Involvement Of L-type Ca2 Research Articles

Mechanisms underlying the cardio-protection of total ginsenosides against myocardial ischemia in rats in vivo and in vitro: Possible involvement of L-type Ca2+ channels, contractility and Ca2+ homeostasis

Here we aimed to observe the effects of total ginsenosides (TG) against isoproterenol (ISO) induced myocardial ischemia (MI) and to explore its underlying mechanisms based on L-type Ca2+ current (ICa-L), intracellular Ca2+ ([Ca2+]i) and contraction in isolated rat myocytes. Rat model of MI was induced by subcutaneously injection of ISO (85 mg/kg) for 2 consecutive days. J-point elevation, heart rate, serum levels of creatine kinase (CK) and lactated dehydrogenase (LDH), and heart morphology changes were observed. Influences of TG on ICa-L, [Ca2+]i and contraction in isolated rat myocytes were observed by the patch-clamp technique and IonOptix detection system. TG significantly reduced J-point elevation, heart rate, serum levels of CK and LDH, and improved heart pathologic morphology. TG decreased ICa-L in concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of 31.65 μg/mL. TG (300 μg/mL) decreased ICa-L of normal and ischemic ventricular myocytes by 64.33 ± 1.28% and 61.29 ± 1.38% respectively. At 30 μg/mL, TG reduced Ca2+ transient by 21.67 ± 0.94% and cell shortening by 38.43 ± 6.49%. This study showed that TG displayed cardioprotective effects on ISO-induced MI rats and the underlying mechanisms may be related to inhibition of ICa-L, damping of [Ca2+]i and decrease of contractility.

Calcium-dependent and calcium-mimicking pathways regulate lead-induced myometrial contraction in water buffaloes

Lead produced dose-dependent contractile effect on buffalo myometrium and it was more marked on tonicity and amplitude while almost negligible on phasic contractions and frequency. In the presence of Ca2+-free Ringer Locke solution (RLS) and nifedipine, lead-induced uterotonic effect was significantly attenuated with decrease in Emax thus, suggesting major role of extracellular Ca2+ in uterotonic effect of lead and involvement of L-type Ca2+-channels in mediating this response. Direct excitatory effect of cyclopiazonic acid (CPA) on myometrium indicates possible Ca2+-influx via store operated calcium channels (SOCC). Lead failed to evoke any excitatory effect in the concurrent presence of nifedipine (1μM) and CPA (10μM), thus intracellular calcium seems to have negligible role in lead-induced myometrial contraction. In Ca2+-free high K+ (80 mM) depolarizing solution, lead-induced excitatory effect was attenuated by GF 109203X; which suggests permeation of lead through L-type Ca2+ channels to replace Ca2+ which directly activates protein kinase C (PKC) to produce excitatory effect. Thus lead seems to interact with calcium in buffalo myometrium in a dynamic fashion and exerts calcium-depending and calcium-mimicking effects; and therefore, lead is likely to have adverse effect on reproductive functions in buffaloes.

Multiple Ca2+ channel-dependent components in growth hormone secretion from rat anterior pituitary somatotrophs.

The involvement of L-type Ca(2+) channels in both 'basal' and 'stimulated' growth hormone (GH) secretion is well established; however, knowledge regarding the involvement of non-L-type Ca(2+) channels is lacking. We investigated whether non-L-type Ca(2+) channels regulate GH secretion from anterior pituitary (AP) cells. To this end, GH secretion was monitored from dissociated AP cells, which were incubated for 15min with 2mm K(+) ('basal' secretion) or 60mm K(+) ('stimulated' secretion). The role of non-L-type Ca(2+) influx was investigated using specific channel blockers, including ω-agatoxin-IVA, ω-conotoxin GVIA or SNX-482, to block P/Q-, N- or R-type Ca(2+) channels, respectively. Our results demonstrate that P/Q-, N- and R-type Ca(2+) channels contributed 21.2±1.9%, 20.2±7.6% and 11.4±1.8%, respectively, to 'basal' GH secretion and 18.3±1.0%, 24.4±5.4% and 14.2±4.8%, respectively, to 'stimulated' GH secretion. After treatment with a 'cocktail' that comprised the previously described non-L-type blockers, non-L-type Ca(2+) channels contributed 50.9±0.4% and 45.5±2.0% to 'basal' and 'stimulated' GH secretion, respectively. Similarly, based on the effects of nifedipine (10μM), L-type Ca(2+) channels contributed 34.2±3.7% and 54.7±4.1% to 'basal' and 'stimulated' GH secretion, respectively. Interestingly, the relative contributions of L-type/non-L-type Ca(2+) channels to 'stimulated' GH secretion were well correlated with the relative contributions of L-type/non-L-type Ca(2+) channels to voltage-gated Ca(2+) influx in AP cells. Finally, we demonstrated that compartmentalisation of Ca(2+) channels is important for GH secretion. Lipid raft disruption (methyl-β-cyclodextrin, 10mm) abrogated the compartmentalisation of Ca(2+) channels and substantially reduced 'basal' and 'stimulated' GH secretion by 43.2±3.4% and 58.4±4.0%, respectively. In summary, we have demonstrated that multiple Ca(2+) channel-dependent pathways regulate GH secretion. The proper function of these pathways depends on their compartmentalisation within AP cell membranes.

Estresse crônico melhora a função miocárdica sem alterar a atividade do canal-L para Ca+2 em ratos

Chronic stress is associated with cardiac remodeling; however the mechanisms have yet to be clarified. The purpose of this study was test the hypothesis that chronic stress promotes cardiac dysfunction associated to L-type calcium Ca2+ channel activity depression. Thirty-day-old male Wistar rats (70 - 100 g) were distributed into two groups: control (C) and chronic stress (St). The stress was consistently maintained at immobilization during 15 weeks, 5 times per week, 1h per day. The cardiac function was evaluated by left ventricular performance through echocardiography and by ventricular isolated papillary muscle. The myocardial papillary muscle activity was assessed at baseline conditions and with inotropic maneuvers such as: post-rest contraction and increases in extracellular Ca2+ concentration, in presence or absence of specific blockers L-type calcium channels. The stress was characterized for adrenal glands hypertrophy, increase of systemic corticosterone level and arterial hypertension. The chronic stress provided left ventricular hypertrophy. The left ventricular and baseline myocardial function did not change with chronic stress. However, it improved the response of the papillary muscle in relation to positive inotropic stimulation. This function improvement was not associated with the L-type Ca2+ channel. Chronic stress produced cardiac hypertrophy; however, in the study of papillary muscle, the positive inotropic maneuvers potentiated cardiac function in stressed rats, without involvement of L-type Ca2+ channel. Thus, the responsible mechanisms remain unclear with respect to Ca2+ influx alterations.

Minor contribution of cytosolic Ca2+ transients to the pacemaker rhythm in guinea pig sinoatrial node cells

The question of the extent to which cytosolic Ca(2+) affects sinoatrial node pacemaker activity has been discussed for decades. We examined this issue by analyzing two mathematical pacemaker models, based on the "Ca(2+) clock" (C) and "membrane clock" (M) hypotheses, together with patch-clamp experiments in isolated guinea pig sinoatrial node cells. By applying lead potential analysis to the models, the C mechanism, which is dependent on potentiation of Na(+)/Ca(2+) exchange current via spontaneous Ca(2+) release from the sarcoplasmic reticulum (SR) during diastole, was found to overlap M mechanisms in the C model. Rapid suppression of pacemaker rhythm was observed in the C model by chelating intracellular Ca(2+), whereas the M model was unaffected. Experimental rupturing of the perforated-patch membrane to allow rapid equilibration of the cytosol with 10 mM BAPTA pipette solution, however, failed to decrease the rate of spontaneous action potential within ∼30 s, whereas contraction ceased within ∼3 s. The spontaneous rhythm also remained intact within a few minutes when SR Ca(2+) dynamics were acutely disrupted using high doses of SR blockers. These experimental results suggested that rapid disruption of normal Ca(2+) dynamics would not markedly affect spontaneous activity. Experimental prolongation of the action potentials, as well as slowing of the Ca(2+)-mediated inactivation of the L-type Ca(2+) currents induced by BAPTA, were well explained by assuming Ca(2+) chelation, even in the proximity of the channel pore in addition to the bulk cytosol in the M model. Taken together, the experimental and model findings strongly suggest that the C mechanism explicitly described by the C model can hardly be applied to guinea pig sinoatrial node cells. The possible involvement of L-type Ca(2+) current rundown induced secondarily through inhibition of Ca(2+)/calmodulin kinase II and/or Ca(2+)-stimulated adenylyl cyclase was discussed as underlying the disruption of spontaneous activity after prolonged intracellular Ca(2+) concentration reduction for >5 min.

Trifolirhizin and desmethylicaritin, active components of roots of Sophora flavescens AIT., induced vascular relaxation via NO/cGMP signaling

Trifolirihizin and desmethylicaritin isolated from roots of Sophora flavescens Ait. induced relaxation of the phenylephrine-precontracted aorta in a dose-dependent manner, respectively, which were disappeared by removal of functional endothelium. Pretreatment of the aortic tissues with NG-nitro-L-arginine methylester (L-NAME), or 1H-[1,2,4]-oxadiazole-[4,3-¥á]-quinoxalin-1-one (ODQ) inhibited the relaxation induced by trifolirhizin and desmethylicaritin, respectively. Trifolirhizin-induced relaxations were also markedly attenuated by addition of verapamil, diltiazem, or atropine. Desmethylicaritin-induced vascular relaxation was also markedly attenuated by addition of verapamil or diltiazem but not by atropine. These results suggest that trifolirhizin dilates vascular smooth muscle via endothelium-dependent NO/cGMP signaling pathways possible involvement of L-type Ca2+ channels and/or muscarinic receptor. However, the mechanism of desmethylicaritin-induced vascular relaxation was dependent on endothelium-dependent NO/cGMP signaling pathway without involvement of muscarinic receptor

Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.

Seasonality of dihydropyridine receptor binding in the heart of an anoxia-tolerant vertebrate, the crucian carp (Carassius carassius L.).

Prolonged anoxia tolerance of facultative anaerobes is based on metabolic depression and thus on controlled reduction of energy-utilizing processes. One proposed survival mechanism is the closing of ion channels to decrease energetic cost of ion pumping (Hochachka PW. Science 231: 234-241, 1986). To test this hypothesis, the involvement of L-type Ca2+ channels in seasonal anoxia tolerance of the vertebrate heart was examined by determining the number of [methyl-3H]PN200-110 (a ligand of L-type Ca2+ channel alpha-subunit) binding sites of the cardiac tissue and the density of Ca2+ current in ventricular myocytes of an anoxia-resistant fish species, the crucian carp. In their natural environment, the fish were exposed for > 3 mo of hypoxia (O2 < 2.5 mg/l) followed by almost 8 wk of anoxia that resulted in abrupt depletion of cardiac glycogen stores in late spring. Unexpectedly, however, the number of [methyl-3H]PN200-110 binding sites did not decline in hypoxia/anoxia as predicted by the channel arrest hypothesis but remained constant for most of the year. However, in early summer, the number of [methyl-3H]PN200-110 binding sites doubled for a period of approximately 2 mo, which functionally appeared as a 74% larger Ca2+ current density. Thus the anoxia tolerance of the carp heart cannot be based on downregulation of Ca2+ channel units in myocytes but is likely to depend on suppressed heart rate, i.e., regulation of the heart at the systemic level, and direct depressive effects of low temperature on Ca2+ current to achieve savings in cardiac work load and ion pumping. The summer peak in the number of functional Ca2+ channels indicates a short period of high cardiac activity possibly associated with reproduction and active perfusion of tissues after the winter stresses.

Lacidipine decreases the honeybee venom-induced vasoconstriction of the isolated porcine coronary artery

The venom of the honeybee Apis mellifera induces cardiovascular dysfunction. As its effects on coronary arteries have not yet been described, we studied the effects of the whole honeybee venom (non-volatile part) in the isolated porcine left anterior descending coronary artery (LAD) and the influence of L-type Ca2+ channel blocker, lacidipine, upon the venom effects in LAD. The venom produced concentration dependent contractions (7 - 70 μg/ml) of the porcine LAD; maximal effect of the venom was approximately the same as the effect of 30 mM KCl. Lacidipine concentration dependently (0.1-10 μM) and significantly (P ≤ 0.05) decreased the venom-induced vasoconstriction. The results indicate the involvement of L-type Ca2+ channels in coronary contraction, induced by bee venom.

O-163 - involvement of L-type Ca2+ channels in basic fibroblast growth factor-induced neurite morphogenesis in cultured rat hippocampal neurons

O-163 - involvement of L-type Ca2+ channels in basic fibroblast growth factor-induced neurite morphogenesis in cultured rat hippocampal neurons

IA-type K+ channel blockers promote survival of cortical neurons in culture: involvement of L-type Ca2+ channels.

Dendrotoxin (DTX), a well characterized IA-type potassium channel blocker, directly added to the culture medium had no effect on survival of cultured cortical neurons at 6 or 14 days in vitro. On the contrary, neurons exposed to DTX remained in better condition than untreated ones. In an attempt to demonstrate the mechanisms by which DTX may affect neuronal survival we studied its effect in co-cultures of cortical neurons and astrocytes submitted to successive medium changes. After the second change of medium, at 9 days in vitro, the neuronal number in controls decreased by 43%, while in cultures receiving astrocyte-conditioned medium the cell loss was significantly reduced (15%, p < 0.01) with respect to control conditions. When DTX was added to the culture medium neuronal loss was also significantly prevented (25% for 1 microM DTX, p < 0.01) with respect to control conditions. 4-Aminopyridine (4-AP) and 21 mM K+ also preserved neurons. The L-type calcium channel antagonist nifedipine (5 microM) abolished the protective effect of DTX and 4-AP. These results show that K+ channel blockade induces protection against damage produced by repetitive medium change and that this effect is mediated by L-type Ca2+ channels.

Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Using a range of Ca2+ channel blockers we have investigated the Ca2+ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca2+ concentration ([Ca2+]i) and glutamate release from cultured rat cerebellar granule cells. omega-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca2+]i rise or on glutamate release, ruling out a significant role for N-type Ca2+ channels. Nifedipine substantially inhibited the initial transient rise in [Ca2+]i and the plateau phase of the [Ca2+]i rise and glutamate release, suggesting the involvement of L-type Ca2+ channels. Both omega-agatoxin and omega-conotoxin-MVIIC also inhibited the transient rise in [Ca2+]i and glutamate release but not the plateau phase of the [Ca2+]i rise. The inhibitions by nifedipine were not increased by coaddition of omega-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca2+ currents that are sensitive to nifedipine, omega-agatoxin-IVA, and also omega-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca2+]i rise or glutamate release to distinct P-, Q-, or O-type Ca2+ currents.