Inhibition Of T-type Calcium Channels Research Articles

The role of T-type calcium channels in elderly human vascular function: A pilot randomized controlled trial.

Endothelial dysfunction develops with age and may precede cardiovascular disease. Animal data suggest that T-type calcium channels play an important role in endothelial function, but data from humans are lacking. This study included 15 healthy, sedentary, elderly males for a double blinded, randomized controlled trial. For 8weeks, they were given 40mg/day of either efonidipine (L- and T-type calcium channel blocker (CCB)) or nifedipine (L-type CCB). Vascular function was evaluated by graded femoral arterial infusions of acetylcholine (ACh; endothelium-dependent vasodilator) and sodium nitroprusside (endothelium-independent vasodilator) both with and without co-infusion of N-acetylcysteine (NAC; antioxidant). We measured leg blood flow and mean arterial pressure and calculated leg vascular conductance to evaluate the leg vascular responses. Despite no significant change in blood pressure in either group, we observed higher leg blood flow responses (Δ 0.43±0.45l/min, P=0.006) and leg vascular conductance (Δ 5.38±5.67ml/min/mmHg, P=0.005) to intra-arterial ACh after efonidipine, whereas there was no change in the nifedipine group, and no differences between groups. We found no upregulation of endothelial nitric oxide synthase in vastus lateralis muscle biopsies within or between groups. Smooth muscle cell responsiveness was unaltered by efonidipine or nifedipine. Intravenous co-infusion of NAC did not affect endothelium-dependent vasodilatation in either of the CCB groups. These results suggest that 8weeks' inhibition of T- and L-type calcium channels augments endothelium-dependent vasodilatory function in healthy elderly males. Further studies are required to elucidate if T-type calcium channel inhibition can counteract endothelial dysfunction.

KTt-45, a T-type calcium channel blocker, acts as an anticancer agent by inducing apoptosis on HeLa cervical cancer cell line

The abnormal expression in the T-type calcium channels is involved in various cancer types, thus inhibiting T-type calcium channels is one of approaches in cancer treatment. The fact that KTt-45 acted as a T-type calcium channel inhibitor as well as a pain-relief agent prompts us to address if KTt-45 plays any role against cancer cells. The results showed that KTt-45 caused cytotoxic effects towards HeLa cervical, Raji lymphoma, MCF-7 breast cancer, and A549 lung cancer cell lines with IC50 values less than 100 μM, in which highly selective toxicity was against HeLa cells (IC50 = 37.4 μM, SI > 3.2). Strikingly, the KTt-45 induced an accumulation of cytoplasmic vacuoles after 48 h treatment and mitochondrial-dependent apoptosis activation as evidenced by morphological features, chromatin condensation, nuclear fragmentation, and significant activation of caspase-9 as well as caspase-3. In conclusion, KTt-45 could inhibit cell growth and trigger mitochondrial-dependent apoptosis in HeLa cervical cancer cells. The results, taken together, strongly demonstrated that KTt-45 is a potential agent for further study on anticancer drug development which not only targets cancer cells but also helps to relieve neuropathic pain in cancer patients.

ERG potassium channels and T-type calcium channels contribute to the pacemaker and atrioventricular conduction in zebrafish larvae.

Bradyarrhythmias result from inhibition of automaticity, prolonged repolarization, or slow conduction in the heart. The ERG channels mediate the repolarizing current IKr in the cardiac action potential, whereas T-type calcium channels (TTCC) are involved in the sinoatrial pacemaker and atrioventricular conduction in mammals. Zebrafish have become a valuable research model for human cardiac electrophysiology and disease. Here, we investigate the contribution of ERG channels and TTCCs to the pacemaker and atrioventricular conduction in zebrafish larvae and determine the mechanisms causing atrioventricular block. Zebrafish larvae expressing ratiometric fluorescent Ca2+ biosensors in the heart were used to measure Ca2+ levels and rhythm in beating hearts invivo, concurrently with contraction and hemodynamics. The atrioventricular delay (the time between the start of atrial and ventricular Ca2+ transients) was used to measure impulse conduction velocity and distinguished between slow conduction and prolonged refractoriness as the cause of the conduction block. ERG blockers caused bradycardia and atrioventricular block by prolonging the refractory period in the atrioventricular canal and in working ventricular myocytes. In contrast, inhibition of TTCCs caused bradycardia and second-degree block (Mobitz type I) by slowing atrioventricular conduction. TTCC block did not affect ventricular contractility, despite being highly expressed in cardiomyocytes. Concomitant measurement of Ca2+ levels and ventricular size showed mechano-mechanical coupling: increased preload resulted in a stronger heart contraction invivo. ERG channels and TTCCs influence the heart rate and atrioventricular conduction in zebrafish larvae. The zebrafish lines expressing Ca2+ biosensors in the heart allow us to investigate physiological feedback mechanisms and complex arrhythmias.

Nanomedicine-based co-delivery of a calcium channel inhibitor and a small molecule targeting CD47 for lung cancer immunotherapy

Pro-tumoral macrophages in lung tumors present a significant challenge in immunotherapy. Here, we introduce a pH-responsive nanomedicine approach for activating anti-tumoral macrophages and dendritic cells. Using a layered double hydroxide nanosheet carrier, we co-deliver a T-type calcium channel inhibitor (TTA-Q6) and a CD47 inhibitor (RRX-001) into lung tumors. In the tumor acidic environment, TTA-Q6 is released, disrupting cancer cell calcium uptake, causing endoplasmic reticulum stress and inducing calreticulin transfer to the cell surface. Surface calreticulin activates macrophages and triggers dendritic cell maturation, promoting effective antigen presentation and therefore activating antitumor T cells. Simultaneously, RRX-001 reduces CD47 protein levels, aiding in preventing immune escape by calreticulin-rich cancer cells. In lung tumor models in male mice, this combined approach shows anti-tumor effects and immunity against tumor re-exposure, highlighting its potential for lung cancer immunotherapy.

Modulation of FDG Uptake by Cell Cycle Synchronization Using a T-Type Calcium Channel Inhibitor.

We investigated whether cell cycle synchronization induced by the T-type calcium channel inhibitor mibefradil could increase tumoral 2-[18F] fluoro-2-deoxy-d-glucose (FDG) uptake in vitro and in vivo. Human prostate cancer cells (PC-3) were treated with 10 μM mibefradil for 24, 48, and 72 h to induce G1 arrest. Cell cycle distribution was analyzed at 0, 4, 8, 12, 15, 18, and 24 h after mibefradil withdrawal. Cellular uptake was measured after incubating cells with [3H] Deoxy-d-Glucose (DDG) for 1 h at the same time points used in the cell cycle analysis. The correlation between [3H] DDG uptake and each cell cycle phase was evaluated in the early (0-12 h) and late phases (15-24 h) of synchronization. In vivo FDG PET imaging was performed in PC-3-bearing mice at baseline, 24 h, and 48 h after mibefradil treatment. The G0/G1 fraction of PC-3 cells was significantly increased from 33.1% ± 0.2% to 60.9% ± 0.8% after 24 h mibefradil treatment, whereas the S and G2/M fractions were decreased from 36.3% ± 1.4% to 23.2% ± 1.1% and from 29.7% ± 1.3% to 14.9% ± 0.9%, respectively, which were similar to the results by serum starvation. Mibefradil treatment for 24, 48, and 72 h increased the number of cells in S phase at 18-24 h after withdrawal; however, only the 72 h treatment increased [3H] DDG uptake (145.8 ± 5.8% of control at 24 h after withdrawal). [3H] DDG uptake was positively correlated with the size of the S phase fraction and negatively correlated with the size of the G0/G1 fraction in the late phase of synchronization. DDG uptake was significantly increased by mibefradil-induced cell cycle synchronization and correlated with the sizes of cell cycle fractions. In vivo FDG PET imaging also demonstrated a significant increase in tumor uptake after mibefradil treatment. Quantified tumor FDG uptake (%ID/g) increased from 4.13 ± 2.10 to 4.7 ± 2.16 at 24 h, and 5.95 ± 2.57 at 48 h (p < 0.05). Cell cycle synchronization could be used to increase the diagnostic sensitivity of clinical FDG positron emission tomography.

T-type Calcium Channel Blocker, NNC 55-0396, Suppress Cell Proliferation and Promote Apoptosis in SNU-1 Gastric Cancer Cells

Accumulating evidence reports that T-type calcium channels play crucial roles in tumor formation and development. However, the roles of inhibiting calcium channels in tumor cells with various inhibitors in tumor progression remain unclear. This study aimed to investigate the cytotoxic and apoptotic effects of NNC 55-0396, a T-type calcium channel inhibitor, against SNU-1 gastric cancer cells. Cytotoxic and apoptotic effects of NNC 55-0396 were evaluated by the XTT assay and flow cytometry. The results showed that NNC 55-0396 had concentration-dependent cytotoxicity in SNU-1 cells and its the half-maximal inhibitory concentration (IC50) value was calculated as 4.17 µM. The results of the Annexin V experiments also showed that this inhibitor significantly increased apoptosis in SNU-1 cells. In conclusion, these results demonstrated that NNC 55-0396 induces cytotoxic effects by increasing apoptosis in gastric cancer cells. However, further research is required for its use as a possible therapeutic agent in the treatment of gastric cancer.

Inhibition of T-Type Calcium Channels With TTA-P2 Reduces Chronic Neuropathic Pain Following Spinal Cord Injury in Rats

Spinal cord injury (SCI)-induced neuropathic pain (SCI-NP) develops in up to 60 to 70% of people affected by traumatic SCI, leading to a major decline in quality of life and increased risk for depression, anxiety, and addiction. Gabapentin and pregabalin, together with antidepressant drugs, are commonly prescribed to treat SCI-NP, but their efficacy is unsatisfactory. The limited efficacy of current pharmacological treatments for SCI-NP likely reflects our limited knowledge of the underlying mechanism(s) responsible for driving the maintenance of SCI-NP. The leading hypothesis in the field supports a major role for spontaneously active injured nociceptors in driving the maintenance of SCI-NP. Recent data from our laboratory provided additional support for this hypothesis and identified the T-type calcium channels as key players in driving the spontaneous activity of SCI-nociceptors, thus providing a rational pharmacological target to treat SCI-NP. To test whether T-type calcium channels contribute to the maintenance of SCI-NP, male and female SCI and sham rats were treated with TTA-P2 (a blocker of T-type calcium channels) to determine its effects on mechanical hypersensitivity (as measured with the von Frey filaments) and spontaneous ongoing pain (as measured with the conditioned place preference paradigm), and compared them to the effects of gabapentin, a blocker of high voltage–activated calcium channels. We found that both TTA-P2 and gabapentin reduced mechanical hypersensitivity in male and females SCI rats, but surprisingly only TTA-P2 reduced spontaneous ongoing pain in male SCI rats. PerspectivesSCI-induced neuropathic pain, and in particular the spontaneous ongoing pain component, is notoriously very difficult to treat. Our data provide evidence that inhibition of T-type calcium channels reduces spontaneous ongoing pain in SCI rats, supporting a clinically relevant role for T-type channels in the maintenance of SCI-induced neuropathic pain.

Structurally modified Cyclovirobuxine-D Buxus alkaloids as effective analgesic agents through Cav3.2 T-Type calcium channel inhibition

Cyclovirobuxine-D (CVB-D) is a Buxus alkaloid and a major active constituent in the Chinese medicinal herb Buxus microphylls. Traditionally, the natural alkaloid cyclovirobuxine-D has a long history of use as a traditional Chinese medicine for cardiovascular diseases as well as to treat a wide variety of medical conditions. As we found that CVB-D inhibited T-type calcium channels, we designed and synthesized a variety of fragments and analogues and evaluated them for the first time as new Cav3.2 inhibitors. Compounds 2–7 exhibited potency against Cav 3.2 channels, and two of them were more active than their parent molecules. As a result of the in vivo experiments, both compounds 3 and 4 showed significantly reduced writhes in the acetic acid-induced writhing test. Studies of molecular modeling have identified possible mechanism(s) of Cav3.2 binding. Moreover, the relationship between structure and activity was studied in a preliminary manner. Our results indicated that compounds 3 and 4 could play an important role in the discovery and development of novel analgesics.

Discovery of pimozide derivatives as novel T-type calcium channel inhibitors with little binding affinity to dopamine D2 receptors for treatment of somatic and visceral pain

T-type Ca2+ channels (T-channels), particularly Cav3.2 and Cav3.1 isoforms, are promising targets for treating various diseases including intractable pain. Given the potent inhibitory activity of pimozide, an antipsychotic, against T-channels, we conducted structure–activity relationship studies of pimozide derivatives, and identified several compounds including 3a, 3s, and 4 that had potency comparable to that of pimozide in inhibiting T-channels, but little binding affinity to dopamine D2 receptors. The introduction of a phenylbutyl group on the benzoimidazole nuclei of pimozide was considered a key structural modification to reduce the binding affinity to D2 receptors. Those pimozide derivatives potently suppressed T-channel-dependent somatic and visceral pain in mice, without causing any motor dysfunctions attributable to D2 receptor blockade, including catalepsy. The present study thus provides an avenue to develop novel selective T-channel inhibitors available for pain management via the structural modification of existing medicines.

Icetexane diterpenoids as Cav3.2 T-type calcium channel inhibitors from Salvia prattii and analgesic effect of their Semi-synthesized derivatives

Ten new icetexane diterpenoids, salpratins E–N (1–10) and a known analogue (11) were characterized from Salvia prattii Hemsl. Structurally, 1 is the first 19(4 → 3)-abeo-icetexane diterpenoid featuring with a 6/7/6 ring system. The structures of isolated compounds were determined by comprehensive analyses of spectroscopic data, ECD calculation, and single-crystal X-ray diffraction. Biological studies initially revealed that 1, 7, 10, and 11 are notable Cav3.2 T-type Ca2+ channel (TTCC) inhibitors with IC50 values of 2.9, 5.1, 2.3, and 3.2 μM, respectively. Five icetexane related derivatives (13–17) were synthesized from an abietane type precursor, (+)-carnosic acid (12), for the purpose of overcoming the poor water solubility of aforementioned active compounds and further investigating diverse diterpenes with valuable activity. Among them, 13 and 14 showed potent inhibitions on Cav3.2, having IC50 values of 6.7 and 2.4 μM, respectively. Significantly, they exhibited dose-dependent (1, 3, and 10 mg/kg) and comparable analgesic effects as that of Z944, a TTCCs inhibitor under clinical trial for pain management, in the mouse acetic acid writhing test. These findings further enrich structural diversity and bioactivity of Salvia diterpenoids, as well as provide promising structural templates for the development of Cav3.2 analgesics.

The Tarantula Toxin ω-Avsp1a Specifically Inhibits Human CaV3.1 and CaV3.3 via the Extracellular S3-S4 Loop of the Domain 1 Voltage-Sensor

Inhibition of T-type calcium channels (CaV3) prevents development of diseases related to cardiovascular and nerve systems. Further, knockout animal studies have revealed that some diseases are mediated by specific subtypes of CaV3. However, subtype-specific CaV3 inhibitors for therapeutic purposes or for studying the physiological roles of CaV3 subtypes are missing. To bridge this gap, we employed our spider venom library and uncovered that Avicularia spec. (“Amazonas Purple”, Peru) tarantula venom inhibited specific T-type CaV channel subtypes. By using chromatographic and mass-spectrometric techniques, we isolated and sequenced the active toxin ω-Avsp1a, a C-terminally amidated 36 residue peptide with a molecular weight of 4224.91 Da, which comprised the major peak in the venom. Both native (4.1 μM) and synthetic ω-Avsp1a (10 μM) inhibited 90% of CaV3.1 and CaV3.3, but only 25% of CaV3.2 currents. In order to investigate the toxin binding site, we generated a range of chimeric channels from the less sensitive CaV3.2 and more sensitive CaV3.3. Our results suggest that domain-1 of CaV3.3 is important for the inhibitory effect of ω-Avsp1a on T-type calcium channels. Further studies revealed that a leucine of T-type calcium channels is crucial for the inhibitory effect of ω-Avsp1a.

Putative Synthetic Cannabinoids MEPIRAPIM, 5F-BEPIRAPIM (NNL-2), and Their Analogues Are T-Type Calcium Channel (CaV3) Inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are a large and growing class of new psychoactive substances (NPSs). Two recently identified compounds, MEPIRAPIM and 5F-BEPIRAPIM (NNL-2), have not been confirmed as agonists of either cannabinoid receptor subtype but share structural similarities with both SCRAs and a class of T-type calcium channel (CaV3) inhibitors under development as new treatments for epilepsy and pain. In this study, MEPIRAPIM and 5F-BEPIRAPIM and 10 systematic analogues were synthesized, analytically characterized, and pharmacologically evaluated using in vitro cannabinoid receptor and CaV3 assays. Several compounds showed micromolar affinities for CB1 and/or CB2, with several functioning as low potency agonists of CB1 and CB2 in a membrane potential assay. 5F-BEPIRAPIM and four other derivatives were identified as potential CaV3 inhibitors through a functional calcium flux assay (>70% inhibition), which was further confirmed using whole-cell patch-clamp electrophysiology. Additionally, MEPIRAPIM and 5F-BEPIRAPIM were evaluated in vivo using a cannabimimetic mouse model. Despite detections of MEPIRAPIM and 5F-BEPIRAPIM in the NPS market, only the highest MEPIRAPIM dose (30 mg/kg) elicited a mild hypothermic response in mice, with no hypothermia observed for 5F-BEPIRAPIM, suggesting minimal central CB1 receptor activity.

T-Type Calcium Channel Inhibitors Induce Apoptosis in Medulloblastoma Cells Associated with Altered Metabolic Activity

Medulloblastoma (MB) is the most common malignant paediatric brain tumour. In our previous studies, we developed a novel 3D assay for MB cells that was used to screen a panel of plasma membrane calcium channel modulators for their effect on the 3D growth of D341 MB cells. These studies identified T-type (CaV3) channel inhibitors, mibefradil and NNC-55–0396 (NNC) as selective inhibitors of MB cell growth. Mibefradil was originally approved for the treatment of hypertension and angina pectoris, and recently successfully completed a phase I trial for recurrent high-grade glioma. NNC is an analogue of mibefradil with multiple advantages compared to mibefradil that makes it attractive for potential future clinical trials. T-type channels have a unique low voltage-dependent activation/inactivation, and many studies suggest that they have a direct regulatory role in controlling Ca2+ signalling in non-excitable tissues, including cancers. In our previous study, we also identified overexpression of CaV3.2 gene in MB tissues compared to normal brain tissues. In this study, we aimed to characterise the effect of mibefradil and NNC on MB cells and elucidate their mechanism of action. This study demonstrates that the induction of toxicity in MB cells is selective to T-type but not to L-type Ca2+ channel inhibitors. Addition of CaV3 inhibitors to vincristine sensitised MB cells to this MB chemotherapeutic agent, suggesting an additive effect. Furthermore, CaV3 inhibitors induced cell death in MB cells via apoptosis. Supported by proteomics data and cellular assays, apoptotic cell death was associated with reduced mitochondrial membrane potential and reduced ATP levels, which suggests that both compounds alter the metabolism of MB cells. This study offers new insights into the action of mibefradil and NNC and will pave the way to test these molecules or their analogues in pre-clinical MB models alone and in combination with vincristine to assess their suitability as a potential MB therapy.

Endothelium-derived hydrogen sulfide acts as a hyperpolarizing factor and exerts neuroprotective effects via activation of large-conductance Ca2+ -activated K+ channels.

Endothelium-derived hyperpolarizing factor (EDHF) has been suggested as a therapeutic target for vascular protection against ischaemic brain injury. However, the molecular entity of EDHF and its action on neurons remains unclear. This study was undertaken to demonstrate whether the hydrogen sulfide (H2 S) acts as EDHF and exerts neuroprotective effect via large-conductance Ca2+ -activated K+ (BKCa /KCa 1.1) channels. The whole-cell patch-clamp technology was used to record the changes of BKCa currents in rat neurons induced by EDHF. The cerebral ischaemia/reperfusion model of mice and oxygen-glucose deprivation/reoxygenation (OGD/R) model of neurons were used to explore the neuroprotection of EDHF by activating BKCa channels in these neurons. Increases of BKCa currents and membrane hyperpolarization in hippocampal neurons induced by EDHF could be markedly inhibited by BKCa channel inhibitor iberiotoxin or endothelial H2 S synthase inhibitor propargylglycine. The H2 S donor, NaHS-induced BKCa current and membrane hyperpolarization in neurons were also inhibited by iberiotoxin, suggesting that H2 S acts as EDHF and activates the neuronal BKCa channels. Besides, we found that the protective effect of endothelium-derived H2 S against mice cerebral ischaemia/reperfusion injury was disrupted by iberiotoxin. Importantly, the inhibitory effect of NaHS or BKCa channel opener on OGD/R-induced neuron injury and the increment of intracellular Ca2+ level could be inhibited by iberiotoxin but enhanced by co-application with L-type but not T-type calcium channel inhibitor. Endothelium-derived H2 S acts as EDHF and exerts neuroprotective effects via activating the BKCa channels and then inhibiting the T-type calcium channels in hippocampal neurons.

The mineralocorticoid receptor leads to increased expression of EGFR and T-type calcium channels that support HL-1 cell hypertrophy

The EGF receptor (EGFR) has been extensively studied in tumor biology and recently a role in cardiovascular pathophysiology was suggested. The mineralocorticoid receptor (MR) is an important effector of the renin–angiotensin–aldosterone-system and elicits pathophysiological effects in the cardiovascular system; however, the underlying molecular mechanisms are unclear. Our aim was to investigate the importance of EGFR for MR-mediated cardiovascular pathophysiology because MR is known to induce EGFR expression. We identified a SNP within the EGFR promoter that modulates MR-induced EGFR expression. In RNA-sequencing and qPCR experiments in heart tissue of EGFR KO and WT mice, changes in EGFR abundance led to differential expression of cardiac ion channels, especially of the T-type calcium channel CACNA1H. Accordingly, CACNA1H expression was increased in WT mice after in vivo MR activation by aldosterone but not in respective EGFR KO mice. Aldosterone- and EGF-responsiveness of CACNA1H expression was confirmed in HL-1 cells by Western blot and by measuring peak current density of T-type calcium channels. Aldosterone-induced CACNA1H protein expression could be abrogated by the EGFR inhibitor AG1478. Furthermore, inhibition of T-type calcium channels with mibefradil or ML218 reduced diameter, volume and BNP levels in HL-1 cells. In conclusion the MR regulates EGFR and CACNA1H expression, which has an effect on HL-1 cell diameter, and the extent of this regulation seems to depend on the SNP-216 (G/T) genotype. This suggests that the EGFR may be an intermediate for MR-mediated cardiovascular changes and that SNP analysis can help identify subgroups of patients that will benefit most from MR antagonists.

Inhibitory Mechanism of the Isoflavone Derivative Genistein in the Human CaV3.3 Channel.

Regulation of cellular excitability and oscillatory behavior of resting membrane potential in nerve cells are largely mediated by the low-voltage activated T-type calcium channels. This calcium channel family is constituted by three isoforms, namely, CaV3.1, CaV3.2, and CaV3.3, that are largely distributed in the nervous system and other parts of the body. Dysfunction of T-type calcium channels is associated with a wide range of pathophysiologies including epilepsy, neuropathic pain, cardiac problems, and major depressive disorders. Due to their pharmacological relevance, finding molecular agents able to modulate the channel's function may provide therapeutic means to ameliorate their related disorders. Here we used electrophysiological experiments to show that genistein, a canonical tyrosine kinase inhibitor, reduces the activity of the human CaV3.3 channel in a concentration-dependent manner. The inhibitory effect of genistein is independent of tyrosine kinase modulation and does not affect the voltage-dependent gating of the channel. Subsequently, we used computational methods to identify plausible molecular poses for the interaction of genistein and the CaV3.3 channel. Starting from different molecular poses, we carried out all-atom molecular dynamics (MD) simulations to identify the interacting determinants for the CaV3.3/genistein complex formation. Our extensive (microsecond-length) simulations suggest specific binding interactions that seem to stabilize the protein/inhibitor complex. Furthermore, our results from the unbiased MD simulations are in good agreement with the recently solved cryoelectron microscopy structure of the CaV3.1/Z944 complex in terms of both the location of the ligand binding site and the role of several equivalent amino acid residues. Proposed interacting complex loci were subsequently tested and corroborated by electrophysiological experiments using another naturally occurring isoflavone derivative, daidzein. Thus, by using a combination of in vitro and in silico techniques, we have identified interacting determinants relevant to the CaV3.3/genistein complex formation and propose that genistein directly blocks the function of the human CaV3.3 channel as a result of such interaction. Specifically, we proposed that a combination of polar interactions involving the three hydroxyl groups of genistein and an aromatic interaction with the fused rings are the main binding interactions in the complex formation. Our results pave the way for the rational development of improved and novel low-voltage activated T-type calcium channel inhibitors.

Mibefradil and Flunarizine, Two T-Type Calcium Channel Inhibitors, Protect Mice against Lipopolysaccharide-Induced Acute Lung Injury.

Recent studies have illuminated that blocking Ca2+ influx into effector cells is an attractive therapeutic strategy for lung injury. We hypothesize that T-type calcium channel may be a potential therapeutic target for acute lung injury (ALI). In this study, the pharmacological activity of mibefradil (a classical T-type calcium channel inhibitor) was assessed in a mouse model of lipopolysaccharide- (LPS-) induced ALI. In LPS challenged mice, mibefradil (20 and 40 mg/kg) dramatically decreased the total cell number, as well as the productions of TNF-α and IL-6 in bronchoalveolar lavage fluid (BALF). Mibefradil also suppressed total protein concentration in BALF, attenuated Evans blue extravasation, MPO activity, and NF-κB activation in lung tissue. Furthermore, flunarizine, a widely prescripted antimigraine agent with potent inhibition on T-type channel, was also found to protect mice against lung injury. These data demonstrated that T-type calcium channel inhibitors may be beneficial for treating acute lung injury. The important role of T-type calcium channel in the acute lung injury is encouraged to be further investigated.

T-type calcium channel blockade induces apoptosis in C2C12 myotubes and skeletal muscle via endoplasmic reticulum stress activation.

Loss of T-type calcium channel (TCC) function has been reported to result in decreased cell viability and impaired muscle regeneration, but the underlying mechanisms remain largely unknown. We previously found that expression of TCC is reduced in aged pelvic floor muscle of multiple vaginal delivery mice, and this is related to endoplasmic reticulum stress (ERS) activation and autophagy flux blockade. In the present work, we further investigated the effects of TCC function loss on C2C12 myotubes and skeletal muscle, which is mediated by promotion of ERS and ultimately contributes to mitochondrial-related apoptotic cell death. We found that application of a TCC inhibitor induced mitochondria-related apoptosis in a dose-dependent manner and also reduced mitochondrial transmembrane potential (MMP), induced mito-ROS generation, and enhanced expression of mitochondrial apoptosis proteins. Functional inhibition of TCC induced ERS, resulting in disorder of Ca2+ homeostasis in endoplasmic reticulum, and ultimately leading to cell apoptosis in C2C12 myotubes. Tibialis anterior muscles of T-type α1H channel knockout mice displayed a smaller skeletal muscle fiber size and elevated ERS-mediated apoptosis signaling. Our data point to a novel mechanism whereby TCC blockade leads to ERS activation and terminal mitochondrial-related apoptotic events in C2C12 myotubes and skeletal muscles.

Contribution of T-Type Calcium Channels to Spinal Cord Injury-Induced Hyperexcitability of Nociceptors.

A hyperexcitable state and spontaneous activity of nociceptors have been suggested to play a critical role in the development of chronic neuropathic pain following spinal cord injury (SCI). In male rats, we employed the action potential-clamp technique to determine the underlying ionic mechanisms responsible for driving SCI-nociceptors to a hyperexcitable state and for triggering their spontaneous activity. We found that the increased activity of low voltage activated T-type calcium channels induced by the injury sustains the bulk (∼60-70%) of the inward current active at subthreshold voltages during the interspike interval in SCI-nociceptors, with a modest contribution (∼10-15%) from tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. In current-clamp recordings, inhibition of T-type calcium channels with 1 μm TTA-P2 reduced both the spontaneous and the evoked firing in response to current injections in SCI-nociceptors to a level similar to sham-nociceptors. Electrophysiology in vitro was then combined with the conditioned place preference (CPP) paradigm to determine the relationship between the increased activity of T-type channels in SCI-nociceptors and chronic neuropathic pain following SCI. The size of the interspike T-type calcium current recorded from nociceptors isolated from SCI rats showing TTA-P2-induced CPP (responders) was ∼6 fold greater than the interspike T-type calcium current recorded from nociceptors isolated from SCI rats without TTA-P2-induced CPP (non-responders). Taken together, our data suggest that the increased activity of T-type calcium channels induced by the injury plays a primary role in driving SCI-nociceptors to a hyperexcitable state and contributes to chronic neuropathic pain following SCI.SIGNIFICANCE STATEMENT Chronic neuropathic pain is a major comorbidity of spinal cord injury (SCI), affecting up to 70-80% of patients. Anticonvulsant and tricyclic antidepressant drugs are first line analgesics used to treat SCI-induced neuropathic pain, but their efficacy is very limited. A hyperexcitable state and spontaneous activity of SCI-nociceptors have been proposed as a possible underlying cause for the development of chronic neuropathic pain following SCI. Here, we show that the increased activity of T-type calcium channels induced by the injury plays a major role in driving SCI-nociceptors to a hyperexcitable state and for promoting their spontaneous activity, suggesting that T-type calcium channels may represent a pharmacological target to treat SCI-induced neuropathic pain.

606 Small molecule blockade of T-type calcium channels by DX416 inhibits itch and reduces corresponding inflammation in acute and chronic itch mouse models

The processing of itch signals is regulated by many different ion channels. Among those are T-type calcium channels which are important regulators that contribute to the firing behavior of pruriceptors, or itch sensing neurons. The human genome encodes three different T-type channels: Cav3.1, Cav3.2, Cav3.3. It has been shown recently that pharmacological blockade of T-type calcium channels inhibits acute itch and that immune cells can express Cav 3 channels with roles in effector function. Thus, we hypothesized that local administration of the novel small molecule T-Type calcium channel inhibitor, DX416, would decrease cutaneous itch both through reducing pruriceptors signals triggering itch and decreasing local inflammatory immune responses potentiating itch.