Blockade Of L-type Ca2 Research Articles

Blockade of L-Type Ca2+ Channel Activity Alleviates Oligodendrocyte Pathology following Brain Injury in Male Rats.

A growing body of studies suggests that Ca2+ signaling controls a variety of biological processes in brain elements. Activation of L-type voltage-operated Ca2+ channels (VOCCs) plays a role in the development of oligodendrocyte (OL) lineage loss, and indicates that the blocking of these channels may be an effective way to inhibit OL lineage cell loss. For this study, 10.5-day-old male Sprague-Dawley rats were used to generate cerebellar tissue slices. The slice tissues were cultured and randomly allocated to one of four groups (six each) and treated as follows: Group I, (sham control); Group II, 0.1% dimethyl sulfoxide (DMSO) only (vehicle control); Group III, injury (INJ); Group IV, (INJ and treatment with NIF). The injury was simulated by exposing the slice tissues to 20 min of oxygen-glucose deprivation (OGD). At 3 days post-treatment, the survival, apoptosis, and proliferation of the OL lineages were measured and compared. Results: In the INJ group, there was a decrease in mature myelin basic protein+ OLs (MBP+ OLs) and their precursors, NG2+ OPCs (Nerve-glia antigen 2+ oligodendrocyte precursor cell), compared with controls. A significant elevation was observed in the NG2+ OPCs and apoptotic MBP+ OLs as confirmed by a TUNEL assay. However, the cell proliferation rate was decreased in NG2+ OPCs. NIF increased OL survival as measured by apoptosis rate in both OL lineages and preserved the rate of proliferation in the NG2+ OPCs. Conclusions: Activation of L-type VOCCs may contribute to OL pathology in association with reduced mitosis of OPCs following brain injury as a strategy to treat demyelinating diseases.

Ca2+ channel blockade reduces cocaine\u2019s vasoconstriction and neurotoxicity in the prefrontal cortex

Cocaine profoundly affects both cerebral blood vessels and neuronal activity in the brain. The vasoconstrictive effects of cocaine, concurrently with its effects on neuronal [Ca2+]i accumulation are likely to jeopardize neuronal tissue that in the prefrontal cortex (PFC) could contribute to impaired self-regulation and compulsive cocaine consumption. Here we used optical imaging to study the cerebrovascular and neuronal effects of acute cocaine (1 mg/kg i.v.) and to examine whether selective blockade of L-type Ca2+ channels by Nifedipine (NIF) (0.5 mg/kg i.v.) would alleviate cocaine’s effects on hemodynamics (measured with cerebral blood volume, HbT), oxygenation (measured with oxygenated hemoglobin, HbO2) and neuronal [Ca2+]i, which were concomitantly measured in the PFC of naive rats. Our results show that in the PFC acute cocaine significantly reduced flow delivery (HbT), increased neuronal [Ca2+]i accumulation and profoundly reduced tissue oxygenation (HbO2) and these effects were significantly attenuated by NIF pretreatment. They also show that cocaine-induced vasoconstriction is distinct from its increase of neuronal [Ca2+]i accumulation though both of them contribute to hypoxemia and both effects were attenuated by NIF. These results provide evidence that blockade of voltage-gated L-type Ca2+ channels might be beneficial in preventing vasoconstriction and neurotoxic effects of cocaine and give support for further clinical investigations to determine their value in reducing cocaine’s neurotoxicity in cocaine use disorders.

Role of Ca V 3.1 Channels in Myogenic Tone and Blood Pressure Regulation in Mouse Mesenteric Arteries

The myogenic response is a fundamental mechanism whereby intraluminal pressure elicits arterial constriction pursuant to maintenance of tissue perfusion. Smooth muscle [Ca2+] is a key determinant of constriction, a process intimately tied to L-type (CaV1.2) Ca2+ channel activity. While important, other Ca2+ channels are expressed and could contribute to pressure regulation within defined voltage ranges. In this regard, this study examined the role of T-type Ca2+ channels, using mesenteric arterioles from wild type (WT) and CaV3.1-/- mice. Using pressure myography, experiments first revealed that CaV3.1-/- arterioles displayed decidedly less myogenic tone than WT, particular at lower pressures (20 – 60 mmHg) where membrane potential is hyperpolarized. We next showed with rapid Ca2+ imaging that reduced myogenic tone in CaV3.1-/- arteries resulted from diminished Ca2+ waves generation, asynchronous events dependent upon Ca2+ release from the sarcoplasmic reticulum. In contrast, blockade of L-type Ca2+ channels with nifedipine (200 nM) had no effect on Ca2+ waves, akin to previous findings. The proximity ligation assay confirmed a close association (<40 nm) between IP3R and CaV3.1, in WT and not knockout arterioles. In whole animals, this diminishment of myogenic tone coincided with a reduction in systemic blood pressure at rest. Intriguingly, this hypotension response did not impact blood pressure response to chronic AngII infusion. Taken together, this data suggests that CaV3.1 channels are contributors to tone development at low intraluminal pressures and that this effect is indirect through a Ca2+-induced Ca2+ release mechanism involving the sarcoplasmic reticulum. Further experiments are being extended to define the role of CaV3.1 in other pathophysiological models.

Berberine Mediated Positive Inotropic Effects on Rat Hearts via a Ca2+-Dependent Mechanism.

Previous studies showed that berberine, an alkaloid from Coptis Chinensis Franch, might exert a positive inotropic effect on the heart. However, the underlying mechanisms were unclear. Here, we reported that berberine at 10–20 µM increased the left ventricular (LV) developed pressure and the maximal rate of the pressure rising, and it increased the maximal rate of the pressure descending at 20 µM in Langendorff-perfused isolated rat hearts. These effects diminished with the concentration of berberine increasing to 50 µM. In the concentration range of 50–300 µM, berberine increased the isometric tension of isolated left ventricular muscle (LVM) strips with or without electrical stimulations, and it (30–300 µM) also increased the intracellular Ca2+ level in the isolated LV myocytes. The removal of extracellular Ca2+ hindered the berberine-induced increases in the tension of LVM strips and the intracellular Ca2+ level of LV myocytes. These suggested that berberine might exert its positive inotropic effects via enhancing Ca2+ influx. The blockade of L-type Ca2+ channels (LTCCs) with nifedipine significantly attenuated 300 μM berberine-induced tension increase in LVM strips but not the increase in the intracellular Ca2+ level. Berberine (300 μM) further increased the LVM tension following the treatment with the LTCC opener FPL-64716 (10 μM), indicating an LTCC-independent effect of berberine. Lowering extracellular Na+ attenuated the berberine-induced increases in both the tension of LVM strips and the intracellular Ca2+ level of LV myocytes. In conclusion, berberine might exert a positive inotropic effect on the isolated rat heart by enhancing the Ca2+ influx in LV myocytes; these were extracellular Na+-dependent.

Blockade of L-type Ca2+ channel attenuates doxorubicin-induced cardiomyopathy via suppression of CaMKII-NF-\u03baB pathway

Ca2+/calmodulin-dependent protein kinase II (CaMKII) and nuclear factor-kappa B (NF-κB) play crucial roles in pathogenesis of doxorubicin (DOX)-induced cardiomyopathy. Their activities are regulated by intracellular Ca2+. We hypothesized that blockade of L-type Ca2+ channel (LTCC) could attenuate DOX-induced cardiomyopathy by regulating CaMKII and NF-κB. DOX activated CaMKII and NF-κB through their phosphorylation and increased cleaved caspase 3 in cardiomyocytes. Pharmacological blockade or gene knockdown of LTCC by nifedipine or small interfering RNA, respectively, suppressed DOX-induced phosphorylation of CaMKII and NF-κB and apoptosis in cardiomyocytes, accompanied by decreasing intracellular Ca2+ concentration. Autocamtide 2-related inhibitory peptide (AIP), a selective CaMKII inhibitor, inhibited DOX-induced phosphorylation of NF-κB and cardiomyocyte apoptosis. Inhibition of NF-κB activity by ammonium pyrrolidinedithiocarbamate (PDTC) suppressed DOX-induced cardiomyocyte apoptosis. DOX-treatment (18 mg/kg via intravenous 3 injections over 1 week) increased phosphorylation of CaMKII and NF-κB in mouse hearts. Nifedipine (10 mg/kg/day) significantly suppressed DOX-induced phosphorylation of CaMKII and NF-κB and cardiomyocyte injury and apoptosis in mouse hearts. Moreover, it attenuated DOX-induced left ventricular dysfunction and dilatation. Our findings suggest that blockade of LTCC attenuates DOX-induced cardiomyocyte apoptosis via suppressing intracellular Ca2+ elevation and activation of CaMKII-NF-κB pathway. LTCC blockers might be potential therapeutic agents against DOX-induced cardiomyopathy.

Blockade of nifedipine‐sensitive Ca2+ channels decreases manganese accumulation on the dopamine neurons

Manganese (Mn) is an essential trace element involved in many physiological processes. Mn is transported into the cells through a number of transporters. Overexposure to Mn leads to its accumulation in the basal ganglia where it induces circuit dysregulation and a Parkinson-like syndrome referred to as manganism. The mechanisms by which Mn regulates the intrinsic firing behaviors of dopamine neurons and how Mn overexposure dysregulates this activity are not well understood. In this study, we investigated Mn's modulation of the excitability, intrinsic firing activity, Ca2+ homeostasis, and voltage-gated Ca2 channel expression in mouse dopamine neurons. We found that Mn increases the spontaneous firing activity of dopamine neuron via a concentration-dependent mechanism (10–100mM). Analysis of action potential morphology revealed that Mn truncated the amplitude and narrowed the width of action potentials and reduced coefficients of variation of the interspike interval. Unexpectedly, we found neither removal of external Ca2+ (Ca2+-free), chelation of intracellular Ca2+, nor combined chelation of internal and external Ca2+ altered Mn-regulation of action potential in dopamine neurons. Therefore, we asked whether Mn alters Ca2+ currents via the voltage-gated Ca2+ channels in the dopamine neurons. We found that Mn suppressed the peak amplitude of Ca2+ channel-mediated currents, and blockade of Ca2+ channels prevented Mn-stimulation of firing rate, reduction of action potential amplitude in dopamine neurons, and reduced 54Mn uptake in the CaV1-2, CaV1-3 expressing cells (p< 0.05, n=8–10). Finally, in vivo magnetic resonance imaging (MEMRI) revealed nifidepine-blockade of Ca2+ channels decreased Mn accumulation in the midbrain region of C57BL mice (p<0.05, n=6–8). Our data suggest a potential mechanism by which Mn can induce defects in basal ganglia function, namely a marked increase the excitability of dopaminergic neurons. Mn increases the spontaneous firing activity of dopamine neurons. Blockade of L-type Ca2+ channels inhibited Mn-stimulation of spontaneous firing activity of dopamine neurons. Manganese enhanced magnetic resonance imaging (MEMRI) provides evidence of an in vivo calcium-dependent mechanism involved in midbrain Mn accumulation. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Multiple target of hAmylin on rat primary hippocampal neurons

Alzheimer's disease (AD) and type II diabetes mellitus (DM2) are the most common aging-related diseases and are characterized by β-amyloid and amylin accumulation, respectively. Multiple studies have indicated a strong correlation between these two diseases. Amylin oligomerization in the brain appears to be a novel risk factor for developing AD. Although amylin aggregation has been demonstrated to induce cytotoxicity in neurons through altering Ca2+ homeostasis, the underlying mechanisms have not been fully explored. In this study, we investigated the effects of amylin on rat hippocampal neurons using calcium imaging and whole-cell patch clamp recordings. We demonstrated that the amylin receptor antagonist AC187 abolished the Ca2+ response induced by low concentrations of human amylin (hAmylin). However, the Ca2+ response induced by higher concentrations of hAmylin was independent of the amylin receptor. This effect was dependent on extracellular Ca2+. Additionally, blockade of L-type Ca2+ channels partially reduced hAmylin-induced Ca2+ response. In whole-cell recordings, hAmylin depolarized the membrane potential. Moreover, application of the transient receptor potential (TRP) channel antagonist ruthenium red (RR) attenuated the hAmylin-induced increase in Ca2+. Single-cell RT-PCR demonstrated that transient receptor potential vanilloid 4 (TRPV4) mRNA was expressed in most of the hAmylin-responsive neurons. In addition, selective knockdown of TRPV4 channels inhibited the hAmylin-evoked Ca2+ response. These results indicated that different concentrations of hAmylin act through different pathways. The amylin receptor mediates the excitatory effects of low concentrations of hAmylin. In contrast, for high concentrations of hAmylin, hAmylin aggregates precipitated on the neuronal membrane, activated TRPV4 channels and subsequently triggered membrane voltage-gated calcium channel opening followed by membrane depolarization. Therefore, our data suggest that TRPV4 is a key molecular mediator for the cytotoxic effects of hAmylin on hippocampal neurons.

Differential zinc permeation and blockade of L-type Ca2 + channel isoforms Cav1.2 and Cav1.3

Certain voltage-activated Ca2+ channels have been reported to act as potential zinc entry routes. However, it remains to be determined whether zinc can permeate individual Ca2+ channel isoforms. We expressed recombinant Ca2+ channel isoforms in Xenopus oocytes and attempted to record zinc currents from them using a two-electrode voltage clamp method. We found that, in an extracellular zinc solution, inward currents arising from zinc permeation could be recorded from Xenopus oocytes expressing L-type Cav1.2 or Cav1.3 isoforms, but not from oocytes expressing Cav2.2, Cav2.3, Cav3.1, or Cav3.2. Zinc currents through Cav1.2 and Cav1.3 were blocked by nimodipine, but enhanced by (±)Bay K8644, supporting the finding that zinc can permeate both L-type Cav1.2 and Cav1.3 channel isoforms. We also examined the blocking effects of low concentrations of zinc on Ca2+ currents through the L-type channel isoforms. Low micro-molar zinc potently blocked Ca2+ currents through Cav1.2 and Cav1.3 with different sensitivities (IC50 for Cav1.2 and Cav1.3=18.4 and 34.1μM) and de-accelerated the activation and inactivation kinetics in a concentration-dependent manner. Notably, mild acidifications of the external zinc solution increased zinc currents through Cav1.2 and Cav1.3, with the increment level for Cav1.3 being greater than that for Cav1.2. In overall, we provide evidence that Cav1.2 and Cav1.3 isoforms are capable of potentially functioning as zinc permeation routes, through which zinc entry can be differentially augmented by mild acidifications.

Retina derived relaxation is mediated by Kir channels and the inhibition of Ca2+ sensitization in isolated bovine retinal arteries

Retinal relaxing factor (RRF) has recently been identified as a novel paracrine regulator of retinal circulation acting differently from well known mediators of the endothelium and the retina. Herein, we aimed to characterize the relaxing mechanism of the retina, i.e. RRF, by evaluating the role of Ca+2-dependent and -independent signaling mechanisms as well as inward rectifier K+ (Kir) channels. Retinal relaxation was determined by placing a piece of retinal tissue just on top of the precontracted bovine retinal arteries mounted in a wire myograph. The retina produced a complete relaxation response, which display a biphasic character, in depolarized arteries contracted by L-type Ca2+ channel agonist, Bay k 8644. Blockade of L-type Ca2+ channel by nifedipine, inhibition of sarcoplasmic reticulum Ca2+-ATPase by cyclopiazonic acid or removal of extracellular Ca2+ did not influence the prominent relaxation to the retina. Originally, retinal relaxation was found to be unaffected from the inhibition of myosin light chain kinase by ML7, whereas, completely abolished in the presence of myosin light chain phosphatase (MLCP) inhibitor, Calyculin A. Moreover, the inhibition of Rho kinase by its putative inhibitor, Y-27632 displayed comparable relaxant effects to RRF in retinal arteries precontracted either by prostaglandin F2α or K+, and augmented the moderate response to the retina in K+ precontracted arteries. In addition, retinal relaxation was significantly inhibited and lost its biphasic character in the presence of Kir channel blocker, Ba2+. Our results suggested that inhibition of Ca2+ sensitization through the activation of MLCP, possibly via interfering with Rho kinase, and the opening of Kir channels are likely to be involved in the inhibitory influence of RRF on the retinal arteries.

Vasorelaxant effects of mercury on rat thoracic aorta

Mercury, a heavy metal, is widespread and persistent in the environment and has been elucidated as a possible risk factor in cardiovascular diseases. Mercury has been reported to selectively impair the nitric oxide (NO) pathway in the vascular endothelium as a consequence of oxidative stress. Conversely, mercury per se causes endothelium-dependent vasorelaxation at lower concentration via the NO pathway. Little is known about the effects of mercury per se on other endothelial mediators. To elucidate possible mechanisms involved in this action, isometric tension was measured in aortic rings precontracted with phenylephrine (10 µM) from Wistar rats. Responses to increasing concentrations of inorganic mercuric chloride (10(-12)-10(-5) M) were obtained in the presence and absence of endothelium. Inorganic mercury produced a biphasic response in endothelium-intact aortic rings and produced only vasoconstriction in endothelium-denuded aortic rings. To study the possible underlying mechanisms for the biphasic response of mercury, increasing concentrations of mercuric chloride (10(-12)-10(-5) M) were used before and after N(G)-nitro-L-arginine methyl ester (L-NAME (10(-4) M)), glybenclamide (10(-5) M), superoxide dismutase (10 U/ml) + catalase (100 U/ml), and nifedipine (10(-4) M) treatment. Results suggest that mercury produces endothelium-dependent relaxation at low concentration mediated by endothelial-generated NO and endothelium-derived hyperpolarizing factor and endothelium-independent contraction resulting from the blockade of L-type Ca(2+) channels by generation of free radicals.

Endocannabinoid system as a potential mechanism for n-3 long-chain polyunsaturated fatty acid mediated cardiovascular protection

The presence of an active and functioning endocannabinoid (EC) system within cardiovascular tissues implies that this system has either a physiological or pathophysiological role (or both), and there is a substantial literature to support the notion that, in the main, they are protective in the setting of various CVD states. Moreover, there is an equally extensive literature to demonstrate the cardio- and vasculo-protective effects of n-3 long-chain (LC)-PUFA. It is now becoming evident that there appears to be a close relationship between dietary intervention with n-3 LC-PUFA and changes in tissue levels of EC, raising the question as to whether or not EC may, at least in part, play a role in mediating the cardio-and vasculo-protective effects of n-3 LC-PUFA. This brief review summarises the current understanding of how both EC and n-3 LC-PUFA exert their protective effects in three major cardiovascular disorders (hypertension, atherosclerosis and acute myocardial infarction) and attempts to identify the similarities and differences that may indicate common or integrated mechanisms. From the data available, it is unlikely that in hypertension EC mediate any beneficial effects of n-3 LC-PUFA, since they do not share common mechanisms of blood pressure reduction. However, inhibition of inflammation is an effect shared by EC and n-3 LC-PUFA in the setting of both atherosclerosis and myocardial reperfusion injury, while blockade of L-type Ca2+ channels is one of the possible common mechanisms for their antiarrhythmic effects. Although both EC and n-3 LC-PUFA demonstrate vasculo- and cardio-protection, the literature overwhelmingly shows that n-3 LC-PUFA decrease tissue levels of EC through formation of EC–n-3 LC-PUFA conjugates, which is counter-intuitive to an argument that EC may mediate the effects of n-3 LC-PUFA. However, the discovery that these conjugates have a greater affinity for cannabinoid receptors than the native EC provides a fascinating avenue for further research into novel approaches for the treatment and prevention of atherosclerosis and myocardial injury following ischaemia/reperfusion.

Extracellular Ca2+ entry and mobilization of inositol trisphosphate-dependent Ca2+ stores modulate histamine and electrical field stimulation induced contractions of the guinea-pig prostate

This investigation aimed to examine the source of Ca2+ mobilization that leads to the contractile response to either exogenously added histamine (1μM–1mM) or electrical field stimulation (10Hz, 0.5ms, 60V). Removal of extracellular Ca2+ by removal of Ca2+ from the bathing medium reduced histamine (1mM) induced responses by 34% and responses induced by electrical field stimulation by 94%. Similarly, blockade of L-type Ca2+ channels by nifedipine (1μM) reduced histamine (1mM) induced responses by 43% and responses induced by electrical field stimulation by 77%. Application of cyclopiazonic acid (CPA) (10μM) to inhibit Ca2+ reuptake to the sarcoplasmic reticulum enhanced both histamine-induced and electrical field stimulation induced responses to a small degree, while the addition of the inosotol triphosphate (IP3) receptor antagonist, 2-aminophenoxyethane borane (2-APB) (100μM) inhibited histamine induced responses by 70% and electrical field stimulation induced responses by 57%. Ryanodine (1μM) did not affect contractile responses to either histamine or electrical field stimulation, either in the absence or presence of 2-APB (100μM). During both histamine and electrical field stimulation induced contractions, prostate smooth muscle generates IP3 receptor mediated Ca2+ release in conjunction with Ca2+ entry from the extracellular environment. Ryanodine receptors on the other hand, appear not to play a role in this physiological mechanism.

Sustained Glutamate Receptor Activation Down-regulates GABAB Receptors by Shifting the Balance from Recycling to Lysosomal Degradation

Metabotropic GABA(B) receptors are abundantly expressed at glutamatergic synapses where they control excitability of the synapse. Here, we tested the hypothesis that glutamatergic neurotransmission may regulate GABA(B) receptors. We found that application of glutamate to cultured cortical neurons led to rapid down-regulation of GABA(B) receptors via lysosomal degradation. This effect was mimicked by selective activation of AMPA receptors and further accelerated by coactivation of group I metabotropic glutamate receptors. Inhibition of NMDA receptors, blockade of L-type Ca(2+) channels, and removal of extracellular Ca(2+) prevented glutamate-induced down-regulation of GABA(B) receptors, indicating that Ca(2+) influx plays a critical role. We further established that glutamate-induced down-regulation depends on the internalization of GABA(B) receptors. Glutamate did not affect the rate of GABA(B) receptor endocytosis but led to reduced recycling of the receptors back to the plasma membrane. Blockade of lysosomal activity rescued receptor recycling, indicating that glutamate redirects GABA(B) receptors from the recycling to the degradation pathway. In conclusion, the data indicate that sustained activation of AMPA receptors down-regulates GABA(B) receptors by sorting endocytosed GABA(B) receptors preferentially to lysosomes for degradation on the expense of recycling. This mechanism may relieve glutamatergic synapses from GABA(B) receptor-mediated inhibition resulting in increased synaptic excitability.

Carbachol-Induced Long-Term Synaptic Depression Is Enhanced During Senescence at Hippocampal CA3–CA1 Synapses

Dysregulation of the cholinergic transmitter system is a hallmark of Alzheimer's disease and contributes to an age-associated decline in memory performance. The current study examined the influence of carbachol, a cholinergic receptor agonist, on synaptic transmission over the course of aging. Extracellular excitatory postsynaptic field potentials were recorded from CA3-CA1 synapses in acute hippocampal slices obtained from young adult (5-8 mo) and aged (22-24 mo) male Fischer 344 rats. Bath application of carbachol elicited a transient depression of synaptic transmission, which was followed by a long-lasting depression (CCh-LTD) observed 90 min after carbachol cessation in both age groups. However, the magnitude of CCh-LTD was significantly larger in senescent animals and was attenuated by N-methyl-D-aspartate receptor blockade in aged animals. Blockade of L-type Ca(2+) channels inhibited CCh-LTD to a greater extent in aged animals compared to young adults. Finally, the expression of CCh-LTD was dependent on protein synthesis. The results indicate that altered Ca(2+) homeostasis or muscarinic activation of Ca(2+) signaling contribute to the enhanced CCh-LTD during senescence.

Overlap in the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.

We have previously shown that elimination of buffer Ca2+ markedly reduced maximum 5-HT-induced contractions. We have now investigated the effect of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists on 5-HT- and K+-induced contractions in rat aorta to explore the possibility of a relationship between blockade of L-type Ca2+ channels and 5-HT2 receptor antagonism. Sodium nitroprusside, felodipine, nifedipine, diltiazem, cinnarizine, verapamil, ritanserin, cyproheptadine, ketanserin and mianserin inhibited 5-HT-induced contractions of rat aorta with mean IC50 values (concentration (M) resulting in 50% inhibition) of 2.2 x 10(-11), 6.6 x 10(-11), 1.5 x 10(-9), 1.7 x 10(-9), 3.2 x 10(-7), 5.4 x 10(-7), 9.7 x 10(-10), 1.9 x 10(-8), 5.0 x 10(-7) and 6.4 x 10(-7), respectively. The same compounds antagonized K+-induced rat aortic contractions with the rank order of potency (mean IC50, M): felodipine (7.0 x 10(-11)) > nifedipine (4.8 x 10(-9)) > sodium nitroprusside (4.1 x 10(-8)) > verapamil (5.5 x 10(-8)) > cyproheptadine (6.2 x 10(-8)) > diltiazem (4.1 x 10(-7)) > cinnarizine (1.3 x 10(-6)) > ritanserin (1.8 x 10(-6)) > ketanserin (9.0 x 10(-6)) > mianserin (2.0 x 10(-5)). These data are indicative of a highly significant correlation (r=0.81, P=0.03) between potency against 5-HT-induced contraction and that against contractile response to K+ depolarization, and suggest overlap of the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.

Combined L-/T-Type Calcium Channel Blockers

Opening of the voltage-dependent Ca2+ channels permits influx of Ca2+ across the plasma membrane, triggering diverse physiological processes. These channels are widely distributed in the cardiovascular system, constituting the main route for Ca2+ entry essential for excitation and contraction. Ten unique α1 subunits, grouped in 3 families (CaV1, CaV2, and CaV3), that encode the low-voltage–activated T-type and the high-voltage–activated L-, N-, P/Q- and R-type Ca2+ channels, have been identified.1,2 L-type Ca2+ channels are predominantly expressed in the hearts and peripheral vasculature and serve as the preferred molecular target of the initial Ca2+ channel antagonists in the treatment of hypertension.3 Recently, a growing body of evidence has accumulated depicting important roles of T-type Ca2+ channels in the regulation of cardiovascular function, such as generation of pacemaker potential and regulation of arterial resistance.3,4 T-type Ca2+ channels are found in various cell types, including neurons, cardiomyocytes, vascular smooth muscle cells, and endocrine cells, where they participate in a variety of physiological processes, such as low-threshold Ca2+ spike generation, action potential firing, pacemaking, impulse conduction, maintenance of myogenic tone, cell proliferation, and hormone secretion.1 In addition to their predominant role in the regulation of vascular function, T-type Ca2+ channels are also involved in cardiomyocyte growth and survival.3 T-type Ca2+ channel blockers are capable of interrupting certain pathological hypertrophic signaling pathways, including calcineurin-mediated nuclear factor of activated T cells–3 activation.1 The importance of voltage-dependent Ca2+ channels is demonstrated by the clinical efficacy of Ca2+ channels blockers in certain disease conditions, as well as the widespread distribution and function of these channels.1 Three classes of chemically distinct L-type Ca2+ channel blockers have been widely used clinically depending on their biophysical and conformation-dependent interactions …

(S)-reticuline induces vasorelaxation through the blockade of L-type Ca2+ channels

In Brazil, various species of the genus Ocotea are used in folk medicine for treating several diseases. The chemical characterization of this plant showed the presence of alkaloids belonging to the benzyltetrahydroisoquinoline family, the major component of which is (S)-reticuline. The present study investigated whether (S)-reticuline exerts an inhibitory effect on smooth muscle L-type Ca(2+) channels. Tension measurements and patch clamp techniques were utilized to study the effects of (S)-reticuline. Whole-cell Ca(2+) currents were measured using the A7r5 smooth muscle cell line. (S)-reticuline antagonized CaCl(2)- and KCl-induced contractions and elicited vasorelaxation. It also reduced the voltage-activated peak amplitude of I (Ca,L) in a concentration-dependent manner. (S)-reticuline did not change the characteristics of current density vs. voltage relationship. (S)-reticuline shifted leftwards the steady-state inactivation curve of I (Ca,L). The application of dibutyryl cyclic adenosine monophosphate to the cell decreased the amplitude of Ca(2+) currents. In cells pretreated with forskolin, an adenylate cyclase activator, the addition of (S)-reticuline caused further inhibition of the Ca(2+) currents suggesting an additive effect. The results obtained show that (S)-reticuline elicits vasorelaxation probably due to the blockade of the L-type voltage-dependent Ca(2+) current in rat aorta. The reported effect may contribute to the potential cardioprotective efficacy of (S)-reticuline.

Effect of clomiphene on [Ca2+]i rises and cell viability in rabbit corneal epithelial cells

AbstractThe effect of clomiphene a first‐line therapy for WHO group II (eu‐estrogenic) infertility on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in rabbit corneal epithelial cells (SIRC). This study examined whether clomiphene altered [Ca2+]i levels and caused cell death in SIRC cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura‐2 and WST‐1, respectively. Clomiphene at concentrations ≥5 µM increased [Ca2+]i in a concentration‐dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The clomiphene‐induced Ca2+ influx was insensitive to blockade of L‐type Ca2+ channel blockers. In Ca2+‐free medium, after pretreatment with 1 µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), clomiphene failed to increase [Ca2+]i. Inhibition of phospholipase C with 2 µM U73122 did not change clomiphene‐induced [Ca2+]i rises. At concentrations of 0.5–20 µM, clomiphene killed cells in a concentration‐dependent manner. The cytotoxic effect of 15 µM clomiphene was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in SIRC cells, clomiphene‐induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from non‐L‐type Ca2+ channels. Clomiphene‐caused cytotoxicity was not mediated by a preceding [Ca2+]i rise. Drug Dev Res 69:272–278, 2008 ©2008 Wiley‐Liss, Inc.

Cardiomyocyte Ca2+ overload in atrial tachycardia: is blockade of L-type Ca2+ channels a promising approach to prevent electrical remodeling and arrhythmogenesis?

Electrical remodeling paradigm has important implications for the understanding of atrial fibrillation (AF) and improvement of current treatment. Cardiomyocyte Ca(2+) overload is generally accepted as the initiating signal for the tachycardia-induced changes in atrial electrical properties (electrical remodeling). The precise role of cardiomyocyte Ca(2+) overload in AF-related ion channel alterations that contribute to AF maintenance is not fully understood. Clinically, patients with AF are often treated with Ca(2+) channel blockers such as verapamil to control their ventricular rate and to improve the success rate of cardioversion procedures. However, verapamil may produce an increased L-type Ca(2+) channel current (I(Ca,L)) that may reinforce Ca(2+) overload thereby promoting AF in the atrium. Ca(2+) channel blockers which target T-type Ca(2+) channels in addition to I(Ca,L) (for instance, efonidipine) may be more efficient at preventing Ca(2+) overload and arrhythmogenic electrical remodeling, but the potential benefits of these drugs have usually been tested in experimental models where drug administration preceded the initiation of electrical remodeling. Studies in animal models with established atrial tachycardia remodeling and in patients with AF are clearly warranted to prove the efficacy of Ca(2+) channel blockers that additionally target T-type Ca(2+) channels.

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability in OC2 human oral cancer cells. [Ca2+]i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The tamoxifen-induced Ca2+ influx was sensitive to blockade of L-type Ca2+ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca2+-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca2+]i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca2+]i rises, in a nongenomic manner, by causing Ca2+ release from the endoplasmic reticulum, and Ca2+ influx from L-type Ca2+ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca2+]i rise.