Voltage-gated calcium channel blockers deregulate macroautophagy in cardiomyocytes

Voltage-gated calcium channel blockers are widely used for the management of cardiovascular diseases, however little is known about their effects on cardiac cells in vitro.We challenged neonatal ventricular cardiomyocytes (CMs) with therapeutic L-type and T-type Ca²⁺ channel blockers (nifedipine and mibefradil, respectively), and measured their effects on cell stress and survival, using fluorescent microscopy, Q-PCR and Western blot. Both nifedipine and mibefradil induced a low-level and partially transient up-regulation of three key mediators of the Unfolded Protein Response (UPR), indicative of endoplasmic (ER) reticulum stress. Furthermore, nifedipine triggered the activation of macroautophagy, as evidenced by increased lipidation of microtubule-associated protein 1 light chain 3 (LC3), decreased levels of polyubiquitin-binding protein p62/SQSTM1 and ubiquitinated protein aggregates, that was followed by cell death. In contrast, mibefradil inhibited CMs constitutive macroautophagy and did not promote cell death. The siRNA-mediated gene silencing approach confirmed the pharmacological findings for T-type channels.We conclude that L-type and T-type Ca²⁺ channel blockers induce ER stress, which is divergently transduced into macroautophagy induction and inhibition, respectively, with relevance for cell viability. Our work identifies VGCCs as novel regulators of autophagy in the heart muscle and provides new insights into the effects of VGCC blockers on CMs homeostasis, that may underlie both noxious and cardioprotective effects.     

N-type calcium channels are involved in the dopamine releasing effect of nicotine

Mouse striatum was incubated with [³H]dopamine ([³H]DA) and superfused with and the tritium efflux induced by nicotine, electrical stimulation, or simultaneous nicotine and electrical stimulation was measured, to characterize the role of different Ca²⁺ channels in the transmitter release. Nicotine stimulation and electrical stimulation exerted additive effects on tritium efflux. Separation of the released radioactivity on alumina columns indicated that nicotine or electrical stimulation increases the release of [³H]DA and that the outflow of³H-labeled metabolites was similar with the two different stimulation procedures. Removal of Ca²⁺ from the superfusate resulted in a marked reduction in the tritium release evoked by nicotine, whereas the electrical stimulation-evoked tritium release was completely dependent on external Ca²⁺. The L-and N-type calcium channel blockers omega-conotoxin GVIA and Cd²⁺ inhibited the tritium release from the striatum evoked by either nicotine or electrical stimulation, whereas the L-type and T-type channel blockers diltiazem and Ni²⁺ did not alter release of [³H]DA. We conclude that N-type voltage-sensitive Ca²⁺ channels participate in striatal dopamine release, and we speculate that nicotinic receptor-operated ion channels permeable to cations such as Ca²⁺ and N-type voltage-sensitive calcium channels may simultaneously open up, and they additively increase free intracellular Ca²⁺ concentration.     

The role of voltage-gated Ca2+ channels in neurite growth of cultured chromaffin cells induced by extremely low frequency (ELF) magnetic field stimulation

The ion Ca²⁺ has been shown to play an important role in a wide variety of cellular functions, one of them being related to cell differentiation in which nerve growth factor (NGF) is involved. Chromaffin cells obtained from adrenals of 2- to 3-day-old rats were cultured for 7 days. During this time, these cells were subjected to the application of either NGF or extremely low frequency magnetic fields (ELF MF). Since this induced cell differentiation toward neuronal-like cells, the mechanism by which this occurred was studied. When the L-Ca²⁺ channel blocker nifedipine was applied simultaneously with ELF MF, this differentiation did not take place, but it did when an N-Ca²⁺ channel blocker was used. In contrast, none of the Ca²⁺ channel blockers prevented differentiation in the presence of NGF. In addition, Bay K-8644, an L-Ca²⁺ channel agonist, increased both the percentage of differentiated cells and neurite length in the presence of ELF MF. This effect was much weaker in the presence of NGF. [³H]-noradrenaline release was reduced by nifedipine, suggesting an important role for L-Ca²⁺ channels in neurotransmitter release. Total high voltage Ca²⁺ currents were significantly increased in ELF MF-treated cells with NGF, but these currents in ELF MF-treated cells were more sensitive to nifedipine. Amperometric analysis of catecholamine release revealed that the KCl-induced activity of cells stimulated to differentiate by ELF MF is highly sensitive to L-type Ca²⁺ channel blockers. A possible mechanism to explain the way in which the application of magnetic fields can induce differentation of chromaffin cells into neuronal-like cells is proposed.     

L-Type Calcium Channel Mediates Anticonvulsant Effect of Cannabinoids in Acute and Chronic Murine Models of Seizure

The anticonvulsant activities of cannabinoid compounds have been shown in various models of seizure and epilepsy. At least, part of antiseizure effects of cannabinoid compounds is mediated through calcium (Ca²⁺) channels. The L-type Ca²⁺ channels have been shown to be important in various epilepsy models. However, there is no data regarding the role of L-type Ca²⁺ channels in protective action of cannabinoids on acute and chronic models of seizure. In this study, the effects of cannabinoid compounds and L-type Ca²⁺ channels blockers, either alone or in combination were investigated using acute model of pentylenetetrazole (PTZ)-induced seizure in mice and chronic model electrical kindling of amygdala in rats. Pretreatment of mice with both cannabinoid CB1 receptor agonist arachidonyl-2′-chloroethylamide (ACEA) and endocannabinoid degradating enzyme inhibitor cyclohexylcarbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597) produced a protective effect against PTZ-induced seizure. Administration of various doses of the two L-type Ca²⁺ channel blockers verapamil and diltiazem did not alter PTZ-induced seizure threshold. However, co-administration of verapamil and either ACEA or URB597 attenuated the protective effect of cannabinoid compounds against PTZ-induced seizure. Also, pretreatment of mice with diltiazem blocked the anticonvulsant activity of both ACEA and URB597. Moreover, (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55,212-2), the non-selective cannabinoid CB1 and CB2 receptor agonist showed anticonvulsant effect in amygdala-kindled rats. However, co-administration of WIN55,212-2 and verapamil attenuated the protective properties of WIN55,212-2. Our results showed that the anticonvulsant activity of cannabinoid compounds is mediated, at least in part, by L-type Ca²⁺ channels in these two models of convulsion and epilepsy.     

T-type Ca2+ channel blockers suppress the growth of human cancer cells

In order to further clarify the role of T-type Ca²⁺ channels in cell proliferation, we have measured the growth inhibition of human cancer cells by using our potent T-type Ca²⁺ channel blockers. As a result, KYS05090, a most potent T-type Ca²⁺ channel blocker, was found to be as potent as doxorubicin against some human cancer cells without acute toxicity. Therefore, this letter provides the biological results that T-type calcium channel is important in regulating the important cellular phenotype transition leading to cell proliferation, and thus novel T-type Ca²⁺ channel blocker presents new prospects for cancer treatment.     

A non-invasive vibrating calcium-selective electrode measures acetylcholine-induced calcium flux across the sarcolemma of a smooth muscle

To determine possible sources of Ca²⁺ during excitation-contraction coupling in smooth muscle, a vibrating Ca²⁺-selective electrode was used to measure Ca²⁺ flux during the process of contraction. The smooth muscle model was the longitudinal muscle of the body wall of a sea cucumberSclerodactyla briareus. Because acetylcholine caused slow contractions of the muscle that were inhibited by Ca²⁺ channel blockers diltiazem and verapamil in earlier mechanical studies, we chose a vibrating Ca²⁺-selective electrode as our method to test the hypothesis that acetylcholine may be stimulating Ca²⁺ influx across the sarcolemma, providing a Ca²⁺ source during excitation-contraction coupling. Acetylcholine treatment stimulated a net Ca²⁺ efflux that was both dose and time dependent. We then tested two L-type Ca²⁺ channel blockers, diltiazem and verapamil, and two non-specific Ca²⁺ blockers, cobalt (Co²⁺) and lanthanum (La³⁺) on acetylcholine-induced Ca²⁺ flux. All four Ca²⁺ blockers tested potently inhibited Ca²⁺ efflux induced by physiological doses of acetylcholine. We propose that the acetylcholine-induced Ca²⁺ efflux was the result of, first, Ca²⁺ influx through voltage-sensitive L-type Ca²⁺ channels, then the rapid extrusion of Ca²⁺ by an outwardly directed carrier such as the Na–Ca exchanger as suggested by Li⁺ substitution experiments. The vibrating Ca²⁺ electrode has provided new insights on the active and complex role the sarcolemma plays in Ca²⁺ homeostasis and regulating Ca²⁺ redistribution during excitation-contraction coupling.Abbreviations ACh acetylcholine - E-C coupling excitation-contraction coupling - LMBW longitudinal muscle of the body wall     

Epigallocatechin-3-gallate elicits Ca spike in MCF-7 breast cancer cells: Essential role of Cav3.2 channels

We used MCF-7 human breast cancer cells that endogenously express Cav3.1 and Cav3.2 T-type Ca²⁺ channels toward a mechanistic study on the effect of EGCG on [Ca²⁺]_i. Confocal Ca²⁺ imaging showed that EGCG induces a [Ca²⁺]_i spike which is due to extracellular Ca²⁺ entry and is sensitive to catalase and to low-specificity (mibefradil) and high-specificity (Z944) T-type Ca²⁺channel blockers. siRNA knockdown of T-type Ca²⁺ channels indicated the involvement of Cav3.2 but not Cav3.1. Application of EGCG to HEK cells expressing either Cav3.2 or Cav3.1 induced enhancement of Cav3.2 and inhibition of Cav3.1 channel activity. Measurements of K⁺ currents in MCF-7 cells showed a reversible, catalase-sensitive inhibitory effect of EGCG, while siRNA for the Kv1.1 K⁺ channel induced a reduction of the EGCG [Ca²⁺]_i spike. siRNA for Cav3.2 reduced EGCG cytotoxicity to MCF-7 cells, as measured by calcein viability assay. Together, data suggest that EGCG promotes the activation of Cav3.2 channels through K⁺ current inhibition leading to membrane depolarization, and in addition increases Cav3.2 currents. Cav3.2 channels are in part responsible for EGCG inhibition of MCF-7 viability, suggesting that deregulation of [Ca²⁺]_i by EGCG may be relevant in breast cancer treatment.     

Molecular Characterization of a Novel Family of Low Voltage-Activated, T-Type, Calcium Channels

Low voltage-activated, T-type, calcium channels are thought to be involved in pacemaker activity, low threshold Ca²⁺ spikes, neuronal oscillations and resonance, and rebound burst firing. Mutations in T-type channel genes may be a contributing factor to neurological and cardiovascular disorders, such as epilepsy, arrhythmia, and hypertension. Due to the lack of selective blockers, little is known about their structure or molecular biology. This review discusses our recent findings on the cloning, chromosomal localization, and functional expression, of two novel channels, 1G and 1H. The biophysical properties of these cloned channels (distinctive voltage dependence, kinetics, and single channel conductance) demonstrates that these channels are members of the T-type Ca²⁺ channel family.     

Muscarinic acetylcholine receptor compounds alter net Ca<Superscript>2+</Superscript> flux and contractility in an invertebrate smooth muscle

Responses of a holothurian smooth muscle to a range of muscarinic (M₁ to M₅) acetylcholine receptor (mAChR) agonists and antagonists were surveyed using calcium (Ca²⁺)-selective electrodes and a mechanical recording technique. Most of the mAChR agonists and antagonists tested increased both contractility and net Ca²⁺ efflux, with M₁-specific agents like oxotremorine M being the most potent in their action. To investigate the possible sources of Ca²⁺ used during mAChR activation, agents that disrupt intracellular Ca²⁺ ion sequestration [cyclopiazonic acid (CPA), caffeine, ryanodine], the phosphoinositide signaling pathway [lithium chloride (LiCl)], and L-type Ca²⁺ channels (diltiazem and verapamil) were used to challenge contractions induced by oxotremorine M. These contractions were blocked by treatment with CPA, caffeine, LiCl, and by channel blockers, diltiazem and verapamil, but were unaltered by ryanodine. Our data suggest that this smooth muscle had an M_1,3,5-like receptor that was associated with the phosphoinositide signaling pathway that relied on intracellular Ca²⁺ stores, but secondarily used extracellular Ca²⁺ via the opening of L-type channels.     

Ligand-activated signal transduction in the 2-cell embryo

Platelet-activating factor (PAF) is an autocrine trophic/survival factor for the preimplantation embryo. PAF induced an increase in intracellular calcium concentration ([Ca2+]i) in the 2-cell embryo that had an absolute requirement for external calcium. L-type calcium channel blockers (diltiazem, verapamil, and nimodipine) significantly inhibited PAF-induced Ca2+ transients, but inhibitors of P/Q type (omega-agatoxin; omega-conotoxin MVIIC), N-type (omega-conotoxin GVIA), T-type (pimozide), and store-operated channels (SKF 96365 and econazole) did not block the transient. mRNA and protein for the alpha1-C subunit of L-type channels was expressed in the 2-cell embryo. The L-type calcium channel agonist (+/-) BAY K 8644 induced [Ca2+]i transients and, PAF and BAY K 8644 each caused mutual heterologous desensitization of each other's responses. Depolarization of the embryo (75 mM KCl) induced a [Ca2+]i transient that was inhibited by diltiazem and verapamil. Whole-cell patch-clamp measurements detected a voltage-gated channel (blocked by diltiazem, verapamil, and nifedipine) that was desensitized by prior responses of embryos to exogenous or embryo-derived PAF. Replacement of media Ca2+ with Mn2+ allowed Mn2+ influx to be observed directly; activation of a diltiazem-sensitive influx channel was an early response to PAF. The activation of a voltage-gated L-type calcium channel in the 2-cell embryo is required for normal signal transduction to an embryonic trophic factor.     

T-type Ca2+ channels and autoregulation of local blood flow

L-type voltage gated Ca²⁺ channels are considered to be the primary source of calcium influx during the myogenic response. However, many vascular beds also express T-type voltage gated Ca²⁺ channels. Recent studies suggest that these channels may also play a role in autoregulation. At low pressures (40–80 mmHg) T-type channels affect myogenic responses in cerebral and mesenteric vascular beds. T-type channels also seem to be involved in skeletal muscle autoregulation. This review discusses the expression and role of T-type voltage gated Ca²⁺ channels in the autoregulation of several different vascular beds. Lack of specific pharmacological inhibitors has been a huge challenge in the field. Now the research has been strengthened by genetically modified models such as mice lacking expression of T-type voltage gated Ca²⁺ channels (Ca_V3.1 and Ca_V3.2). Hopefully, these new tools will help further elucidate the role of voltage gated T-type Ca²⁺ channels in autoregulation and vascular function.     

Ca2+ channels involved in endothelin-induced mitogenic response in carotid artery vascular smooth muscle cells

Endothelin (ET)-1 activates twotypes of Ca²⁺-permeable nonselective cation channels(NSCC-1 and NSCC-2) and a store-operated Ca²⁺ channel(SOCC) in rabbit internal carotid artery (ICA) vascular smooth musclecells (VSMCs) in addition to the voltage-operated Ca²⁺channel (VOCC). These channels can be discriminated using the Ca²⁺ channel blockers SK&F-96365 and LOE-908. SK&F-96365 issensitive to NSCC-2 and SOCC, and LOE-908 is sensitive to NSCC-1 andNSCC-2. On the basis of sensitivity to nifedipine, a specific blocker of the L-type VOCC, VOCCs have a minor role in ET-1-inducedmitogenesis. Both LOE-908 and SK&F-96365 inhibited ET-1-inducedmitogenesis in a concentration-dependent manner, and the combination ofLOE-908 and SK&F-96365 abolished it. The IC₅₀ values ofthese blockers for ET-1-induced mitogenesis correlated well with thoseof the ET-1-induced intracellular free Ca²⁺concentration responses. These results indicate that the inhibitory action of these blockers on ET-1-induced mitogenesis may bemediated by blockade of NSCC-1, NSCC-2, and SOCC. Collectively,extracellular Ca²⁺ influx through NSCC-1, NSCC-2, and SOCCmay be essential for ET-1-induced mitogenesis in ICA VSMCs.

     

Ca2+-conductances in cultured rat retinal pigment epithelial cells

Membrane conductances for Ca²⁺ in cultured rat pigment epithelial cells were studied in the whole-cell configuration of the patch-clamp technique using barium (10 mM) as a charge carrier. Two types of voltage-dependent and verapamiland diltiazem-sensitive Ba²⁺ currents were observed. First, a nearly sustained current was activated by depolarization to potentials more positive than — 30mV and blocked by nifedipine (1 μM). This current was observed in cells of primary cultures less than 13 days old. Second, a transient nifedipine (1 μM) insensitive current was activated by depolarization to potentials more positive than — 55mV in cultures which were more than 13 days old. This current was not carried by sodium and blocked by 1 μM tetrodotoxin (TTX). In summary, cultured rat retinal pigment epithelial cells in younger primary cultures express Ba²⁺ currents indicating the presence of L-type Ca²⁺ channels. In order primary cultures a low-voltage activated channel was observed with properties different from T-type calcium channels or TTX-sensitive calcium conducting sodium channels. © 1994 Wiley-Liss, Inc.     

Increase of atrial ANP release by 2,3-butanedione monoxime in beating rabbit atria

2,3-Butanedione monoxime (BDM) is a chemical phosphatase and has been known to dissociate mechanical contraction in the excitation–contraction coupling via inhibition of myofibrillar ATPase. BDM has also been found to decrease sarcolemmal L-type Ca²⁺ channel activity and intracellular Ca²⁺ in cardiac myocytes. It has been shown that Ca²⁺ entry via L-type Ca²⁺ channels decreased atrial myocyte atrial natriuretic peptide (ANP) release. The purpose of the present study was to address the effects of BDM in the regulation of ANP release. Experiments were performed in perfused beating rabbit atria. BDM accentuated atrial myocyte ANP release concomitantly with a decrease in atrial stroke volume and pulse pressure in a concentration-dependent manner. The BDM-induced activation of ANP release was attenuated by the treatment with nifedipine, an inhibitor of L-type Ca²⁺ channels. BDM further decreased atrial stroke volume and pulse pressure in the presence of nifedipine. Blockade of function of the sarcoplasmic reticulum with thapsigargin plus ryanodine slightly but not significantly attenuated the BDM-induced activation of ANP release. These data show that BDM is a potent stimulator for the ANP release and also suggest that the mechanism by which BDM activates atrial myocyte ANP release is related to inhibition of the L-type Ca²⁺ channel activity. The present finding also suggests that the effects of ANP released may be considered in an occasion of uncoupling by BDM of the excitation–contraction coupling of cardiomyocytes.     

Identification of a sphingosine-sensitive Ca2+ channel in the plasma membrane of Leishmania mexicana

The disruption of the intracellular Ca²⁺ homeostasis of Leishmania mexicana represents a major target for the action of drugs, such as amiodarone and miltefosine. However, little is known about the mechanism of Ca²⁺ entry to these cells. Here we show the presence of a Ca²⁺ channel in the plasma membrane of these parasites. This channel has many characteristics similar to the human L-type voltage-gated Ca²⁺ channel. Thus, Ca²⁺ entry is blocked by verapamil, nifedipine and diltiazem while Bay K 8644 opened this channel. However, different to its human counterpart, sphingosine was able to open this channel, while other well known sphingolipids had no effect. This fact could have important pharmacological implications.     

The effects of calcium channel blockade on proliferation and differentiation of cardiac progenitor cells

Cardiogenesis depends on a tightly regulated balance between proliferation and differentiation of cardiac progenitor cells (CPCs) and their cardiomyocyte descendants. While exposure of early mouse embryos to Ca²⁺ channel antagonists has been associated with abnormal cardiac morphogenesis, less is known about the consequences of Ca²⁺ channel blockade on proliferation and differentiation of CPCs at the cellular level. Here we showed that at embryonic day (E) 11.5, the murine ventricles express several L-type and T-type Ca²⁺ channel isoforms, and that the dihydropyridine Ca²⁺ channel antagonist, nifedipine, blunts isoproterenol induced increases in intracellular Ca²⁺. Nifedipine mediated Ca²⁺ channel blockade was associated with a reduction in cell cycle activity of E11.5 CPCs and impaired assembly of the cardiomyocyte contractile apparatus. Furthermore, in cell transplantation experiments, systemic administration of nifedipine to adult mice receiving transplanted E11.5 ventricular cells (containing CPCs and cardiomyocytes) was associated with smaller graft sizes compared to vehicle treated control animals. These data suggest that intracellular Ca²⁺ is a critical regulator of the balance between CPC proliferation and differentiation and demonstrate that interactions between pharmacological drugs and transplanted cells could have a significant impact on the effectiveness of cell based therapies for myocardial repair.     

Cardiomyocyte-like cells differentiated in vitro from embryonic carcinoma cells P19 are characterized by functional expression of adrenoceptors and Ca2+ channels

Summary P19 embryonal carcinoma cells were differentiated via embryolike aggregates (embryoid bodies) into spontaneously beating myocytes. During the whole process of differentiation the functional expression of cardiac-specific receptors and ionic channels was characterized by measuring the chronotropic reactivity, action potentials, and ionic currents in response to various cardioactive drugs. Positive chronotropic effects obtained at different maximal effective concentrations of adrenoceptor-mediated agonists indicated differential adrenoceptor expression during the in vitro development of cardiomyocyte-like cells. No cardiac-specific response was obtained with the muscarinic cholinoceptor agonist carbachol. Single beating cells were enzymatically isolated and investigated by the patch-clamp technique. Pacemaker action potentials similar to those of embryonal cardiomyocytes exhibited amplitudes ranging from 50 to 85 mV. The action potentials were synchronous to the mechanical contractions and, comparable to the chronotropic effects, were modulated by BayK 8644, isradipine, and adrenaline. The functional expression of L-type Ca²⁺ channels was demonstrated by the Ca²⁺ channel blockers isradipine, nisoldipine, gallopamil, and diltiazem causing negative chronotropic responses, as well as by the Ca²⁺ channel activator BayK 8644 causing positive chronotropic responses. These effects gradually increased with time of differentiation. The expression of L-type Ca²⁺ channels and of nicotinic acetylcholine receptors was confirmed in voltage-clamp experiments. The study demonstrates that P19 embryonal carcinoma cells can be induced to differentiate into cardiomyocyte-like cells comparable to embryonal and neonatal heart cells lacking the muscarinic cholinoceptor response only.     

Properties of calcium channels in cardiac muscle and vascular smooth muscle

The voltage-dependent slow channels in the myocardial cell membrane are the major pathway by which Ca²⁺ ions enter the cell during excitation for initiation and regulation of the force of contraction of cardiac muscle. The slow channels have some special properties, including functional dependence on metabolic energy, selective blockade by acidosis, and regulation by the intracellular cyclic nucleotide levels. Because of these special properties of the slow channels, Ca²⁺ influx into the myocardial cell can be controlled by extrinsic factors (such as autonomic nerve stimulation or circulating hormones) and by intrinsic factors (such as cellular pH or ATP level). The slow Ca²⁺ channels of the heart are regulated by cAMP in a stimulatory fashion. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a slow channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_si, Ca²⁺ influx, and contraction. The myocardial slow Ca²⁺ channels are also regulated by cGMP, in a manner that is opposite to that of CAMP. The effect of cGMP is presumably mediated by means of phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the slow channel. Preliminary data suggest that calmodulin also may play a role in regulation of the myocardial slow Ca²⁺ channels, possibly mediated by the Ca²⁺-calmodulin-protein kinase and phosphorylation of some regulatory-type of protein. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of extrinsic and intrinsic factors.VSM cells contain two types of Ca²⁺ channels: slow (L-type) Ca²⁺ channels and fast (T-type) Ca²⁺ channels. Although regulation of voltage-dependent Ca²⁺ slow channels of VSM cells have not been fully clarified yet, we have made some progress towards answering this question. Slow (L-type, high-threshold) Ca²⁺ channels may be modified by phosphorylation of the channel protein or an associated regulatory protein. In contrast to cardiac muscle where cAMP and cGMP have antagonistic effects on Ca²⁺ slow channel activity, in VSM, cAMP and cGMP have similar effects, namely inhibition of the Ca²⁺ slow channels. Thus, any agent that elevates cAMP or cGMP will inhibit Ca²⁺ influx, and thereby act to produce vasodilation. The Ca²⁺ slow channels require ATP for activity, with a K_0.5 of about 0.3 mM. C-kinase may stimulate the Ca²⁺ slow channels by phosphorylation. G-protein may have a direct action on the Ca²⁺ channels, and may mediate the effects of activation of some receptors. These mechanisms of Ca²⁺ channel regulation may be invoked during exposure to agonists or drugs, which change second messenger levels, thereby controlling vascular tone.