Comparisons of different stages of chick embryonic development by the physiological regulatory response to hyposmotic challenge

Cardiac myocytes isolated and cultured from 11 day chick embryos present a Ca(2+)-dependent regulatory volume decrease (RVD) when exposed to hyposmotic stimulus. The RVD of myocytes from different embryonic stages were analyzed to evaluate their physiological performance through development. Among the several embryonic stages analyzed (6, 11, 16 and 19 days) only 19 day cardiac myocytes present a greater RVD when compared with 11 day (considered as control), the other ages showed no difference in the regulatory response. As it is known that RVD is Ca(2+) dependent, we decided to investigate the transient free Ca(2+) response during the hyposmotic swelling of the 11 and 19 day stages. The 11 day cardiac myocyte showed a transient 40% increase in intracellular free Ca(2+) when submitted to hyposmotic solutions, and the free Ca(2+) returned to baseline levels while the cells remained in hyposmotic buffer. However, the intracellular free Ca(2+) transient in the 19 day cells during hyposmotic challenge increases 100% and instead of returning to baseline levels, declines to 55% above control, well after the 11 day transient has returned to baseline. Also, quantitative fluorescence microscopy revealed that 19 day cardiac myocytes have more sarcoplasmic reticulum (SR) Ca(2+) ATPase sites per cell as compared to the 11 day cells. Our findings suggest that 19 day cells have more developed intracellular Ca(2+) stores (SR). By evoking the mechanism of Ca(2+) induced Ca(2+) release, the cells have more free Ca(2+) available for signaling the RVD during hyposmotic swelling.     

Vascular smooth muscle contraction evoked by cell volume modulation: role of the cytoskeleton network.

Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca(2+) channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca(2+)-free medium by only 30-50%. Several research groups reported cell volume-dependent cytoskeleton network rearrangements. This study examines the role of cytoskeleton proteins in cell volume-dependent contraction of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat thoracic aorta. Hyperosmotic shrinkage and hyposmotic swelling were triggered by modulation of medium osmolality; isosmotic shrinkage was induced by VSMR transfer from hypo- to isosmotic medium. The relative content of globular (G) and fibrillar (F) actin was estimated by fluorescence microscopy. Hyperosmotic shrinkage and hyposmotic swelling led to elevation of the F-actin/G-actin ratio by 2.5- and 1.8-fold respectively. Contraction of shrunken and swollen VSMR was insensitive to modulators of microtubules such as vinblastine, colchicine and docetaxel. Microfilament disassembly by cytochalasin B resulted in dramatic attenuation of the maximal amplitude of contraction of hyperosmotically-shrunken and hyposmotically-swollen VSMR, and almost completely abolished the contraction triggered by isosmotic shrinkage. These data suggest that both L-type Ca(2+) channel-mediated contraction of swollen vascular SMC and Ca(2+)(o)-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.     

Calcium and cytoskeleton signaling during cell volume regulation in isolated nematocytes of Aiptasia mutabilis (Cnidaria: Anthozoa)

Cell volume regulation has not been completely clarified in Coelenterates. The present investigation focuses on cell volume regulation under anisosmotic conditions, both hyposmotic and hypertonic, and on the underlying signals in nematocytes isolated from the Coelenterate Aiptasia mutabilis living in sea water. Nematocytes, once isolated from acontia, that were submitted to either hyposmotic (35%) and hypertonic shock (45%) show RVD and RVI capabilities, respectively. In order to ascertain the role of Ca2+ in triggering such regulatory mechanisms and the possible involvement of cytoskeleton components, tests were performed by employing either Ca2+ free conditions, Gd3+ as Ca2+ channel blockers, TFP as calmodulin inhibitor, colchicine as microtubule inhibitor and cytochalasin B as microfilament polymerization inhibitor. Results show that isolated nematocytes of A. mutabilis can regulate their volume upon both hyposmotic and hypertonic challenge. Ca2+ both from external medium and from internal stores is needed to perform RVD mechanisms, whereas, intracellular Ca2+ seems to be mainly involved in RVI. Moreover cytoskeletal components may play an important role since a significant RVD and RVI inhibition was observed in treated cells. On the basis of our observations further studies are warranted to further verify the role of signals, including phosphatases and phosphorylases, in cell volume regulation of primitive eukaryotic cells.     

An apoplastic Ca2+ sensor regulates internal Ca2+ release in aequorin-transformed tobacco cells

Removal of Ca(2+) from tobacco suspension cell medium has two immediate effects on cytosolic Ca(2+) fluxes: (i) externally derived Ca(2+) influx (occurring in response to cold shock or hypo-osmotic shock) is inhibited, and (ii) organellar Ca(2+) release (induced by a fungally derived defense elicitor, caffeine, or hypo-osmotic shock) is elevated. We show here that the enhanced release of internal Ca(2+) is likely due to increased discharge from a caffeine-sensitive store in response to a signal transduced from an extracellular Ca(2+) sensor. Thus, chelation of extracellular Ca(2+) in the absence of any other stimulus directly activates release of intracellular Ca(2+) into the cytosol. Evidence that this chelator-activated Ca(2+) flux is dependent on a signaling pathway includes its abrogation by prior treatment with caffeine, and its inhibition by protein kinase inhibitors (K252a and staurosporine) and anion channel blockers (niflumate and anthracene-9-carboxylate). An unexpected characteristic of tobacco cell adaptation to low external Ca(2+) was the emergence of a new Ca(2+) compartment that was inaccessible to external EGTA, yet responsive to the usual stimulants of extracellular Ca(2+) entry. Thus, cells that are exposed to EGTA for 20 min lose sensitivity to caffeine and defense elicitors, indicating that their intracellular Ca(2+) pools have been depleted. Surprisingly, these same cells simultaneously regain their ability to respond to stimuli that usually activate extracellular Ca(2+) influx even though all external Ca(2+) is chelated. Because this gradual restoration of Ca(2+) influx can be inhibited by the same kinase inhibitors that block EGTA-activated Ca(2+) release, we propose that chelator-activated Ca(2+) release from internal stores leads to deposition of this Ca(2+) into a novel EGTA- and caffeine-insensitive compartment that can subsequently be activated by stimulants of extracellular Ca(2+) entry.     

Biphasic effects of hyposmotic challenge on excitation-contraction coupling in rat ventricular myocytes

The effects of short (1 min) and long (7-10 min) exposure to hyposmotic solution on excitation-contraction coupling in rat ventricular myocytes were studied. After short exposure, the action potential duration at 90% repolarization (APD(90)), the intracellular Ca(2+) concentration ([Ca(2+)](i)) transient amplitude, and contraction increased, whereas the L-type Ca(2+) current (I(Ca, L)) amplitude decreased. Fractional sarcoplasmic reticulum (SR) Ca(2+) release increased but SR Ca(2+) load did not. After a long exposure, I(Ca,L), APD(90), [Ca(2+)](i) transient amplitude, and contraction decreased. The abbreviation of APD(90) was partially reversed by 50 microM DIDS, which is consistent with the participation of Cl(-) current activated by swelling. After 10-min exposure to hyposmotic solution in cells labeled with di-8-aminonaphthylethenylpyridinium, t-tubule patterning remained intact, suggesting the loss of de-t-tubulation was not responsible for the fall in I(Ca,L). After long exposure, Ca(2+) load of the SR was not increased, and swelling had no effect on the site-specific phosphorylation of phospholamban, but fractional SR Ca(2+) release was depressed. The initial positive inotropic response to hyposmotic challenge may be accounted for by enhanced coupling between Ca(2+) entry and release. The negative inotropic effect of prolonged exposure can be accounted for by shortening of the action potential duration and a fall in the I(Ca,L) amplitude.     

Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

Increases in global Ca(2+) in the endothelium are a crucial step in releasing relaxing factors to modulate arterial tone. In the present study we investigated spontaneous Ca(2+) events in endothelial cells, and the contribution of smooth muscle cells to these Ca(2+) events, in pressurized rat mesenteric resistance arteries. Spontaneous Ca(2+) events were observed under resting conditions in 34% of cells. These Ca(2+) events were absent in arteries preincubated with either cyclopiazonic acid or U-73122, but were unaffected by ryanodine or nicotinamide. Stimulation of smooth muscle cell depolarization and contraction with either phenylephrine or high concentrations of KCl significantly increased the frequency of endothelial cell Ca(2+) events. The putative gap junction uncouplers carbenoxolone and 18alpha-glycyrrhetinic acid each inhibited spontaneous and evoked Ca(2+) events, and the movement of calcein from endothelial to smooth muscle cells. In addition, spontaneous Ca(2+) events were diminished by nifedipine, lowering extracellular Ca(2+) levels, or by blockers of non-selective Ca(2+) influx pathways. These findings suggest that in pressurized rat mesenteric arteries, spontaneous Ca(2+) events in the endothelial cells appear to originate from endoplasmic reticulum IP(3) receptors, and are subject to regulation by surrounding smooth muscle cells via myoendothelial gap junctions, even under basal conditions.     

Volume-activated K(+)(Rb(+)) efflux in lactating rat mammary tissue

The effect of cell swelling, induced by a hyposmotic shock, on K(+)(Rb(+)) efflux from lactating rat mammary tissue explants has been studied. A hyposmotic challenge increased the fractional release of K(+)(Rb(+)) from mammary tissue in the absence and presence of the loop-diuretic bumetanide (100 microM). However, the volume-sensitive moiety of K(+)(Rb(+)) efflux was proportionately larger when bumetanide was present in the incubation medium. On the other hand, a hyposmotic shock appeared to reduce the bumetanide-sensitive component of K(+)(Rb(+)) efflux. The increase in K(+)(Rb(+)) efflux, induced by cell swelling, was dependent upon the extent of the hyposmotic challenge. In the presence of bumetanide, substituting Cl(-) with NO(3)(-) reduced the initial increase in volume-sensitive K(+)(Rb(+)) efflux. However, volume-sensitive K(+)(Rb(+)) release was prolonged in the presence of NO(3)(-). Volume-activated K(+)(Rb(+)) efflux from rat mammary tissue explants was inhibited by quinine. Cell swelling increased the intracellular concentration of Ca(2+) in a fashion which depended on the presence of extracellular Ca(2+). However, removing extracellular Ca(2+) did not inhibit volume-activated K(+)(Rb(+)) efflux from rat mammary tissue explants. The results are consistent with the presence of volume-activated K(+) channels in lactating rat mammary tissue. Volume-activated K(+) efflux may play a central role in mammary cell volume regulation.     

Differential coupling of alpha7 and non-alpha7 nicotinic acetylcholine receptors to calcium-induced calcium release and voltage-operated calcium channels in PC12 cells

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that can modulate various neuronal processes by altering intracellular Ca(2+) levels. Following nAChR stimulation Ca(2+) can enter cells either directly, through the intrinsic ion channel, or indirectly following voltage-operated Ca(2+) channel (VOCC) activation; Ca(2+) levels can subsequently be amplified via Ca(2+)-induced Ca(2+) release from intracellular stores. We have used subtype-selective nAChR agonists to investigate the Ca(2+) sources contributing to alpha7 and non-alpha7 nAChR-mediated increases in intracellular Ca(2+) in PC12 cells. Application of the alpha7 nAChR positive allosteric modulator PNU 120596 (10 mum), in conjunction with the alpha7 nAChR agonist, compound A [(R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide), 10 nm], produces a rapid increase in fluo-3 fluorescence that is prevented by the selective alpha7 nAChR antagonist alpha-bungarotoxin. The non-alpha7 nAChR agonist 5-Iodo-A-85380 produces alpha-bungarotoxin-insensitive increases in intracellular Ca(2+) (EC(50) = 11.2 mum). Using these selective agonists or KCl in conjunction with general and selective VOCC inhibitors, we demonstrate that the primary route of Ca(2+) entry following either non-alpha7 nAChR activation or KCl stimulation is via L-type VOCCs. In contrast, the alpha7 nAChR-mediated response is unaffected by VOCC blockers but is inhibited by modulators of intracellular Ca(2+) stores. These results indicate that alpha7 and non-alpha7 nAChRs are differentially coupled to Ca(2+)-induced Ca(2+) release and VOCCs, respectively.     

Evidence that IP3 and ryanodine-sensitive intra-cellular Ca2+ stores are not involved in acute hyposmotically-induced prolactin release in Tilapia.

Prolactin (PRL) cells from the euryhaline tilapia, Oreochromis mossambicus, behave like osmoreceptors by responding directly to reductions in medium osmolality with increased secretion of the osmoregulatory hormone PRL. Extracellular Ca(2+) is essential for the transduction of a hyposmotic stimulus into PRL release. In the current study, the presence and possible role of intracellular Ca(2+) stores during hyposmotic stimulation was investigated using pharmacological approaches. Changes in intracellular Ca(2+) concentration were measured with fura-2 in isolated PRL cells. Intracellular Ca(2+) stores were depleted in dispersed PRL cells with thapsigargin (1 microM) or cyclopiazonic acid (CPA, 10 microM). Pre-incubation with thapsigargin prevented the rise in [Ca(2+)](i) induced by lysophosphatidic acid (LPA, 1 microM), an activator of the IP(3) signalling cascade, but did not prevent the hyposmotically-induced rise in [Ca(2+)](i) in medium with normal [Ca(2+)] (2mM). Pre-treatment with CPA produced similar results. Prolactin release from dispersed cells followed a pattern that paralleled observed changes in [Ca(2+)](i). CPA inhibited LPA-induced prolactin release but not hyposmotically-induced release. Xestospongin C (1microM), an inhibitor of IP(3) receptors, had no effect on hyposmotically-induced PRL release. Pre-exposure to caffeine (10mM) or ryanodine (1microM) did not prevent a hyposmotically-induced rise in [Ca(2+)](i). Taken together these results indicate the presence of IP(3) and ryanodine-sensitive Ca(2+) stores in tilapia PRL cells. However, the rapid rise in intracellular [Ca(2+)] needed for acute PRL release in response to hyposmotic medium can occur independently of these intracellular Ca(2+) stores.     

Biphasic modulation of ryanodine receptors by sulfhydryl oxidation in rat ventricular myocytes

To understand better the modulation of ryanodine receptors (RyRs) during oxidative stress, the effect of 4,4'-dithiodipyridine (DTDP), a cell-permeant and thiol-reactive oxidant, on global Ca(2+) signal and spontaneous Ca(2+) sparks of rat ventricular myocytes was investigated. It was shown that a brief Ca(2+) transient was elicited by DTDP, when its concentration was raised to 100 microM DTDP. In addition a dose-dependent increase of cytoplasmic free Zn(2+) concentration was induced by DTDP. An increase of the frequency of spontaneous Ca(2+) sparks appeared at 3 microM DTDP, whereas higher concentration of DTDP caused a biphasic change of the frequency in both intact and permeabilized myocytes. Consistent with the biphasic effect, caffeine-induced Ca(2+) transients were similarly affected. Because DTDP did not reduce the free Ca(2+) concentration in the sarcoplasmic reticulum lumen, it is likely that the effects of DTDP on the frequency and caffeine-induced Ca(2+) transients are due mainly to sulfhydryl oxidation-induced activation and subsequent inactivation of RyRs. Unlike the frequency, the spatio-temporal properties of Ca(2+) sparks were not influenced by DTDP. The finding that DTDP does not affect the duration of Ca(2+) sparks is inconsistent with that the DTDP-induced increase of the open time of reconstituted RyR channels. The mechanism underlying this discrepancy, especially the possible role of the interaction between arrayed RyRs in myocytes, is discussed. This study suggests that, even if oxidative stress is mild enough not to cause intracellular Ca(2+) accumulation, it may affect signaling pathways through directly modulating the RyR or its complex and in turn changing the frequency of spontaneous Ca(2+) sparks. Thus, the functional importance of moderate oxidative stress should not be overlooked.     

Anion channels influence ECC by modulating L-type Ca(2+) channel in ventricular myocytes.

Anion channels are extensively expressed in the heart, but their roles in cardiac excitation-contraction coupling (ECC) are poorly understood. We, therefore, investigated the effects of anion channels on cardiac ventricular ECC. Edge detection, fura 2 fluorescence measurements, and whole cell patch-clamp techniques were used to measure cell shortening, the intracellular Ca(2+) transient, and the L-type Ca(2+) current (I(Ca,L)) in single rat ventricular myocytes. The anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid reversibly inhibited the Ca(2+) transients and cell shortening in a dose-dependent manner. Comparable results were observed when the majority of the extracellular Cl(-) was replaced with the relatively impermeant anions glutamate (Glt(-)) and aspartate (Asp(-)). NPPB and niflumic acid or the Cl(-) substitutes did not affect the resting intracellular Ca(2+) concentration but significantly inhibited I(Ca,L). In contrast, replacement of extracellular Cl(-) with the permeant anions NO, SCN(-), and Br(-) supported the ECC and I(Ca,L), which were still sensitive to blockade by NPPB. Exposure of cardiac ventricular myocytes to a hypotonic bath solution enhanced the amplitude of cell shortening and supported I(Ca,L), whereas hypertonic stress depressed the contraction and I(Ca,L). Moreover, cardiac contraction was completely abolished by NPPB (50 microM) under hypotonic conditions. It is concluded that a swelling-activated anion channel may be involved in the regulation of cardiac ECC through modulating L-type Ca(2+) channel activity.     

cADP ribose and [Ca(2+)](i) regulation in rat cardiac myocytes

cADP ribose (cADPR)-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) responses were assessed in acutely dissociated adult rat ventricular myocytes using real-time confocal microscopy. In quiescent single myocytes, injection of cADPR (0.1-10 microM) induced sustained, concentration-dependent [Ca(2+)](i) responses ranging from 50 to 500 nM, which were completely inhibited by 20 microM 8-amino-cADPR, a specific blocker of the cADPR receptor. In myocytes displaying spontaneous [Ca(2+)](i) waves, increasing concentrations of cADPR increased wave frequency up to approximately 250% of control. In electrically paced myocytes (0.5 Hz, 5-ms duration), cADPR increased the amplitude of [Ca(2+)](i) transients in a concentration-dependent fashion, up to 150% of control. Administration of 8-amino-cADPR inhibited both spontaneous waves as well as [Ca(2+)](i) responses to electrical stimulation, even in the absence of exogenous cADPR. However, subsequent [Ca(2+)](i) responses to 5 mM caffeine were only partially inhibited by 8-amino-cADPR. In contrast, even under conditions where ryanodine receptor (RyR) channels were blocked with ryanodine, high cADPR concentrations still induced an [Ca(2+)](i) response. These results indicate that in cardiac myocytes, cADPR induces Ca(2+) release from the sarcoplasmic reticulum through both RyR channels and via mechanisms independent of RyR channels.     

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

本研究旨在观察胍丁胺 (agmatine ,Agm)对分离大鼠心室肌细胞内游离钙浓度 ( [Ca2 +]i)的影响。用酶解方法分离大鼠心室肌细胞 ,用Fluo 3 AM负载 ,然后用激光共聚焦法测定单个心室肌细胞 [Ca2 +]i 的荧光强度 (fluorescenceintensity ,FI) ,结果以FI或相对荧光强度 (F/F0 % )表示。实验结果表明 ,在正常台氏液 (含钙 1 0mmol/L)和无钙台氏液中 ,单个大鼠心室肌细胞的荧光密度分别为 12 8 8± 13 8和 119 6± 13 6,两者无差异。Agm 0 1、1和 10mmol/L浓度依赖性地显著降低细胞的钙浓度 ;在正常台氏液中加入EGTA 3mmol/L ,Agm同样降低细胞的钙浓度。KCl 60mmol/L ,PE 3 0 μmol/L ,和Bay K 864 410 μmol/L均升高心室肌细胞的[Ca2 +]i。Agm同样降低高浓度KCl、Bay K 864 4和PE诱发的心室肌细胞 [Ca2 +]i 升高。当细胞外液钙浓度由 1mmol/L增加到 10mmol/L时 ,诱发心室肌细胞钙超载 ,同时部分心室肌细胞产生可传播的钙波 (Ca2 +wave) ,Agm 1mmol/L降低钙波的传播速度和持续时间 ,最终阻断钙波。以上结果提示 ,Agm对心室肌细胞的胞浆[Ca2 +]i具有抑制作用 ,此作用通过阻断电压依赖性钙通道而实现 ;并可能与抑制大鼠心室肌细胞内钙释放有关     

Exercise training corrects control of spontaneous calcium waves in hearts from myocardial infarction heart failure rats

Impaired cardiac control of intracellular diastolic Ca(2+) gives rise to arrhythmias. Whereas exercise training corrects abnormal cyclic Ca(2+) handling in heart failure, the effect on diastolic Ca(2+) remains unstudied. Here, we studied the effect of exercise training on the generation and propagation of spontaneous diastolic Ca(2+) waves in failing cardiomyocytes. Post-myocardial infarction heart failure was induced in Sprague-Dawley rats by coronary artery ligation. Echocardiography confirmed left ventricular infarctions of 40 ± 5%, whereas heart failure was indicated by increased left ventricular end-diastolic pressures, decreased contraction-relaxation rates, and pathological hypertrophy. Spontaneous Ca(2+) waves were imaged by laser linescanning confocal microscopy (488 nm excitation/505-530 nm emission) in 2 μM Fluo-3-loaded cardiomyocytes at 37°C and extracellular Ca(2+) of 1.2 and 5.0 mM. These studies showed that spontaneous Ca(2+) wave frequency was higher at 5.0 mM than 1.2 mM extracellular Ca(2+) in all rats, but failing cardiomyocytes generated 50% (P < 0.01) more waves compared to sham-operated controls at Ca(2+) 1.2 and 5.0 mM. Exercise training reduced the frequency of spontaneous waves at both 1.2 and 5.0 mM Ca(2+) (P < 0.05), although complete normalization was not achieved. Exercise training also increased the aborted/completed ratio of waves at 1.2 mM Ca(2+) (P < 0.01), but not 5.0 mM. Finally, we repeated these studies after inhibiting the nitric oxide synthase with L-NAME. No differential effects were found; thus, mediation did not involve the nitric oxide synthase. In conclusion, exercise training improved the cardiomyocyte control of diastolic Ca(2+) by reducing the Ca(2+) wave frequency and by improving the ability to abort spontaneous Ca(2+) waves after their generation, but before cell-wide propagation.     

Dynamic regulation of sarcoplasmic reticulum Ca(2+) content and release by luminal Ca(2+)-sensitive leak in rat ventricular myocytes.

In cardiac muscle, excitation-contraction (E-C) coupling is determined by the ability of the sarcoplasmic reticulum (SR) to store and release Ca(2+). It has been hypothesized that the Ca(2+) sequestration and release mechanisms might be functionally linked to optimize the E-C coupling process. To explore the relationships between the loading status of the SR and functional state of the Ca(2+) release mechanism, we examined the effects of changes in SR Ca(2+) content on spontaneous Ca(2+) sparks in saponin-permeabilized and patch-clamped rat ventricular myocytes. SR Ca(2+) content was manipulated by pharmacologically altering the capacities of either Ca(2+) uptake or leak. Ca(2+) sparks were recorded using a confocal microscope and Fluo-3 and were quantified considering missed events. SR Ca(2+) content was assessed by application of caffeine. Exposure of permeabilized cells to anti-phospholamban antibodies elevated the SR Ca(2+) content and increased the frequency of sparks. Suppression of the SR Ca(2+) pump by thapsigargin lowered [Ca(2+)](SR) and reduced the frequency of sparks. The ryanodine receptor (RyR) blockers tetracaine and Mg(2+) transiently suppressed the frequency of sparks. Upon washout of the drugs, sparking activity transiently overshot control levels. Low doses of caffeine transiently potentiated sparking activity upon application and transiently depressed the sparks upon removal. In patch-clamped cardiac myocytes, exposure to caffeine produced only a transient increase in the probability of sparks induced by depolarization. We interpret these results in terms of a novel dynamic control scheme for SR Ca(2+) cycling. A central element of this scheme is a luminal Ca(2+) sensor that links the functional activity of RyRs to the loading state of the SR, allowing cells to auto-regulate the size and functional state of their SR Ca(2+) pool. These results are important for understanding the regulation of intracellular Ca(2+) release and contractility in cardiac muscle.     

Caffeine-induced arrhythmias in murine hearts parallel changes in cellular Ca(2+) homeostasis

Heart failure leading to ventricular arrhythmogenesis is a major cause of clinical mortality and has been associated with a leak of sarcoplasmic reticular Ca(2+) into the cytosol due to increased open probabilities in cardiac ryanodine receptor Ca(2+)-release channels. Caffeine similarly increases such open probabilities, and so we explored its arrhythmogenic effects on intact murine hearts. A clinically established programmed electrical stimulation protocol adapted for studies of isolated intact mouse hearts demonstrated that caffeine (1 mM) increased the frequency of ventricular tachycardia from 0 to 100% yet left electrogram duration and latency unchanged during programmed electrical stimulation, thereby excluding slowed conduction as a cause of arrhythmogenesis. We then used fluorescence measurements of intracellular Ca(2+) concentration in isolated mouse ventricular cells to investigate parallel changes in Ca(2+) homeostasis associated with these arrhythmias. Both caffeine (1 mM) and FK506 (30 microM) reduced electrically evoked cytosolic Ca(2+) transients yet increased the frequency of spontaneous Ca(2+)-release events. Diltiazem (1 microM) but not nifedipine (1 microM) pretreatment suppressed these increases in frequency. Identical concentrations of diltiazem but not nifedipine correspondingly suppressed the arrhythmogenic effects of caffeine in whole hearts. These findings thus directly implicate spontaneous Ca(2+) waves in triggered arrhythmogenesis in intact hearts.     

The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles

Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca(2+)](i) of the SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH) induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs. The frequency of the Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular Ca(2+) or in the presence of Ni(2+), the frequency of the Ca(2+) oscillations declined and the airway relaxed. By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching. Each KCl-induced Ca(2+) oscillation consisted of a large Ca(2+) wave that was preceded by multiple localized Ca(2+) transients. KCl-induced responses were resistant to neurotransmitter blockers but were abolished by Ni(2+) or nifedipine and the absence of extracellular Ca(2+). Caffeine abolished the contractile effects of 5-HT, ACH, and KCl. These results indicate that (a) 5-HT and ACH induce airway SMC contraction by initiating Ca(2+) oscillations, (b) KCl induces Ca(2+) transients and twitching by overloading and releasing Ca(2+) from intracellular stores, (c) a sustained, Ni(2+)-sensitive, influx of Ca(2+) mediates the refilling of stores to maintain Ca(2+) oscillations and, in turn, SMC contraction, and (d) the magnitude of sustained airway SMC contraction is regulated by the frequency of Ca(2+) oscillations.     

Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).     

Biphasic effects of cell volume on excitation-contraction coupling in rabbit ventricular myocytes

We studied the effects of osmotic swelling on the components of excitation-contraction coupling in ventricular myocytes. Myocyte volume rapidly increased 30% in hyposmotic (0.6T) solution and was constant thereafter. Cell shortening transiently increased 31% after 4 min in 0.6T but then decreased to 68% of control after 20 min. In parallel, the L-type Ca(2+) current (I(Ca-L)) transiently increased 10% and then declined to 70% of control. Similar biphasic effects on shortening were observed under current clamp. In contrast, action potential duration was unchanged at 4 min but decreased to 72% of control after 20 min. Ca(2+) transients were measured with fura 2-AM. The emission ratio with excitation at 340 and 380 nm (f(340)/f(380)) decreased by 12% after 3 min in 0.6T, whereas shortening and I(Ca-L) increased at the same time. After 8 min, shortening, I(Ca-L), and the f(340)/f(380) ratio decreased 28, 25, and 59%, respectively. The results suggest that osmotic swelling causes biphasic changes in I(Ca-L) that contribute to its biphasic effects on contraction. In addition, swelling initially appears to reduce the Ca(2+) transient initiated by a given I(Ca-L), and later, both I(Ca-L) and the Ca(2+) transient are inhibited.