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Ginger has been widely used as a flavor, food, and traditional medicine for centuries. 6‐Gingerol (6‐Gin) is the active components of ginger and offers some beneficial effects on cardiovascular diseases. Here, the effects of 6‐Gin on L‐type Ca2+ current (ICa‐L), contractility, and the Ca2+ transients of rat cardiomyocytes, were investigated via patch‐clamp technique and the Ion Optix system. The 6‐Gin decreased the ICa‐L of normal and ischemic ventricular myocytes by 58.17 ± 1.05% and 55.22 ± 1.34%, respectively. 6‐Gin decreased ICa‐L in a concentration‐dependent manner with a half‐maximal inhibitory concentration (IC50) of 31.25 μmol/L. At 300 μmol/L, 6‐Gin reduced the cell shortening by 48.87 ± 5.44% and the transients by 42.5 ± 9.79%. The results indicate that the molecular mechanisms underlying the cardio‐protective effects of 6‐Gin may because of a decreasing of intracellular Ca2+ via the inhibition of ICa‐L and contractility in rat cardiomyocytes.

Fil: Gonzalez, Betina. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Houssay. Instituto de Investigaciones Farmacologicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquimica. Instituto de Investigaciones Farmacologicas; Argentina

Recent studies have suggested that transmitter release facilitation at synapses is largely mediated by presynaptic Ca2+ current facilitation, but the exact contribution of Ca2+ current facilitation has not been determined quantitatively. Here, we determine the contribution of Ca2+ current facilitation, and of an increase in the residual free Ca2+ concentration ([Ca2+]i) in the nerve terminal, to paired-pulse facilitation of transmitter release at the calyx of Held. Under conditions of low release probability imposed by brief presynaptic voltage-clamp steps, transmitter release facilitation at short interstimulus intervals (4 ms) was 227 ± 31% of control, Ca2+ current facilitation was 113 ± 4% of control, and the peak residual [Ca2+]i was 252 ± 18 nm over baseline. By inferring the ‘local’[Ca2+]i transients that drive transmitter release during these voltage-clamp stimuli with the help of a kinetic release model, we estimate that Ca2+ current facilitation contributes to ∼40% to paired-pulse facilitation of transmitter release. The remaining component of facilitation strongly depends on the build-up, and on the decay of the residual free [Ca2+]i, but cannot be explained by linear summation of the residual free [Ca2+]i, and the back-calculated ‘local’[Ca2+]i signal, which only accounts for ∼10% of the total release facilitation. Further voltage-clamp experiments designed to compensate for Ca2+ current facilitation demonstrated that about half of the observed transmitter release facilitation remains in the absence of Ca2+ current facilitation. Our results indicate that paired-pulse facilitation of transmitter release at the calyx of Held is driven by at least two distinct mechanisms: Ca2+ current facilitation, and a mechanism independent of Ca2+ current facilitation that closely tracks the time course of residual free [Ca2+]i.

Aldosterone is involved in a variety of pathophysiological processes that ultimately cause cardiovascular diseases. Despite this, the physiological role of aldosterone in heart function remains elusive. We took advantage of transgenic mouse models characterized by a renal salt-losing (SL) or salt-retaining (SR) phenotype, thus exhibiting chronically high or low plasma aldosterone levels, respectively, to investigate the chronic effects of aldosterone in cardiomyocytes devoid of pathology. On a diet containing normal levels of salt, these animals do not develop any evidence of cardiovascular disease. Using the whole cell patch-clamp technique on freshly isolated adult ventricular cardiomyocytes, we observed that the amplitude of L-type Ca2+ currents (ICa) correlates with plasma aldosterone levels. Larger values of ICa are associated with high aldosterone concentrations in SL models, whereas smaller values of ICa were observed in the SR model. Neither the time- nor the voltage-dependent properties of ICa varied measurably. In parallel, we determined whether modulation of ICa by blood concentration of aldosterone has a major physiological impact on the excitation–contraction coupling of the cardiomyocytes. Action potential duration, [Ca2+]i transient amplitude and contraction are increased in the SL model and decreased in the SR model. In conclusion, we demonstrate that the blood concentration of aldosterone exerts chronic regulation of ICa in mouse cardiomyocytes. This regulation has important consequences for excitation–contraction coupling and, potentially, for other Ca2+-regulated functions in cardiomyocytes.

Currents arising from T-type Ca2+ channels in nucleus reticularis thalami (nRT) play a critical role in generation of low-amplitude oscillatory bursting involving mutually interconnected cortical and thalamic neurones, and are implicated in the state of arousal and sleep, as well as seizures. Here we show in brain slices from young rats that two kinetically different T-type Ca2+ currents exist in nRT neurones, with a slowly inactivating current expressed only on proximal dendrites, and fast inactivating current predominantly expressed on soma. Nickel was about twofold more potent in blocking fast (IC50 64 μm) than slow current (IC50 107 μm). The halogenated volatile anaesthetic enflurane blocked both currents, but only the slowly inactivating current was affected in voltage-dependent fashion. Slow dendritic current was essential for generation of low-threshold Ca2+ spikes (LTS), and both enflurane and nickel also suppressed LTS and neuronal burst firing at concentrations that blocked isolated T currents. Differential kinetic properties of T currents expressed in cell soma and proximal dendrites of nRT neurones indicate that various subcellular compartments may exhibit different membrane properties in response to small membrane depolarizations. Furthermore, since blockade of two different T currents in nRT neurones by enflurane and other volatile anaesthetics occurs within concentrations that are relevant during clinical anaesthesia, our findings suggest that these actions could contribute to some important clinical effects of anaesthetics.

Outer hair cells (OHC) serve as electromechanical amplifiers that guarantee the unique sensitivity and frequency selectivity of the mammalian cochlea. It is unknown whether the afferent fibres connected to adult OHCs are functional. If so, voltage-activated Ca2+ channels would be required for afferent synaptic transmission. In neonatal OHCs, Ca2+ channels seem to play a role in maturation since OHCs from Cav1.3-deficient (Cav1.3-/-) mice degenerate shortly after the onset of hearing. We therefore studied whole-cell Ca2+ currents in outer hair cells aged between postnatal day 1 (P1) and P8. OHCs showed a rapidly activating inward current that was 1.8 times larger with 10 mM Ba2+ as charge carrier (IBa) than with equimolar Ca2+ (ICa). IBa started activating at -50 mV with Vmax = -1.9 +/- 6.9 mV, V0.5 = -15.0 +/- 7.1 mV and k = 8.2 +/- 1.1 mV (n = 34). The peak IBa showed negligible inactivation (3.6 % after 300 ms) whereas the ICa (10 mM Ca2+) was inactivated by 50.7 %. OHC IBa was reduced by 33.5 +/- 10.3 % (n = 14) with 10 microM nifedipine and increased to 178.5 +/- 57.8 % (n = 14) by 5 microM Bay K 8644. A dose-response curve for nifedipine revealed an IC50 of 2.3 microM, a Hill coefficient of 2.7 and a maximum block of 36 %. Average IBa density in OHCs was 24.4 +/- 10.8 pA pF-1 (n = 105) which is only 38 % of the value in inner hair cells. Single cell RT-PCR revealed expression of Cav1.3 in OHCs. In OHCs from Cav1.3-/- mice, Ba2+ current density was reduced to 0.6 +/- 0.5 pA pF-1 (n = 9) indicating that > 97 % of the Ca2+ channel current in OHCs flows through Cav1.3.

Different subtypes of voltage-dependent Ca(2+) currents in native neurones are essential in coupling action potential firing to Ca(2+) influx. For most of these currents, the underlying Ca(2+) channel subunits have been identified on the basis of pharmacological and biophysical similarities. In contrast, the molecular basis of R-type Ca(2+) currents remains controversial. We have therefore examined the contribution of the Ca(V)2.3 (alpha(1E)) subunits to R-type currents in different types of central neurones using wild-type mice and mice in which the Ca(V)2.3 subunit gene was deleted. In hippocampal CA1 pyramidal cells and dentate granule neurones, as well as neocortical neurones of wild-type mice, Ca(2+) current components resistant to the combined application of omega-conotoxin GVIA and MVIIC, omega-agatoxin IVa and nifedipine (I(Ca,R)) were detected that were composed of a large R-type and a smaller T-type component. In Ca(V)2.3-deficient mice, I(Ca,R) was considerably reduced in CA1 neurones (79 %) and cortical neurones (87 %), with less reduction occurring in dentate granule neurones (47 %). Analysis of tail currents revealed that the reduction of I(Ca,R) is due to a selective reduction of the rapidly deactivating R-type current component in CA1 and cortical neurones. In all cell types, I(Ca,R) was highly sensitive to Ni(2+) (100 microM: 71-86 % block). A selective antagonist of cloned Ca(V)2.3 channels, the spider toxin SNX-482, partially inhibited I(Ca,R) at concentrations up to 300 nM in dentate granule cells and cortical neurones (50 and 57 % block, EC(50) 30 and 47 nM, respectively). I(Ca,R) in CA1 neurones was significantly less sensitive to SNX-482 (27 % block, 300 nM SNX-482). Taken together, our results show clearly that Ca(V)2.3 subunits underlie a significant fraction of I(Ca,R) in different types of central neurones. They also indicate that Ca(V)2.3 subunits may give rise to Ca(2+) currents with differing pharmacological properties in native neurones.

Ca2+ signals in cardiac muscle cells are composed of spatially limited elementary events termed Ca2+ sparks. Several studies have also indicated that Ca2+ signals smaller than Ca2+ sparks can be elicited. These signals have been termed Ca2+ quarks and were proposed to result from the opening of a single Ca2+ release channel of the sarcoplasmic reticulum. We used laser-scanning confocal microscopy to examine the subcellular properties of Na+ current (I(Na))- and L-type Ca2+ current (I(Ca,L))-induced Ca2+ transients in voltage-clamped ventricular myocytes isolated from guinea-pigs. Both currents, I(Na) and I(Ca,L), evoked substantial, global Ca2+ transients. To examine the spatiotemporal properties of such Ca2+ signals, we performed power spectral analysis of these Ca2+ transients and found that both lacked spatial frequency components characteristic for Ca2+ sparks. The application of 10 microM verapamil to partially block L-type Ca2+ current reduced the corresponding Ca2+ transients down to individual Ca2+ sparks. In contrast, I(Na)-induced Ca2+ responses were still spatially homogeneous and lacked Ca2+ sparks even for small current amplitudes. By using high resistance patch pipettes (4 MOmega) to exaggerate the loss of voltage control during I(Na), Ca2+ sparks appeared superimposed on a homogeneous Ca2+ release component and were exclusively triggered during the flow of I(Na). In the presence of 10 microM ryanodine both I(Ca,L) and I(Na) elicited small, residual Ca2+ transients that were spatially homogeneous but displayed distinctively different temporal profiles. We conclude that I(Na) is indeed able to cause Ca2+ release in guinea-pig ventricular myocytes. In contrast to I(Ca,L)-induced Ca2+ transients, which are built up from the recruitment of individual Ca2+ sparks, the I(Na)-evoked cellular responses were always homogeneous, indicating that their underlying elementary Ca2+ release event is distinct from the Ca2+ spark. Thus, I(Na)-induced Ca2+ transients are composed of smaller Ca2+ signals, most likely Ca2+ quarks.

Agmatine, an imidazoline deriviatives, suppress the vasopressor sympathetic outflow to produce hypotension. This effect has been known to be mediated in part by suppressing sympathetic outflow via acting imidazoline I2 receptors (IR2) at postganglionic sympathetic neurons. But, the cellular mechanism of IR1-induced inhibition of noradrenaline (NA) release is still unknown. We therefore, investigated the effect of IR1 activation on voltage-dependent Ca2+ channels which is known to play an pivotal role in regulating NA in rat coeliac ganglion neurons, using the conventional whole-cell patch-clamp method. In the presence of rauwolscine (3 uM), which blocks a2-adrenoceptor (Ra2), agmatine inhibited voltage-dependent Ca2+ current (ICa) by about 30%. This agmatine-induced inhibition was almost completely prevented by efaroxan (10 uM) which blocks IR1 as well as Ra2. In addition, w-conotoxin (CgTx) GVIA (1 uM) occluded agmatine-induced inhibition of ICa, but, agmatine-induced ICa inhibition was not affected by p...

Adenosine causes voltage- and non-voltage-dependent inhibition of high voltage-activated (HVA) Ca2+ currents in Xenopus laevis embryo spinal neurons. As this inhibition can be blocked by 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) and mimicked by N6-cyclopentyladenosine (CPA) it appears to be mediated by A1 receptors. Agents active at A2 receptors either were without effect or could be blocked by DPCPX. AMP had no agonist action on these receptors. By using omega-conotoxin GVIA we found that adenosine inhibited an N-type Ca2+ current as well as a further unidentified HVA current that was insensitive to dihydropyridines, omega-agatoxin TK and omega-conotoxin MVIIC. Both types of current were subject to voltage- and non-voltage-dependent inhibition. We used CPA and DPCPX to test whether A1 receptors regulated spinal motor pattern generation in spinalized Xenopus embryos. DPCPX caused a near doubling of, while CPA greatly shortened, the length of swimming episodes. In addition, DPCPX slowed, while CPA greatly speeded up, the rate of run-down of motor activity. Our results demonstrate a novel action of A1 receptors in modulating spinal motor activity. Furthermore they confirm that adenosine is produced continually throughout swimming episodes and acts to cause the eventual termination of activity.

Expression of rat alpha1G, human alpha1H and rat alpha1I subunits of voltage-activated Ca2 + channels in HEK-293 cells yields robust Ca2 + inward currents with 1.25 mM Ca2 + as the charge carrier. Both similarities and marked differences are found between their biophysical properties. Currents induced by expression of alpha1G show the fastest activation and inactivation kinetics. The alpha1H and alpha1I currents activate and inactivate up to 1.5- and 5-fold slower, respectively. No differences in the voltage dependence of steady state inactivation are detected. Currents induced by expression of alpha1G and alpha1H deactivate with time constants of up to 6 ms at a test potential of - 80 mV, but currents induced by alpha1I deactivate about three-fold faster. Recovery from short-term inactivation is more than three-fold slower for currents induced by alpha1H and alpha1I in comparison to alpha1G. In contrast to these characteristics, reactivation after long-term inactivation was fastest for currents arising from expression of alpha1I and slowest in cells expressing alpha1H calcium channels. The calcium inward current induced by expression of alpha1I is increased by positive prepulses while currents induced by alpha1H and alpha1G show little ( < 5%) or no facilitation. The data thus provide a characteristic fingerprint of each channel's activity, which may allow correlation of the alpha1G, alpha1H and alpha1I induced currents with their in vivo counterparts.

Previously it was found that the amplitude of Ca currents in CA1 hippocampal neurons increases after adrenalectomy (ADX) of young adult rats. Preliminary data suggested that this effect of ADX is age-dependent. In the present study we therefore investigated the effect of ADX on Ca currents in three age groups: rats that were 1, 3, or 6 months old. It appeared that ADX of the youngest age group resulted in a selective enhancement of sustained, high threshold Ca currents. By contrast, ADX of the oldest rats enhanced only the low threshold, transient Ca current amplitude. The age dependency of the ADX-induced effects can be explained by developmental changes in Ca current properties in the adrenally intact rats with which ADX animals were compared. Data from acutely dissociated cells, which lack most of their dendrites, suggest that the ADX-induced changes of Ca current properties are at least partly targeted at currents generated in the distal dendrites. No significant changes were observed with age or after ADX for the mRNA expression of the alpha 1D Ca channel subunit, which forms the ionpore of the sustained high threshold L-type Ca channel. We can therefore presently not exclude that the altered amplitude of Ca current with age and ADX is due to changes in ion channel function rather than in the total number of these channels.

We recently showed that cyclooxygenase 1 (COX-1)-derived prostaglandin E2 (PGE2) signaling through PGE2 type 1 receptors (EP1R) in the SFO is required for ROS-mediated hypertension induced by slow-...

1. In guinea-pig ventricular cells, the concentration of ionized cytosolic calcium ([Ca2+]o) was estimated from the fluorescence of 100 microM K5-indo-1. At 36 degrees C and 2 mM [Ca2+]o, the Ca2+ load of the cells was varied by 1 Hz trains of conditioning clamp pulses to -30 mV (low Ca2+ load), 0 mV (intermediate Ca2+ load) and paired pulses (high Ca2+ load). After seven pulses potentiation was steady and short test pulses to 0 mV were tested for their efficacy in triggering [Ca2+]c transients. The influx of trigger Ca2+ was graded by varying the test-pulse duration between 1 and 180 ms. 2. After a 3 min rest period, [Ca2+]c was 100 +/- 20 nM (mean +/- S.E.M.) and 2 ms test pulses were unable to induce [Ca2+]c transients. Test pulses of 2 ms duration, however, induced [Ca2+]c transients after potentiation with single or paired pulses. 3. At high cellular Ca2+ load, the amplitude of the [Ca2+]c transients (delta[Ca2+]c) gradually increased with pulse durations up to 8 ms. Pulse durations between 8 and 160 ms, however, did not further increase delta[Ca2+]c as if the largest part of the [Ca2+]c transient was due to regenerative contribution of Ca(2+)-induced Ca2+ release. 4. Pulses of 160 ms duration induced 'saturating' responses whose amplitudes delta[Ca2+]c, t = infinity decreased from 938 +/- 120 nM at high Ca2+ load, to 610 +/- 90 and 350 +/- 120 nM at intermediate and low Ca2+ loads, respectively. 5. Delta[Ca2+]c was more sensitive to the duration of Ca2+ influx at low or intermediate Ca2+ loads than at high Ca2+ load. When delta[Ca2+]c was plotted against the test-pulse duration, 50% of delta[Ca2+]c, t = infinity was found to be at 9 +/- 2 ms (low), 4.6 +/- 1 ms (intermediate) or 1.8 +/- 0.5 ms pulses (high Ca2+ load). Correspondingly, the efficacy of 2 ms test pulses in triggering [Ca2+]c transients increased with the Ca2+ load. 6. At high Ca2+ load, [Ca2+]c peaked nearly independently of pulse duration at 19 +/- 3 ms. At intermediate or low Ca2+ load, time to peak increased with pulse duration. 7. The results confirm the theory that sarcoplasmic reticulum (SR) Ca2+ release contributes an amount to the [Ca2+]c transient that increases with the cellular Ca2+ load. The results are compatible with the hypothesis that SR Ca2+ release can be activated by both Ca2+ influx and by SR Ca2+ release and that the latter mechanism constitutes a positive feedback, the amplification of which increases with the amount of releasable Ca2+.

Background Studies on animal models of Hirschsprung’s disease (HD) suggest that L-type Ca2+ channels are down-regulated in the aganglionic bowel segment, however, this has yet to be confirmed in HD patients. The objective of this study was to test the hypothesis that L-type Ca2+ current density is decreased in smooth muscle cells (SMC) obtained from the aganglionic bowel segment of patients with HD in comparison with those from the ganglionic segment. Methods Smooth muscle cells were freshly isolated from colon samples obtained from HD patients undergoing pull-through surgery. L-type Ca2+ currents were recorded using the perforated patch configuration of the whole cell voltage clamp technique and the expression levels of CACNA1C transcripts (which encode L-type Ca2+ channels) in the ganglionic and aganglionic bowel segments were compared using real-time quantitative PCR. Key Results All SMC displayed robust currents that had activation/inactivation kinetics typical of L-type Ca2+ current, were inhibited by nifedipine and enhanced by the L-type Ca2+ channel agonists FPL 64176 and Bay K 8644. Moreover, FPL 64176 activated currents were also inhibited by nifedipine. However, there was no significant difference in L-type Ca2+ current density, CACNA1C subunit expression or sensitivity to the pharmacological agents noted above, between SMC isolated from the ganglionic and aganglionic regions of the HD colon. Conclusions & Inferences In contrast to studies on genetic animal models of HD, L-type Ca2+ currents are not down-regulated in the aganglionic bowel segment of HD patients and are therefore unlikely to account for the impaired colonic peristalsis observed in these patients.

1. The characteristics of a transient inward Ca2+ current (IT) underlying low-threshold Ca2+ potentials were studied in projection cells of the cat and rat dorsal lateral geniculate nucleus (LGN) in vitro using the single-electrode voltage-clamp technique. 2. In cat LGN slices perfused at 25 degrees C with a solution which included 1 mM-Ca2+ and 3 mM-Mg2+, IT could be evoked by depolarizing voltage steps to -55 mV from a holding potential (Vh) of -95 mV and was abolished by reducing [Ca2+]o from 1 to 0.1 mM. IT was also blocked by 8 mM-Mg2+ and 500 microM-Ni2+, but 500 microM-Cd2+ was a significantly less effective antagonist. 3. The inactivation of IT, which occurred at Vh positive to -65 mV, was removed as Vh approached -100 mV. The process of inactivation removal was also time dependent, with 800-1000 ms needed for total removal. Activation curves for IT showed a threshold of -70 mV and illustrated that IT was extremely voltage sensitive over the voltage range from -65 to -55 mV. 4. The decay phase of IT followed a single-exponential time course with a time constant of decay which was voltage sensitive and ranged from 20 to 100 ms. The mean peak conductance increase associated with IT was 8.4 nS (+/-0.9, S.E.M.). 5. In more 'physiological' conditions (35 degrees C and 1.5 mM-Ca2+, 1 mM-Mg2+) the voltage dependence of activation and inactivation were unaffected. However, the development and decay of IT proceeded more rapidly and only 500-600 ms were needed for total removal of inactivation. Under these conditions, the use of voltage ramps showed that depolarization rates of greater than 30 mV/s were necessary for IT activation. 6. The use of multiple voltage-step protocols illustrated that the process of inactivation removal was rapidly reversed by brief returns to a Vh of -50 mV. Furthermore, any delay in IT activation, once the LGN cell membrane potential was in the IT activation range, resulted in a current of reduced amplitude. 7. Although IT in rat LGN cells was briefer and had a shorter latency to peak, it was otherwise similar to that seen in cat LGN cells. 8. The characteristics of IT are very similar to those of the T-type Ca2+ currents of other excitable membranes. The properties of IT are discussed with respect to its role in generating the low-threshold Ca2+ potentials which are central to the oscillatory behaviour of thalamic projection cells.