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Biomedical subjects

S Wray

Publications and source records attributed to S Wray.

At least 19 recordsLinked to original sources
PubMed2005

Distribution of AQP2 and AQP3 water channels in human tissue microarrays.

The objective of this investigation was to use semi-quantitative immunohistochemistry to determine the distribution and expression levels of AQP2 and AQP3 proteins in normal human Tissue MicroArrays. Expression of the vasopressin regulated AQP2 was observed in a limited number of tissues. AQP2 was prominent in the apical and subapical plasma membranes of cortical and medullary renal collecting ducts. Surprisingly, weak AQP2 immunoreactivity was also noted in pancreatic islets, fallopian tubes and peripheral nerves. AQP2 was also localized to selected parts of the central nervous system (ependymal cell layer, subcortical white matter, hippocampus, spinal cord) and selected cells in the gastrointestinal system (antral and oxyntic gastric mucosa, small intestine and colon). These findings corroborate the restricted tissue distribution of AQP2. AQP3 was strongly expressed in many of the human tissues examined particularly in basolateral membranes of the distal nephron (medullary collecting ducts), distal colon, upper airway epithelia, transitional epithelium of the urinary bladder, tracheal, bronchial and nasopharyngeal epithelium, stratified squamous epithelial cells of the esophagus, and anus. AQP3 was moderately expressed in basolateral membranes of prostatic tubuloalveolar epithelium, pancreatic ducts, uterine endometrium, choroid plexus, articular chondrocytes, subchondral osteoblasts and synovium. Low AQP3 levels were also detected in skeletal muscle, cardiac muscle, gastric pits, seminiferous tubules, lymphoid vessels, salivary and endocrine glands, amniotic membranes, placenta and ovary. The abundance of basolateral AQP3 in epithelial tissues and its expression in many non-epithelial cells suggests that this aquaglyceroporin is a major participant in barrier hydration and water and osmolyte homeostasis in the human body.

Aquaporin 2↗
PubMed2005

Developmental changes in GABA receptor subunit composition within the gonadotrophin-releasing hormone-1 neuronal system.

It is becoming increasingly evident that GABA plays an important role in the regulation of gonadotrophin-releasing hormone (GnRH)-1 neurones via the GABAA receptor. The aim of the present study was to characterise expression of the GABAA receptor within the GnRH-1 system across development. The expression pattern of five GABAAalpha subunits and one GABAAbeta subunit was first examined within individual GnRH-1 neurones by the polymerase chain reaction. A significant increase in the expression of GABAAalpha2 and a significant decrease in the expression of GABAAalpha6 over time were found. Of the other subunits examined, two (alpha1 and alpha3) showed no differences in expression and two (alpha4 and beta3) showed variable low incidence of expression. Given the reciprocal relationship of alpha2 and alpha6 expression, we hypothesised that there is a developmental switch in the expression of these subunits in GnRH-1 neurones. To investigate this hypothesis, single- and double-label immunocytochemistry for GABAAalpha2 and alpha6 and GnRH-1 was performed in tissue from ages E12.5 to adulthood, as well as in nasal explants. We show that GABAAalpha2 and alpha6 are present in the GnRH-1 neuronal system both in vivo and in vitro and that the levels of expression are altered as a function of age.

Aging↗
PubMed2004

Membrane cholesterol regulates smooth muscle phasic contraction.

The regulation of contractile activity in smooth muscle cells involves rapid discrimination and processing of a multitude of simultaneous signals impinging on the membrane before an integrated functional response can be generated. The sarcolemma of smooth muscle cells is segregated into caveolar regions-largely identical with cholesterol-rich membrane rafts-and actin-attachment sites, localized in non-raft, glycerophospholipid regions. Here we demonstrate that selective extraction of cholesterol abolishes membrane segregation and disassembles caveolae. Simultaneous measurements of force and [Ca2+]i in rat ureters demonstrated that extraction of cholesterol resulted in inhibition of both force and intracellular Ca2+ signals. Considering the major structural reorganization of cholesterol-depleted sarcolemma, it is intriguing to note that decreased levels of membrane cholesterol are accompanied by a highly specific inhibition of phasic, but not tonic contractions. This implies that signalling cascades that ultimately lead to either phasic or tonic response may be spatially segregated in the plane of the sarcolemma. Replenishment of cholesterol restores normal contractile behavior. In addition, the tissue function is re-established by inhibiting the large-conductance K(+)-channel. Sucrose gradient ultracentrifugation in combination with Western blotting analysis demonstrates that its alpha-subunit is associated with detergent-resistant membranes, suggesting that the channel might be localized within the membrane rafts in vivo. These findings are important in understanding the complex signalling pathways in smooth muscle and conditions such as premature labor and hypertension.

Animals↗
PubMed2004

Characterization of contractile activity and intracellular Ca2+ signalling in mouse myometrium.

OBJECTIVE: To characterize the contractile responses of mouse myometrium, the associated calcium (Ca2+) changes and the role of the sarcoplasmic reticulum (SR), and to better understand excitation contraction coupling in this tissue. METHODS: Strips of longitudinal myometrium were used, and Ca2+ was measured after loading with Indo-1. RESULTS: Intracellular Ca2+ transients, produced by Ca2+ entry, preceded phasic spontaneous contractions. Depolarization with high potassium concentration significantly increased the amplitude of the contractions and transformed the pattern of activity from phasic to tonic, with accompanying changes in intracellular Ca2+ concentration ([Ca2+]i). Oxytocin significantly stimulated contractile activity and [Ca2+]i above the level occurring spontaneously. Thus all forms of contractile activity were closely correlated with Ca2+. When the SR was emptied using a blocker of the SR calcium-adenosinetriphosphatase, cyclopiazonic acid, spontaneous Ca2+ and force transients increased greatly in frequency and amplitude. Ryanodine, a blocker of Ca(2+)-induced Ca2+ release (CICR), did not impair activity. In the absence of external Ca2+, oxytocin was able to release Ca2+ from the SR through IP3 but produced only a small increase in force, demonstrating a requirement for Ca2+ entry as part of the mechanism of agonist action. CONCLUSION: Mouse myometrium, (1) produces contractile activity reflecting changes in [Ca2+]i irrespective of the stimulus, (2) has a significant SR Ca2+ content releasable by agonists but not CICR, (3) has an SR acting to inhibit spontaneous activity, and (4) behaves qualitatively similarly to human and rat myometrium in major aspects of excitation contraction coupling and is therefore a useful model tissue.

Animals↗
PubMed2003

Obstructed labour.

Obstructed labour is an important cause of maternal deaths in communities in which undernutrition in childhood is common resulting in small pelves in women, and in which there is no easy access to functioning health facilities with the capability of carrying out operative deliveries. Obstructed labour also causes significant maternal morbidity in the short term (notably infection) and long term (notably obstetric fistulas). Fetal death from asphyxia is also common. There are differences in the behaviour of the uterus during obstructed labour, depending on whether the woman has delivered previously. The pattern in primigravid women (typically diminishing contractility with risk of infection and fistula) may result from tissue acidosis, whereas in parous women, contractility may be maintained with the risk of uterine rupture. Ultimately, tackling the problem of obstructed labour will require universal adequate nutritional intake from childhood and the ability to access adequately equipped and staffed clinical facilities when problems arise in labour. These seem still rather distant aspirations. In the meantime, strategies should be implemented to encourage early recognition of prolonged labour and appropriate clinical responses. The sequelae of obstructed labour can be an enormous source of human misery and the prevention of obstetric fistulas, and skilled treatment if they do occur, are important priorities in regions where obstructed labour is still common.

Cervical Ripening↗
PubMed2002

Development of gonadotropin-releasing hormone-1 neurons.

Gonadotropin releasing hormone-1 (GnRH-1) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain during prenatal development. Once within the brain, GnRH-1 cells become integral components of the CNS-pituitary-gonadal axis, essential for reproductive maturation and maintenance of reproductive function in adults. This review focuses on the lineage and development of the GnRH-1 neuroendocrine system. Although the migration of these cells from nose to brain has been well documented in a variety of species, many questions remain concerning the melecules and cues directing GnRH-1 cell differentiation, migration, axon targeting, and establishment and control of GnRH-1 secretion. These process most likely involve multiple and redundant cues because if these mechanisms fail, reproduction dysfunction will ensue and guarantee that this defect does not remain in the gene pool.

Animals↗
PubMed2002

17-Beta-estradiol regulates expression of genes that function in macrophage activation and cholesterol homeostasis.

Macrophage activation and cholesterol processing can be affected by changes in estrogen concentrations. However, there is a paucity of information about the genes and mechanisms regulating this estrogen effect. In primary monocyte-derived macrophages we detected transcript and protein for estrogen receptor beta (ERbeta). Determination of genes regulated by estrogen was completed using cDNA arrays and semiquantitative RT-PCR on RNA isolated from macrophages cultured in serum free media containing (5-10) x 10(-9)M 17-beta-estradiol and subsequently deprived of estrogen for a 24h period. The data indicate that the transcript levels of interleukin 1 receptor antagonist (IL-1ra), beta 2-microglobulin, annexin XI and the LXR(alpha) receptor significantly increased and that Ly-GDI transcript levels significantly decreased after estrogen withdrawal; data congruent with estrogen depletion regulating macrophage inflammatory and biochemical processes. Treatment of THP-1 cells with phorbol 12-myristate-13-acetate in the presence or absence of estrogen indicate that differentiation to a macrophage-like cell type was a prerequisite for production of the estrogen response. In addition, experiments using cycloheximide treatment, that blocks nascent protein synthesis, indicated that estrogen withdrawal affected the transcript levels of LXR(alpha) and IL-1ra through dissimilar pathways.

Blotting, Western↗
PubMed2002

Lipopolysaccharide-induced oestrogen receptor regulation in the paraventricular hypothalamic nucleus of lewis and Fischer rats.

Oestrogen receptor (ER) regulation of gene transcription in neurosecretory and pituitary cells has been proposed as an important mechanism for increased hypothalamic-pituitary-adrenal (HPA) axis responses in females of several mammalian species, including humans. Inbred female Fischer (F344/N) and Lewis (LEW/N) rats have similar oestrogen levels, although Fischer rats exhibit hyper- and Lewis rats hypo-HPA axis responses. The blunted HPA axis response of Lewis rats has been associated with their blunted hypothalamic corticotropin releasing hormone (CRH) expression. To determine if the female CRH expression deficiency in Lewis rats is associated with defective ER expression and regulation, hypothalamic paraventricular nucleus (PVN) transcript levels of CRH and ER were determined under basal conditions and after immune challenge. Microdissected PVN were obtained from control and lipopolysaccharide (LPS) treated Lewis and Fischer rats and CRH, ERalpha and beta mRNA levels were determined by semiquantitative reverse-transcriptase-polymerase chain reaction. In addition, ERalpha and beta protein levels were determined by semiquantitative Western blots. ERalpha and beta mRNA and protein levels in the PVN of control Fischer rats were significantly higher than in control Lewis rats. ERalpha and beta mRNA and protein levels in Fischer rats were reduced by LPS administration at the time of maximal CRH mRNA levels but did not change in Lewis rats, an effect independent of oestrogen levels. These data indicate that defective neuroendocrine HPA axis responses are associated with defective ER expression and regulation in Lewis PVN despite oestrogen concentrations.

Animals↗
PubMed2002

Molecular mechanisms for migration of placodally derived GnRH neurons.

Gonadotropin-releasing hormone (GnRH) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain along nasal axons. GnRH neurons appear to diverge from olfactory sensory cells during early stages of nasal placode differentiation. However, GnRH neurons rely on olfactory/vomeronasal axons as their pathway to the central nervous system (CNS). A novel factor, termed nasal embryonic luteinizing hormone-releasing hormone factor (NELF), was discovered in a differential screen of migrating versus nonmigrating GnRH neurons. NELF is expressed in olfactory sensory cells and GnRH cells in nasal areas. Antisense experiments demonstrated that knock-down of NELF decreased olfactory axon outgrowth and GnRH neuronal migration. These results indicate that NELF plays a role as a guidance molecule for olfactory axon projections and migration of GnRH cells. We hypothesize that NELF acts via a homophilic interaction and that NELF expression is critical for reproduction by insuring that GnRH cells reach the CNS. Furthermore, down-regulation of NELF on GnRH cells as they enter the telencephalon may allow GnRH cells to distinguish a different pathway(s) in the CNS (from those leading to olfactory regions) and thereby facilitate establishment of the appropriate adult-like GnRH distribution.

Animals↗
PubMed2002

Effect of inhibiting the sarcoplasmic reticulum on spontaneous and oxytocin-induced contractions of human myometrium.

OBJECTIVE: 1. To assess the contribution of the sarcoplasmic reticulum calcium store in the generation of uterine smooth muscle contractions; 2. to evaluate the contribution of calcium induced calcium release or ryanodine gated calcium channels to myometrial force production. DESIGN: Laboratory scientific study. METHODS: Myometrial strips were obtained from women undergoing elective prelabour caesarean section at term. These were loaded with the calcium sensitive indicator Indo-1 allowing simultaneous assessment of intracellular calcium concentrations and force production. The effect of exposing the strips to ryanodine (which abolishes calcium induced calcium release), caffeine (which activates calcium induced calcium release) and cyclopiazonic acid (which abolishes the sarcoplasmic reticulum calcium store) was examined. RESULTS: Exposure to ryanodine had no appreciable effect on either the amplitude or the duration of the myometrial calcium and force transients but did increase the frequency of contractions (139+/-5%). Caffeine did not potentiate force. Cyclopiazonic acid increased frequency, duration and amplitude of both calcium and force transients. The ability of oxytocin to provoke calcium and force transients in the absence of extracellular calcium was abolished by cyclopiazonic acid but not by ryanodine. CONCLUSIONS: These results demonstrate that calcium induced calcium release does not play a significant role in human myometrium and that no functioning role for the ryanodine receptors in human myometrial tissue could be shown. These data suggest that the sarcoplasmic reticulum may act to limit contractions and act as a calcium sink, rather than to amplify contractions.

Adult↗
PubMed2001

Simultaneous measurements of changes in sarcoplasmic reticulum and cytosolic.

1. The role of the sarcoplasmic reticulum (SR) was investigated in spontaneous and agonist-induced uterine Ca2+ transients, by combining low- (mag-fluo-4) and high-affinity (fura-2) indicators to measure intraluminal SR ([Ca2+]L) and cytosolic ([Ca2+]i) calcium concentration, simultaneously, in single smooth muscle cells from pregnant rat uterus. 2. Carbachol or ATP, in the absence of extracellular Ca2+, decreased [Ca2+]L and increased [Ca2+]i. Although some replenishment (around 50 %) occurred in its absence, extracellular Ca2+ was required for full replenishment of the SR Ca2+. 3. In 4/15 cells, ATP evoked oscillations of [Ca2+]i. These were accompanied by successive release and re-uptake of SR Ca2+. Inhibition of the SR Ca2+-ATPase with thapsigargin abolished the oscillations and luminal changes. 4. Spontaneous [Ca2+]i transients produced no detectable changes in [Ca2+]L. The larger [Ca2+]i transients evoked by high-K+ depolarisation increased [Ca2+]L. Spontaneous activity was inhibited when [Ca2+]L was increased. 5. These data show that it is possible to simultaneously measure SR and cytosolic [Ca2+], and to investigate their response to agonist application and spontaneous activity.

Animals↗
PubMed2001

pH regulation and buffering power in gastric smooth muscle.

Intracellular pH can have profound effects on tissue function, but little is known about how pH is regulated, buffered or affects the function of gastric smooth muscle. As the pH of gastric myocytes may alter with pathophysiological disturbance of the gastric lining, or reduction in blood flow to the stomach, these parameters were investigated. Intracellular pH was measured in strips of corpus from rats and guinea-pigs and pH perturbed by the addition of Na butyrate. pH regulation was investigated using pharmacological inhibitors and ionic substitutions. Resting pH was found to be around 7.0, and buffering power relatively high, compared to other muscles in both species. In the guinea-pig amiloride, EIPA and HOE694 prevented pH regulation from an acid load, but amiloride- and EIPA-insensitive pH-regulating mechanisms were found in the rat. The pH-regulatory mechanism present in the rat was also insensitive to DIDS, SITS and removal of external Cl-, but inhibited by Na+ substitution and HOE694. Acidification reduced gastric tone in both species. We conclude that pH alteration will significantly affect gastric contractility, despite a high capacity of the tissue to buffer and regulate pH change. The sensitivity to NHE inhibitors differs between rat and guinea-pig, suggesting that Na+/H+ exchanger isoform expression differs between gastric tissue.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

The effects of inhibiting Rho-associated kinase with Y-27632 on force and intracellular calcium in human myometrium.

Recent work has indicated that smooth muscle force production may be influenced by pathways not dependent upon the Ca2+-calmodulin phosphorylation of light chains. Few studies, however, have examined the importance of these pathways in intact muscles that contract phasically rather than tonically. Therefore, to determine whether the Ca2+-independent Rho-A and associated kinase (ROK) pathway can affect contractions of the intact human myometrium, we used Y-27632 to inhibit ROK. Three types of contractile activity were examined: spontaneous and those elicited by oxytocin and by depolarisation by high K+. Y-27632 decreased force significantly under all three conditions, without changing intracellular [Ca2+]. However, the effects on force were only large when the uterus was producing force tonically rather than phasically. This suggests that the Rho-A-ROK pathway may not be a potent modulator of force in the human myometrium under physiological conditions.

Amides↗
PubMed2001

Nasal embryonic LHRH factor (NELF) expression within the CNS and PNS of the rodent.

A novel protein (NELF) was identified screening embryonic luteinizing hormone releasing hormone (LHRH) neurons at different migrational states. Experiments in vitro revealed that NELF functions in olfactory axon outgrowth and subsequently alters LHRH neuronal migration. NELF was not restricted to LHRH neurons in the developing rodent. Multiple CNS and PNS tissues expressed this gene. To characterize the specific regions that express NELF in situ hybridization histochemistry was performed. Within the CNS, cells in the cortex, hippocampus, thalamus and olfactory regions express NELF pre- and postnatally.

Journal Article↗
PubMed2001

Development of luteinizing hormone releasing hormone neurones.

This review concentrates on some of the recent discoveries and future questions relevant to the development of the neuroendocrine luteinizing hormone releasing hormone (LHRH) cells. Neuroendocrine LHRH cells originate outside the central nervous system, in the nasal placode, and thereafter migrate into the forebrain during prenatal development. It is this population of LHRH cells that is responsible for reproductive function, becoming integral members of the hypothalamo-pituitary-gonadal axis postnatally. Disruption of the development of this system results in reproductive dysfunction. Increasing our understanding of LHRH neuroendocrine cells establishes conditions where we can look with greater precision at the mechanisms controlling reproductive development, both activation and failure. In addition, the ability to manipulate the molecular and cellular biology of the LHRH system opens the route to understanding critical neurobiological issues such as phenotypic commitment, axonal path finding and mechanisms involved in neuronal migration. Each of the topics is discussed in turn and potential mechanisms controlling the development of the neuroendocrine LHRH system are indicated.

Animals↗
PubMed2001

The physiological basis of uterine contractility: a short review.

In this review we discuss our current understanding of the cellular basis of uterine contractility, highlighting those areas requiring further study. It is clear that the basic processes of excitation-contraction coupling lie within the myometrial cell, and that these may be modified by agonists. Pacemaker activity, however, remains a mystery. The contribution of extracellular calcium entry to contraction is shown to be vital, whilst the role of the sarcoplasmic reticulum remains controversial. Much current experimental focus is on pathways controlling and regulating contraction, and we discuss sensitisation mechanisms and question their role in intact uterine preparations. Experimental Physiology (2001) 86.2, 239-246.

Animals↗
PubMed2000

Midline nasal tissue influences nestin expression in nasal-placode-derived luteinizing hormone-releasing hormone neurons during development.

Neurons differentiating into the luteinizing hormone-releasing hormone (LHRH) neuroendocrine phenotype are derived from the nasal placode. Cells within the vomeronasal organ anlage that turn on LHRH gene and peptide expression subsequently migrate into the forebrain where they influence reproductive function. The molecular and cellular cues regulating differentiation and migration of these cells are unknown. Discovery of developmental markers can indicate proteins directing or associated with differentiation. Analysis of such markers after manipulation of external cues can elucidate important extracellular differentiation signals. Embryonic LHRH neurons were examined in vivo for Mash-1 and nestin, two factors that delineate precursor populations in PNS and forebrain CNS cells. Nestin, but not Mash-1, was detected in early expressing LHRH cells in the vomeronasal organ anlage. These results were duplicated in LHRH neurons maintained in vitro in nasal explants. Such LHRH cells expressed nestin mRNA but not Mash-1 mRNA and were also negative for three other olfactory epithelial developmental transcription factors, Math4A, Math4C/neurogenin1, and NeuroD mRNA. Experimental manipulation of nasal explants revealed dual expression of nestin protein and LHRH in cells proximal to the vomeronasal organ anlage that was dependent upon midline cartilaginous/mesenchymal tissues. Prolonged nestin expression in LHRH cells after midline removal is consistent with nasal midline tissues modulating differentiation of LHRH neurons from the nasal placode.

Animals↗