Biomedical subjects

S Wray

Publications and source records attributed to S Wray.

At least 37 records · Page 2Linked to original sources

PubMed2000

Novel gene expressed in nasal region influences outgrowth of olfactory axons and migration of luteinizing hormone-releasing hormone (LHRH) neurons.

Although a variety of cues have been implicated in axonal targeting during embryogenesis and regeneration, the precise mechanisms guiding olfactory axons remain unclear. Appropriate olfactory axon pathfinding is essential for functional chemoreceptive and pheromone receptive systems. Olfactory axon pathfinding is also necessary for establishment of the neuroendocrine LHRH system, cells critical for reproductive function. LHRH cells exhibit neurophilic migration moving from the nasal region along olfactory axons into the brain. Factors involved in the migration of these neuroendocrine cells are as yet unresolved. We report identification of a novel factor termed nasal embryonic LHRH factor (NELF) that was discovered in a differential screen of migrating versus nonmigrating primary LHRH neurons. NELF is expressed in PNS and CNS tissues during embryonic development, including olfactory sensory cells and LHRH cells. NELF antisense experiments indicate that a reduction in NELF expression decreases olfactory axon outgrowth and the number of LHRH neurons that migrate out of the nasal tissue. These results demonstrate that NELF plays a role as a common guidance molecule for olfactory axon projections and subsequently, either directly or indirectly, in the neurophilic migration of LHRH cells.

Amino Acid Sequence↗

PubMed2000

Interactions between Ca(2+) and H(+) and functional consequences in vascular smooth muscle.

Ca(2+) and H(+) ions can profoundly alter vascular tone. In many physiological and pathological processes, changes in the concentration of both ions occur. Thus, to understand the processes and mechanisms that modify force, it is necessary to understand what changes occur in these ions and, importantly, how they interact with each other. In this minireview, we highlight the quantitatively important mechanisms involved in the contractile responses of vascular tissues to pH change and discuss the cellular and molecular reasons underlying these responses.

Actin Cytoskeleton↗

PubMed2000

The effects of inhibiting myosin light chain kinase on contraction and calcium signalling in human and rat myometrium.

The effect of inhibiting myosin light chain kinase on contractions of human and rat myometrium has been investigated, to determine whether force can be produced independently of myosin phosphorylation. Two inhibitors were used, wortmannin and ML-9, and their effects on spontaneous, high-K-depolarization-induced and oxytocin-induced force studied. Both inhibitors reduced and then abolished uterine force, irrespective of how it was produced; this was the case for both human and rat myometrium, and pregnant and non-pregnant tissue. The effects of wortmannin on intracellular [Ca2+] and inward Ca2+ current were examined. The data showed that the reduction in force produced by wortmannin occurs without a reduction of either the Ca2+ current or [Ca2+]. It is concluded that, under normal physiological conditions, myosin light chain kinase phosphorylation of myosin is essential for uterine force production and that there is little or no role for alternative force-producing pathways.

Adolescent↗

PubMed2000

Ectopic expression of luteinizing hormone-releasing hormone and peripherin in the respiratory epithelium of mice lacking transcription factor AP-2alpha.

The vertebrate transcription factor activator protein-2 (AP-2alpha) is involved in craniofacial morphogenesis. In the nasal placode AP-2alpha expression delineates presumptive respiratory epithelia from olfactory epithelia, with AP-2alpha expression restricted to the anterior region of the respiratory epithelium (absent from the olfactory epithelium) at later stages. To address the role AP-2alpha plays in differentiation of cell groups in the nasal placode, the spatiotemporal expression pattern of four markers normally associated with olfactory epithelial structures was analyzed in mice lacking AP-2alpha. These markers were the intermediate filament protein peripherin, the neuropeptide luteinizing hormone-releasing hormone (LHRH), the neural cell adhesion molecule (NCAM) and the olfactory transcription factor Olf-1. Development of cells expressing these markers was similar in both genotypes until embryonic day 12.5 (E12.5), indicating that the main olfactory epithelium and olfactory pit formation was normal. At E13.5 in mutant mice, ectopic LHRH neurons and peripherin axons were detected in respiratory epithelial areas, areas devoid of Olf-1 and NCAM staining. Over the next few days, an increase in total nasal LHRH neurons occurred. The increase in nasal LHRH neurons could be accounted for by LHRH neurons arising and migrating out of respiratory epithelial regions on peripherin-positive fibers. These results indicate that AP-2alpha is not essential for the separation of the olfactory and respiratory epithelium from the nasal placode and is consistent with AP-2alpha preventing recapitulation of developmental programs within the respiratory epithelium that lead to expression of LHRH and peripherin phenotypes.

Animals↗

PubMed2000

Two olfactory placode derived galanin subpopulations: luteinizing hormone-releasing hormone neurones and vomeronasal cells.

In adult rodents, the peptide galanin is expressed in a subpopulation of hypothalamic luteinizing hormone-releasing hormone (LHRH) neurones in an activity-dependent manner. In this investigation, we examined whether galanin mRNA expression in mice was activated coincident with LHRH mRNA expression, as LHRH neurones differentiate from the olfactory placode. Using in situ hybridization, we show (i) that galanin mRNA is coexpressed in LHRH neurones prenatally, (ii) that there is a decrease in galanin mRNA expression relative to LHRH mRNA expression once LHRH mRNA positive/galanin mRNA positive neurones migrate out of the olfactory pit and into the nasal septum, and (iii) the presence of a novel population of galanin mRNA positive/LHRH mRNA negative expressing neurones in the olfactory pit/vomeronasal organ which do not migrate into the central nervous systenm (CNS). This study demonstrates that there are at least two populations of galanin mRNA expressing neurones arising from the olfactory placode; one that remains in nasal regions, is LHRH mRNA negative and whose function is unknown, and one which is coexpressed with LHRH. In addition, the temporal expression of galanin mRNA in LHRH cells indicates that initial activation and subsequent inactivation of galanin mRNA expression is independent of synaptic CNS connections.

Animals↗

PubMed2000

A comparison of the contractile properties of human myometrium obtained from the upper and lower uterine segments.

This study compared the contractile characteristics of myometrium taken from upper and lower uterine segments. Biopsies were obtained from women undergoing classical caesarean section. Myometrial strips were dissected and mounted in an organ bath, and the contractions were recorded. The cross sectional area of the contractile elements within the strips was measured enabling strips of differing dimensions to be compared. There were no significant differences in the contractile rate and force production produced by myometrium from the upper and lower segments. This study demonstrated that for contractile studies, the use of lower segment is appropriate. The results fail to demonstrate any functional regionality of the human uterus in terms of contractility.

Female↗

PubMed2000

Luteinizing hormone-releasing hormone (LHRH) biosynthesis and secretion in embryonic LHRH.

Evidence indicates that LH-releasing hormone (LHRH) neurons can exhibit neuroendocrine secretory properties before entrance into the central nervous system. In this study, we evaluated LHRH biosynthesis and secretion in embryonic LHRH neurons maintained in nasal explants. Using ELISA and calcium imaging techniques, peptide content and single neuron activities were examined. LHRH neurons maintained for 7-10 days in vitro were found to possess a similar amount of LHRH/cell as the equivalent aged LHRH cells in vivo (postnatal day 1). LHRH peptide was measured in the medium of these relatively young cultures, and 40 mM KCl stimulated a 4-fold increase in LHRH secretion. KCl enhanced medium also resulted in a significant increase in LHRH content per culture (24.5 pg vs. 32.3). A similar effect was observed after muscimol-enhanced media (32.2 pg). Both agents also stimulated a substantial rise in intracellular calcium. Pretreatment of cultures with tetrodotoxin partially blocked the affects of muscimol on both peptide content and calcium activity, but not KCl. Calcium-depleted medium blocked the effects of KCl yet only attenuated the effects of muscimol. Treatment of cultures with cycloheximide blocked the effects of both muscimol and KCl. These results indicate that developing LHRH neurons are capable of synthesizing, secreting, and rapidly replenishing stores of LHRH peptide.

Animals↗

PubMed2000

Transcription factor activator protein-2 is required for continued luteinizing hormone-releasing hormone expression in the forebrain of developing mice.

LHRH is the neuropeptide responsible for reproductive function. Prenatally, LHRH expression begins when neurons are in the olfactory pit and continues as these cells migrate into the brain. Thus, LHRH neurons maintain neuropeptide expression through very distinct environments. The regulatory interactions that control onset and continued expression of the LHRH phenotype are unknown. To begin to address this question primary LHRH neurons were removed from nasal explants at different ages. A complementary DNA (cDNA) subtraction screen was performed comparing a 3.5-days in vitro LHRH neuron [approximately embryonic day 15 (E15) in vivo] to two 10.5-days in vitro LHRH neurons (approximately postnatal day 1 in vivo). The transcription factor activator protein-2 (AP-2alpha) was differentially expressed and was present in the developmentally younger LHRH neuron. In vivo analysis revealed that LHRH neurons expressed AP-2 as they migrated across the cribriform plate and into the forebrain beginning on E13.5, but that coexpression of LHRH and AP-2 was no longer detected in postnatal day 1 animals. This suggested a regulatory role for AP-2 in LHRH neurons. Analysis of animals lacking AP-2alpha revealed a dramatic decrease in forebrain LHRH neurons between E13.5 and E14.5, correlating with normal onset of AP-2 expression in LHRH neurons as they entered the central nervous system. Nasal cells robustly expressing LHRH were still present on E 14.5. The continued presence of forebrain LHRH cells is proposed based on a second marker, galanin, and lack of increased apoptotic/necrotic cells in this region. A decrease in LHRH messenger RNA in forebrain neurons indicates regulation of LHRH occurred at the transcriptional or posttranscriptional level in mutant animals. These results indicate a developmentally restricted involvement of the transcription factor AP-2 in LHRH expression once the LHRH neurons have migrated into the forebrain, but before establishment of an adult-like distribution.

Animals↗

PubMed1999

The relationship between the action potential, intracellular calcium and force in intact phasic, guinea-pig uretic smooth muscle.

1. We investigated the relationship between the action potential, Ca2+ and phasic force in intact guinea-pig ureter, following physiological activation. 2. The action potential elicited a Ca2+ transient consisting of three components: a fast increment, associated with the first action potential spike, a slower increment, associated with subsequent spikes and the initial part of the plateau component, and a steady-state phase associated with the plateau. 3. Prolongation of the plateau, by agonists, prolonged the third component of the Ca2+ transient and increased force amplitude and duration. 4. The force-Ca2+ relationship during phasic contractions showed hysteresis; more force was produced as Ca2+ declined than when it rose. Paired pulse stimuli suggested that the delay between Ca2+ and force was not due to mechanical properties. Wortmannin, which has been shown to selectively inhibit force and myosin light chain (MLC) phosphorylation in the guinea-pig ureter, did not affect electrical activity or Ca2+ but significantly increased the delay, suggesting that myosin phosphorylation is a major contributor to it. 5. Prolongation of the duration of the [Ca2+]i transient, at unchanged amplitude, increased force. The rise of [Ca2+]i did not limit the rate of contraction. Slowing of the rate of [Ca2+]i rise abolished the hysteresis between Ca2+ and force. 6. Cooling reduced force, increased the delay and hysteresis between Ca2+ and force, but did not affect the rate of rise of Ca2+. The reduction in force could be compensated, by increasing the duration of the Ca2+ transient. 7. We suggest that in vivo, steady-state force-Ca2+ relationships are not applicable in phasic smooth muscles. Furthermore, agonists increase force mainly by prolonging the action potential, which increases the duration of the [Ca2+] signal.

Action Potentials↗

PubMed1999

The role of the sarcoplasmic reticulum as a Ca2+ sink in rat uterine smooth muscle cells.

1. The mechanisms responsible for removing calcium ions from the cytoplasm were investigated in single rat uterine myocytes using indo-1. 2. Trains of depolarizing voltage-clamp pulses increased [Ca2+]i. The rate of decay of [Ca2+]i was slowed by inhibition of the sarcoplasmic reticulum (SR) Ca2+-ATPase with cyclopiazonic acid (CPA). However, if the sarcolemmal Na+-Ca2+ exchanger and Ca2+-ATPase were inhibited then recovery of [Ca2+]i was abolished showing that the SR Ca2+-ATPase alone cannot produce decay of [Ca2+]i. 3. In another series of experiments, Ca2+ release from the SR was induced with carbachol in a Ca2+-free solution. Under these conditions responses to repeated applications of carbachol could be obtained. In the presence of CPA, however, only the first application was effective. This suggests that the SR Ca2+-ATPase sequesters a significant amount of Ca2+ into the SR. 4. CPA slowed the rate of decay of [Ca2+]i following carbachol addition by > 50 %. Again, however, after a brief transient fall, decay was abolished when the Na+-Ca2+ exchanger and sarcolemmal Ca2+-ATPase were inhibited. 5. These data show that, although the SR Ca2+-ATPase contributes to the decay of [Ca2+]i, it cannot function effectively in the absence of Ca2+ removal from the cell. These data are discussed in the context of the superficial buffer barrier model in which Ca2+ is taken up into the SR and then released very close to sarcolemmal Ca2+ extrusion sites, i.e. the SR acting in series with the surface membrane extrusion mechanisms. We also suggest that the amount of filling of the SR influences the rate of Ca2+ removal.

Animals↗

PubMed1999

In vivo pH and metabolite changes during a single contraction in rat uterine smooth muscle.

1. We have used 31P NMR spectroscopy to measure metabolites and pHi at three periods during a phasic contraction of the uterus, in vivo, to determine whether they change as a consequence of contraction. The regular uterine contractions were recorded via a balloon catheter in the uterine lumen. Each phasic contraction was divided into three parts: the period between contractions (rest), the development of force (up) and the relaxation of force (down). The NMR data were summed separately from each of these three periods over 20-40 successive contractions. 2. Significant changes in ATP, phosphocreatine (PCr) and inorganic phosphate (Pi) occurred during the contraction. [ATP] fell from 2.0 to 1.6 mM and [PCr] from 2.6 to 2.0 mM during the up period, while [Pi] increased from 2.2 to 2.8 mM. Recovery of ATP and PCr occurred during the relaxation part of the contraction, whereas Pi did not fully recover until the contraction was complete. 3. Significant acidification from pH 7.28 +/- 0.02 at rest to 7.16 +/- 0.02, occurred with contraction. This acidification is greater than that previously reported for in vitro uterine preparations. Measurements of uterine blood flow show that it decreased with contraction. Therefore, ischaemia, in addition to the metabolic consequences of contraction, may account for the larger acidification observed in vivo. 4. Lowering pHi in an in vitro uterine preparation by a similar level to that found in vivo produced a significant reduction of the phasic contractions. Thus we propose that these changes, especially the fall in pHi during force development, feed back negatively on the contraction to limit its strength, and may help prevent uterine ischaemia and fetal hypoxia during labour.

Adenosine Triphosphate↗

PubMed1999

The effect of cyclopiazonic acid on excitation-contraction coupling in guinea-pig ureteric smooth muscle: role of the sarcoplasmic reticulum.

1. We have investigated the effect of cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase on excitation-contraction (EC) coupling in guinea-pig ureter, by measuring membrane currents, action potentials, intracellular [Ca2+] and force. 2. CPA (20 micrometers) significantly enhanced the amplitude and duration of phasic contractions of ureteric smooth muscle associated with action potentials. This was accompanied by an increase in the duration of the intracellular Ca2+ transient in intact tissue and single cells but not their amplitude. However, CPA also slowed the rate of rise, and fall, of the force 1|1|Phiand1Phi Ca2+ transients. 3. Membrane potential recordings showed that CPA produced a small depolarization and a large increase in the duration of the plateau phase of the action potential. 4. Patch-clamp studies showed marked inhibition of outward potassium current in the presence of CPA and an inhibition of spontaneous transient outward currents (STOCs). CPA had no effect on inward Ca2+ current. 5. These data suggest that the SR plays a major role in modulating the excitability of the ureter, particularly via curtailing the action potential duration. This in turn will shorten the Ca2+ transient and decrease force. This negative action on developed force predominates over any small role it may play in initiating force in the guinea-pig ureter.

Animals↗

PubMed1999

Intracellular calcium stores and agonist-induced contractions in isolated human myometrium.

OBJECTIVE: We hypothesized that the release of calcium from intracellular stores contributes to the contractions produced by the agonists oxytocin, carbachol, and prostaglandin F(2 )(alpha ) in human myometrium. STUDY DESIGN: Strips of myometrium were obtained at cesarean section and hysterectomy. The strips were loaded with the calcium-sensitive dye Indo-1 to enable simultaneous measurement of tension and intracellular calcium levels. Agonist-induced responses in the presence and absence of extracellular calcium were studied. RESULTS: Strips of myometrium were obtained from 48 women not in labor undergoing cesarean section and 6 women not pregnant undergoing hysterectomy. An increase in intracellular calcium level after agonist stimulation invariably preceded an increase in tension. Intracellular calcium level returned to baseline before myometrial relaxation. Oxytocin, carbachol, and prostaglandin F(2)(alpha) all gave both force and intracellular calcium responses in the absence of extracellular calcium, although both these responses were only 26% to 40% of the maximal response when extracellular calcium was present. CONCLUSIONS: Release of calcium from internal stores induced by oxytocin, carbachol, and prostaglandin F(2)(alpha) may contribute to agonist-induced myometrial force production.

Adult↗

PubMed1999

Luteinizing hormone-releasing hormone quantified in tissues and slice explant cultures of postnatal rat hypothalami.

LH-releasing hormone (LHRH) peptide from postnatal rat preoptic area (POA)/hypothalamic tissues in vivo and slice explant cultures maintained in vitro was quantitated using an enzyme-linked immunosorbant assay. Moreover, messenger RNA (mRNA) copy number was calculated in LHRH neurons maintained in culture using in situ hybridization histochemistry with autoradiographic film analysis. POA/hypothalami from postnatal day 5-6 pups averaged 1250 pg of LHRH, with approximately 28% of peptide residing within rostral tissues where most LHRH perikarya reside. Explant cultures maintained 18 days in vitro contained 30.4-92.0 pg/slice with a whole animal total of 244.8 pg. Considering cell numbers in vivo and in vitro, LHRH neurons in whole animal produce 1.0 pg of LHRH/cell, whereas those in culture average 2.0 pg/cell. Furthermore, LHRH mRNA copies/cell in organotypic culture was estimated conservatively at 1410 copies/cell, a relatively high number. This work shows that, compared with whole animal, cultures have substantial LHRH stores, indicating maturation of synthetic activity and/or formation of new terminals in vitro. High LHRH mRNA copy number also suggests a high rate of peptide biosynthesis. Our analysis, demonstrating the dynamic potential of LHRH neurons, suggests that subtle changes in LHRH mRNA expression in all cells or a subpopulation can dramatically alter the LHRH system biosynthetic capacity.

Animals↗

PubMed1999

Modulation of force induced by pH in the guinea-pig uterus examined at two stages of the oestrous cycle.

Changes in pH have a marked influence on uterine contractility. Changes in uterine pH occur during pregnancy and labour, when marked endocrine changes are occurring. As hormonal status can also influence contractility, this study investigated whether pH-induced modulation of uterine force in influenced by hormonal changes. The effects of altering intracellular and extracellular pH on uterine contractions were studied in guinea-pigs on day 7 (high progesterone) and day 15 (low progesterone) of the oestrous cycle. Resting values of pH were significantly more acidic on day 15 compared with day 7, and more force was produced on day 15. Changing external pH produced similar changes in intracellular pH and force on both days. External acidification was associated with a large increase in force. In contrast, intracellular acidification, at constant external pH, reduced force. In conclusion, the stage of the oestrous cycle has a large effect on resting pH in the myometrium but only small effects on the pH-induced modulation of force, and the link between pH and force is complex.

Analysis of Variance↗

PubMed1998

Properties of voltage-activated [Ca2+]i transients in single smooth muscle cells isolated from pregnant rat uterus.

1. The intracellular calcium concentration ([Ca2+]i) was measured at 35 degrees C using the fluorescent indicator indo-1 in patch-clamped, single uterine myocytes from pregnant rats to investigate the relationship between depolarization, Ca2+ current (ICa) and [Ca2+]i. 2. Membrane depolarization activated ICa and produced a [Ca2+]i transient. The rapid increase in [Ca2+]i occurred at the same time as the inward ICa. Both ICa and the increase in [Ca2+]i were abolished by nifedipine (10 microM). 3. When the membrane potential was held at -80 mV the threshold depolarization for an increase in [Ca2+]i was about -55 to -50 mV. As the magnitude of the depolarization was increased to about 0 mV there was an increase in the size of both ICa and the increase in [Ca2+]i. As the magnitude of the depolarization was further increased both ICa and the [Ca2+]i increase declined. 4. When the depolarizing pulses were applied at 3 Hz to mimic normal action potentials then the individual [Ca2+]i transients did not fully relax and a tetanic rise of [Ca2+]i was observed. Under these conditions, there was not a simple relationship between the magnitude of the Ca2+ response and Ca2+ entry. When pairs of depolarizing pulses were applied, the increase in [Ca2+]i produced by the second pulse was larger (in relation to the magnitude of the L-type Ca2+ current) than that produced by the first pulse. This facilitation was abolished by both ryanodine and cyclopiazonic acid suggesting a role for release from intracellular stores. 5. We conclude that the L-type Ca2+ current is the major source of Ca2+ ions entering the cell to produce the [Ca2+]i transient on depolarization. The magnitude of the increase in [Ca2+]i may, however, be amplified by Ca2+-induced Ca2+ release.

Animals↗

PubMed1998

Contribution of sarcoplasmic reticular calcium to smooth muscle contractile activation: gestational dependence in isolated rat uterus.

1. The contribution of Ca2+ released from the sarcoplasmic reticulum (SR) to smooth muscle contractile activation remains poorly understood. By simultaneously monitoring cytosolic [Ca2+] ([Ca2+]i) and force in isolated rat uterine smooth muscle, we report the influence of SR Ca2+ release on contractility during conditions (a) of altered SR Ca2+ homeostasis and (b) where the only source of activating Ca2+ was derived from the SR. 2. In myometria of non-pregnant rats, ryanodine (1-50 microM), a modulator of calcium-induced calcium release (CICR), had no effect on the spontaneous [Ca2+]i or force transients. However, depletion of SR Ca2+ by inhibiting the SR Ca2+-ATPase (with cyclopiazonic acid (CPA), 20 microM) resulted in an enhancement of spontaneous [Ca2+]i and force transients. 3. In myometria of pregnant rats, although ryanodine had no effect in 40% of tissues studied it produced a small but significant enhancement of the integrated spontaneous [Ca2+]i and force transient in 60% of cases. The potentiating effects of CPA were enhanced in myometria of pregnant rats compared with non-pregnant rats, often resulting in maintained [Ca2+]i increases and contraction. 4. In zero external Ca2+, agonist-induced SR Ca2+ release resulted in transient increases in [Ca2+]i and force. The magnitude of these agonist-induced [Ca2+]i and force changes were significantly enhanced in myometria of pregnant rats. No evidence for agonist-induced Ca2+-independent force production was observed. 5. These results indicate that CICR plays little role in SR Ca2+ release from the myometrium, and that there are gestational-dependent alterations in the ability of SR Ca2+ mobilization to contribute to contractile activation. The implications of these findings for the co-ordination of myometrial [Ca2+]i signalling and contractility are discussed.

Animals↗

PubMed1998

Hypoxia and smooth muscle function: key regulatory events during metabolic stress.

Hypoxia rapidly reduces force in many smooth muscles and we review recent data that shed light on the mechanisms involved. As many regulated cellular processes are integrated to co-ordinate smooth muscle contractility, the processes responsible for decreased force output with altered metabolism are also likely to be many, acting in concert, rather than the actions of one altered parameter. Nevertheless the aim of this study is to elucidate the hierarchical series of events that contribute to reduced smooth muscle force production during altered metabolism. We conclude that in many phasic smooth muscles the decrease in force can be attributed to impaired electro-mechanical coupling whereby the Ca2+ transient is reduced. A direct effect of hypoxia on the Ca2+ channel may be of key importance. In tonic vascular smooth muscles KATP channels may also play a role in the integrated functional responses to hypoxia. There are also many examples of force being reduced, in tonically activated preparations, without a fall in steady-state Ca2+; indeed it usually increases. We examine the roles of altered [ATP], pH, myosin phosphorylation, inorganic phosphate and proteolytic activity on the [Ca2+]-force relationship during hypoxia. We find no defining force-inhibitory role for any one factor acting alone, and suggest that force most probably falls as a result of the combination of myriad factors.

Animals↗