Effect of cilostazol, a phosphodiesterase type III inhibitor, on histamine-induced increase in [Ca2+]i and force in middle cerebral artery of the rabbit

1. The effect of cilostazol, an inhibitor of phosphodiesterase type III (PDE III), on the contraction induced by histamine was studied by making simultaneous measurements of isometric force and the intracellular concentration of Ca2+ ([Ca2+]i) in endothelium-denuded muscle strips from the peripheral part of the middle cerebral artery of the rabbit. 2. High K+ (80 mM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (10 microM) did not modify the resting [Ca2+]i, but it did significantly decrease the tonic contraction induced by high K+ without a corresponding change in the [Ca2+]i response. 3. Histamine (3 microM) produced a phasic, followed by a tonic increase in both [Ca2+]i and force. Cilostazol (3 and 10 microM) significantly reduced both the phasic and tonic increases in [Ca2+]i and force induced by histamine, in a concentration-dependent manner. 4. Rp-adenosine-3':5'-cyclic monophosphorothioate (Rp-cAMPS, 0.1 mM), a PDE-resistant inhibitor of protein kinase A (and as such a cyclic AMP antagonist), did not modify the increases in [Ca2+]i and force induced by histamine alone, but it did significantly decrease the cilostazol-induced inhibition of the histamine-induced responses. 5. In Ca2+-free solution containing 2 mM EGTA, both histamine (3 microM) and caffeine (10 mM) transiently increased [Ca2+]i and force. Cilostazol (1-10 microM) (i) significantly reduced the increases in [Ca2+]i and force induced by histamine, and (ii) significantly reduced the increase in force but not the increase in [Ca2+]i induced by caffeine. 6. In ryanodine-treated strips, which had functionally lost the histamine-sensitive Ca2+ storage sites, histamine (3 microM) slowly increased [Ca2+]i and force. Cilostazol (3 and 10 microM) lowered the resting [Ca2+]i, but did not modify the histamine-induced increase in [Ca2+]i, suggesting that functional Ca2+ storage sites are required for the cilostazol-induced inhibition of histamine-induced Ca2+ mobilization. 7. The [Ca2+]i-force relationship was obtained in ryanodine-treated strips by applying ascending concentrations of Ca2+ (0.16-2.6 mM) in Ca2+-free solution containing 100 mM K+. Histamine (3 microM) shifted the [Ca2+]i-force relationship to the left and increased the maximum Ca2+-induced force. Under the same conditions, whether in the presence or absence of 3 microM histamine, cilostazol (3-10 microM) shifted the [Ca2+]i-force relationship to the right without producing a change in the maximum Ca2+-induced force. 8. It is concluded that, in smooth muscle of the peripheral part of the rabbit middle cerebral artery, cilostazol attenuates the histamine-induced contraction both by inhibiting histamine-induced Ca2+ mobilization and by reducing the myofilament Ca2+ sensitivity. It is suggested that the increase in the cellular concentration of cyclic AMP that will follow the inhibition of PDE III may play an important role in the cilostazol-induced inhibition of the histamine-contraction.     

Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world

We measured [Ca2+]i and [Na+]i in isolated transgenic (TG) mouse myocytes overexpressing the Na+-Ca2+ exchanger and in wild-type (WT) myocytes. In TG myocytes, the peak systolic level and amplitude of electrically stimulated (ES) [Ca2+]i transients (0.25 Hz) were not significantly different from those in WT myocytes, but the time to peak [Ca2+]i was significantly prolonged. The decline of ES [Ca2+]i transients was significantly accelerated in TG myocytes. The decline of a long-duration (4-s) caffeine-induced [Ca2+]i transient was markedly faster in TG myocytes, and [Na+]i was identical in TG and WT myocytes, indicating that the overexpressed Na+-Ca2+ exchanger is functionally active. The decline of a short-duration (100-ms) caffeine-induced [Ca2+]i transient in 0 Na+/0 Ca2+ solution did not differ between the two groups, suggesting that the sarcoplasmic reticulum (SR) Ca2+-ATPase function is not altered by overexpression of the Na+-Ca2+ exchanger. There was no difference in L-type Ca2+ current density in WT and TG myocytes. However, the sensitivity of ES [Ca2+]i transients to nifedipine was reduced in TG myocytes. This maintenance of [Ca2+]i transients in nifedipine was inhibited by Ni2+ and required SR Ca2+ content, consistent with enhanced Ca2+ influx by reverse Na+-Ca2+ exchange, and the resulting Ca2+-induced Ca2+ release from SR. The rate of rise of [Ca2+]i transients in nifedipine in TG myocytes was much slower than when both the L-type Ca2+ current and the Na+-Ca2+ exchange current function together. In TG myocytes, action potential amplitude and action potential duration at 50% repolarization were reduced, and action potential duration at 90% repolarization was increased, relative to WT myocytes. These data suggest that under these conditions, overexpression of the Na+-Ca2+ exchanger in TG myocytes accelerates the decline of [Ca2+]i during relaxation, indicating enhanced forward Na+-Ca2+ exchanger function. Increased Ca2+ influx also appears to occur, consistent with enhanced reverse function. These findings provide support for the physiological importance of both these modes of Na+-Ca2+ exchange.     

Histamine-induced calcium released from cultured human mucosal microvascular endothelial cells from nasal inferior turbinate

Changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human mucosal microvascular endothelial cells (HMMECs) from nasal inferior turbinate were measured using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy. Histamine caused a transient increase in intracellular free Ca2+ in cell populations and in individual cells, followed by a decrease to a sustained elevation. Histamine (100 microM) elevated [Ca2+]i in HMMECs up to 563 +/- 20 nM from a resting level of 60 +/- 45 nM (means +/- SD, n = 31). Promethazine (a histamine H1 receptor antagonist) inhibited [Ca2+]i increase during histamine stimulation, whereas cimetidine (a H2 receptor antagonist) and thioperamide (a H3 receptor antagonist) showed no inhibition. These results suggest that the histamine increase [Ca2+]i in HMMECs induces both a Ca2+ release from stores and a Ca2+ influx through activation of the H1 receptor.     

Co-ordinated changes in cAMP, phosphorylated phospholamban, Ca2+ and contraction following beta-adrenergic stimulation of rat heart

Concentration-dependent changes in cyclic AMP (cAMP), site-specific phosphorylation of phospholamban, the intracellular calcium ([Ca2+]i) transient and contraction were measured in isolated rat ventricular myocytes exposed to the beta-adrenoceptor agonist isoprenaline. Cyclic AMP was measured by [125I]-cAMP scintillation proximity assay, phosphorylation of phospholamban at Ser16 and Thr17 was assessed using a pair of site-specific polyclonal antibodies, and [Ca2+]i was monitored with the fluorescent dye fura 2. Cyclic AMP rose to twice basal levels in the presence of 10(-6) M isoprenaline. The maximum increase in phosphorylation at Ser16 and Thr17 of phospholamban was seen at 10(-7) M isoprenaline. At this stage Ser16 phosphorylation was six times higher, and Thr17 phosphorylation was three times higher than that recorded in the absence of isoprenaline. Phosphorylation at Ser16 correlated more closely with changes in the [Ca2+]i transient and contraction than did phosphorylation at Thr17. This is the first study of its kind to measure simultaneous changes in cAMP, the phosphorylation of phospholamban, the [Ca2+]i transient and contraction over a range of concentrations of beta-agonist. The results suggest that phosphorylation of phospholamban at Thr17 is of lesser physiological relevance to the effects of beta-adrenergic stimulation on the heart than phosphorylation at Ser16.     

Tonic regulation of excitation-contraction coupling by basal protein kinase C activity in isolated cardiac myocytes

A high-speed imaging technique was used to investigate the effects of inhibitors and activators of protein kinase C (PKC) on the [Ca2+]i transients and contraction of fura-2 loaded rat ventricular cardiac myocytes. The amplitude of the [Ca2+]i transient was reduced following treatment with 100 nM phorbol 12,13-dibutyrate (PDBu), whereas the PKC inhibitors staurosporine (0.5 microM) and calphostin C (10 microM) increased [Ca2+]i transient amplitude, elevated basal [Ca2+]i and slowed the decay of the [Ca2+]i transient. These changes were paralleled by similar alterations in the rate and extent of cell shortening. The activity of nitrendipine-sensitive Ca2+ channels was monitored indirectly as the rate of Mn2+ quench of cytosolic fura-2 in electrically-paced cells. PDBu reduced Mn2+ influx by six-fold, whereas staurosporine and calphostin C increased the influx rate by eight-fold and seven-fold over basal quench, respectively. The caffeine releasable Ca2+ pool was reduced in the presence of PDBu and increased transiently in presence of staurosporine. The effects of PKC activation and inhibition on sarcoplasmic reticulum Ca2+ content may be secondary to alterations of sarcolemmal Ca2+ influx. However, the PKC inhibitors also decreased the rate of sarcoplasmic reticulum Ca2+ uptake in permeabilized myocytes, suggesting that a direct effect of PKC on the sarcoplasmic reticulum may contribute to the prolongation of the [Ca2+]i transient under these conditions. The present work demonstrates that basal PKC activity has a potent depressant effect, mediated primarily through inhibition of sarcolemmal Ca2+ influx, which may play a key role in setting the basal tone of cardiac muscle.     

Effects of Sho-saiko-to, San''o-shashin-to and Scutellariae Radix on intracellular Ca2+ mobilization in C6 rat glioma cells

Glial cells have a role in maintaining the function of neural cells. This study was undertaken to clarify the effects of baicalin and baicalein, flavonoids isolated from an important medicinal plant Scutellariae Radix (the root of Scutellaria baicalensis Georgi), on glial cell function using C6 rat glioma cells. Baicalin and baicalein caused concentration-dependent inhibition of a histamine-induced increase in intracellular Ca2+ concentrations ([Ca2+]i). The potency of baicalein was significantly greater than that of baicalin. The noradrenaline- and carbachol-induced increase in [Ca2+]i was also inhibited by baicalein and both drugs inhibited histamine-induced accumulation of total [3H]inositol phosphates, consistent with their inhibition of the increase in [Ca2+]i. These results suggest that baicalin and baicalein inhibit [Ca2+]i elevation by reducing phospholipase C activity. The inhibitory effects of baicalin and baicalein on [Ca2+]i elevation might be important in the interpretation of their pharmacological action on glial cells, such as inhibition of Ca2(+)-required enzyme phospholipase A2.     

Different mechanisms for [Ca2+]i oscillations induced by carbachol and high concentrations of [Ca2+]o in the rat ventricular myocyte

1. The purpose of the present study was to explore the different mechanisms of [Ca2+]i oscillations induced by high concentrations of either carbachol (CCh) or extracellular Ca2+ ([Ca2+]o). First, we compared the oscillations induced by CCh at concentrations of 100-300 micromol/L and [Ca2+]o (5 mmol/L) in the single rat ventricular myocyte. Second, we studied CCh- and [Ca2+]o-induced [Ca2+]i oscillations following either interference with the production of inositol trisphosphate (IP3), reductions in cytosolic Ca2+ ([Ca2+]i), inhibition of Ca2+ influx and Na+-Ca2+ exchange or depletion of Ca2+ from its intracellular store. 2. The [Ca2+]i oscillations induced by CCh were frequent and were superimposed on [Ca2+]i transients in electrically stimulated cells, whereas those induced by high [Ca2+]o were occasional and occurred in quiescent cells and between [Ca2+]i transients in electrically stimulated cells. In both cases, [Ca2+]i oscillations were preceded by an increase in resting levels of [Ca2+]i. 3. Carbachol-induced [Ca2+]i oscillations were accompanied by an increase in amplitude and prolongation of the time of decline to 80% of the peak of the [Ca2+]i transient, while high [Ca2+]o-induced [Ca2+]i oscillations were the opposite. 4. A reduction of [Ca2+]o to 0.1 mmol/L and treatment with Ni2+ or ryanodine or 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid AM (BAPTA-AM) abolished the [Ca2+]i oscillations induced by both CCh and high [Ca2+]o. 5. The calcium channel blockers verapamil and nifedipine and inhibitors of phospholipase C (neomycin and U-73122) abolished the [Ca2+]i oscillations induced by CCh; Li+ accelerated the onset of the [Ca2+]i oscillations induced by CCh. 6. These observations suggest that the mechanisms responsible for the [Ca2+]i oscillations induced by CCh and high [Ca2+]o are different from each other. Other than an increase in extracellular Ca2+ influx as a mechanism common for both CCh- and high [Ca2+]o-induced [Ca2+]i oscillations, the CCh-induced [Ca2+]i oscillations involve influx of Ca2+ via L-type Ca2+ channels, Na+-Ca2+ exchange, mobilization of intracellular Ca2+ and IP3 production.     

Fenspiride inhibits histamine-induced responses in a lung epithelial cell line

Using the human lung epithelial WI26VA4 cell line, we investigated the capacity of fenspiride, an anti-inflammatory drug with anti-bronchoconstrictor properties, to interfere with histamine-induced intracellular Ca2+ increase and eicosanoid formation. Histamine and a histamine H1 receptor agonist elicited a rapid and transient intracellular Ca2+ increase (0-60 s) in fluo 3-loaded WI26VA4 cells. This response was antagonized by the histamine H1 receptor antagonist, diphenhydramine, the histamine H2 receptor antagonist, cimetidine, having no effect. Fenspiride (10(-7)-10(-5) M) inhibited the histamine H1 receptor-induced Ca2+ increase. In addition, histamine induced a biphasic increase in arachidonic acid release. The initial rise (0-30 s), a rapid and transient arachidonic acid release, was responsible for the histamine-induced intracellular Ca2+ increase. In the second phase release (15-60 min), a sustained arachidonic acid release appeared to be associated with the formation of cyclooxygenase and lipoxygenase metabolites. Fenspiride (10(-5) M) abolished both phases of histamine-induced arachidonic acid release. These results suggest that anti-inflammatory and antibronchoconstrictor properties of fenspiride may result from the inhibition of these effects of histamine.     

Exposure of the cryptic Arg-Gly-Asp sequence in thrombospondin-1 by protein disulfide isomerase

OBJECTIVES: Human cardiac muscle from failing heart shows a decrease in active tension development and a rise in diastolic tension at stimulation frequencies above 50-60 beats/min due to both systolic and diastolic dysfunction. We have investigated underlying changes in cellular [Ca2+]i regulation. METHODS: Single ventricular myocytes were isolated enzymatically from the explanted hearts of transplant recipients with ischemic cardiomyopathy (nhearts = 5 ncells = 15) or dilated cardiomyopathy (nhearts = 6, ncells = 19). Cells were studied during whole-cell patch clamp with fluo-3 and fura-red as [Ca2+]i indicators (36 +/- 1 degrees C). RESULTS: In current clamp mode (action potential recording), the amplitude of Ca2+ release from the sarcoplasmic reticulum (SR) decreased at stimulation frequencies above 0.5 Hz; this decrease was more pronounced for cells from dilated cardiomyopathy. Diastolic [Ca2+]i increased at 1 and 2 Hz for both groups. Action potential duration (APD90) decreased with frequency in all cells; in addition there was a drop in plateau potential of 10 +/- 1 mV for cells from ischemic cardiomyopathy and of 13 +/- 2 mV for cells from dilated cardiomyopathy. In voltage clamp mode the L-type Ca2+ current showed reversible decrease during stimulation at 1 and 2 Hz. Recovery from inactivation during a double pulse protocol was slow (75 +/- 3% at 500 ms, 89 +/- 3% at 1000 ms) and followed the decay of the [Ca2+]i transient. CONCLUSIONS: The negative force-frequency relation of the failing human heart is due to a decrease in Ca2+ release of the cardiac myocytes at frequencies > or = 0.5 Hz, more pronounced in dilated than in ischemic cardiomyopathy. Inhibition of ICaL at higher frequencies, at least partially related to an increase in diastolic [Ca2+]i, will contribute to this negative staircase because of a decrease in the trigger for Ca2+ release, and of decreased loading of the SR.     

Involvement of extracellular and intracellular calcium sources in TRH-induced alpha-MSH secretion from frog melanotrope cells

The stimulatory effect of thyrotropin-releasing hormone (TRH) on alpha-melanocyte stimulating hormone (MSH) secretion from the frog pars intermedia is mediated through the phospholipase C (PLC) pathway but requires extracellular Ca2+. The aim of the present study was to investigate the respective contribution of extracellular and intracellular Ca2+ in the action of TRH on cytosolic calcium concentration ([Ca2+]i) and alpha-MSH release. In normal conditions, TRH (10(-7) M; 5 s) evoked two types of Ca2+ responses: in 63% of the cells, TRH caused a sustained and biphasic increase in [Ca2+]i while in 37% of the cells, TRH only induced a transient response. In the presence of EGTA or Ni2+, the stimulatory effect of TRH on [Ca2+]i and alpha-MSH secretion was totally suppressed. Nifedipine (10(-6) M) reduced by approximately 50% the amplitude of the two types of Ca2+ responses whereas omega-conotoxin GVIA (10(-7) M) suppressed the plateau-phase of the sustained response indicating that the activation of L-type Ca2+-channels (LCC) is required for initiation of the Ca2+ response while N-type Ca2+-channels (NCC) are involved in the second phase of the response. Paradoxically, neither nifedipine nor omega-conotoxin GVIA had any effect on TRH-induced alpha-MSH secretion. The PLC inhibitor U-73122 (10(-6) M) significantly reduced the transient increase in [Ca2+]i and totally suppressed the sustained phase of the Ca2+ response but had no effect on TRH-induced alpha-MSH secretion. The stimulatory effect of TRH on PLC activity was not effected by nifedipine and omega-conotoxin GVIA but was abolished in Ca2+-free medium. Ryanodine had no effect on the TRH-induced stimulation of [Ca2+]i and alpha-MSH secretion. Concomitant administration of nifedipine/omega-conotoxin GVIA or U-73122/omega-conotoxin GVIA markedly reduced the response to TRH but did not affect TRH-evoked alpha-MSH release. In contrast, concomitant administration of U-73122 and nifedipine significantly reduced the effect of TRH on both [Ca2+]i and alpha-MSH release. Taken together, these data indicate that, in melanotrope cells, activation of TRH receptors induces an initial Ca2+ influx through nifedipine- and omega-conotoxin-insensitive, Ni2+-sensitive Ca2+-channels which subsequently activates LCC and causes Ca2+ mobilization from intracellular pools by enhancing PLC activity. Activation of the PLC causes Ca2+ entry through NCC which is responsible for the plateau-phase of sustained Ca2+ response. Although nifedipine and U-73122, separately used, were devoid of effect on secretory response, Ca2+ entry through LCC and mobilization of intracellular Ca2+ are both involved in TRH-evoked alpha-MSH release because only one source of Ca2+ is sufficient for inducing maximal hormone release. In contrast, the Ca2+ influx through NCC does not contribute to TRH-induced alpha-MSH secretion.     

Complications associated with rapid caffeine application to cardiac myocytes that are not voltage clamped

The rapid application of caffeine to cardiac myocytes is commonly used to assess changes in the Ca2+ content of the sarcoplasmic reticulum (SR) and to study other parameters of intracellular Ca2+ regulation. Here we examined the effects of rapid caffeine application on membrane potential, intracellular Ca2+, and cell shortening in ventricular myocytes (rat, rabbit, guinea pig, dog) and atrial myocytes (rabbit) that were not voltage clamped. Conditioning pacing was used to achieve a steady-state level of SR Ca2+ loading prior to caffeine (10 mM) application. Caffeine transiently depolarized myocytes as expected from activation of forward Na+-Ca2+ exchange. However, we also found in each species (50% rat, 36% rabbit ventricular, 53% rabbit atrial, 56% guinea pig, 31% dog) that the caffeine-induced depolarization could also trigger an action potential. Caffeine-triggered potentials were completely blocked by thapsigargin (1 microM). The Ca2+ transient and contraction that accompanied caffeine-triggered action potentials had a larger magnitude and slower rate of decline (or relaxation) than occurred during caffeine-induced subthreshold depolarizations. Thus, the use of rapid caffeine application to study SR function and [Ca2+]i regulation in myocytes that are not voltage clamped can yield erroneous results.     

Histamine receptor subtypes mediating hyperpolarization in the isolated, perfused rat mesenteric pre-arteriolar bed

Histamine is a general dilator of rat blood vessels. We investigated the relative contribution of receptor subtypes to the rat mesenteric dilator responses initiated by histamine and related agonists. Histamine initiated dose, and endothelium-dependent, dilation of constricted mesenteric beds with an ED50 of 0.4 +/- 0.1 nmol. The ED50 was increased 10-fold by 0.1 microM chlorpheniramine (a histamine H1-receptor selective antagonist). Histamine H2 receptor blockade with tiotidine (0.1 microM) slightly decreased, while thioperamide (1 microM), a selective histamine H3 receptor antagonist, did not block histamine-induced dilation. Mesenteric bed dilation initiated by histamine H2 receptor selective agonists, amthamine and dimaprit, were antagonized markedly by tiotidine. However, the dilation initiated by the putative histamine H3 receptor selective agonists, R(-)- or S(+)-alpha-methylhistamine and imetit were not affected by thioperamide (1 microM). Histamine H2- and H3-receptor mediated dilator effects were endothelium-independent and were blocked by either excess (80 mM) extracellular K+, or 1 mM tetrabutylammonium (a non-selective K+ channel blocker), as well as by 1 microM dequalinium, a non-peptide blocker of the small conductance Ca2+-activated (SKCa) K+ channels. We conclude that (i) histamine H1 receptor subtype predominantly mediates endothelium-dependent dilator effect of histamine, and (ii) vascular hyperpolarization through opening of K+ channels (SKCa) mediate the dilator responses to histamine H2 receptor (amthamine and dimaprit) and the putative histamine H3 receptor (R(-)-alpha-methylhistamine and imetit) agonists.     

Cytoplasmic sodium, calcium and free energy change of the Na+/Ca2+-exchanger in rat ventricular myocytes

The relationship between changing driving force of the Na+/Ca2+-exchanger (deltaG(exch)) and associated cytosolic calcium fluxes was studied in rat ventricular myocytes. DeltaG(exch) was abruptly reversed by the reduction of extracellular sodium ([Na+]o) with or without sustained depolarization by the elevation of potassium ([K+]o). Cytosolic sodium ([Na+]i) and calcium ([Ca2+]i) were measured with SBFI and indo-1 respectively and the time course of recovery of deltaG(exch) was calculated. Following abrupt reversal of deltaG(exch) from +4.1 to -9.2 kJ/mol [Na+]i exponentially decreased from 9.6-2.5 mmol/l (t(1/2) about 30 s) and [Ca2+]i transiently increased to a peak value after about 30 s. Negative values of deltaG(exch) were associated with an increase and positive values with a decrease of [Ca2+]i. Equilibrium (deltaG(exch) = 0) was reached after about 30 s coinciding with the time to peak [Ca2+]i. After 180 s deltaG(exch) reached a new steady state at +3.5 kJ/mol. Inhibition of SR with ryanodine or thapsigargin reduced the amplitude of the [Ca2+]i transient and shifted its peak to 80 s, but did not affect the time course of [Na+]i changes. In the presence of ryanodine or thapsigargin the time required for deltaG(exch) to recover to equilibrium was also shifted to 80 s. When we changed the deltaG(exch) to the same extent by the reduction of [Na+]o in combination with a sustained depolarization, [Na+]i decreased less and the amplitude of [Ca2+]i transient was much enhanced. This increase of [Ca2+]i was completely abolished by verapamil. DeltaG(exch) only recovered to a little above equilibrium (+1 kJ/mol). Inhibition of the Na+/K+-ATPase with ouabain entirely prevented the decrease of [Na+]i and caused a much larger increase of [Ca2+]i, which remained elevated; deltaG(exch) recovered to equilibrium and never returned to positive values. The rate of change of total cytosolic calcium was related to deltaG(exch), despite the fact that the calcium flux associated with the exchanger itself contributed only about 10%; SR related flux contributed by about 90% to the rate of change of total cytosolic calcium. In summary, reduction of [Na+]o causes reversal of the Na+/Ca2+-exchanger and its driving force deltaG(exch), a transient increase of [Ca2+]i and a decrease of [Na+]i. The influx of calcium associated with reversed deltaG(exch) triggers the release of calcium from SR. Both the decrease of [Na+]i and the increase of [Ca2+]i contribute to the recovery of deltaG(exch) to equilibrium. The time at which deltaG(exch) reaches equilibrium always coincides with the time to peak of [Ca2+]i transient. Activation of the Na+/K+-ATPase is required to reduce [Na+]i and recover deltaG(exch) to positive values in order to reduce [Ca2+]i. We conclude that deltaG(exch) is a major regulator of cytosolic calcium by interaction with SR.     

New advances in sex preselection

The effects of peroxynitrite (ONOO-) on cultured cardiac myocytes were examined by simultaneous measurements of intracellular Ca2+ ([Ca2+]i) and contractile function. On exposure to 0.2 mM ONOO-, [Ca2+]i increased to beyond the systolic level within 5 min with a concomitant decrease in spontaneous contraction of myocytes followed by complete arrest. Addition of a L-type Ca2+ channel blocker or removal of extracellular Ca2+ prevented the ONOO(-)-induced increase in [Ca2+]i, indicating that the increase in [Ca2+]i was caused by the enhanced influx of Ca2+ through the plasma membrane and not by the enhanced release from sarcoplasmic reticulum (SR). Plasma membrane fluidity and concentration of the thiobarbiturate acid-reactive substance (TBARS) in the cells remained unchanged by the ONOO- treatment. The complete cessation of contraction of myocytes persisted even under the massive increase in [Ca2+]i, which was induced by an additional saponin (5 microM) treatment. In conclusion, ONOO- increases [Ca2+]i in myocytes through disturbance of Ca2+ transport systems in the plasma membrane and impairs contractile protein.     

Ca2+ transients in cardiac myocytes measured with high and low affinity Ca2+ indicators

Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.     

Withdrawal of acetylcholine elicits Ca2+-induced delayed afterdepolarizations in cat atrial myocytes

BACKGROUND: Recent experiments in atrial myocytes indicate that withdrawal of cholinergic agonist can directly increase Ca2+ influx via L-type Ca2+ current and stimulate Ca2+ uptake into the sarcoplasmic reticulum (SR), thereby increasing intracellular Ca2+. Overload of cellular Ca2+ within the SR can initiate various types of atrial dysrhythmias. The present study was designed to determine whether withdrawal of acetylcholine (ACh) can elicit Ca2+-induced delayed afterdepolarizations (DADs) in atrial myocytes. METHODS AND RESULTS: A nystatin perforated-patch whole-cell method and fluorescence microscopy (indo 1) were used to measure electrical activities and intracellular free Ca2+ ([Ca2+]i), respectively. Withdrawal of ACh (1 micromol/L) increased action potential duration, shifted plateau voltage toward positive, and generated DADs that initiated spontaneous action potentials. Voltage-clamp analysis revealed that withdrawal of ACh elicited a rebound stimulation of L-type Ca2+ current (I(Ca,L)) (+45%) and Na/Ca exchange current (I(NaCa)) (+16%) and the appearance of transient inward current (I(ti)) and spontaneous [Ca2+]i transients. Each of these changes induced by withdrawal of ACh was abolished by Rp-cAMPs (50 to 100 micromol/L) or H-89 (2 micromol/L), inhibitors of cAMP-dependent protein kinase A. Ryanodine (1 micromol/L) abolished I(NaCa) and the appearance of I(ti) without decreasing the rebound stimulation of I(Ca,L) elicited by withdrawal of ACh. CONCLUSIONS: Withdrawal of ACh can elicit cAMP-mediated stimulation of Ca2+ influx via I(Ca,L) and uptake of SR Ca2+. As a result, cellular Ca2+ overload causes enhanced SR Ca2+ release and the initiation of DADs. These mechanisms may generate triggered and/or spontaneous atrial depolarizations elicited by withdrawal of vagal nerve activity.     

Protective effect of l-cis-diltiazem on hypercontracture of rat myocytes induced by veratridine

The protective effect of l-cis-diltiazem, the stereoisomer of d-cis-diltiazem, was studied against the veratridine-induced hypercontracture of rat myocytes. Veratridine increased both [Na+]i and [Ca2+]i, but did not cause hypercontracture in the absence of extracellular Ca2+. Both l-cis-diltiazem (0.1-10 microM) and d-cis-diltiazem (10-30 microM) inhibited the hypercontracture and the increase in [Ca2+]i in a concentration-dependent manner. However, l-cis-diltiazem did not exert a negative inotropic effect in K+ (20 mM)-depolarized rat papillary muscles even at a dose of 10 microM. As seen in the case of tetrodotoxin, l-cis-diltiazem and d-cis-diltiazem also suppressed the increase in [Na+]i. The results show that l-cis-diltiazem prevents the veratridine-induced hypercontracture of myocytes by suppression of the [Ca2+]i increase. The attenuation of the [Ca2+]i increase by l-cis-diltiazem was not dependent on inhibition of Ca2+ channels, but was partly due to inhibition of excessive Na+ entry via veratridine-modified Na+ channels.     

Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication

We have previously reported that angiotensin II (ANG II) induces oscillations in the cytoplasmic calcium concentration ([Ca2+]i) of pulmonary vascular myocytes. The present work was undertaken to investigate the effect of ANG II in comparison with ATP and caffeine on membrane currents and to explore the relation between these membrane currents and [Ca2+]i. In cells clamped at -60 mV, ANG II (10 microM) or ATP (100 microM) induced an oscillatory inward current. Caffeine (5 mM) induced only one transient inward current. In control conditions, the reversal potential (Erev) of these currents was close to the equilibrium potential for Cl- ions (Ecl = -2.1 mV) and was shifted towards more positive values in low-Cl- solutions. Niflumic acid (10-50 microM) and DIDS (0.25-1 mM) inhibited this inward current. Combined recordings of membrane current and [Ca2+]i by indo-1 microspectrofluorimetry revealed that ANG II- and ATP-induced currents occurred simultaneously with oscillations in [Ca2+]i whereas the caffeine-induced current was accompanied by only one transient increase in [Ca2+]i. Niflumic acid (25 microM) had no effect on agonist-induced [Ca2+]i responses, whereas thapsigargin (1 microM) abolished both membrane current and the [Ca2+]i response. Heparin (5 mg/ml in the pipette solution) inhibited both [Ca2+]i responses and membrane currents induced by ANG II and ATP, but not by caffeine. In pulmonary arterial strips, ANG II-induced contraction was inhibited by niflumic acid (25 microM) or nifedipine (1 microM) to the same extent and the two substances did not have an additive effect. This study demonstrates that, in pulmonary vascular smooth muscle, ANG II, as well as ATP, activate an oscillatory calcium dependent chloride current which is triggered by cyclic increases in [Ca2+]i and that both oscillatory phenomena are primarily IP3-mediated. It is suggested that ANG II-induced oscillatory chloride current could depolarise the cell membrane leading to activation of voltage-operated Ca2+ channels. The resulting Ca2+ influx contributes to the component of ANG II-induced contraction that is equally sensitive to chloride or calcium channel blockade.     

Histamine causes Ca2+ entry via both a store-operated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca2+ enters the cell through a (capacitative) Ca2+ entry pathway activated as a consequence of intracellular Ca2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 microM) caused a rapid but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+, only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca(2+)-free medium to deplete the internal Ca2+ stores, histamine caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Re-introduction of external Ca2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H1-receptor antagonist, mepyramine (2 microM). A comparison was made between store-dependent Ca2+ entry consequent upon store mobilization with histamine in Ca(2+)-free medium and plateau phase Ca2+ entry resulting from stimulation with histamine in Ca(2+)-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P < 0.0001) at the same concentration of histamine (10 microM). It is concluded that histamine causes Ca2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca2+ entry through other pathways.     

Differential roles of two types of voltage-gated Ca2+ channels in the dendrites of rat cerebellar Purkinje neurons

The distribution and function of voltage-gated Ca2+ channels in Purkinje neurons in rat cerebellar slices were studied using simultaneous Ca2+ imaging and whole-cell patch clamp recording techniques. Voltage-gated Ca2+ channels were activated by applying depolarizing voltage steps through the pipette attached at the soma in a voltage-clamp mode in the presence of tetrodotoxin. Poor space clamp due to extensive arborization of the dendrites allowed the dendrites to fire Ca2+ spikes. Ca2+ imaging with Fura-2 injected through the pipette, showed a steady [Ca2+]i increase at the soma and transient, spike-linked [Ca2+]i jumps in the dendrites. omega-Agatoxin-IVA (200 nM) abolished the depolarization-induced Ca2+ spikes, the spike-linked [Ca2+]i increase in the dendrites, and the steady [Ca2+]i increase at the soma. omega-Conotoxin-GVIA (5 microM) and nifedipine (3 microM) had no significant effect on the depolarization-induced responses. In the presence of 4-aminopyridine(2 mM) and omega-Agatoxin-IVA, transient [Ca2+]i increases remained in the dendrites. Low concentrations of Ni2+(100 microM) reversibly suppressed this [Ca2+]i increase. The voltage for half-maximal activation and inactivation of this component were lower than -50 mV and -31 mV, respectively. In normal conditions, low concentration of Ni2+ slowed the onset of the Ca2+ spike without changing the time course of the spikes or the amplitude of the accompanying [Ca2+]i increase. These results show that omega-Agatoxin-IVA-sensitive Ca2+ channels are distributed both in the soma and the dendrites, and are responsible for dendritic Ca2+ spikes, whereas low-voltage activated, Ni2+-sensitive Ca2+ channels are distributed in the whole dendrites including both thick and fine branches, and provide boosting current for spike generation.