Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes.

Previous studies in adult myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated abnormal contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) homeostasis and decreased sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2) expression and activity, but sarcoplasmic reticulum Ca(2+) leak was unchanged. In the present study, we investigated whether SERCA2 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. Compared with sham-operated hearts, 3-wk MI hearts exhibited significantly higher left ventricular end-diastolic and end-systolic volumes but lower fractional shortening and ejection fraction, as measured by M-mode echocardiography. Seventy-two hours after adenovirus-mediated gene transfer, SERCA2 overexpression in 3-wk MI myocytes did not affect Na(+)-Ca(2+) exchanger expression but restored the depressed SERCA2 levels toward those measured in sham myocytes. In addition, the reduced sarcoplasmic reticulum Ca(2+) uptake in MI myocytes was improved to normal levels by SERCA2 overexpression. At extracellular Ca(2+) concentration of 5 mM, the subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored to normal by SERCA2 overexpression. However, at 0.6 mM extracellular Ca(2+) concentration, the supernormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were exacerbated by SERCA2 overexpression. We conclude that SERCA2 overexpression was only partially effective in ameliorating contraction and [Ca(2+)](i) transient abnormalities in our rat model of ischemic cardiomyopathy. We suggest that other Ca(2+) transport pathways, e.g., Na(+)-Ca(2+) exchanger, may also play an important role in contractile and [Ca(2+)](i) homeostatic abnormalities in MI myocytes.     

Effects of sprint training on contractility and [Ca(2+)](i) transients in adult rat myocytes.

The effects of 6-8 wk of high-intensity sprint training (HIST) on rat myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients were investigated. Compared with sedentary (Sed) myocytes, HIST induced a modest (5%) but significant (P < 0.0005) increase in cell length with no changes in cell width. In addition, the percentage of myosin heavy chain alpha-isoenzyme increased significantly (P < 0.02) from 0.566 +/- 0.077% in Sed rats to 0.871 +/- 0.006% in HIST rats. At all three (0.6, 1.8, and 5 mM) extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, maximal shortening amplitudes and maximal shortening velocities were significantly (P < 0.0001) lower and half-times of relaxation were significantly (P < 0.005) longer in HIST myocytes. HIST myocytes had significantly (P < 0.0001) higher diastolic [Ca(2+)](i) levels. Compared with Sed myocytes, systolic [Ca(2+)](i) levels in HIST myocytes were higher at 0.6 mM [Ca(2+)](o), similar at 1.8 mM [Ca(2+)](o), and lower at 5 mM [Ca(2+)](o). The amplitudes of [Ca(2+)](i) transients were significantly (P < 0.0001) lower in HIST myocytes. Half-times of [Ca(2+)](i) transient decline, an estimate of sarcoplasmic reticulum (SR) Ca(2+) uptake activity, were not different between Sed and HIST myocytes. Compared with Sed hearts, Western blots demonstrated a significant (P < 0.03) threefold decrease in Na(+)/Ca(2+) exchanger, but SR Ca(2+)-ATPase and calsequestrin protein levels were unchanged in HIST hearts. We conclude that HIST effected diminished myocyte contractile function and [Ca(2+)](i) transient amplitudes under the conditions studied. We speculate that downregulation of Na(+)/Ca(2+) exchanger may partly account for the decreased contractility in HIST myocytes.     

Sustained overexpression of IGF-1 prevents age-dependent decrease in charge movement and intracellular Ca(2+) in mouse skeletal muscle

In this work we tested the hypothesis that transgenic sustained overexpression of IGF-1 prevents age-dependent decreases in charge movement and intracellular Ca(2+) in skeletal muscle fibers. To this end, short flexor digitorum brevis (FDB) muscle fibers from 5-7- and 21-24-month-old FVB (wild-type) and S1S2 (IGF-1 transgenic) mice were studied. Fibers were voltage-clamped in the whole-cell configuration of the patch-clamp technique according to described procedures (Wang, Z. M., M. L. Messi, and O. Delbono. 1999. Biophys. J. 77:2709-2716). Charge movement and intracellular Ca(2+) concentration were recorded simultaneously. The maximum charge movement (Q(max)) recorded in young wild-type and transgenic mice was (mean +/- SEM, in nC microF(-1)): 52 +/- 2.1 (n = 46) and 54 +/- 1.9 (n = 38) (non-significant, ns), respectively, whereas in old wild-type and old transgenic mice the values were 36 +/- 2.1 (n = 32) and 49 +/- 2.3 (n = 35), respectively (p < 0.01). The peak intracellular calcium [Ca(2+)](i) recorded in young wild-type and transgenic mice was (in muM): 14.5 +/- 0.9 and 16 +/- 2.1 (ns), whereas in old wild-type and transgenic mice the values were 9.9 +/- 0.1 and 14 +/- 1.1 (p < 0.01), respectively. No significant changes in the voltage distribution or steepness of the Q-V or [Ca(2+)]-V relationship were found. These data support the concept that overexpression of IGF-1 in skeletal muscle prevents age-dependent reduction in charge movement and peak [Ca(2+)](i).     

Measurement of sub-membrane [Ca2+] in adult myofibers and cytosolic [Ca2+] in myotubes from normal and mdx mice using the Ca2+ indicator FFP-18

The hypothesis that intracellular Ca(2+) is elevated in dystrophic (mdx) skeletal muscle due to increased Ca(2+) influx is controversial. As the sub-sarcolemmal Ca(2+) ([Ca(2+)](mem)) should be even higher than the global cytosolic Ca(2+) in the presence of increased Ca(2+) influx, we investigated [Ca(2+)](mem) levels in collagenase-isolated adult flexor digitorum brevis (FDB) myofibres and myotubes of mdx and normal mice with the near-membrane Ca(2+) indicator FFP-18. Confocal imaging showed strong localization of FFP-18 to the sarcolemma only. No significant difference in [Ca(2+)](mem) was found in FDB myofibres of normal (77.3+/-3.8 nM, n=68) and mdx (79.3+/-5.6 nM, n=21, p=0.89) mice using FFP-18. Increasing external Ca(2+) to 18 mM did not significantly affect [Ca(2+)](mem) in either the normal or mdx myofibres. In the myotubes, the FFP-18 was non-selectively incorporated, distributing throughout the cytoplasm, and FFP-18-derived [Ca(2+)] values were similar to values obtained with Fura-2. Nevertheless, in the mdx myotubes, the [Ca(2+)] measured with FFP-18 increased linearly to a level approximately 2.75 times that of controls as the time of culture was prolonged. In older mdx myotubes (>or=8 days in culture), 18 mM extracellular Ca(2+) increased the steady state cytosolic [Ca(2+)] to approximately 22 times greater level than controls. This study suggests that the sub-sarcolemmal Ca(2+) homeostasis is well maintained in isolated adult mdx myofibers and also further supports the hypothesis that cytosolic Ca(2+) handling is compromised in mdx myotubes.     

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

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

Effect of diethylstilbestrol (DES) on intracellular Ca(2+) levels in renal tubular cells

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca(2+) indicator. DES (10-50 microM) evoked [Ca(2+)](i) increases in a concentration-dependent manner. Extracellular Ca(2+) removal inhibited 45 +/- 5% of the Ca(2+) response. In Ca(2+)-free medium, pretreatment with 50 microM DES abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca(2+)](i) signals. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 50 microM DES in Ca(2+)-free medium, suggesting that DES may induce capacitative Ca(2+) entry. 17beta-Estradiol (2-20 microM) increased [Ca(2+)](i), but 100 microM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 microM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca(2+) release. DES (20 microM) also increased [Ca(2+)](i) in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca(2+)](i) increases by releasing intracellular Ca(2+) and triggering extracellular Ca(2+) entry in renal tubular cells.     

Mitochondrial calcium oscillations in C2C12 myotubes

Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) was monitored in C2C12 skeletal muscle cells stably expressing the Ca(2+)-sensitive photoprotein aequorin targeted to mitochondria. In myotubes, KCl-induced depolarization caused a peak of 3.03 +/- 0.14 micrometer [Ca(2+)](m) followed by an oscillatory second phase (5.1 +/- 0.1 per min). Chelation of extracellular Ca(2+) or blockade of the voltage-operated Ca(2+) channel attenuated both phases of the KCl response. The inhibitor of the sarcoplasmic reticulum Ca(2+)-ATPase, cyclopiazonic acid, reduced the amplitude of the KCl-induced [Ca(2+)](m) peak and prevented the oscillations, suggesting that these were generated intracellularly. No such [Ca(2+)](m) oscillations occurred with the nicotinic agonist carbachol, cyclopiazonic acid alone, or the purinergic agonist ATP. In contrast, caffeine produced an oscillatory behavior, indicating a role of ryanodine receptors as mediators of the oscillations. The [Ca(2+)](m) response was desensitized when cells were exposed to two consecutive challenges with KCl separated by a 5-min wash, whereas a second pulse of carbachol potentiated [Ca(2+)](m), indicating differences in intracellular Ca(2+) redistribution. Cross-desensitization between KCl and carbachol and cross-potentiation between carbachol and KCl were observed. These results suggest that close contacts between mitochondria and sarcoplasmic reticulum exist permitting Ca(2+) exchanges during KCl depolarization. These newly demonstrated dynamic changes in [Ca(2+)](m) in stimulated skeletal muscle cells might contribute to the understanding of physiological and pathological processes in muscular disorders.     

Ca(2+)](i) signaling in renal arterial smooth muscle cells of pregnant rat is enhanced during inhibition of NOS

Vascular resistance and arterial pressure are reduced during normal pregnancy, but dangerously elevated during pregnancy-induced hypertension (PIH), and changes in nitric oxide (NO) synthesis have been hypothesized as one potential cause. In support of this hypothesis, chronic inhibition of NO synthesis in pregnant rats has been shown to cause significant increases in renal vascular resistance and hypertension; however, the cellular mechanisms involved are unclear. We tested the hypothesis that the pregnancy-associated changes in renal vascular resistance reflect changes in contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) of renal arterial smooth muscle. Smooth muscle cells were isolated from renal interlobular arteries of virgin and pregnant Sprague-Dawley rats untreated or treated with the NO synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 4 mg. kg(-1). day(-1) for 5 days), then loaded with fura 2. In cells of virgin rats incubated in Hanks' solution (1 mM Ca(2+)), the basal [Ca(2+)](i) was 86 +/- 6 nM. Phenylephrine (Phe, 10(-5) M) caused a transient increase in [Ca(2+)](i) to 417 +/- 11 nM and maintained an increase to 183 +/- 8 nM and 32 +/- 3% cell contraction. Membrane depolarization by 51 mM KCl, which stimulates Ca(2+) entry from the extracellular space, caused maintained increase in [Ca(2+)](i) to 292 +/- 12 nM and 31 +/- 2% contraction. The maintained Phe- and KCl-induced [Ca(2+)](i) and contractions were reduced in pregnant rats but significantly enhanced in pregnant rats treated with L-NAME. Phe- and KCl-induced contraction and [Ca(2+)](i) were not significantly different between untreated and L-NAME-treated virgin rats or between untreated and L-NAME + L-arginine treated pregnant rats. In Ca(2+)-free Hanks', application of Phe or caffeine (10 mM), to stimulate Ca(2+) release from the intracellular stores, caused a transient increase in [Ca(2+)](i) and a small cell contraction that were not significantly different among the different groups. Thus renal interlobular smooth muscle of normal pregnant rats exhibits reduction in [Ca(2+)](i) signaling that involves Ca(2+) entry from the extracellular space but not Ca(2+) release from the intracellular stores. The reduced renal smooth muscle cell contraction and [Ca(2+)](i) in pregnant rats may explain the decreased renal vascular resistance associated with normal pregnancy, whereas the enhanced cell contraction and [Ca(2+)](i) during inhibition of NO synthesis in pregnant rats may, in part, explain the increased renal vascular resistance associated with PIH.     

Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes

In this study, we examined the acute effects of thyroid hormones (TH) T(3) and T(4), leading to improvement of myocardial function through activation of Ca(2+) extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca(2+)](i)) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T(3) or T(4) rapidly (within 10 sec) decreased [Ca(2+)](i) to its control level. Similar results were obtained when [Ca(2+)](i) was elevated by decreasing extracellular Na(+) concentration, causing backward influx of Ca(2+) through Na(+)/Ca(2+) exchanger, or by administration of caffeine, releasing Ca(2+) from the sarcoplasmic reticulum (SR). Under these conditions, T(3) or T(4) decreased [Ca(2+)](i). T(3) and T(4) also exhibited protective effects during ischemia. T(3) or T(4) presence during hypoxia for 120 min in culture medium restricted the increase of [Ca(2+)](i) and prevented the pathological effects of its overload. An inhibitor of SR Ca(2+)-ATPase (SERCA2a), thapsigargin, increases [Ca(2+)](i) and in its presence neither T(3) nor T(4) had any effect on the [Ca(2+)](i) level. The reduction of [Ca(2+)](i) level by T(3) and T(4) was also blocked in the presence of H-89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca(2+)](i) was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca(2+) transport into the SR.     

K(+) channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle

We investigated the role of K(+) channels in the regulation of baseline intracellular free Ca(2+) concentration ([Ca(2+)](i)), alpha-adrenoreceptor-mediated Ca(2+) signaling, and capacitative Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). Inhibition of voltage-gated K(+) channels with 4-aminopyridine (4-AP) increased the membrane potential and the resting [Ca(2+)](i) but attenuated the amplitude and frequency of the [Ca(2+)](i) oscillations induced by the alpha-agonist phenylephrine (PE). Inhibition of Ca(2+)-activated K(+) channels (with charybdotoxin) and inhibition (with glibenclamide) or activation of ATP-sensitive K(+) channels (with lemakalim) had no effect on resting [Ca(2+)](i) or PE-induced [Ca(2+)](i) oscillations. Thapsigargin was used to deplete sarcoplasmic reticulum Ca(2+) stores in the absence of extracellular Ca(2+). Under these conditions, 4-AP attenuated the peak and sustained components of capacitative Ca(2+) entry, which was observed when extracellular Ca(2+) was restored. Capacitative Ca(2+) entry was unaffected by charybdotoxin, glibenclamide, or lemakalim. In isolated pulmonary arterial rings, 4-AP increased resting tension and caused a leftward shift in the KCl dose-response curve. In contrast, 4-AP decreased PE-induced contraction, causing a rightward shift in the PE dose-response curve. These results indicate that voltage-gated K(+) channel inhibition increases resting [Ca(2+)](i) and tone in PASMCs but attenuates the response to PE, likely via inhibition of capacitative Ca(2+) entry.     

L-type Ca(2+) channels, resting [Ca(2+)](i), and ET-1-induced responses in chronically hypoxic pulmonary myocytes

In the lung, chronic hypoxia (CH) causes pulmonary arterial smooth muscle cell (PASMC) depolarization, elevated endothelin-1 (ET-1), and vasoconstriction. We determined whether, during CH, depolarization-driven activation of L-type Ca(2+) channels contributes to 1) maintenance of resting intracellular Ca(2+) concentration ([Ca(2+)](i)), 2) increased [Ca(2+)](i) in response to ET-1 (10(-8) M), and 3) ET-1-induced contraction. Using indo 1 microfluorescence, we determined that resting [Ca(2+)](i) in PASMCs from intrapulmonary arteries of rats exposed to 10% O(2) for 21 days was 293.9 +/- 25.2 nM (vs. 153.6 +/- 28.7 nM in normoxia). Resting [Ca(2+)](i) was decreased after extracellular Ca(2+) removal but not with nifedipine (10(-6) M), an L-type Ca(2+) channel antagonist. After CH, the ET-1-induced increase in [Ca(2+)](i) was reduced and was abolished after extracellular Ca(2+) removal or nifedipine. Removal of extracellular Ca(2+) reduced ET-1-induced tension; however, nifedipine had only a slight effect. These data indicate that maintenance of resting [Ca(2+)](i) in PASMCs from chronically hypoxic rats does not require activation of L-type Ca(2+) channels and suggest that ET-1-induced contraction occurs by a mechanism primarily independent of changes in [Ca(2+)](i).     

Pyruvate potentiates inotropic effects of isoproterenol and Ca(2+) in rabbit cardiac muscle preparations

Catecholamines and elevated extracellular Ca(2+) concentration ([Ca(2+)](o)) augment contractile force by increased Ca(2+) influx and subsequent increased sarcoplasmic reticulum (SR) Ca(2+) release. We tested the hypothesis that pyruvate potentiates Ca(2+) release and inotropic response to isoproterenol and elevated [Ca(2+)](o), since this might be of potential importance in a clinical setting to circumvent deleterious effects on energy demand during application of catecholamines. Therefore, we investigated isometrically contracting myocardial preparations from rabbit hearts at 37 degrees C, pH 7.4, and a stimulation frequency of 1 Hz. At a [Ca(2+)](o) of 1.25 mM, pyruvate (10 mM) alone increased developed force (F(dev)) from 1.89 +/- 0.42 to 3.62 +/- 0.62 (SE) mN/mm(2) (n = 8, P < 0.05) and isoproterenol (10(-6) M) alone increased F(dev) from 2.06 +/- 0. 55 to 25.11 +/- 2.1 mN/mm(2) (P < 0.05), whereas the combination of isoproterenol and pyruvate increased F(dev) overproportionally from 1.89 +/- 0.42 to 33.31 +/- 3.18 mN/mm(2) (P < 0.05). In a separate series of experiments, we assessed SR Ca(2+) content by means of rapid cooling contractures and observed that, despite no further increase in F(dev) by increasing [Ca(2+)](o) from 8 to 16 mM, 10 mM pyruvate could still increase F(dev) from 26.4 +/- 6.8 to 29.7 +/- 7. 1 mN/mm(2) (P < 0.05, n = 9) as well as the Ca(2+) load of the SR. The results show that the positive inotropic effects of pyruvate potentiate the inotropic effects of isoproterenol or Ca(2+), because in the presence of pyruvate, Ca(2+) and isoproterenol induced larger increases in inotropy than can be calculated by mere addition of the individual effects.     

Calcium release domains in mammalian skeletal muscle studied with two-photon imaging and spot detection techniques

The spatiotemporal characteristics of the Ca(2+) release process in mouse skeletal muscle were investigated in enzymatically dissociated fibers from flexor digitorum brevis (FDB) muscles, using a custom-made two-photon microscope with laser scanning imaging (TPLSM) and spot detection capabilities. A two-microelectrode configuration was used to electrically stimulate the muscle fibers, to record action potentials (APs), and to control their myoplasmic composition. We used 125 muM of the low-affinity Ca(2+) indicator Oregon green 488 BAPTA-5N (OGB-5N), and 5 or 10 mM of the Ca(2+) chelator EGTA (pCa 7) in order to arrest fiber contraction and to constrain changes in the [Ca(2+)] close to the release sites. Image and spot data showed that the resting distribution of OGB-5N fluorescence was homogeneous along the fiber, except for narrow peaks ( approximately 23% above the bulk fluorescence) centered at the Z-lines, as evidenced by their nonoverlapping localization with respect to di-8-ANEPPS staining of the transverse tubules (T-tubules). Using spot detection, localized Ca(2+) transients evoked by AP stimulation were recorded from adjacent longitudinal positions 100 nm apart. The largest and fastest DeltaF/F transients were detected at sites flanking the Z-lines and colocalized with T-tubules; the smallest and slowest were detected at the M-line, whereas transients at the Z-line showed intermediate features. Three-dimensional reconstructions demonstrate the creation of two AP-evoked Ca(2+) release domains per sarcomere, which flank the Z-line and colocalize with T-tubules. In the presence of 10 mM intracellular EGTA, these domains are formed in approximately 1.4 ms and dissipate within approximately 4 ms, after the peak of the AP. Their full-width at half-maximum (FWHM), measured at the time that Ca(2+) transients peaked at T-tubule locations, was 0.62 mum, similar to the 0.61 mum measured for di-8-ANEPPS profiles. Both these values exceed the limit of resolution of the optical system, but their similarity suggests that at high [EGTA] the Ca(2+) domains in adult mammalian muscle fibers are confined to Ca(2+) release sites located at the junctional sarcoplasmic reticulum (SR).     

Enhanced [Ca2+]i in renal arterial smooth muscle cells of pregnant rats with reduced uterine perfusion pressure

Reduction of uterine perfusion pressure (RUPP) during late pregnancy has been suggested to trigger increases in renal vascular resistance and lead to hypertension of pregnancy. We investigated whether the increased renal vascular resistance associated with RUPP in late pregnancy reflects increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) and contraction of renal arterial smooth muscle. Single smooth muscle cells were isolated from renal interlobular arteries of normal pregnant Sprague-Dawley rats and a rat model of RUPP during late pregnancy. The cells were loaded with fura 2 and both cell length and [Ca(2+)](i) were measured. In cells of normal pregnant rats incubated in Hanks' solution (1 mM Ca(2+)), ANG II (10(-7) M) caused an initial increase in [Ca(2+)](i) to 414 +/- 13 nM, a maintained increase to 149 +/- 8 nM, and 21 +/- 1% cell contraction. In RUPP rats, the initial ANG II-induced [Ca(2+)](i) (431 +/- 18 nM) was not different from pregnant rats, but both the maintained [Ca(2+)](i) (225 +/- 9 nM) and cell contraction (48 +/- 2%) were increased. Membrane depolarization by 51 mM KCl and the Ca(2+) channel agonist BAY K 8644 (10(-6) M), which stimulate Ca(2+) entry from the extracellular space, caused maintained increases in [Ca(2+)](i) and cell contraction that were greater in RUPP rats than control pregnant rats. In Ca(2+)-free (2 mM EGTA) Hanks' solution, the ANG II- and caffeine (10 mM)-induced [Ca(2+)](i) transient and cell contraction were not different between normal pregnant and RUPP rats, suggesting no difference in Ca(2+) release from the intracellular stores. The enhanced maintained ANG II-, KCl- and BAY K 8644-induced [Ca(2+)](i) and cell contraction in RUPP rats compared with normal pregnant rats suggest enhanced Ca(2+) entry mechanisms of smooth muscle contraction in resistance renal arteries and may explain the increased renal vascular resistance associated with hypertension of pregnancy.     

Virtual electrodes and the induction of fibrillation in Langendorff-perfused rabbit ventricles: the role of intracellular calcium

A strong premature electrical stimulus (S(2)) induces both virtual anodes and virtual cathodes. The effects of virtual electrodes on intracellular Ca(2+) concentration ([Ca(2+)](i)) transients and ventricular fibrillation thresholds (VFTs) are unclear. We studied 16 isolated, Langendorff-perfused rabbit hearts with simultaneous voltage and [Ca(2+)](i) optical mapping and for vulnerable window determination. After baseline pacing (S(1)), a monophasic (10 ms anodal or cathodal) or biphasic (5 ms-5 ms) S(2) was applied to the left ventricular epicardium. Virtual electrode polarizations and [Ca(2+)](i) varied depending on the S(2) polarity. Relative to the level of [Ca(2+)](i) during the S(1) beat, the [Ca(2+)](i) level 40 ms after the onset of monophasic S(2) increased by 36+/-8% at virtual anodes and 20+/-5% at virtual cathodes (P<0.01), compared with 25+/-5% at both virtual cathode-anode and anode-cathode sites for biphasic S(2). The VFT was significantly higher and the vulnerable window significantly narrower for biphasic S(2) than for either anodal or cathodal S(2) (n=7, P<0.01). Treatment with thapsigargin and ryanodine (n=6) significantly prolonged the action potential duration compared with control (255+/-22 vs. 189+/-6 ms, P<0.05) and eliminated the difference in VFT between monophasic and biphasic S(2), although VFT was lower for both cases. We conclude that virtual anodes caused a greater increase in [Ca(2+)](i) than virtual cathodes. Monophasic S(2) is associated with lower VFT than biphasic S(2), but this difference was eliminated by the inhibition of the sarcoplasmic reticulum function and the prolongation of the action potential duration. However, the inhibition of the sarcoplasmic reticulum function also reduced VFT, indicating that the [Ca(2+)](i) dynamics modulate, but are not essential, to ventricular vulnerability.     

Effects of docosahexaenoic acid on [Ca(2+)](i) increase induced by doxorubicin in ventricular rat cardiomyocytes

The clinical use of doxorubicin (DXR) is limited by cardiotoxicity partially due to interference with intracellular Ca(2+) homeostasis and involving the activation of the sarcoplasmic reticulum (SR) Ca(2+) release channels. It is known that docosahexaenoic acid (DHA) is able to potentiate the sensitivity of cancer cells to DXR. The aim of our study was to further evaluate the effects of DHA on [Ca(2+)](i) overload induced by DXR in adult rat ventricular cardiomyocytes in order to verify if DHA interferes with DXR-induced cardiotoxicity too. [Ca(2+)](i) was measured by microfluorimetry. Our data demonstrated that 100 microM DXR induced a statistically significant [Ca(2+)](i)-increase in cardiomyocytes perfused with CaCl(2) Krebs solution (from 135.7 +/- 15 nM to 560.2 +/- 49 nM, n = 9, p < 0.01) and with Ca(2+)-free Krebs solution (from 89.3 +/- 15 nM to 551.1 +/- 35 nM, n = 9, p < 0.01). Treatment with 10 microM DHA for 20 min significantly suppressed DXR [Ca(2+)](i)- increase in cells perfused with CaCl(2) Krebs solution (142.3 +/- 12 nM, n = 9, p < 0.01) and in Ca(2+)-free procedures (100.4 +/- 12 nM, n = 9, p < 0.01). Caffeine 10 mM significantly increased [Ca(2+)](i) in cardiomyocytes perfused with CaCl(2) Krebs solution (from 135.7 +/- 15 nM to 979.2 +/- 17.8 nM, n = 9, p < 0.01) and with Ca(2+)-free Krebs solution (from 89.3 +/- 15 nM to 891.1 +/- 30 nM, n = 9, p < 0.01). Treatment with 10 microM DHA for 20 min suppressed caffeine [Ca(2+)](i)-increase in cardiomyocytes perfused with CaCl(2) Krebs solution (174.2 +/- 28 nM, n = 9, p < 0.01) and in Ca(2+)-free procedures (161.9 +/- 34 nM, n = 9, p < 0.01). In conclusion, our results suggest that DHA is able to prevent acute modifications of calcium homeostasis induced by DXR probably interfering with SR Ca(2+) release channels.     

CaMKII-dependent reactivation of SR Ca(2+) uptake and contractile recovery during intracellular acidosis

In hearts, intracellular acidosis disturbs contractile performance by decreasing myofibrillar Ca(2+) response, but contraction recovers at prolonged acidosis. We examined the mechanism and physiological implication of the contractile recovery during acidosis in rat ventricular myocytes. During the initial 4 min of acidosis, the twitch cell shortening decreased from 2.3 +/- 0.3% of diastolic length to 0.2 +/- 0.1% (means +/- SE, P < 0.05, n = 14), but in nine of these cells, contractile function spontaneously recovered to 1.5 +/- 0.3% at 10 min (P < 0.05 vs. that at 4 min). During the depression phase, both the diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)) and Ca(2+) transient (CaT) amplitude increased, and the twitch [Ca(2+)](i) decline prolonged significantly (P < 0.05). In the cells that recovered, a further increase in CaT amplitude and a reacceleration of twitch [Ca(2+)](i) decline were observed. The increase in diastolic [Ca(2+)](i) was less extensive than the increase in the cells that did not recover (n = 5). Blockade of sarcoplasmic reticulum (SR) function by ryanodine (10 microM) and thapsigargin (1 microM) or a selective inhibitor of Ca(2+)-calmodulin kinase II, 2-[N- (2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methyl benzylamine (1 microM) completely abolished the reacceleration of twitch [Ca(2+)](i) decline and almost eliminated the contractile recovery. We concluded that during prolonged acidosis, Ca(2+)-calmodulin kinase II-dependent reactivation of SR Ca(2+) uptake could increase SR Ca(2+) content and CaT amplitude. This recovery can compensate for the decreased myofibrillar Ca(2+) response, but may also cause Ca(2+) overload after returning to physiological pH(i).     

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.     

In situ SR function in postinfarction myocytes.

Previous studies have shown lower systolic intracellular Ca(2+) concentrations ([Ca(2+)](i)) and reduced sarcoplasmic reticulum (SR)-releasable Ca(2+) contents in myocytes isolated from rat hearts 3 wk after moderate myocardial infarction (MI). Ca(2+) entry via L-type Ca(2+) channels was normal, but that via reverse Na(+)/Ca(2+) exchange was depressed in 3-wk MI myocytes. To elucidate mechanisms of reduced SR Ca(2+) contents in MI myocytes, we measured SR Ca(2+) uptake and SR Ca(2+) leak in situ, i.e., in intact cardiac myocytes. For sham and MI myocytes, we first demonstrated that caffeine application to release SR Ca(2+) and inhibit SR Ca(2+) uptake resulted in a 10-fold prolongation of half-time (t(1/2)) of [Ca(2+)](i) transient decline compared with that measured during a normal twitch. These observations indicate that early decline of the [Ca(2+)](i) transient during a twitch in rat myocytes was primarily mediated by SR Ca(2+)-ATPase and that the t(1/2) of [Ca(2+)](i) decline is a measure of SR Ca(2+) uptake in situ. At 5.0 mM extracellular Ca(2+), systolic [Ca(2+)](i) was significantly (P 相似文献   

Increased Ca(2+) sparks and sarcoplasmic reticulum Ca(2+) stores potentially determine the spontaneous activity of pulmonary vein cardiomyocytes

Pulmonary veins (PVs) contain cardiomyocytes with spontaneous activity that may be responsible for PV arrhythmia. Abnormal Ca(2+) regulation is known to contribute to PV arrhythmogenesis. The purpose of this study was to investigate whether PV cardiomyocytes with spontaneous activity have different intracellular Ca(2+) ([Ca(2+)](i)) transients, Ca(2+) sparks and responses to isoproterenol and ryanodine receptor modulators (magnesium and FK506) than do PV cardiomyocytes without spontaneous activity and left atrial (LA) cardiomyocytes. Through fluorescence and confocal microscopy, we evaluated the [Ca(2+)](i) transients and Ca(2+) sparks in isolated rabbit PV and LA cardiomyocytes. PV cardiomyocytes with spontaneous activity had larger [Ca(2+)](i) transients and sarcoplasmic reticulum (SR) Ca(2+) stores than PV cardiomyocytes without spontaneous activity or LA cardiomyocytes. PV cardiomyocytes with spontaneous activity also had a higher incidence and frequency of Ca(2+) sparks, and had Ca(2+) sparks with larger amplitudes than other cardiomyocytes. Magnesium (5.4 mM) reduced the [Ca(2+)](i) transient amplitude and beating rate in PV cardiomyocytes with spontaneous activity. However, in contrast with other cardiomyocytes, low doses (1.8 mM) of magnesium did not reduce the [Ca(2+)](i) transients amplitude in PV cardiomyocytes with spontaneous activity. FK506 (1 muM) diminished the SR Ca(2+) stores in PV cardiomyocytes with spontaneous activity to a lesser extent than that in other cardiomyocytes. Isoproterenol (10 nM) increased the [Ca(2+)](i) transient amplitude to a lesser extent in LA cardiomyocytes than in PV cardiomyocytes with or without spontaneous activity. In conclusion, our results suggest that enhanced [Ca(2+)](i) transients, increased Ca(2+) sparks and SR Ca(2+) stores may contribute to the spontaneous activity of PV cardiomyocytes.