Voltage-dependent Ca Channel Blocker Research Articles

Abstract MP17: Angiotensin II Inhibits Renin Secretion Directly By Eliciting Robust Calcium Oscillations In Juxtaglomerular Cells

Background: Renin secretion from the juxtaglomerular (JG) cells controls circulating Angiotensin II (AngII), a critical blood pressure determinant. AngII is believed to suppress renin release from JG cells to prevent excessive blood pressure elevation. However, a direct negative feedback mechanism has yet to be established in JG cells. In JG cells, Ca 2+ is a crucial second messenger that governs renin secretion by suppressing cAMP, a potent renin secretion stimulator. Thus, in contrast to most secretory cells, renin secretion from JG cells is inversely related to the intracellular Ca 2+ level. However, the role of AngII in modulating JG cell Ca 2+ dynamics remains unclear. Objective: Define intracellular Ca 2+ responses to AngII and identify the critical channels to suppress renin secretion in JG cells. Methods: Kidney slices from mice expressing JG-specific GCaMP6f, a genetically encoded Ca 2+ indicator under the control of the Ren1 c promoter, were imaged ex vivo on a widefield fluorescent microscope for 30 min per experiment (n = 5). In each experiment, slices were continuously perfused with buffer containing variable Ca 2+ and AngII concentrations, ± specific Ca 2+ channel inhibitors. Images were captured at 10 Hz and analyzed using Mesmerize Ca 2+ imaging software to identify and plot fluorescent intensity over time for each JG cell. Additional kidney slices were incubated in 24-well plates with buffer containing vehicle or AngII ± Ca 2+ channel inhibitors for 30 min. ELISA determined renin concentration. Results: 1) AngII dose-dependently evoked robust and periodic Ca 2+ -oscillations (Ca 2+ spikes) in JG cells (mean spikes/min; 50 pM: 5.4, 300 pM: 7.7, 3 nM: 13.2, and 300 nM: 17.6). 2) AngII-elicited Ca 2+ activity was markedly reduced with “Ca 2+ -free” buffer. 3) L-type voltage-dependent Ca 2+ channel (VDCC) blocker nifedipine moderately decreased AngII-elicited Ca 2+ spikes, while T-type VDCC blocker TTA-P2 did not. 4) Blocking endoplasmic reticulum (ER) Ca 2+ -release drastically reduced Ca 2+ activity. 5) AngII dose-dependently decreased renin secretion in kidney slices, congruent with the AngII-elicited Ca 2+ activity in imaging studies. Conclusion: AngII dose-dependently and directly elicited large, periodic Ca 2+ -oscillations in JG cells that suppressed renin release. Both extracellular Ca 2+ influx and intracellular Ca 2+ release from ER stores are required for sustained Ca 2+ activity. L-type, but not T-type, Ca 2+ channels participate in JG cell Ca 2+ -oscillations.

Quantitative real-time measurement of endothelin-1-induced contraction in single non-activated hepatic stellate cells.

Although quiescent hepatic stellate cells (HSCs) have been suggested to regulate hepatic blood flow, there is no direct evidence that quiescent HSCs display contractile abilities. Here, we developed a new method to quantitatively measure the contraction of single isolated HSCs and evaluated whether endothelin-1 (ET-1) induced contraction of HSCs in a non-activated state. HSCs isolated from mice were seeded on collagen gel containing fluorescent beads. The beads around a single HSC were observed gravitating toward the cell upon contraction. By recording the movement of each bead by fluorescent microscopy, the real-time contraction of HSCs was quantitatively evaluated. ET-1 induced a slow contraction of non-activated HSCs, which was inhibited by the non-muscle myosin II inhibitor blebbistatin, the calmodulin inhibitor W-7, and the ETA receptor antagonist ambrisentan. ET-1-induced contraction was also largely reduced in Ca2+-free conditions, but sustained contraction still remained. The tonic contraction was further diminished by the Rho-kinase inhibitor H-1152. The mRNA expression of P/Q-type voltage-dependent Ca2+ channels (VDCC), as well as STIM and Orai, constituents of store-operated channels (SOCs), was observed in mouse non-activated HSCs. ET-1-induced contraction was not affected by amlodipine, a VDCC blocker, whereas it was partly reduced by Gd3+ and amiloride, non-selective cation channel blockers. However, neither YM-58483 nor SKF-96365, which inhibit SOCs, had any effects on the contraction. These results suggest that ET-1 leads to Ca2+-influx through cation channels other than SOCs and produces myosin II-mediated contraction of non-activated HSCs via ETA receptors, as well as via mechanisms involving Ca2+-calmodulin and Rho kinase.

Opposite Roles in Short-Term Plasticity for N-Type and P/Q-Type Voltage-Dependent Calcium Channels in GABAergic Neuronal Connections in the Rat Cerebral Cortex.

Neurotransmitter release is triggered by Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs). Distinct expression patterns of VDCC subtypes localized on the synaptic terminal affect intracellular Ca2+ dynamics induced by action potential-triggered Ca2+ influx. However, it has been unknown whether the expression pattern of VDCC subtypes depends on each axon terminal or neuronal subtype. Furthermore, little information is available on how these VDCC subtypes regulate the release probability of neurotransmitters. To address these questions, we performed multiple whole-cell patch-clamp recordings from GABAergic neurons in the insular cortex of either the male or the female rat. The paired-pulse ratio (PPR; 50 ms interstimulus interval) varied widely among inhibitory connections between GABAergic neurons. The PPR of unitary IPSCs was enhanced by ω-conotoxin GVIA (CgTx; 3 μm), an N-type VDCC blocker, whereas blockade of P/Q-type VDCCs by ω-agatoxin IVA (AgTx, 200 nm) decreased the PPR. In the presence of CgTx, application of 4 mm [Ca2+]o or of roscovitine, a P/Q-type activator, increased the PPR. These results suggest that the recruitment of P/Q-type VDCCs increases the PPR, whereas N-type VDCCs suppress the PPR. Furthermore, we found that charybdotoxin or apamin, blockers of Ca2+-dependent K+ channels, with AgTx increased the PPR, suggesting that Ca2+-dependent K+ channels are coupled to N-type VDCCs and suppress the PPR in GABAergic neuronal terminals. Variance-mean analysis with changing [Ca2+]o showed a negative correlation between the PPR and release probability in GABAergic synapses. These results suggest that GABAergic neurons differentially express N-type and/or P/Q-type VDCCs and that these VDCCs regulate the GABA release probability in distinct manners.SIGNIFICANCE STATEMENT GABAergic neuronal axons target multiple neurons and release GABA triggered by Ca2+ influx via voltage-dependent Ca2+ channels (VDCCs), including N-type and P/Q-type channels. Little is known about VDCC expression patterns in GABAergic synaptic terminals and their role in short-term plasticity. We focused on inhibitory synaptic connections between GABAergic neurons in the cerebral cortex using multiple whole-cell patch-clamp recordings and found different expression patterns of VDCCs in the synaptic terminals branched from a single presynaptic neuron. Furthermore, we observed facilitative and depressive short-term plasticity of IPSCs mediated by P/Q-type and N-type VDCCs, respectively. These results suggest that VDCC expression patterns regulate distinctive types of synaptic transmission in each GABAergic axon terminal even though they are branched from a common presynaptic neuron.

Afferent Activity is Greatly Increased by Spontaneous Phasic Contractions in Mouse Ex Vivo Urinary Bladder

Spontaneous phasic contractions (SPCs) are transient, non‐voiding contractions during urinary bladder filling that hallmark many bladder pathologies. However, the origin and physiological importance of SPCs are largely unknown. Using an ex vivo mouse urinary bladder preparation, we examined the relationship between SPCs and afferent nerve activity. Continuous bladder filling (30 µl/min) alone increased baseline afferent activity (~100 Hz per 4 mmHg pressure). Strikingly, over the same pressure range, SPCs evoked bursts of afferent activity that were more than 3‐fold greater than baseline afferent activity (>350 Hz) and often contained large‐amplitude action potentials not normally observed in the absence of SPCs. The frequency of afferent bursts correlated with the SPC rate of rise, and peaked prior to the associated SPC. Inhibition of neuronal Na+ channels blocked afferent nerve activity without altering SPC amplitude, duration or rate of rise. In contrast, inhibition of smooth muscle voltage‐dependent Ca2+ channels (VDCCs) abolished SPCs as well as all associated afferent bursts, indicating afferent bursts coupled directly to the smooth muscle contractile machinery. However, In the presence of VDCC blockers, mechanical deformation of the bladder evoked SPC‐like events and afferent bursts with an amplitude and SPC/afferent ratio typical of “natural” SPCs. This supports the concept that mechanical distention of the bladder by SPCs generates bursts of sensory nerve activity that dominate bladder afferent outflow. These findings suggest that SPC‐dependent bursts of afferent activity may convey sensory information about bladder fullness to the CNS, and thus contribute to the regulation of micturition frequency. Supported by NIH R37DK053832.

Distinct roles of L- and T-type voltage-dependent Ca2+ channels in regulation of lymphatic vessel contractile activity.

Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of collecting lymphatic vessels through which lymph is propelled back into the blood circulation. This rhythmic contractile activity (i.e. lymphatic pumping) increases in rate with increase in luminal pressure and relies on activation of nifedipine-sensitive voltage-dependent Ca(2+) channels (VDCCs). Despite their importance, these channels have not been characterized in lymphatic vessels. We used pressure- and wire-myography as well as intracellular microelectrode electrophysiology to characterize the pharmacological and electrophysiological properties of L-type and T-type VDCCs in rat mesenteric lymphatic vessels and evaluated their particular role in the regulation of lymphatic pumping by stretch. We complemented our study with PCR and confocal immunofluorescence imaging to investigate the expression and localization of these channels in lymphatic vessels. Our data suggest a delineating role of VDCCs in stretch-induced lymphatic vessel contractions, as the stretch-induced increase in force of lymphatic vessel contractions was significantly attenuated in the presence of L-type VDCC blockers nifedipine and diltiazem, while the stretch-induced increase in contraction frequency was significantly decreased by the T-type VDCC blockers mibefradil and nickel. The latter effect was correlated with a hyperpolarization. We propose that activation of T-type VDCCs depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L-type VDCCs, which drive the strength of contractions.

Distinct Ca2+ channels maintain a high motility state of the sperm that may be needed for penetration of egg jelly of the newt, Cynops pyrrhogaster

Activation state of sperm motility named "hyperactivation" enables mammalian sperm to progress through the oviductal matrix, although a similar state of sperm motility is unknown in non-mammalian vertebrates at fertilization. Here, we found a high motility state of the sperm in the newt Cynops pyrrhogaster. It was predominantly caused in egg jelly extract (JE) and characterized by a high wave velocity of the undulating membrane (UM) that was significantly higher at the posterior midpiece. An insemination assay suggested that the high motility state might be needed for sperm to penetrate the egg jelly, which is the accumulated oviductal matrix. Specific characteristics of the high motility state were completely abrogated by a high concentration of verapamil, which blocks the L-type and T-type voltage-dependent Ca(2+) channels (VDCCs). Mibefradil, a dominant blocker of T-type VDCCs, suppressed the wave of the UM at the posterior midpiece with separate wave propagation from both the anterior midpiece and the posterior principal piece. In addition, nitrendipine, a dominant L-type VDCC blocker, weakened the wave of the UM, especially in the anterior midpiece. Live Ca(2+) imaging showed that, compared with the intact sperm in the JE, the relative intracellular Ca(2+) level changed especially in the anterior and posterior ends of the midpiece of the blocker-treated sperm. These suggest that different types of Ca(2+) channels mediate the intracellular Ca(2+) level predominantly in the anterior and posterior ends of the midpiece to maintain the high motility state of the newt sperm.

Phorbol Ester Modulation of Ca2+ Channels Mediates Nociceptive Transmission in Dorsal Horn Neurones

Phorbol esters are analogues of diacylglycerol which activate C1 domain proteins, such as protein kinase C (PKC). Phorbol ester/PKC pathways have been proposed as potential therapeutic targets for chronic pain states, potentially by phosphorylating proteins involved in nociception, such as voltage-dependent Ca2+ channels (VDCCs). In this brief report, we investigate the potential involvement of CaV2 VDCC subtypes in functional effects of the phorbol ester, phorbol 12-myristate 13-acetate (PMA) on nociceptive transmission in the spinal cord. Effects of PMA and of selective pharmacological blockers of CaV2 VDCC subtypes on nociceptive transmission at laminae II dorsal horn neurones were examined in mouse spinal cord slices. Experiments were extended to CaV2.3(−/−) mice to complement pharmacological studies. PMA increased the mean frequency of spontaneous postsynaptic currents (sPSCs) in dorsal horn neurones, without an effect on event amplitude or half-width. sPSC frequency was reduced by selective VDCC blockers, ω-agatoxin-IVA (AgTX; CaV2.1), ω-conotoxin-GVIA (CTX; CaV2.2) or SNX-482 (CaV2.3). PMA effects were attenuated in the presence of each VDCC blocker and, also, in CaV2.3(−/−) mice. These initial data demonstrate that PMA increases nociceptive transmission at dorsal horn neurones via actions on different CaV2 subtypes suggesting potential anti-nociceptive targets in this system.

Chronic hypoxia selectively enhances L- and T-type voltage-dependent Ca2+ channel activity in pulmonary artery by upregulating Cav1.2 and Cav3.2

Hypoxia-induced pulmonary hypertension (HPH) is characterized by sustained pulmonary vasoconstriction and vascular remodeling, both of which are mediated by pulmonary artery smooth muscle cell (PASMC) contraction and proliferation, respectively. An increase in cytosolic Ca²⁺ concentration ([Ca²⁺]cyt) is a major trigger for pulmonary vasoconstriction and an important stimulus for cell proliferation in PASMCs. Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) is an important pathway for the regulation of [Ca²⁺]cyt. The potential role for L- and T-type VDCC in the development of HPH is still unclear. Using a hypoxic-induced pulmonary hypertension mouse model, we undertook this study to identify if VDCC in pulmonary artery (PA) are functionally upregulated and determine which type of VDCC are altered in HPH. Mice subjected to chronic hypoxia developed pulmonary hypertension within 4 wk, and high-K⁺- and U-46619-induced contraction of PA was greater in chronic hypoxic mice than that in normoxic control mice. Additionally, we demonstrate that high-K⁺- and U-46619-induced Ca²⁺ influx in PASMC is significantly increased in the hypoxic group. The VDCC activator, Bay K8864, induced greater contraction of the PA of hypoxic mice than in that of normoxic mice in isometric force measurements. L-type and T-type VDCC blockers significantly attenuated absolute contraction of the PA in hypoxic mice. Chronic hypoxia did not increase high-K⁺- and U-46619-induced contraction of mesenteric artery (MA). Compared with MA, PA displayed higher expression of calcium channel voltage-dependent L-type α1C-subunit (Cav1.2) and T-type α1H-subunit (Cav3.2) upon exposure to chronic hypoxia. In conclusion, both L-type and T-type VDCC were functionally upregulated in PA, but not MA, in HPH mice, which could result from selectively increased expression of Cav1.2 and Cav3.2.

Voltage-dependent N-type Ca2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by angiotensin II in mice

N-type voltage-dependent Ca2+channels (VDCCs), expressed predominantly in the nervous system, play pivotal roles in sympathetic regulation of the circulatory system. Although N-type VDCCs are also reportedly expressed in the vasculature, their pathophysiological role is obscure. We demonstrated that oxidative stress-related endothelial dysfunction induced by angiotensin (Ang) II is suppressed in mice lacking the N-type VDCC α1B subunit (Cav 2.2). Impairment of endothelium-dependent relaxation of the thoracic aorta observed following Ang II treatment in wild-type (WT) mice was significantly attenuated in the Ang II-treated Cav 2.2-deficient mice, despite the comparable increase of the blood pressure in the two groups of mice. The thoracic aorta of the Cav 2.2-deficient mice showed a smaller positive area of oxidative stress markers as compared to the WT mice. The Ang II-induced endothelial dysfunction was also suppressed by cilnidipine, an L/N-type VDCC blocker, but not by amlodipine, an L-type VDCC blocker; however, this unique effect of cilnidipine was completely abolished in the Cav 2.2-deficient mice. Furthermore, selective inhibition of N-type VDCCs by ω-conotoxin GVIA dramatically suppressed the production of reactive oxygen species (ROS) as well as agonist-induced Ca2+ influx in the vascular endothelial cells. These results suggest that N-type VDCCs expressed in the vascular endothelial cells contribute to ROS production and endothelial dysfunction observed in Ang II-treated hypertensive mice.

Functional characterization of voltage-dependent Ca2+ channels in mouse pulmonary arterial smooth muscle cells: divergent effect of ROS

Electromechanical coupling via membrane depolarization-mediated activation of voltage-dependent Ca(2+) channels (VDCC) is an important mechanism in regulating pulmonary vascular tone, while mouse is an animal model often used to study pathogenic mechanisms of pulmonary vascular disease. The function of VDCC in mouse pulmonary artery (PA) smooth muscle cells (PASMC), however, has not been characterized, and their functional role in reactive oxygen species (ROS)-mediated regulation of vascular function remains unclear. In this study, we characterized the electrophysiological and pharmacological properties of VDCC in PASMC and the divergent effects of ROS produced by xanthine oxidase (XO) and hypoxanthine (HX) on VDCC in PA and mesenteric artery (MA). Our data show that removal of extracellular Ca(2+) or application of nifedipine, a dihydropyridine VDCC blocker, both significantly inhibited 80 mM K(+)-mediated PA contraction. In freshly dissociated PASMC, the maximum inward Ca(2+) currents were -2.6 ± 0.2 pA/pF at +10 mV (with a holding potential of -70 mV). Window currents were between -40 and +10 mV with a peak at -15.4 mV. Nifedipine inhibited currents with an IC(50) of 0.023 μM, and 1 μM Bay K8644, a dihydropyridine VDCC agonist, increased the inward currents by 61%. XO/HX attenuated 60 mM K(+)-mediated increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) due to Ca(2+) influx through VDCC in PASMC. Exposure to XO/HX caused relaxation in PA preconstricted by 80 mM K(+) but not in aorta and MA. In contrast, H(2)O(2) inhibited high K(+)-mediated increase in [Ca(2+)](cyt) and caused relaxation in both PA and MA. Indeed, RT-PCR and Western blot analysis revealed significantly lower expression of Ca(V)1.3 in MA compared with PA. Thus our study characterized the properties of VDCC and demonstrates that ROS differentially regulate vascular contraction by regulating VDCC in PA and systemic arteries.

L-type calcium channels play a crucial role in the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

L-type voltage-dependent Ca2+ channels (VDCCL) play an important role in the maintenance of intracellular calcium homeostasis, and influence multiple cellular processes. They have been confirmed to contribute to the functional activities of osteoblasts. Recently, VDCCL expression was reported in mesenchymal stem cells (MSCs), but the role of VDCCL in MSCs is still undetermined. The aim of this study was to determine whether VDCCL may be regarded as a new regulator in the proliferation and osteogenic differentiation of rat MSC (rMSCs). In this study, we examined functional Ca2+ currents (ICa) and mRNA expression of VDCCL in rMSCs, and then suppressed VDCCL using nifedipine (Nif), a VDCCL blocker, to investigate its role in rMSCs. The proliferation and osteogenic differentiation of MSCs were analyzed by MTT, flow cytometry, alkaline phosphatase (ALP), Alizarin Red S staining, RT-PCR, and real-time PCR assays. We found that Nif exerts antiproliferative and apoptosis-inducing effects on rMSCs. ALP activity and mineralized nodules were significantly decreased after Nif treatment. Moreover, the mRNA levels of the osteogenic markers, osteocalcin (OCN), bone sialoprotein (BSP), and runt-related transcription factor 2 (Runx2), were also down-regulated. In addition, we transfected α1C-siRNA into the cells to further confirm the role of VDCCL in rMSCs, and a similar effect on osteogenesis was found. These results suggest that VDCCL plays a crucial role in the proliferation and osteogenic differentiation of rMSCs.

N- and L-Type Voltage-Dependent Ca2+ Channels Contribute to the Generation of After-Discharges in the Spinal Ventral Root After Cessation of Noxious Mechanical Stimulation

Voltage-dependent Ca2+ channels (VDCCs) play a crucial role in the spinal pain transduction. We previously reported that nociceptive mechanical stimuli to the rat hindpaw evoked two types of ventral root discharges that increased during stimulation (during-discharges) and after cessation of stimulation (after-discharges). To explore the involvement of VDCCs in these ventral root discharges, several VDCC blockers were applied directly to the surface of the spinal cord. Spinalized rats were laminectomized. The fifth lumbar ventral root was sectioned and used for multi-unit efferent discharges recording. An agar pool was constructed on the first lumbar vertebra for drug application. Ethosuximide (a T-type VDCC blocker) had no effect on ventral root discharges. ω-Conotoxin GVIA (an N-type VDCC blocker) preferentially suppressed after-discharges. ω-Agatoxin IVA (a P/Q-type VDCC blocker), diltiazem, and verapamil (L-type VDCC blockers) nonselectively depressed both during- and after-discharges. The more selective L-type VDCC blocker nicardipine depressed only after-discharges and the depression was exhibited when nicardipine was microinjected into the dorsal horn, but not into the ventral horn. These findings suggested that N- and L-type VDCCs in the dorsal horn were involved in the generation of afterdischarges and these blockers might be useful for treatment of persistent pain that involves the spinal pathway.

Neuroprotection by lomerizine, a prophylactic drug for migraine, against hydrogen peroxide-induced hippocampal neurotoxicity

Migraine is one of the risk factor for ischemic stroke. The purpose of this study was to examine the effect of lomerizine, a prophylactic drug for migraine, on H(2)O(2)-induced cell death of hippocampal neurons. Cytosolic Ca(2+) concentration was measured using fura-2 as a Ca(2+) indicator. Cell death was estimated by trypan blue exclusion. In rat-cultured hippocampal neurons, the addition of H(2)O(2) induced biphasic Ca(2+) elevations and cell death. The H(2)O(2)-induced biphasic Ca(2+) elevations and cell death only occurred when extracellular Ca(2+) was present. The biphasic Ca(2+) elevation was mediated by Ca(2+) influx through the plasma membrane, but not Ca(2+) release from the intracellular Ca(2+) store. Both the early and late phases of H(2)O(2)-induced Ca(2+) influx were reduced by either a T- or L-type voltage-dependent Ca(2+) channel (VDCC) blocker, lomerizine. In fact, L-type VDCC (α(1C) subunit) and T-type VDCC (α(1G) subunit) mRNA were expressed in rat hippocampal neurons. Although an L-type VDCC blocker, nifedipine, partly suppressed the late phase of Ca(2+) influx in response to H(2)O(2), a T-type VDCC blocker, mibefradil, reduced both phases of Ca(2+) influx. Moreover, lomerizine and mibefradil strongly reduced H(2)O(2)-induced cell death, and nifedipine weakly reduced it. These findings suggest that the inhibition of H(2)O(2)-induced Ca(2+) influx through T-type VDCC seems to be important in the protective effect of lomerizine against oxidative stress. It is possible that lomerizine may be a useful drug for prophylactic treatment of migraine, because migraine is a risk factor for ischemic stroke.

Effects of neuropathy on high-voltage-activated Ca 2+ current in sensory neurones

Voltage-dependent Ca 2+ channels (VDCCs) have emerged as targets to treat neuropathic pain; however, amongst VDCCs, the precise role of the Ca V2.3 subtype in nociception remains unproven. Here, we investigate the effects of partial sciatic nerve ligation (PSNL) on Ca 2+ currents in small/medium diameter dorsal root ganglia (DRG) neurones isolated from Ca V2.3(−/−) knock-out and wild-type (WT) mice. DRG neurones from Ca V2.3(−/−) mice had significantly reduced sensitivity to SNX-482 versus WT mice. DRGs from Ca V2.3(−/−) mice also had increased sensitivity to the Ca V2.2 VDCC blocker ω-conotoxin. In WT mice, PSNL caused a significant increase in ω-conotoxin-sensitivity and a reduction in SNX-482-sensitivity. In Ca V2.3(−/−) mice, PSNL caused a significant reduction in ω-conotoxin-sensitivity and an increase in nifedipine sensitivity. PSNL-induced changes in Ca 2+ current were not accompanied by effects on voltage-dependence of activation in either Ca V2.3(−/−) or WT mice. These data suggest that Ca V2.3 subunits contribute, but do not fully underlie, drug-resistant (R-type) Ca 2+ current in these cells. In WT mice, PSNL caused adaptive changes in Ca V2.2- and Ca V2.3-mediated Ca 2+ currents, supporting roles for these VDCCs in nociception during neuropathy. In Ca V2.3(−/−) mice, PSNL-induced changes in Ca V1 and Ca V2.2 Ca 2+ current, consistent with alternative adaptive mechanisms occurring in the absence of Ca V2.3 subunits.

T-type Ca2+ Channels Promote Oxygenation-induced Closure of the Rat Ductus Arteriosus Not Only by Vasoconstriction but Also by Neointima Formation

The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca(2+) influx through several membrane Ca(2+) channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca(2+) channel (VDCC) in DA closure remains unclear. Here we found that the expression of alpha1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that alpha1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by alpha1G overexpression. Moreover, it was decreased by adding alpha1G-specific small interfering RNAs or using R(-)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca(2+) concentration of DA SMCs was significantly decreased by adding alpha1G-specific siRNAs or using R(-)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(-)-efonidipine (10(-6) m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(-)-efonidipine (10(-6) m) significantly attenuated oxygenation-induced vasoconstriction by approximately 27% using a vascular ring of fetal DA at term. Finally, R(-)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially alpha1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.

Modulators of Voltage-Dependent Calcium Channels for the Treatment of Nervous System Diseases

Voltage-dependent Ca(2+) channels (VDCCs) play important roles in physiological functions and pathological processes of the nervous system. Given that the precise regulation of Ca(2+) signaling is important for neuronal processes such as action potential generation, transmitter release, and synaptic plasticity, alterations in Ca(2+) current through VDCCs affect the functions of neurons and circuits. Central nervous system (CNS) diseases, including pain, epilepsy, seizure, anxiety, depression, dementia, and stroke, are characterized by an altered balance between excitatory and inhibitory neuronal functions. An efficient way of controlling such diseases is to block or modulate VDCC function. An effective strategy to reduce the likelihood of adverse effects is to develop agents that selectively control the VDCC isoform/subunit involved in the mechanism of the disease in question. This review provides an overview of knowledge on VDCCs, traditional and newly developed therapeutic fields, clinical fields, and the diverse medicinal chemistry of traditional and newly developed VDCC blockers in the CNS based on the scientific and patent literature.

Tributyltin-induced Ca 2+ mobilization via L-type voltage-dependent Ca 2+ channels in PC12 cells

The effects of tributyltin (TBT) on cytosolic Ca 2+ concentration ([Ca 2+] c) and cell viability were investigated in nerve growth factor-differentiated PC12 cells. TBT concentration dependently increased [Ca 2+] c with an EC 50 value of 0.07 μM. This effect was markedly reduced by removal of the extracellular Ca 2+ or membrane depolarization with a high K + medium, but unaffected by thapsigargin causing depletion of intracellular Ca 2+ stores. The L-type voltage-dependent Ca 2+ channel (VDCC) blocker nicardipine blocked the effect of TBT, but the N-type VDCC blocker ω-conotoxin did not. TBT decreased the number of viable cells with an EC 50 value of 0.09 μM. The TBT-induced cell death was prevented by nicardipine or by chelating the cytosolic Ca 2+ with BAPTA-AM, but not by ω-conotoxin. The results show that TBT causes an increase in [Ca 2+] c via activating L-type VDCCs, and support the idea that the organotin-induced cell death arises through Ca 2+ mobilization via L-type VDCCs.

Toluene Can Perturb the Neuronal Voltage-Dependent Ca2+ Channels Involved in the Middle-Ear Reflex

Numerous laboratory-based data have shown the ability of toluene (Tol) to exacerbate noise-induced hearing loss. However, the mechanism responsible for the synergistic effects of a coexposure to noise and Tol has not yet been completely elucidated. Recent investigations in rats have focused on quantifying the anticholinergic effects of certain aromatic solvents and have demonstrated that these solvents can cancel the protective role played by the middle-ear reflex (MER). Voltage-dependent Ca(2+) channels (VDCCs) regulate acetylcholine release in the central synaptic network and control muscular excitation/contraction processes as well. In order to identify the prevailing action of Tol in the central or peripheral compartment of the MER arc, two VDCC antagonists were injected into the common carotid trunk: omega-conotoxin MVIIC, which blocks only the neuronal N- and P/Q-type Ca(2+) channels, or verapamil, which inhibits the muscular L-type Ca(2+) channels. Rats were also implanted with an electrode on the round window membrane to measure the cochlear microphonic potential (CMP) elicited with a band noise centered at 4 kHz and emitted at 85 dB sound pressure level. The variations in CMP recorded during the test compound injection showed that Tol has similar effects to those induced by omega-conotoxin, the neuronal VDCC blocker. The response obtained with the verapamil injection was broader than those obtained with Tol or conotoxin. This investigation therefore revealed that Tol can mimic the effects of VDCC blockers. The antagonist effects of Tol would be closer to neuronal than to muscular blockers and would be presumably located at the level of the integrator centers of the reflex.

Glutamate preconditioning prevents neuronal death induced by combined oxygen–glucose deprivation in cultured cortical neurons

The study of ischemic tolerance is critical in the development of strategies for the treatment of ischemic stroke. We used the oxygen and glucose deprivation (OGD) paradigm in cultured cortical neurons as an in vitro approach to elucidate the mechanism of protection conferred by glutamate preconditioning. Pretreatment of neurons with N-methyl-d-aspartate (NMDA) receptor antagonists prevented OGD-induced cell death whereas α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor and voltage-dependent Ca++ channel (VDCC) blockers were without effect. Neurons preconditioned with glutamate exhibited resistant to damage induced by OGD. The ischemic tolerance depended on the duration of preconditioning exposure and the interval between preconditioning exposure and test challenge. Protective efficacy was blocked by the NMDA or AMPA receptor antagonists but not by the VDCC blocker. Furthermore, neuroprotective effect was not seen if extracellular Ca++ was omitted or removed with EGTA. Pretreatment with staurosporin and 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)] amino-N-(4-chlorocinnamyl)-N-methylbenzylamine (KN93) but not 2-(4-Morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one (LY294002) or 1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio] butadiene (U0126) significantly reduced ischemic tolerance. Preconditioning increased phosphorylated levels of cAMP responsive element binding protein (CREB) and pretreatment with CRE-decoy oligonucleotide completely blocked preconditioning-induced increase in cell viability. Importantly, glutamate preconditioning increased Bcl-2 expression that was blocked by KN93, staurosporin and CRE-decoy oligonucleotide. These results suggest that preconditioning with glutamate conferred neuroprotection against subsequent OGD by inducing p-CREB-mediated Bcl-2 expression.

L‐type calcium channels are involved in fast endocytosis at the mouse neuromuscular junction

We used fluorescence microscopy of FM dyes-labeled synaptic vesicles and electrophysiological recordings to examine the functional characteristics of vesicle recycling and study how different types of voltage-dependent Ca(2+) channels (VDCCs) regulate the coupling of exocytosis and endocytosis at mouse neuromuscular junction. Our results demonstrate the presence of at least two different pools of recycling vesicles: a high-probability release pool (i.e. a fast destaining vesicle pool), which is preferentially loaded during the first 5 s (250 action potentials) at 50 Hz; and a low-probability release pool (i.e. a slow destaining vesicle pool), which is loaded during prolonged stimulation and keeps on refilling after end of stimulation. Our results suggest that a fast recycling pool mediates neurotransmitter release when vesicle use is minimal (i.e. during brief high-frequency stimulation), while vesicle mobilization from a reserve pool is the prevailing mechanism when the level of synaptic activity increases. We observed that specific N- and L-type VDCC blockers had no effect on evoked transmitter release upon low-frequency stimulation (5 Hz). However, at high-frequency stimulation (50 Hz), L-type Ca(2+) channel blocker increased FM2-10 destaining and at the same time diminished quantal release. Furthermore, when L-type channels were blocked, FM2-10 loading during stimulation was diminished, while the amount of endocytosis after stimulation was increased. Our experiments suggest that L-type VDCCs promote endocytosis of synaptic vesicles, directing the newly formed vesicles to a high-probability release pool where they compete against unused vesicles.