The role of chloride in taurine transport across the human placental brush-border membrane. Taurine, a sulfated beta-amino acid, is conditionally essential during development. A maternal supply of taurine is necessary for normal fetal growth and neurologic development, suggesting the importance of efficient placental transfer. Uptake by the brush-border membrane (BBM) in several other tissues has been shown to be via a selective Na(+)-dependent carrier mechanism which also has a specific anion requirement. Using BBM vesicles purified from the human placenta, we have confirmed the presence of Na(+)-dependent, carrier-mediated taurine transport with an apparent Km of 4.00 +/- 0.22 microM and a Vmax of 11.72-0.36 pmol mg-1 protein 20 s-1. Anion dependence was examined under voltage-clamped conditions, in order to minimize the contribution of membrane potential to transport. Uptake was significantly reduced when anions such as thiocyanate, gluconate, or nitrate were substituted for Cl-. In addition, a Cl(-)-gradient alone (under Na(+)-equilibrated conditions) could energize uphill transport as evidenced by accelerated uptake (3.13 +/- 0.8 pmol mg-1 protein 20 s-1) and an overshoot compared to Na+, Cl- equilibrated conditions (0.60 +/- 0.06 pmol mg-1 protein 20 s-1). A Cl(-)-gradient (Na(+)-equilibrated) also stimulated uptake of [3H]taurine against its concentration gradient. Analysis of uptake in the presence of varying concentrations of external Cl- suggested that 1 Cl- ion is involved in Na+/taurine cotransport. We conclude that Na(+)-dependent taurine uptake in the placental BBM has a selective anion requirement for optimum transport. This process is electrogenic and involves a stoichiometry of 2:1:1 for Na+/Cl-/taurine symport.


Identification of a putative collagen-binding protein from chicken skeletal muscle as glycogen phosphorylase. We have purified and generated antisera to a 95 kDa skeletal muscle protein that constitutes the largest mass fraction of gelatin-agarose binding proteins in skeletal muscle. Preliminary results indicated that this 95 kDa chicken skeletal muscle protein bound strongly to gelatin-agarose and type IV collagen-agarose, suggesting a possible function in muscle cell adhesion to collagen. However, N-terminal sequencing of proteolytic fragments of the 95 kDa protein indicates that it is the chicken skeletal muscle form of glycogen phosphorylase, the binding of which to gelatin-agarose is unlikely to be biologically relevant. Further characterization showed that the skeletal muscle form of glycogen phosphorylase is immunologically distinct from the liver and brain forms in the chicken, and suggests that, unlike mammalian skeletal muscle, chicken skeletal muscle may have two phosphorylase isoforms. Furthermore, immunolocalization data and solubility characteristics of glycogen phosphorylase in muscle extraction experiments suggest the enzyme may interact strongly with an unidentified component of the muscle cytoskeleton. Thus, this study yields a novel purification technique for skeletal muscle glycogen phosphorylase, provides new information on the distribution and isoforms of glycogen phosphorylase, and provides a caveat for using gelatin affinity chromatography as a primary step in purifying collagen-binding proteins from skeletal muscle.


Endothelins and sarafotoxins: effects on motility, binding properties and phosphoinositide hydrolysis during the estrous cycle of the rat uterus. The effects of four peptides of the endothelin/sarafotoxin (ET/SRTX) family on the motility of the rat uterus were examined during the different stages of the estrous cycle. ET-1, ET-3, SRTX-b and SRTX-c showed similar effects on the contraction of the uterus: a slight increase in the maximum tension of the spontaneous rhythmic contractions, a suppression of the relaxation phase of these contractions and an increase in their rate. All three effects were concentration dependent. Of the four peptides, ET-1 and SRTX-b showed the highest potency and efficacy, suggesting that among the various peptides of this family so far studied, ET-1 and SRTX-b are the two full agonists. The rank order of susceptibility of the different stages was, in most cases: proestrus greater than estrus greater than metestrus. Freshly excised diestrus uteri showed no spontaneous contractions and did not respond to any of the peptides. The binding potency of ET-1 and SRTX-b to uterine membranes was similar at the various estrous stages, but their maximal binding decreased gradually from proestrus to diestrus. All four peptides induced phosphoinositide (PI) hydrolysis in uterine slices at all four different stages, with ET-1 and SRTX-b again being more potent than ET-3 or SRTX-c. The maximal PI hydrolysis correlated with the increased rate of the rhythmic contractions. It is suggested that the reaction of the rat uterus to the ET/SRTX peptides depends on its hormonal status and that ET may act in concert with steroid hormones in the modulation of the estrous cycle.


Investigation of the active site of human placenta glutathione transferase pi by means of a spin-labelled glutathione analogue. A spin-labelled analogue of glutathione (sl-glutathione) has been used in order to characterize the active site of human placenta glutathione transferase pi. The sl-glutathione shows a competitive inhibition towards glutathione (Ki = 14 microM). Binding of sl-glutathione to the enzyme, followed by electron paramagnetic resonance spectroscopy, gives a Kd of 3 microM and two identical binding sites for dimeric unit. Inhibition of the enzyme, by modification of the Cys-47 residue, completely prevents the binding of sl-glutathione. The same results are obtained by monitoring the binding of glutathione by means of fluorescence spectroscopy. It is concluded that integrity of the thiolate of Cys-47 is necessary to maintain an active conformation of the enzyme able to efficiently bind glutathione into the active site.


Study of the structure of arrestin (S-antigen) from bovine photoreceptors by FTIR spectroscopy. Fourier transform-infrared spectroscopy has been used for the study of the secondary structure of arrestin from bovine retina rod cells. Spectra have been obtained in H2O and in D2O media. Resolution enhancement of the amide I secondary structure-sensitive overlapped component bands has been achieved by means of Fourier self-deconvolution and Fourier derivation. In order to obtain a quantitative estimation of the proportion of amino acid residues involved in each type of secondary structure, bands at the resolved frequencies have been curve-fitted to the deconvolved amide I contour by means of a least-squares best-fitting iterative program. The analysis of the results suggests that the secondary structure of arrestin comprises 56-63% of extended strands, 12-19% of turns and bends, 15% of alpha-helices and 10% of undefined and irregular segments.


Comparative study of the effects of amphotericin B on the glucose metabolism in Saccharomyces cerevisiae in K(+)- and Na(+)-rich media. In order to elucidate the effects of amphotericin B (AMB) on the glycolytic pathway, the metabolism of [1-13C]glucose in glucose-grown repressed Saccharomyces cerevisiae was studied. The cells were aerobically suspended in pyrophosphate solutions of high potassium concentration with or without 10(-6) M amphotericin B and measurements were made using 1H-, 13C-NMR spectroscopy and biochemical methods. The results were compared with those obtained under the same experimental conditions but in a medium rich in sodium salts containing the same antibiotic concentration. In general the presence of 10(-6) M AMB reduces the glucose consumption and the ethanol production while favouring the glycerol and trehalose formation. These effects are greatly reduced when a high K+ concentration was used. The AMB effects on the glucose consumption and the production of ethanol, glycerol and trehalose, observed in a suspension rich in Na+, can be fairly well explained by the leakage of K+ through AMB membrane channels. This outflux induces a substantial decrease in the activity of some K(+)-dependent enzymes, such as aldolase, phosphofructokinase and pyruvate kinase. The intensities of the glutamate C2 and C4 signals are higher with a suspension rich in Na+ than with a suspension rich in K+, suggesting that the Krebs cycle operates more effectively in a solution rich in Na+. In the absence of AMB, the passive diffusion of glycerol through the cell membrane is relatively slow and apparently depends on the ionic external medium: it is more efficient in solutions with a high K+ than with a high Na+ concentration. In the presence of 10(-6) M AMB, the glycerol C1,3 resonance drastically decreases at 20 min and then disappears in the noise. This rapid disappearance suggests that glycerol can easily pass through the pores arising from the interaction of AMB with the membrane sterols. However, the rate of pore formation is slow, independent of the external medium (Na+ or K+) and this process is not completed within 20 min.


Increased hydrogen peroxide concentration in human tumor cells due to a nitroxide free radical. Evidence is presented that the nitroxide free radical, TEMPO, at concentrations commonly used to prevent oxidative damage, increases the intracellular hydrogen peroxide concentration. To investigate the origin of this increased hydrogen peroxide concentration, we have incubated various human tumor cell lines with compounds interfering with the generation of active oxygen metabolites. Sodium azide, inhibitor of the respiratory chain, the iron-chelating agent desferrioxamine, superoxide dismutase and catalase had no effect on the hydrogen peroxide concentration. Metyrapone, inhibitor of the cytochrome P450 system, was demonstrated to decrease, but not completely prevent, the hydrogen peroxide production. N-ethylmaleimide, a sulphydryl-bond alkylating agent, was able to completely prevent the increased hydrogen peroxide production. We conclude that, by increasing the cellular hydrogen peroxide concentration, TEMPO exerts a pro-oxidant effect. This increase in hydrogen peroxide production seems to be mediated by the induction of oxidase activity in the cytochrome P450 system, but other cellular systems involved in electron transport may also play a role.


Modulation of the activity of amino acid transport system L by phorbol esters and calmodulin antagonists in a human placental choriocarcinoma cell line. We investigated the regulation of the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line by agents which are known to modulate the activities of three different classes of protein kinases, A-kinase, C-kinase and CaM-kinase. The system L activity was measured by determining the uptake of leucine in these cells, grown as confluent monolayers. Leucine uptake in these cells was predominantly Na(+)-independent, was stimulated by lowering the extracellular pH and was inhibited by hydrophobic neutral amino acids. These characteristics demonstrate that uptake of leucine in this cell line occurs primarily via system L. Treatment of the cells with cholera toxin and forskolin, agents which are known to elevate intracellular cAMP levels, did not have any effect on the activity of system L. 4 beta-Phorbol 12-myristate 13-acetate, an activator of C-kinase, but not the inactive analogue 4 alpha-phorbol 12,13-didecanoate, caused a significant stimulation of system L. The involvement of C-kinase in the phorbol ester-induced stimulation was supported by the finding that staurosporine, an inhibitor of C-kinase, effectively blocked the stimulation. Calmodulin antagonists, calmidazolium, W-7 and CGS 9343 B stimulated system L activity markedly. The potency of these antagonists was in the following order: calmidazolium greater than CGS 9343 B greater than W-7. This stimulatory effect was specific for system L because systems A and ASC were not stimulated by these agents. The stimulation caused by these agents was primarily due to an increase in the maximal velocity, the apparent Km of the system being only minimally affected. It is concluded that the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line is stimulated by C-kinase, inhibited by CaM-kinase and unaffected by A-kinase.


Antiglucocorticoid treatment disrupts endocrine cycle and nocturnal sleep pattern. Mifepriston (RU 486) is a steroid antagonist which binds with high affinity to glucocorticoid receptors (GR), and also to progesterone receptors. The antiglucocorticoid action of Mifepriston in man has been demonstrated by blockade of the negative feedback action of endogenous cortisol and by antagonism of the effects of exogenously administered dexamethasone. In the present study Mifepriston was administered to a normal male volunteer at 14.00 h and its effects on pituitary-adrenal activity and nocturnal sleep pattern were recorded. Mifepriston caused a large rise in plasma ACTH levels during the morning hours in comparison to untreated male control subjects. Plasma ACTH levels in the Mifepriston treated subject at 7.00 h were threefold greater than in the control subjects (104.4 pg/ml vs. 37.6 +/- 13.9 pg/ml; mean +/- SD). Subsequently the cortisol secretion was enhanced and the rise was advanced by about 60 minutes compared to controls. The main effects of Mifepriston on EEG sleep pattern were a dramatic disruption of sleep quality with a prolonged sleep onset latency, increased nocturnal awakenings and a considerable reduction of both slow wave sleep (SWS) and REM sleep. After Mifepriston, SWS was reduced by about 80% in comparison to placebo, and REM sleep was reduced by more than 50%. While the present data were collected from only a single subject the effects observed were so pronounced that tentative conclusions seem to be justified: The well-established pharmacological properties of Mifepriston as a glucocorticoid antagonist are reflected by its action on sleep physiology since it influences sleep in a direction opposite to that produced by cortisol.(ABSTRACT TRUNCATED AT 250 WORDS)


Age-related alterations in gonadotropin, adrenocorticotropin and growth hormone secretion. This review addresses some relevant aspects of the aging of the neuroendocrine system, particularly the reproductive and the adrenocortical axis. Deterioration of the reproductive function, one of the most striking endocrine alterations occurring in aging, is related to a complex interplay of factors. They comprise alterations occurring at the level of all the three components of the reproductive axis, the gonads, the pituitary and the brain, acting synergistically to disrupt the normal pulsatile release of gonadotropins. Particular relevance is given to the neurotoxic action of estrogens during the constant estrous phase occurring in aged female rodents, at the level of hypothalamic nuclei regulating gonadotropin secretion. This effect, to be found also in women during the anovulatory period of the perimenopause, would worsen the dysregulation of the central mechanisms controlling the reproductive function. The activity of the adrenocortical (HPA) axis increases with advancing age in rodents but also, although less strikingly, in humans. The main alteration which can be evidenced in both species is a delayed post-stimulus decline in plasma corticosteroid levels, indicating a diminished sensitivity to glucocorticoids of HPA axis feedback regulation in the elderly. Increased exposure to the highly catabolic adrenal glucocorticoids appears to be associated to a loss of cerebral neurons, particularly in the hippocampus, and the emergence of cognitive deficits in the aged rats. The relevance of experimental data performed in rodents to healthy and pathological human aging is extensively discussed. Finally, this chapter considers the age-related impairment in growth hormone secretion, a common finding of all the animal species investigated so far. The etiology of the hyposomatotropism of aging is namely linked to a progressive defect in growth hormone releasing hormone-producing hypothalamic neurons, although alterations of somatostatin-producing neurons have also been described. This background knowledge makes the use of neuroactive compounds aimed at restoring the physiologic function of hypothalamic hypophysiotropic hormones a rational approach to rectify the alterations of the neuroendocrine system occurring in elderly individuals.


G-proteins and the role of second messengers in the regulation of the human neutrophil. The ideas discussed above clearly point to increasingly complex and interactive transduction mechanisms for the regulation of the neutrophil. The central challenges to be met include the following: 1. Better assays are needed for the study of physiological parameters such as adherence, aggregation, shape change, and cytoskeletal rearrangements, assays which are not prohibitively complex and expensive while still allowing for more detailed physiological observations. 2. The neutrophil receptors need to be characterized in greater detail at the molecular level. Protein purification, sequencing, and cloning approaches are needed. Given the inherent shortcomings of working with the neutrophil system due to the presence of proteases and the problems of obtaining sufficient amounts of plasma membranes as source material for receptor purification, this is a difficult task. Advances in micropurification and sequencing may alleviate some of the difficulties here. 3. The size and complexity of the G-protein family continue to expand. However, as pointed out earlier, stimulus-responsive enzymes without G-protein-associated regulation, and G-proteins without clearly identified targets, remain. A better definition and description of the G-protein family will be required if cellular regulation is to be understood at the molecular level. In terms of second messengers and their role in cellular regulation, the main questions which remain to be answered concern identification of the precise pathways which are important to cellular regulation. In order to understand the complex cascades of arachidonate metabolism, phospholipid turnover, and calcium homeostasis, it is all the more important that the manner in which second messengers may regulate particular cell functions be better understood. An omission in this review is the role of kinases in cellular regulation. Activation of kinase C (through calcium and diacylglycerol) and kinase A (through cAMP) has been demonstrated. The substrates for these kinases have been described by various investigators. However, relating phosphorylation changes in a particular protein to the activity of the protein, and assignment of activity to particular physiological roles, has not been satisfactorily accomplished and remains a challenge for the future.


Effects of cyclic nucleotides on granulocytes. The survey of agents that stimulate increases of cAMP and cGMP revealed many similarities but some differences between basophils, mast cells, and neutrophils. With procedures now available for isolation of eosinophils, it will be of great interest to learn how their cyclic nucleotides are regulated. In the granulocytes studied to date, most functions are inhibited by cAMP. In blood basophils and lung mast cells, but apparently not in rat peritoneal mast cells, cGMP can promote release reactions. Neutrophil functions are regulated by cAMP to variable degrees, O2- generation being the most sensitive and phagocytosis perhaps the least. cAMP controls cell surface receptor- and Ca(2+)-dependent events but not those signaled by PKC activation. Very few cytokines have been analyzed for their effects on cyclic nucleotides. LIF and GM-CSF increase cGMP, but more studies are needed to determine whether this effect is relevant to the biological effects of the cytokines. It is conceivable that the clinical efficacy of cytokines could be enhanced by the co-administration of agents tailored to enhance the cytokines' desirable effects on cyclic nucleotides. The study of cAMP, cGMP, and other signaling systems will certainly provide material for exciting research for a considerable time.


[Gammagraphic study of nodular adrenal hyperplasia]. A common cause of the Cushing's Syndrome (CS) is nodular suprarenal hyperplasia (NSH), which is characterized by the presence of nodes in both suprarenal glands. Its pathophysiology is not well known and its diagnosis is quite difficult due to the heterogenicity of the biochemical and radiological data. We analyzed the suprarenal gammagraphies (SRG) of 7 patients with anatomopathological diagnosis of NSH. Bilateral uptake was observed in all cases and in five patients, such bilateral uptake presented a clear asymmetry. We believe that these observations demonstrate a bilateral suprarenal affectation and are of great use in order to orientate the diagnosis of NSH and, especially, in order to differentiate it from other suprarenal diseases causing CS, like adenomas, in which uptake is unilateral.


A novel platelet-derived renal vasoconstrictor agent in normotensives and essential hypertensives. Platelet homogenates from 200 ml blood of essential hypertensives (n = 28) and normotensives (n = 13) were deproteinized and separated by gel chromatography. The fractions obtained were then tested for vasopressor activity in the isolated perfused rat kidney. In both normotensives and hypertensives, two vasopressor fractions appeared. There was no difference in vasopressor activity in the first vasoactive fraction between normotensives and hypertensives. In the second vasoactive fraction, the hypertensive patients showed a significant higher activity than the normotensive subjects (increase in perfusion pressure by 35.9 +/- 11.5 vs. 6.8 +/- 5.3 mmHg, p less than 0.01). This vasopressor fraction was not inhibited by saralasin, phentolamine, ketanserin, nitroprusside and daltroban and was effective after pretreatment with indomethacin and reserpine and in enzymatically deendothelialized kidneys. The effect was reduced by nifedipine and unchanged by heating the fraction at 100 degrees C and by incubation with proteinase K. It is concluded that a yet unidentified platelet-derived vasopressor agent may contribute to the enhanced vasoconstriction in essential hypertension.


Two roles of Ca2+ in agonist stimulated Ca2+ oscillations. We propose a mechanism for agonist-stimulated Ca2+ oscillations that involves two roles for cytosolic Ca2+: (a) inhibition of inositol-1,4,5-trisphosphate (IP3) stimulated Ca2+ release from the endoplasmic reticulum (ER) and (b) stimulation of the production of IP3 through its action on phospholipase C (PLC), via a Gq protein related mechanism. Relying on quantitative experiments by Parker, I., and I. Ivorra (1990. Proc. Natl. Acad. Sci. USA. 87:260-264) on the inhibition of Ca2+ release from the ER using caged-IP3, we develop a kinetic model of inhibition that allows us to simulate closely their experiments. The model assumes that the ER IP3 receptor is a tetramer of independent subunits that can bind both Ca2+ and IP3. Upon incorporation of the action of Ca2+ on PLC that leads to production of IP3, we observe in-phase-oscillations of Ca2+ and IP3 at intermediate values of agonist stimulation. The oscillations occur on a time scale of 10-20 s, which is comparable to the time scale for inhibition in Xenopus oocytes. Analysis of the mechanism shows that Ca(2+)-inhibition of IP3-stimulated Ca2+ release from the ER is an essential step in the mechanism. We also find that the effect of Ca2+ on PLC can lead to an indirect increase of cytosolic Ca2+, superficially resembling "Ca(2+)-induced Ca(2+)-release." The mechanism that we propose appears to be consistent with recent experiments on REF52 cells by Harootunian, A. T., J. P. Y. Kao, S. Paranjape, and R. Y. Tsien. (1991. Science [Wash. DC]. 251:75-78.) and we propose additional experiments to help test its underlying assumptions.


A new mode for heme-heme interactions in hemoglobin associated with distal perturbations. The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.


Male sterilization.

Male sterilization. This review of recent information and advances in the area of male sterilization deals with recent epidemiologic studies that discuss potential ill effects hypothesized to be a result of vasectomy, including carcinoma of the prostate and carcinoma of the testicle. Rebuttals to these hypotheses are presented. Recent advances in techniques of vasectomy including the "no-scalpel" vasectomy technique and open-ended vasectomy are presented, including the rationale for their use. A good deal of attention is given to postvasectomy follow-up, particularly the use of the technique of measuring numbers of ejaculations following vasectomy rather than the period of time afterward to determine when a man is sterile. The final two sections deal with complications of vasectomy and the most recent percentages on reversing vasectomy with particular reference to return of sperm to the ejaculate and pregnancy rates.


Intrauterine contraception.

Intrauterine contraception. Currently 85 million women use an intrauterine device (IUD), making it the most widely used, reliable, reversible contraceptive method worldwide. Although the exact mechanisms by which copper-bearing IUDs produce contraceptive action are not completely defined, recent evidence indicates that they act primarily to prevent sperm from fertilizing ova. The future of the IUD is brighter than it has been for the past 20 years. The latest generation of IUDs, such as the TCu 380A (Paragard, GynoPharma, Somerville, NJ), are safer and more effective than ever. In World Health Organization large, multicenter trials, pregnancy rates for the TCu 380A are 1.0, 1.4, 1.6, and 1.8 at 3, 5, 7, and 9 years of use, respectively. The ectopic pregnancy rates and removal for pelvic inflammatory disease are very low. The device may soon be the major IUD available in most countries. The acceptability of IUD use can be increased by good clinical management, sympathetic counseling, careful client selection, proper device selection, careful insertion, timing of insertion, and regular follow-up with quick access to medical care.


Human papillomavirus and the pathogenesis of vulvar carcinoma. The pathogenesis of vulvar and cervical cancer are thought to be similar and to be related to a sexually transmitted agent, which, in recent years, has been demonstrated to be human papillomavirus. These two neoplasms differ dramatically in age distribution and relationship to precursor lesions, making a common etiology unlikely. The apparent discrepancies can be explained by a hypothesis that implicates human papillomavirus as an etiologic factor for the majority of cervical carcinomas but for only a small proportion of vulvar carcinomas. Most vulvar carcinomas occur in older women and are not related to human papillomavirus, whereas a subset of vulvar carcinomas occur in young women and are related to this virus. Characterization of two distinct types of vulvar carcinoma may clarify associated risk factors and may have important implications in the clinical management of this disease.


Pathology of the cervix.

Pathology of the cervix. An increasing number of cervical neoplasia studies implicate human papillomavirus (HPV) in the carcinogenic process, perhaps as a necessary factor but not the sole factor. Many different HPV types infect the cervix and the pattern of types is complex, even in women with a normal cervix or a low-grade cervical intraepithelial neoplasia lesion. Although certain types (eg, HPV 18) are particularly likely to be associated with more aggressive cervical cancer types, the results regarding the prognostic value of specific HPV types are still equivocal. Studies of latent HPV infection have yielded varying results, but an HPV detection rate of 31% in swabs from women attending a student health service has been reported. To solve many of the questions raised about HPV in relation to cervical pathology, it is evident that there is a need for large, prospective studies. It is also likely that the continuing development and improvement of serologic assay and a further understanding of the immune response to HPV may prove to be of major importance in the diagnosis and treatment of cervical neoplastic lesions.


Kinetics of the association of several tritiated polychlorinated dibenzo-p-dioxin and dibenzofuran congeners with hepatic cytosolic Ah receptor from the Wistar rat. Several tritiated chlorinated dibenzo-p-dioxins and dibenzofurans have been prepared with specific activities in the range of 30-50 Ci/mmol in order to investigate the effects of structure on the kinetics of their association with the rat cytosolic Ah receptor. The compounds were 2,3,7-trichlorodibenzo-p-dioxin (TrCDD), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1,2,3,7,8-pentchlorodibenzo-p-dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,7,8-TCDF, and 1,2,3,7,8-pentachlorodibenzofuran (PeCDF). Although these congeners differed by up to 2 orders of magnitude in their biochemical and toxic potencies, their affinities for the Ah receptor as determined by conventional Scatchard analysis varied by less than 2-fold (range of KD values 5.0-9.3 nM). The rate of association of these ligands with the Ah receptor was studied at several temperatures, taking into account the competing thermal inactivation of the unbound receptor. The equilibrium constants (KD) were also obtained as the ratio of the rate constants for dissociation and formation, respectively, of the receptor-ligand complexes. The following conclusions were derived from the kinetic studies: (1) 2,3,7-TrCDD and 1,2,7,8-TCDF bound significantly more slowly to the Ah receptor than the other radioligands at all temperatures (13.5-37 degrees C), and this paralleled the lower biochemical potencies of the congeners; (2) the KD values obtained kinetically were in the subnanomolar range, with the smallest KD values observed for those ligands which bound most rapidly to the receptor; and (3) the temperature dependence of the KD values indicated that receptor-ligand association was favorable both enthalpically and entropically.


Interleukin-4 stimulates immunoglobulin secretion by Epstein-Barr virus (EBV)-activated tonsillar B cells, and by EBV-transformed lymphoblastoid B cell lines without increasing cell division. Freshly prepared Epstein-Barr virus-transformed B lymphoblastoid cell lines derived from five different donors were tested for their responses to recombinant human interleukin-4 and to low molecular weight B cell growth factor. In the absence of either cytokine, all five lines secreted immunoglobulin of more than one isotype (IgM, IgG, and IgA, but not IgE). Stimulation with interleukin-4 resulted in a significant increase in immunoglobulin secretion, but did not enhance cell division measured by tritiated-thymidine uptake or cell counts. In contrast, low molecular weight B cell growth factor increased both immunoglobulin secretion and cell division. The increase in immunoglobulin secretion stimulated by interleukin-4 occurred for each of the different isotypes (IgM, IgG and IgA) produced by the unstimulated line. No IgE secretion was detected for any of the five lines. It was also found that low (5 units/ml), but not high (100 units/ml), concentrations of interleukin-4 increased IgM, IgG and IgA secretion by tonsillar B cells polyclonally activated with Epstein-Barr virus. Again, no IgE was detected at any time. These results suggest that interleukin-4 can function as a late-acting B cell differentiation factor as well as a growth factor for human B cells.


Site-specific DNA damage by phenylhydrazine and phenelzine in the presence of Cu(II) ion or Fe(III) complexes: roles of active oxygen species and carbon radicals. Phenylhydrazine cleaved isolated DNA in the presence of Cu(II), Mn(III), hemin, Fe(III)-EDTA, or peroxidase/H2O2, while phenelzine cleaved in the presence of Cu(II). DNA cleavage by phenylhydrazine in the presence of Mn(III), hemin, or Fe(III)-EDTA occurred without marked site specificity. Inhibitory effects of scavengers of hydroxyl free radical (.OH) on the DNA damage suggest the involvement of .OH. On the other hand, Cu(II)-mediated DNA cleavage by phenylhydrazine or phenelzine was inhibited by catalase and bathocuproine, a Cu(I)-specific chelator, but not by .OH scavengers. The predominant cleavage site was the thymine residue of 5'-GTC-3' sequence. Since the cleavage pattern was similar to that induced by Cu(I) plus H2O2 but not to that induced by Cu(II) plus H2O2, it is speculated that the copper-oxygen complex derived from the reaction of H2O2 with Cu(I) participates in DNA damage by phenylhydrazine or phenelzine in the presence of Cu(II). A comparison between scavenger effects on the DNA damage and those on radical production detected with ESR suggests that carbon-centered radicals (phenyl radical, 2-phenylethyl radical) do not play an important role in Cu(II)-, hemin-, or Fe(III)-EDTA-mediated DNA damage by phenylhydrazine or phenelzine of relatively low concentrations (less than 0.5 mM). However, during the oxidation of a high concentration (10 mM) of phenylhydrazine by ferricyanide, phenyl radical seemed to cause DNA damage, especially the breakage of the deoxyribose phosphate backbone. The possibility that active oxygen species (copper-oxygen complex, .OH) are more important in DNA damage induced by hydrazines in vivo than carbon-centered radicals is discussed.


Carpal tunnel syndrome: a review.

Carpal tunnel syndrome: a review. Carpal tunnel syndrome involves classic symptoms of numbness and paresthesias in the radial 3-1/2 digits, most frequently nocturnal, and pain associated with this distribution. Thenar weakness and autonomic dysfunction rarely are seen in this syndrome except in advanced cases. Provocative tests on physical examination such as the wrist flexion test and the local percussion sensitivity test over the median nerve can be extremely helpful in determining and confirming the diagnosis. Nerve conduction velocity and electromyographic studies of the median nerve and its compression can be helpful especially in difficult cases involving a complex differential diagnosis. It has been clearly documented that a negative NCV/EMG study by itself, does not exclude the possibility of carpal tunnel syndrome. The hallmark of the diagnosis remains the history and a careful physical examination. Treatment initially consisting of wrist splint immobilization and steroid injection into the carpal canal can provide initial relief and elimination of symptoms on a long-term basis in several patient groups. Patients without any resolution of symptoms after two to three months of conservative treatment or those with symptoms of greater than one year's duration generally can be considered candidates for surgical decompression of the carpal canal. The likelihood of operative treatment being required for resolution of symptoms is heightened if the patient is involved in daily manual repetitive activities of the hand and/or wrist. Surgical decompression can be accomplished by either a limited open technique or the new endoscopic released technique.(ABSTRACT TRUNCATED AT 250 WORDS)


Opposite effects of mild and severe stress on in vitro activation of rat peripheral blood lymphocytes. The effects of short-term handling and different durations of immobilization on serum levels of catecholamines, ACTH, prolactin, and corticosterone and in vitro functions of lymphocytes were examined in rats. The results show that changes in the immune response of peripheral blood lymphocytes (PBL) depend on the intensity of the stressor: Short (1 min) handling of cannulated rats induced an enhanced stimulation of PBL to respond to T and B cell mitogens, whereas immobilization of the same animals led to suppression, dependent on the time this stressor was applied. The decrease in the mitogen reactivity of PBL after 120 min of immobilization was reversible within 24 h, and could be largely prevented by adrenalectomy, confirming that factors released by this gland are mainly responsible for immunosuppression. In contrast to PBL, spleen cells showed an enhanced mitogen response to immobilization and adrenalectomy, indicating that the immune response is differently regulated in the various compartments of the immune system. Possible correlations of the various effects with changes in stress hormone levels are discussed.


Modulation of immune status by a conditioned aversive stimulus: evidence for the involvement of endogenous opioids. Recent research has shown that presentations of an unconditioned aversive stimulus, such as electric shock, can induce alterations of immune function in rats. Furthermore, it has been demonstrated that an innocuous stimulus paired with an unconditioned aversive stimulus can acquire immunomodulatory properties. Research has suggested that endogenous opioid activity is responsible for the alterations of immune function by unconditioned aversive stimulation. The present study evaluated the effect of administration of opiate receptor antagonists, naltrexone and N-methylnaltrexone, on the immunomodulatory effect of a conditioned stimulus (CS) that had been paired with electric footshock. Naltrexone dose-dependently attenuated the CS-induced suppression of the in vitro proliferative response of splenic lymphocytes to concanavalin A, lipopolysaccharide, and a combination of ionomycin and phorbol myristate acetate. Naltrexone also attenuated the CS-induced reduction in natural-killer cell activity. In contrast, the quaternary form of naltrexone, N-methylnaltrexone, did not significantly attenuate the CS-induced immunomodulatory effects. Collectively, these findings indicate that endogenous opioid activity is involved in CS-induced alterations of immune function. Moreover, the lack of effectiveness of N-methylnaltrexone in attenuating the CS-induced immunomodulatory effect suggests that the opioid receptors involved in the effect are located in the central nervous system.


The distribution of immunoreactive topoisomerase II Protein in human tissues and neoplasms. Western blotting experiments with topoisomerase II antiserum were performed to measure type II topoisomerase content in human tissues. In this study, antiserum produced against the C-terminus of recombinant HeLa topoisomerase II recognized both p170 and p180 forms of the enzyme. Antisera specifically made against p180 selectively recognized that enzyme form. By using these antisera, a remarkable difference in topoisomerase content between the tissues studied was observed. Tissues with a high proportion of cycling cells contained predominantly the p170 form of the enzyme, but also smaller amounts of p180. In terminally differentiated organs or in neoplasms with a low percentage of cycling cells, topoisomerase II immunoprotein was not detected in whole tissue extracts. In term human placenta, this was because this tissue contained predominantly the p180 enzyme form, yet the concentration of this form of the enzyme was apparently in too low a concentration to be detected by the antibody in crude extracts. It was readily observed after concentration by chromatography on hydroxylapatite.


Antagonism of TGF-alpha receptor binding and TGF-alpha induced stimulation of cell proliferation by methyl pheophorbides. Over 4,500 natural product extracts were screened for their abilities to inhibit binding of radiolabeled TGF-alpha to A431 cells; several plant extracts were identified as potential leads with IC50 values of less than 30 micrograms/mL. The active components of one extract were purified to homogeneity and identified as the porphyrin structures, methyl pheophorbides a and b. These compounds inhibited both TGF-alpha receptor binding and the TGF-alpha induced proliferation of NRK-49F cells in soft agar. To construct a structure-function relationship, a series of commercially available porphyrin derivatives was evaluated. The most potent compound, hematoporphyrin IX, inhibited TGF-alpha functions in a dose-dependent fashion with IC50 values slightly lower than the methyl pheophorbides. Further studies revealed that inhibition of TGF-alpha binding was light dependent and that inhibition did not involve direct competition of porphyrins for the TGF-alpha binding site. To determine the specificity of inhibition, the porphyrins were tested in a number of other receptor-ligand assays. TNF-alpha and beta-adrenoceptor bindings were unaffected, whereas IL-1 beta binding to EL-4 membranes and platelet-derived growth factor induced thymidine incorporation in NIH-3T3 cells were both antagonized by the most active porphyrins. Inhibition of TGF-beta binding to NRK-49F cells and TGF-beta-induced growth of AKR-2B cells was also observed. In summary, we report that methyl pheophorbides are naturally occurring, photodynamic antagonists of TGF-alpha, and although the inhibitory properties of these molecules were not confined to TGF-alpha alone, some level of receptor selectivity was observed.


Fungemia caused by Hansenula anomala: successful treatment with fluconazole. Fungemia, due to Hansenula anomala, developed in an adult patient with small cell lung cancer who received anti-cancer chemotherapy and plasmapheresis for a sensori-motor neuropathy complication. Treatment with intravenous infusion of fluconazole in addition to the removal of the central venous catheter was successful in treating the fungemia. Pathogenic Hansenula anomala infections are rare, but reports of this infection have been increasing. The use of fluconazole treatment for this infection has not been reported in the literature, and this is the first case of an adult infection of Hansenula anomala in Japan.


Fabry's disease.

Fabry's disease. A 20-year-old man presented with generalized acquired anhidrosis and heat intolerance which was confirmed by a sweat test. Other clinical features included severe pain of the extremities and cutaneous angiokeratomas. On electronmicroscopy, granules specific for Fabry's disease were observed in the endothelial cells. Biochemical examination revealed a decreased level of serum alpha-galactosidase A. These findings confirmed the diagnosis of Fabry's disease.


Siblings of 21-hydroxylase deficiency (non-salt-losing) with aldosterone hypersecretion. We describe siblings with the non-salt-losing form of 21-hydroxylase deficiency who had hypersecretion of aldosterone and plasma renin activity (PRA). Blood pressure and serum electrolytes in both cases were normal despite the aldosterone hypersecretion. Aldosterone secretion was elevated markedly with ACTH administration and with sodium deprivation and/or volume depletion during ACTH suppression by dexamethasone. With suppression by dexamethasone, aldosterone hypersecretion was decreased with lowering of the steroids proximal to the block in the biosynthetic pathway. However, urinary sodium excretion was decreased. These results suggest that the biosynthetic pathway for aldosterone production was preserved. Furthermore, aldosterone hypersecretion and high PRA may serve to compensate for the sodium loss which results in turn from the overproduction of the sodium-losing steroids, such as progesterone and 17 alpha-hydroxyprogesterone which are aldosterone antagonists.


Treatment of herpesvirus infections in HIV-infected individuals. To discuss strategies available for the treatment of herpesvirus infections in individuals infected with HIV. Information was obtained from controlled and uncontrolled clinical trials, abstracts, conference proceedings, and review articles. Emphasis was placed on controlled investigations in subjects infected with HIV. Data from human studies were extracted by the author and evaluated according to the patient population studied, sample size, dosage regimen, and therapeutic response. Herpes group viruses are common opportunistic pathogens in HIV-infected individuals. Zoster, caused by varicella-zoster virus (VZV), is an early indication of the loss of cell-mediated immunity and HIV disease progression. Anorectal mucocutaneous disease is the most common manifestation caused by herpes simplex virus (HSV). Acyclovir is the drug of choice for treatment of both VZV and HSV infections. Cytomegalovirus (CMV) is the most common life-threatening viral infection in patients with AIDS; retinitis is the most frequent clinical manifestation. The response rate of CMV retinitis to initial treatment with either ganciclovir or foscarnet is equivalent, approximately 60-90 percent. Recent data suggest that the survival benefit may be greater with foscarnet. Advances in the development and application of antiviral drugs for herpes group viruses have made treatment and, in some cases, prevention of infections possible. Future efforts, aimed at earlier intervention and suppression of latent virus, may offer additional improvement in quality of life for the HIV-infected individual.


[Probe into internal relation between classification of the differentiation-syndrome in traditional Chinese medicine and serum copper and zinc in lung cancer]. This article probed into the internal relations and significance between differentiation of syndrome of traditional Chinese medicine (TCM) and trace elements--copper and zinc in lung cancer patients. The serum copper and zinc of 95 patients with lung cancer and 82 healthy persons were measured. According to differentiation of syndrome of TCM types of 95 lung cancer patients were divided, and the relations with their levels of serum copper, zinc and the ratio of copper/zinc compared respectively. The authors found that there were some inner links among the differentiation syndromes and levels of serum copper, zinc and its ratio. The result showed that the level of Cu/Zn ratio could reflect increase and decrease of body resistance and pathogenic factors the level of Cu/Zn ratio was more significant than that of copper and zinc. The authors suggested that the ratio of Cu/Zn could be used as the criteria of differentiation of syndrome of TCM. It is clinically significant to combine the level of copper, zinc and its ratio with differentiation of syndrome of TCM to evaluate the severity and prognosis of the patients and to direct the treatment of them with TCM.


Comparative studies on the effect of 4-APP(4-aminopyrazolopyrimidine) on hamster and rat adrenal cortex. The study aimed to compare, by means of stereologic methods, the reactivity of the adrenal cortex of the hamster and the rat to prolonged treatment with 4-aminopyrazolo-(3,4d)pyrimidine (4-APP), a drug reducing hepatic secretion of plasma lipoprotein. Adult female hamsters, intact or cortisone suppressed, were administered i.p. daily with 0.5 mg 4-APP for 5 days while intact female rats received 1 mg of the drug per dose. 4-APP resulted in a loss of body weight, with the more profound effect in the hamster. In the rat 4-APP did not change the adrenal gland weight, the volume of the adrenocortical zones, the average volume of adrenocortical cells and the number of parenchymal cells in the gland. Moreover, serum ACTH and corticosterone levels in the rat remained unchanged. In the intact hamster 4-APP decreased the adrenal gland weight, the volume of the zona fasciculata and enhanced the serum cortisol level. In steroid suppressed hamsters 4-APP lowered the adrenal weight, the volume of fasciculata and reticularis zones, the average volume of the fasciculata cells and the number of parenchymal cells in the gland. These findings may suggest the direct inhibitory effect of low 4-APP doses on the hamster adrenal cortex and clearly demonstrate higher susceptibility of the hamster adrenal cortex, if compared with the rat, to 4-aminopyrazolo-pyrimidine.


A murine leukemia virus derived retroviral vector with a rat VL30 packaging psi sequence. A rat VL30 packaging Psi sequence of 168 nucleotides has been characterized. It bears little or no homology to the Murine Leukemia Virus (MLV) Psi sequence, is much smaller in size and at least as efficient for packaging. The use of this small VL30 Psi sequence in MLV derived retroviral vectors should render even more unlikely recombinations possibly generating replication competent MLV viruses. Thus, utilization of the rat VL30 Psi sequence should improve the biological safety of MLV derived vectors for human gene transfer.


T cell development and selection in the thymus. T cell receptor (TCR) transgenic mice have been useful models to study the selection of lymphocytes during T cell development. They also have raised new questions with regard to allelic exclusion of T cell receptor genes and mechanisms determining the CD4/CD8 phenotype of mature T cells. Our data indicate that exclusion of beta and alpha TCR alleles occurs by different mechanisms: the expression of beta TCR genes as cell surface proteins in the absence of alpha, gamma or delta TCR chains apparently suppresses effectively further rearrangement of the beta TCR locus in spite of the presence of an active recombination machinery in these cells. In contrast an alpha TCR surface protein has little effect on further alpha TCR rearrangement which only ceases after positive selection of alpha beta T cells. This enables a developing T cell to test various alpha TCR chains with one beta TCR chain in the formation of a selectable receptor. Further data support the concept that different signals instruct developing T cells to either become CD4+8- helper or CD4-8+ killer cells: CD4+8+ cells with high levels of a class I MHC specific TCR were shown to result exclusively from positive selection and developed in vitro in the absence of selecting ligands in CD4-8+ but not CD4+8- T cells.


A specific B2-bradykinin receptor antagonist HOE 140 abolishes the antihypertrophic effect of ramipril. To evaluate the role of bradykinin in the antihypertrophic effect of the angiotensin-converting enzyme (ACE) inhibitor, ramipril, we investigated the influence of HOE 140, a specific B2-receptor antagonist, on the effects of ramipril on left ventricular hypertrophy (LVH) in rats with aortic banding. Ramipril at a dose of 1 mg kg-1 day-1 for 6 weeks prevented the increase in blood pressure and development of LVH after aortic banding; plasma ACE activity was significantly inhibited. A lower dose of ramipril (10 micrograms kg-1 day-1 for 6 weeks) had no effect on the increase in blood pressure or on plasma ACE activity, but prevented LVH after aortic banding. The antihypertrophic effects of the higher and the lower dose ramipril, as well as the antihypertensive action of the higher dose of ramipril were abolished by the coadministration of HOE 140 (500 micrograms kg-1 day-1). The present data show for the first time that the beneficial effects of an ACE-inhibitor on LVH in rats with hypertension caused by aortic banding can be prevented by a specific B2-receptor antagonist.


Human big-endothelin-1 and endothelin-1 release prostacyclin via the activation of ET1 receptors in the rat perfused lung. Although ET1 and ET2 binding sites were found in rat lung membranes, a selective ET1 receptor antagonist, BQ-123 (10 microM), did not displace [125I]-endothelin-1 ([125I]ET-1) from ET2 sites, illustrating the selectivity of the angatonist for ET1 receptors. In rat perfused lungs, BQ-123 (1 microM) markedly reduced the prostacyclin (PGI2) releasing properties of endothelin-1 (ET-1: 5 nM) and human big-ET-1 (100 nM) suggesting that both peptides induce the release of PGI2 via the selective activation of ET1 receptors.


Investigation of peripheral neuropathy.

Investigation of peripheral neuropathy. Much exciting work has been carried out on the peripheral nervous system during the past 20 years and has led to a greater understanding of peripheral nerve disorders and to improved diagnosis and treatment. This article sets out a systematic approach to the diagnosis of the patient with peripheral neuropathy.


Role of nitric oxide in non-adrenergic, non-cholinergic inhibitory junction potentials in canine ileocolonic sphincter. 1. Electrical field stimulation causes neurally-mediated relaxation of the ileocolonic sphincter that is due to activation of non-adrenergic and non-cholinergic (NANC) nerves. Recent studies have suggested that nitric oxide (NO) is the neurotransmitter that mediates relaxation. 2. Using intracellular recording techniques, we have tested whether NANC inhibitory junction potentials (i.j.ps) in the canine ileocolonic sphincter are also mediated by NO. 3. Electrical field stimulation elicited excitatory and inhibitory junction potentials: e.j.ps were blocked by atropine (10(-6) M) and tetrodotoxin (TTX; 10(-6) M); i.j.ps were also blocked by TTX and partially blocked by apamin (10(-6) M). I.j.ps were unaffected by atropine, phentolamine and propranolol (all at 10(-6) M). 4. The arginine analogues, L-NG-nitroarginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), decreased the amplitude of i.j.ps and L-arginine, but not D-arginine, partially restored the i.j.ps. 5. I.j.ps were also inhibited by oxyhaemoglobin (1%), but not by methaemoglobin. 6. Exogenous NO (10(-7) M to 3 x 10(-5) M) caused concentration-dependent hyperpolarizations that were similar in amplitude to the NANC nerve-evoked i.j.ps. Hyperpolarizations to NO were unaffected by L-NAME, but were blocked by oxyhaemoglobin. 7. Tetrodotoxin, L-NAME and oxyhaemoglobin all caused depolarization of resting membrane potential. 8. The specific guanosine 3':5'-cyclic monophosphate phosphodiesterase inhibitor, M&B 22948, caused hyperpolarization, increased the maximum level of hyperpolarization reached during i.j.ps, and increased the duration of i.j.ps. 9. These data further support the hypothesis that NANC neurotransmission in the ileocolonic sphincter is mediated by NO or an NO-releasing compound. The data also suggest that tonic release of NO, possibly from spontaneous firing of NANC nerves, may regulate resting membrane potential and tone in this sphincter.


Investigation of the prostaglandin E (EP-) receptor subtype mediating relaxation of the rabbit jugular vein. 1. Prostaglandin E2 (PGE2) relaxes circular smooth muscle of the rabbit isolated jugular vein at very low concentrations (mean pIC50 against histamine-induced contraction = 9.34). This effect is not blocked by the EP1-receptor antagonist, AH 6809 (2 microM). 2. From a group of prostaglandin E analogues examined, 16,16-dimethyl PGE2, misoprostol, 11-deoxy PGE2-1-alcohol and 11-deoxy PGE1 were highly potent relaxant agents, whereas 17-phenyl-omega-trinor PGE2, MB 28767 and butaprost had low potency and sulprostone and oxoprostol were virtually inactive. 3. Comparison of the jugular vein data with published data for inhibitory agonist potencies on the cat trachea (EP2 preparation) and the field-stimulated guinea-pig vas deferens (EP3) indicates that the EP-receptor in the rabbit jugular vein is closest to the EP2 subtype. However, the correlation is not entirely convincing. For example, butaprost, 16,16-dimethyl PGE2 and 11-deoxy PGE1 are of similar potency on the cat trachea, whereas butaprost is about 300 times less potent than the other two analogues on the jugular vein. The existence of more than one EP2-receptor appears possible. 4. It was felt that the activity of butaprost required further investigation in view of the claim that it is a specific EP2-receptor agonist. We have shown that butaprost has very low inhibitory activity on the guinea-pig vas deferens, a very sensitive EP3-receptor containing preparation. However, on the chick ileum, the original EP3 preparation, butaprost showed potent contractile activity (pEC25 approximately 8.0).In addition, its maximum response was lower than that of PGE2; lower maxima were also found for sulprostone, MB 28767 and oxoprostol, but not for ICI 80205, 16,16-dimethyl PGE2 and 17-phenyl-omega-trinor PGE2. The maximal response to a combination of either sulprostone and butaprost or sulprostone and PGE2 was similar to that achieved by PGE2 alone. Analysis of the interaction between sulprostone and PGE2 appears to exclude a partial agonist action for sulprostone. Furthermore neither sulprostone nor butaprost appear to have inhibitory activity on the ileum. AH 6809 at 2 pM produced only a small shift of the PGE2 log concentration-response curve.5. It is likely that contraction of the longitudinal smooth muscle of the chick ileum is mediated by (at least) two EP-receptor subtypes; activation of only one receptor system does not induce the maximum response (i.e. the acetylcholine maximum) of the preparation. One receptor could be an EP3 subtype, at which sulprostone exerts a selective agonist action. The other receptor is unlikely to be an EP, subtype, because of the high agonist potency of butaprost, the low agonist potency of iloprost, and the low antagonist potency of AH 6809. An alternative hypothesis is that the chick ileum contains a novel EP-receptor subtype in addition to an EP3-receptor.


Involvement of beta 2-adrenoceptors in the regional haemodynamic responses to bradykinin in conscious rats. 1. Bradykinin can release neuronal calcitonin gene-related peptide (CGRP) and adrenal medullary catecholamines, both of which could contribute to its cardiovascular effects in vivo. Therefore, in the main experiment, regional haemodynamic responses to bolus injections of bradykinin (3 nmol kg-1, i.v.) were assessed in the same chronically-instrumented, conscious, Long Evans rats in the absence and in the presence of human alpha-CGRP [8-37] or ICI 118551, antagonists of CGRP1-receptors and beta 2-adrenoceptors, respectively. The selected doses of these antagonists caused specific inhibition of responses mediated by exogenous human alpha-CGRP and beta 2-adrenoceptor agonists, respectively. 2. Bradykinin administered alone as an i.v. bolus had a slight pressor effect accompanied by a marked tachycardia. There were early (at about 30 s) increases in flow and conductance in the mesenteric vascular bed, and delayed (at about 90 s), but qualitatively similar, changes in the hindquarters vascular bed. There were only slight increases in flow and conductance in the renal vascular bed. 3. Human alpha-CGRP [8-37] had no statistically significant effects on the responses to bolus doses of bradykinin. However, in the presence of ICI 118551, the pressor effect of bradykinin was significantly enhanced while its tachycardic effect was significantly suppressed. The hindquarters vasodilator effect of bradykinin was converted to a vasoconstriction and there was a slight renal vasoconstriction, but the mesenteric vasodilator effect of bradykinin was unchanged by ICI 118551. 4. In subsidiary experiments, in other animals, it was found that infusion of bradykinin (36 nmol kg-1 min-1) elicited a pattern of haemodynamic responses similar to that seen with bolus injections and, as in the latter case, the hindquarters hyperaemic vasodilation was inhibited by ICI 118551. In the presence of mecamylamine (at a dose sufficient to block reflex heart rate responses to rises or falls in arterial blood pressure) bolus injection or infusion of bradykinin still elicited increases in renal, mesenteric and hindquarters blood flow. However, in additional experiments in adrenal demedullated rats (n = 4) the hindquarters hyperaemic effect of bradykinin was absent, although the mesenteric hyperaemic effect remained. 5. The results indicate that the increase in hindquarters blood flow following administration of bradykinin in vivo is largely due to activation of beta 2-adrenoceptors by catecholamines released subsequent to direct stimulation of the adrenal medulla by the peptide. However, the bradykinin-induced increase in mesenteric blood flow does not depend on this mechanism.


Effect of alpha-human atrial natriuretic peptide on the synthesis of dopamine in the rat kidney. 1. The present study has examined the influence of alpha-human atrial natriuretic peptide (alpha-hANP) on the synthesis of dopamine and its deamination into 3,4-dihydroxyphenylacetic acid (DOPAC) in rat kidney slices loaded with exogenous L-dihydroxyphenylalanine (L-DOPA). 2. alpha-hANP (3.3 and 330 nM) was found to produce a marked reduction (63-78% reduction) in the time-dependent accumulation of newly-formed dopamine and of its deaminated metabolite DOPAC in kidney slices loaded with 10 microM L-DOPA. alpha-hANP (330 nM) was also found to decrease the accumulation of newly-formed dopamine (45-66% reduction) and DOPAC (38-61% reduction) in experiments in which increasing concentrations (1-100 microM) of L-DOPA were used. This inhibitory effect was found to be potentiated by zaprinast (M&B 22,948; 10 microM), a guanosine cyclic 3',5'-monophosphate (cyclic GMP) phosphodiesterase inhibitor. Alone, zaprinast also decreased the accumulation of both dopamine (54-71% reduction) and DOPAC (73-92% reduction). 3. In kidney homogenates, alpha-hANP (330 nM) was found to affect neither the formation of dopamine nor its deamination to DOPAC. 4. Both alpha-hANP (330 nM) and zaprinast (10 microM) were found not to affect the formation of dopamine and DOPAC in kidney slices obtained from rats on a high salt diet during the previous 6 weeks. A similar situation was also found to occur when kidney slices obtained from 24-months old rats were used.5. The results obtained suggest that the inhibitory effect of alpha-hANP on the renal synthesis of dopamine is dependent on the activation of a membrane-operated mechanism, coupled to the enzyme guanylate cyclase, controlling the entry of L-DOPA into the cells.


Effect of treatment with vitamin D3 on the responses of the duodenum of spontaneously hypertensive rats to bradykinin and to potassium. 1. The diet of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) and Wistar (NWR) rats was supplemented with either 2% calcium lactate in the drinking water or 12.5 micrograms vitamin D3 100 g-1 body weight daily by gavage, for 14 days. 2. The blood pressure of the SHR treated with either calcium or vitamin D decreased to the same levels as that of WKY and NWR. 3. The response to bradykinin of the SHR isolated duodenum, which is predominantly contractile, upon treatment with vitamin D (but not with calcium), became predominantly relaxant, approaching the normal behavior of the WKY and NWR duodenum. 4. The relaxant responses of the SHR and WKY duodenum to potassium were smaller than those of NWR, but treatment with vitamin D increased the response in all three rat strains. 5. It is concluded that, besides sharing the hypotensive effect of calcium, vitamin D treatment of SHR has an effect on the duodenum smooth muscle which might be due to calmodulin-dependent activation of calcium-dependent potassium channels.


Effectiveness of GABAB antagonists in inhibiting baclofen-induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat. 1. The purpose of the present experiments was to assess the activities of GABAB receptor antagonists in mammalian isolated melanotrophs. 2. Cytosolic free Ca concentration ([Ca2+]i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura-2. 3. (-)-Baclofen lowered [Ca2+]i in a concentration-dependent manner with an EC50 of 0.96 microM. The reduction in [Ca2+]i produced by (-)-baclofen at a maximally effective concentration (100 microM) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nM), or by perifusion with a nominally Ca-free solution. 4. The GABAB receptor antagonists, 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2-hydroxy saclofen, phaclofen and 4-amino-3-(5-methoxybenzo[b]furan-2-yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca2+]i produced by (-)-baclofen (3 microM). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 microM, compared to 120 to 400 microM for the others. CGP 35348 acted competitively. 5. CGP 35348 alone had no effect on basal [Ca2+]i, or on the changes in [Ca2+]i produced by dopamine (10 nM) or the specific GABAA receptor agonist, muscimol (10 microM). 6. The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABAB receptors in melanotrophs.


Direct and indirect stimulations of cyclic AMP formation in human brain. 1. Adenosine 3':5'-cyclic monophosphate (cyclic AMP) formation, by use of an [3H]-adenine prelabelling assay, was measured in fragments of human cerebral cortex, taken in the course of various neurosurgical procedures. 2. Large accumulations of [3H]-cyclic AMP due to isoprenaline, noradrenaline and adenosine and small effects due to forskolin were observed. Histamine, 5-hydroxytryptamine (5-HT) and the excitatory amino acids glutamate and quisqualate were ineffective. 3. The response to noradrenaline consisted of two components; a direct beta-adrenoceptor response and an enhancement mediated by an alpha-adrenoceptor which appears to be similar to that in rat cerebral cortex. 4. The response to isoprenaline was also potentiated by histamine H1 receptor stimulation but the direct effect of 2-chloroadenosine was not altered by histamine, 5-HT or quisqualate. 5. It is concluded that some, but not all, of the indirect modulations of cyclic AMP formation previously observed in experimental animal brain exist in human cerebral cortex.


Dexamethasone-induced gastric mucosal damage in the rat: possible role of platelet-activating factor. 1. The aim of the present experiments was to study the possible role of platelet-activating factor (PAF) in mediating gastric mucosal damage induced by dexamethasone in the rat by measuring gastric tissue levels of PAF during dexamethasone-treatment and by investigating the effects of specific PAF receptor antagonists on dexamethasone-induced gastric lesions. PAF-like bioactivity extracted from the rat glandular stomach was determined by a platelet aggregation assay. 2. Dexamethasone treatment (0.4-4 mg kg-1, daily for 1-6 days) produced time- and dose-dependent damage to the glandular mucosa of the stomach as characterized by extensive, uniform hyperaemia with multiple, focal petechiae and erosions. 3. These changes were accompanied by a time-, and dose-dependent increase in PAF content of the glandular stomach. Control rat stomach contained small amounts of PAF (0.14 +/- 0.04 ng per g wet weight), which increased over 40 fold in response to dexamethasone treatment (4 mg kg-1, daily for 6 consecutive days). The presence of PAF-like material in the stomach extract was ascertained by thin-layer chromatography, high performance liquid chromatography and by alkaline hydrolysis. 4. Pretreatment of the animals with one or other of the structurally unrelated PAF receptor antagonists, BN 52021 (10 mg kg-1, i.p.) or BN 50727 (1 mg kg-1, i.p.) significantly reduced dexamethasone-induced gastric damage. In these animals neither petechiae nor erosions were observed. 5. These observations suggest that PAF is a likely endogenous mediator of glucocorticoid-induced gastric mucosal damage in the rat.


Bradykinin stimulation of phosphoinositide hydrolysis in guinea-pig ileum longitudinal muscle. 1. Bradykinin (BK)-induced contraction of ileal smooth muscle is assumed to be due to phosphoinositide hydrolysis but this has never been reported. We have investigated whether BK receptors are linked to this transduction mechanism in guinea-pig ileum longitudinal muscle and determined whether these receptors are equivalent to those labelled in [3H]-BK binding assays. 2. In membranes prepared from longitudinal muscle, [3H]-BK bound to a single class of sites with high affinity. Characterization of the binding with BK analogues indicated that the radioligand selectivity labelled a B2 type receptor. 3. BK significantly elevated tissue levels of [3H]-inositol phosphates in longitudinal muscle slices preincubated with [3H]-myo-inositol. The agonists potencies of BK, Lys-BK, Met-Lys-BK, Tyr5-BK and Tyr8-BK were in agreement with their relative potencies in the binding assay. The B1 receptor agonist des-Arg9-BK, did not stimulate inositol phosphate production. The response to BK was blocked by known B2 receptor antagonists but not by the B1 antagonist des-Arg9, Leu8-BK. 4. BK-induced phosphoinositide hydrolysis was unaffected by exposure of muscle slices to either atropine or indomethacin. 5. The results indicate that the B2 receptors linked to phosphoinositide turnover in ileal longitudinal muscle exhibit properties similar to those involved in contractile responses. Also, the receptor mediating the phosphoinositide response is likely to be that labelled in the [3H]-BK binding studies.


Correlation between airway epithelium-induced relaxation of rat aorta in the co-axial bioassay and cyclic nucleotide levels. 1. In co-axial bioassays, in the presence of indomethacin, addition of histamine (100 microM) or methacholine (100 microM) to guinea-pig trachea produced an epithelium-dependent relaxation of precontracted rat aorta which was associated with an approximately 2 fold elevation in tissue levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP). Removal of the airway epithelium abolished the histamine-induced relaxation of rat aorta and the associated increase in intracellular cyclic GMP. 2. Epithelium-dependent relaxation was not associated with altered adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in rat aorta. Unstimulated intact or denuded guinea-pig trachea also did not affect the levels of cyclic AMP or cyclic GMP in rat aorta. 3. Methylene blue (10 microM) abolished the methacholine-induced, endothelium-derived relaxing factor (EDRF)-mediated rise in intracellular cyclic GMP in rat endothelium-intact aorta alone. In contrast, methylene blue (10 microM) did not affect the methacholine-induced epithelium-dependent rise in intracellular cyclic GMP in rat endothelium-denuded aorta in the co-axial bioassay. 4. Relaxation of the rat aorta without endothelium was associated with increased levels of cyclic GMP (but not cyclic AMP) in response to sodium nitroprusside (5 nM) and of cyclic AMP (but not cyclic GMP) in response to isoprenaline (1 microM). 5. These results provide evidence that the postulated epithelium-derived inhibitory factor (EpDIF) may produce relaxation of vascular tissue via elevation in cyclic GMP levels. Furthermore, some data suggest that EpDIF may act by stimulation of the particulate, rather than the soluble form of guanylate cyclase.


The 5-HT4 receptor subtype inhibits K+ current in colliculi neurones via activation of a cyclic AMP-dependent protein kinase. 1. The aim of the present study was to examine the effect of 5-hydroxytryptamine (5-HT) on K+ current in primary culture of mouse colliculi neurones and to identify the 5-HT receptor subtype that could be involved in this effect. 2. The voltage-activated K+ current of the neurones was partially blocked by 8-bromo adenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP). This effect was mimicked by 5-HT and the action of 5-HT could be antagonized by H7, a non specific protein kinase inhibitor, and by PKI, the specific cyclic AMP-dependent protein kinase blocker. 3. A similar cyclic AMP-dependent blockade of the K+ current was found with renzapride (BRL 24,924) and other 5-HT4 receptor agonists such as cisapride, BIMU 8, zacopride and 5-methoxytryptamine (5-MeOT). ICS 205,930, the classical 5-HT4 receptor blocker, could not be used in this study because it inhibited the studied K+ current by itself. However, the novel 5-HT4 receptor antagonist, DAU 6285 blocked the effects of 5-HT and renzapride on the K+ current. 4. The current was insensitive to the 5-HT1 and 5-HT3 receptor agonists (8-hydroxy-2-(di-n-propylamino) tetralin, RU 24,969, carboxamidotryptamine, 2-CH3-5-HT) as well as to 5-HT1, 5-HT2 and 5-HT3 antagonists (methiothepin, ketanserin, ondansetron [GR 38,032]). Moreover, these antagonists did not affect the actions of the tested 5-HT4 receptor agonists. 5. The present results show that part of the voltage-activated K+ current in mouse colliculi neurones is cyclic AMP-sensitive and the blockade of the current by 5-HT involves the 5-HT4 receptor subtype.The putative implication of 5-HT4 receptors in neuronal plasticity, via a blockade of K+ channels, is discussed.


Inhibitory effects of certain enantiomeric cannabinoids in the mouse vas deferens and the myenteric plexus preparation of guinea-pig small intestine. 1. The psychoactive cannabinoids (-)-delta 9-tetrahydrocannabinol ((-)-delta 9-THC) and the 1,1-dimethyl-heptyl homologue of (-)-11-hydroxy-delta 8-tetrahydrocannabinol ((-)-DMH) both inhibited electrically-evoked contractions of the mouse isolated vas deferens and the myenteric plexus-longitudinal muscle preparation of the guinea-pig small intestine. 2. Concentrations of (-)-delta 9-THC and (-)-DMH that decreased twitch heights by 50% were 6.3 and 0.15 nM respectively in the mouse vas deferens and 60 nM and 1.4 nM respectively in the myenteric plexus preparation. (-)-DMH was about 40 times more potent than (-)-delta 9-THC in both preparations, supporting the notion that their mode of action in each tissue is the same. 3. The psychically inactive cannabinoid, (+)-DMH, had no inhibitory effect in the mouse vas deferens at a concentration of 30 nM, showing it to be at least 1000 times less potent than (-)-DMH. In the myenteric plexus preparation, (+)-DMH was about 500 times less potent than its (-)-enantiomer. 4. The inhibitory effects of sub-maximal concentrations of (-)-delta 9-THC were not attenuated by 300 nM naloxone. 5. The findings that (-)-delta 9-THC and (-)-DMH are highly potent as inhibitors of the twitch response of the mouse vas deferens and guinea-pig myenteric plexus preparation and that DMH shows considerable stereoselectivity suggest that the inhibitory effects of cannabinoids in these preparations are mediated by cannabinoid receptors.


Studies on the cardiac actions of flosequinan in vitro. 1. We have investigated the in vitro cardiac actions of flosequinan and of its major metabolite in man, BTS 53554. 2. Positive inotropic activity was seen with flosequinan in guinea-pig isolated ventricles, the threshold concentration for effect being less than 1 x 10(-5) M. BTS 53554 was approximately half as potent as the parent compound. 3. In guinea-pig working whole hearts flosequinan increased left ventricular dp/dtmax, indicating a positive inotropic action. This effect was accompanied by increases in heart rate, cardiac output and stroke volume. 4. The virtual complete inhibition of inotropic responses to flosequinan and BTS 53554 by carbachol suggests that these responses are adenosine 3':5'-cyclic monophosphate (cyclic AMP)-mediated. 5. Flosequinan was shown to increase calcium inward current in guinea-pig ventricle, an action consistent with a cyclic AMP involvement in the response. 6. The inotropic activity of flosequinan was not potentiated by the selective phosphodiesterase (PDE) III inhibitor SK&F 94120, a result which indicates that flosequinan does not increase cyclic AMP concentrations via stimulation of adenylate cyclase. 7. Flosequinan inotropic responses were potentiated by rolipram, a selective PDE IV inhibitor, a result consistent with flosequinan being itself a PDE III inhibitor. 8. Biochemical studies with purified enzymes confirmed that flosequinan and BTS 53554 are relatively selective inhibitors of PDE III. 9. A comparison of pharmacological and biochemical data for both flosequinan and BTS 53554 indicates that their PDE III inhibitory potency is sufficient to account for their inotropic activity.


Comparative effects of BRL 38227, nitrendipine and isoprenaline on carbachol- and histamine-stimulated phosphoinositide metabolism in airway smooth muscle. 1. The ability of BRL 38227 and nitrendipine to affect muscarinic agonist and histamine-stimulated [3H]-inositol phosphate accumulation in slices of bovine tracheal smooth muscle has been studied and compared with the established inhibitory effects of isoprenaline on this pathway. 2. Pre-addition of BRL 38227 (5 microM), nitrendipine (1 microM) or isoprenaline (10 microM) significantly inhibited the subsequent inositol phosphate response to histamine at all concentrations studied (10- 1000 microM). BRL 38227 and nitrendipine also significantly inhibited the [3H]-inositol phosphate response to low (1 microM), but not high (100 microM) concentrations of carbachol. Isoprenaline had no effect at any concentration of carbachol studied. 3. Nitrendipine (IC50 = 95 nM) and BRL 38227 (IC50 = 322 nM) caused concentration-related inhibitions of the inositol phosphate response to histamine (100 microM). Similar maximal inhibitions were caused by each agent (55-58%). Inhibitory effect of BRL 38227 was reduced in potency (IC50 = 5.5 microM), but not magnitude, in the presence of glibenclamide (0.5 microM). 4. Time-course studies comparing the effects of BRL 38227 addition 15 min before, and 10 min after histamine challenge showed that for pre-addition a distinct (less than 2 min) lag occurred following histamine addition before the inhibitory effect of BRL 38227 was manifest. In contrast, when BRL 38227 was added 10 min after histamine, an inhibitory effect was immediately apparent. 5. Further evidence for an initial, 'protected' phase of inositol phosphate accumulation was provided by the finding that BRL 38227 pre-addition had no effect on the early (0-300 s) time-course of inositol 1,4,5-trisphosphate mass accumulation. 6. The inhibitory effect of BRL 38227, but not that of nitrendipine or isoprenaline, on histaminestimulated [3H]-inositol phosphate accumulation was completely prevented in the presence of an elevated extracellular K+ (65 mM) concentration. 7. The results demonstrate that membrane hyperpolarization, and/or blockade of voltage-operated Ca2"-channels can regulate agonist-stimulated phosphoinositide metabolism in airway smooth muscle. The possible contribution of this regulatory mechanism to the relaxant properties of these agents is discussed.


Imidazoline binding sites in human placenta: evidence for heterogeneity and a search for physiological function. 1. An alpha 2-adrenoceptor antagonist, idazoxan, that binds to both alpha 2-adrenoceptors and to imidazoline sites (IR), has been used to characterize human placental IR. Human placenta is shown to be the richest source of IR (1800 +/- 100 fmol mg-1 protein; Kd 38.9 +/- 3.4 nM). 2. Primary cells derived from human placenta and grown in monolayers, also displayed a high density of receptors (3209 +/- 136 fmol mg-1 in cytotrophoblasts and 3642 +/- 144 fmol mg-1 protein in syncytiotrophoblast enriched cell culture). 3. [3H]-idazoxan did not show binding characteristics of alpha 2-adrenoceptors in human placental membranes or human trophoblastic cells, thus making it a ligand of choice to study the imidazoline site. The tissue appeared to be lacking alpha 2-adrenoceptors in that other alpha 2-adrenoceptor ligands, [3H]-rauwolscine and [3H]-clonidine, do not bind to alpha 2-adrenoceptors in human placenta. 4. IRs are localized on the cell surface, as determined by the release of bound [3H]-idazoxan from cells, when washed with high ionic/acidic medium. 5. Imidazoline receptors of human placenta display high affinity for amiloride (72 +/- 27 nM). The high affinity was used as a criterion to classify IR to IRa subtype (placenta, rabbit kidney, rabbit liver and rabbit adipose cells) as opposed to the IRb subtype which display low affinity for amiloride (greater than 2 microM, in all the other tissues).6. Several novel ligands comprising a guanido functional group attached to an aromatic residue (e.g. benziliden-amino-guanidine (BAG), guanido pyrole) display pronounced selectivity for IR over the M2-adrenoceptors as the affinity of BAG is about 40 fold higher (Kd= 18.9 +/- 13.8 nM in human placenta), than the affinity for M2-adrenoceptors (Kd = 768 +/- 299 nM in human platelets). Imidazoline sites bind selectively BAG and other guanido ligands thus indicating a distinct structural requirement at its site of binding.7. K+ channel blockers and monovalent ions (e.g. Cs' and NH4+) interfere with idazoxan binding to IR, indicating a possible involvement of IR in K+ transport.


The effect of pertussis toxin on beta-adrenoceptor responses in isolated cardiac myocytes from noradrenaline-treated guinea-pigs and patients with cardiac failure. 1. A decreased responsiveness to the positive inotropic effects of beta-adrenoceptor agonists is a characteristic of human heart failure. We have investigated the involvement of inhibitory guanine nucleotide binding proteins (G-proteins) in this process using pertussis toxin treatment of isolated cardiac myocytes. 2. Myocytes isolated from failing human myocardium had a reduced maximum contractile response to isoprenaline relative to that for maximally stimulating concentrations of calcium, giving an isoprenaline/calcium ratio of 0.71 +/- 0.06 (n = 7 patients). This was significantly lower than in myocytes from non-failing myocardium, where the isoprenaline/calcium ratio was 1.16 +/- 0.07 (n = 6, P less than 0.001). Responses to high calcium were unchanged. 3. Myocytes were treated with pertussis toxin for 3-5 h at 35 degrees C. Successful inactivation of inhibitory G-proteins by pertussis toxin treatment was indicated by a loss of responsiveness to 10 microM adenosine (in the presence of submaximal isoprenaline). 4. Following pertussis toxin treatment of the myocytes from failing human heart the isoprenaline/calcium ratio increased to 1.43 +/- 0.27 (n = 7, P less than 0.05). Pertussis toxin treatment had no effect upon the maximum calcium contraction. The isoprenaline/calcium ratio in myocytes from non-failing human ventricle was not affected by the toxin treatment (n = 3). These observations support the hypothesis that increased inhibitory G-protein levels or activities contribute to beta-adrenoceptor desensitization in human heart failure. 5. beta-Adrenoceptor desensitization in human heart failure is thought to be secondary to raised noradrenaline levels in these patients. Experiments were repeated on myocytes isolated from hearts of guinea-pigs which had been chronically infused with noradrenaline. The isoprenaline/calcium ratio of these myocytes was reduced below that of myocytes from sham-operated animals (0.65 0.04, n = 6 compared with 0.88 +/- 0.02, n = 7, P<0.001).6. Pertussis toxin treatment (2 h at 35 degrees C) increased the isoprenaline/calcium ratio to 1.02 0.02 (n = 6,P<0.01) in myocytes from noradrenaline-treated guinea-pigs. This effect of pertussis toxin treatment was not seen in myocytes from sham-operated guinea-pig hearts.7. Incubation at 35 degrees C for similar periods in the absence of pertussis toxin also restored the isoprenaline/calcium ratio towards normal in the myocytes from both failing human and noradrenaline-treated guinea-pig hearts, although the effect was significantly smaller than that produced by the toxin. Myocytes kept at room temperature (22 degrees C) showed no such evidence of resensitization over periods up to 6h.8. These observations are consistent with the hypothesis that raised catecholamine levels result in increased inhibitory G-protein levels and functional P-adrenoceptor desensitization in heart failure.


Modulation of vascular tone by endothelin-1: role of preload, endothelial integrity and concentration of endothelin-1. 1. Endothelin-1 (ET-1) has been shown to exert both arterial relaxant and constrictor effects. To examine the mechanisms of these divergent effects, rat aortic rings were suspended in an organ bath (baseline preload, 5 g) and exposed to ET-1 (10(-11) to 10(-7) M). ET-1 contracted these rings in a concentration-dependent fashion. 2. When aortic rings were contracted with noradrenaline (NA) to 1 g of tension, ET-1 caused further contraction of these rings. In rings precontracted to 2 to 4 g of tension, low concentrations of ET-1 (10(-11) to 10(-9) M) caused a significant relaxation, but high concentrations (greater than or equal to 5 x 10(-9) M) caused a marked contraction, indicating both relaxant and contractile effects of ET-1 depending on the preload and ET-1 concentration. 3. To determine the mechanism of ET-1-induced relaxation, aortic rings were pretreated with the cyclo-oxygenase inhibitor indomethacin, NG-monomethyl-L-arginine (L-NMMA) an inhibitor of synthesis of endothelium-derived relaxing factor (EDRF), or oxyhaemoglobin (Hb) which decreases the activity of EDRF, prior to their exposure to ET-1. Both indomethacin and L-NMMA markedly (P less than 0.01) attenuated ET-1-induced relaxation, whereas Hb totally abolished it. Removal of the endothelium from aortic rings also abolished ET-1-mediated relaxation. 4. The relaxant effect of ET-1 in NA-precontracted rings was associated with marked accumulation of guanosine 3':5'-cyclic monophosphate (cyclic GMP), whereas ET-1-induced contraction of quiescent rings was not. 5.In manually stretched rings (4 g of tension), ET-l caused only concentration-dependent contraction, but no cyclic GMP accumulation.6. Thus, ET-1 contracts rat aortic rings with intact endothelium and those which are passively stretched. However, stimulation of rat aortic rings with NA to modest tension alters the contractile effect of ET-1 to a potent relaxant effect. The ET-l-mediated relaxation in this setting appears to be endothelium-dependent and is related to release of both cyclo-oxygenase products and EDRF.


Adenosine A1-receptor stimulation of inositol phospholipid hydrolysis and calcium mobilisation in DDT1 MF-2 cells. 1. The effect of adenosine receptor-stimulation on inositol phospholipid hydrolysis and calcium mobilization has been investigated in the hamster vas deferens smooth muscle cell line DDT1 MF-2. 2. Adenosine receptor stimulation increased the accumulation of total [3H]-inositol phosphates in DDT1 MF-2 cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was N6-cyclopentyladenosine greater than 5'-N-ethylcarboxamidoadenosine greater than 2-chloroadenosine greater than adenosine. 3. The response to 2-chloroadenosine was antagonized by the antagonists 8-cyclopentyl-1,3-dipropylxanthine (KD 1.2 nM), PD 115,199 (KD 39 nM) and 8-phenyltheophylline (KD 31 nM). 4. The inositol phosphate response to 2-chloradenosine (10 microM) was not significantly altered when the extracellular Ca2+ ion concentration was reduced from 2.4 mM to 1.2 mM or 0.6 mM. Under calcium-free conditions, however, a reduced but still significant response to 2-chloroadenosine was evident (39 +/- 10% of the response in calcium-containing medium). 5. The 5-lipoxygenase inhibitor AA861 (10 and 100 microM) inhibited the inositol phosphate response to 2-chloroadenosine by 40 +/- 9% and 60 +/- 4% respectively. The cyclo-oxygenase inhibitor, indomethacin, however, was without significant effect at 1 microM. 6. 2-Chloroadenosine stimulated an increase in intracellular free Ca2+ ion concentration in fura-2 loaded DDT1 MF-2 cells in calcium-free medium containing 0.1 mM EGTA, which could be inhibited by the adenosine A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.1 microM). 7. These data suggest that adenosine A1-receptor stimulation results in inositol phospholipid hydrolysis and calcium mobilization from intracellular stores in DDT1 MF-2 cells.


Morphine 6-glucuronide: a metabolite of morphine with greater emetic potency than morphine in the ferret. 1. The emetic potencies of morphine and its metabolite morphine 6-glucuronide have been determined in the ferret by constructing dose-response curves for mean total retches and vomits for subcutaneous doses of 0.05 mg kg-1 to 5 mg kg-1. Morphine 6-glucuronide induced retching and vomiting at lower doses than morphine and at a maximal dose induced more retching and vomiting than morphine. 2. The emesis induced by both morphine and morphine 6-glucuronide was abolished by the preadministration of naloxone (0.5 mg kg-1 s.c.). 3. The 5-HT3 receptor antagonists granisetron and ondansetron (1 mg kg-1, s.c.) failed to abolish or reduce emesis induced by either compound. 4. At a high-dose (5 mg kg-1), morphine but not morphine 6-glucuronide failed to induce emesis and abolished the emesis induced by the cytotoxic drug, cyclophosphamide (200 mg kg-1, i.p.). 5. Preliminary pharmacokinetic studies of intravenous and subcutaneous morphine and morphine 6-glucuronide revealed that morphine 6-glucuronide accounts for less than 1% of the metabolic product of morphine in the ferret. Peak plasma levels of the two compounds after their subcutaneous administration were obtained within 10 min. The metabolic profile of morphine was not dose-dependent. There was no relationship between plasma level and emetic response for either compound.


Comparison of pyrrolidinyl and piperidinyl benzamides for their anticonvulsant activity and inhibitory action on sodium channel. 1. A pair of benzamide analogues containing a pyrrolidinyl or piperidinyl group was examined for their anticonvulsant activity against the electroshock-induced seizures in mice and the ability to block the voltage-gated Na channel in N1E-115 cells, in comparison with the prototype compound, U-54494A, (+/-)-cis-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-ben zam ide , a potent anticonvulsant and a Na channel blocker. 2. The pyrrolidinyl benzamide (U-49524E) was found to be effective against the electroshock-induced seizures (ED50 = 35 mg kg-1, i.p.) whereas the benzamide with a piperidinyl moiety (U-49132E) was inactive (ED50 greater than 100 mg kg-1). 3. Using whole-cell patch clamp techniques we found that U-49132E was several times less potent, with an IC50 of 396 microM as compared to 118 microM for U-49524E at the holding potential of -80 mV, and was much slower in blocking Na channels with a half-time of 10.7 +/- 1.1 min vs. 2.2 +/- 0.4 min for its counterpart. 4. Qualitatively, their general modes of interaction with Na channels were similar to each other and to that of U-54494A in that they interacted with the resting and slowly-inactivated states of the channels and exhibited a use-dependent inhibition because of a slow recovery from the inactivated state in the presence of the drugs. 5. Comparison of their physicochemical properties, shows the less potent and slowly acting U-49132E is more hydrophobic and bulkier than U-49524E, but has the same pKa. This suggests that the drugs approach the Na channel through a narrow and hydrophilic pathway.6. Overall, this study underscores the importance of inhibiting the Na channel to the anticonvulsant activity of the benzamide compounds.


Haemodynamic effects of a new dihydropyridine calcium entry blocker, S-12968-(-), in a rat model of cardiovascular calcium overload. 1. The haemodynamic effects of S-12968-(-), a new dihydropyridine calcium entry blocker (enantiomer of S-11568), were compared with those of the stereoisomer, S-12967-(+), nifedipine, and sodium nitroprusside. 2. A first experiment was performed in conscious, young male rats chronically implanted with femoral artery and vein cannula and repeated in rats previously treated with vitamin D3 and nicotine. Such treatment produces marked vascular calcium overload, especially of the compliance arteries, with no overt sign of toxicity as far as can be judged from the plasma profile. 3. In conscious rats the hypotensive effects of S-12968-(-), nifedipine and sodium nitroprusside were of similar potency. The falls in blood pressure produced by nifedipine and sodium nitroprusside were accompanied by reflex tachycardia which was less marked in the vascular calcium overload model. S-12968-(-) did not induce reflex tachycardia. S-12967-(+) increased blood pressure in both models. 4. A second experiment was performed in open-chest pentobarbitone-anaesthetized rats with electromagnetic flowprobes on the ascending aorta. In controls the falls in blood pressure produced by low doses (0.1 and 0.3 mg kg-1, i.v.) of S-12968-(-) were accompanied by falls in total peripheral resistance. The higher dose (1 mg kg-1, i.v.) of S-12968-(-) produced no change in total peripheral resistance, and in rats pretreated with vitamin D3 and nicotine, cardiac output fell. 5. In conclusion, S-12968-(-) appears to have a dual action and to lower blood pressure at higher doses at least in part by a cardiac effect. This phenomenon is more pronounced in rats pretreated with vitamin D3 and nicotine.6. S-12967-(+) resembles a calcium channel activator in this model.


(S)-homoquisqualate: a potent agonist at the glutamate metabotropic receptor. The synthetic quisqualate analogue, (S)-homoquisqualate was examined for activity at the glutamate metabotropic receptor, in relation to its ability to stimulate phosphoinositide hydrolysis in rat pup cerebro-cortical slices. The compound produced a robust increase in hydrolysis (EC50 = 50.2 +/- 1.6 microM), which, in common with responses to quisqualate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD), was antagonized uncompetitively by L-2-amino-3-phosphonopropionate (L-AP3). In contrast to quisqualate which exhibits low efficacy, (S)-homoquisqualate behaves as a full agonist at the metabotropic receptor.


Apparent block of K+ currents in mouse motor nerve terminals by tetrodotoxin, mu-conotoxin and reduced external sodium. 1. In mouse triangularis sterni nerve-muscle preparations, reduced extracellular Na+ concentrations and low concentrations of the Na+ channel blocking toxins tetrodotoxin (TTX, 18-36 nM) and mu-conotoxin GIIIB (0.4-2.0 microM) selectively decreased the amplitude of the component of perineural waveforms associated with nerve terminal K+ currents, without affecting the main Na+ spike. 2. Intracellular recording of endplate potentials (e.p.ps) and miniature endplate potentials (m.e.p.ps) from triangularis sterni preparations revealed that TTX and mu-conotoxin GIIIB depressed the evoked quantal release of acetylcholine without significant effects on m.e.p.p. amplitude, frequency or time constant of decay. 3. The apparent block of K+ current by low concentrations of TTX and mu-conotoxin is probably not a direct effect on K+ channels but results from a decrease in the passive depolarization of nerve terminals following blockade of a small proportion of axonal Na+ channels.


Differential inotropic effects of flosequinan in ventricular muscle from normal ferrets versus patients with end-stage heart failure. 1. In right ventricular papillary muscles from control ferrets, flosequinan (10(-7)-10(-4) M) produced a concentration-dependent positive inotropic effect (10(-5) M = 153 +/- 24, 10(-4) M = 198 +/- 44% increase in isometric tension; control tension = 100%; n = 11) associated with a corresponding increase in amplitude of the intracellular Ca2+ ([Ca2+]i) transient recorded with aequorin (10(-5) M = 133 +/- 11, 10(-4) M = 187 +/- 36% increase in [Ca2+]i transient; n = 11). 2. The positive inotropic effect of flosequinan in control ferret ventricular muscle was neither blocked by propranolol (6 x 10(-7) M), nor associated with the abbreviation of the [Ca2+]i transient and contraction that is typical of catecholamines. 3. Neither flosequinan (n = 12) nor BTS 53 554, its sulphone metabolite (n = 6) produced a positive inotropic effect or altered the time course of contraction in myocardium from the hearts of patients with end-stage failure. 4. In contrast to milrinone, which produces a positive inotropic effect via phosphodiesterase inhibition, the unresponsiveness of myopathic human myocardium to flosequinan was not restored after intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels were increased by prior treatment with forskolin (n = 13). 5. Taken together, these data indicate that flosequinan has a direct positive inotropic effect that is Ca(2+)-dependent, but independent of changes in intracellular cyclic AMP concentrations. 6. The positive inotropic effect may be species-dependent or altered by the presence of hypertrophy and/or heart failure. However, when used therapeutically in patients with severe heart failure, our data suggest that flosequinan should not adversely affect myocardial oxygen consumption through direct or catecholamine-mediated actions on the heart.


The effect of epithelium removal on leukotriene E4-induced histamine hyperresponsiveness in guinea-pig tracheal smooth muscle. 1. Removal of the epithelium resulted in a threefold increase in guinea-pig tracheal sensitivity to histamine without increasing the maximal response. 2. Preincubation of epithelially-denuded guinea-pig tracheal smooth muscle with leukotriene E4 (LTE4) in vitro increased the subsequent maximal response of the tissues to histamine. The sensitivity of the tissues to histamine was unaffected by LTE4 pretreatment. 3. Pretreatment of the epithelially-denuded tissues with the LTE4-analogue, 20-COOH LTE4, did not affect the maximal response to histamine. 4. LTE4 pretreatment increased the maximal response of the epithelially-denuded tissues to substance P (SP) but did not affect the maximal response to carbachol, KCl nor to the beta-adrenoceptor agonist, isoprenaline. 5. LTE4-induced airway histamine hyperresponsiveness was blocked by indomethacin (5 microM), GR32191 (3 microM) and atropine (1 microM). 6. Both LTE4 and U46619 pretreatment increased the contractile response of tracheal smooth muscle to electrical field stimulation. 7. It is proposed that LTE4 induces an increased maximal response of epithelially-denuded guinea-pig airway smooth muscle to both histamine and substance P via a facilitation of cholinergic neurotransmission, which is dependent upon the secondary generation of prostanoid mediator(s) acting on TP-receptors situated on cholinergic nerve terminals. Further, it is suggested that the increased maximal response of the epithelially-intact tissues to both histamine and substance P, after LTE4 pretreatment, may be suppressed by an epithelially-derived factor.