Inhibition Of ICa Research Articles

The C-terminus of Rad is required for membrane localization and L-type calcium channel regulation.

L-type CaV1.2 current (ICa,L) links electrical excitation to contraction in cardiac myocytes. ICa,L is tightly regulated to control cardiac output. Rad is a Ras-related, monomeric protein that binds to L-type calcium channel β subunits (CaVβ) to promote inhibition of ICa,L. In addition to CaVβ interaction conferred by the Rad core motif, the highly conserved Rad C-terminus can direct membrane association in vitro and inhibition of ICa,L in immortalized cell lines. In this work, we test the hypothesis that in cardiomyocytes the polybasic C-terminus of Rad confers t-tubular localization, and that membrane targeting is required for Rad-dependent ICa,L regulation. We introduced a 3xFlag epitope to the N-terminus of the endogenous mouse Rrad gene to facilitate analysis of subcellular localization. Full-length 3xFlag-Rad (Flag-Rad) mice were compared with a second transgenic mouse model, in which the extended polybasic C-termini of 3xFlag-Rad was truncated at alanine 277 (Flag-RadΔCT). Ventricular cardiomyocytes were isolated for anti-Flag-Rad immunocytochemistry and ex vivo electrophysiology. Full-length Flag-Rad showed a repeating t-tubular pattern whereas Flag-RadΔCT failed to display membrane association. ICa,L in Flag-RadΔCT cardiomyocytes showed a hyperpolarized activation midpoint and an increase in maximal conductance. Additionally, current decay was faster in Flag-RadΔCT cells. Myocardial ICa,L in a Rad C-terminal deletion model phenocopies ICa,L modulated in response to β-AR stimulation. Mechanistically, the polybasic Rad C-terminus confers CaV1.2 regulation via membrane association. Interfering with Rad membrane association constitutes a specific target for boosting heart function as a treatment for heart failure with reduced ejection fraction.

Mitochondrial dysfunction in chromaffin cells from the R6/1 mouse model of Huntington's disease: Impact on exocytosis and calcium current regulation

From a pathogenic perspective, Huntington's disease (HD) is being considered as a synaptopathy. As such, alterations in brain neurotransmitter release occur. As the activity of the sympathoadrenal axis is centrally controlled, deficits in the exocytotic release of catecholamine release may also occur. In fact, in chromaffin cells (CCs) of the adrenal medulla of the R6/1 model of HD, decrease of secretion and altered kinetics of the exocytotic fusion pore have been reported. Those alterations could be linked to mitochondrial deficits occurring in peripheral CCs, similar to those described in brain mitochondria. Here we have inquired about alterations in mitochondrial structure and function and their impact on exocytosis and calcium channel currents (ICa). We have monitored various parameters linked to those events, in wild type (WT) and the R6/1 mouse model of HD at a pre-disease stage (2 months age, 2 m), and when motor deficits are present (7 months age, 7 m). In isolated CCs from 7 m and in the adrenal medulla of R6/1 mice, we found the following alterations (with respect 7 m WT mice): (i) augmented fragmented mitochondria and oxidative stress with increased oxidized glutathione; (ii) decreased basal and maximal respiration; (iii) diminution of ATP cell levels; (iv) mitochondrial depolarization; (v) drastic decrease of catecholamine release with poorer potentiation by protonophore FCCP; (vi) decreased ICa inhibition by FCCP; and (vii) lesser potentiation by BayK8644 of ICa and smaller prolongation of current deactivation. Of note was the fact several of these alterations were already manifested in CCs from 2 m R6/1 mice at pre-disease stages. Based on those results, a plausible hypothesis can be raised in the sense that altered mitochondrial function seems to be an early primary event in HD pathogenesis. This is in line with an increasing number of mitochondrial, metabolic, and inflammatory alterations being recently reported in various HD peripheral tissues.

Acute Detubulation of Ventricular Myocytes Amplifies the Inhibitory Effect of Cholinergic Agonist on Intracellular Ca2+ Transients.

Muscarinic receptors expressed in cardiac myocytes play a critical role in the regulation of heart function by the parasympathetic nervous system. How the structural organization of cardiac myocytes affects the regulation of Ca2+ handling by muscarinic receptors is not well-defined. Using confocal Ca2+ imaging, patch-clamp techniques, and immunocytochemistry, the relationship between t-tubule density and cholinergic regulation of intracellular Ca2+ in normal murine ventricular myocytes and myocytes with acute disruption of the t-tubule system caused by formamide treatment was studied. The inhibitory effect of muscarinic receptor agonist carbachol (CCh, 10 μM) on the amplitude of Ca2+ transients, evoked by field-stimulation in the presence of 100 nM isoproterenol (Iso), a β-adrenergic agonist, was directly proportional to the level of myocyte detubulation. The timing of the maximal rate of fluorescence increase of fluo-4, a Ca2+-sensitive dye, was used to classify image pixels into the regions functionally coupled or uncoupled to the sarcolemmal Ca2+ influx (ICa). CCh decreased the fraction of coupled regions and suppressed Ca2+ propagation from sarcolemma inside the cell. Formamide treatment reduced ICa density and decreased sarcoplasmic reticulum (SR) Ca2+ content. CCh did not change SR Ca2+ content in Iso-stimulated control and formamide-treated myocytes. CCh inhibited peak ICa recorded in the presence of Iso by ∼20% in both the control and detubulated myocytes. Reducing ICa amplitude up to 40% by changing the voltage step levels from 0 to –25 mV decreased Ca2+ transients in formamide-treated but not in control myocytes in the presence of Iso. CCh inhibited CaMKII activity, whereas CaMKII inhibition with KN93 mimicked the effect of CCh on Ca2+ transients in formamide-treated myocytes. It was concluded that the downregulation of t-tubules coupled with the diminished efficiency of excitation–contraction coupling, increases the sensitivity of Ca2+ release and propagation to muscarinic receptor-mediated inhibition of both ICa and CaMKII activity.

B-PO04-012 ACUTE TESTOSTERONE ADMINISTRATION DOES NOT SHORTEN ACTION POTENTIAL DURATION IN LANGENDORFF PERFUSED RABBIT VENTRICLES

A previous study showed that testosterone (T) rapidly shortens the action potential duration (APD) with EC50=2.1 to 8.7 nmol/L in guinea pig ventricular myocytes due to non-transcriptional enhancement of IKs and suppression of ICa,L. These findings have not been reproduced in intact rabbit ventricles. To test the hypothesis that T and dihydroxytestosterone (D) acutely shorten epicardial APD in rabbit ventricles. We performed optical mapping studies in 6 female and 7 male Langendorff-perfused rabbit hearts. Blebbistatin (10-20 μM) was used to suppress motion. Right atrium was paced during mapping. After baseline recording, mapping was done with T (N=10) or D (N=3). In T group, either high (3 μM) or low (1 to 100 nM) doses were applied to 5 and 4 hearts respectively, and 1 received both low and high doses. In D group, 1 received only low (3 to 100 nM), and 2 received low and then high dose (1 μM). In one heart we determined the QT interval recorded with widely spaced bipolar electrodes by using Tyrode’s solution only, without optical dye (RH237) or blebbistatin. At pacing cycle length (PCL) 350 ms, 300 ms, and 250 ms, there were no significant differences of APD (ms) at baseline vs high dose T (182 ± 15 vs 186 ± 5, p=0.73; 160 ± 24 vs 159 ± 20, p=0.95, 152 ± 23 vs 149 ± 15 ms, p=0.71, respectively). High-dose D tends to lengthen APD (ms) in 2 hearts at PCL 350 ms (161 vs 179 and 143 vs 153), 300 ms (152 vs 167 and 139 vs 148), and 250 ms (139 vs 158 and 129 vs 137). APDs (ms) at baseline vs low-dose T were (144 ± 9 vs 147 ± 8 at 1nM, p=0.23 and 150 ± 8 at 10 nM, p=0.05). APDs at baseline vs low dose D were 146 ± 9 vs 153 ± 10 at 3 nM (p=0.34), 162 ± 15 at 10 nM (p=0.34), and 160 ± 11 at 100 nM (p=0.19) at PCL 300 ms. APD significantly lengthened after 100 nM of T (154 ± 12 ms at 100 nM, p=0.01). In one rabbit heart without RH237 or blebbistatin, QT interval (ms) at PCL 280 ms did not shorten after T (170 at baseline, 172 at 10 nM, and 176 at 100 nM). When separately analyzed, neither male nor female hearts showed APD shortening with T or D. T and D do not shorten and may lengthen ventricular repolarization in normal male or female rabbit hearts. These findings do not support non-transcriptional regulation of cardiac ion currents by T or D to shorten APD.

The Influence of Cochlear Implant-Based Electric Stimulation on the Electrophysiological Characteristics of Cultured Spiral Ganglion Neurons.

Background Cochlear implant-based electrical stimulation may be an important reason to induce the residual hearing loss after cochlear implantation. In our previous study, we found that charge-balanced biphasic electrical stimulation inhibited the neurite growth of spiral ganglion neurons (SGNs) and decreased Schwann cell density in vitro. In this study, we want to know whether cochlear implant-based electrical stimulation can induce the change of electrical activity in cultured SGNs. Methods Spiral ganglion neuron electrical stimulation in vitro model is established using the devices delivering cochlear implant-based electrical stimulation. After 48 h treatment by 50 μA or 100 μA electrical stimulation, the action potential (AP) and voltage depended calcium current (ICa) of SGNs are recorded using whole-cell electrophysiological method. Results The results show that the ICa of SGNs is decreased significantly in 50 μA and 100 μA electrical stimulation groups. The reversal potential of ICa is nearly +80 mV in control SGN, but the reversal potential decreases to +50 mV in 50 μA and 100 μA electrical stimulation groups. Interestingly, the AP amplitude, the AP latency, and the AP duration of SGNs have no statistically significant differences in all three groups. Conclusion Our study suggests cochlear implant-based electrical stimulation only significantly inhibit the ICa of cultured SGNs but has no effect on the firing of AP, and the relation of ICa inhibition and SGN damage induced by electrical stimulation and its mechanism needs to be further studied.

Pharmacological Inhibition of Glucose‐6‐phosphate Dehydrogenase Produces Inhibition of L‐type Calcium Channel Current in Arterial Smooth Muscle Cells

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme in pentose phosphate pathway (PPP). G6PD is involved in cellular redox regulation by reducing NADP+ to NADPH, a primary cellular reducing agent, and PPP produces ribose 5‐phosphate, a substrate necessary for nucleotide synthesis. We used two inhibitors of G6PD, 6‐aminonicotinamide (6‐AN) and dehydroepiandrosterone (DHEA), to clarify involvement of G6PD in the regulation of L‐type Ca2+ channel current (ICa,L) in arterial smooth muscle cells (ASMCs). 6‐AN is converted to 6‐ANADP in the cell to competitively inhibit NADP+ binding to G6PD. DHEA is an adrenal steroid hormone with the highest serum concentration among steroid hormones with its sulfate ester DHEAS. DHEA inhibits G6PD in a non‐competitive manner. The potency of DHEAS to inhibit G6PD is about 1/100 of DHEA. DHEA induced relaxation of high K+‐induced contraction in arterial strips excised from carotid artery and aorta of rat in a dose‐dependent manner. In contrast, DHEAS did not produce the relaxation. In previous meetings we reported that DHEA inhibits ICa,L in voltage‐dependent and ‐independent mechanisms in ASMCs. 6‐AN produced voltage‐independent inhibition of ICa,L recorded with 10 mM Ba2+ as the charge carrier in A7r5 ASMCs. When constant depolarization step was repetitively applied from hyperpolarized HP, 6‐AN (3 mM) gradually induced inhibition of ICa,L decreasing the peak amplitude of ICa,L (ICa,L, peak) by 25% in 5 min and 45% in 10 min. The inhibition was not reversible by washout of 6‐AN. This was in contrast to DHEA‐induced inhibition of ICa,L which exhibits rapid onset and rapid recovery by washout. 6‐AN little affected the time course of inactivation of ICa,L during depolarization steps, while DHEA markedly accelerated the time course. When I‐V relationship was obtained with 5 min interval before and during application of 6‐AN, ICa,L did not significantly change in control, but decreased in 5 min in the presence of 6‐AN and the inhibition was increased by another 5 min application of 6‐AN. In the presence of 6‐AN, 0.1 mM DHEA rapidly decreased ICa,L,peak and markedly accelerated the time course of inactivation in a reversible manner. Inhibition of G6PD by 6‐AN and DHEA could inhibit ICa,L,peak by thiol‐oxidation of CaV1.2 channel proteins via decrease of NADPH. DHEA additionally augmented voltage‐dependent inactivation of ICa,L in a mode of open channel block produced by Ca2+ channel blockers.Support or Funding InformationNational Heart, Lung, and Blood Institute Grant RO1‐HL‐085352 and ROI‐HL‐132574 to S.A.G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of ICa,L by DHEA in Aortic Smooth Muscle Cells: Voltage‐dependency, Modulation by GPCR Signaling and Inhibition of Glucose‐6‐phosphate Dehydrogenase

Dehydroepiandrosterone (DHEA) is a hormone abundantly released from adrenal cortex as DHEA‐sulfate (DHEA‐S). DHEA‐S is released also in brain. DHEA and epiandrosterone (EPI), a metabolite of DHEA, produces relaxation of high‐K+ induced contraction of aortic smooth muscle (ASM). EPI increases voltage‐dependent inactivation (voltage‐dependent inhibition, VDI) of ICa,L in ventricular myocytes. Whole‐cell ICa,L was recorded as IBa from ASM cells (ASMCs) of A7r5 cell line and enzymatically isolated bovine coronary ASMCs to study mechanism and modulation of DHEA‐induced inhibition of ICa,L. DHEA produced VDI; acceleration of time course of inactivation, increase of inhibition by depolarization of holding potential (HP) and leftward shift of steady state inactivation curve; f∞, 2s‐V, obtained by 2 s conditioning pulse and f∞, HP‐V as a function HP. In addition, DHEA produced voltage‐independent inhibition (VIDI) represented by inhibition of peak amplitude (ICa,L,peak) of ICa,L elicited by pulses from hyperpolarized HPs. Window ICa,L (IWD) supplies Ca2+ in the E‐C coupling of ASM. IWD obtained from I–V of ICa,L,peak and f∞, HP‐V fitted well with recorded IWD and they were strongly inhibited by DHEA. Efficacy of DHEA‐induced VDI was reduced by rightward shift of f∞, 2s‐V curve produced by depletion of cholesterol by MβCD. Blocking of GPCR signaling by intracellular dialysis of GDP‐β‐S also shifted f∞, 2s‐V to the right and it produced decrease of DHEA‐induced inhibition of ICa,L at depolarized HP (VDI). DHEA and EPI are inhibitors of glucose‐phosphate‐dehydrogenase (G6PD), a redox‐related pentose‐phosphate pathway enzyme. 6‐Aminonicotinamide (6‐AN), another inhibitor of G6PD produced VIDI without inducing VDI. DHEA‐S that does not inhibit G6PD induced small but significant VDI at an order higher concentration of DHEA without generating VIDI. GPCR signaling and membrane cholesterol are indirectly involved in the modulation of DHEA‐induced VDI by affecting basal voltage‐dependent inactivation. The occurrence of VIDI by 6‐AN indicates that G6PD inhibition underlies DHEA‐induced VIDI. DHEA‐induced inhibition of IWD by VDI and VIDI contributes for DHEA‐induced relaxation of ASM.Support or Funding InformationNational Heart, Lung, and Blood Institute Grant RO1 HL‐085352 to S. A. Gupte.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Retinoic acid inhibits neuronal voltage-gated calcium channels

Retinoic acid is the active metabolite of vitamin A and regulates several important cellular processes by activating retinoic acid receptors (RAR) and retinoid X receptors (RXR). These receptors generally act as transcription factors, though non-genomic actions of both retinoic acid and the receptors have also been reported. One such nongenomic effect includes the modulation of Ca2+ levels during homeostatic synaptic plasticity in the hippocampus. Retinoic acid can thus affect Ca2+ signaling and can potentially control both synaptic plasticity and neuronal firing. However, whether retinoic acid can regulate voltage-gated Ca2+ channels (either via genomic or nongenomic actions), which are fundamental to these processes, has not yet been studied in detail.Here we demonstrate the effects of retinoic acid on the biophysical properties of voltage-gated Ca2+ channels in cultured invertebrate motorneurons. Overnight exposure to physiological concentrations of retinoic acid significantly inhibited the voltage-gated Ca2+ current (ICa) in an isomer-dependent manner. Specifically, all-trans retinoic acid (atRA), but not 9-cis RA (9cRA), depolarized the voltage of half-maximal activation of ICa. AtRA also reduced the rate of channel activation and delayed recovery from inactivation. We provide evidence that both L-type and non-L-type voltage-gated Ca2+ channels are affected by atRA, as both nifedipine-sensitive and nifedipine-resistant ICa were inhibited in these neurons. These effects of retinoic acid are thought to be at least partially mediated by the retinoid receptors, as treatment of the neurons with synthetic RAR and RXR agonists produced a similar inhibition of ICa.

Regulation of Ca2 + signaling by acute hypoxia and acidosis in rat neonatal cardiomyocytes

Ischemic heart disease is an arrhythmogenic condition, accompanied by hypoxia, acidosis, and impaired Ca2+ signaling. Here we report on effects of acute hypoxia and acidification in rat neonatal cardiomyocytes cultures. ResultsTwo populations of neonatal cardiomyocyte were identified based on inactivation kinetics of L-type ICa: rapidly-inactivating ICa (τ~20ms) myocytes (prevalent in 3–4-day cultures), and slow-inactivating ICa (τ≥40ms) myocytes (dominant in 7-day cultures). Acute hypoxia (pO2<5mmHg for 50–100s) suppressed ICa reversibly in both cell-types to different extent and with different kinetics. This disparity disappeared when Ba2+ was the channel charge carrier, or when the intracellular Ca2+ buffering capacity was increased by dialysis of high concentrations of EGTA and BAPTA, suggesting critical role for calcium-dependent inactivation. Suppressive effect of acute acidosis on ICa (~40%, pH6.7), on the other hand, was not cell-type dependent. Isoproterenol enhanced ICa in both cell-types, but protected only against suppressive effects of acidosis and not hypoxia. Hypoxia and acidosis suppressed global Ca2+ transients by ~20%, but suppression was larger, ~35%, at the RyR2 microdomains, using GCaMP6-FKBP targeted probe. Hypoxia and acidosis also suppressed mitochondrial Ca2+ uptake by 40% and 10%, respectively, using mitochondrial targeted Ca2+ biosensor (mito-GCaMP6). ConclusionOur studies suggest that acute hypoxia suppresses ICa in rapidly inactivating cell population by a mechanism involving Ca2+-dependent inactivation, while compromised mitochondrial Ca2+ uptake seems also to contribute to ICa suppression in slowly inactivating cell population. Proximity of cellular Ca2+ pools to sarcolemmal Ca2+ channels may contribute to the variability of inactivation kinetics of ICa in the two cell populations, while acidosis suppression of ICa appears mediated by proton-induced block of the calcium channel.

Bud extracts from Salix caprea L. inhibit voltage gated calcium channels and catecholamines secretion in mouse chromaffin cells

BackgroundSalix caprea L. is an ornamental plant with prominent antioxidant activity. In the last decades Salix caprea bud extracts (SCBEs) have been used for the treatment of oxidative stress related disorders. PurposeA large part of cellular functions depends on the amount of intracellular Ca2+ concentration which in turn is mainly determined by Ca2+ ions movements across plasma membrane as well as by Ca2+ released from the stores. For better evaluating the mechanism of action of SCBEs, we focused on the effect of SCBEs on voltage gated Ca2+ channels (VGCCs) functioning and related catecholamines secretion in mouse chromaffin cells (MCCs). These latter are neuroendocrine cells that share a wide variety of functions with neurons. They are particularly interesting for studying the relationship between VGCCs activation and catecholamines secretion both in control and under stressful conditions. Study design and methodsWe focused on the effect of SCBEs on VGCCs being these latter considered one of the main pathway of Ca2+ influx across plasma membrane. Ca2+ currents and capacitance changes were measured in patch clamp experiments performed in voltage clamp configuration. ResultsWe show that SCBEs inhibited VGCCs in a dose dependent manner. On average, the saturating concentration of SCBEs (SCBEsmax) is able to block 36% of the maximum Ca2+ current amplitude (ICa) without selectivity for L (ICa, L) or non-L type (ICa, non-L) Ca2+ channels. Furthermore, ICa inhibition is not followed by alteration of VGCCs gating kinetics, but is responsible for a marked decrease of Ca2+ dependent catecholamines secretion. ConclusionWe conclude that the ability of SCBEs to inhibit VGCCs function, known to be potentiated during oxidative stress, could contribute to the already known antioxidant properties of Salix caprea L. We finally suggest that the inhibitory effect of SCBEs on catecholamines secretion may contribute to treat stress dependent cellular dysfunctions.

Metabolic inhibition reduces cardiac L-type Ca2+ channel current due to acidification caused by ATP hydrolysis.

Metabolic stress evoked by myocardial ischemia leads to impairment of cardiac excitation and contractility. We studied the mechanisms by which metabolic inhibition affects the activity of L-type Ca2+ channels (LTCCs) in frog ventricular myocytes. Metabolic inhibition induced by the protonophore FCCP (as well as by 2,4- dinitrophenol, sodium azide or antimycin A) resulted in a dose-dependent reduction of LTCC current (ICa,L) which was more pronounced during β-adrenergic stimulation with isoprenaline. ICa,L was still reduced by metabolic inhibition even in the presence of 3 mM intracellular ATP, or when the cell was dialysed with cAMP or ATP-γ-S to induce irreversible thiophosphorylation of LTCCs, indicating that reduction in ICa,L is not due to ATP depletion and/or reduced phosphorylation of the channels. However, the effect of metabolic inhibition on ICa,L was strongly attenuated when the mitochondrial F1F0-ATP-synthase was blocked by oligomycin or when the cells were dialysed with the non-hydrolysable ATP analogue AMP-PCP. Moreover, increasing the intracellular pH buffering capacity or intracellular dialysis of the myocytes with an alkaline solution strongly attenuated the inhibitory effect of FCCP on ICa,L. Thus, our data demonstrate that metabolic inhibition leads to excessive ATP hydrolysis by the mitochondrial F1F0-ATP-synthase operating in the reverse mode and this results in intracellular acidosis causing the suppression of ICa,L. Limiting ATP break-down by F1F0-ATP-synthase and the consecutive development of intracellular acidosis might thus represent a potential therapeutic approach for maintaining a normal cardiac function during ischemia.

DHEA‐induced Voltage‐dependent Inhibition of ICa,L Is Augmented by Cholesterol Depletion and Down‐regulated by GPCR Blockade in Aortic Smooth Muscle Cells

Dehydroepiandrosterone (DHEA) is a steroid hormone synthesized from cholesterol and abundantly secreted as DHEA sulfate from the adrenal gland. DHEA produces relaxation of systemic and pulmonary arterial smooth muscles (ASM) and epiandrosterone, a metabolite of DHEA, causes voltage‐dependent inhibition (VDI) of ICa,L in ventricular myocytes. Here, we characterized DHEA‐induced VDI of ICa,L recorded with Ba2+ as a charge carrier by whole cell clamp and studied underlying mechanism and modulation by signal transduction in ASM cells (ASMCs) from bovine coronary artery and A7r5 cells from rat aorta. DHEA decreased peak amplitude of ICa,L rapidly, reversibly and dose‐dependently and accelerated time course of current decay decreasing the ratio of current amplitude at 500 ms (r500) to peak amplitude and shifted steady‐state inactivation curve (f∞‐V) to the left. DHEA‐induced inhibition of ICa,L became prominent by depolarization of holding potential; e.g. at −30 mV, the inhibition was 29% at 10 μM and 89% at 100 μM. Blockade of GPCR by dialysis of GDP βS shifted both f∞‐V and I–V relationships to the depolarizing direction, and decreased 10 μM‐DHEA‐induced VDI obtained at HP of −40 mV by 40%. Depletion of membrane cholesterol by methyl‐b‐cyclodextrin increased r500 and shifted f∞‐V to the right. Conversely, loading PEG‐cholesterol, a non‐ionic amphiphile, to the membrane decreased r500 and shifted f∞‐V to the left. In the cholesterol‐depleted cells, DHEA produced more potent decrease of r500 and larger negative shift of f∞‐V than in control. Therefore, DHEA‐induced VDI of ICa,L in ASMCs is due, at least partly, to cholesterol‐like action of DHEA on CaV1.2 in the membrane lipid bilayer. GPCR‐mediated signaling regulates voltage‐dependence of basal ICa,L and indirectly augments DHEA‐induced VDI of ICa,L in ASMCs.

Ion channel mechanisms of rat tail artery contraction-relaxation by menthol involving, respectively, TRPM8 activation and L-type Ca2+ channel inhibition

Transient receptor potential melastatin 8 (TRPM8) is the principal cold and menthol receptor channel. Characterized primarily for its cold-sensing role in sensory neurons, it is expressed and functional in several nonneuronal tissues, including vasculature. We previously demonstrated that menthol causes variable mechanical responses (vasoconstriction, vasodilatation, or biphasic reactions) in isolated arteries, depending on vascular tone. Here we aimed to dissect the specific ion channel mechanisms and corresponding Ca2+ signaling pathways underlying such complex responses to menthol and other TRPM8 ligands in rat tail artery myocytes using patch-clamp electrophysiology, confocal Ca2+ imaging, and ratiometric Ca2+ recording. Menthol (300 μM, a concentration typically used to induce TRPM8 currents) strongly inhibited L-type Ca2+ channel current (L-ICa) in isolated myocytes, especially its sustained component, most relevant for depolarization-induced vasoconstriction. In contraction studies, with nifedipine present (10 μM) to abolish L-ICa contribution to phenylephrine (PE)-induced vasoconstrictions of vascular rings, a marked increase in tone was observed with menthol, similar to resting (i.e., without α-adrenoceptor stimulation by PE) conditions, when L-type channels were mostly deactivated. Menthol-induced increases in PE-induced vasoconstrictions could be inhibited both by the TRPM8 antagonist AMTB (thus confirming the specific role of TRPM8) and by cyclopiazonic acid treatment to deplete Ca2+ stores, pointing to a major contribution of Ca2+ release from the sarcoplasmic reticulum in these contractile responses. Immunocytochemical analysis has indeed revealed colocalization of TRPM8 and InsP3 receptors. Moreover, menthol Ca2+ responses, which were somewhat reduced under Ca2+-free conditions, were strongly reduced by cyclopiazonic acid treatment to deplete Ca2+ store, whereas caffeine-induced Ca2+ responses were blunted in the presence of menthol. Finally, two other common TRPM8 agonists, WS-12 and icilin, also inhibited L-ICa With respect to L-ICa inhibition, WS-12 is the most selective agonist. It augmented PE-induced contractions, whereas any secondary phase of vasorelaxation (as with menthol) was completely lacking. Thus TRPM8 channels are functionally active in rat tail artery myocytes and play a distinct direct stimulatory role in control of vascular tone. However, indirect effects of TRPM8 agonists, which are unrelated to TRPM8, are mediated by inhibition of L-type Ca2+ channels and largely obscure TRPM8-mediated vasoconstriction. These findings will promote our understanding of the vascular TRPM8 role, especially the well-known hypotensive effect of menthol, and may also have certain translational implications (e.g., in cardiovascular surgery, organ storage, transplantation, and Raynaud's phenomenon).

Dopamine D1 receptor modulation of calcium channel currents in horizontal cells of mouse retina.

Horizontal cells form the first laterally interacting network of inhibitory interneurons in the retina. Dopamine released onto horizontal cells under photic and circadian control modulates horizontal cell function. Using isolated, identified horizontal cells from a connexin-57-iCre × ROSA26-tdTomato transgenic mouse line, we investigated dopaminergic modulation of calcium channel currents (ICa) with whole cell patch-clamp techniques. Dopamine (10 μM) blocked 27% of steady-state ICa, an action blunted to 9% in the presence of the L-type Ca channel blocker verapamil (50 μM). The dopamine type 1 receptor (D1R) agonist SKF38393 (20 μM) inhibited ICa by 24%. The D1R antagonist SCH23390 (20 μM) reduced dopamine and SKF38393 inhibition. Dopamine slowed ICa activation, blocking ICa by 38% early in a voltage step. Enhanced early inhibition of ICa was eliminated by applying voltage prepulses to +120 mV for 100 ms, increasing ICa by 31% and 11% for early and steady-state currents, respectively. Voltage-dependent facilitation of ICa and block of dopamine inhibition after preincubation with a Gβγ-blocking peptide suggested involvement of Gβγ proteins in the D1R-mediated modulation. When the G protein activator guanosine 5'-O-(3-thiotriphosphate) (GTPγS) was added intracellularly, ICa was smaller and showed the same slowed kinetics seen during D1R activation. With GTPγS in the pipette, additional block of ICa by dopamine was only 6%. Strong depolarizing voltage prepulses restored the GTPγS-reduced early ICa amplitude by 36% and steady-state ICa amplitude by 3%. These results suggest that dopaminergic inhibition of ICa via D1Rs is primarily mediated through the action of Gβγ proteins in horizontal cells.

Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes

Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca(2+) cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca(2+) signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca(2+) current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy.

The inhibition of high-voltage-activated calcium current by activation of MrgC11 involves phospholipase C-dependent mechanisms

High-voltage-activated (HVA) calcium channels play an important role in synaptic transmission. Activation of Mas-related G-protein-coupled receptor subtype C (MrgC; mouse MrgC11, rat homolog rMrgC) inhibits HVA calcium current (ICa) in small-diameter dorsal root ganglion (DRG) neurons, but the intracellular signaling cascade underlying MrgC agonist-induced inhibition of HVA ICa in native DRG neurons remains unclear. To address this question, we conducted patch-clamp recordings in MrgA3-eGFP-wild-type mice, in which most MrgA3-eGFP+ DRG neurons co-express MrgC11 and can be identified for recording. We found that the inhibition of HVA ICa by JHU58 (0.001–100nM, a dipeptide, MrgC-selective agonist) was significantly reduced by pretreatment with a phospholipase C (PLC) inhibitor (U73122, 1μM), but not by its inactive analog (U73343) or vehicle. Further, in rats that had undergone spinal nerve injury, pretreatment with intrathecal U73122 nearly abolished the inhibition of mechanical hypersensitivity by intrathecal JHU58. The inhibition of HVA ICa in MrgA3-eGFP+ neurons by JHU58 (100nM) was partially reduced by pretreatment with a Gβγ blocker (gallein, 100μM). However, applying a depolarizing prepulse and blocking the Gαi and Gαs pathways with pertussis toxin (PTX) (0.5μg/mL) and cholera toxin (CTX) (0.5μg/mL), respectively, had no effect. These findings suggest that activation of MrgC11 may inhibit HVA ICa in mouse DRG neurons through a voltage-independent mechanism that involves activation of the PLC, but not Gαi or Gαs, pathway.

Pegylated Cholesterol and Methyl-Beta-Cyclodextrin are Modulators of L-Type Calcium Channel Current and Decrease Membrane Capacitance in Vascular Smooth Muscle Cells

Loading of pegylated cholesterol (PEG-cholesterol) by pretreatment of several hours decreases current density and augments voltage-dependent inactivation of L-type Ca channel current (ICa,L) and depletion of cholesterol by similar long pretreatment with methyl-beta-cyclodextrin (MbCD) increases the ICa,L density in A7r5 aortic smooth muscle cells. On the other hand, dehydroepiandrosterone (DHEA), a cholesterol-derived steroid hormone, rapidly induces voltage-dependent inhibition of ICa,L. If endocytosis and exocytosis of CaV1.2-containing membrane vesicles are involved in these modulations, they should induce a change in membrane capacitance (Cm). Cm could be affected also by a change of thickness and dielectric constant of lipid bilayer. Here we studied acute effects of PEG-cholesterol, MbCD and DHEA on Cm of isolated bovine coronary artery smooth muscle cells by whole-cell patch clamp technique. Ramp steps of 5 or 10 ms were applied repetitively before, during and after application of the modulators. Control Cm was 18.3±4.5 pF (mean±SD.). PEG-cholesterol and MbCD induced gradual and small decrease of Cm and their washout decelerated the rate of the decrease. Cm after 10 min of wash-in normalized by the initial value was: control without modulator, 1.01±0.02 (mean±SD.); 1 mM PEG-cholesterol, 0.96±0.02 (p<0.01, compared with control); 10 mM MbCD, 0.90±0.05 (p<0.0001); 0.1 mM DHEA, 1.00±0.03. Since the change of Cm is small, it little affects qualitative conclusion of the modulation of ICa,L density obtained by the pretreatment experiments with PEG-cholesterol and MbCD. The small but significant decrease of Cm reflects structural changes of the lipid bilayer which may be involved in the modulation of ICa,L by PEG-cholesterol and MbCD.

Inhibition of L-type Ca2+ current by ginsenoside Rd in rat ventricular myocytes

BackgroundGinsenoside Rd (GSRd), one of the most abundant ingredients of Panax ginseng, protects the heart via multiple mechanisms including the inhibition of Ca2+ influx. We intended to explore the effects of GSRd on L-type Ca2+ current (ICa,L) and define the mechanism of the suppression of ICa,L by GSRd. MethodsPerforated-patch recording and whole-cell voltage clamp techniques were applied in isolated rat ventricular myocytes. Results(1) GSRd reduced ICa,L peak amplitude in a concentration-dependent manner [half-maximal inhibitory concentration (IC50) = 32.4 ± 7.1 μmol/L] and up-shifted the current–voltage (I–V) curve. (2) GSRd (30 μmol/L) significantly changed the steady-state activation curve of ICa,L (V0.5: −19.12 ± 0.68 vs. −16.26 ± 0.38 mV; n = 5, p < 0.05) and slowed down the recovery of ICa,L from inactivation [the time content (ζ) from 91 ms to 136 ms, n = 5, p < 0.01]. (3) A more significant inhibitive effect of GSRd (100 μmol/L) was identified in perforated-patch recording when compared with whole-cell recording [65.7 ± 3.2% (n = 10) vs. 31.4 ± 5.2% (n = 5), p < 0.01]. (4) Pertussis toxin (Giprotein inhibitor) completely abolished the ICa,L inhibition induced by GSRd. There was a significant difference in inhibition potency between the two cyclic adenosine monophosphate elevating agents (isoprenaline and forskolin) prestimulation [55 ± 7.8% (n = 5) vs. 17.2 ± 3.5% (n = 5), p < 0.01]. (5) 1H-[1,2,4]Oxadiazolo[4,3-a]-quinoxalin-1-one (a guanylate cyclase inhibitor) and N-acetyl-l-cysteine (a nitric oxide scavenger) partly recovered the ICa,L inhibition induced by GSRd. (6) Phorbol-12-myristate-13-acetate (a protein kinase C activator) and GF109203X (a protein kinase C inhibitor) did not contribute to the inhibition of GSRd. ConclusionThese findings suggest that GSRd could inhibit ICa,L through pertussis toxin-sensitive G protein (Gi) and a nitric oxide–cyclic guanosine monophosphate-dependent mechanism.

Somatostatin receptor subtype 4 modulates L-type calcium channels via Gβγ and PKC signaling in rat retinal ganglion cells

Somatostatin subtype-4 receptors (sst4) inhibit L-type calcium channel currents (ICa) in retinal ganglion cells (RGCs). Here we identify the signaling pathways involved in sst4 stimulation leading to suppression of ICa in RGCs. Whole cell patch clamp recordings were made on isolated immunopanned RGCs using barium as a charge carrier to isolate ICa. Application of the selective sst4 agonist, L-803 (10 nM), reduced ICa by 41.2%. Pretreatment of cells with pertussis toxin (Gi/o inhibitor) did not prevent the action of L-803, which reduced ICa by 34.7%. To determine the involvement of Gβγ subunits after sst4 activation, depolarizing pre-pulse facilitation paradigms were used to remove voltage-dependent inhibition of calcium channels. Pre-pulse facilitation did not reverse the inhibitory effects of L-803 on ICa (8.4 vs. 8.8% reductions, ctrl vs. L-803); however, pharmacologic inhibition of Gβγ reduced ICa suppression by L-803 (23.0%, P < 0.05). Inhibition of PKC (GF109203X; GFX) showed a concentration-dependent effect in preventing the action of L-803 on ICa (1 μM GFX, 34.3%; 5 μM GFX, 14.6%, P < 0.05). When both PKC and Gβγ were inhibited, the effects of L-803 on ICa were blocked (1.8%, P < 0.05). These results suggest that sst4 stimulation modulates RGC calcium channels via Gβγ and PKC activation. Since reducing intracellular Ca2+ is known to be neuroprotective in RGCs, modulating these sst4 signaling pathways may provide insights to the discovery of unique therapeutic targets to reduce intracellular Ca2+ levels in RGCs.

Alterations of contractions and L-type Ca2+ currents by murrayafoline-A in rat ventricular myocytes

We examined the effects of murrayafoline-A (1-methoxy-3-methylcarbazole, Mu-A), which is isolated from the dried roots of Glycosmis stenocarpa, on cell shortenings and L-type Ca2+ currents (ICa,L) in rat ventricular myocytes. Cell shortenings and ICa,L were measured using the video edge detection method and patch-clamp techniques, respectively. Mu-A transiently increased cell shortenings in a concentration-dependent manner with an EC50 of ~20μM. The maximal effect of Mu-A, approximately 175% of the control, was observed at ≥100μM. The positive inotropic effect of Mu-A (25μM) reached a maximum after ~2-min exposures, and then decayed after a ~1-min steady-state. During the Mu-A-induced positive inotropy, the rate of contraction was accelerated, whereas the rate of relaxation was not significantly altered. To understand the possible mechanism for the Mu-A-induced positive inotropy, the ICa,L was assessed. Mu-A transiently enhanced the ICa,L. Concentration-dependence of the increase in ICa,L by Mu-A was similar to that of positive inotropic effect of Mu-A. The maximal effect of Mu-A (25μM) on ICa,L was observed at 2−3min after the application of Mu-A. A partial inhibition of ICa,L using verapamil (1μM) induced a right shift of concentration-response curve of the positive inotropic effect of Mu-A and significantly attenuated the effect. These results suggest that Mu-A may transiently enhance contractility, at least in part, by increasing the Ca2+ influx through the L-type Ca2+ channels in rat ventricular myocytes.