Temporal aspects of excitation-contraction coupling in airway smooth muscle.

In airway smooth muscle (ASM), ACh induces propagating intracellular Ca2+ concentration ([Ca2+]i) oscillations (5-30 Hz). We hypothesized that, in ASM, coupling of elevations and reductions in [Ca2+]i to force generation and relaxation (excitation-contraction coupling) is slower than ACh-induced [Ca2+]i oscillations, leading to stable force generation. When we used real-time confocal imaging, the delay between elevated [Ca2+]i and contraction in intact porcine ASM cells was found to be approximately 450 ms. In beta-escin-permeabilized ASM strips, photolytic release of caged Ca2+ resulted in force generation after approximately 800 ms. When calmodulin (CaM) was added, this delay was shortened to approximately 500 ms. In the presence of exogenous CaM and 100 microM Ca2+, photolytic release of caged ATP led to force generation after approximately 80 ms. These results indicated significant delays due to CaM mobilization and Ca2+-CaM activation of myosin light chain kinase but much shorter delays introduced by myosin light chain kinase-induced phosphorylation of the regulatory myosin light chain MLC20 and cross-bridge recruitment. This was confirmed by prior thiophosphorylation of MLC20, in which force generation occurred approximately 50 ms after photolytic release of caged ATP, approximating the delay introduced by cross-bridge recruitment alone. The time required to reach maximum steady-state force was >15 s. Rapid chelation of [Ca2+]i after photolytic release of caged diazo-2 resulted in relaxation after a delay of approximately 1.2 s and 50% reduction in force after approximately 57 s. We conclude that in ASM cells agonist-induced [Ca2+]i oscillations are temporally and spatially integrated during excitation-contraction coupling, resulting in stable force production.     

Mechanism of ACh-induced asynchronous calcium waves and tonic contraction in porcine tracheal muscle bundle

Stimulation of the tracheal muscle bundle by acetylcholine (ACh) results in the generation of asynchronous repetitive Ca2+ waves (ACW) in intact tracheal smooth muscle (TSM) cells. We showed previously that ACW underlie cholinergic excitation-contraction coupling in porcine TSM and that Ca2+ entry through the L-type voltage-gated Ca2+ channel (VGCC) contributes partially to maintenance of the ACW. However, the mechanism of the ACW remains undefined. In this study, we pharmacologically characterized the mechanism of ACh-induced ACW in the intact porcine tracheal muscle bundle. We found that inhibition of receptor-operated channels/store-operated channels (ROC/SOC) by SKF-96365 completely abolished the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Blockade of Na+/Ca2+ exchange with KB-R7943 or 2',4'-dichlorobenzamil or removal of extracellular Na+ resulted in nearly complete inhibition of the nifedipine-insensitive component of ACh-mediated ACW and tonic contraction. Inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase by cyclopiazonic acid abolished the ongoing ACW. Application of 2-aminoethoxydiphenyl borate (2-APB) or xestospongin C to inhibit the inositol 1,4,5-trisphosphate-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels produced no effect on ACh-mediated ACW and tonic contraction. However, pretreatment with caffeine or ryanodine inhibited ACh-induced ACW. Furthermore, application of procaine or tetracaine prevented the generation and abolished the ongoing ACh-mediated ACW and tonic contraction. Collectively, these results indicate that the ACh-stimulated ACW in porcine TSM are produced by repetitive cycles of Ca2+ release from SR through 2-APB- and xestospongin C-insensitive Ca2+ release channels, and plasmalemmal Ca2+ entry involving reverse-mode Na+/Ca2+ exchange, ROC/SOC, and L-type VGCC is required to refill the SR via SERCA to support the ongoing ACW.     

Spatial and temporal aspects of ACh-induced [Ca2+]i oscillations in porcine tracheal smooth muscle

This study evaluated the relationship between regional elevation in intracellular calcium concentration ([Ca2+]i) induced by acetylcholine (ACh) and the global cellular responses in porcine tracheal smooth muscle (TSM) cells. Regional (approximately 1.5 microm3) and global (whole cell) changes in [Ca2+]i were measured in fluo-3 loaded TSM cells using real-time confocal microscopy. Regional responses appeared as propagating [Ca2+]i oscillations whereas global responses reflected the spatiotemporal integration of these regional responses. Within a region, [Ca2+]i oscillations were 'biphasic' with initial higher frequencies, followed by slower steady-state oscillations. With increasing ACh concentration, the peak (maximum value relative to 0 nM) of regional [Ca2+]i oscillations remained relatively constant, whereas both frequency and propagation velocity increased. In contrast, the global spatiotemporal integration of the regional oscillatory responses appeared as a concentration-dependent increase in peak as well as mean cellular [Ca2+]i. We conclude that the significance of ACh-induced [Ca2+]i oscillations lies in the establishment of mean [Ca2+]i level for slower Ca2+-dependent physiological processes via modulation of oscillation frequency and propagation velocity.     

Desynchronising effect of the endothelium on intracellular Ca2+ concentration dynamics in vascular smooth muscle cells of rat mesenteric arteries

The kinetics of the intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle cells (VSMCs) in rat small mesenteric arteries was investigated by confocal laser scanning microscopy using the fluorescent Ca2+ indicator fluo-3 AM. One micromole noradrenaline (NA) induced randomly distributed transient elevations of [Ca2+]i in several single VSMCs which were weakly temporally coupled. Higher NA concentrations of 3 or 10 microM, however, induced strongly synchronised [Ca2+]i oscillations in VSMCs. In preparations with intact endothelium, the synchronisation of [Ca2+]i signals was attenuated by acetylcholine (ACh) but augmented by the NO synthase antagonist L-NAME, pointing to a desynchronising effect of the endothelium even under basal conditions. In preparations with or without intact endothelium sodium nitroprusside (SNP) as well as the gap-junction uncoupler heptanol reversibly desynchronised the [Ca2+]i transients. The effect of ACh but not that of SNP was influenced by L-NAME. Propagated intracellular [Ca2+]i waves had a velocity of 25 microm/s. The phase shift of [Ca2+]i oscillations between single VSMCs were maximally 2s and independent of the distance of up to 90 microm between individual cells. Therefore, we consider intercellular [Ca2+]i waves to be too slow to account for the synchronisation of [Ca2+]i oscillations.We conclude that the coupling of [Ca2+]i signals in vascular smooth muscle cells is not constant but highly regulated by NA and by endothelium derived NO. Oscillations of vessel contraction at high sympathetic tone may be induced by synchronisation of [Ca2+]i transients of distinct VSMCs whereas endothelium derived NO inhibits vasomotion by desynchronising [Ca2+]i transients of single VSMCs.     

Endothelial cell Ca2+ increases are independent of membrane potential in pressurized rat mesenteric arteries

In rat mesenteric arteries, the ability of ACh to evoke hyperpolarization of smooth muscle cells and consummate dilatation relies on an increase in endothelial cell cytosolic free [Ca2+] and activation of Ca2+-activated K+ channels (KCa). The time course of average and spatially organized rises in endothelial cell [Ca2+]i and concomitant effects on membrane potential were investigated in individual cells of pressurized arteries and isolated sheets of native cells stimulated with ACh. In both cases, ACh stimulated a sustained and oscillating rise in endothelial cell [Ca2+]i. Overall, the oscillations remained asynchronous between cells, yet occasionally localized intercellular coordination became evident. In pressurized arteries, repetitive waves of Ca2+ moved longitudinally across endothelial cells, and depended on Ca2+-store refilling. The rise in endothelial cell Ca2+ was associated with sustained hyperpolarization of endothelial cells in both preparations. This hyperpolarization was also evident when recording from smooth muscle cells in pressurized arteries, and from resting membrane potential, selective inhibition of small-conductance K Ca (SK Ca) with apamin (50 nM) was sufficient to inhibit this response. In the presence of phenylephrine-tone, both apamin and the selective inhibitor of intermediate conductance K Ca (IK Ca) TRAM-34 (1 microM) were required to inhibit the non-nitric oxide-mediated dilatation to ACh. When hyperpolarization of endothelial cells was fully prevented either with inhibitors of K Ca or in KCl (35 mM)-depolarized cells, both the time course and frequency of oscillations in endothelial cell [Ca2+]i to ACh were unaffected. Together, these data show that although a rise in endothelial cell [Ca2+]i stimulates hyperpolarization, depletion of intracellular stores with ACh stimulates Ca2+-influx which is not significantly influenced by the increase in cellular electrochemical gradient for Ca2+ caused by that hyperpolarization.     

Calyculin-A disrupts subplasmalemmal junction and recurring Ca2+ waves in vascular smooth muscle

Excitation-contraction coupling (E-C coupling) in phenylephrine(PE)-stimulated rabbit inferior vena cava (IVC) depends on the generation of asynchronous recurring Ca2+ waves in the in situ vascular smooth muscle cells (VSMC). Previous studies by our group have implicated a putative non-selective cationic store-operated channel and the reverse-mode Na+-Ca2+ exchange in refilling of the intracellular Ca2+ store via the sarco/endoplasmic reticulum ATPase (SERCA) and the maintenance of the recurring Ca2+ waves. We hypothesize that for the proper functioning of these three Ca2+ translocators in the process of SR refilling, the plasma membrane (PM) and the underlying superficial sarcoplasmic reticulum (SR) form specialized PM-SR junctions, which are essential for the maintenance of the recurring Ca2+ waves. In order to test this hypothesis, calyculin-A, a serine/threonine phosphatase inhibitor that has been demonstrated to result in the disruption of the PM-SR junctions was used. In the control rabbit IVC, electron microscopy of the in situ VSMC indicates that 14.2+/-0.7% of the PM is closely apposed by the prominent superficial SR network, forming numerous flattened PM-SR junctional cytoplasmic spaces. In the control IVC stimulation with 5 microM PE resulted in sustained recurring Ca2+ waves with a frequency of 0.42+/-0.02 Hz. In calyculin-A treated rabbit IVC, a concentration-dependent dissociation of the superficial SR and loss of PM-SR junctions was observed. This progressive loss of the PM-SR junctions occurs over the same concentration range as the inhibition of PE-induced recurring Ca2+ waves. These findings offer support for the hypothesis that the presence of the PM-SR junctions is required for the generation of asynchronous recurring Ca2+ waves, which underlie excitation-contraction coupling in the VSMC of the rabbit IVC.     

Asynchronous calcium waves in smooth muscle cells

Asynchronous Ca2+ waves or wave-like [Ca2+]i oscillations constitute a specialized form of agonist-induced Ca2+ signaling that is observed in a variety of smooth muscle cell types. Functionally, it is involved in the contractile regulation of the smooth muscle cells as it signals for tonic contraction in certain smooth muscle cells while causing relaxation in others. Mechanistically, repetitive Ca2+ waves are produced by repetitive cycles of sarcoplasmic reticulum Ca2+ release followed by Ca2+ uptake. Plasmalemmal Ca2+ entry mechanisms are important for providing the additional Ca2+ necessary to maintain proper refilling of the sarcoplasmic reticulum Ca2+ store and support ongoing Ca2+ waves. In this paper, we will review the phenomenon of asynchronous Ca2+ waves in smooth muscle and discuss the scientific and clinical significance of this new understanding.     

Muscarinic receptor-mediated increase in cytoplasmic free Ca2+ in isolated bovine adrenal medullary cells. Effects of TMB-8 and phorbol ester TPA

The change in cytoplasmic free calcium, [Ca2+]i in isolated bovine adrenal medullary cells during stimulation by acetylcholine (ACh) in Ca2+-free incubation medium was measured using the fluorescent Ca2+ indicator quin2. ACh (1-100 microM) caused an increase in [Ca2+]i by mobilization of Ca2+ from the intracellular pool. Nicotine (10 microM) did not increase [Ca2+]i in the absence of extracellular Ca2+. Pretreatment of the cells with atropine (10 microM) completely inhibited ACh-induced increase in [Ca2+]i, whereas pretreatment with hexamethonium (100 microM) did not. The intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), inhibited ACh-induced increase in [Ca2+]i. The activator of protein kinase C 12-O-tetradecanoylphorbol-13-acetate (TPA), but not its 'inactive' analog 4 alpha-phorbol-12,13-didecanoate (PDD), also inhibited ACh-induced increase in [Ca2+]i. These findings suggest that in bovine adrenal medullary cells, stimulation of muscarinic ACh receptor causes an increase in [Ca2+]i by mobilizing Ca2+ from the intracellular pool and that protein kinase C is involved in 'termination' or 'down regulation' of this response.     

Intercellular communication: role of gap junctions in establishing the pattern of ATP-elicited Ca2+ oscillations and Ca2+-dependent currents in freshly isolated aortic smooth muscle cells

Cytosolic-free [Ca2+] was evaluated in freshly dissociated smooth muscle cells from mouse thoracic aorta by the ratio of Fura Red and Fluo 4 emitted fluorescence using confocal microscopy. The role of intercellular communication in forming and shaping ATP-elicited responses was demonstrated. Extracellular ATP (250 microM) elicited [Ca2+]i transient responses, sustained [Ca2+]i rise, periodic [Ca2+]i oscillations and aperiodic repetitive [Ca2+]i transients. Quantity of smooth muscle cells in the preparation responding to ATP with periodical [Ca2+]i oscillations depended on the density of isolated cells on the cover slip. ATP-elicited bursts of [Ca2+]i spikes in 66+/-7% of cells in dense and in 33+/-8.5% of cells in non-dense preparations. The number of cells responding to ATP with bursts of [Ca2+]i spikes decreased from 55+/-5% (n=84) to 14+/-3% (n=141) in dense preparations pretreated with carbenoxolone. Simultaneous measurement of [Ca2+]i and ion currents revealed a correlation between [Ca2+]i and current oscillations. ATP-elicited bursts of current spikes in 76% of cells regrouped in small clusters and in 9% of isolated cells. Clustered cells responding to ATP with current oscillations had higher membrane capacity than clustered cells with transient and sustained ATP-elicited responses. Lucifer Yellow (1% in 130 mM KCl) injected into one of clustered cells was transferred to the neighboring cell only when ATP-elicited oscillations. Fast application of carbenoxolone (100 microM) inhibited ATP (250 microM) elicited Ca2+-dependent current oscillations. Taken together these results suggest that the probability of ATP (250 microM) triggered cytosolic [Ca2+]i oscillations accompanied with K+ and Cl- current oscillations increased with the coupling of smooth muscle cells.     

Genistein inhibits contractile force, intracellular Ca2+ increase and Ca2+ oscillations induced by serotonin in rat aortic smooth muscle

The soy-derived isoflavones genistein and daidzein affect the contractile state of different kinds of smooth muscle. We describe acute effects of genistein and daidzein on contractile force and intracellular Ca2+ concentration ([Ca2+]i) in in situ smooth muscle of rat aorta. Serotonin (5-HT) (2 microM) or a depolarizing high K+ solution produced the contraction of aortic rings, which were immediately relaxed by 20 microM genistein and by 20 microM daidzein. Accordingly, both 5-HT and a high K+ solution increased the [Ca2+]i in in situ smooth muscle cells. Genistein strongly inhibited the [Ca2+]i increase evoked by 5-HT (74.0 +/- 7.3%, n = 11, p < 0.05), and had a smaller effect on high K+ induced [Ca2+]i increase (19.9 +/- 4.0%, n = 7, p < 0.05). The K+ channels blocker tetraethylammonium (TEA) (0.5 mM) diminished genistein effects on 5-HT-induced [Ca2+]i increase. Interestingly, during prolonged application of 5-HT, the [Ca2+]i oscillated and a short (90 s) preincubation with genistein (20 microM) significantly diminished the frequency of the oscillations. This effect was totally abolished by TEA. In conclusion, in rat aortic smooth muscle, genistein is capable of diminishing the increase in [Ca2+]i and in force evoked by 5-HT and high K+ solution, and of decreasing the frequency of [Ca2+]i oscillations induced by 5-HT. The short time required by genistein, and the relaxing effect of daidzein suggest that tyrosine kinases inhibition is not involved. The small inhibiting effect of genistein on the [Ca2+]i increase evoked by high K+ and the effect of TEA point to the activation by genistein of calcium-activated K+ channels.     

Role of M2 muscarinic receptors in airway smooth muscle contraction

Airway smooth muscle expresses both M2 and M3 muscarinic receptors with the majority of the receptors of the M2 subtype. Activation of M3 receptors, which couple to Gq, initiates contraction of airway smooth muscle while activation of M2 receptors, which couple to Gi, inhibits beta-adrenergic mediated relaxation. Increased sensitivity to intracellular Ca2+ is an important mechanism for agonist-induced contraction of airway smooth muscle but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization by acetylcholine (ACh) and endothelin-1 (ET-1) in porcine tracheal smooth muscle by measuring contractions at constant [Ca2+] in strips permeabilized with Staphylococcal alpha-toxin. Both ACh and ET-1 contracted airway smooth muscle at constant [Ca2+]. Pretreatment with pertussis toxin for 18-20 hours reduced ACh contractions, but had no effect on those of ET-1 or GTPgammaS. We conclude that the M2 muscarinic receptor contributes to airway smooth muscle contraction at constant [Ca2+] via the heterotrimeric G-protein Gi.     

Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate.

Intercellular Ca2+ signaling in primary cultures of glial cells was investigated with digital fluorescence video imaging. Mechanical stimulation of a single cell induced a wave of increased [Ca2+]i that was communicated to surrounding cells. This was followed by asynchronous Ca2+ oscillations in some cells. Similar communicated Ca2+ responses occurred in the absence of extracellular Ca2+, despite an initial decrease in [Ca2+]i in the stimulated cell. Mechanical stimulation in the presence of glutamate induced a typical communicated Ca2+ wave through cells undergoing asynchronous Ca2+ oscillations in response to glutamate. The coexistence of communicated Ca2+ waves and asynchronous Ca2+ oscillations suggests distinct mechanisms for intra- and intercellular Ca2+ signaling. This intercellular signaling may coordinate cooperative glial function.     

Increased secretion of leukemia inhibitory factor by immature airway smooth muscle cells enhances intracellular signaling and airway contractility

Airway smooth muscle cells (ASMC) play a major role in airway inflammation, hyperresponsiveness, and obstruction in asthma. However, very little is known regarding the relation between inflammatory mediators and cytokines and immature ASMC. The aim of this study was to evaluate 1) the secretion of leukemia inhibitory factor (LIF) (an IL-6 family neurotrophic cytokine) by ASMC; 2) intracellular calcium concentration ([Ca(2+)](i)) signaling; and 3) the effect of LIF on mast cell chemotaxis and rat airway contractility. Immature and adult human ASMC were cultured. ELISA and real-time PCR were performed to assess LIF protein secretion and mRNA production, [methyl-(3)H]thymidine incorporation to quantify ASMC DNA synthesis, a Boyden chamber to evaluate the effect of LIF on mast cell chemotaxis, microspectroflurimetry using indo-1 (at baseline and after stimulation bradykinin, U-46619, histamine, and acetylcholine, in the presence or absence of LIF or TNF-alpha) for [Ca(2+)](i) signaling, and isolated rat pup tracheae to determine the effect of LIF on airway contractility to ACh. TNF-alpha-stimulated immature ASMC produce more LIF mRNA and protein than adult ASMC, although this cytokine induces a moderate increase in DNA synthesis (+20%) in adult ASMC only. Human recombinant LIF exerts no chemotactic effect on human mast cells. In immature ASMC, ACh-induced [Ca(2+)](i) response was enhanced twofold after incubation with LIF, whereas TNF-alpha increased the [Ca(2+)](i) to U-46619 threefold. In TNF-alpha-exposed adult ASMC, [Ca(2+)](i) responses to ACh were of greater magnitude (sixfold increase) than in immature ASMC. Human recombinant LIF increased contractility to ACh by 50% in immature, isolated rat tracheae. Stimulated immature human ASMC greatly secrete LIF, thus potentially contributing to neuroimmune airway inflammation and subsequent remodeling. Increased LIF secretion enhances airway reactivity and [Ca(2+)](i) signaling.     

Ca(2+) oscillations, gradients, and homeostasis in vascular smooth muscle

Vascular smooth muscle shows both plasticity and heterogeneity with respect to Ca(2+) signaling. Physiological perturbations in cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) may take the form of a uniform maintained rise, a transient uniform [Ca(2+)](i) elevation, a transient localized rise in [Ca(2+)](i) (also known as spark and puff), a transient propagated wave of localized [Ca(2+)](i) elevation (Ca(2+) wave), recurring asynchronous Ca(2+) waves, or recurring synchronized Ca(2+) waves dependent on the type of blood vessel and the nature of stimulation. In this overview, evidence is presented which demonstrates that interactions of ion transporters located in the membranes of the cell, sarcoplasmic reticulum, and mitochondria form the basis of this plasticity of Ca(2+) signaling. We focus in particular on how the junctional complexes of plasmalemma and superficial sarcoplasmic reticulum, through the generation of local cytoplasmic Ca(2+) gradients, maintain [Ca(2+)](i) oscillations, couple these to either contraction or relaxation, and promote Ca(2+) cycling during homeostasis.     

The effects of isoproterenol on intracellular calcium concentration

beta-Adrenergic agonist, isoproterenol (ISO), is a potent relaxant of tracheal smooth muscle and inhibits carbachol-induced contraction. The effect of ISO on intracellular free Ca2+ concentration ([Ca2+]i) was examined in bovine tracheal smooth muscle strips, employing aequorin as Ca2+ indicator. Surprisingly, 10 microM ISO induces a 5-fold increase in [Ca2+]i which then gradually declines but still remains higher than basal after 1 h of stimulation. The ISO-induced increase in [Ca2+]i is dose-dependent, and the ED50 is approximately 50 nM. The ISO-induced increase in [Ca2+]i is inhibited by a beta-receptor blocker, propranolol, not by an alpha-blocker, phentolamine. The ISO-induced rise in [Ca2+]i is dependent on extracellular Ca2+. Forskolin, an adenylate cyclase activator, and vasoactive intestinal peptide, which is known to stimulate adenylate cyclase via a specific receptor in this tissue, have similar effects on [Ca2+]i, suggesting that a rise in cyclic AMP concentration mediates this effect of ISO on [Ca2+]i. Pretreatment of muscle with 10 microM ISO inhibits both the initial Ca2+ transient and the contractile response induced by 0.3 microM carbachol. Conversely, in carbachol-pretreated muscle strips, addition of ISO causes a fall rather than a rise in [Ca2+]i, and an inhibition of contraction. These results indicate that ISO has effects on cellular Ca2+ metabolism at more than a single site in bovine tracheal smooth muscle, that these effects are different in control and carbachol-pretreated muscle, and that the relaxing effect of ISO is not due solely to its effect on Ca2+ metabolism.     

Sarcomere mechanics in uniform and non-uniform cardiac muscle: a link between pump function and arrhythmias

Starling's Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac muscle. These Ca2+ waves can reversibly be induced in muscle with non-uniform excitation contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). These observations suggest the unifying hypothesis that force feedback to Ca2+ binding by Tn-C is responsible for Starling's Law and the ESPVR in uniform myocardium and leads in non-uniform myocardium to a surge of Ca2+ released by the myofilaments during relaxation, which initiates arrhythmogenic propagating Ca2+ release by the SR.     

Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP-gamma-S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura-2) and measurement of the Ca2(+)-dependent Cl- current (patch-clamp whole-cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1-0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)-dependent Cl- current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl- current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP-gamma-S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl- current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.     

Efficacy of muscarinic stimulation and mode of excitation-contraction coupling in bovine trachealis muscle

We have compared the efficacy of cromakalim and nifedipine to inhibit acetylcholine (ACh) and pilocarpine-induced tonic contractions in control preparations and in tissues where a fraction of the muscarinic receptor population had been removed by alkylation with phenoxybenzamine (PBZ). Both agonists induced contractions by stimulating pharmacologically similar receptors, probably of the M3 muscarinic subtype. The receptor reserve was larger, and the coupling between stimulation and contraction (E-C coupling) more efficient when ACh was the stimulating agonist. For stimulations that produced equal levels of muscle response, cromakalim was more efficacious in inhibiting contractions induced by pilocarpine. The efficacy of cromakalim in relaxing contractions induced by ACh increased when the number of functional receptors decreased. Cromakalim and nifedipine decreased the efficiency of E-C coupling for ACh and pilocarpine. Cromakalim efficacy decreased in a sigmoid manner when stimulating concentrations of ACh (and receptor occupancy) increased, and there was an inverse relationship between receptor occupancy by ACh and cromakalim efficacy. In the presence of TEA, a K+ channel blocker, nifedipine almost completely inhibited contractions induced by the M3 muscarinic agonist bethanechol. These data indicate that in bovine tracheal smooth muscle, electro-mechanical coupling is an inherent part of muscarinic E-C coupling, but its functional expression is dependent upon the efficacy of stimulation. The data also suggest that the M3 receptor is coupled to a cellular pathway linked with the activation of K+ channels that exerts a potent functional antagonism against activation of voltage-dependent Ca2+ entry.