The roles of pH in regulation of uterine contraction in the laying hens.

In the laying hens, the uterus (shell gland) plays essential roles in calcium transfer for calcification of the eggshell and expulsion of the egg through the vagina for oviposition. Much is known about the effects of pH changes on eggshell production of the uterus. However, very little is understood about the effects of pH changes on uterine contractility. We investigated the effects of pH changes on uterine contraction in the laying hens. The laying hens were humanely killed, and strips of uterine smooth muscles were isolated. Isometric force was measured and the effects of intracellular and extracellular pH changes studied. The results show that alterations of pH clearly have marked effects on force in the hen uterus. Both intracellular and extracellular acidifications significantly decreased uterine activity, whether it arises spontaneously or in the presence of agonists such as prostaglandin F(2alpha) and arachidonic acid. Alkalinization produced the opposite effects. Thus, changes in pH can regulate uterine contraction. This insight into pH regulation of the uterine activity provides a focus for egg production management directed at physiological and pathological oviposition in the laying hens.

Mechanisms of uterine contractility in laying hens.

The physiological basis of uterine contractility in laying hens is not well understood, but a better understanding is important for understanding the mechanisms governing egg laying. The characteristics of uterine contractility arising spontaneously or by prostaglandin F(2alpha) (PGF(2alpha)) stimulation were therefore examined and the underlying mechanisms investigated. Uterine strips were isolated from laying hens 4h before oviposition and force measured. These strips remained healthy in vitro and produced regular spontaneous contractions. The contractions were phasic and could be recorded for several hours. Exposure to nifedipine, the specific L-type Ca channel blocker, led to the abolition of force. The contraction amplitude and frequency were significantly increased when Bay K8644, an agonist of L-type Ca channels, was applied or when the concentration of extracellular Ca was elevated. Spontaneous contractions were also significantly inhibited by wortmannin, the specific inhibitor of myosin light chain kinase (MLCK). When 1 microM PGF(2alpha) was applied to spontaneously contracting uterus, it significantly increased their amplitude and frequency of the contractions. As with spontaneous contractions, PGF(2alpha)-induced force production was abolished by nifedipine and wortmannin. In the absence of extracellular Ca, a small but tonic force was generated upon application of PGF(2alpha) which was not affected by wortmannin. Thus, extracellular Ca entry and MLCK phosphorylation are essential for uterine force production occurring spontaneously or by PGF(2alpha) stimulation. Our data supports the conclusion that the pathway dependent on extracellular Ca entry and MLCK phosphorylation predominates during PGF(2alpha) stimulation but suggests some involvement of an alternative force-producing pathway, presumably Ca-sensitization.

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.

Electrophysiological characterization and functional importance of calcium-activated chloride channel in rat uterine myocytes.

In order to better understand the mechanisms underlying excitation of the uterus, we have elucidated the characteristics and functional importance of Ca(2+)-activated Cl(-) currents ( I(Cl-Ca)) in pregnant rat myometrium. In 101/320 freshly isolated myocytes, there was a slowly inactivating tail current (162+/-48 pA) upon repolarization following depolarising steps. This current has a reversal potential close to that for chloride, and was shifted when [Cl(-)] was altered. It was activated by Ca(2+) (but not Ba(2+)) entry through L-type Ca(2+) channels, enhanced by the Ca(2+) channel agonist Bay K8644 (2 microM), and inhibited by the Cl(-) channel blockers, niflumic acid (10 microM) and anthracene-9-carboxylic acid (9-AC, 100 microM). We therefore conclude that the pregnant rat myometrium contains Ca(2+)-activated Cl(-) channels producing inward current in ~30% of its cells. When these channels were inhibited by niflumic acid or 9-AC in intact tissues, the frequency of spontaneous contractions, was significantly reduced. Niflumic acid was also shown to inhibit oxytocin-induced contractions and Ca(2+) transients. Neither 9-AC nor niflumic acid had any effect on high-K-invoked contractions. Taken together these data suggest that Ca(2+)-activated Cl(-) channels are activated by Ca(2+) entry and play a functionally important role in myometrium, probably by contributing to membrane potential and firing frequency (pacemakers) in these cells.

The effects of metabolic inhibition on intracellular calcium and contractility of human myometrium.

OBJECTIVE: Hypoxia occurs in the uterus during labour and may contribute to dysfunctional labours. We wanted to establish its effects on pregnant human myometrium and elucidate the mechanisms involved. DESIGN: Scientific study. SETTING: University Hospital and laboratories. POPULATION OR SAMPLE: Term pregnant women. METHODS: We measured contractions and intracellular [Ca(2+)] ([Ca(2+)](i)), in biopsies from term pregnant women undergoing elective caesarean section, and used cyanide to block oxidative phosphorylation. MAIN OUTCOME MEASURES: Changes in contractility and calcium. RESULTS: Although basal levels of [Ca(2+)](i) and tone rose, spontaneous and agonist-induced Ca(2+) transients and phasic contractions were rapidly reduced and abolished by cyanide. Neither stimulation of the uterus with oxytocin nor the Ca channel agonist, Bay K8644, prevented the changes produced by cyanide. The tonic force produced by depolarising the myometrium was also decreased by cyanide, but slowly recovered towards control levels, whereas [Ca(2+)](i) was maintained throughout. Similar data were obtained when nitrogen, rather than cyanide, was applied to the depolarised uterus. CONCLUSIONS: Impairment of oxidative phosphorylation is a potent depressor of phasic activity in human myometrium, irrespective of how it is produced, and our data suggest its effects lie at and beyond the surface membrane. Stimulation of the hypoxic uterus was not effective, which may explain the unpredictability of oxytocin application in some dysfunctional labours.

Calcium signaling and uterine contractility.

Changes in Ca(2+) signals within the myometrium have important functional consequences, as they determine contractility. We show that the basic phasic nature of uterine contractions, which is essential for successful labor, is critically dependent on Ca(2+) influx through voltage-gated L-type Ca(2+) channels, and hence in turn, on membrane potential. Thus changes in ion channel expression around term will play an important role in governing uterine excitability and contractility. There remains uncertainty about which channels are present in human myometrium and the nature of the pacemaker mechanism that initiates the action potential. The sarcoplasmic reticulum may augment, to a small extent, the necessary increase in [Ca(2+)] for contraction when agonists stimulate the uterus, but its main role appears to be to control excitability, acting as a negative feedback mechanism to limit contractions. Myosin light chain kinase activity and phosphorylation of myosin are essential components in the pathway of uterine contraction, once Ca(2+) has been elevated. Modulation of myosin light chain phosphatase activity can also influence contractions, but the effects are small compared with those modulating myosin light chain kinase. Ca(2+)-sensitizing pathways may not be utilized much in modulating normal phasic uterine activity, and caution is needed in extrapolating from in vitro experiments to in vivo conditions, especially because there may be redundant pathways. There is a need to study appropriate physiologic preparations, but these are not always available (eg, preterm laboring myometrium) and to combine functional studies with modern molecular approaches, to advance our understanding to a new level, from which better therapeutics will be developed.

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.

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.

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.

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.