Channel Inhibitor Verapamil Research Articles

The cystic fibrosis treatment Trikafta affects the growth, viability, and cell wall of Aspergillus fumigatus biofilms.

PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.

Personalized medicine in the dish to prevent calcium leak associated with short-coupled polymorphic ventricular tachycardia in patient-derived cardiomyocytes

BackgroundPolymorphic ventricular tachycardia (PMVT) is a rare genetic disease associated with structurally normal hearts which in 8% of cases can lead to sudden cardiac death, typically exercise-induced. We previously showed a link between the RyR2-H29D mutation and a clinical phenotype of short-coupled PMVT at rest using patient-specific hiPSC-derived cardiomyocytes (hiPSC-CMs). In the present study, we evaluated the effects of clinical and experimental anti-arrhythmic drugs on the intracellular Ca2+ handling, contractile and molecular properties in PMVT hiPSC-CMs in order to model a personalized medicine approach in vitro.MethodsPreviously, a blood sample from a patient carrying the RyR2-H29D mutation was collected and reprogrammed into several clones of RyR2-H29D hiPSCs, and in addition we generated an isogenic control by reverting the RyR2-H29D mutation using CRIPSR/Cas9 technology. Here, we tested 4 drugs with anti-arrhythmic properties: propranolol, verapamil, flecainide, and the Rycal S107. We performed fluorescence confocal microscopy, video-image-based analyses and biochemical analyses to investigate the impact of these drugs on the functional and molecular features of the PMVT RyR2-H29D hiPSC-CMs.ResultsThe voltage-dependent Ca2+ channel inhibitor verapamil did not prevent the aberrant release of sarcoplasmic reticulum (SR) Ca2+ in the RyR2-H29D hiPSC-CMs, whereas it was prevented by S107, flecainide or propranolol. Cardiac tissue comprised of RyR2-H29D hiPSC-CMs exhibited aberrant contractile properties that were largely prevented by S107, flecainide and propranolol. These 3 drugs also recovered synchronous contraction in RyR2-H29D cardiac tissue, while verapamil did not. At the biochemical level, S107 was the only drug able to restore calstabin2 binding to RyR2 as observed in the isogenic control.ConclusionsBy testing 4 drugs on patient-specific PMVT hiPSC-CMs, we concluded that S107 and flecainide are the most potent molecules in terms of preventing the abnormal SR Ca2+ release and contractile properties in RyR2-H29D hiPSC-CMs, whereas the effect of propranolol is partial, and verapamil appears ineffective. In contrast with the 3 other drugs, S107 was able to prevent a major post-translational modification of RyR2-H29D mutant channels, the loss of calstabin2 binding to RyR2. Using patient-specific hiPSC and CRISPR/Cas9 technologies, we showed that S107 is the most efficient in vitro candidate for treating the short-coupled PMVT at rest.

Pharmacological Studies on the Ca2+ Influx Pathways in Platelet-Activating Factor (PAF)-Induced Mouse Urinary Bladder Smooth Muscle Contraction.

Platelet-activating factor (PAF) not only acts as a mediator of platelet aggregation, inflammation, and allergy responses but also as a constrictor of various smooth muscle (SM) tissues, including gastrointestinal, tracheal/bronchial, and pregnancy uterine SMs. Previously, we reported that PAF induces basal tension increase (BTI) and oscillatory contraction (OC) in mouse urinary bladder SM (UBSM). In this study, we examined the Ca2+ influx pathways involved in PAF-induced BTI and OC in the mouse UBSM. PAF (10-6 M) induced BTI and OC in mouse UBSM. However, the PAF-induced BTI and OC were completely suppressed by extracellular Ca2+ removal. PAF-induced BTI and OC frequencies were markedly suppressed by voltage-dependent Ca2+ channel (VDCC) inhibitors (verapamil (10-5 M), diltiazem (10-5 M), and nifedipine (10-7 M)). However, these VDCC inhibitors had a minor effect on the PAF-induced OC amplitude. The PAF-induced OC amplitude in the presence of verapamil (10-5 M) was strongly suppressed by SKF-96365 (3 × 10-5 M), an inhibitor of receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC), but not by LOE-908 (3 × 10-5 M) (an inhibitor of ROCC). Overall, PAF-induced BTI and OC in mouse UBSM depend on Ca2+ influx and the main Ca2+ influx pathways in PAF-induced BTI and OC may be VDCC and SOCC. Of note, VDCC may be involved in PAF-induced BTI and OC frequency, and SOCC might be involved in PAF-induced OC amplitude.

GhIQD10 interacts with GhCaM7 to control cotton fiber elongation via calcium signaling

IQ67-domain (IQD) proteins function in plant defense and in organ development. The mechanisms by which they influence cotton fiber development are unknown. In the present study, GhIQD10 was expressed mainly in the transition period of cotton fiber development, and GhIQD10-overexpression lines showed shorter fibers. GhIQD10 interacted with GhCaM7 and the interaction was inhibited by Ca2+. In in vitro ovule culture, Ca2+ rescued the shorter-fiber phenotype of GhIQD10-overexpression lines, which were insensitive to the Ca2+ channel inhibitor verapamil and the Ca2+ pool release channel blocker 2-aminoethoxydiphenyl borate. We conclude that GhIQD10 affects cotton fiber elongation via Ca2+ signaling by interacting with GhCaM7. Brassinosteroid (BR) biosynthesis and signaling genes were up-regulated in GhIQD10-overexpression lines. Fiber development in these lines was not affected by epibrassinolide or the BR biosynthesis inhibitor brassinozole, indicating that the influence of GhIQD10 on fiber elongation was not associated with BR.

Salt stress-induced H2O2 and Ca2+ mediate K+/Na+ homeostasis in Pyropia haitanensis

Maintaining K+/Na+ homeostasis and redox homeostasis is crucial for the tolerance of P. haitanensis to hypersalinity. However, the precise link between the signaling role of reactive oxygen species (ROS) and K+/Na+ homeostasis remains poorly characterized. In this study, we analyze hydrogen peroxide (H2O2) production and flux as well as H2O2 effects on K+, Na+, and Ca2+ transport in P. haitanensis under hypersaline condition. An exposure to hypersaline stress (110‰, 15 min) rapidly increased the H2O2 content and efflux in P. haitanensis cells, which was counteracted by the rapid increase in superoxide dismutase activity and activation of defense responses. The enhanced Na+ efflux and Ca2+ influx induced by salt stress were substantially suppressed by an NADPH oxidase inhibitor (DPI) or an ROS scavenger (DMTU). Additionally, Na+ efflux decreased in response to a plasma membrane Ca2+-permeable channel inhibitor (verapamil). This suggested that NADPH oxidase-mediated H2O2 production may promote Na+ efflux via the Ca2+-dependent Na+/H+ antiporter system in salt-stressed P. haitanensis thalli. Moreover, salt-induced H2O2 accumulation also enhanced K+ efflux, which was alleviated by exogenous Ca2+. H2O2 and Ca2+ may independently mediate K+ homeostasis in P. haitanensis. H2O2-induced K+ leakage may induce cells to switch from normal metabolic activities to those associated with adaptation and repair. The present results provide new insight for clarifying the relationship between salt-induced ROS signaling and ion homeostasis in intertidal seaweed species.

Ion channel inhibition against COVID-19: A novel target for clinical investigation

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease (COVID-19), has been classified by the World Health Organization (WHO) as an ongoing pandemic. Owing to the global emergency, there is an unmet need to identify effective and scalable therapeutic options to attenuate the early stages of virus infection. SARS-CoV-2 has different stages of the infection cycle. The first phase is characterized by infection and replication of the virus within the host cells. The last phase occurs with cytokine storm leading to cellular apoptosis [1]. We posit the rationale for ion channel inhibition as a novel therapeutic target to counteract SARS-CoV-2 infection and replication. Within this framework, we discuss the potential clinical role of the ion channel inhibitors amiodarone and verapamil against COVID-19 that deserves clinical investigations.

Identification of potassium and calcium channel inhibitors as modulators of polyomavirus endosomal trafficking

During virus entry, members of the Polyomaviridae transit the endolysosomal network en route to the endoplasmic reticulum (ER), from which degraded capsids escape into the cytoplasm and enter the nucleus. Emerging evidence suggests that viruses require both endosomal acidification and the correct ionic balance of K+ and Ca2+ ions in endosomes for correct virus trafficking and genome release. Here, using two polyomaviruses with different capsid architectures, namely Simian virus 40 (SV40) and Merkel cell polyomavirus (MCPyV), we describe methods to rapidly quantify virus infection using IncuCyte ZOOM imaging analysis, and use this system to investigate the role of both K+ and Ca2+ channels during the early stages of virus entry. Using broad spectrum blockers of both K+ and Ca2+ channels to specifically target host cell ion channel functionality, we show that MCPyV, but not SV40 can be inhibited by K+ channel modulators, whilst both viruses are restricted by the broad spectrum Ca2+ channel inhibitor verapamil. Using a panel of more specific Ca2+ blockers, we show that both MCPyV and SV40 are dependent on the activity of two-pore Ca2+ channels (TPCs), as the TPC-specific blocker tetrandrine prevented capsid disassembly and nuclear transport required for virus entry. We therefore reveal a novel target to restrict the entry of polyomaviruses, which given the known role of TPCs during endolysosomal-ER fusion, is likely to be applicable to other viruses that transit this pathway.

P2575Excess and deficiency of serotonin in embryogenesis affects the contractility of the right ventricle myocardium

Abstract Background Congenital heart disease (CHD) is one of the most common pathologies among congenital malformations in children. The incidence of CHD is currently more than 30% of all malformations. The most frequent and severe complication of CHD is pulmonary hypertension (PH). PH worsens the CHD prognosis, affects the survival rate of children, and increases the risk of disability and mortality. Experimental studies demonstrate a high correlation between the PH degree, concentrations of serotonin and its metabolites in the blood. The purpose of our study was to investigate the effect of excess and lack of serotonin in the embryonic period on the functioning of the heart in early postnatal ontogenesis. Methods Study was conducted according to “Principles of laboratory animal care”. The study was carried out on pregnant female Wistar rats and their offspring at the age of 14 days. Myocardial contractility in vitro was studied on myocardial strips of right ventricle. The amplitude-time characteristics of the contraction were calculated by the method of S. Laer 1998. The following series of experiments were conducted: 1. Pregnant females, starting from the 11th day of pregnancy for 10 days were injected intraperitoneally: group 1 - serotonin synthesis inhibitor (p-chlorophenylalanine) at a dose of 100 mg/kg; group 2 - selective serotonin reuptake inhibitor (fluoxetine) at a dose of 50 mg/kg; group 3 - (control group) - saline solution. Inotropic myocardial function was studied in the offsprings of each group at the age of 14 days. The reactions of the contraction of myocardial stripes of rat pups were evaluated in 2 batches: first on epinephrine and then on the calcium channel inhibitor (verapamil) at concentrations of 2.5 μM and in the reverse order. Results Our experiment demonstrates that the reaction of inotropic function of the right ventricle on epinephrine in 2 weeks of age rats is not affected by changes in serotonin metabolism in the embryonic period of development. Against the background of a pronounced positive effect from the use of epinephrine, verapamil was used to inhibit calcium channels. We observed a significant reduction in the force of contraction by 83.41±3.14% in the control group, by 71.42±2.95% in the group with an excess of serotonin and by 63.97±7.93% in group with serotonin deficiency. In the next series of experiments, verapamil was first applied, resulting the decrease of contraction strength by 78.58±1.71% in the control group, by 80.81±7.33% in the myocardium of rats with an excess of serotonin and by 67.70±4, 66% in the group with serotonin deficiency. Subsequent exposure of epinephrine led to an increase in myocardial contractility equally in all groups at 132–135%. Conclusion Violation of serotonin metabolism during embryogenesis affects the regulation of contractility (force of contraction) of the right ventricle. Acknowledgement/Funding This work was supported by the Ministry of Science and Education of Republic of Kazakhstan [grant number AP05136034]

Regulation mechanism of ρ-Hydroxybenzoic acid on grapevine root secretion characteristics

ρ-Hydroxybenzoic acid (4-HBA) is a major autotoxin that can regulate grapevine secretion characteristics and cause replant disease. In our study, we aimed to investigate the involvement of various signal transduction pathways (calcium, Ca2+ and reactive oxygen species) in 4-HBA-induced grapevine root secretions. The roots of grapevine seedlings were treated with specific calcium channel inhibitor (Verapamil, Vp), and inhibitors of superoxide dismutase (SOD) (diethyldithiocarbamate, DETC) and NADPH oxidase (diphenylene iodonium, DPI). The distribution of Ca2+ in the root tip tissues was observed, and the phenylalanine ammonia-lyase (PAL) activity and phenolic acid content (ρ-Hydroxybenzoic acid and salicylic acid) in the secretions were measured. The results showed that 1000 μM of 4-HBA had a significant allelopathic effect on seedling growth, leading to Ca2+ flow from the cell wall to the cytoplasm, and that Vp hindered this process. The treatments with Vp and 4-HBA or DETC and 4-HBA decreased the PAL activity of roots and 4-HBA content in root exudates compared to the 4-HBA treatment. The salicylic acid (SA) content in root exudates decreased after co-treating with Vp and 4-HBA, DPI and 4-HBA. This result indicates that Ca2+ and SOD play major roles in 4-HBA induced root secretions.

Telemetered common marmosets is useful for the assessment of electrocardiogram parameters changes induced by multiple cardiac ion channel inhibitors.

The objective of this study is to assess the response of telemetered common marmosets to multiple cardiac ion channel inhibitors and to clarify the usefulness of this animal model in evaluating the effects of drug candidates on electrocardiogram (ECG). Six multiple cardiac ion channel inhibitors (sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine) were orally administered to telemetered common marmosets and changes in QTc, PR interval and QRS duration were evaluated. Drugs plasma levels were determined to compare the sensitivity in common marmosets to that in humans. QTc prolongation was observed in the marmosets dosed with sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine. PR prolongation was noted after flecainide and verapamil administration, and QRS widening occurred following treatment with flecainide and quinidine. Drugs plasma levels associated with ECG changes in marmosets were similar to those in humans, except for verapamil-induced QTc prolongation. Verapamil-induced change is suggested due to body temperature decrease. These results indicate that telemetered common marmoset is a useful animal for evaluation of the ECG effects of multiple cardiac ion channel inhibitors and the influence of body temperature change should be considered in the assessment.

Hypotonic stress promotes ATP release, reactive oxygen species production and cell proliferation via TRPV4 activation in rheumatoid arthritis rat synovial fibroblasts

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune-disease with complex and unclear etiology. Hypotonicity of synovial fluid is a typical characteristic of RA, which may play pivotal roles in RA pathogenesis. In this work, we studied the responses of RA synovial fibroblasts to hypotonic stress in vitro and further explored the underlying mechanisms. Data showed that hyposmotic solutions significantly triggered increases in cytosolic calcium concentration ([Ca2+]c) of synoviocytes. Subsequently, it caused rapid release of ATP, as well as remarkable production of intracellular reactive oxygen species (ROS). Meanwhile, hypotonic stimulus promoted the proliferation of synovial fibroblasts. These effects were almost abolished by calcium-free buffer and significantly inhibited by gadolinium (III) chloride (a mechanosensitive Ca2+ channel blocker) and ruthenium red (a transient receptor potential vanilloid 4 (TRPV4) blocker). 4α-phorbol 12,13-didecanoate, a specific agonist of TRPV4, also mimicked hypotonic shock-induced responses shown above. In contrast, voltage-gated channel inhibitors verapamil and nifedipine had little influences on these responses. Furthermore, RT-PCR and western blotting evidently detected TRPV4 expression at mRNA and protein level in isolated synoviocytes. Taken together, our results indicated that hypotonic stimulus resulted in ATP release, ROS production, and cell proliferation depending on Ca2+ entry through activation of TRPV4 channel in synoviocytes.

Abstract P196: Chemerin Activates Procontractile Pathways in Isolated Arteries

In the vasculature, the adipokine chemerin is present in perivascular adipose tissue and causes arterial contraction through activation of the heptahelical receptor ChemR23. We presently test the hypothesis that ChemR23 is linked to procontractile signaling pathways in isolated arteries. The isolated, endothelium-denuded thoracic aorta of both male and female rats was used for measurement of isometric contraction and biochemical analyses. The peptide analog chemerin-9 (1 nm -3 μM) caused concentration-dependent contraction in both isolated aorta from male and female, with a maximum of ~80% of a phenylephrine (10 μM) contraction. The L type voltage dependent calcium channel inhibitors verapamil (1 μM) and nifedipine (1 μM), as well as the protein kinase C (PKC) inhibitor chelerythrine chloride (10 μM), abolished chemerin-9 induced contraction relative to vehicle-incubated tissues (male and female). Next most effective in inhibiting chemerin-9-induced contraction were the Rho Kinase inhibitors Y27632 (1 μM) and fasudil (1 μM), both effective in male and female aorta. The p38 MAPK inhibitor SB203850 (10 μM) and phosphoinositide-3-kinase inhibitor LY294002 (10 μM) were significantly less effective in reducing chemerin-9-induced contraction in both sexes, reducing maximum contraction in male and female by less than 50%. Finally, the phospholipase C inhibitor U73122 (5 μM) and Erk MAPK inhibitor PD098059 (1 μM) did not shift or reduce chemerin-9-induced contraction compared to control. Western analyses of chemerin-9-contracted tissues validated activation of phosphoinositide-3-kinase and lack of activation of the Erk MAPKs. These data are the first to associate ChemR23 with L type channel and Rho Kinase activation, and support the hypothesis that ChemR23 taps into some but not all of the well-recognized contractile pathways. This is important knowledge for understanding the mechanisms by which chemerin could support obesity-associated hypertension

ASSA13-03-46 The Effects of Verapamil On SK2 Channel In Myocardium Cells From Human Chronic Atrial Fibrillation

<h3>Objective</h3> Small conductance calcium-activated potassium channel (SK channel) plays an important role in heart. Four subtype of SK channel are classified according to their sensitive to apamin. Electrophysiological properties of SK2 channel are voltage-independent as well as solely activated by changes in intracellular calcium concentration. This study investigated the alteration of SK2 channel currents in cAF patients. Moreover, L-type calcium channel inhibitor verapamil were adopted to observe the change of SK2 channel. We plan to explore the functional relationship between L-type calcium channel and SK2 channels. <h3>Methods</h3> Single human cardiac myocyte was isolated by two-step enzyme digestion. The whole-cell patch clamp technique was used to record the SK2 current in isolated cardiomyocytes from patients with siuns rhythm (SR) or chronic atrial fibrillation (cAF). Verapamil (10 μmol/L) and apamin (100 nmol/L) were added into bath solution respectively to observe the change of current. <h3>Results</h3> The current density (pA/pF) of SK2 channel as well as the ration in the integrated inward currents were up-regulated significantly in cAF groups. At wide range of test potentials, L-type calcium channel inhibitor verapamil reduced SK2 channel currents in cardiomyocytes from patients with SR or cAF. Compared with control group, the apamin-sensitive currents density of the verapamil group showed a significant decrease in human atrial myocytes. It indicates that part of SK2 current can be inhibited by verapamil. <h3>Conclusions</h3> It suggested that the up-regulation of SK2 currents underlies the occurrence and maintenance of cAF. There may be functionally coupling between SK2 channels and L-type calcium channels in human myocardium cells. The regulation of L-type calcium channel involves the remodelling of SK2 channels in the atrial myocytes from SR and cAF patients. This work was supported by the National Natural Science Foundation of China (No. 30870903) and the Natural Science Foundation of Luzhou.

Calcium handling and ventricular tachyarrhythmias

Pharmacologic modification of cellular calcium handling recently moved into focus as an alternative for prevention and treatment of ventricular tachyarrhythmias. Calcium overload and spontaneous calcium release from the sarcoplasmatic reticulum are regarded as possible initiations of early and delayed afterdepolarization thereby triggering ventricular arrhythmias. In chronic heart failure, calcium overload is more likely to occur compared with healthy hearts, which is one explantation for the increased vulnerability in this condition. L-type calcium channel, sodium-calcium-exchanger (NCX), and ryanodine receptor are crucial for calcium homeostasis and therefore represent potential targets for antiarrhythmic drug therapy. Experimental studies have proven beneficial effects for all these three mechanisms in prevention and suppression of tachyarrhythmias. However, clinical data is mainly available for the L-type calcium channel inhibitor verapamil. Therefore, it is still a long way to clinical employment of drugs modifying cellular calcium handling for antiarrhythmic therapy.

Effects of calcium channel inhibitors on the hypothermic response to oxotremorine in normo and hypercholinergic rats

The Flinders Sensitive Line of rats (FSL) has been selectively bred to have increased sensitivity to cholinergic drugs. Typically, these rats react with twice as great a hypothermic effect to muscarinic agonists such as oxotremorine, as do similarly bred Flinders Resistant Line rats (FRL). We compared the effects of three chemically different calcium channel inhibitors (diltiazem, nicardipine and verapamil) on the hypothermia induced in FRL and FSL rats by oxotremorine (0.2 mg kg-1 s.c.). Each drug was injected i.p. in a dose of 20 mumol kg-1 30 min before oxotremorine. Methylatropine (2 mg kg-1 s.c.) was administered 15 min before oxotremorine to block the peripheral effects of the agonist. The hypothermic effect of oxotremorine in FSL rats was antagonized by nicardipine and diltiazem. In contrast, verapamil failed to influence the hypothermic response in FSL rats. Verapamil significantly (P less than 0.05) augmented oxotremorine hypothermia in FRL rats. Diltiazem and nicardipine were without effect on oxotremorine-induced hypothermia in FRL rats. There were no significant changes in temperature in separate groups of FRL and FSL rats treated with calcium channel inhibitors alone.

Involvement of sarcoplasmic reticulum in changing intracellular calcium due to Na+/K+-ATPase inhibition in cardiomyocytes

Earlier studies have demonstrated that ouabain-induced increase in [Ca2+]i, as a consequence of sarcolemma (SL) Na+/K+-ATPase inhibition, is associated with activation of both the SL Na+/Ca2+ exchanger and SL Ca2+ channels. In view of the importance of sarcoplasmic reticulum (SR) in the regulation of [Ca2+]i, this study examined the role of SR in ouabain-induced increase in [Ca2+]i in both quiescent and KCl-depolarized cardiomyocytes. For this purpose, adult rat cardiomyocytes were loaded with fura-2 and ouabain-induced changes in [Ca2+]i were monitored upon treatment with or without different agents that are known to influence Ca2+ handling by the intracellular organelles. Ouabain not only increased the basal [Ca2+]i and augmented KCl-induced increase in [Ca2+]i but also produced similar effects on the ATP-induced increase in [Ca2+]i. Treatments of cardiomyocytes with caffeine, ryanodine, or cyclopiazonic acid, which affect SR Ca2+ stores, attenuated the ouabain-induced increase in basal Ca2+ as well as augmentation of the KCl response. Both ryanodine and cyclopiazonic acid produced additional effects, when used in combination with a SL Ca2+ channel inhibitor (verapamil), but not with a Na+/Ca2+ exchange inhibitor (KB-R7943). Inhibitors of Ca2+/calmodulin kinase, protein kinase A, and inositol-3-phosphate receptors were also observed to depress the ouabain-induced increase in [Ca2+]i in cardiomyocytes. On the other hand, mitochondrial Ca2+ transport inhibitors did not exert any effect on the ouabain-induced alterations in [Ca2+]i in cardiomyocytes. Furthermore, ouabain did not show any direct effect on the Ca2+ uptake and Ca2+ release activities of SR or mitochondria. These results suggest an indirect involvement of SR Ca2+ stores in the ouabain-induced increase in [Ca2+]i in cardiomyocytes and indicate the participation of both Ca2+-induced Ca2+ release and regulatory mechanisms in this action.

Verapamil inhibits calcification and matrix vesicle activity of bovine vascular smooth muscle cells

Calcium channel activity in vascular smooth muscle cells is a critical component during vascular calcification and formation of matrix vesicles. Here, we examined whether the blockade of L-type calcium channels inhibits these functions. Bovine vascular smooth muscle cells or rat aorta organ cultures were incubated in media known to promote calcification and treated with the L-type calcium channel inhibitors verapamil, nifedipine, or nimodipine. The phenylalkylamine, verapamil, significantly decreased calcification of the vascular smooth muscle cells and rat aorta, in a dose-dependent manner, whereas the dihydropyridines, nifedipine and nimodipine, had no effect. Furthermore, verapamil, but not nifedipine, significantly decreased the alkaline phosphatase activity of bovine vascular smooth muscle cells. Verapamil pretreatment of the cells also inhibited matrix vesicle alkaline phosphatase activity and reduced the ability of these matrix vesicles to subsequently calcify on a type I collagen extracellular matrix scaffold. As L-type channels are blocked by verapamil and dihydropyridines, we suggest that verapamil inhibits vascular smooth muscle mineralization and matrix vesicle activity by mechanisms other than the simple blockade of this calcium channel activity.

Mediation of EDHF-induced reduction of smooth muscle [Ca(2+)](i) and arteriolar dilation by K(+) channels, 5,6-EET, and gap junctions.

To characterize the role of K(+) channels, the cytochrome P-450 (CYP) metabolite 5,6-EET, and gap junctions in modulation of arteriolar myogenic tone by a non-nitric oxide nonprostaglandin mediator, termed "endothelium-dependent hyperpolarizing factor" (EDHF), released to acetylcholine (ACh) in skeletal muscle arterioles. In isolated rat gracilis arterioles, simultaneous changes in smooth muscle (aSM) [Ca(2+)](i) (assessed by changes in fura-2 ratiometric signal, R(Ca)) and diameter were measured in response to ACh in the presence of indomethacin and L-NAME. ACh, the K(ATP) channel opener pinacidil, and the Ca(2+) channel inhibitor verapamil elicited comparable decreases in aSM [Ca(2+)](i) (max.: -32 +/- 3%, 29 +/- 3%, and -30 +/- 3%, respectively) and arteriolar dilations (max.: 90 +/- 4%, 96 +/- 2%, and 95 +/- 2%, respectively). ACh-induced responses were inhibited by KCl-depolarization, K(Ca) channel blockers (TEA, charybdotoxin), or gap junction inhibitors (18alpha-glycyrrhetinic acid, hyperosmolar sucrose). The K(ATP) channel inhibitor glibenclamide, the K(IR) channel inhibitor barium chloride, or the CYP inhibitor 17-octadecynoic acid (ODYA) were without effect. The putative EDHF analogue 5,6-EET elicited constrictions in the presence of the endothelium that could be prevented by indomethacin or a TxA(2) receptor antagonist, whereas in the absence of the endothelium, EDHF elicited only small, charybdotoxin-insensitive decreases in aSM R(Ca) and dilations (max.: -8 +/- 2% and 27 +/- 4%, respectively). In skeletal muscle arterioles, EDHF 1) substantially and rapidly reduces myogenic tone by decreasing aSM [Ca(2+)](i) via opening K(Ca) channels, 2) it is unlikely to be 5,6-EET or other CYP metabolites, but 3) requires functional gap junctions.

Calcium Channel Inhibitor, Verapamil, Inhibits the Voltage-Dependent K+ Channels in Rabbit Coronary Smooth Muscle Cells

We investigated the effect of the phenylalkylamine Ca(2+) channel inhibitor verapamil on voltage-dependent K(+) (Kv) channels in rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Verapamil reduced the Kv current amplitude in a concentration-dependent manner. The apparent K(d) value for Kv channel inhibition was 0.82 microM. Although verapamil had no effect on the activation kinetics, it accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation by verapamil were 2.20+/-0.02 microM(-1) s(-1), and 1.79+/-0.26 s(-1), respectively. The steady-state activation and inactivation curves were unaffected by verapamil. The application of train pulses increased the verapamil-induced Kv channel inhibition. Furthermore, verapamil increased the recovery time constant, suggesting that the inhibitory effect of this agent was use-dependent. The inhibitory effect of verapamil was not affected by intracellular and extracellular Ca(2+)-free conditions. Another Ca(2+) channel inhibitor, nifedipine (10 microM) did not affect the Kv current, and did not alter the inhibitory effect of verapamil. Based on these results, we concluded that verapamil inhibited Kv current in a state-, time-, and use-dependent manner, independent of Ca(2+) channel inhibition.

Slow Channel Inhibitor Verapamil in the Treatment of Arrhythmia

Slow Channel Inhibitor Verapamil in the Treatment of Arrhythmia