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In this review, we give a state-of-the-art account of uterine contractility, focussing on excitation-contraction (electro-mechanical) coupling ( ECC). This will show how electrophysiological data and intracellular calcium measurements can be related to more modern techniques such as confocal microscopy and molecular biology, to advance our understanding of mechanical output and its modulation in the smooth muscle of the uterus, the myometrium. This new knowledge and understanding, for example concerning the role of the sarcoplasmic reticulum ( SR), or stretch-activated K channels, when linked to biochemical and molecular pathways, provides a clearer and better informed basis for the development of new drugs and targets. These are urgently needed to combat dysfunctions in excitation-contraction coupling that are clinically challenging, such as preterm labour, slow to progress labours and post-partum haemorrhage. It remains the case that scientific progress still needs to be made in areas such as pacemaking and understanding interactions between the uterine environment and ion channel activity. [ABSTRACT FROM AUTHOR]

2- and 3-dimensional confocal imaging of Fluo-4 loaded ureteric microvesells in situ allowed us to demonstrate distinct morphology, Ca2+ signalling and contractility in myocytes of arcade arterioles and pericytes of arcade venules. In myocytes and pericytes, Ca2+ signals arise exclusively from Ca2+ release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate receptors. Ca2+ transients in pericytes are less oscillatory, slower and longer-lasting than those in myocytes. The data obtained suggest differences in the mechanisms controlling local blood flow in precapillary arterioles and postcapillary venules. [ABSTRACT FROM AUTHOR]

The role of caveolins, signature proteins of caveolae, in arterial Ca2+ regulation is unknown. We investigated modulation of Ca2+ homeostasis by caveolin-1 and caveolin-3 using smooth muscle cells from rat cerebral resistance arteries. Membrane current and Ca2+ transients were simultaneously measured with voltage-clamped single cells. Membrane depolarization triggered Ca2+ current and increased intracellular Ca2+ concentration ([Ca2+]i). After repolarization, elevated [Ca2+]i returned to the resting level. Ca2+ removal rate was determined from the declining phase of the Ca2+ transient. Application of caveolin-1 antibody or caveolin-1 scaffolding domain peptide, corresponding to amino acid residues 82-101 of caveolin-1, significantly slowed Ca2+ removal rate at a measured [Ca2+]i of 250 nM, with little effect at a measured [Ca2+]i of 600 nM. Application of caveolin-3 antibody or caveolin-3 scaffolding domain peptide, corresponding to amino acid residues 55-74 of caveolin-3, also significantly slowed Ca2+ removal rate at a measured [Ca2+]i of 250 nM, with little effect at a measured [Ca2+]i of 600 nM. Likewise, application of calmodulin inhibitory peptide, autocamtide-2-related inhibitory peptide, and cyclosporine A, inhibitors for calmodulin, Ca2+/calmodulin-dependent protein kinase II, and calcineurin, also significantly inhibited Ca2+ removal rate at a measured [Ca2+]i of 250 nM but not at 600 nM. Application of cyclopiazonic acid, a sarcoplasmic reticulum Ca2+ ATPase inhibitor, also significantly inhibited Ca2+ removal rate at a measured [Ca2+]i of 250 nM but not at 600 nM. Our results suggest that caveolin-1 and caveolin-3 are important in Ca2+ removal of resistance artery smooth muscle cells. [ABSTRACT FROM AUTHOR]

In excitable tissues the refractory period is a critical control mechanism preventing hyperactivity and undesirable tetani, by preventing subsequent stimuli eliciting action potentials and Ca2+ entry. In ureteric smooth muscle, peristaltic waves that occur as invading pacemaker potentials produce long-lasting action potentials (300–800 ms) and extraordinarily long (more than 10 s) refractory periods, which prevent urine reflux and kidney damage. For smooth muscles neither the mechanisms underlying the refractory period nor the link between excitability and refractoriness are properly understood. Here we show that a negative feedback process, which depends on Ca2+ loading the sarcoplasmic reticulum (SR) during the action potential and on the subsequent activation of local releases of Ca2+ from the SR (sparks), stimulating plasmalemmal Ca2+-sensitive K+ (BK) channels, determines the refractory period of the action potential. As sparks gradually reduce the Ca2+ load in the SR, electrical inhibition is released, the refractory period is terminated and peristaltic contractions occur again. The refractory period can be manipulated, for example from 10 s to 100 s, by altering the Ca2+ content of the SR or release mechanism or by inhibiting BK channels. This insight into the control of excitability and hence function provides a focus for therapies directed at pathologies of smooth muscle. [ABSTRACT FROM AUTHOR]

Presents a corrected reprint of an article in "Nature" periodical concerning action potential refractory period in ureter smooth muscle.

: ObjectiveTo 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.: MethodsStrips of longitudinal myometrium were used, and Ca2+ was measured after loading with Indo-1.: ResultsIntracellular 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 Ca2+-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.: ConclusionMouse 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. [Copyright &y& Elsevier]

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 α-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. [ABSTRACT FROM AUTHOR]

Examines the role of myosin light chain (MLC) phosphorylation in facilitating in vivo filament reassembly after mechanical perturbation. Necessity of phosphorylation in thick filament formation; Use of a potent inhibitor of MLC; Postoscillation filament density.

Recent data have shown Ca2+-dependent activation of Rho-kinase by sustained depolarization of arterial smooth muscle. Visceral smooth muscles, however, contract phasically in response to action potentials and it is unclear whether Ca2+-dependent or -independent Rho-kinase activation occurs. We have therefore investigated this, under physiologically relevant conditions, in intact ureter. Action potentials, ionic currents, Ca2+ transients, myosin light chain (MLC) phosphorylation and phasic contraction evoked by action potentials in guinea-pig and rat ureter were investigated. In rat, but not guinea-pig ureter, three Rho-kinase inhibitors, Y-27632, HA-1077 and H-1152, significantly decreased phasic contractions and Ca2+ transients. Voltage- and current-clamp data showed that Rho-kinase inhibition reduced the plateau component of the action potential, inhibited Ca2+-channels and, indirectly, Ca2+-activated Cl- channels. The Ca2+ channel agonist Bay K8644 could reverse these effects. The K+ channel blocker TEA could also reverse the inhibitory effect of Y-27632 on the action potential and Ca2+ transient. Ca2+ transients and inward current, activated by carbachol-induced sarcoplasmic reticulum Ca2+ release, were not affected by Rho-kinase inhibition. Rho-kinase inhibition produced a Ca2+-independent increase in the relaxation rate of contraction, associated with acceleration of MLC dephosphorylation, which was sensitive to calyculin A. These data show for the first time that: (1) Rho-kinase has major effects on Ca2+ signalling associated with the action potential, (2) this effect is species dependent and (3) Rho-kinase controls relaxation of phasic contraction of myogenic origin. Thus Rho-kinase can modulate phasic smooth muscle in the absence of agonist, and the mechanisms are both Ca2+-dependent,... [ABSTRACT FROM AUTHOR]