Calcium Signaling in the Ventricular Myocardium of the Goto-Kakizaki Type 2 Diabetic Rat.

The association between diabetes mellitus (DM) and high mortality linked to cardiovascular disease (CVD) is a major concern worldwide. Clinical and preclinical studies have demonstrated a variety of diastolic and systolic dysfunctions in patients with type 2 diabetes mellitus (T2DM) with the severity of abnormalities depending on the patients' age and duration of diabetes. The cellular basis of hemodynamic dysfunction in a type 2 diabetic heart is still not well understood. The aim of this review is to evaluate our current understanding of contractile dysfunction and disturbances of Ca2+ transport in the Goto-Kakizaki (GK) diabetic rat heart. The GK rat is a widely used nonobese, nonhypertensive genetic model of T2DM which is characterized by insulin resistance, elevated blood glucose, alterations in blood lipid profile, and cardiac dysfunction.

Evidence that a Ca2+ sparks/STOCs coupling mechanism is responsible for the inhibitory effect of caffeine on electro-mechanical coupling in guinea pig ureteric smooth muscle.

Recent studies have highlighted the role of the sarcoplasmic reticulum (SR) in controlling excitability, Ca2+ signalling and contractility in smooth muscle. Caffeine, an agonist of ryanodine receptors (RyRs) on the SR has been previously shown to effect Ca2+ signalling but its effects on excitability and contractility are not so clear. We have studied the effects of low concentration of caffeine (1 mM) on Ca2+ signalling, action potential and contractility of guinea pig ureteric smooth muscle. Caffeine produced reversible inhibition of the action potentials, Ca2+ transients and phasic contractions evoked by electrical stimulation. It had no effect on the inward Ca2+ current or Ca2+ transient but increased the amplitude and the frequency of spontaneous transient outward currents (STOCs) in voltage clamped ureteric myocytes, suggesting Ca2+-activated K+ channels (BK) are affected by it. In isolated cells and cells in situ caffeine produced an increase in the frequency and the amplitude of Ca2+ sparks as well the number of spark discharging sites per cell. Inhibition of Ca2+ sparks by ryanodine (50 microM) or SR Ca2+-ATPase (SERCA) cyclopiazonic acid (CPA, 20 microM) or BKCa channels by iberiotoxin (200 nM) or TEA (1 mM), fully reversed the inhibitory effect of caffeine on Ca2+ transients and force evoked by electrical field stimulation (EFS). These data suggest that the inhibitory effect of caffeine on the action potential, Ca2+ transients and force in ureteric smooth muscle is caused by activation of Ca2+ sparks/STOCs coupling mechanism.

A new technique for simultaneous and in situ measurements of Ca2+ signals in arteriolar smooth muscle and endothelial cells.

We report here the first local and global Ca(2+) measurements made from in situ terminal arterioles. The advantages of the method are that there is minimal disturbance to the vessels, which retain their relationship to the tissue they are supplying (rat ureter) and the small size of vessel that can be studied. Good loading with the Ca(2+) indicator, Fluo-4 was obtained, and confocal sectioning through the tissue enabled vascular smooth muscle and endothelial cells to be clearly seen, along with red blood cells, nerve endings and the ureteric smooth muscle cells. We find the terminal arterioles to be extremely active, both spontaneously and in response to nor-adrenaline stimulation, with Ca(2+) sparks occurring in the vascular myocytes and Ca(2+) puffs in the endothelial cells. Even under resting conditions, endothelial cells produced oscillations and waves, which could pass from cell to cell, whereas the vascular myocytes only produced waves in response to agonist stimulation, and with no increase in the frequency of Ca(2+) sparks, and no spread from cell to cell. We compare our data to those obtained in dissected intact vessels and single cells. We conclude that this approach is a convenient and useful method for studying inter- and intracellular Ca(2+) signalling events and communication between cell types, particularly in very small vessels.

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 caffeine on chronotropic correlations in the myocardium of the rat and guinea pig]. / Vliianie kofeina na khronoinotropnye vzaimootnosheniia v miokarde krysy i morskoi svinki.

Force-frequency relations in the isolated papillary muscles of rats and guinea-pigs were compared using isometric force measurement technique. Stimulation frequency varied between 0.33 and 4 Hz. Under normal conditions the rat papillary muscle exhibits a negative force-frequency dependence which differs from ventricular preparations in many other mammals. Caffeine (10 mmol/l) introduced into the bath solution abolishes a negative force-frequency dependence in the papillary muscle of rat. During incubation of the rat ventricular preparations in the caffeine-containing solution the force-frequency relations measured on these muscles display the same behaviour as in guinea pig preparations. Caffeine has induced no changes in the force-frequency relations of guinea-pig ventricular preparations. A conclusion is made that caffeine-sensitive intracellular calcium stores participate in species-determined differences in force-frequency relations of the ventricular muscles.

Towards a computational reconstruction of the electrodynamics of premature and full term human labour.

We apply virtual tissue engineering to the full term human uterus with a view to reconstruction of the spatiotemporal patterns of electrical activity of the myometrium that control mechanical activity via intracellular calcium. The three-dimensional geometry of the gravid uterus has been reconstructed from segmented in vivo magnetic resonance imaging as well as ex vivo diffusion tensor magnetic resonance imaging to resolve fine scale tissue architecture. A late-pregnancy uterine smooth muscle cell model is constructed and bursting analysed using continuation algorithms. These cell models are incorporated into partial differential equation models for tissue synchronisation and propagation. The ultimate objective is to develop a quantitative and predictive understanding of the mechanisms that initiate and regulate labour.

Depletion of membrane cholesterol eliminates the Ca2+-activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes.

Changes in membrane cholesterol content have potent effects on cell signalling and contractility in rat myometrium and other smooth muscles. We have previously shown that depletion of cholesterol with methyl-beta-cyclodextrin (MCD) disrupts caveolar microdomains. The aim of this work was to determine the mechanism underlying the increase in Ca(2+) signalling and contractility occurring in the myometrium with MCD. Patch clamp data obtained on freshly isolated myocytes from the uterus of day 19-21 rats showed that outward K(+) current was significantly reduced by MCD. Membrane capacitance was also reduced. Cholesterol-saturated MCD had no effect on the amplitude of outward current suggesting that the reduction in the outward current was due to cholesterol depletion induced by MCD rather than a direct inhibitory action of MCD on the K(+) channels. Confocal visualization of the membrane bound indicator Calcium Green C18, revealed internalization of the surface membrane with MCD treatment. Large conductance, Ca(2+)-sensitive K(+) channel proteins have been shown to localize to caveolae. When these channels were blocked by iberiotoxin outward current was significantly reduced in the uterine myocytes; MCD treatment reduced the density of outward current. Following reduction of outward current by MCD pretreatment, iberiotoxin was unable to produce any additional decrease in the current, suggesting a common target. MCD treatment also increased the amplitude and frequency of spontaneous rises in cytosolic Ca(2+) level ([Ca(2+)](i) transients) in isolated myocytes. In intact rat myometrium, MCD treatment increased Ca(2+) signalling and contractility, consistent with previous findings, and this effect was also found to be reduced by BK channel inhibition. These data suggest that (1) disruption of cholesterol-rich microdomains and caveolae by MCD leads to a decrease in the BK channel current thus increasing cell excitability, and (2) the changes in membrane excitability produced by MCD underlie the changes found in Ca(2+) signalling and uterine contractility.

Vimentin-positive, c-kit-negative interstitial cells in human and rat uterus: a role in pacemaking?

The mechanism underlying spontaneous pacemaker potential in the uterus is not clearly understood. Several spontaneously active smooth muscles have interstitial cells of Cajal (ICCs) or ICC-like cells. We therefore examined cells from freshly dispersed uterine muscle strips (from pregnant human and rat myometrium) and in situ uterine preparations to determine the cell types present. Both preparations revealed numerous ICC-like cells; they were multipolar, with spider-like projections and enlarged central regions. These cells were readily distinguished from uterine myocytes by their morphology and ultrastructure, i.e., no myofilaments, numerous mitochondria, caveolae, and filaments. In addition, the ICC-like cells were noncontractile. These cells were negative to c-kit, a classic marker for ICCs. They stained positive for the intermediate filament, vimentin, a marker for cells of mesenchymal origin but not differentiated myocytes. The ICC-like cells had a more or less stable resting membrane potential of -58+/-7 mV compared with smooth-muscle cells, -65+/-13 mV, and produced outward current in response to voltage clamp pulses. However, in contrast with uterine myocytes, inward currents were not observed. This is the first description of ICC-like cells in myometrium and their role in the uterus is discussed, as possible inhibitors of intrinsic smooth-muscle activity.

Ca2+ entry, efflux and release in smooth muscle.

Control of smooth muscle is vital for health. The major route to contraction is a rise in intracellular [Ca2+], determined by the entry and efflux of Ca2+ and release and re-uptake into the sarcoplasmic reticulum (SR). We review these processes in myometrium, to better understand excitation-contraction coupling and develop strategies for preventing problematic labours. The main mechanism of elevating [Ca2+] is voltage-gated L-type channels, due to pacemaker activity, which can be modulated by agonists. The rise of [Ca2+] produces Ca-calmodulin and activates MLCK. This phosphorylates myosin and force results. Without Ca2+ entry uterine contraction fails. The Na/Ca exchanger (NCX) and plasma membrane Ca-ATPase (PMCA) remove Ca2+, with contributions of 30% and 70% respectively. Studies with PMCA-4 knockout mice show that it contributes to reducing [Ca2+] and relaxation. The SR contributes to relaxation by vectorially releasing Ca2+ to the efflux pathways, and thereby increasing their rates. Agonists binding produces IP3 which can release Ca from the SR but inhibition of SR Ca2+ release increases contractions and Ca2+ transients. It is suggested that SR Ca2+ targets K+ channels on the surface membrane and thereby feedback to inhibit excitability and contraction.

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.

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.

Ca2+ entry, efflux and release in smooth muscle

Control of smooth muscle is vital for health. The major route to contraction is a rise in intracellular [Ca2+], determined by the entry and efflux of Ca2+ and release and re-uptake into the sarcoplasmic reticulum (SR). We review these processes in myometrium, to better understand excitation-contraction coupling and develop strategies for preventing problematic labours. The main mechanism of elevating [Ca2+] is voltage-gated L-type channels, due to pacemaker activity, which can be modulated by agonists. The rise of [Ca2+] produces Ca-calmodulin and activates MLCK. This phosphorylates myosin and force results. Without Ca2+ entry uterine contraction fails. The Na/Ca exchanger (NCX) and plasma membrane Ca-ATPase (PMCA) remove Ca2+, with contributions of 30 percet and 70 percet respectively. Studies with PMCA-4 knockout mice show that it contributes to reducing [Ca2+] and relaxation. The SR contributes to relaxation by vectorially releasing Ca2+ to the efflux pathways, and thereby increasing their rates. Agonists binding produces IP3 which can release Ca from the SR but inhibition of SR Ca2+ release increases contractions and Ca2+ transients. It is suggested that SR Ca2+ targets K+ channels on the surface membrane and thereby feedback to inhibit excitability and contraction.