Biomedical subjects

S Wray

Publications and source records attributed to S Wray.

At least 73 records · Page 4Linked to original sources

PubMed1997

Simultaneous measurements of electrical activity, intracellular [Ca2+] and force in intact smooth muscle.

Although both electrical activity and changes in intracellular [Ca2+] are known to be important determinants of smooth muscle force, little is known about the relationship between the three in intact muscle. This is due to a lack of simultaneous measurements of these parameters. In this paper we describe how we have combined the sucrose gap technique with microspectrophotometry and force recording in rat ureteric smooth muscle. We have investigated the timecourses of the changes in these parameters following physiological stimulation i.e. the action potential. The results of this paper show that it is possible to simultaneously measure electrical activity, [Ca] and force in intact smooth muscle and that (i) about a third of the change in Ca2+ occurs on the upstroke of the action potential and the remainder during the plateau. (ii) 50% of the decline in [Ca2+] occurs after the repolarization of the action potential and (iii) the kinetics of force development and relaxation are significantly slower than those of Ca2+, suggesting that [Ca2+] is not rate-limiting. These are the first such simultaneous measurements in any smooth muscle.

Action Potentials↗

PubMed1997

External alkalinization decreases intracellular Ca++ and spontaneous contractions in pregnant rat myometrium.

OBJECTIVES: As plasma pH rises during pregnancy, the effect of raising external pH on spontaneous contractions in pregnant rat myometrium was investigated to test the hypothesis that elevated external pH depresses contraction. STUDY DESIGN: Strips of longitudinal myometrium were loaded with SNARF (seminaphthorhodafluor) or Indo-1 for simultaneous intracellular pH or Ca++ and force measurements. Results were obtained from a minimum of five animals in each group, and significant differences were tested for by paired Student t tests. RESULTS: Raising the external pH significantly reduced spontaneous force and calcium transient in the pregnant uterus. Raising the external pH led to a slow rise in intracellular pH, but this could not account for the functional effect. K+ rubidium 86-labeled efflux rates were unaffected by external pH, suggesting no hyperpolarization. The Ca++ channel agonist Bay K8644 (5 mumol/L) restored contractions abolished by raised external pH. CONCLUSIONS: Raised external pH reduces spontaneous contractions in the pregnant rat uterus, probably by an external effect on Ca++ entry. This effect may contribute to uterine quiescence before term.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1997

Agonist mobilization of sarcoplasmic reticular calcium in smooth muscle: functional coupling to the plasmalemmal Na+/Ca2+ exchanger?

There is a close association of peripheral sarcoplasmic reticulum (SR), containing IP3 receptors, and regions of the plasma membrane enriched in the Na+/Ca2+ exchanger in smooth muscle. We have tested the possibility in rat uterine smooth muscle that Ca2+ released from the SR is preferentially removed from the cytosol by the Na+/Ca2+ exchanger. In Ca(2+)-free solution, carbachol stimulation of myometria of non-pregnant rats resulted in transient increases in [Ca2+]i and force due entirely to the release of SR Ca2+. Inhibition of Na+/Ca2+ exchange by removal of extracellular Na+ did not alter the agonist-induced transients suggesting that Na+/Ca2+ exchange was not involved in the removal of SR released Ca2+. However, in myometria of pregnant rats, Na+/Ca2+ exchange inhibition resulted in changes in the agonist-induced [Ca2+]i transient profiles. The peak amplitude, duration and integral of carbachol-induced [Ca+]i transients were enhanced in Ca(2+)-free/Na(+)-free solution without significantly affecting force transients. The lower rate of decay of [Ca2+]i transients in Na(+)-free solution leads us to suggest that up to 35% of the SR released Ca2+ may be extruded by the Na+/Ca2+ exchanger in myometria of pregnant rats. Thus, in uterine smooth muscle, there is a gestational-dependent coupling of SR releasable Ca2+ and plasmalemmal Na+/Ca2+ exchange activity.

Animals↗

PubMed1996

GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants.

In previous work, we showed a robust gamma-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic mice in vivo and study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures. In vivo, glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around -50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABAA) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl- conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pit in vivo.

Acetylcholine↗

PubMed1996

Cell-specific expression of the human gonadotropin-releasing hormone gene in transgenic animals.

We have previously demonstrated that 1131 base pairs (bp) of the human gonadotropin-releasing hormone (hGnRH) gene promoter can target simian virus 40 T antigen expression to GnRH neurons in transgenic mice. In these animals, GnRH neurons were transformed before they migrated to their final location in the rostral hypothalamus, complicating an analysis of cell-specific expression. To localize regions of the hGnRH promoter that are important for cell-specific expression, we created transgenic mice with various 5'-flanking regions of the hGnRH gene fused to the luciferase reporter gene. When 3828 or 1131 bp of the hGnRH promoter 5'-flanking DNA were used (-3828/+5LUC and -1131/+5LUC, respectively), luciferase expression in adult transgenic mice was observed in the rostral hypothalamus and olfactory tissues, regions which have been shown to be loci of GnRH-expressing neurons. Luciferase expression was not observed in other brain or peripheral tissues. Double-labeled in situ hybridization further demonstrated that luciferase expression was invariably colocalized with GnRH expression. When transgenic animals were created with a construct consisting of 484 bp of the hGnRH 5'-flanking DNA fused to the luciferase gene (-484/+5LUC), luciferase expression was not observed in the hypothalamus or in olfactory tissues. This is the first report localizing DNA sequences responsible for cell-specific expression of the GnRH gene in vivo.

Age Factors↗

PubMed1996

An investigation into the mechanism whereby pH affects tension in guinea-pig ureteric smooth muscle.

1. We have altered intracellular (pHi) and extracellular pH (pHo) in the smooth muscle of guinea-pig ureter and determined the effects on evoked phasic contractions. In order to investigate the mechanisms underlying the effects of pH alteration, intracellular Ca2+ ([Ca2+]i), pHi, electrical activity and force were measured. 2. Intracellular acidification, produced by the weak acid butyrate, application of CO2 at constant pHo or removal of weak bases, greatly increased phasic contractions. Alkalinization with weak bases or by removal of CO2 inhibited contractions. The results were similar whether Hepes or CO2-HCO3-buffered the solutions. 3. Phasic contractions were preceded by intracellular Ca2+ transients in the ureter. Acidification of the cytoplasm led to an increase in the amplitude of the Ca2+ transient, and alkalinization decreased its magnitude. 4. In the ureter the action potential leads to Ca2+ influx, therefore electrophysiological recordings of its configuration were made during alteration of pHi. Acidification led to the action potential duration and amplitude being increased, whereas alkalinization shortened the action potential and reduced its amplitude. 5. As the effects of acidification on the action potential resembled the effects of blocking of K+ channels, we investigated whether pHi alteration was able to alter tension when K+ channels were blocked by tetraethylammonium. Acidification was unable to potentiate force under these conditions nor did alkalinization decrease force. 6. External pH over the range 6.8-8.0 had little or no effect on pHi, phasic contractions and [Ca2+]i. Tonic contractions were enhanced, however, when pHo was increased. 7. These data suggest that pHi alteration in the guinea-pig ureter modulates the action potential, probably by alteration of K+ currents. Subsequent changes in [Ca2+]i and contraction then occur. A potentiating effect of acidic pH on force is not common in muscle, but may be a characteristic of the smooth muscle of the urinary tract. Changes of pHo had little effect on phasic force or pHi, but modulated tonic contractions. The possible physiological significance of these results is discussed.

Action Potentials↗

PubMed1996

Simultaneous measurement of intracellular pH, calcium, and tension in rat mesenteric vessels: effects of extracellular pH.

In rat mesenteric vessels changes in external pH (pHo) alter tension. Changes in intracellular pH (pHi) and calcium ([Ca2+]i) have both been suggested to underlie the tension changes. As no simultaneous measurements of pHi and [Ca2+]i had been made, however, the relative importance and temporal relationship between pHi [Ca2+]i and tension were unknown. In order therefore to gain a clearer understanding of the mechanisms involved, we have made simultaneous measurements of these parameters using carboxy-SNARF and INDO-1. Raising pHo caused rises in pHi, Ca2+ and tension. The increases in pHi preceded increases in [Ca2+]i, which preceded the increases in tension. Similar but opposite effects were observed when pHo was decreased. We conclude that the change in [Ca2+]i upon alteration of pHo is subsequent to that of pHi.

Animals↗

PubMed1996

Identification of a novel protein containing two C2 domains selectively expressed in the rat brain and kidney.

We have isolated and characterized a rat brain cDNA clone which encodes a new protein of 474 amino acids in length which contains two C2 domains structurally homologous to those present in synaptotagmins. The overall amino acid identity in C2 domains between this protein and the synaptotagmins is 36-44%. This protein also contains 3 putative consensus sequences for phosphorylation by cAMP-dependent protein kinase. RNA blot hybridization revealed a 3.0 kb transcript abundantly expressed only in the rat brain and the kidney. Thus, we called this brain/kidney protein (B/K). In situ hybridization and Northern blot analyses showed that the B/K transcript was found in forebrain including the olfactory bulb, cerebral cortex, hippocampus, and hypothalamus. In the kidney, high levels of B/K transcript were expressed in the papillary region of the inner medulla, the inner stripe of the outer medulla and the cortex. The selective expression in forebrain and kidney suggests that B/K may be involved in similar cAMP-dependent processes at these very different sites.

Amino Acid Sequence↗

PubMed1996

Stimulus-dependent modulation of smooth muscle intracellular calcium and force by altered intracellular pH.

Measurements of simultaneous force and intracellular Ca2+ concentration ([Ca2+]i) in rat uterine smooth muscle have been made to elucidate the mechanisms involved when force produced spontaneously, by high-K+ depolarization or carbachol is altered by a change of intracellular pH (pHi). Rises in force and [Ca2+]i were closely correlated for all forms of contraction, with the Ca2+ transient peaking before force. In spontaneously active preparations, alkalinization significantly increased, and acidification decreased, force and [Ca2+]i. Inhibition of the sarcoplasmic reticulum ATPase (cyclopiazonic acid) did not affect these changes, whereas removal of external Ca2+ abolished both responses, suggesting that the effect of pHi is on Ca2+ entry. Alkalinization caused a prolongation of the action potential complex, associated with a potentiation of contractile activity. Acidification produced hyperpolarization and abolition of action potentials and spontaneous activity, but did not prevent brief applications of carbachol or high-K+ from producing depolarization and increasing force, suggesting no impairment of the mechanism of generation of the action potential. For depolarized preparations, acidification increased tonic force and [Ca2+]i; the increase in the calcium signal persisted in zero-external calcium. In the presence of carbachol, acidification transiently increased force and [Ca2+]i, followed by a reduction in both. It is concluded that changes in pHi act at more than one step in excitation-contraction coupling and that changes in [Ca2+]i can account for most of the changes in uterine force.

Animals↗

PubMed1995

Major difference between rat and guinea-pig ureter in the ability of agonists and caffeine to release Ca2+ and influence force.

1. We have investigated the internal Ca2+ store and its ability to affect contraction by simultaneously measuring force and Ca2+ in the ureter from guinea-pig and rat. Both species responded in a similar manner to electrical stimulation and depolarization with high-K+, generating plateau-type action potentials and increasing intracellular calcium ([Ca2+]i) and force. 2. In the guinea-pig, carbachol had no effect on [Ca2+]i and force in the resting ureter. In contrast, resting rat ureter always responded with a large [Ca2+]i rise and maintained force to carbachol in Ca(2+)-containing solution, and in Ca(2+)-free solution it showed a transient increase in [Ca2+]i and force. This Ca2+ release and force development was also present in both polarized and high-K(+)-depolarized preparations and was insensitive to nifedipine, suggesting the presence of a receptor-coupled pathway of Ca2+ release in rat ureter. 3. Caffeine was able to produce a release of Ca2+ from the internal store of guinea-pig ureter and elicit contraction. However, rat ureter failed to respond to caffeine. In the presence of La3+, the caffeine response in the guinea-pig ureter and carbachol response in the rat ureter, elicited in Ca(2+)-free solutions, were always increased and prolonged and could be repeatedly evoked, suggesting similarity in Ca2+ uptake behaviour of the store in both species. 4. Ryanodine blocked the caffeine responses of the guinea-pig ureter elicited both in Ca(2+)-containing and Ca(2+)-free solutions, both in the absence and presence of La3+. However, ryanodine failed to prevent the rat ureter responding to carbachol, suggesting that carbachol was releasing Ca2+ from a ryanodine-insensitive channel in the sarcoplasmic reticulum (SR). 5. Cyclopiazonic acid, which inhibits the SR Ca(2+)-ATPase, abolished the effects of both caffeine and carbachol in Ca(2+)-free solutions in guinea-pig and rat, respectively. 6. We conclude that there is a major difference in the mechanisms of Ca2+ release in the internal Ca2+ store of smooth muscle from guinea-pig and rat ureter. The data suggest that the guinea-pig store is purely a calcium-induced calcium release (CICR)-type store and that the rat store is a pure receptor-operated Ca2+ store.

Animals↗

PubMed1995

The effects of extracellular pH and calcium change on force and intracellular calcium in rat vascular smooth muscle.

1. In order to investigate the mechanism whereby changes in external pH (pHo) alter tone in rat mesenteric resistance vessels, we have made simultaneous measurements of tension and intracellular Ca2+ [Ca2+]i. Strips of mesenteric artery were loaded with the Ca(2+)-sensitive indicator indo-1 and superfused with physiological salt solution at pH 7.4 and 37 degrees C. 2. An increase of pHo from 7.4 to 7.9 produced an increase in tension. This was accompanied by an increase in [Ca2+]i in resting and high-K(+)-depolarized vessels. Acidification to 6.9 reduced tension and was associated with a fall in [Ca2+]i. Over the pHi range examined, 6.6-7.9, parallel changes in [Ca2+]i and tension were found in K(+)-activated vessels. 3. In contrast to the relatively slow change in [Ca2+]i, pHi and tension with change of pHo, depolarization produced rapid changes in [Ca2+]i and tension, consistent with a more direct action on Ca2+ mobilization. 4. Reducing the external [Ca2+] below 1 mM produced a pronounced fall in [Ca2+]i and force. Changes in [Ca2+]i, produced by alteration of external [Ca2+] (Cao2+) were used to examine the relation between [Ca2+]i and tension. A linear relation was found. Alteration of pHo to 6.9 or 7.9 did not significantly change this relation. When the tension data were normalized to their own maxima, no shift in the tension-Ca2+ relation occurred, suggesting little or no effect of pH on the Ca2+ sensitivity of force production by the contractile proteins. 5. To determine further whether the changes in [Ca2+]i produced by alteration of pHo could account for all the changes observed in tension, [Ca2+]i was restored to control levels while maintaining an altered pHo. When this was done, restoration of [Ca2+]i led to restoration of force. Thus, in this preparation, the changes in [Ca2+]i produced by altering pHo in depolarized vessels can account for the changes in vascular tone.

Animals↗

PubMed1995

Electrical and synaptic properties of embryonic luteinizing hormone-releasing hormone neurons in explant cultures.

Voltage- and ligand-activated channels in embryonic neurons containing luteinizing hormone-releasing hormone (LHRH) were studied by patch-pipette, whole-cell current and voltage clamp techniques. LHRH neurons were maintained in explant cultures derived from olfactory pit regions of embryonic mice. Cells were marked intracellularly with Lucifer yellow following recording. Sixty-two cells were unequivocally identified as LHRH neurons by Lucifer yellow and LHRH immunocytochemistry. The cultured LHRH neurons had resting potentials around -50 mV, exhibited spontaneous discharges generated by intrinsic and/or synaptic activities and contained a time-dependent inward rectifier (Iir). Voltage clamp analysis of ionic currents in the LHRH neuron soma revealed a tetrodotoxin-sensitive Na+ current (INa) and two major types of K+ currents, a transient current (IA), a delayed rectifier current (IK) and low- and high-voltage-activated Ca2+ currents. Spontaneous depolarizing synaptic potentials and depolarizations induced by direct application of gamma-aminobutyrate were both inhibited by picrotoxin or bicuculline, demonstrating the presence of functional gamma-aminobutyrate type A synapses on these neurons. Responses to glutamate were found in LHRH neurons in older cultures. Thus, embryonic LHRH neurons not yet positioned in their postnatal environment in the forebrain contained a highly differentiated repertoire of voltage- and ligand-gated channels.

Action Potentials↗

PubMed1995

A 31P NMR investigation into the effects of repeated vascular occlusion on uterine metabolites, intracellular pH and force, in vivo.

Little is known about the metabolic effects of ischaemia on high energy phosphates in vivo in smooth muscle. We have developed a method for reversibly occluding the uterine artery, which allows simultaneous measurement of uterine metabolites using 31P NMR spectroscopy, and intra-uterine pressure, in vivo during ischaemia. We have investigated the effects of repeated ischaemia on metabolites, intracellular pH and contractions in anaesthetized rats. Occlusion produced an immediate drop in uterine blood flow and decreased contractions. Although contractions recovered upon reperfusion after both occlusions, the contractile activity was less after the second period of occlusion, suggesting less resistance after a prior ischaemic period. Significant falls in [ATP] and [phosphocreatine] and an increase in [P(i)] occurred during both occlusions. These were all reversed within 30 min of reperfusion. There was a large drop in intracellular pH produced by occlusion, which was rapidly reversed upon reperfusion. The changes in metabolites and intracellular pH were similar during the repeated ischaemic period, to those occurring during the first ischaemic period suggesting no alteration in energy production or utilization had occurred, with prior exposure to ischaemia. The significance of these results to the functioning of the uterus in labour is briefly discussed.

Adenosine Triphosphate↗

PubMed1995

The effect of metabolic inhibition on rat uterine intracellular pH and its role in contractile failure.

Although intracellular pH (pHi) is expected to change during inhibition of oxidative phosphorylation and to affect force, there have been no simultaneous measurements of its effect on pHi and force in smooth muscle. Therefore, we have investigated the relationship between force and pHi in strips of longitudinal rat myometrium, loaded with the pH-sensitive indicator carboxy-SNARF and simultaneously measured tension. The application of cyanide produced an abolition of spontaneous contractions and a rapid initial fall in pHi. In 9/19 preparations pHi then started to return to resting values but there was no corresponding restoration of force. Cyanide and weak base were simultaneously added to uterine preparations to prevent any acidification; force still fell. Addition of cyanide to depolarized preparations also produced an acidification and a fall in force. Depolarization of preparations in which spontaneous force had been abolished by cyanide often produced a transient rise in force, despite further acidification of the cytoplasm. Cyanide produced an acidification in zero Ca(2+)-containing solution, similar to that in the presence of Ca2+ indicating little role for changes in [Ca2+] in producing the acidification. It is concluded that cyanide decreases pHi and force in the uterus, but that there is not a simple relationship between the two.

Animals↗

PubMed1995

An investigation of intrinsic buffering power in rat vascular smooth muscle cells.

Intrinsic buffering power (beta i) has been measured in vascular strips and single cells from rat mesenteric artery. Intracellular pH (pHi) regulation was inhibited to prevent overestimation of beta i due to acid extrusion or entry via regulatory processes. At resting values of pHi (7.0-7.2), a mean value of 41 +/- 4 mM/pH unit for beta i was found. beta i increased approximately fivefold from 30 to 150 mM/pH unit over the pHi range 7.5-6.5. The mean data relating beta i to pHi could be described by relating beta i to buffer concentrations and pKa. This gave a value of 310 mM for buffer concentration and a pKa of 6.0. As changes in pHi are known to have marked effects on vascular tone then the increase in beta i as pHi falls may be considered as a means of attenuating pHi decreases, before pH regulation restores pHi to resting levels.

Animals↗

PubMed1995

Changes of pH affect calcium currents but not outward potassium currents in rat myometrial cells.

Spontaneous contraction of uterine smooth muscle is enhanced by alkalinization and depressed by acidification. We have investigated the ionic currents responsible for this in single myometrial cells. Intracellular acidification (20 mM butyrate) at constant external pH depressed the magnitude of the calcium current to 58+/-6% of control, but had little effect on outward currents. Similar but slower effects were also observed when the extracellular pH was lowered to 6.9 (56+/-9% of control). Correspondingly, when the intracellular or extracellular pH was elevated (20 mM NH4Cl or pH 7.9 respectively) the calcium current magnitude increased (165+/-15% in NH4Cl; 136+/-2% at pH 7.9) and there was, again, no effect on the outward currents. These observations are consistent with the effects of pH on spontaneous contractile activity being due to an effect on the membrane calcium current.

Animals↗

PubMed1995

Changes in intracellular pH close to term and their possible significance to labour.

The contractile pattern of uterine smooth muscle is markedly altered by even modest manipulations of intracellular pH (pHi); an acidification can abolish contractions, while alkalinization increases contractility. As at the end of gestation the uterus changes from being relatively quiescent to highly contractile, we investigated whether pHi changes with pregnancy in women. Isolated tissue was loaded with the pH-sensitive fluorophore, carboxy-SNARF. We found a significant alkalinization over the last few weeks of pregnancy. Such an increase in pHi will increase contractility of the uterus, and may therefore contribute to the mechanisms ensuring strong and efficient contractions occur during labour.

Adult↗

PubMed1995

Molecular characterization of a neuronal-specific protein that stimulates the activity of Cdk5.

Cyclin-dependent kinase, Cdk5, has been identified in neural tissue in connection with neurofilament and tau protein phosphorylation. This report describes the characterization of a 62-kDa protein that copurifies with Cdk5 from rat spinal cord homogenates. Dissociation of the protein from neural Cdk5 is concomitant with a reversible loss in kinase activity. Amino acid sequence information from tryptic peptide fragments was used to clone the complementary DNA from rat brain. A single full-length cDNA was characterized coding for a 67.5-kDa protein (p67). Exogenously expressed p67 stimulated Cdk5 kinase activity in vitro in a dose-dependent manner and when presented as an affinity matrix, selectively adsorbed Cdk5 from a cleared rat brain homogenate. In situ hybridization analysis of E18 rat embryos and adult rat brain demonstrated that p67 transcript expression is restricted to neural tissue. Immunohistochemical staining with an amino-terminal peptide-specific antibody further indicated that p67 is exclusively expressed in neurons. Localization in vivo and in cultured rat hippocampal neurons showed that p67 is highly enriched in axons. We propose that p67, by virtue of its regulation of Cdk5, participates in the dynamics of axonal architecture through the modulation of phosphorylation of cytoskeletal components.

Amino Acid Sequence↗