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.     

Effects of the nitric oxide donor sodium nitroprusside on intracellular pH and contraction in hypertrophied myocytes

BACKGROUND: We compared the effects of the nitric oxide donor sodium nitroprusside (SNP) on intracellular pH (pHi), intracellular calcium concentration ([Ca2+]i) transients, and cell contraction in hypertrophied adult ventricular myocytes from aortic-banded rats and age-matched controls. METHODS AND RESULTS: pHi was measured in individual myocytes with SNARF-1, and [Ca2+]i transients were measured with indo 1 simultaneously with cell motion. Experiments were performed at 37 degrees C in myocytes paced at 0.5 Hz in HEPES-buffered solution (extracellular pH = 7.40). At baseline, calibrated pHi, diastolic and systolic [Ca2+]i values, and the amplitude of cell contraction were similar in hypertrophied and control myocytes. Exposure of the control myocytes to 10(-6) mol/L SNP caused a decrease in the amplitude of cell contraction (72 +/- 7% of baseline, P < .05) that was associated with a decrease in pHi (-0.10 +/- 0.03 U, P < .05) with no change in peak systolic [Ca2+]i. In contrast, in the hypertrophied myocytes exposure to SNP did not decrease the amplitude of cell contraction or cause intracellular acidification (-0.01 +/- 0.01 U, NS). The cGMP analogue 8-bromo-cGMP depressed cell shortening and pHi in the control myocytes but failed to modify cell contraction or pHi in the hypertrophied cells. To examine the effects of SNP on Na(+)-H+ exchange during recovery from intracellular acidosis, cells were exposed to a pulse and washout of NH4Cl. SNP significantly depressed the rate of recovery from intracellular acidosis in the control cells compared with the rate in hypertrophied cells. CONCLUSIONS: SNP and 8-bromo-cGMP cause a negative inotropic effect and depress the rate of recovery from intracellular acidification that is mediated by Na(+)-H+ exchange in normal adult rat myocytes. In contrast, SNP and 8-bromo-cGMP do not modify cell contraction or pHi in hypertrophied myocytes.     

Calcium dynamics in single spines during coincident pre- and postsynaptic activity depend on relative timing of back-propagating action potentials and subthreshold excitatory postsynaptic potentials

We compared the transient increase of Ca2+ in single spines on basal dendrites of rat neocortical layer 5 pyramidal neurons evoked by subthreshold excitatory postsynaptic potentials (EPSPs) and back-propagating action potentials (APs) by using calcium fluorescence imaging. AP-evoked Ca2+ transients were detected in both the spines and in the adjacent dendritic shaft, whereas Ca2+ transients evoked by single EPSPs were largely restricted to a single active spine head. Calcium transients elicited in the active spines by a single AP or EPSP, in spines up to 80 micro(m) for the soma, were of comparable amplitude. The Ca2+ transient in an active spine evoked by pairing an EPSP and a back-propagating AP separated by a time interval of 50 ms was larger if the AP followed the EPSP than if it preceded it. This difference reflected supra- and sublinear summation of Ca2+ transients, respectively. A comparable dependence of spinous Ca2+ transients on relative timing was observed also when short bursts of APs and EPSPs were paired. These results indicate that the amplitude of the spinous Ca2+ transients during coincident pre- and postsynaptic activity depended critically on the relative order of subthreshold EPSPs and back-propagating APs. Thus, in neocortical neurons the amplitude of spinous Ca2+ transients could encode small time differences between pre- and postsynaptic activity.     

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.     

Rapid effects of cytochalasin-D on contraction and intracellular calcium in single rat ventricular myocytes

Contraction and intracellular calcium ([Ca2+]i) transients were recorded using a video edge detector and fluorescence spectrophotometry, respectively, in rat ventricular myocytes at 22-24 degreesC stimulated at a frequency of 1 Hz. Application of the F-actin disrupter cytochalasin-D (Cyt-D) caused a large reduction in the amplitude of contraction and a small increase in the [Ca2+]i transient. These responses began within a few seconds of application and were complete after 2 min of exposure. Phase-plane relationships of contraction and [Ca2+]i were consistent with cytochalasin-D causing a decrease in myofilament responsiveness to Ca2+.     

Changes in mitochondrial calcium concentration during the cardiac contraction cycle

OBJECTIVE: The aim was to examine whether mitochondrial Ca2+ fluxes are high enough to change mitochondrial and cytosolic calcium concentration during the contraction cycle. METHODS: Isolated guinea pig ventricular myocytes were stimulated with paired voltage clamp pulses until contractions were maximal (2 mM [Ca2+]o, 36 degrees C). At defined times of diastole or systole, the cells were shock frozen. Electron-probe microanalysis measured the concentration of total calcium in mitochondria (sigma Ca(mito)) and surrounding cytosol (sigma Cac). Other experiments were performed to evaluate DNP sensitive mitochondrial Ca2+ uptake from depolarisation induced [Ca2+]c transients (K5indo-1 fluorescence). RESULTS: At end of diastole, sigma Ca(mito) was 446 mumol.litre-1. During systole, sigma Ca(mito) increased with a 20 ms delay. A peak sigma Ca(mito) of 1050 mumol.litre-1 was measured 40 ms after start of systole, while 95 ms after start of systole sigma Ca(mito) had fallen to 530 mumol.litre-1. From the changes in sigma Ca(mito) the rates of net mitochondrial Ca2+ flux were estimated at 100 nmol.s-1 x mg-1 protein for Ca2+ influx and 36 nmol.s-1 x mg-1 protein for Ca2+ egress. Decay of sigma Ca(mito) was coupled to a rise in sigma Na(mito). sigma Cl(mito) and sigma K(mito) rose and fell in parallel with sigma Ca(mito), suggesting Ca2+ activation of mitochondrial anion and cation channels. Activation of the non-specific permeability can be excluded. Block of mitochondrial Ca2+ uptake with DNP (100 microM) or FCCP (10 microM) increased the amplitude of the [Ca2+]c transients for 1-3 min by about 50%; evaluation of mitochondrial Ca2+ uptake from DNP sensitive difference signals, however, was hampered by sequestration of mitochondrial Ca2+ into the sarcoplasmic reticulum. CONCLUSIONS: Mitochondrial calcium content changes during each individual contraction cycle; a substantial amount of calcium is taken up during the systole and released during later systole and diastole.     

Spontaneous electrical and calcium oscillations in unstimulated pituitary gonadotrophs

Single pituitary cells often fire spontaneous action potentials (APs), which are believed to underlie spiking fluctuations in cytosolic calcium concentration ([Ca2+]i). To address how these basal [Ca2+]i fluctuations depend on changes in plasma membrane voltage (V), simultaneous measurements of V and [Ca2+]i were performed in rat pituitary gonadotrophs. The data show that each [Ca2+]i spike is produced by the Ca2+ entry during a single AP. Using these and previously obtained patch-clamp data, we develop a quantitative mathematical model of this plasma membrane oscillator and the accompanying spatiotemporal [Ca2+]i oscillations. The model demonstrates that AP-induced [Ca2+]i spiking is prominent only in a thin shell layer neighboring the cell surface. This localized [Ca2+]i spike transiently activates the Ca2(+)- dependent K+ current resulting in a sharp afterhyperpolarization following each voltage spike. In accord with experimental observations, the model shows that the frequency and amplitude of the voltage spikes are highly sensitive to current injection and to the blocking of the Ca(2+)-sensitive current. Computations also predict that leaving the membrane channels intact, the firing rate can be modified by changing the Ca2+ handling parameters: the Ca2+ diffusion rate, the Ca2+ buffering capacity, and the plasma membrane Ca2+ pump rate. Finally, the model suggests reasons that spontaneous APs were seen in some gonadotrophs but not in others. This model provides a basis for further exploring how plasma membrane electrical activity is involved in the control of cytosolic calcium level in unstimulated as well as agonist-stimulated gonadotrophs.     

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes

Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca2+ entry via Na-Ca exchange current (INaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1-5 day), juvenile (JV, 10-14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD90: 108-378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (QExout) and inward (QExin) Ni2+-sensitive charge movement in response to AP prolongation. QExout and QExin measured during age-appropriate APs declined postnatally [QEXout: NB (2 day) 0.19 +/- 0.02, JV (10 day) 0.10 +/- 0.01, AD 0.04 +/- 0.002; QEXin: NB -0. 2 +/- 0.01, JV -0.11 +/- 0.02; AD -0.04 +/- 0.003 pC/pF] despite the significantly shorter APD90 of newborn myocytes (NB 122 +/- 10; AD 268 +/- 22 ms). When Ca2+ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 +/- 0.5, JV 1.2 +/- 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth.     

JTT-501, a novel oral antidiabetic agent, improves insulin resistance in genetic and non-genetic insulin-resistant models

The effects of histamine on the intracellular Ca2+ concentration ([Ca2+]i), action potential and membrane currents were assessed in single atrial myocytes prepared from guinea-pigs. Histamine caused a concentration-dependent increase in the [Ca2+]i transient in indol/AM loaded myocytes when stimulated electrically at 0.5 Hz. However, the maximum increase in [Ca2+]i transient produced by histamine was less than 50% of that elicited by isoprenaline. The histamine-induced increase in [Ca2+]i transient was significantly inhibited by chlorpheniramine, but not by cimetidine. Pretreatment with nifedipine nearly completely suppressed the histamine-induced increase in [Ca2+]i transient. Cyclopiazonic acid did not affect the histamine response. In the whole-cell current-clamp mode of the patch-clamp method, both histamine and isoprenaline prolonged action potential duration (APD) in atrial myocytes. In the presence of Co2+ or nifedipine, the isoprenaline-induced APD prolongation was abolished and an APD shortening effect was manifested, while histamine still increased APD. The APD prolongation elicited by histamine was reversed by chlorpheniramine. In the voltage-clamp mode, the histamine-sensitive membrane current was inwardly rectifying and reversed close to the calculated value of the K+ equilibrium potential. Histamine had no apparent effect on L-type Ca2+ current, in contrast to the pronounced effect of isoprenaline. These results indicate that in guinea-pig atrial myocytes stimulation of H1-receptors with histamine does not directly activate Ca2+ channels but causes an elevation of [Ca2+]i transient by increasing Ca2+ influx through the channels during the prolonged repolarization of action potentials resulting from inhibition of the outward K+ current.     

Monensin-induced reversal of positive force-frequency relationship in cardiac muscle: role of intracellular sodium in rest-dependent potentiation of contraction

We have investigated the role of a rest-dependent inotropic factor in determining species-related differences in cardiac force-frequency relationships (FFR). Isolated rat, rabbit or guinea-pig papillary muscles, as well as guinea-pig ventricular myocytes were superfused with 1.8 mM Ca2+ Tyrode. In rat muscles, isometric force amplitude decreased, while in rabbit or guinea-pig muscles force increased with frequency (0.02-1 Hz). Paired-pulse pacing potentiated contraction markedly at all frequencies in rabbit muscles, but not at low frequencies in rat muscles. We tested the hypothesis that high intracellular Na+ levels (Nai) are responsible for negative FFR. The ionophore monensin increased Nai, reversed the FFR of rabbit and guinea-pig muscles from positive to negative, by increasing force mostly at low frequencies, and decreased the paired-pulse potentiation of contraction at low frequencies. Monensin added during rest also reversed rest-induced decay. In isolated myocytes, monensin had qualitatively similar effects on cell shortening as well as on Cai transients. Monensin also decreased the action potential duration (APD) but did not change the pattern of its variation with frequency. Cells intracellularly dialyzed with 20 mM Na+ via a patch pipette also showed rest potentiation of the Cai transients, in contrast to cells dialyzed with 10 mM Na+, which showed rest decay of the transients. APD was also shorter in myocytes dialyzed with 20 mM Na+ than in those dialyzed with lower Na+. The results indicate that in the presence of high Nai, sarcoplasmic reticular Ca2+ load is increased during diastole, possibly via reverse-mode Na+/Ca2+ exchange, and therefore that Nai is an important factor determining the FFR. In addition, the data suggest that short APDs in preparations showing negative FFR may be partly a consequence of increased Nai.     

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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.     

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.     

The Raf-MEK-ERK cascade represents a common pathway for alteration of intracellular calcium by Ras and protein kinase C in cardiac myocytes

Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade, may participate in the development of cardiac hypertrophy, a condition characterized by diminished and prolonged contractile calcium transients. To directly examine the influence of this pathway on intracellular calcium ([Ca2+]i), cardiac myocytes were cotransfected with effectors of this pathway and with green fluorescent protein, which allowed the living transfected myocytes to be identified and examined for [Ca2+]i via indo-1. Transfection with constitutively active Ras (Ha-RasV12) increased cell size, decreased expression of the myofibrils and the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+]i transients. Similar effects on [Ca2+]i were obtained with Ha-RasV12S35, a Ras mutant that selectively couples to Raf, and with constitutively active Raf. In contrast, Ha-RasV12C40, a Ras mutant that activates the phosphatidylinositol 3-kinase pathway, had a lesser effect. The PKC-activating phorbol ester, phorbol 12-myristate 13-acetate, also prolonged the contractile [Ca2+]i transients. Cotransfection with dnMEK inhibited the effects of Ha-RasV12, Raf, and phorbol 12-myristate 13-acetate on [Ca2+]i. The effects of Ha-RasV12 and Raf on [Ca2+]i were also counteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+]i via the Raf-MEK-ERK cascade, and this pathway may represent a critical determinant of cardiac physiological function.     

Fat malabsorption in cystic fibrosis patients receiving enzyme replacement therapy is due to impaired intestinal uptake of long-chain fatty acids

BACKGROUND: Recombinant human growth hormone (GH) improves in vivo cardiac function in rats with postinfarction heart failure (MI). We examined the effects of growth hormone (14 days of 3.5 mg. kg-1. d-1 begun 4 weeks after MI) on contractile reserve in left ventricular myocytes from rats with chronic postinfarction heart failure. METHODS AND RESULTS: Cell shortening and [Ca2+]i were measured with the indicator fluo 3 in myocytes from MI, MI+GH, control, and normal animals treated with GH (C+GH) under stimulation at 0.5 Hz at 37 degrees C. Cell length was similar in MI and MI+GH rats (150+/-5 and 157+/-5 microm) and was greater in these groups than in the control and C+GH groups (140+/-4 and 139+/-4 microm, P<0.05). At baseline perfusate calcium of 1.2 mmol/L, myocyte fractional shortening and [Ca2+]i transients were similar among the 4 groups. We then assessed contractile reserve by measuring the increase in myocyte fractional shortening in the presence of high-perfusate calcium of 3.5 mmol/L. In the control and C+GH groups, myocyte fractional shortening and peak systolic [Ca2+]i were similarly increased in the presence of high-perfusate calcium. In the presence of high-perfusate calcium, both myocyte fractional shortening and peak systolic [Ca2+]i were depressed in the MI compared with the control groups. In contrast, myocyte fractional shortening (14.1+/-.9% versus 11.1+/-.9%, P<0.05) and peak systolic [Ca2+]i (647+/-43 versus 509+/-37 nmol/L, P<0.05) were significantly higher in MI+GH than in MI rats and were comparable to controls. Left ventricular myocyte expression of sarcoplasmic reticulum Ca2+ ATPase 2 (SERCA-2) and left ventricular SERCA-2 protein levels were increased in MI+GH compared with MI rats. CONCLUSIONS: Calcium-dependent contractile reserve is depressed in myocytes from rats with postinfarction heart failure. Long-term growth hormone therapy increases contractile reserve by restoring normal augmentation of systolic [Ca2+]i in myocytes from rats with postinfarction heart failure.     

Na+/Ca2+ exchanger modulates kainate-triggered Ca2+ signaling in Bergmann glial cells in situ

The role of sodium-calcium exchanger in calcium homeostasis in Bergmann glial cells in situ was investigated by monitoring cytoplasmic calcium ([Ca2+]i) and sodium ([Na+]i) concentrations. The [Ca2+]i and [Na+]i transients were measured either separately by using fluorescent indicators fura-2 and SBFI, respectively, or simultaneously using the indicators fluo-3 and SBFI. Since the removal of extracellular Na+ induced a relatively small (approximately 50 nM) elevation of [Ca2+]i, the Na+/Ca2+ exchanger seems to play a minor role in regulation of resting [Ca2+]i. In contrast, kainate-triggered [Ca2+]i increase was significantly suppressed by lowering of the extracellular Na+ concentration ([Na+]o). In addition, manipulations with [Na+]o dramatically affected the recovery of the kainate-induced [Ca2+]i transients. Simultaneous recordings of [Ca2+]i and [Na+]i revealed that kainate-evoked [Ca2+]i transients were accompanied with an increase in [Na+]i. Moreover, kainate induced significantly larger [Ca2+]i and smaller [Na+]i transients under current-clamp conditions as compared to those recorded when the membrane voltage was clamped at -70 mV. The above results demonstrate that the Na(+)-Ca2+ exchanger is operative in Bergmann glial cells in situ and is able to modulate dynamically the amplitude and kinetics of [Ca2+]i signals associated with an activation of ionotropic glutamate receptors.     

The effect of inhibition of myosin light chain kinase by Wortmannin on intracellular [Ca2+], electrical activity and force in phasic smooth muscle

To investigate the role of myosin light chain kinase (MLCK) in phasic contractions of intact smooth muscle, we have applied Wortmannin, an MLCK inhibitor, to strips of guinea-pig ureter. Simultaneous measurements of electrical activity, intracellular [Ca2+] ([Ca2+]i) and phasic force showed that Wortmannin (1-4 microM) abolishes force with little or no change in [Ca2+]i and electrical activity. High-K+-induced force production was also abolished by Wortmannin. The effects of Wortmannin were dose dependent - at lower concentrations (100 nM) Wortmannin reduced phasic contractility by 40-50%. It also significantly increased the delay between the Ca2+ peak and force production. These data show that, in phasic smooth muscle, inhibition of MLCK causes contraction to fail, despite normal electrical activity and Ca2+ transients. Our results also indicate that Wortmannin has no secondary effects and that other means of producing force, independent of myosin phosphorylation, are negligible in this tissue. The increased lag between the rise of Ca2+ and force production when MLCK is inhibited was surprising and suggests that post-phosphorylation steps may play a larger role in the delay than was previously considered.     

Salicylic acid induces a cytosolic Ca2+ elevation in yeast

Cytosolic free calcium ion concentration ([Ca2+]cyt) after a salicylic acid (SA)-stimulus was monitored in cells of the yeast Saccharomyces cerevisiae expressing apoaequorin, which constitutes a Ca(2+)-sensitive luminescent protein, aequorin, when combined with coelenterazine. SA induced a transient [Ca2+]cyt elevation that was dependent on the concentration of SA and pH of the SA solution. The SA-induced [Ca2+]cyt elevation was not reduced in Ca(2+)-deficient medium, suggesting that Ca2+ was mobilized from an intracellular Ca2+ store(s). Benzoic acid, butyric acid and sorbic acid did not induced a [Ca2+]cyt elevation.     

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

The effects of endurance run training on Na+-dependent Ca2+ regulation in rat left ventricular myocytes were examined. Myocytes were isolated from sedentary and trained rats and loaded with fura 2. Contractile dynamics and fluorescence ratio transients were recorded during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degreesC. Resting and peak cytosolic Ca2+ concentration ([Ca2+]c) did not change with exercise training. However, resting and peak [Ca2+]c increased significantly in both groups during 5 min of continuous pacing, although diastolic [Ca2+]c in the trained group was less susceptible to this elevation of intracellular Ca2+. Run training also significantly reduced the rate of [Ca2+]c decay during relaxation. Myocytes were then exposed to 10 mM caffeine in the absence of external Na+ or Ca2+ to trigger sarcoplasmic reticular Ca2+ release and to suppress cellular Ca2+ efflux. This maneuver elicited an elevated steady-state [Ca2+]c. External Na+ was then added, and the rate of [Ca2+]c clearance was determined. Run training significantly reduced the rate of Na+-dependent clearance of [Ca2+]c during the caffeine-induced contractures. These data demonstrate that the removal of cytosolic Ca2+ was depressed with exercise training under these experimental conditions and may be specifically reflective of a training-induced decrease in the rate of cytosolic Ca2+ removal via Na+/Ca2+ exchange and/or in the amount of Ca2+ moved across the sarcolemma during a contraction.     

Voltage-activated intracellular calcium transients in thalamic relay cells and interneurons

The dynamics of intracellular calcium concentration ([Ca2+]i) following activation of low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ currents were studied in identified relay neurons and interneurons of the rat dorsal lateral geniculate nucleus (LGNd) in situ using Ca2+ imaging and patch-clamp techniques. In relay neurons, [Ca2+]i transients associated with the LVA Ca2+ current showed a fairly homogeneous somatodendritic distribution, whereas HVA transients significantly decreased to 65% of the somatic value at 60 microns dendritic distance. In interneurons, LVA transients significantly increased to 239% of the somatic value at 60 microns dendritic distance, whereas HVA transients were not significantly different in the soma and dendrites. These results indicate differences in [Ca2+]i dynamics, which may reflect a heterogeneous distribution of Ca2+ channels contributing to subcellular compartmentation in the two types of thalamic neurons.     

Local control of excitation-contraction coupling in rat heart cells

1. Cytosolic free calcium ion concentration ([Ca2+]i) and whole-cell L-type Ca2+ channel currents were measured during excitation-contraction (E-C) coupling in single voltage-clamped rat cardiac ventricular cells. The measurements were used to compute the total cellular efflux of calcium ions through sarcoplasmic reticulum (SR) Ca2+ release channels (FSR,rel) and the influx of Ca2+ via L-type Ca2+ channels (FICa). 2. FSR,rel was elicited by depolarizing voltage-clamp pulses 200 ms in duration to membrane potentials from -30 to +80 mV. Over this range, peak FSR,rel had a bell-shaped dependence on clamp pulse potential. In all cells, the 'gain' of the system, measured as the ratio, FSR,rel(max)/FICa(max), declined from about 16, at 0 mV, to much lower values as clamp pulse voltage was made progressively more positive. We named this phenomenon of change in gain as a function of membrane potential, 'variable gain'. At clamp pulse potentials in the range -30 to 0 mV, the gain differed from cell to cell, being constant at about 16 in some cells, but decreasing from high values (approximately 65) at -20 mV in others. 3. At clamp pulse potentials at which Ca2+ influx (FICa) was maintained, FSR,rel also had a small maintained component. When macroscopic Ca2+ influx was brief (1-2 ms, during 'tails' of FICa), FSR,rel rose rapidly to a peak after repolarization and then declined with a half-time of about 9 ms (typically). 4. The rising phase of [Ca2+]i transients could be interrupted by stopping Ca2+ influx rapidly (by voltage clamp). We therefore termed this phenomenon 'interrupted SR Ca2+ release'.(ABSTRACT TRUNCATED AT 250 WORDS)