Dihydropyridine Binding Sites Research Articles

Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cav channels.

Novel Labdane Diterpenes-Based Synthetic Derivatives: Identification of a Bifunctional Vasodilator That Inhibits CaV1.2 and Stimulates KCa1.1 Channels.

Sesquiterpenes such as leucodin and the labdane-type diterpene manool are natural compounds endowed with remarkably in vitro vasorelaxant and in vivo hypotensive activities. Given their structural similarity with the sesquiterpene lactone (+)-sclareolide, this molecule was selected as a scaffold to develop novel vasoactive agents. Functional, electrophysiology, and molecular dynamics studies were performed. The opening of the five-member lactone ring in the (+)-sclareolide provided a series of labdane-based small molecules, promoting a significant in vitro vasorelaxant effect. Electrophysiology data identified 7 as a CaV1.2 channel blocker and a KCa1.1 channel stimulator. These activities were also confirmed in the intact vascular tissue. The significant antagonism caused by the CaV1.2 channel agonist Bay K 8644 suggested that 7 might interact with the dihydropyridine binding site. Docking and molecular dynamic simulations provided the molecular basis of the CaV1.2 channel blockade and KCa1.1 channel stimulation produced by 7. Finally, 7 reduced coronary perfusion pressure and heart rate, while prolonging conduction and refractoriness of the atrioventricular node, likely because of its Ca2+ antagonism. Taken together, these data indicate that the labdane scaffold represents a valuable starting point for the development of new vasorelaxant agents endowed with negative chronotropic properties and targeting key pathways involved in the pathophysiology of hypertension and ischemic cardiomyopathy.

L-Type Calcium Channel Blockers: A Potential Novel Therapeutic Approach to Drug Dependence.

This review describes interactions between compounds, primarily dihydropyridines, that block L-type calcium channels and drugs that cause dependence, and the potential importance of these interactions. The main dependence-inducing drugs covered are alcohol, psychostimulants, opioids, and nicotine. In preclinical studies, L-type calcium channel blockers prevent or reduce important components of dependence on these drugs, particularly their reinforcing actions and the withdrawal syndromes. The channel blockers also reduce the development of tolerance and/or sensitization, and they have no intrinsic dependence liability. In some instances, their effects include reversal of brain changes established during drug dependence. Prolonged treatment with alcohol, opioids, psychostimulant drugs, or nicotine causes upregulation of dihydropyridine binding sites. Few clinical studies have been carried out so far, and reports are conflicting, although there is some evidence of effectiveness of L-channel blockers in opioid withdrawal. However, the doses of L-type channel blockers used clinically so far have necessarily been limited by potential cardiovascular problems and may not have provided sufficient central levels of the drugs to affect neuronal dihydropyridine binding sites. New L-type calcium channel blocking compounds are being developed with more selective actions on subtypes of L-channel. The preclinical evidence suggests that L-type calcium channels may play a crucial role in the development of dependence to different types of drugs. Mechanisms for this are proposed, including changes in the activity of mesolimbic dopamine neurons, genomic effects, and alterations in synaptic plasticity. Newly developed, more selective L-type calcium channel blockers could be of considerable value in the treatment of drug dependence. SIGNIFICANCE STATEMENT: Dependence on drugs is a very serious health problem with little effective treatment. Preclinical evidence shows drugs that block particular calcium channels, the L-type, reduce dependence-related effects of alcohol, opioids, psychostimulants, and nicotine. Clinical studies have been restricted by potential cardiovascular side effects, but new, more selective L-channel blockers are becoming available. L-channel blockers have no intrinsic dependence liability, and laboratory evidence suggests they reverse previously developed effects of dependence-inducing drugs. They could provide a novel approach to addiction treatment.

Co-existence of a fast tetrodotoxin-sensitive low voltage-activated non selective Na/Ca/K channel and of T-type Ca channels in rat isolated pulmonary vein cardiomyocytes

Ectopic foci in pulmonary veins (PV) are involved in the onset of atrial fibrillation. Norepinephrine (NE) induces an automatic activity occurring in bursts in rat PV cardiomyocytes (CM) which depends on Ca (upstroke) and Na (inter-burst) channels. To characterize low voltage-activated (LVA) Ca currents in rat PVCM susceptible to trigger ectopic foci. Whole-cell ICa (5 mM Ca) was recorded from -100 mV with classical Na- and K-free solutions at room temperature in enzymatically isolated PVCM from male Wistar rats. A fast LVA ICa (FLVA-ICa; −1.5 ± 0.2 pA/pF, n = 45; 26% of peak ICa, max recorded at + 20 mV) was activated between −55 and −20 mV in ∼56% of PVCM and blocked by 10 μM tetrodotoxin (TTX). In that, it was similar to the reported TTX-sensitive Ca current. However, it was markedly increased by addition of NaCl (1 or 3 mM) or KCl (5 or 10 mM). Permeability ratios P’Ca/PNa and P’Ca/PK calculated for bi-ionic conditions were respectively 2.25 ± 0.51 (n = 6) and 1.88 ± 0.25 (n = 14), and not different from a value of 2. NE (10 μM, n = 13) increased FLVA-ICa and negatively shifted its activation threshold, an effect partially reversed by 5 μM Atenolol. It was partially blocked by 5 μM Nifedipine (n = 5) and increased by 300 nM BayK8644 (n = 6), but not blocked by Ranolazine (10 μM, n = 6). NiCl2 (40 μM, n = 9) and TTA-A2 (10 or 100 nM, n = 11) increased FLVA-ICa. Neither Ba nor Sr alone could permeate the FLVA channel or block Ca influx but revealed a large slower activating and inactivating LVA Ca current (SLVA-ICa), present in 10 out of 80 PVCM and absent in LACM. SLVA-ICa was partially blocked by 100 nM TTA-A2. The ionic channel underlying FLVA-ICa is likely a fast voltage-gated non-selective channel permeable to Na/Ca/K with a dihydropyridine binding site. SLVA-ICa might correspond to Ca influx through Cav3.1 channels. The respective role of these channels to triggering ectopic foci in the PV myocardial sleeve remained to be determined.

Low voltage-activated channels in rat pulmonary vein cardiomyocytes: coexistence of a non-selective cationic channel and of T-type Ca channels.

In rat pulmonary vein (PV) cardiomyocytes (CM), norepinephrine (NE) induces an automatic activity consisting of bursts of slow action potentials which depend on Ca2+ (upstroke) and Na+ (inter-burst) channels. Our objective was to characterize low voltage-activated (LVA) currents in rat PVCM susceptible to trigger this activity. Whole-cell ICa (5 mM Ca2+) was recorded from - 100 mV with classical Na+- and K+-free solutions. A fast LVA ICa (FLVA-ICa), present in ≈ 56% of PVCM between ~ - 50 to - 20 mV, was blocked by 10 μM TTX and markedly increased by addition of NaCl (1 or 3 mM) or KCl (5 or 10 mM). Permeability ratios P'Ca/PNa and P'Ca/PK calculated for bi-ionic conditions were respectively 2.25 ± 0.51 and 1.88 ± 0.25, and not different from a value of 2. FLVA-ICa was increased by 10 μM NE and 300 nM BayK8644, decreased by 5 μM nifedipine but not blocked by ranolazine (10 μM). NiCl2 (40 μM) and TTA-A2 (10 or 100 nM) increased FLVA-ICa. Similar results were obtained in left atrial (LA) CM. Neither Ba2+ nor Sr2+ alone could permeate the FLVA channel or block Ca2+ influx but revealed a large slower activating and inactivating LVA Ca2+ current (SLVA-ICa), present in 10 out of 80 PVCM, absent in LACM, and partially inhibited by 100 nM TTA-A2. Therefore, the ionic channel underlying FLVA-ICa is likely a fast voltage-gated non-selective channel with a dihydropyridine binding site. SLVA-ICa might correspond to Ca2+ influx through Cav3.x channels and contribute to triggering NE-induced automatic activity in the PV myocardial sleeve.

Activated L-Type Calcium Channels Inhibit Chemosensitized Nematocyst Discharge from Sea Anemone Tentacles.

Because in vivo nematocyst discharge requires extracellular Ca2+, Ca2+ channels have been suspected to be involved; but their identity and role have not been revealed. The majority of nematocysts that discharge from sea anemone tentacles are under the control of sensitizing chemoreceptors for N-acetylated sugars (e.g., N-acetylneuraminic acid). Activated chemoreceptors predispose contact-sensitive mechanoreceptors to trigger discharge. We show that activating L-type Ca2+ channels inhibits N-acetylneuraminic acid-sensitized discharge, contrary to a previous suggestion. In addition, inhibiting L-type channels increases sensitivity to N-acetylneuraminic acid. Specifically, we show that the L-type Ca2+ channel activator (-)-Bay K 8644 dose-dependently inhibits N-acetylneuraminic acid-sensitized discharge, as does raising ambient Ca2+ levels. We also show that lowering extracellular Ca2+ levels or adding any of several selective and chemically distinct L-type Ca2+ channel blockers, including dihydropyridines, dose-dependently increases N-acetylneuraminic acid sensitivity and broadens the dynamic range of N-acetylneuraminic acid sensitization. Consistent with these functional findings, Aiptasia pallida expresses an L-type Ca2+ channel α subunit transcript that encodes a conserved dihydropyridine-binding site. Phylogenetic analysis confirms a close relationship of the Aiptasia Ca2+ channel α subunit sequence between anemones, anthozoans, and cnidarians that extends into protostomal and deuterostomal bilaterians. We conclude that L-type Ca2+ channel activity modulates N-acetylneuraminic acid-sensitized nematocyst discharge in a push-pull manner depending on channel activity state. Our findings suggest that L-type channel activation promotes chemosensory desensitization, and we predict that N-acetylneuraminic acid chemoreceptor signaling will activate L-type channels.

Characterization of a Dihydropyridine‐insensitive Cav1.3 Channel

The L‐type voltage‐gated calcium channels (L‐VGCCs), Cav1.2 and Cav1.3, play an important role in different physiological processes such as action potential firing, exocytosis and muscle contraction. Due to a lack of selectivity for the dihydropyridine (DHP) class of L‐VGCC blockers, a Cav1.3 mutant that is insensitive to DHPs (Cav1.3/DHPi) has been used to dissect the role of Cav1.3 in glucose‐stimulated insulin secretion and other cellular activities. However, the Cav1.3/DHPi mutant channel has not been biophysically characterized. Our lab created the Cav1.3/DHPi by mutating Thr1033 and Gln1037 that are located in segment 5 of domain III (IIIS5) of the rat neuronal Cav1.3 clone (accession no. AF30010.1) to Tyr and Met, respectively. Ba2+ current (IBa) elicited by different voltage steps from tsA cells expressing Cav channels with and without the presence of drugs was measured. The voltage‐dependent activation and inactivation of Cav1.3/DHPi (V1/2 inact.= ‐26.6±1.2mV, V1/2 act.= ‐22.3±1.1mV) resembles that of wild type Cav1.3 (Cav1.3/WT) (V1/2 inact.= ‐36.0±1.3mV, V1/2 act.= ‐29.8±1.5mV). Cav1.3/DHPi is over 100 fold more insensitive to nifedipine compared to Cav1.3/WT (IC50 for Cav1.3/WT= ~0.55μM; IC50 for Cav1.3/DHPi=~70μM). However, the non‐DHP blockers, diltiazem (500μM) and the recently debated Cav1.3 selective blocker, compound 8 (50μM) achieved comparable inhibition of Cav1.3/WT and Cav1.3/DHPi‐mediated current. Our findings showed that Thr1033 and Gln1037 are not only critical, but also specific for DHP block of Cav1.3. The Cav1.3/DHPi mutant is a useful tool to screen for Cav1.3 specific blockers by removing the DHP binding site which is common to both Cav1.2 and Cav1.3.

Effects of cannabidiol on contractions and calcium signaling in rat ventricular myocytes

Cannabidiol (CBD), a major nonpsychotropic cannabinoid found in Cannabis plant, has been shown to influence cardiovascular functions under various physiological and pathological conditions. In the present study, the effects of CBD on contractility and electrophysiological properties of rat ventricular myocytes were investigated. Video edge detection was used to measure myocyte shortening. Intracellular Ca2+ was measured in cells loaded with the Ca2+ sensitive fluorescent indicator fura-2 AM. Whole-cell patch clamp was used to measure action potential and Ca2+ currents. Radioligand binding was employed to study pharmacological characteristics of CBD binding. CBD (1μM) caused a significant decrease in the amplitudes of electrically evoked myocyte shortening and Ca2+ transients. However, the amplitudes of caffeine-evoked Ca2+ transients and the rate of recovery of electrically evoked Ca2+ transients following caffeine application were not altered. CBD (1μM) significantly decreased the duration of APs. Further studies on L-type Ca2+ channels indicated that CBD inhibits these channels with IC50 of 0.1μM in a voltage-independent manner. Radioligand studies indicated that the specific binding of [3H]Isradipine, was not altered significantly by CBD. The results suggest that CBD depresses myocyte contractility by suppressing L-type Ca2+ channels at a site different than dihydropyridine binding site and inhibits excitation–contraction coupling in cardiomyocytes.

Renoprotective Effects of the L-/T-type Calcium Channel Blocker Benidipine in Patients with Hypertension

The renoprotective effects of benidipine, a calcium channel blocker (CCB) developed in Japan, are reviewed herein. Benidipine has a sustained antihypertensive effect independent of its blood concentration since it binds to dihydropyridine (DHP) receptors via a "membrane approach" (approach to the cell membrane followed by long retention at the DHP binding site). Benidipine dilates glomerular afferent and efferent arterioles equally through inhibition of Ttype Ca channels. Thus, it may cause a decrease of intraglomerular pressure and is superior to CCBs (capable of inhibiting only L-type Ca channels) in terms of suppression of proteinuria. Additionally, benidipine suppresses worsening of renal function more powerfully than CCBs (suppressing only L-type Ca channels), allowing better prognosis as to renal function. The inhibitory effect of benidipine on T-type calcium channels results in the suppression of aldosterone formation in the adrenal glands and of oxidative stress induced by aldosterone. Thus, the aldosterone-inhibitory and antioxidant activities of benidipine mediated by inhibition of T-type calcium channels would result in renoprotection and suppression of disease progression in hypertensive patients with chronic kidney disease (CKD). If such patients have proteinuria, renin-angiotensin system (RAS) inhibitors are used as first-line drugs, but benidipine, as an L-/T-type CCB, is recommended when they require some concomitant drugs. Moreover, the superiority of RAS inhibitors has not been demonstrated in hypertensive patients with CKD and without proteinuria. Thus, in such patients, benidipine should be considered as a first-line antihypertensive drug.

Antagonism of L-type Ca2+ channels CaV1.3 and CaV1.2 by 1,4-dihydropyrimidines and 4H-pyrans as dihydropyridine mimics

The L-type calcium channel (LTCC) CaV1.3 is regarded as a new potential therapeutic target for Parkinson’s disease. Calcium influx through CaV1.3 LTCC during autonomous pacemaking in adult dopaminergic neurons of the substantia nigra pars compacta is related to the generation of mitochondrial oxidative stress in animal models. Development of a CaV1.3 antagonist selective over CaV1.2 is essential because CaV1.2 pore-forming subunits are the predominant form of LTCCs and are abundant in the central nervous and cardiovascular systems. We have explored 1,4-dihydropyrimidines and 4H-pyrans to identify potent and selective antagonists of CaV1.3 relative to CaV1.2 LTCCs. A library of 36 dihydropyridine (DHP)-mimic 1,4-dihydropyrimidines and 4H-pyrans was synthesized, and promising chiral compounds were resolved. The antagonism studies of CaV1.3 and CaV1.2 LTCCs using DHP mimic compounds showed that dihydropyrimidines and 4H-pyrans are effective antagonists of DHPs for CaV1.3 LTCCs. Some 1,4-dihydropyrimidines are more selective than isradipine for CaV1.3 over CaV1.2, shown here by both calcium flux and patch-clamp electrophysiology experiments, where the ratio of antagonism is around 2–3. These results support the hypothesis that the modified hydrogen bonding donor/acceptors in DHP-mimic dihydropyrimidines and 4H-pyrans can interact differently with DHP binding sites, but, in addition, the data suggest that the binding sites of DHP in CaV1.3 and CaV1.2 LTCCs are very similar.

In-vivo binding of (+)-[3H]PN 200-110 to peripheral tissues and brain of spontaneously hypertensive rats: effect of lacidipine.

The time-course of dihydropyridine receptor occupancy by lacidipine and its relationship with pharmacological activity has been studied in spontaneously hypertensive rat (SHR), as measured by the inhibition of specific (+)-[3H]PN 200-110 binding in-vivo. After oral administration of doses active in reducing blood pressure, lacidipine did not show tissue target differences in respect to binding sites labelled by (+)-[3H]PN 200-110 in cerebral cortex, heart, ileum, bladder and thoracic aorta. The relative occupancy of receptors in heart 60 min after oral administration of 1 mg kg-1 lacidipine was 75%. After 12 h, when lacidipine was still effective in reducing blood pressure in SHR, a low (15%) but detectable proportion of receptors was still occupied by the drug. The percentage decrease of blood pressure was linear with the percentage of receptor occupancy obtained by different doses of lacidipine; that is, there was a close correspondence between ED25 for decrease in blood pressure (0.33 mg kg-1) and ED25 for inhibition of (+)-[3H]PN 200-110 specific binding in the heart (0.36 mg kg-1). The long-lasting effect of lacidipine on blood pressure might be explained by its selective interaction with dihydropyridine binding sites labelled in-vivo by (+)-[3H]PN 200-110.

Inhibition of Recombinant L-Type Voltage-Gated Calcium Channels by Positive Allosteric Modulators of GABAA Receptors

Benzodiazepines (BDZs) depress neuronal excitability via positive allosteric modulation of inhibitory GABA(A) receptors (GABA(A)R). BDZs and other positive GABA(A)R modulators, including barbiturates, ethanol, and neurosteroids, can also inhibit L-type voltage-gated calcium channels (L-VGCCs), which could contribute to reduced neuronal excitability. Because neuronal L-VGCC function is up-regulated after long-term GABA(A)R modulator exposure, an interaction with L-VGCCs may also play a role in physical dependence. The current studies assessed the effects of BDZs (diazepam, flurazepam, and desalkylflurazepam), allopregnanolone, pentobarbital, and ethanol on whole-cell Ba(2+) currents through recombinant neuronal Ca(v)1.2 and Ca(v)1.3 L-VGCCs expressed with β(3) and α(2)δ-1 in HEK293T cells. Allopregnanolone was the most potent inhibitor (IC(50), ∼10 μM), followed by BDZs (IC(50), ∼50 μM), pentobarbital (IC(50), 0.3-1 mM), and ethanol (IC(50), ∼300 mM). Ca(v)1.3 channels were less sensitive to pentobarbital inhibition than Ca(v)1.2 channels, similar to dihydropyridine (DHP) L-VGCC antagonists. All GABA(A)R modulators induced a negative shift in the steady-state inactivation curve of Ca(v)1.3 channels, but only BDZs and pentobarbital induced a negative shift in Ca(v)1.2 channel inactivation. Mutation of the high-affinity DHP binding site (T1039Y and Q1043M) in Ca(v)1.2 channels reduced pentobarbital potency. Despite the structural similarity between benzothiazepines and BDZs, mutation of an amino acid important for diltiazem potency (I1150A) did not affect diazepam potency. Although L-VGCC inhibition by BDZs occurred at concentrations that are possibly too high to be clinically relevant and is not likely to play a role in the up-regulation of L-VGCCs during long-term treatment, pentobarbital and ethanol inhibited L-VGCCs at clinically relevant concentrations.

Antagonism of 4-substituted 1,4-dihydropyridine-3,5-dicarboxylates toward voltage-dependent L-type Ca2+ channels CaV1.3 and CaV1.2

L-type Ca2+ channels in mammalian brain neurons have either a CaV1.2 or CaV1.3 pore-forming subunit. Recently, it was shown that CaV1.3 Ca2+ channels underlie autonomous pacemaking in adult dopaminergic neurons in the substantia nigra pars compacta, and this reliance renders them sensitive to toxins used to create animal models of Parkinson’s disease. Antagonism of these channels with the dihydropyridine antihypertensive drug isradipine diminishes the reliance on Ca2+ and the sensitivity of these neurons to toxins, pointing to a potential neuroprotective strategy. However, for neuroprotection without an antihypertensive side effect, selective CaV1.3 channel antagonists are required. In an attempt to identify potent and selective antagonists of CaV1.3 channels, 124 dihydropyridines (4-substituted-1,4-dihydropyridine-3,5-dicarboxylic diesters) were synthesized. The antagonism of heterologously expressed CaV1.2 and CaV1.3 channels was then tested using electrophysiological approaches and the FLIPR Calcium 4 assay. Despite the large diversity in substitution on the dihydropyridine scaffold, the most CaV1.3 selectivity was only about twofold. These results support a highly similar dihydropyridine binding site at both CaV1.2 and CaV1.3 channels and suggests that other classes of compounds need to be identified for CaV1.3 selectivity.

Cellular calcium regulatory machinery of vasorelaxation elicited by petasin

1. The present study examined the cytosolic Ca(2+) regulatory machinery involved in the vasorelaxation produced by petasin, a sesquiterpene isolated from Petasites formosanus. 2. Aortic rings isolated from Sprague-Dawley rats were exposed to petasin (0.01-100 micromol/L) to elucidate its vascular effects on isometric contraction elicited by vasoconstrictors, as well as the contribution of the endothelium and Ca(2+) to the responses observed. In addition, L-type voltage-dependent Ca(2+) channel (VDCC) activity and [Ca(2+)](i) were determined in cultured vascular smooth muscle cells (VSMCs) from Sprague-Dawley rats in the presence of 1-100 micromol/L petasin using whole-cell patch-clamp recording and the fluorescent probe fura-2/AM. The effects of petasin on vascular responses were compared between aortic rings from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. 3. Petasin reduced isometric contraction elicited by KCl or the L-type Ca(2+) channel opener BayK 8644 (IC(50) 3.0 +/- 0.4 and 4.1 +/- 1.1 micromol/L, respectively) in aortic rings isolated from Sprague-Dawley rats, independent of the endothelium. In addition, petasin triggered a rightward shift in the concentration-response curve to KCl while reducing the maximal response by 82%. In Ca(2+)-depleted and high K(+)-depolarized aortic rings, 1-100 micromol/L petasin pretreatment attenuated the Ca(2+)-induced contraction in a concentration-dependent manner. 4. In cultured VSMCs, whole-cell patch-clamp recording revealed that petasin inhibited VDCC activity. Measurement of [Ca(2+)](i) using fura-2/AM fluorescence indicated that petasin suppressed the KCl-induced increase in [Ca(2+)](i). However, receptor binding assays failed to identify any significant interaction between petasin and the dihydropyridine binding sites of the L-type VDCC. 5. In aortic rings from SHR and WKY rats, petasin inhibited Ca(2+)-induced contractions in Ca(2+)-depleted and high K(+)-depolarized solution with a more pronounced effect in rings from SHR. 6. Together, the results suggest that direct Ca(2+) antagonism of L-type VDCC in vascular smooth muscle may account, at least in part, for petasin-induced vasorelaxation. The more pronounced effect of the sesquiterpene in blood vessels from SHR suggests its possible therapeutic potential in the mangement of hypertension.

Chronic Alcohol Consumption Blunts β-Adrenergic Responsiveness in Left Ventricular Cardiomyocytes

Alcoholic cardiomyopathy (ACM) develops from long-term, excessive consumption of alcohol. Initially, ACM is asymptomatic but continued alcohol abuse leads to reductions in cardiac contractility, the onset of arrhythmias, chamber dilation and congestive heart failure. This study was carried out to examine the effects of chronic alcohol on basal and β-adrenergic-stimulated properties of Ca2+ transients during excitation-contraction (E-C) coupling. Rats were pair-fed DeCarli and Lieber control and alcohol liquid diets for 120 days prior to isolating left ventricular myocytes. Under basal conditions, there was no change in the amplitude of electrically-triggered [Ca2+]i transients (Control, 296±21nM vs. Alcoholic, 260±18 nM) or contraction (Control, 11.9±0.6 μm, Alcoholic,12.7±1.2 μm. However, a blunted inotropic response (increase over basal: Control, 90±19% vs. Alcoholic 39±10 %) was observed in the presence of submaximal isoproterenol stimulation. In addition, maximal isoproterenol and forskolin stimulation do not improve the inotropic response of the alcoholic myocytes, suggesting a functional impairment in the initial Ca2+ release steps of E-C coupling. Consistent with the reduced [Ca2+]i transient amplitude, the Ca2+ current (ICa,L) responses to isoproterenol were also markedly reduced in cardiomyocytes from alcohol-fed animals. Surprisingly, measurement of L-type calcium channel expression by dihydropyridine (DHP) binding and real-time PCR, revealed an increased number of DHP binding sites (Control Bmax = 197 ± 60 fmol/mg vs. Alcoholic Bmax = 335 ± 45 fmol/mg, P < 0.05) and α1C subunit expression (Alcoholic 2-ΔΔCt1.69 ± 0.03, Control 2-ΔΔCt0.96 ± 0.02, P< 0.0005), respectively. This loss of L-type calcium channel activity, accompanied by an increased channel expression with chronic alcohol consumption may be a precipitating factor in alcoholic heart disease, leading to the onset of other adaptive mechanisms and, eventually, the clinical syndrome of heart failure.

The role of calcium in apoptosis induced by 7β‐hydroxycholesterol and cholesterol‐5β,6β‐epoxide

Oxysterols, such as 7beta-hydroxy-cholesterol (7beta-OH) and cholesterol-5beta,6beta-epoxide (beta-epoxide), may have a central role in promoting atherogenesis. This is thought to be predominantly due to their ability to induce apoptosis in cells of the vascular wall and in monocytes/macrophages. Although there has been extensive research regarding the mechanisms through which oxysterols induce apoptosis, much remains to be clarified. Given that experimental evidence has long associated alterations of calcium (Ca(2+)) homeostasis to apoptotic cell death, the aim of the present study was to determine the influence of intracellular Ca(2+) changes on apoptosis induced by 7beta-OH and beta-epoxide. Ca(2+) responses in differentiated U937 cells were assessed by epifluorescence video microscopy, using the ratiometric dye fura-2. Over 15-min exposure of differentiated U937 cells to 30 muM of 7beta-OH induced a slow but significant rise in fura-2 ratio. The Ca(2+) channel blocker nifedipine and the chelating agent EGTA blocked the increase in cytoplasmic Ca(2+). Moreover, dihydropyridine (DHP) binding sites identified with BODIPY-FLX-DHP were blocked following pretreatment with nifedipine, indicating that the influx of Ca(2+) occurred through L-type channels. However, following long-term incubation with 7beta-OH, elevated levels of cytoplasmic Ca(2+) were not maintained and nifedipine did not provide protection against apoptotic cell death. Our results indicate that the increase in Ca(2+) may be an initial trigger of 7beta-OH-induced apoptosis, but following chronic exposure to the oxysterol, the influence of Ca(2+) on apoptotic cell death appears to be less significant. In contrast, Ca(2+) did not appear to be involved in beta-epoxide-induced apoptosis.

Effect of serum withdrawal on the contribution of L‐type calcium channels (CaV1.2) to intracellular Ca2+ responses and chemotaxis in cultured human vascular smooth muscle cells

Vascular smooth muscle cell (VSMC) chemotaxis is fundamental to atherosclerosis and intimal hyperplasia. An increase in intracellular Ca2+ [Ca2+]i is an important signal in chemotaxis, but the role of L-type calcium channels (CaV1.2) in this response in human vascular smooth muscle cells (hVSMC) has not been examined. hVSMC were grown from explant cultures of saphenous vein. Confluent hVSMC at passage 3 were studied after culture in medium containing 15% foetal calf serum (FCS) (randomly cycling) or following serum deprivation for up to 7 days. Smooth muscle alpha-actin was measured by immunoblotting and immunofluorescence microscopy. [Ca2+]i was measured using fura 2 fluorimetry. Chemotaxis was measured using a modified Boyden chamber technique and cell attachment to gelatin-coated plates was also quantified. The number and affinity of dihydropyridine-binding sites was assessed using [5-methyl-3H]PN 200-110 binding. In randomly cycling cells, the calcium channel agonist, Bay K 8644a and 100 mM KCl did not affect [Ca2+]i. In addition, the rise in [Ca2+]i induced by platelet-derived growth factor-BB (PDGF) was unaffected by the CaV1.2 antagonists, amlodipine and verapamil. In randomly cycling cells amlodipine did not affect PDGF-induced migration. In serum-deprived cells, smooth muscle alpha-actin was increased and Bay K 8644a and 100 mM KCl increased [Ca2+]i. PDGF-induced rises in [Ca2+]i were also inhibited by amlodipine and verapamil. The ability of Bay K 8644a to increase [Ca2+]i and verapamil to inhibit PDGF-induced rises in [Ca2+]i was evident within 3 days after serum withdrawal. In serum-deprived hVSMC Bay K 8644a induced chemotaxis and amlodipine inhibited PDGF-induced migration. Cell attachment in the presence of PDGF was unaffected by amlodipine in either randomly cycling or serum-deprived hVSMC. Serum withdrawal was associated with a decrease in the maximum number of dihydropyridine-binding sites (B(max)) and a decrease in affinity (K(D)). Serum deprivation of hVSMC results in increased expression of smooth muscle alpha-actin, a marker of more differentiated status, and increased [Ca2+]i responses and chemotaxis mediated by CaV1.2. These observations may have important implications for understanding the therapeutic benefits of calcium channel antagonists in cardiovascular disease.

Abrupt hypokalemia with paralysis from a clinician?s perspective

Monogenic hypokalemic periodic paralysis (HypoPP) is an autosomal dominant genetic disease. HypoPP is more common in males than females and has a reduced penetrance in females. Attacks generally begin during adolescence. They are aggravated by exercise, sleep, stress, alcohol, and meals rich in carbohydrates and salt. The episodes last 324h. Attacks often feature a prodrome of stiffness or heaviness in the lower extremities. Some patients report that they can abort their attacks with mild exercise. Various preventive measures have been suggested, including a low-carbohydrate, low sodium diet, acetazolamide, dichlorphenamide, spironolactone, and triamterene, though none of these are very effective. Acute attacks are generally treated with potassium every 30min until strength returns. In patients with severe episodes who also experience gastrointestinal symptoms, parenteral potassium may be necessary. The electrocardiogram should also be monitored. The paralysis is due to abnormal muscle membrane excitability, commonly arising from mutations in the muscle calcium channel alpha1 subunit gene (CACNA1S) on chromosome 1q3132 or in a smaller number of cases, from a mutation in the skeletal muscle sodium channel SCN4A. The alpha1 subunit of the calcium channel contains the dihydropyridine binding site (receptor) that acts as a pore for conducting calcium ions in the T tubule. The mutation results in a reduction of the calcium current in the T tubule. During attacks, there is an influx of potassium into muscle cells and the cells become electrically refractile. The patients have an increased sensitivity to insulin-related effects on potassium. Exactly how the potassium is shifted from the extracellular to the intracellular space as a result of impaired function of the calcium channel dihydropyridine receptor is not entirely clear.

High-voltage-activated calcium channels in Muller cells acutely isolated from tiger salamander retina.

Muller cells mediate retinal function by stabilizing the ionic environment and signal glial network activity via calcium waves. Using whole-cell patch clamp recording, we describe a high-voltage-activated, slowly inactivating Ca channel current in isolated salamander Muller cells that has unusual pharmacological properties. The Ca channel current has an activation midpoint of approximately -8 mV and an inactivation midpoint of approximately -26 mV in 10 mM Ba2+. The time constant for inactivation is approximately 380 ms at potentials positive to zero. The current is blocked by Cd2+ with an EC50 of <100 nM. nisoldipine (10 microM) blocks approximately 50%, while nifedipine (1 microM), diltiazem (20 microM), and verapamil (50 microM) each block one-third of the current. In contrast to its typical actions, BayK 8644 blocks the current by approximately 25%. Blockers of other Ca channel subtypes were also tested: omega-agatoxin IVA (200 nM) blocked only 13% of the Ca channel current, while omega-conotoxin GVIA (1 microM) blocked 84% of the current. Immnohistochemistry supported the presence of alpha1A, alpha1B, alpha1C, and alpha1D Ca channel subunits. Mapping of dihydropyridine-binding sites with DM-BODIPY revealed a distribution of channels over the entire membrane of the Muller cell with a higher density at the apical region. Overall, these observations suggest either the presence of a mix of L- and N-type Ca channels or a single, unconventional HVA Ca channel subtype sharing L- and N-type Ca channel characteristics.

Disruption of intestinal motility by a calcium channel-stimulating autoantibody in type 1 diabetes

Background & Aims : Autonomic neuropathy, including gastrointestinal dysfunction, is a common complication of type 1 diabetes; however, its cause is uncertain. This study aimed to test whether functional autoantibodies cause the gastrointestinal dysfunction. Methods : We used isolated mouse colon undergoing colonic migrating motor complex (MMC) activity to test for autoantibodies that disrupt colonic motility. Purified immunoglobulin G (IgG) from patients with type 1 diabetes or from controls was tested either in vitro or after passive transfer. Pharmacological studies of the interaction between the IgG and L-type calcium channel activator (Bay K8644) and inhibitors (nicardipine and verapamil) were performed. The effect of IgG on nerve-evoked contraction of the vas deferens longitudinal smooth muscle was also assessed. Results : MMC activity was disrupted by IgG (0.2 mg/mL) from 8 of 16 patients with type 1 diabetes but not by control IgG. Passive transfer of diabetic IgG to mice also disrupted MMCs, showing access to the antigen in vivo. The acute effect of the autoantibody was mimicked by the dihydropyridine agonist, Bay K8644 (2–10 nmol/L), and both Bay K8644 and the autoantibody competitively inhibited the effect on MMC contraction of the dihydropyridine antagonist, nicardipine. Diabetic IgG, but not control IgG, altered the nerve-evoked contractile activity of vas deferens smooth muscle effects mimicked by Bay K8644. Conclusions : A novel functional autoantibody that activates smooth muscle L-type calcium channels at the dihydropyridine binding site is produced specifically by patients with type 1 diabetes and may mediate gastrointestinal and autonomic dysfunction in these patients.