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S Wray

Publications and source records attributed to S Wray.

At least 55 records · Page 3Linked to original sources
PubMed1998

GABA inhibits migration of luteinizing hormone-releasing hormone neurons in embryonic olfactory explants.

During development, a subpopulation of olfactory neurons transiently expresses GABA. The spatiotemporal pattern of GABAergic expression coincides with migration of luteinizing hormone-releasing hormone (LHRH) neurons from the olfactory pit to the CNS. In this investigation, we evaluated the role of GABAergic input on LHRH neuronal migration using olfactory explants, previously shown to exhibit outgrowth of olfactory axons, migration of LHRH neurons in association with a subset of these axons, and the presence of the olfactory-derived GABAergic neuronal population. GABAA receptor antagonists bicuculline (10(-5) M) or picrotoxin (10(-4) M) had no effect on the length of peripherin-immunoreactive olfactory fibers or LHRH cell number. However, LHRH cell migration, as determined by the distance immunopositive cells migrated from olfactory pits, was significantly increased by these perturbations. Addition of tetrodotoxin (10(-6) M), to inhibit Na+-transduced electrical activity, also significantly enhanced LHRH migration. The most robust effect observed was dramatic inhibition of LHRH cell migration in explants cultured in the presence of the GABAA receptor agonist muscimol (10(-4) M). This study demonstrates that GABAergic activity in nasal regions can have profound effects on migration of LHRH neurons and suggests that GABA participates in appropriate timing of LHRH neuronal migration into the developing brain.

Animals↗
PubMed1998

The mechanism of Ca2+ release from the SR of permeabilised guinea-pig and rat ureteric smooth muscle.

Recent work has indicated that there is a major difference in the Ca2+ store of smooth muscle from rat and guinea-pig ureter; with the rat store being agonist-sensitive but ryanodine insensitive and the guinea-pig store being ryanodine sensitive but agonist insensitive [Th. V. Burdyga, M.J. Taggart, S. Wray, J. Physiol. 489 (1995) 327-335]. We have therefore examined directly the mechanism of Ca2+ release from the internal Ca2+ store (SR). Following permeabilisation with alpha-toxin or beta-escin the SR was Ca(2+)-loaded before application of carbachol or caffeine. Only carbachol evoked a transient contraction in rat ureter. The carbachol-induced contraction was blocked by heparin and cyclopiazonic acid (CPA) but not ryanodine. Only caffeine produced contraction in guinea-pig ureter, and this was blocked by ryanodine. Direct application of IP3 caused a small transient contraction in rat but not guinea-pig ureter. We conclude that rat ureter possesses only an IP3 sensitive store while guinea-pig ureter only has a ryanodine sensitive store.

Animals↗
PubMed1998

The effects of metabolic inhibition on force, Ca2+ and pHi in guinea-pig ureteric smooth muscle.

The effect of metabolic inhibition on the contractile function of adult guinea-pig ureter has been investigated. Strips of ureteric smooth muscle were loaded with Indo-1 or SNARF to measure intracellular [Ca2+] ([Ca2+]i) or pH (pHi) simultaneously with force. Inhibiting oxidative phosphorylation with cyanide rapidly reduced phasic contractions and the associated Ca2+ transients, after initial transient increases. The effects of cyanide were reversible and related to the amount of contractile activity undertaken. Inhibition of glycolysis with iodoacetate abolished all force. In high-K+-depolarised preparations, cyanide reduced the tonic contraction, but this was not accompanied by a reduction in [Ca2+]i, suggesting a desensitisation of the myofilaments. Cyanide produced a fall in pHi, which may underlie the initial transient increase in force. These data suggest that metabolic inhibition reduces force in the ureter by affecting both excitation and hence the Ca2+ transient, and at the myofilaments to reduce their sensitivity to Ca2+. Thus when oxidative metabolism is impaired contractile dysfunction may arise in the ureter.

Animals↗
PubMed1998

The effects of changing intracellular pH on calcium and potassium currents in smooth muscle cells from the guinea-pig ureter.

Guinea-pig ureteric smooth muscle is unusual in that intracellular acidification increases and alkalinization decreases force production. To help elucidate the mechanism underlying these effects on force we have investigated the effects of changing intracellular pH on both calcium and potassium currents in single cells isolated from the guinea-pig ureter to determine their possible role in force development. Depolarization to +40 mV resulted in a fast transient outward current which was inhibited by 4-aminopyridine but not tetraethylammonium. Intracellular alkalinization (20 mM trimethylamine) increased this current to 179 +/- 24% of the control and resulted in the development of a slowly activating large outward current which was inhibited by tetraethylammonium and washout. Acidification (40 mM sodium butyrate) decreased the fast transient outward current to 58 +/- 3% of the control and did not produce a slowly activating current. When potassium was replaced by caesium in the pipette solution, depolarization to 0 mV resulted in an inward calcium current which was abolished by nifedipine. Intracellular alkalinization increased this current to 126 +/- 11% of the control whereas acidification had the opposite effect, decreasing it to 55 +/- 10%. Furthermore, current-clamp experiments showed that intracellular alkalinization inhibited the amplitude of the action potential, therefore decreasing excitability of the cell. From our results, we suggest that the predominant effects of intracellular pH on force production in the guinea-pig ureter are mediated via the modulation of outward potassium currents (thereby reducing excitability of the tissue) rather than the effects on the inward calcium current.

Animals↗
PubMed1998

Intracellular Na+ measurements in smooth muscle using SBFI--changes in [Na+], Ca2+ and force in normal and Na(+)-loaded ureter.

Our understanding of the control and effects of intracellular [Na+] ([Na+]i) in intact smooth muscle is limited by the lack of data concerning [Na+]i. The initial aim of this work was therefore to investigate the suitability of using the Na+-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na+]i in ureteric smooth muscle. Resting [Na+]i was found to be around 10 mM and rose to 25 mM when the Na+-K+-ATPase was inhibited by ouabain. This relatively low [Na+]i in the absence of Na+-K+-ATPase suggests that other cellular processes, such as Na+-Ca2+ exchange, play a role in maintaining [Na+]i under these conditions. Simultaneous measurements of [Na+]i or [Ca2+] i and force showed that Na+-Ca2+ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na+ exit and hence Ca2+ entry, the larger the contraction. In addition the Na+-Ca2+ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca2+] and [Na+]i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na+-Ca2+ exchange may play a functional role in the ureter.

Animals↗
PubMed1998

Developmental changes in intracellular pH buffering power in smooth muscle.

Intracellular pH (pHi) is known to modulate contraction. Neonatal tissues can differ from adult tissue in contractile response to stimuli known to alter pHi e.g. hypoxia. Changes of pH are attenuated by buffering, thus any difference in buffering power (beta) between tissues could affect their functional response to pHi perturbation. Similarly the extent to which any extracellular pH (pHo) alteration is transmitted into a pHi change will also influence function. We have therefore determined the intrinsic beta and effect of pHo change on pHi in neonatal and adult ureteric, uterine and gastric smooth muscles using the pH-sensitive fluorophore carboxy-SNARF. beta was found to be similar in the three adult tissues, but there were significant differences between neonatal tissues. In contrast, we found little difference in the amount of pHi change produced by pHo change between neonatal and adult tissues from the same smooth muscle, but a difference between smooth muscles. These data highlight significant differences between smooth muscles and their developmental state, which may contribute to different degrees of protection when pH is perturbed.

Animals↗
PubMed1998

The in vivo relationship between blood flow, contractions, pH and metabolites in the rat uterus.

Little is known about the relationship between smooth muscle contractile activity and its blood supply. We have therefore investigated this in the rat uterus, using laser-Doppler flow measurement and intra-uterine pressure recordings. We found an inverse linear relationship between flow and contractile activity. There was no evidence for a critical level of flow, above which function is maintained and below which it declines; even small reductions in blood flow decreased uterine force. Force was rapidly restored upon reperfusion. Reactive hyperaemia was absent from all but 6 of the 41 preparations studied. We used 31P nuclear magnetic resonance (NMR) spectroscopy to measure concentrations of adenosine triphosphate (ATP), phosphocreatine (PCr), inorganic phosphate (Pi) and intracellular pH (pHi) simultaneously with force and flow. Reductions in flow were associated with significant reductions in [ATP], [PCr] and pHi, and an increase in [Pi]. These changes were related to flow significantly and linearly and their effects on force may be additive. These data show that uterine smooth muscle is closely dependent upon its blood supply for maintaining both normal force production and metabolite levels. Consequently, even small decrements in flow may have deleterious functional effects.

Adenosine Triphosphate↗
PubMed1998

Developmental and species differences in the response of the ureter to metabolic inhibition.

The effect of inhibiting oxidative phosphorylation on electrically stimulated phasic and high-K+ depolarization-induced tonic contractions in ureteric smooth muscle has been investigated. Intracellular [Ca2+] and pH were monitored fluorimetrically with simultaneous tension measurement, in adult and neonatal rat and guinea-pig ureter. Little difference was found in the response of adult or neonatal rat ureters; cyanide abolished phasic contractions and intracellular Ca2+ transients. The contractions of the adult guinea-pig ureter were also reduced by cyanide, but not as much as those of the adult rat. Neonatal guinea-pig was, however, remarkably resistant to the effects of cyanide, with force and Ca2+ transients remaining at control levels after an initial transient dip. These differences between tissues were not apparent when a high K+ concentration was used to depolarize tissues and produce maintained [Ca2+]i and force changes; cyanide reduced force but not [Ca2+]i in all preparations. Intracellular pH decreased in all preparations with inhibition of oxidative phosphorylation, but this did not correlate with changes in contraction. It is concluded that there are both species and developmental differences in the response to metabolic inhibition of the ureter which lead to differing changes in contractile activity.

Animals↗
PubMed1998

The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle.

To investigate the role of myosin light chain kinase (MLCK) in phasic contractions of intact smooth muscle, we have applied Wortmannin, an MLCK inhibitor, to strips of guinea-pig ureter. Simultaneous measurements of electrical activity, intracellular [Ca2+] ([Ca2+]i) and phasic force showed that Wortmannin (1-4 microM) abolishes force with little or no change in [Ca2+]i and electrical activity. High-K+-induced force production was also abolished by Wortmannin. The effects of Wortmannin were dose dependent - at lower concentrations (100 nM) Wortmannin reduced phasic contractility by 40-50%. It also significantly increased the delay between the Ca2+ peak and force production. These data show that, in phasic smooth muscle, inhibition of MLCK causes contraction to fail, despite normal electrical activity and Ca2+ transients. Our results also indicate that Wortmannin has no secondary effects and that other means of producing force, independent of myosin phosphorylation, are negligible in this tissue. The increased lag between the rise of Ca2+ and force production when MLCK is inhibited was surprising and suggests that post-phosphorylation steps may play a larger role in the delay than was previously considered.

Action Potentials↗
PubMed1998

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats.

In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca2+]i) is determined by Ca2+ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca2+ pump inhibitor, carboxyeosin, on the rate of the [Ca2+]i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca2+]i simultaneously with transmembrane calcium current (ICa). [Ca2+]i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca2+]i removal was calculated from the falling phase of the [Ca2+]i transient. Pre-treatment of the cells with 2 microM carboxyeosin led to a marked decrease in the rate of [Ca2+]i transient decay, suggesting that the sarcolemmal Ca2+ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca2+]i decay, indicating an important role for the Na+/Ca2+ exchange. When both the sarcolemmal Ca2+ pump and Na+/Ca2+ exchange were inhibited the cell failed to restore [Ca2+]i after the stimulation. Comparison of the rate constants of [Ca2+]i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca2+]i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na+/Ca2+ exchanger and the intracellular calcium stores.

Animals↗
PubMed1998

A review of the actions and control of intracellular pH in vascular smooth muscle.

OBJECTIVE: This review is an account of the physiological issues involved in the effects of pH on vascular smooth muscle tone. The following criteria were considered when reviewing the literature: (i) the type of smooth muscle, i.e. either tonic or phasic, (ii) the source of the smooth muscle i.e. pulmonary, systemic, large artery, resistance artery, vein or cell line, (iii) the effects of changing intracellular or extracellular pH alone, (iv) the acute or chronic effects of altered pH (v) the influence of extracellular pH on intracellular pH and (vi) the influence of altered intracellular pH on basal or agonist induced tone. Studies of the effects of pH on the individual intracellular components of vascular tone, specifically sarcoplasmic reticulum and contractile proteins function are considered. Finally, the pH sensitivity of molecular components that contribute to smooth muscle cell tone are reviewed. CONCLUSIONS: There appear to be distinct differences in the response of large arteries and resistance arteries to altered intracellular pH which may be based on the different properties of the smooth muscle within the wall of each blood vessel. Similarly, systemic and pulmonary vessels may respond differently, but no systematic study exists to allow a more definitive conclusion. Factors controlling intracellular pH such as intracellular buffering power and sarcolemmal pH regulating mechanisms may differ across the vascular bed and may contribute to some of the differences observed in response to altered extracellular pH. Finally, few studies have examined the pH sensitivity the intracellular processes involved in basal tone and pharmaco-mechanical coupling in vascular smooth muscle. More information concerning these latter aspects of smooth muscle function is required to progress the understanding of the modulator action on pH on vascular tone.

Arteries↗
PubMed1998

Luteinizing hormone releasing hormone (LHRH) neurons maintained in nasal explants decrease LHRH messenger ribonucleic acid levels after activation of GABA(A) receptors.

Inhibition of the LHRH system appears to play an important role in preventing precocious activation of the hypothalamic-pituitary-gonadal axis. Evidence points to gamma-aminobutyric acid (GABA) as the major negative regulator of postnatal LHRH neuronal activity. Changes in LHRH messenger RNA (mRNA) levels after alterations of GABAergic activity have been reported in vivo. However, the extent to which GABA acts directly on LHRH neurons to effect LHRH mRNA levels has been difficult to ascertain. The present work evaluates the effect of GABAergic activity, via GABA(A) receptors, on LHRH neuropeptide gene expression in LHRH neurons maintained in olfactory explants generated from E11.5 mouse embryos. These explants maintain large numbers of primary LHRH neurons that migrate from bilateral olfactory pits in a directed manner. Using in situ hybridization histochemistry and single cell analysis, we report dramatic alterations in LHRH mRNA levels. Inhibition of spontaneous synaptic activity by GABA(A) antagonists, bicuculline (10(-5) M) or picrotoxin (10(-4) M), or of electrical activity by tetrodotoxin (TTX, 10(-6) M) significantly increased LHRH mRNA levels. In contrast, LHRH mRNA levels decreased in explants cultured with the GABA(A) receptor agonist, muscimol (10(-4) M), or KCl (50 mM). The observed responses suggest that LHRH neurons possess functional pathways linking GABA(A) receptors to repression of neuropeptide gene expression and indicate that gene expression in embryonic LHRH neurons, outside the CNS, is highly responsive to alterations in neuronal activity.

Animals↗
PubMed1997

Luteinizing hormone-releasing hormone (LHRH) neurons maintained in hypothalamic slice explant cultures exhibit a rapid LHRH mRNA turnover rate.

Evidence indicates that neuropeptide gene expression is tightly coupled to biosynthesis and secretion. Moreover, rhythmic gene expression often accompanies rhythmic secretion. Luteinizing hormone-releasing hormone (LHRH) neurosecretion, which regulates gonadal function, is pulsatile, with interpulse intervals of approximately 1 hr and pulse decays of <30 min in rats. As a basis for a rapid fall in peptide secretion, we hypothesize that LHRH mRNA levels rapidly decay. To address this hypothesis, we examined LHRH mRNA turnover in primary postnatal LHRH neurons maintained in long-term hypothalamic/preoptic area slice explant cultures, using in situ hybridization histochemistry (ISHH). Relative LHRH mRNA content per cell was quantitated by single-cell analysis after transcription inhibition with 5, 6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) or actinomycin D. Cultures were maintained in serum-free medium with tetrodotoxin to suppress spontaneous electrical activity and hence assess only intrinsic cellular activity. A plot of LHRH mRNA level per cell versus DRB treatment time showed a rapid initial decay of LHRH mRNA (t1/2, 5-13 min), followed by a slower decay rate (t1/2, 329-344 hr). LHRH cell number after drug treatment as determined by immunocytochemistry did not change. Comparison of mammalian LHRH mRNA 3'-untranslated regions showed two conserved regions. These data indicate that, in primary LHRH neurons, LHRH mRNA has an intrinsically high rate of turnover and a mRNA stabilization component. Foremost, decay of LHRH mRNA, the fastest reported for a neuropeptide to date, corresponds to the decay of LHRH peptide pulses.

Animals↗
PubMed1997

The role of the sarcolemmal Ca(2+)-ATPase in the pH transients associated with contraction in rat smooth muscle.

1. We have investigated the origin of the intracellular acid pH transients that accompany myometrial contraction. Intra- and extracellular pH were measured with SNARF and intracellular Ca2+ concentration ([Ca2+]i) with indo-1. 2. An intracellular acidification accompanied spontaneous contractions and those elicited by KCl depolarization or the addition of the agonists carbachol or prostaglandin F2 alpha. The size of the acidification increased with the magnitude of the contraction. 3. The intracellular acidification was accompanied by an extracellular alkalinization, showing that it results from proton movement across the surface membrane. Furthermore, it was decreased either by addition of Cd2+ (20 nM, an inhibitor of the sarcolemmal Ca(2+)-ATPase) or by elevating [Ca2+]o. 4. Extracellular alkalinization increased the magnitude of the rise of [Ca2+]i and force produced by KCl. 5. An intracellular acidification was also associated with contraction in the portal vein and ureter. 6. We conclude that the sarcolemmal Ca(2+)-ATPase produces a significant intracellular acidification while removing Ca2+. Both the acidification and decrease of [Ca2+]i will promote relaxation. Since Ca2+ and protons have opposite effects on many cellular processes, this dual regulation by these two ions may be of general importance.

Animals↗
PubMed1997

Neuronal dopamine subpopulations maintained in hypothalamic slice explant cultures exhibit distinct tyrosine hydroxylase mRNA turnover rates.

Changes in mRNA stability have been shown to regulate critical intracellular processes. In this investigation, we studied tyrosine hydroxylase (TH) mRNA turnover in functionally and anatomically distinct dopaminergic (DA) populations of the rat hypothalamus. To this end, long-term slice explant cultures from postnatal, preoptic area/hypothalami, containing three anatomically discrete DA populations, were generated and maintained under defined conditions. The organotypic cultures were treated with the transcription inhibitors 5,6-dichloro-1-D-ribofuranosylbenzimidazole or actinomycin D and processed for in situ hybridization histochemistry. Relative TH mRNA content per cell was quantitated. Single-cell analysis showed marked differences in basal TH mRNA turnover rates between DA neuronal populations. Anterior and midhypothalamic DA neurons exhibited half-time turnovers of 9-12 and 11-23 hr, respectively. In contrast, in the caudal hypothalamus, DA neurons of the arcuate nucleus had a significantly lower baseline level and more rapid turnover (6-7 hr) of TH mRNA. This investigation shows that basal turnover of a phenotypic mRNA, TH mRNA in DA neurons, is not an intrinsic property of the phenotypic marker. Furthermore, we found that destabilization of TH mRNA in the caudal hypothalamus corresponds to the known rhythmic output displayed by arcuate DA cells and, as such, may be critical for normal function of this population. We propose that intrinsic differences in the post-transcriptional regulation of TH permits neuronal subpopulations, which subserve different physiological functions, an additional mechanism to control DA biosynthesis in response to their unique needs.

Animals↗
PubMed1997

An alternative gonadotropin-releasing hormone (GnRH) RNA splicing product found in cultured GnRH neurons and mouse hypothalamus.

Gonadotropin-releasing hormone (GnRH) is encoded by the proGnRH gene which contains four exons and three introns. In this study, two immortalized GnRH-expressing cell lines (Gn11 and NLT) were characterized. The NLT and Gn11 cells, derived from a same brain tumor in a transgenic mouse, display neuronal morphology and neuron-specific markers. However, NLT cells secrete much higher levels of GnRH than Gn11 cells. To delineate the mechanism underlying this difference, reverse transcriptase-polymerase chain reaction and RNase protection assays were performed to examine proGnRH gene expression. While the mature proGnRH mRNA was predominately expressed in NLT cells, Gn11 cells express an abundant short transcript. Sequence analysis revealed that this short transcript contains exons 1, 3, and 4, but not exon 2, which encodes the GnRH decapeptide. RNase protection assays demonstrated that NLT cells express much higher levels of mature proGnRH mRNA than Gn11 cells. The lower level of GnRH secreting capacity in Gn11 cells is due, in part, to decreased expression of mature proGnRH mRNA. When proGnRH gene expression in the mouse brain was examined, the same short splicing variant was observed in the olfactory area and preoptic area-anterior hypothalamus. But the prevalent transcript in these regions was the mature proGnRH mRNA. In contrast, only the mature proGnRH mRNA was found in the caudal hypothalamus. These results suggest that alternative splicing may be one of the mechanisms regulating proGnRH gene expression in the animal brain.

Alternative Splicing↗
PubMed1997

Effect of metabolic inhibition on intracellular Ca2+, phosphorylation of myosin regulatory light chain and force in rat smooth muscle.

1. The effect of the inhibition of oxidative phosphorylation on intracellular calcium concentration ([Ca2+]i), phosphorylation of the 20 kDa regulatory light chain of myosin (MLC20) and contractility was investigated in isolated longitudinal smooth muscle from rat uteri. 2. Cyanide (2 mM) application to normally polarized preparations resulted in an elevation of basal [Ca2+]i but an inhibition of [Ca2+]i transients and the accompanying contractions. 3. Depolarization with high-K+ solution (40 mM KCI) resulted in elevation of [Ca2+]i and maintained force production. Phosphorylation of MLC20 was transiently increased followed by a steady-state augmentation above resting levels. 4. Carbachol (100 microM) produced a transient elevation of [Ca2+]i and force of depolarized tissues followed by a steady-state augmentation of both parameters. PGF2 alpha (1 microM) did not significantly potentiate [Ca2+]i or force in depolarized preparations. Both carbachol and PGF2 alpha potentiated phosphorylation of MLC20 in depolarized tissues. 5. Addition of cyanide to depolarized preparations, in the presence or absence of carbachol or PGF2 alpha, resulted in significant attenuation of force under each condition. The magnitude and normalized rates of force inhibition by cyanide were not significantly different for each stimulus condition. MLC20 phosphorylation levels were unaltered by cyanide treatment. However, cyanide increased the maintained level of [Ca2+]i under each experimental protocol. 6. It is concluded that the inhibition of oxidative phosphorylation with cyanide results in dissociation of both the [Ca2+]i-force and MLC20 phosphorylation-force relationships in rat uterine smooth muscle.

Animals↗