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S Wray

Publications and source records attributed to S Wray.

At least 109 records · Page 6Linked to original sources
PubMed1993

Simultaneous measurement of intracellular pH and contraction in uterine smooth muscle.

Changes in intracellular pH (pHi) are thought to produce large changes in force production in the uterus. There have however, been no simultaneous measurements of pHi and force in the uterus and therefore no direct information is available about the relation between the two. We have used carboxy-SNARF (a pH-sensitive fluorophore) in small strips of longitudinal myometrium and obtained simultaneous measurements of pHi and force. SNARF did not alter contractile function, and continuous measurements of pHi could be made for 2 hours. The mean resting pHi (7.16) was similar to that reported previously. Application of weak bases rapidly raised pHi, in a concentration-dependent manner, followed by a gradual restoration of pHi to resting levels. Alkalinization greatly increased the frequency of contractions, often accompanied by a small increase in their amplitude. Removal of base produced a rebound acidification which transiently abolished contractions. Direct acidification of the cytoplasm, by application of weak acid, also abolished contractions. However the alkalinization which accompanied removal of acid, produced variable effects on force.

Animals↗
PubMed1993

Effects of metabolic inhibition and changes of intracellular pH on potassium permeability and contraction of rat uterus.

1. We have investigated the role of changes of potassium efflux in the inhibition of uterine force produced by cyanide. K+ efflux (86Rb) was measured from pregnant and non-pregnant rat myometrial strips during metabolic inhibition with cyanide and following manoeuvres to displace intracellular pH (pHi). 2. Cyanide greatly reduced or abolished spontaneous contractions. If the membrane was depolarized directly at this stage (by elevating external K+) then contraction redeveloped. This suggests that the initial depression of force is due to a failure of membrane excitation. 3. Cyanide reversibly increased 86Rb efflux (30-35%) in both pregnant and nonpregnant uteri and contraction was reduced. The increase in 86Rb efflux with cyanide was not secondary to changes of membrane potential as it also occurred in both high-K+ and Ca(2+)-free solutions. 4. Glibenclamide (20 microM), an antagonist of K+ATP channels, reduced the cyanide-evoked increase of 86Rb efflux by about 50%. The glibenclamide-insensitive component of efflux persisted in a Ca(2+)-free solution. Despite its action on 86Rb efflux, glibenclamide did not restore contraction. 5. Intracellular pH falls during metabolic inhibition. We therefore investigated whether reducing pHi (in the absence of cyanide) had an effect on 86Rb efflux. Application of the weak acid butyrate (60 mM, at constant external pH, 7.4) had no significant effect on 86Rb efflux. Thus it is unlikely that the acidification in hypoxia contributes to the increased K+ efflux. 6. Intracellular alkalinization produced by the weak base trimethylamine (60 mM) increased the frequency of uterine contraction and the 86Rb efflux. However, there was no effect on the 86Rb efflux in a Ca(2+)-free solution. The increased efflux is therefore presumably a consequence of the increased frequency. 7. It is concluded that metabolic inhibition produced by cyanide, produces an increase in K+ efflux from the myometrium. Part of this efflux is glibenclamide sensitive. This increased K+ efflux will lead to hyperpolarization of the myometrial membrane and thus decrease excitation. Thus reduced surface membrane excitability will contribute to the fall of force in hypoxia; specifically it may cause the initial loss of spontaneous contractions in the uterus.

Adenosine Triphosphate↗
PubMed1993

Effects of pH and inorganic phosphate on force production in alpha-toxin-permeabilized isolated rat uterine smooth muscle.

1. Strips of longitudinal smooth muscle isolated from rat uterus were permeabilized using crude alpha-toxin from the bacterium Staphylococcus aureus. This treatment rendered the surface membrane permeable to small molecular weight substances. Simultaneous measurements of tension and calcium concentration ([Ca2+]) (using indo-1 fluorescence) were used to investigate the effects of pH and inorganic phosphate concentration ([Pi]) on Ca(2+)-activated force generated by the contractile proteins. 2. Raising the [Pi] from 1 to 11 mM at a pH of 7.2 depressed both maximal and submaximal Ca(2+)-activated force. This effect of Pi was concentration dependent having the majority of its effect by 6 mM. 3. Further experiments at a submaximal [Ca2+] showed that Ca(2+)-activated force was enhanced by raising [Pi] from 6 to 11 mM suggesting that Pi increased the Ca2+ sensitivity of tension production. Based on these results, calculations indicate that the apparent affinity constant of Ca2+ for the contractile proteins increased from 4 x 10(6) M-1 to 6 x 10(6) M-1 on raising [Pi] from 1 to 11 mM. 4. Lowering pH from 7.2 to 6.7 at a [Pi] of 1 mM potentiated Ca(2+)-activated force with a small depression in the apparent Ca2+ sensitivity of tension production. This effect of pH on maximum (100 microM Ca2+) and submaximum (0.3 microM Ca2+) Ca(2+)-activated force was observed over a range of acidic pHs (7.0-6.7). 5. Increasing pH from 7.2 to 7.7 at a [Pi] of 1 mM depressed Ca(2+)-activated force with no effect on Ca2+ sensitivity of tension production. 6. Spontaneous contractions in intact rat myometrium are abolished under hypoxic conditions. Under these same conditions intracellular [Pi] rises and pH falls. The results of this study suggest that taken individually neither the effect of a rise in [Pi] nor a fall in pH on Ca(2+)-activated force generated by the contractile proteins can account for the effect of hypoxia on spontaneous contractions.

Animals↗
PubMed1993

Changes of intracellular pH in rat mesenteric vascular smooth muscle with high-K+ depolarization.

1. In mesenteric vascular smooth muscle cells changes in extracellular pH are rapidly transmitted to the cytoplasm. The mechanism involved is unknown, but it may be due to a high proton permeability of the surface membrane, in which case changes in membrane potential would alter the driving force for proton entry. We have therefore examined the voltage sensitivity of intracellular pH (pHi) in these cells. 2. Strips of mesenteric resistance vessels were loaded with SNARF-1 to monitor pHi and tension was simultaneously measured. Tissues were superfused with oxygenated solutions at 37 degrees C and pH 7.4. Isosmotic substitution of K+ for Na+ was used to depolarize the preparations. 3. pHi was found to be sensitive to alteration of [K+]. Depolarization of the tissue with K+ caused contraction and produced transient increases in pHi. When pHi regulation was blocked there was no significant change in the size of the alkalinization induced by high K+, thus it is unlikely that the results can be explained by voltage sensitivity of pHi regulating mechanisms. 4. In nominally Ca(2+)-free solution, the tissue does not contract and the alkalinization with high K+ was significantly greater than that occurring in 3 mM Ca2+. 5. There was a rapid acidification, when pHi regulation was blocked, which is consistent with a high proton permeability. 6. The effects of membrane potential on pHi have been modelled and show that they can be accounted for by effects of voltage on H+ influx through a proton channel. The effects of changing external pH on H+ influx also fit the model. Estimation of the proton permeability gave a high value (0.4 cm s-1). 7. The results presented demonstrate (i) a voltage sensitivity of pHi in mesenteric vascular smooth muscle cells and (ii) a particularly high permeability of the membrane to protons. The physiological significance of these findings is discussed.

Animals↗
PubMed1993

Occurrence of intracellular pH transients during spontaneous contractions in rat uterine smooth muscle.

1. Intracellular pH (pHi) and contraction have been measured simultaneously in isolated longitudinal rat myometrium. Tissue was loaded with the pH-sensitive fluorophore carboxy-SNARF and superfused with oxygenated, buffered salt solution at 37 degrees C and pH 7.4. 2. Small (0.04 pH unit) transient intracellular acidifications followed each spontaneous uterine contraction. The pHi transients lagged contraction by 15 s. Removal of external Ca2+ abolished both spontaneous contractions and the pHi changes. 3. Increasing uterine contraction by depolarization (40 mM K+) or agonist (50 microM carbachol) produced a larger acidification (0.18 pH unit). 4. The acidification with contraction does not appear to be due to increased glycolysis as no increase in lactate efflux occurred with high-K+ stimulation. 5. Uterine contraction is known to be sensitive to changes in pHi. Comparable pHi changes to the pHi transients seen during spontaneous contraction were induced by application and withdrawal of weak acids and bases. The resulting pHi changes (0.02-0.08 pH unit) produced effects on contraction frequency and amplitude, alkalinization augmenting and acidification depressing contractile activity, suggesting that the pHi transients with contraction are functionally important.

Animals↗
PubMed1993

Uterine contraction and physiological mechanisms of modulation.

Control of the smooth muscle in the uterus (the myometrium), is of vital importance during pregnancy and parturition. It is therefore understandable that several physiological mechanisms (neuronal, hormonal, metabolic, and mechanical) play a role in the control of myometrial activity. As our knowledge of the mechanism of uterine contraction has increased much in recent years, it is now possible to begin to explain, in some detail, how the contractile activity may be modulated. A detailed account of the mechanism of contraction in the uterus is therefore given, followed by examples of modulation of this process for each of the four physiological methods listed above. Examples have been chosen to illustrate either general or particular mechanisms of modulation. The goal of many working in this field is to understand these processes and thus prevent preterm labor and uterine dysfunction in term labor, which are still significant clinical problems.

Animals↗
PubMed1993

Changes in frequency and force production of the human myometrium with alteration of pH and metabolism.

Changes in uterine metabolism and intracellular pH occur with uterine activity and these may in turn alter function. We have investigated the effects of changes in intracellular pH and metabolic inhibition on the spontaneous activity of isolated nonpregnant and pregnant human myometrial strips at 37 degrees C. Intracellular alkalinization resulted in a marked increase in the frequency of contractions in both nonpregnant and pregnant preparations. Intracellular acidification reduced force production in pregnant and nonpregnant preparations. The effects of pH were concentration dependent. Metabolic inhibition (2 mmol cyanide l-1) abolished spontaneous contractions in nonpregnant preparations (ten of ten) but pregnant preparations were able to maintain activity, although at a reduced level (eleven of fifteen). Possible mechanisms involved and relevance to uterine dysfunction are discussed.

Adult↗
PubMed1993

Effects of hypoxia on force produced by agonists and depolarization and arising spontaneously in the rat uterus.

The effects of cyanide and nitrogen on contractile activity in rat uteri was investigated. Hypoxia significantly reduced contractile activity produced either spontaneously, or by application of carbachol (50 mumol l-1) or oxytocin (20 nmol l-1) in preparations from pregnant and nonpregnant rats. Hypoxia had, however, significantly smaller effects on agonist-evoked than on spontaneous contractions. Application of agonists under hypoxic conditions restored some degree of force to preparations in which spontaneous activity had been abolished. This result suggests that the loss of spontaneous contractions was, in part, due to decreased excitability of the uterus, rather than to an impairment of the contractile machinery. Hypoxia significantly decreased the force produced by depolarization of the uterus. The effects of hypoxia on contraction produced by agonists or depolarization were not significantly different, suggesting that a similar mechanism may maintain force under these conditions, and that this mechanism does not occur during spontaneous activity. Lowering the external Ca2+ concentration to 0.1 mmol l-1 resulted in production of significantly less force in the presence or absence of agonist. The ability of hypoxia to decrease agonist-induced force was found not to be due to the intracellular acidification it produces. It was concluded that uterine hypoxia may decrease uterine contractions in vivo and a possible role in dystocia during labour was discussed.

Animals↗
PubMed1993

Extracellular pH signals affect rat vascular tone by rapid transduction into intracellular pH changes.

1. Changes in extracellular pH (pHo) are known to produce large effects on vascular tone, but the mechanisms involved are not understood. As changes in intracellular pH (pHi) can also affect vascular tone, we have investigated the effects of changing pHo upon both pHi and tone. 2. Strips of rat mesenteric resistance vessels were loaded with the pH-sensitive fluorophore SNARF 1; thus tension and pHi could be simultaneously measured as pHo was altered. 3. Whenever pHo was altered there was a corresponding alteration of pHi. Furthermore, when pHo was changed the pHi change was more rapid than that reported to occur in other cells. The time to half-peak intracellular response was 38 +/- 4 s (n = 11). The induced pHi change was also less attenuated than in many other cells studied. Thus a ratio of 0.73 was obtained for the change in pHi per unit pHo change (n = 7). 4. An increase in pHi produced by an increase in pHo was accompanied by an increase in tension in the vascular strips. In other experiments pHi was increased at constant pHo by the addition of the weak base trimethylamine (40 mM). This also elevated tension in the strips. Conversely when pHo was changed while pHi was held at resting values, no change in vascular tone occurred. 5. It is concluded that the effects of pHo on vascular tone are due to the induced change in intracellular pH, and that the vascular smooth muscle cell is functionally well adapted to respond to changes in tissue pH, thereby allowing blood flow to a tissue to be rapidly altered to meet changing needs.

Animals↗
PubMed1992

The effects of intracellular and extracellular alkalinization on contractions of the isolated rat uterus.

The effects of changing pH on a spontaneously active smooth muscle, the myometrium, is examined. We show, for the first time in any smooth muscle, that the frequency of contraction is greatly increased when intracellular pH is raised. Three weak bases, trimethylamine, diethylamine and ammonium, were used to raise intracellular pH (pHi), at constant external pH, in isolated uteri of pregnant and non-pregnant rats. Each base increased spontaneous uterine contractile activity, particularly by raising the frequency, in a concentration-dependent manner. At the highest concentrations (40-50 mM) frequency was so increased that a maintained contraction resulted. Intracellular alkalinization during a high-K-maintained uterine contraction produced a small, but significant, fall in force. When external pH was increased, the results were greatly influenced by gestational state; in uteri from non-pregnant animals there was no effect whereas uteri from pregnant rats were found to be extremely sensitive to a raised external pH above 7.4; spontaneous contractions were reduced. In pregnant uteri, when both internal and external pH were elevated, spontaneous contractions were immediately reduced, thus the effects of external pH predominated. These findings may have significance in labour.

Alkalies↗
PubMed1992

The effects of metabolic inhibition and acidification on force production in the rat uterus.

The effects of hypoxia and intracellular acidification were examined on the mechanical properties of the non-pregnant and pregnant rat uterus. Isolated uteri were investigated during control conditions and in the presence of cyanide, to simulate hypoxia or the salts of weak acids and bases to change intracellular pH at constant external pH (pH 7.4). Both spontaneous contractions and high-K contractures (i.e. fully activated preparations) were investigated at 37 degrees C. In control solutions both pregnant and non-pregnant uteri were spontaneously active and gave a contracture when bathed with a high-K+ solution. When oxidative phosphorylation was inhibited by cyanide, spontaneous contractions were either greatly reduced or abolished. The effects on spontaneous contractions were more pronounced in the non-pregnant than the pregnant uterus. If high-K solution was added after cyanide had abolished spontaneous contractions, then a small amount of force was produced. High-K-induced contractures were not maintained in the presence of cyanide and quickly fell to baseline levels in both pregnant and non-pregnant uteri. All the effects of cyanide were fully reversible. When only intracellular pH was changed by adding weak acids (50 mM-butyrate or propionate), spontaneous contractions were greatly diminished or abolished in both pregnant and non-pregnant uteri. However, the application of weak acids had no effect upon the KCl-induced contractures in both pregnant and non-pregnant uteri. The effects of cyanide on the uterus include both an acidification and changes in metabolites, e.g. a fall in [ATP]. To investigate the changes in metabolites without a change in pHi, cyanide was applied with the weak base trimethylamine (40-50 mM). 31P Nuclear magnetic resonance spectroscopy was used to show that the alkalinization which occurs with trimethylamine alone abolished the acidification ordinarily associated with cyanide. Spontaneous and high-K-induced contractions were greatly reduced or abolished, i.e. the result was the same as with cyanide alone. This occurred in both pregnant and non-pregnant uteri. It is concluded that spontaneous force production is affected by both changes in metabolites (e.g. decreased [ATP] and increased inorganic phosphate) and pHi, and that both can depress activation of the uterus. Only the changes in metabolites can depress force production in fully activated preparation. The greater effect on force in the non-pregnant uterus compared to the pregnant uterus may be due to the lower initial levels of [ATP] and the greater fall in ATP seen in non-pregnant compared to pregnant uterus, and gestational changes in metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1991

Migratory arrest of gonadotropin-releasing hormone neurons in transgenic mice.

Gonadotropin-releasing hormone (GnRH) is important in reproduction, although the mechanism of central hypogonadism in humans remains unclear. Because the GnRH neuron originates from the olfactory placode and migrates to the hypothalamus during development, central hypogonadism in humans could be caused by failure in normal migration of GnRH neurons to the hypothalamus. We report that in transgenic mice expression of the simian virus 40 T antigen, driven by the promoter of human GnRH gene, resulted in central hypogonadism due to an arrest in neuronal migration during development and tumor formation along the migratory pathway. This system appears to be an important animal model of hypogonadotropic hypogonadism in humans. Additionally, olfactory bulb tumors from these animals were dispersed, and a GnRH-secreting neuronal cell line (GN cell line) was established.

Animals↗
PubMed1991

Cytochrome oxidase content of rat brain during development.

The cytochrome oxidase concentration and content of rat brain during development was measured using a simple new assay for cytochrome a. The cytochrome oxidase concentration increased from 1.2 nmol/g wet wt. of brain at birth to about 5.5 nmol/g in the adult, most of the change occurring between 5 and 25 days after birth.

Animals↗
PubMed1991

Neurochemical anatomy of fetal hippocampus transplanted into large lesion cavities made in the adult rat brain.

The purpose of the present study was to determine whether neurochemicals normally found within neuron somata, fibers, and terminals of the hippocampal formation would also be present in transplanted hippocampal tissue that had developed in lesion cavities made in adult rat brains by aspiration of the hippocampus and overlying dorsolateral neocortex. Embryonic Day 15 or 16 rat brian tissue containing hippocampus with some medial pallial anlage was transplanted into the site of hippocampal aspiration lesions in adult male rats. One hundred ten to one hundred thirty-five days later the brains of these rats were sectioned and processed using the avidin-biotin-horseradish peroxidase immunocytochemical procedure to visualize choline acetyltransferase, met-enkephalin (MENK), neurotensin (NT), somatostatin, substance P, tyrosine hydroxylase (TH), or vasoactive intestinal polypeptide. Sections from two brains were stained using the thiocholine technique for visualization of acetylcholinesterase. All of these substances were found within cell bodies and/or fibers in the transplants. However, several abnormalities were noted. In addition to TH-immunoreactive fibers, TH-immunoreactive cell bodies were found in the transplants. Since TH is not expressed in mature hippocampal or cortical neurons this suggests that mechanisms for suppression of manufacture of this enzyme are lacking or inhibited in the transplants. Further, although all of the peptides were present either in fibers or in both cell bodies and fibers, the density of staining for NT and MENK was less than would be expected for normal hippocampus, and none of the cell bodies or fibers reacting for the peptides exhibited any apparent organization resembling that normally observed in hippocampus or cortex. However, some histological organization was present and the cholinergic markers were associated with this organization. These data suggest that some tropic and/or trophic factor such as nerve growth factor is present in the transplants to guide cholinergic innervation.

Animals↗
PubMed1991

Intracellular pH and buffering power measured in isolated single cells from pregnant rat uterus.

Intracellular pH (pHi) affects smooth muscle function yet there have been few direct pHi measurements. Using the fluorescent indicator, 2',7'-bis-2-(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester, we report here the first measurements of pHi and buffering power (beta) in single, isolated, uterine smooth muscle cells. Mean pHi in cells from pregnant rat uteri was 7.09 +/- 0.08 (+/- S.E.M., n = 16 cells; 37 degrees C), and beta was found to be 12.8 +/- 2.8 mmol/l per pH unit (n = 15). The cells were able to regulate their pHi in the presence of butyrate when perfused in nominally CO2-free solution.

Animals↗
PubMed1991

Maintenance of LHRH and oxytocin neurons in slice explants cultured in serum-free media: effects of tetrodotoxin on gene expression.

A variety of neuroendocrine cells survive and express specific neuropeptide genes for long periods of time in slice explant cultures in the presence of serum. However, before use of these slice explant cultures as experimental models for physiological and pharmacological studies on the regulation of neuropeptide gene expression, it is first necessary to evaluate their characteristics in defined (e.g. serum free) media and to control for the spontaneous electrical and synaptic activity of neurons in these cultures. In this study, brain slices from postnatal day 4 rats were cultured in serum-containing media (SCM) for 12 days to allow thinning, and then maintained in a serum-free, defined media (SFM) for 6 days. Culture slices transferred to SFM appeared healthy and numerous neuroendocrine neurons containing messenger RNA (mRNA) encoding for LHRH and magnocellular neurons containing mRNA encoding for oxytocin (OT) were detected using in situ hybridization histochemistry (ISHH). Each of these neuronal subtypes robustly produced their appropriate gene products as determined by immunocytochemical analysis. Abundant magnocellular OT neurons were found in cultures grown in either SCM or SFM. In contrast, magnocellular vasopressin (VP) neurons were rarely detected under these conditions. Inhibition of spontaneous electrical and synaptic activity in these slice explant cultures was effectively achieved by incubation for the last 2.5 days of culture in the presence of tetrodotoxin (TTX; 10(-6) M). Densitometric single cell analyses after ISHH was performed on both LHRH and OT cells. Comparisons of the density values (corresponding to mRNA levels), from these slice explants, found that: (1) cellular LHRH mRNA levels decreased in the absence of serum, whereas cellular OT mRNA levels did not significantly change under these conditions; (2) the presence of TTX in the media resulted in an overall decrease in cellular LHRH mRNA values in both SCM and SFM, and (3) the OT neurons in these slice cultures appear to be composed of a heterogeneous population, with one cell subtype responding to TTX with an increase in cellular OT mRNA levels. These data show that factors in serum and spontaneous electrical activity can differentially influence mRNA levels of LHRH cells and magnocellular OT neurons in culture.

Action Potentials↗