O2. PDGF-A chain mRNA was unaffected by exposure to 85% O2. Nuclear runoff transcription analysis confirmed increased transcription of PDGF-B chain mRNA, whereas Western blot analysis of lung homogenates suggested consequent increased translation of PDGF-B chain mRNA to PDGF-BB at 7 days of exposure to 85% O2. Combined immunocytochemistry and autoradiography localized PDGF-BB to the major site of cell division, the pulmonary interstitium. We speculate that the early pulmonary fibroblast hyperplasia observed following exposure to 85% O2 is mediated by increased PDGF-B chain gene expression and may also be mediated by changes in PDGF B-type receptor gene expression.


Paraquat-induced lung injury: prevention by N-tert-butyl-alpha-phenylnitrone, a free-radical spin-trapping agent. The herbicide paraquat causes lung injury that is believed to be oxygen-radical mediated. To further characterize this injury and explore new methods of preventing it, we used the spin-trapping agent N-tert-butyl-alpha-phenylnitrone (PBN) to identify the paraquat radical in lung tissue and to reduce the injury resulting from the subsequent generation of reactive oxygen species. The formation of a paraquat free radical by guinea pig lung was detected under anaerobic conditions by electron paramagnetic resonance spectrometry. Infused (25, 50, or 100 mg/kg) into guinea pig lungs (perfused at constant flow with Krebs solution containing 4% bovine serum albumin and ventilated with 95% O2-5% CO2), paraquat produced dose-dependent increases in peak airway pressure (Paw), mean pulmonary arterial perfusion pressure (Ppa), and wet-to-dry (W/D) lung weight ratio. At 100 mg/kg, paraquat increased Paw by 589.6 +/- 59.8% (mean +/- SE, n = 8) and W/D ratio from 5.33 +/- 0.07 to 6.29 +/- 0.11 (P less than 0.001). Pulmonary vascular leak index increased from 0.40 +/- 0.09 to 1.96 +/- 0.45 (P less than 0.02), without changes in pulmonary microvascular pressure. Perfusate concentrations of thromboxane B2 and 6-ketoprostaglandin F1 alpha increased, but indomethacin did not reduce the injury. PBN (2.3 mM) markedly attenuated all evidence of lung injury, which was also reduced by catalase, mannitol, ethanol, and vitamin E.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of indomethacin on PMA-induced pulmonary endothelial enzyme dysfunction in vivo. We investigated the effects of phorbol myristate acetate (PMA) on metabolic pulmonary endothelial ectoenzyme dysfunction. Anesthetized rabbits were placed on total heart bypass, and the single-pass transpulmonary metabolism of [3H]benzoyl-Phe-Ala-Pro (BPAP) by endothelial-bound angiotensin-converting enzyme (ACE) and [14C]adenosine 5'-monophosphate (AMP) by 5'-nucleotidase (NCT) was calculated before and after PMA (10 micrograms/kg iv), a dose that does not produce histologically evident endothelial damage. Under conditions of partial microvascular recruitment (blood flow = 400 ml/min through the entire lung), PMA, but not the vehicle, significantly reduced substrate utilization of both BPAP and adenosine 5'-monophosphate (AMP) and increased the apparent Michaelis constant (Km) values of ACE for BPAP, indicative of metabolic dysfunction. These changes were completely prevented by pretreatment with indomethacin. Under conditions of near full microvascular recruitment (blood flow = 640 ml/min through the left lung only), PMA similarly reduced substrate utilization and increased the apparent Km of ACE for BPAP. In this case, however, indomethacin failed to prevent the observed PMA-induced metabolic dysfunction. We conclude that PMA alters endothelial ectoenzyme substrate metabolism independently from changes in pulmonary blood flow; indomethacin appears to antagonize the effects of PMA under conditions of partial microvascular recruitment only, perhaps by diverting flow to previously unperfused, unexposed to PMA, and hence metabolically healthy vessels.


Calcium-dependent regulation of Cl secretion in tracheal epithelium. In primary cultures of dog tracheal epithelium, isoproterenol produced a transient increase in short-circuit current (Isc) (duration 30 s; maximal increase, 32 +/- 5 microA/cm2). This was followed by a more slowly developing sustained increase (9 +/- 3 microA/cm2), which mimicked the response to N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (DBcAMP). The transient and sustained responses had dissociation constants for isoproterenol of 2 x 10(-8) and 2 x 10(-9) M, respectively. Bradykinin (in the presence of indomethacin), substance P, histamine, and thrombin produced only transient increases in Isc. The time courses of these transients closely paralleled changes in concentration of intracellular Ca ([Ca2+]i) as measured with fura 2. For different mediators, there was a significant correlation between the maximal transient increase in Isc and the maximal increase in [Ca2+]i. The transients in Isc were not associated with elevation of adenosine 3',5'-cyclic monophosphate (cAMP) and were unaffected by pretreatment with DBcAMP, which abolishes the steady-state increase in response to isoproterenol. Both the transient increases in Isc and [Ca2+]i were inhibited by pretreatment with the Ca chelator 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate abolished the transient increases in [Ca2+]i and Isc in response to isoproterenol but not to bradykinin. These results provide evidence that 1) isoproterenol and bradykinin elevate [Ca2+]i by different mechanisms, and 2) Ca elevation is associated with a transient increase in Isc, whereas increased cAMP is associated with a smaller sustained increase.


Tracheal epithelial cell fatty acid composition modulates prostaglandin E2 and cAMP production. Tracheal epithelial (TE) cells from both rabbits and humans, when cultured in defined serum-free media without lipid supplements, develop fatty acid profiles significantly different from freshly isolated epithelium, including a markedly decreased cellular content of arachidonic acid (AA). In rabbit TE cells, supplementation of media with a phospholipid-rich lipoprotein extract (Excyte III) plus 1 microM bovine serum albumin-complexed AA (Excyte/AA) restored the fatty acid composition of the cultured cells more similar to that of native airway epithelium than did supplementation of media with 5% fetal bovine serum (FBS). In human TE cells, Excyte/AA or 5% FBS increased AA content, but neither lipid supplement completely "normalized" the fatty acid profiles. Compared with lipid-unsupplemented cultures, basal production of prostaglandin E2 (PGE2) was increased by approximately four- to eightfold in rabbit and human TE cells supplemented with 5% FBS or Excyte/AA. In Excyte/AA-supplemented human TE cells, PGE2 production induced by 5 microM calcium ionophore A23187 was more than threefold greater than that of companion ionophore-stimulated unsupplemented monolayers, but PGE2 production was similar in both culture conditions in response to 10 microM exogenous AA. Thus increased cellular content and availability of AA, rather than changes in cyclooxygenase activity, appear to be responsible for the elevated PGE2 production in Excyte/AA-supplemented human TE cells. Secondary effects of lipid supplementation were also observed; Excyte/AA-supplemented human TE cells produced significantly less adenosine 3',5'-cyclic monophosphate (cAMP) in response to exogenous PGE2 and isoproterenol than did lipid-unsupplemented cells.(ABSTRACT TRUNCATED AT 250 WORDS)


Endothelin regulation of mucus glycoprotein secretion from feline tracheal submucosal glands. We examined the effects of endothelin on both the trichloroacetic acid precipitable 3H-labeled glycoconjugate release and intracellular Ca2+ concentration ([Ca2+]i]) measured by the usage of fura-2 in submucosal glands isolated from feline trachea. Endothelin-1 produced a significant increase in glycoconjugate release from the isolated glands in a dose-dependent fashion, reaching a response of 161% of the control at 10(-6) M. Atropine, propranolol, phentolamine, or indomethacin did not produce any significant alterations in the ET-1-evoked glycoconjugate secretion from the isolated glands. In contrast, in tracheal explants which contained epithelium, ET-1 produced a significant reduction in the glycoconjugate secretion in a dose-dependent fashion, reaching a response of 59% of the control at 10(-6) M. In the presence of cultured epithelial cells, ET-1 also produced a significant reduction in the glycoconjugate secretion from isolated glands. In isolated glands, ET-1 produced a sustained increase in the [Ca2+]i which was abolished by the removal of Ca2+ from the medium or by the presence of cultured epithelial cells. Pretreatment with indomethacin failed to alter the epithelial inhibitory action evoked by ET-1 in both the glycoconjugate secretion and the [Ca2+]i in isolated glands. ET-2 and ET-3 failed to produce significant alterations in the glycoconjugate secretion or [Ca2+]i. These findings indicate 1) that ET-1 induces mucus glycoprotein secretion via a Ca2+ influx and 2) that it possibly augments the an epithelial action inhibitory to the mucus glycoprotein secretion from airway submucosal glands.


Effect of pharmacological manipulation of endogenous atriopeptin activity on renal function. Atrial stretch causes the release of atriopeptin (AP, ANF) from preformed vesicular storage sites. The circulating hormone acts on unique receptor sites (containing guanylate cyclase) to release guanosine 3',5'-cyclic monophosphate (cGMP) that mediates the natriuresis and vasodilation and probably the suppression of renin, aldosterone, and vasopressin. The biological effects of atriopeptin are transient because of the rapid inactivation of the circulating hormone (by neutral endopeptidase or clearance receptors) or the second messenger (by cGMP-phosphodiesterase). Heart failure due to chronic cardiac volume overload [aortovenocaval (A-V) fistula] exhibits markedly elevated circulating AP blood levels and urinary cGMP levels, accompanied by induction of ventricular AP gene and protein expression and release. Pharmacological manipulation of endogenous AP, either by inhibiting cGMP phosphodiesterase (i.e., mediator prolongation) or neutral endopeptidase (i.e., prolongation of hormone half-life) in A-V fistula animals results in profound natriuresis and diuresis without hypotension. These pharmacological maneuvers bypass the suppressed renal response to exogenous AP seen in heart failure and provide a rational therapeutic strategy based on our understanding of the underlying physiological and pathological mechanisms.


Mechanism of decreased calcitriol degradation in renal failure. Metabolic clearance rate (MCR) of calcitriol is decreased in renal failure, and uremic toxins play a major role in the suppression of calcitriol degradation. In this experiment, we studied the effect of uremic toxins on renal 24- and 26-hydroxylase (HX) activities. Normal rats were infused for 20 h with 30 ml of normal or uremic plasma ultrafiltrates. At the end of infusion, renal enzymes activities were measured by the generations of 1,24,25- and 1,25,26-trihydroxyvitamin D3 10 min after the addition of 25 nM or 1 microM calcitriol. Renal 24-HX activity decreased approximately 50%, whereas 26-HX activity did not decrease in rats infused with uremic plasma ultrafiltrate. The induction of 24-HX activity by 100 ng calcitriol also decreased in rats infused with uremic ultrafiltrate. To examine whether uremic ultrafiltrate could directly inhibit the degradation enzymes, 24- and 26-HX activities were measured in kidney homogenates preincubated for 3 h with either normal or uremic ultrafiltrate. Uremic ultrafiltrate did not directly suppress 24- and 26-HX activities. Furthermore, the disappearance rate of calcitriol was similar for 90 min in kidney homogenates after they were preincubated for 3 h with uremic and normal ultrafiltrates. Because 24-HX synthesis is induced by the calcitriol-receptor complex binding to nuclear chromatin and activating genes coding for the enzyme, we studied the effect of uremic toxins on the binding affinity of calcitriol-receptor complex for DNA-cellulose. Uremic ultrafiltrate significantly reduced the binding affinity of the hormone receptor complex for DNA when the receptor was preincubated with the ultrafiltrate for 3 h.(ABSTRACT TRUNCATED AT 250 WORDS)


Parathyroid hormone inhibits proximal tubule Na(+)-K(+)-ATPase activity. Parathyroid hormone (PTH) decreases the transepithelial transport of Na+ in the proximal tubule, an action ascribed to PTH-inhibited apical Na(+)-H+ exchanger-dependent Na+ entry. We tested the possibility that PTH could also diminish Na(+)-K(+)-ATPase-dependent Na+ exit. To dissociate effects on Na+ entry, studies were performed in a suspension of rat proximal tubules by measuring nystatin-stimulated ouabain-inhibitable O2 consumption (QO2) and monensin-stimulated ouabain-sensitive 86Rb uptake in the absence or presence of bovine PTH-(1-34) fragment. PTH inhibited the percent nystatin-stimulated QO2 in a concentration-dependent manner, with maximal effect at 10(-10) M. PTH-increased cAMP formation was seen at doses higher than 10(-9) M and was maximal at 10(-7) M. Dibutyryl cAMP (10(-4) M) only partially reproduced the PTH action on QO2. Angiotensin II (10(-6) M) blunted the effect of 10(-7) M PTH on QO2, although it did not change 10(-7) M PTH-dependent cAMP generation. The analogues PTH-(3-34) and [Nle8,Nle18,Tyr34]PTH-(3-34)-amide mimicked the effects of PTH-(1-34) on QO2 but did not affect cAMP formation. Monensin-stimulated ouabain-sensitive 86Rb uptake was inhibited by PTH in a dose-dependent manner, with 10(-7) M PTH being maximally inhibitory. Na(+)-K(+)-ATPase activity was also decreased by PTH-(3-34) in a concentration-dependent manner, with maximal effect occurring at 10(-8) M. Agonist-dependent inhibition of Na+ pump was not due to a decrease of mitochondrial activity, because mitochondrial uncoupled QO2 rates were the same in control and PTH-treated tubules.(ABSTRACT TRUNCATED AT 250 WORDS)


Localization of ecto-ATPase in rat kidney and isolated renal cortical membrane vesicles. Brush-border (BBMV) and basolateral membrane vesicles (BLMV) from rat renal cortex exhibit an ecto-ATPase activity that is distinct from other ATPases. We have examined the cellular and regional distribution of this enzyme in rat kidney using antibodies against rat liver ecto-ATPase. In isolated vesicles, the distribution shown by biochemical assays of ATPase activity was confirmed by immunocytochemistry and Western blotting. Indirect immunofluorescence and immunogold labeling showed that brush borders of the S1 and S3 segments of the proximal tubule (PT) were stained, but the S2 segment was negative. Staining was most intense in the S3 segment. The luminal membrane of the initial part of the thin descending limb of Henle also showed a marked staining. Surprisingly, basolateral plasma membranes of PT had no detectable staining. However, the plasma membrane of endothelial cells was heavily stained, both in larger vessels and in peritubular capillaries. Using an antibody against rat thrombomodulin, a marker for endothelial cell plasma membranes, we showed that preparations of BBMV, BLMV, and endocytic vesicles are all contaminated with these membranes. This may explain, at least partially, the biochemically measured ecto-ATPase activity in renal cortical membrane vesicles. Finally, no specific staining in the kidney was found using polyclonal antipeptide antibodies against the "long form" of liver ecto-ATPase, either by immunocytochemistry or by Western blotting. This indicates either that there is no long isoform of the ecto-ATPase in the kidney or that the intracellular domains of the long form are different in the two tissues.