Excitation-Contraction Coupling in Ureteric Smooth Muscle: Mechanisms Driving Ureteric Peristalsis.

The ureter acts as a functional syncytium and is controlled by a propagating plateau-type action potential (AP) which gives rise to a wave of contraction (ureteral peristalsis) via a process called excitation-contraction (E-C)coupling. The second messenger Ca2+ activates Ca2+/calmodulin-dependent myosin light chain kinase-dependent phosphorylation of 20-kDa regulatory light chains of myosin which leads to ureteric contraction. Ca2+ entry from the extracellular space via voltage-gated L-type Ca2+ channels (VGCCs) provides the major source of activator Ca2+, responsible for generation of both the AP and a Ca2+ transient that appears as an intercellular Ca2+ wave. The AP, inward Ca2+ current, Ca2+ transient and twitch contraction are all fully blocked by the selective L-type Ca2+ channel blocker nifedipine. Ca2+ entry via VGCCs, coupled to activation of Ca2+-sensitive K+ (KCa) or Cl- (ClCa) channels, acts as a negative or positive feedback mechanism, respectively, to control excitability and the amplitude and duration of the plateau component of the AP, Ca2+ transient and twitch contraction. The ureter, isolated from the pelvis, is not spontaneously active. However, spontaneous activity can be initiated in the proximal and distal ureter by a variety of biological effectors such as neurotransmitters, paracrine, endocrine and inflammatory factors. Applied agonists depolarise ureteric smooth muscles cells to threshold of AP activation, initiating propagating intercellular AP-mediated Ca2+ waves to produce antegrade and/or retrograde ureteric peristalsis. Several mechanisms have been proposed to describe agonist-induced depolarization of ureteric smooth muscle, which include suppression of K+ channels, stimulation of ClCa current and activation of non-selective cation receptor/store operated channels.

Ca2+ Signalling in Pericytes.

Microcirculation is the generic name for the finest level of the circulatory system and consists of arteriolar and venular networks located upstream and downstream of capillaries, respectively. Anatomically arterioles are surrounded by a monolayer of spindle-shaped smooth muscle cells (myocytes), while terminal branches of precapillary arterioles, capillaries and all sections of postcapillary venules are surrounded by a monolayer of morphologically different perivascular cells (pericytes). Pericytes are essential components of the microvascular vessel wall. Wrapped around endothelial cells, they occupy a strategic position at the interface between the circulating blood and the interstitial space. There are physiological differences in the responses of pericytes and myocytes to vasoactive molecules, which suggest that these two types of vascular cells could have different functional roles in the regulation of local blood flow within the same microvascular bed. Also, pericytes may play different roles in different microcirculatory beds to meet the characteristics of individual organs. Contractile activity of pericytes and myocytes is controlled by changes of cytosolic free Ca2+concentration. In this chapter, we attempt to summarize the results in the field of Ca2+ signalling in pericytes especially in light of their contractile roles in different tissues and organs. We investigate the literature and describe our results regarding sources of Ca2+, relative importance and mechanisms of Ca2+ release and Ca2+ entry in control of the spatio-temporal characteristics of the Ca2+ signals in pericytes, where possible Ca2+ signalling and contractile responses in pericytes are compared to those of myocytes.

Sarcoplasmic reticulum function in smooth muscle.

The sarcoplasmic reticulum (SR) of smooth muscles presents many intriguing facets and questions concerning its roles, especially as these change with development, disease, and modulation of physiological activity. The SR's function was originally perceived to be synthetic and then that of a Ca store for the contractile proteins, acting as a Ca amplification mechanism as it does in striated muscles. Gradually, as investigators have struggled to find a convincing role for Ca-induced Ca release in many smooth muscles, a role in controlling excitability has emerged. This is the Ca spark/spontaneous transient outward current coupling mechanism which reduces excitability and limits contraction. Release of SR Ca occurs in response to inositol 1,4,5-trisphosphate, Ca, and nicotinic acid adenine dinucleotide phosphate, and depletion of SR Ca can initiate Ca entry, the mechanism of which is being investigated but seems to involve Stim and Orai as found in nonexcitable cells. The contribution of the elemental Ca signals from the SR, sparks and puffs, to global Ca signals, i.e., Ca waves and oscillations, is becoming clearer but is far from established. The dynamics of SR Ca release and uptake mechanisms are reviewed along with the control of luminal Ca. We review the growing list of the SR's functions that still includes Ca storage, contraction, and relaxation but has been expanded to encompass Ca homeostasis, generating local and global Ca signals, and contributing to cellular microdomains and signaling in other organelles, including mitochondria, lysosomes, and the nucleus. For an integrated approach, a review of aspects of the SR in health and disease and during development and aging are also included. While the sheer versatility of smooth muscle makes it foolish to have a "one model fits all" approach to this subject, we have tried to synthesize conclusions wherever possible.

Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition.

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 µM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 µM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon).

Calcium signalling in pericytes.

Recent advances in pericyte research have contributed to our understanding of the physiology and pathophysiology of microvessels. The microvasculature consists of arteriolar and venular networks located upstream and downstream of the capillaries. Arterioles are surrounded by a monolayer of spindle-shaped myocytes, while terminal branches of precapillary arterioles, capillaries and all sections of postcapillary venules are encircled by a monolayer of morphologically diverse pericytes. There are physiological differences in the response of pericytes and myocytes to vasoactive molecules, suggesting that these two vascular cell types could have different functional roles in the regulation of local blood flow. The contractile activity of pericytes and myocytes is controlled by changes of cytosolic free Ca(2+) concentration. In this short review, we summarize our results and those of other authors on the contractility of pericytes and their Ca(2+) signalling. We describe results regarding sources of Ca(2+) and mechanisms of Ca(2+) release and Ca(2+) entry in control of the spatiotemporal characteristics of the Ca(2+) signals in pericytes.

Escherichia coli-mediated impairment of ureteric contractility is uropathogenic E. coli specific.

BACKGROUND: Ureters are fundamental for keeping kidneys free from uropathogenic Escherichia coli (UPEC), but we have shown that 2 strains (J96 and 536) can subvert this role and reduce ureteric contractility. To determine whether this is (1) a widespread feature of UPEC, (2) exhibited only by UPEC, and (3) dependent upon type 1 fimbriae, we analyzed strains representing epidemiologically important multilocus sequence types ST131, ST73, and ST95 and non-UPEC E. coli. METHODS: Contractility and calcium transients in intact rat ureters were compared between strains. Mannose and fim mutants were used to investigate the role of type 1 fimbriae. RESULTS: Non-UPEC had no significant effect on contractility, with a mean decrease after 8 hours of 8.8%, compared with 8.8% in controls. UPEC effects on contractility were strain specific, with decreases from 9.47% to 96.7%. Mannose inhibited the effects of the most potent strains (CFT073 and UTI89) but had variable effects among other UPEC strains. Mutation and complementation studies showed that the effects of the UTI89 cystitis isolate were fimH dependent. CONCLUSIONS: We find that (1) non-UPEC do not affect ureteric contractility, (2) impairment of contractility is a common feature of UPEC, and (3) the mechanism varies between strains, but for the most potent UPEC type 1 fimbriae are involved.

How structure, Ca signals, and cellular communications underlie function in precapillary arterioles.

RATIONALE: Precapillary arterioles control blood flow to tissues and their correct function is vital. However, their small size has limited study and little is known concerning the calcium signals in their endothelial and muscle cells and how these relate to function. OBJECTIVE: We aimed to investigate whether these small vessels are specialized in terms of structure and calcium signaling. METHODS AND RESULTS: Using in situ confocal imaging we have studied the ultrastructure, Ca signaling and coordination of contraction in precapillary arterioles in ureter and vas deferens. We have compared the data to that from a small mesenteric artery. In the precapillary arteriole, 1 myocyte covers a approximately 10-microm length, and contraction of this single cell can decrease the diameter of this segment. In the mesenteric artery, more than 20 myocytes are required for this. In the precapillary arteriole, Ca signals arise solely from Ca release from the sarcoplasmic reticulum through inositol 1,4,5-trisphosphate-induced Ca release and not via ryanodine receptors. Agonist-induced Ca signals do not require Ca entry into the cell, do not spread or synchronize with neighboring cells, and are unaffected by endothelial stimulation, thereby allowing local control. This contrasts with the mesenteric artery, where Ca entry and ryanodine receptors are important and stimulation of the endothelium inhibits myocyte Ca signals and contraction. CONCLUSIONS: These data reveal the structural and signaling specializations underlying how blood flow is locally regulated, provide new insight into control of microcirculation, and provide a framework to explain its vulnerability to disease.

TRPM channels in the vasculature.

Recent studies show that mammalian melastatin TRPM nonselective cation channels (TRPM1-8), members of the largest and most diverse TRP subfamily, are widely expressed in the endothelium and vascular smooth muscles. When activated, these channels similarly to other TRPs permit the entry of sodium, calcium and magnesium, thus causing membrane depolarisation. Although membrane depolarisation reduces the driving force for calcium entry via TRPMs as well as other pathways for calcium entry, in smooth muscle myocytes expressing voltage-gated Ca(2+) channels the predominant functional effect is an increase in intracellular Ca(2+) concentration and myocyte contraction. This review focuses on several best documented aspects of vascular functions of TRPMs, including the role of TRPM2 in oxidant stress, regulation of endothelial permeability and cell death, the connection between TRPM4 and myogenic response, significance of TRPM7 for magnesium homeostasis, vessel injury and hypertension, and emerging evidence that the cold and menthol receptor TRPM8 is involved in the regulation of vascular tone.

Modulation of ureteric Ca signaling and contractility in humans and rats by uropathogenic E. coli.

Ascending urinary tract infections, a significant cause of kidney damage, are predominantly caused by uropathogenic Escherichia coli (UPEC). However, the role and mechanism of changes in ureteric function during infection are poorly understood. We therefore investigated the effects of UPEC on Ca signaling and contractions in rat (n = 17) and human (n = 6) ureters. Ca transients and force were measured and effects of UPEC on the urothelium were monitored in live tissues. In both species, luminal exposure of ureters to UPEC strains J96 and 536 caused significant time-dependent decreases in phasic and high K depolarization-induced contractility, associated with decreases in the amplitude and duration of the Ca transients. These changes were significant after 3-5 h and irreversible over the next 5 h. The infection causes increased activity of K channels, causing inhibition of voltage-gated Ca entry, and K channel blockers could reverse the effects of UPEC on ureteric function. A smaller direct effect on Ca entry also occurs. Nonpathogenic E. coli (TG2) or abluminal application of UPEC did not produce changes in Ca signaling or contractility. UPEC exposure also caused significant impairment of urothelial barrier function; luminal application of the Ca channel blocker nifedipine caused a reduction in contractions as it entered the tissue, an effect not observed in untreated ureters. Thus, UPEC impairs ureteric contractility in a Ca-dependent manner, largely caused by stimulation of potassium channels and this mechanism is dependent on host-urothelium interaction.

Morphology, calcium signaling and mechanical activity in human ureter.

PURPOSE: We determined the mechanisms of calcium signaling in the human ureter, and the relationship to peristaltic contractions and bundular structure in living tissue, thereby advancing the understanding of ureteral function in health and obstruction and reflux. MATERIALS AND METHODS: Confocal imaging of 31 ureters was performed and simultaneous force and calcium measurements were made. Immunohistochemistry and Western blotting were also performed. RESULTS: Confocal imaging showed a 3-dimensional network of smooth muscle bundles with no defined longitudinal or circular layers. Fast propagating Ca waves spread throughout the bundles, were closely associated with contraction and depended on L-type Ca channel entry. Immunohistochemistry and Western blotting demonstrated L-type Ca channels, Ca dependent K channels, sarcoplasmic reticulum Ca-adenosine triphosphatase isoforms 2 and 3, inositol triphosphate, and ryanodine receptors. Modulation of Ca and K channel activity was a potent mechanism for affecting Ca and force, whereas manipulation of the sarcoplasmic reticulum had little effect. CONCLUSIONS: To our knowledge this study represents the first measurements of Ca signals in the human ureter obtained during phasic contractions and in response to agonists. Results show that it is controlled by fast propagating Ca waves, which spread rapidly between the muscle bundles, producing regular contractions, and drugs that interfere with excitability or Ca entry through L-type Ca channels have profound effects on Ca signaling and contractility. These data are discussed in relation to the treatment of patients with suspected ureteral dysfunction using Ca entry blockers.

Smooth muscle gap-junctions allow propagation of intercellular Ca2+ waves and vasoconstriction due to Ca2+ based action potentials in rat mesenteric resistance arteries.

The role of vascular gap junctions in the conduction of intercellular Ca2+ and vasoconstriction along small resistance arteries is not entirely understood. Some depolarizing agents trigger conducted vasoconstriction while others only evoke a local depolarization. Here we use a novel technique to investigate the temporal and spatial relationship between intercellular Ca2+ signals generated by smooth muscle action potentials (APs) and vasoconstriction in mesenteric resistance arteries (MA). Pulses of exogenous KCl to depolarize the downstream end (T1) of a 3 mm long artery increased intracellular Ca2+ associated with vasoconstriction. The spatial spread and amplitude of both depended on the duration of the pulse, with only a restricted non-conducting vasoconstriction to a 1 s pulse. While blocking smooth muscle cell (SMC) K+ channels with TEA and activating L-type voltage-gated Ca2+ channels (VGCCs) with BayK 8644 spread was dramatically facilitated, so the 1 s pulse evoked intercellular Ca2+ waves and vasoconstriction that spread along an entire artery segment 3000 µm long. Ca2+ waves spread as nifedipine-sensitive Ca2+ spikes due to SMC action potentials, and evoked vasoconstriction. Both intercellular Ca2+ and vasoconstriction spread at circa 3 mm s-1 and were independent of the endothelium. The spread but not the generation of Ca2+ spikes was reversibly blocked by the gap junction inhibitor 18ß-GA. Thus, smooth muscle gap junctions enable depolarization to spread along resistance arteries, and once regenerative Ca2+-based APs occur, spread along the entire length of an artery followed by widespread vasoconstriction.

In situ calcium signaling: no calcium sparks detected in rat myometrium.

Controlled uterine smooth muscle activity is essential for our reproductive health. While we understand reasonably well the steps that produce contraction following a rise in intracellular [Ca], the mechanism controlling excitability and thus the rise of Ca, is less well understood. Here we examine the role of the internal Ca sore, the sarcoplasmic reticulum (SR), and its relation to surface membrane ion channels. We show that despite having a well-developed SR, the rat uterus does not produce the elemental and local Ca signals, known as Ca sparks. This in turn has consequences for excitability, as the negative feedback loop between these Ca signals and Ca-activated K (BK) channels on the surface membrane is lost. This may be important for producing the powerful long-lasting contractions of the uterus required during labor.

Calcium signalling in smooth muscle.

Calcium signalling in smooth muscles is complex, but our understanding of it has increased markedly in recent years. Thus, progress has been made in relating global Ca2+ signals to changes in force in smooth muscles and understanding the biochemical and molecular mechanisms involved in Ca2+ sensitization, i.e. altering the relation between Ca2+ and force. Attention is now focussed more on the role of the internal Ca2+ store, the sarcoplasmic reticulum (SR), global Ca2+ signals and control of excitability. Modern imaging techniques have shown the elaborate SR network in smooth muscles, along with the expression of IP3 and ryanodine receptors. The role and cross-talk between these two Ca(2+) release mechanisms, as well as possible compartmentalization of the SR Ca2+ store are discussed. The close proximity between SR and surface membrane has long been known but the details of this special region to Ca2+ signalling and the role of local sub-membrane Ca2+ concentrations and membrane microdomains are only now emerging. The activation of K+ and Cl- channels by local Ca2+ signals, can have profound effects on excitability and hence contraction. We examine the evidence for both Ca2+ sparks and puffs in controlling ion channel activity, as well as a fundamental role for Ca2+ sparks in governing the period of inexcitability in smooth muscle, i.e. the refractory period. Finally, the relation between different Ca2+ signals, e.g. sparks, waves and transients, to smooth muscle activity in health and disease is becoming clearer and will be discussed.

On the mechanisms whereby temperature affects excitation-contraction coupling in smooth muscle.

Moderate cooling of smooth muscle can modulate force production and may contribute to pathophysiological conditions, but the mechanisms underlying its effects are poorly understood. Interestingly, cooling increases force in rat ureter, but decreases it in guinea pigs. Therefore, this study used ureteric smooth muscle as a model system to elucidate the mechanisms of the effects of cooling on excitation-contraction coupling. Simultaneous recordings of force, intracellular [Ca(2+)], and electrical activity were made in intact ureter and ionic currents measured in isolated cells. The increase in force amplitude in rat ureter with cooling was found to be due to a significant increase in the duration of the Ca(2+) transient. This in turn was due to a marked prolongation of the action potential. In guinea pigs, both these parameters were much less affected by cooling. Examination of membrane currents revealed that differences in ion channel contribution to the action potential underlie these differences. In particular, cooling potentiated Ca(2+)-activated Cl(-) currents, which are present in rat but not guinea pig ureteric smooth muscle, and prolonged the plateau of the action potential and Ca(2+) entry. The force-Ca(2+) relationship revealed that the increased duration of the Ca(2+) transient was sufficient in the rat, but not in the guinea pig, to overcome kinetic lags produced in both species by cooling and potentiate force. Ca(2+) entry and release processes were largely temperature-insensitive, but the rate of relaxation was very temperature-sensitive. Effects of cooling on myosin light chain phosphatase, confirmed in experiments using calyculin A, appear to be the predominant mechanisms affecting relaxation. Thus, smooth muscle is diverse in its response to temperature, even when experimental variables, such as the mode of stimulation, are removed. Although the biochemical and mechanical events accompanying contraction are likely to be affected in similar ways by temperature, differences in electrical events lead to subsequent differences in these processes between smooth muscles.

Evidence that NO/cGMP/PKG signalling cascade mediates endothelium dependent inhibition of IP3R mediated Ca²âº oscillations in myocytes and pericytes of ureteric microvascular network in situ.

In ureteric microvessels the antagonistic relationship between Ca(2+) signalling in endothelium and Ca(2+) oscillations in myocytes and pericytes of arterioles and venules involves nitric oxide (NO), but the underlying mechanisms are not well understood. In the present study we investigated the effects of carbachol and NO donor SNAP on Ca(2+) signalling and vasomotor responses of arterioles and venules in intact urteric microvascular network in situ using confocal microscopy. Vasomotor responses of arterioles and venules induced by AVP correlated with the occurrence of Ca(2+) oscillations in the myocytes and pericytes and were not abolished by the removal of Ca(2+) from extracellular fluid. Carbachol-induced rise of intracellular Ca(2+) in endothelium was accompanied by the termination of the Ca(2+) oscillations in myocytes and pericytes. This carbachol-induced inhibitory effect on Ca(2+) oscillations in myocytes and pericytes was reversed by ODQ, an inhibitor of soluble guanylyl cyclase (sGC) and by Rp-8-pCPT-cGMPS, an inhibitor of protein kinase G (PKG). Ca(2+) oscillations in myocytes and pericytes were also effectively blocked by NO donor SNAP. An Inhibitory effect of SNAP was markedly enhanced by zaprinast, a selective inhibitor of cGMP-specific phosphodiesterase-5, and reversed by sGC inhibitor, ODQ and PKG inhibitor, Rp-8-pCPT-cGMPS. The cGMP analogue and selective PKG activator 8pCPT-cGMP also induced inhibition of the AVP-induced Ca(2+) oscillations in myocytes and pericytes. SNAP had no effects on Ca(2+) oscillations induced by caffeine in distributing arcade arterioles. Consequently, we conclude that NO- mediated inhibition of Ca(2+) oscillations in myocytes and pericytes predominantly recruits the cGMP/PKG dependent pathway. The inhibitory effect of NO/cGMP/PKG cascade is associated with suppressed Ca(2+) release from the SR of myocytes and pericytes selectively via the inositol triphosphate receptor (IP3R) channels.

Sarcoplasmic reticulum function and contractile consequences in ureteric smooth muscles.

This paper discusses the role of Ca2+-induced Ca2+ release (CICR) and inositol-1,4,5-trisphosphate (InsP3)-induced Ca2+ release (IICR) from the sarcoplasmic reticulum (SR) in the control of contractile activity in the ureter. The Ca2+ store in guinea-pig ureter has been found to be exclusively a CICR type with ryanodine receptors (RyRs) present. In the rat ureter the SR store is exclusively an IICR type with InsP3 receptors (InsP3Rs) present. Guinea-pig ureteric cells in vitro and in situ have been found to generate Ca2+ sparks--small localized, transient releases from RyRs. The sparks are enhanced by caffeine and blocked by emptying the SR. In rat cells Ca2+ puffs occur in response to agonists, representing the opening of InsP3Rs. The puffs can be abolished by heparin or store emptying. These SR Ca2+-release events affect the excitability of the ureteric cells. In guinea-pig cells, spontaneous transient outward currents (STOCs) can be recorded in response to caffeine application (an agonist for RyR), followed by a shortening of the plateau phase of the action potential. This in turn causes a decrease in the amplitude and duration of the contractions of the ureter. If the SR is inhibited then STOCs are abolished, the action potential plateau prolonged and force increased. Thus it is concluded that the SR acts to limit contraction in the guinea-pig ureter. The mechanism underlying this involves its Ca2+ release being directed to Ca2+-activated K+ channels on the surface membrane and causing STOCs and hyperpolarization, and controlling the duration of the action potential. In rat ureter IICR acts to potentiate force via membrane depolarization and increased L-type Ca2+ entry into the cells. Thus the SR can alter cell signalling and excitation-contraction coupling in the ureter, but its precise role is species dependent. The ureter, with its species-dependent expression of either IICR or CICR provides an ideal system (a natural transgenic model) for studying the SR. Eventually, we will be able to apply this knowledge to the human ureter, to increase our understanding of its functioning in health and disease.

Force and myosin light chain phosphorylation in dog airway smooth muscle activated in different ways.

To assess activation mechanisms of dog trachealis muscle and test whether isometric force generation could be separated from myosin light-chain (MLC) phosphorylation, force and phosphorylation were measured in the presence of wortmannin (a light-chain kinase inhibitor) or Y-27632 (a rho-kinase inhibitor) during electrically stimulated tetani and sustained contractures induced by acetylcholine, KCl, or calyculin A, a light-chain phosphatase inhibitor which caused irreversible contractures and both di- and mono-phosphorylation of light chain. Phosphorylation was not much more than half under any circumstances. A nearly constant proportionality between steady force and phosphorylation existed over a 9-fold force range during contractures and 25-sec tetani, except that force correlated best with the di-phosphorylated light chain produced by calyculin A. Phosphorylation was disproportionately higher than force at the outset of tetani, and this disproportion was exaggerated by Y-27632. The results suggest that about half the light chain is sequestered from kinases and that mechanical activation is tightly linked to phosphorylation, except at the outset of stimulation.

Atherosclerosis differentially affects calcium signalling in endothelial cells from aortic arch and thoracic aorta in Apolipoprotein E knockout mice.

Apolipoprotein-E knockout (ApoE(-/-)) mice develop hypercholesterolemia and are a useful model of atherosclerosis. Hypercholesterolemia alters intracellular Ca(2+) signalling in vascular endothelial cells but our understanding of these changes, especially in the early stages of the disease process, is limited. We therefore determined whether carbachol-mediated endothelial Ca(2+) signals differ in plaque-prone aortic arch compared to plaque-resistant thoracic aorta, of wild-type and ApoE(-/-) mice, and how this is affected by age and the presence of hypercholesterolemia. The extent of plaque development was determined using en-face staining with Sudan IV. Tissues were obtained from wild-type and ApoE(-/-) mice at 10 weeks (pre-plaques) and 24 weeks (established plaques). We found that even before development of plaques, significantly increased Ca(2+) responses were observed in arch endothelial cells. Even with aging and plaque formation, ApoE(-/-) thoracic responses were little changed, however a significantly enhanced Ca(2+) response was observed in arch, both adjacent to and away from lesions. In wild-type mice of any age, 1-2% of cells had oscillatory Ca(2+) responses. In young ApoE(-/-) and plaque-free regions of older ApoE(-/-), this is unchanged. However a significant increase in oscillations (~13-15%) occurred in thoracic and arch cells adjacent to lesions in older mice. Our data suggest that Ca(2+) signals in endothelial cells show specific changes both before and with plaque formation, that these changes are greatest in plaque-prone aortic arch cells, and that these changes will contribute to the reported deterioration of endothelium in atherosclerosis.

Store-operated Ca²âº entry and depolarization explain the anomalous behaviour of myometrial SR: effects of SERCA inhibition on electrical activity, Ca²âº and force.

In the myometrium SR Ca(2+) depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca(2+) sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca(2+)-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca(2+) transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 µM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca(2+)]i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca(2+) spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca(2+) and depolarization continued a point was reached when electrical and Ca(2+) spikes and phasic contractions ceased, and a maintained, tonic force and Ca(2+) was produced. Lanthanum, a non-selective blocker of store-operated Ca(2+) entry, but not the L-type Ca(2+) channel blocker nifedipine (1-10 µM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca(2+) depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca(2+) release is coupled to marked Ca(2+) entry, via store operated Ca(2+) channels, leading to depolarization and enhanced electrical and mechanical activity.

Atherosclerosis affects calcium signalling in endothelial cells from apolipoprotein E knockout mice before plaque formation.

Little is known about how hypercholesterolaemia affects Ca(2+) signalling in the vasculature of ApoE(-/-) mice, a model of atherosclerosis. Our objectives were therefore to determine (i) if hypercholesterolaemia alters Ca(2+) signalling in aortic endothelial cells before overt atherosclerotic lesions occur, (ii) how Ca(2+) signals are affected in older plaque-containing mice, and (iii) whether Ca(2+) signalling changes were translated into contractility differences. Using confocal microscopy we found agonist-specific Ca(2+) changes in endothelial cells. ATP responses were unchanged in ApoE(-/-) cells and methyl-ß-cyclodextrin, which lowers cholesterol, was without effect. In contrast, Ca(2+) signals to carbachol were significantly increased in ApoE(-/-) cells, an effect methyl-ß-cyclodextrin reversed. Ca(2+) signals were more oscillatory and store-operated Ca(2+) entry decreased as mice aged and plaques formed. Despite clearly increased Ca(2+) signals, aortic rings pre-contracted with phenylephrine had impaired relaxation to carbachol. This functional deficit increased with age, was not related to ROS generation, and could be partially rescued by methyl-ß-cyclodextrin. In conclusion, carbachol-induced calcium signalling and handling are significantly altered in endothelial cells of ApoE(-/-) mice before plaque development. We speculate that reduction in store-operated Ca(2+) entry may result in less efficient activation of eNOS and thus explain the reduced relaxatory response to CCh, despite the enhanced Ca(2+) response.