Maintenance of Ca(i)(2+)transients during prolonged cardiac arrest aids rapid contractile recovery

We explored the effects of contractile arrest maintained for 24-72 h in the presence of 2,3-butanedione monoxime or a Ca(2+) channel blocker (nifedipine or verapamil) on contractile activity, Ca(i)(2+) transients, and myofibrillar protein content and ultrastructure in long-term cultures of spontaneously beating adult guinea-pig cardiomyocytes. The contractions were not affected by 5 mM 2, 3-butanedione monoxime, but they were strongly or fully suppressed by 10 and 18 mM 2,3-butanedione monoxime, respectively, while the Ca(i)(2+)transients triggered by the maintained spontaneous electrical activity were either not changed at all (5 and 10 mM 2, 3-butanedione monoxime) or decreased only slightly (18 mM 2, 3-butanedione monoxime). The uncoupling of excitation from contraction by 10-18 mM for 24-72 h did not affect the content of the myofibrillar proteins. Confocal laser microscopy showed that these exposures affected the assembly of myofilaments, giving an overall deranged appearance to the myofibrils. In spite of this effect, the cells' contractile activity was readily regained within 15-60 min upon the washout of 2,3-butanedione monoxime. The 24-72-h exposures to 5 microM nifedipine or 10 microM verapamil, which blocked fully both the Ca(i)(2+) transients and contractility, did not affect the myofibrillar protein content nor their assembly. However, the recovery of contractile activity after exposure to a Ca(2+)-channel blocker was significantly slower (several days) than after 2,3-butanedione monoxime exposure. Furthermore, cultures exposed to Ca(2+)-channel blockers also had significantly decreased sensitivity to beta-adrenergic stimulation. Altogether, these data indicate the importance of regular Ca(2+) influx for the maintenance of the functional integrity of adult cardiomyocytes during prolonged periods of contractile arrest.     

Spontaneous beta(2)-adrenergic signaling fails to modulate L-type Ca(2+) current in mouse ventricular myocytes.

A receptor can be activated either by specific ligand-directed changes in conformation or by intrinsic, spontaneous conformational change. In the beta(2)-adrenergic receptor (AR) overexpression transgenic (TG4) murine heart, spontaneously activated beta(2)AR (beta(2)-R*) in the absence of ligands has been evidenced by elevated basal adenylyl cyclase activity and cardiac function. In the present study, we determined whether the signaling mediated by beta(2)-R* differs from that of a ligand-elicited beta(2)AR activation (beta(2)-LR*). In ventricular myocytes from TG4 mice, the properties of L-type Ca(2+) current (I(Ca)), a major effector of beta(2)-LR* signaling, was unaltered, despite a 2.5-fold increase in the basal cAMP level and a 1.9-fold increase in baseline contraction amplitude as compared with that of wild-type (WT) cells. Although the contractile response to beta(2)-R* in TG4 cells was abolished by a beta(2)AR inverse agonist, ICI118,551 (5 x 10(-7) M), or an inhibitory cAMP analog, Rp-CPT-cAMPS (10(-4) M), no change was detected in the simultaneously recorded I(Ca). These results suggest that the increase in basal cAMP due to beta(2)-R*, while increasing contraction amplitude, does not affect I(Ca) characteristics. In contrast, the beta(2)AR agonist, zinterol elicited a substantial augmentation of I(Ca) in both TG4 and WT cells (pertussis toxin-treated), indicating that L-type Ca(2+) channel in these cells can respond to ligand-directed signaling. Furthermore, forskolin, an adenylyl cyclase activator, elicited similar dose-dependent increase in I(Ca) amplitude in WT and TG4 cells, suggesting that the sensitivity of L-type Ca(2+) channel to cAMP-dependent modulation remains intact in TG4 cells. Thus, we conclude that beta(2)-R* bypasses I(Ca) to modulate contraction, and that beta(2)-LR* and beta(2)-R* exhibit different intracellular signaling and target protein specificity.     

Inhibition by Zn2+ of uridine 5'-triphosphate-induced Ca(2+)-influx but not Ca(2+)-mobilization in rat phaeochromocytoma cells.

1. Uridine 5''-triphosphate (UTP)-evoked increase in intracellular Ca2+ concentration ([Ca]i) and release of dopamine were investigated in rat phaeochromocytoma PC12 cells. UTP (1-100 microM) evoked an increase in [Ca]i in a concentration-dependent manner. This response was decreased to about 30% by extracellular Ca(2+)-depletion, but not abolished. This [Ca]i rise was mimicked by 100 microM ATP but not by 100 microM 2-methyl-thio-ATP or alpha,beta-methylene-ATP in the absence of external Ca2+, suggesting that the response was mediated by P2U purinoceptors, a subclass of P2-purinoceptors. 2. The UTP-evoked [Ca]i rise consisted of two components; a transient and a sustained one. When external Ca2+ was removed, the sustained component was abolished while the transient component was decreased by about 70% but did not disappear. These results suggest that UTP induces Ca(2+)-mobilization and, subsequently, Ca(2+)-influx. 3. The UTP-evoked increase in [Ca]i was not affected by Cd2+ (100 and 300 microM) or nicardipine (30 microM), inhibitors of voltage-gated calcium channels, but was significantly inhibited by Zn2+ (10-300 microM) in the presence of external Ca2+. Zn2+, however, did not affect the Ca2+ response to UTP in the absence of external Ca2+. 4. UTP (30 microM-1 mM) evoked the release of dopamine from the cells in a concentration-dependent manner. This dopamine release was abolished by Ca(2+)-depletion or Zn2+ but not by Cd2+ or nicardipine. 5. Taken together, the data demonstrate that UTP stimulates P2U-purinoceptors and induces a rise in [Ca]i both by Ca(2+)-mobilization and Ca(2+)-influx in PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular genetics of Ca(2+) stores and intracellular Ca(2+) signalling.

An increasing number of studies based on recombinant cells and on mouse models that express an altered repertoire of some of the key components of the intracellular Ca(2+) release stores are becoming available as a result of molecular genetics techniques. Information from these studies, together with results from studies of human diseases caused by mutations in genes that encode proteins of the intracellular Ca(2+) stores, are providing a significant advancement in understanding the interactive nature of the molecular machinery that underlies intracellular Ca(2+) signalling and how the different components of the Ca(2+) stores contribute to the regulation of cellular functions.     

Anisodamine inhibits cardiac contraction and intracellular Ca(2+) transients in isolated adult rat ventricular myocytes

Increased cardiac workload often leads to serious complications during cardiac surgery such as pericardiopulmonary bypass. Various agents have been applied to lower peripheral resistance and cardiac workload, one of which, anisodamine, is widely used in Asia. However, the direct action of anisodamine on cardiac contractile property is essentially unknown. This study was designed to examine the influence of anisodamine on ventricular contractile function at the single cardiac myocyte level. Ventricular myocytes from adult rat hearts were stimulated to contract at 0.5 Hz, and mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dL/dt), intracellular Ca(2+) fluorescence intensity change (DeltaFFI) and decay (tau). Anisodamine exhibited a concentration-dependent (10(-12)-10(-6) M) inhibition in PS and DeltaFFI, with maximal inhibitions of 44.7% and 47.2%, respectively. Anisodamine inhibited +/-dL/dt, lowered resting FFI but elicited no effect on TPS/TR(90) and tau. Pretreatment with the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished the inhibitory effect of anisodamine in cell shortening. In addition, anisodamine prevented cholinoceptor agonist carbachol-induced positive cardiac contractile response. This study demonstrated a direct cardiac depressive action of anisodamine at the myocyte level, which may be related to, at least in part, NO production and cholinoceptor antagonism.     

Na(+)/Ca(2+) exchanger inhibition exerts a positive inotropic effect in the rat heart, but fails to influence the contractility of the rabbit heart

BACKGROUND AND PURPOSE: The Na(+)/Ca(2+) exchanger (NCX) may play a key role in myocardial contractility. The operation of the NCX is affected by the action potential (AP) configuration and the intracellular Na(+) concentration. This study examined the effect of selective NCX inhibition by 0.1, 0.3 and 1.0 microM SEA0400 on the myocardial contractility in the setting of different AP configurations and different intracellular Na(+) concentrations in rabbit and rat hearts. EXPERIMENTAL APPROACH: The concentration-dependent effects of SEA0400 on I(Na/Ca) were studied in rat and rabbit ventricular cardiomyocytes using a patch clamp technique. Starling curves were constructed for isolated, Langendorff-perfused rat and rabbit hearts. The cardiac sarcolemmal NCX protein densities of both species were compared by immunohistochemistry. KEY RESULTS: SEA0400 inhibited I(Na/Ca) with similar efficacy in the two species; there was no difference between the inhibitions of the forward or reverse mode of the NCX in either species. SEA0400 increased the systolic and the developed pressure in the rat heart in a concentration-dependent manner, for example, 1.0 microM SEA0400 increased the maximum systolic pressures by 12% relative to the control, whereas it failed to alter the contractility in the rabbit heart. No interspecies difference was found in the cardiac sarcolemmal NCX protein densities. CONCLUSIONS AND IMPLICATIONS: NCX inhibition exerted a positive inotropic effect in the rat heart, but it did not influence the contractility of the rabbit heart. This implies that the AP configuration and the intracellular Na(+) concentration may play an important role in the contractility response to NCX inhibition.     

Dopamine receptor-interacting proteins: the Ca(2+) connection in dopamine signaling

Abnormal activity of the dopamine system has been implicated in several psychiatric and neurological illnesses; however, lack of knowledge about the precise sites of dopamine dysfunction has compromised our ability to improve the efficacy and safety of dopamine-related drugs used in treatment modalities. Recent work suggests that dopamine transmission is regulated via the concerted efforts of a cohort of cytoskeletal, adaptor and signaling proteins called dopamine receptor-interacting proteins (DRIPs). The discovery that two DRIPs, calcyon and neuronal Ca(2+) sensor 1 (NCS-1), are upregulated in schizophrenia highlights the possibility that altered protein interactions and defects in Ca(2+) homeostasis might contribute to abnormalities in the brain dopamine system in neuropsychiatric diseases.     

SEA0400 fails to alter the magnitude of intracellular Ca2+ transients and contractions in Langendorff-perfused guinea pig heart

SEA0400 is a recently developed inhibitor of the Na(+)/Ca(2+) exchanger (NCX) shown to suppress both forward and reverse mode operation of NCX. Present experiments were designed to study the effect of partial blockade of NCX on Ca handling and contractility in Langendorff-perfused guinea pig hearts loaded with the fluorescent Ca-sensitive dye fura-2. Left ventricular pressure and intracellular calcium concentration ([Ca(2+)](i)) were synchronously recorded before and after cumulative superfusion with 0.3 and 1 muM SEA0400. SEA0400 caused no significant change in the systolic and diastolic values of left ventricular pressure and [Ca(2+)](i). Accordingly, pulse pressure and amplitude of the [Ca(2+)](i) transient also remained unchanged in the presence of SEA0400. SEA0400 had no influence either on the time required to reach peak values of pressure and [Ca(2+)](i) or on half relaxation time. On the other hand, both 0.3 and 1 muM SEA0400 significantly increased the decay time constant of [Ca(2+)](i) transients, obtained by fitting its descending limb between 30% and 90% of relaxation, from 127 +/- 7 to 165 +/- 7 and 177 +/- 14 ms, respectively (P < 0.05, n = 6). In contrast to the guinea pig hearts, rat hearts responded to SEA0400 treatment with increased [Ca(2+)](i) transients and contractility. These interspecies differences observed in the effect of SEA0400 can be explained by the known differences in calcium handling between the two species.     

Ca(2+)-dependent inhibition of Ca(2+)-independent contraction in uterine smooth muscle.

Uterine smooth muscle of the rat shows Ca(2+)-independent contraction in response to oxytocin in Ca(2+)-free medium. Micromolar Ca2+ inhibits this contraction. We now tested whether Ca2+ itself is the cause of this inhibition. The ratio of fura-2 fluorescence, the indicator of the intracellular level of Ca2+, was increased in parallel with the degree of inhibition by Ca2+. When inhibition was elicited by Ca2+, EGTA released the inhibition. Comparison of the dose-response curve for oxytocin in Ca(2+)-free solution and that in the medium with 1 microM Ca2+ showed that the inhibition by Ca2+ is non-competitive. EGTA chelation of the intracellular Ca2+ by loading of EGTA as its acetoxymethylester resulted in diminution of inhibition by Ca2+. EGTA suppressed the Ca(2+)-induced contraction but did not affect Ca(2+)-independent contraction. It is concluded that the inhibition is induced by intracellular Ca2+ itself.     

Tributyltin-induced Ca(2+) mobilization via L-type voltage-dependent Ca(2+) channels in PC12 cells

The effects of tributyltin (TBT) on cytosolic Ca(2+) concentration ([Ca(2+)](c)) and cell viability were investigated in nerve growth factor-differentiated PC12 cells. TBT concentration dependently increased [Ca(2+)](c) with an EC(50) value of 0.07μM. This effect was markedly reduced by removal of the extracellular Ca(2+) or membrane depolarization with a high K(+) medium, but unaffected by thapsigargin causing depletion of intracellular Ca(2+) stores. The L-type voltage-dependent Ca(2+) channel (VDCC) blocker nicardipine blocked the effect of TBT, but the N-type VDCC blocker ω-conotoxin did not. TBT decreased the number of viable cells with an EC(50) value of 0.09μM. The TBT-induced cell death was prevented by nicardipine or by chelating the cytosolic Ca(2+) with BAPTA-AM, but not by ω-conotoxin. The results show that TBT causes an increase in [Ca(2+)](c) via activating L-type VDCCs, and support the idea that the organotin-induced cell death arises through Ca(2+) mobilization via L-type VDCCs.     

Differential regulation by Ca(2+) of calmodulin- and PKC-dependent contractile pathways in cat lower oesophageal sphincter

1. In the present investigation we examined the regulation of calmodulin (CaM)- and protein kinase C (PKC)-dependent pathways by cytosolic Ca(2+) in the contraction of cat lower oesophageal sphincter (LES). 2. Force developed in response to increasing doses of acetylcholine (ACh) was directly related to the increase of the [Ca(2+)](i) measured by fura-2. Thapsigargin, which depletes Ca(2+) stores, reduced the contraction and the [Ca(2+)](i). In addition, contraction in response to maximal ACh was reduced by the CaM inhibitor CGS9343B but not by the PKC inhibitor chelerythrine. The contraction in response to submaximal ACh was reduced by chelerythrine but not by CGS9343B. 3. In permeabilized cells, the contraction in response to low Ca(2+) (0.54 microm) was also reduced by CGS9343B. 4. The response to high Ca(2+) (1.0 microm) was reduced by CGS9343B. ACh also inhibited PKC activation induced by diacylglycerol, which activation is inhibited by the N-myristoylated peptide inhibitor derived from pseudosubstrate sequences of PKCalphabetagamma (myr-PKC-alphabetagamma), but not of myr-PKC-alpha. 5. These data are consistent with the view that activated CaM-dependent pathways inhibit PKC-dependent pathways, this switch mechanism might be regulated by Ca(2+) in the LES.     

Mechanisms involved in carbachol-induced Ca(2+) sensitization of contractile elements in rat proximal and distal colon

1. Mechanisms involved in Ca(2+) sensitization of contractile elements induced by the activation of muscarinic receptors in membrane-permeabilized preparations of the rat proximal and distal colon were studied. 2. In alpha-toxin-permeabilized preparations from the rat proximal and distal colon, Ca(2+) induced a rapid phasic and subsequent tonic component. After Ca(2+)-induced contraction reached a plateau, guanosine 5'-triphosphate (GTP) and carbachol (CCh) in the presence of GTP further contracted preparations of both the proximal and distal colon (Ca(2+) sensitization). Y-27632, a rho-kinase inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization more significantly in the proximal colon than in the distal colon. 3. Y-27632 at 10 microm had no effect on Ca(2+)-induced contraction or slightly inhibited phorbol-12,13-dibutyrate-induced Ca(2+) sensitization in either proximal or distal colon. Chelerythrine, a protein kinase C inhibitor, inhibited GTP plus CCh-induced Ca(2+) sensitization in the distal colon, but not in the proximal colon. The component of Ca(2+) sensitization that persisted after the chelerythrine treatment was completely inhibited by Y-27632. 4. In beta-escin-permeabilized preparations of the proximal colon, C3 exoenzyme completely inhibited GTP plus CCh-induced Ca(2+) sensitization, but PKC(19-31) did not. In the distal colon, C3 exoenzyme abolished GTP-induced Ca(2+) sensitization. It inhibited CCh-induced sensitization by 50 % and the remaining component was inhibited by PKC(19-31). 5. These results suggest that both protein kinase C and rho pathways in parallel mediate the Ca(2+) sensitization coupled to activation of muscarinic receptors in the rat distal colon, whereas the rho pathway alone mediates this action in the proximal colon.     

Nebivolol, carvedilol and metoprolol do not influence cardiac Ca(2+) sensitivity.

It has been argued that some beta-adrenoceptor antagonists may directly influence myofibrillar cross-bridge interaction in cardiac skinned fiber preparations of animal models. The present study investigates the effects of nebivolol, metoprolol and carvedilol on tension development of Triton X-100 skinned fibers obtained from human failing myocardium as well as on force of contraction and intracellular Ca(2+) transient in isolated trabeculae. In skinned fiber preparations, none of the beta-adrenoceptor antagonists (10 microM) influenced Ca(2+) sensitivity of tension development or maximal Ca(2+) activated tension (DT(max)): control: EC(50) for Ca(2+): 1.28+/-0.05 microM, DT(max): 14.09+/-0.59 mN/mm(2); nebivolol: 1.36+/-0.1 microM, 14.14+/-0.95 mN/mm(2); carvedilol: 1.32+/-0.11 microM, 13.83+/-0.90 mN/mm(2); metoprolol: 1.34+/-0.14 microM, 13.72+/-0.36 mN/mm(2). Simultaneous measurement of force and Ca(2+) transient in the presence of the beta-adrenoceptor antagonists (3 microM) showed that the decrease in force of contraction was paralleled by a similar decrease in the intracellular Ca(2+) transient. In conclusion, none of the investigated beta-adrenoceptor antagonists influenced Ca(2+) sensitivity of myofibrillar tension development in human failing myocardium.     

Structural domains influencing sensitivity to isothiourea derivative inhibitor KB-R7943 in cardiac Nna(+)/Ca(2+) exchanger

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) is a potent and selective Na(+)/Ca(2+) exchange (NCX) inhibitor that is 3-fold more inhibitory to NCX3 than to NCX1 or NCX2. Here we searched for amino acid residues that may form the KB-R7943 receptor in the exchanger by analyzing the function of chimeras between NCX1 and NCX3 as well as of their site-directed mutants. We found that the highly conserved alpha-2 repeat of the exchanger is almost exclusively responsible for the difference in drug response of the isoforms. Such difference was mostly reproduced by single substitutions of residues in the alpha-2 repeat (V820G or Q826V in NCX1 and A809V or A809I in NCX3), suggesting their importance in drug sensitivity. Cysteine scanning mutagenesis of the alpha-2 repeat of NCX1 identified one residue (Gly833) that caused a large (> or = 30-fold) reduction in drug sensitivity. We found that the Gly-to-Thr substitution caused even larger reduction in drug sensitivity. Interestingly, extracellularly applied KB-R7943 at 0.8 microM markedly inhibited the whole-cell outward exchange current, whereas the drug applied intracellularly at 30 microM did not. These results suggest that KB-R7943 inhibits the exchanger from the external side in intact cells and that a region of the alpha-2 repeat of NCX1 containing Gly833 may participate in the formation of the drug receptor. Because we suggested previously that Gly833 is accessible from the inside of a cell, the results raised an interesting possibility that this residue may alter its position during Na(+)/Ca(2+) exchange in such a way that it becomes accessible to external drug.     

Role of mitochondria in Ca(2+) oscillations and shape of Ca(2+) signals in pancreatic acinar cells

We studied the role of mitochondria in Ca(2+) signals in fura-2 loaded exocrine pancreatic acinar cells. Mitochondrial depolarization in response to carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone or rotenone (assessed by confocal microscopy using rhodamine-123) induced a partial but statistically significant reduction in the decay of Ca(2+) signals under different experimental conditions. Spreading of Ca(2+) waves evoked by the pancreatic secretagogue cholecystokinin cholecystokinin octapeptide was accelerated by mitochondrial inhibitors, whereas the cytosolic Ca(2+) concentration ([Ca(2+)](i)) oscillations in response to physiological levels of this hormone were suppressed by rotenone and carbonylcyanide-p-tryfluoromethoxyphenyl hydrazone. Oligomycin, an inhibitor of mitochondrial ATP synthase, did no affect either propagation of calcium waves nor [Ca(2+)](i) oscillations. Individual mitochondria of rhod-2 loaded acinar cells showed heterogeneous matrix Ca(2+) concentration increases in response to oscillatory and maximal levels of cholecystokinin octapeptide. On the other hand, using Ba(2+) for unequivocal study of capacitative calcium entry we found that mitochondrial inhibitors did not affect this process. Our results show that although the role of mitochondria as a Ca(2+) clearing system in exocrine cells is quantitatively secondary, they play an essential role in the spatial propagation of Ca(2+) waves and in the development of [Ca(2+)](i) oscillations.     

Inhibition of T-type and L-type Ca(2+) currents by aranidipine, a novel dihydropyridine Ca(2+) antagonist

The effects of aranidipine, a novel dihydropyridine Ca(2+) channel antagonist, on membrane currents in guinea pig ventricular myocytes and on action potentials in rabbit sinoatrial node tissue were examined. In myocytes, aranidipine (10 nmol/l to 1 micromol/l) concentration-dependently decreased T-type and L-type Ca(2+) currents. Aranidipine (1 micromol/l) had little effect on K(+) currents. In the sinoatrial node, 0.1 micromol/l aranidipine increased cycle length, and decreased +V(max) and the slope of the phase 4 depolarization. Thus, inhibition of both T-type and L-type Ca(2+) currents by aranidipine may partly explain its potent negative chronotropic activity.     

Influence of hypothyroid state on 45Ca(2+) influx and sensitivity of rat uterus to nifedipine and diltiazem

The influence of methimazole-induced hypothyroidism on spontaneous rhythmic contractions and Ca2+ channel function of rat uterus was examined. Hypothyroidism significantly reduced the amplitude and frequency of spontaneous rhythmic contractions. Nifedipine (10(-12)-10(-6) M) and diltiazem (10(-9)-10(-4) M) caused concentration-related inhibition of the myogenic responses of the oestrogenised rat uterus obtained from both eu- and hypothyroid rats. However, nifedipine was less potent (IC(50); 5.4 x 10(-9) M; n=6) in hypothyroid rat uterus as compared to euthyroid controls (IC(50): 8.13 x 10(-12) M; n=9) to inhibit the rhythmic contractions. Similarly, diltiazem was less potent (IC(50): 4.57 x 10(-6) M; n=9) to inhibit the uterine spontaneous contractions in hypothyroid than in euthyroid rat uterus (IC(50): 6.4 x 10(-8) M; n=6). A similar decrease in the sensitivity to nifedipine and diltiazem for reversal of K+ (100 mM)-induced tonic contraction was observed in uterus obtained from hypothyroid rats compared to the controls. Both nifedipine and diltiazem were less potent for causing concentration-related inhibition of K+-stimulated 45Ca2+ influx in uterine strips taken from the hypothyroid rats. Thus, the IC(50) values of nifedipine (1.83 x 10(-8) M; n=12) and diltiazem (1.8 x 10(-6) M; n=9) were significantly greater in tissues obtained from hypothyroid rats compared to the controls (IC(50) of nifedipine, 1.15 x 10(-11) M; n=12, diltiazem, 8.1 x 10(-8) M; n=8). Nifedipine-sensitive influx of 45Ca2+ - stimulated either by K+ (100 mM) or Bay k8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2'-(trifluromethyl)phenyl]-3-pyridine carboxylic acid methyl ester) (10(-8) M) was significantly less in uterine strips from hypothyroid rats compared to the controls. The results of the present study suggest that the inhibition of uterine rhythmic contractions may be attributable to a reduction in rat myometrial Ca2+ channel function in the hypothyroid state.     

Ca(2+) and insulin-mediated glucose uptake

Insulin stimulates glucose uptake in striated muscle and fat via a complex cascade of signaling events. Insulin resistance in these tissues and type 2 diabetes constitute major and rapidly increasing health problems in society. Recent research implicates an important role of Ca(2+) in insulin-mediated glucose uptake. Maneuvers that increase or decrease Ca(2+) influx also increase or decrease insulin-mediated glucose uptake both in normal and insulin-resistant cells. Ca(2+) appears to act on late steps in the insulin-signaling cascade, that is, the docking and fusion of glucose transporter 4 (GLUT4) vesicles with the plasma membrane. No Ca(2+) sensor in this process has yet been explicitly identified but recent studies point at synaptotagmin VII and the motor protein Myo1c as possible candidates.     

Emodin increases Ca(2+) influx through L-type Ca(2+) channel in guinea pig gallbladder smooth muscle

Emodin is known to be used in the treatment of cholesterol stones and cholecystitis. This study sought to investigate the effects of emodin on the contraction of gallbladder smooth muscle (GBSM), intracellular Ca(2+) concentration and L-type calcium current in GBSM cells. Gallbladder muscle strips were obtained from adult guinea pigs and the resting tension was recorded. Gallbladder smooth muscle cells were isolated by enzymatic digestion. Cells were loaded with fluo-3/AM and [Ca(2+)](i) was determined by a laser confocal microscope. Calcium current was recorded by the whole-cell patch clamp method. Emodin increased the resting tension of GBSM strips in a dose-dependent manner. Emodin elevated [Ca(2+)](i) in GBSM cells, and this effect was attenuated by pretreatment with nifedipine. In addition, Emodin increased L-type calcium current at concentrations of 1 to 30 microM (at +10 mV, 10 microM, 45.1+/-5.2% compared to control, EC(50) =3.11 microM). In the presence of protein kinase C (PKC) inhibitor, Staurosporine, emodin did not significantly affect the calcium current. However, phorbol 12, 13-dibutyrate mimicked emodin in enhancement of the calcium current. These results suggest that emodin promotes gallbladder contraction by increasing Ca(2+) influx through L-type calcium channel via PKC pathway.     

Tetramethylpyrazine elicits disparate responses in cardiac contraction and intracellular Ca(2+) transients in isolated adult rat ventricular myocytes

Tetramethylpyrazine (TMP) is the biologically active ingredient isolated from a popular Chinese medicinal plant, Ligusticum wallichil franchat, which has been used effectively since the 1970s to treat ischemic heart disease, cerebrovascular and thrombotic vascular diseases. The direct action of TMP on cardiac contractile function, however, is largely unclear. This study was designed to examine the effect of TMP on ventricular contractile function at the single cardiac myocyte level. Adult rat ventricular myocytes were isolated and stimulated to contract at 0.5 Hz, and mechanical and intracellular Ca(2+) properties were evaluated using an IonOptix Myocam system. Contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), maximal velocity of shortening/relengthening (+/-dl/dt), resting intracellular Ca(2+) level, Ca(2+)-induced Ca(2+) release (CICR) and decay. TMP (10(-10)-10(-5) M) exhibited an increase in PS with a maximal increase of 30.9%. TMP had no effect on +/-dl/dt, TPS/TR(90) or CICR but lowered resting intracellular Ca(2+) level and slowed intracellular Ca(2+) decay. Pretreatment with either the nonspecific nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME, 100 microM) or inducible NOS inhibitor W1400 effectively abolished the positive effect of TMP on myocyte shortening. Our data demonstrate a direct positive inotropic effect of TMP in cardiac myocytes, which may be related, at least in part, to NO production.