Combination of the CRAC Channel Inhibitor CM4620 and Galactose as a Potential Therapy for Acute Pancreatitis.

Acute pancreatitis (AP) is a life-threatening inflammatory disease with no specific therapy. Excessive cytoplasmic Ca2+ elevation and intracellular ATP depletion are responsible for the initiation of AP. Inhibition of Ca2+ release-activated Ca2+ (CRAC) channels has been proposed as a potential treatment, and currently, a novel selective CRAC channel inhibitor CM4620 (Auxora, CalciMedica) is in Phase 2b human trials. While CM4620 is on track to become the first effective treatment for AP, it does not produce complete protection in animal models. Recently, an alternative approach has suggested reducing ATP depletion with a natural carbohydrate galactose. Here, we have investigated the possibility of using the smallest effective concentration of CM4620 in combination with galactose. Protective effects of CM4620, in the range of 1-100 n m, have been studied against necrosis induced by bile acids, palmitoleic acid, or l-asparaginase. CM4620 markedly protected against necrosis induced by bile acids or asparaginase starting from 50 n m and palmitoleic acid starting from 1 n m. Combining CM4620 and galactose (1 m m) significantly reduced the extent of necrosis to near-control levels. In the palmitoleic acid-alcohol-induced experimental mouse model of AP, CM4620 at a concentration of 0.1 mg/kg alone significantly reduced edema, necrosis, inflammation, and the total histopathological score. A combination of 0.1 mg/kg CM4620 with galactose (100 m m) significantly reduced further necrosis, inflammation, and histopathological score. Our data show that CM4620 can be used at much lower concentrations than reported previously, reducing potential side effects. The novel combination of CM4620 with galactose synergistically targets complementary pathological mechanisms of AP.

Investigating the therapeutic effects of nimodipine on vasogenic cerebral edema and blood-brain barrier impairment in an ischemic stroke rat model.

Vasogenic brain edema, a potentially life-threatening consequence following an acute ischemic stroke, is a major clinical problem. This research aims to explore the therapeutic benefits of nimodipine, a calcium channel blocker, in mitigating vasogenic cerebral edema and preserving blood-brain barrier (BBB) function in an ischemic stroke rat model. In this research, animals underwent the induction of ischemic stroke via a 60-min blockage of the middle cerebral artery and treated with a nonhypotensive dose of nimodipine (1 mg/kg/day) for a duration of five days. The wet/dry method was employed to identify cerebral edema, and the Evans blue dye extravasation technique was used to assess the permeability of the BBB. Furthermore, immunofluorescence staining was utilized to assess the protein expression levels of matrix metalloproteinase-9 (MMP-9) and intercellular adhesion molecule-1 (ICAM-1). The study also examined mitochondrial function by evaluating mitochondrial swelling, succinate dehydrogenase (SDH) activity, the collapse of mitochondrial membrane potential (MMP), and the generation of reactive oxygen species (ROS). Post-stroke administration of nimodipine led to a significant decrease in cerebral edema and maintained the integrity of the BBB. The protective effects observed were associated with a reduction in cell apoptosis as well as decreased expression of MMP-9 and ICAM-1. Furthermore, nimodipine was observed to reduce mitochondrial swelling and ROS levels while simultaneously restoring MMP and SDH activity. These results suggest that nimodipine may reduce cerebral edema and BBB breakdown caused by ischemia/reperfusion. This effect is potentially mediated through the reduction of MMP-9 and ICAM-1 levels and the enhancement of mitochondrial function.

Nimodipine Protects Vascular and Cognitive Function in an Animal Model of Cerebral Small Vessel Disease.

BACKGROUND: Cerebral small vessel disease is a common cause of vascular cognitive impairment and dementia. There is an urgent need for preventative treatments for vascular cognitive impairment and dementia, and reducing vascular dysfunction may provide a therapeutic route. Here, we investigate whether the chronic administration of nimodipine, a central nervous system-selective dihydropyridine calcium channel blocking agent, protects vascular, metabolic, and cognitive function in an animal model of cerebral small vessel disease, the spontaneously hypertensive stroke-prone rat. METHODS: Male spontaneously hypertensive stroke-prone rats were randomly allocated to receive either a placebo (n=24) or nimodipine (n=24) diet between 3 and 6 months of age. Animals were examined daily for any neurological deficits, and vascular function was assessed in terms of neurovascular and neurometabolic coupling at 3 and 6 months of age, and cerebrovascular reactivity at 6 months of age. Cognitive function was evaluated using the novel object recognition test at 6 months of age. RESULTS: Six untreated control animals were terminated prematurely due to strokes, including one due to seizure, but no treated animals experienced strokes and so had a higher survival (P=0.0088). Vascular function was significantly impaired with disease progression, but nimodipine treatment partially preserved neurovascular coupling and neurometabolic coupling, indicated by larger (P<0.001) and more prompt responses (P<0.01), and less habituation upon repeated stimulation (P<0.01). Also, animals treated with nimodipine showed greater cerebrovascular reactivity, indicated by larger dilation of arterioles (P=0.015) and an increase in blood flow velocity (P=0.001). This protection of vascular and metabolic function achieved by nimodipine treatment was associated with better cognitive function (P<0.001) in the treated animals. CONCLUSIONS: Chronic treatment with nimodipine protects from strokes, and vascular and cognitive deficits in spontaneously hypertensive stroke-prone rat. Nimodipine may provide an effective preventive treatment for stroke and cognitive decline in cerebral small vessel disease.

L-type calcium channel regulation of depression, anxiety and anhedonia-related behavioral phenotypes following chronic stress exposure.

Exposure to chronic and unpredictable stressors can precipitate mood-related disorders in humans, particularly in individuals with pre-existing mental health challenges. L-type calcium channels (LTCCs) have been implicated in numerous neuropsychiatric disorders, as LTCC encoding genes have been identified as candidate risk factors for neuropsychiatric illnesses. In these sets of experiments, we sought to examine the ability of LTCC blockade to alter depression, anxiety, and anhedonic-related behavioral responses to chronic unpredictable stress (CUS) exposure in female and male rats. Rats first underwent either 21 days of CUS or no exposure to chronic stressors, serving as home cage controls (HCC). Then rats were examined for anhedonia-related behavior, anxiety and depression-like behavioral responses as measured by the sucrose preference test (SPT), elevated plus maze (EPM), and forced swim test (FST). CUS exposed females and males showed anhedonic and anxiogenic-like behavioral responses on the SPT and EPM, respectively, when compared to HCCs. In female and male rats, systemic administration of the LTCC blocker isradipine (0.4 mg/kg and 1.2 mg/kg, I.P.) attenuated the CUS-induced decrease in sucrose preference and reversed the CUS-induced decrease in open arm time. In the FST, systemic isradipine decreased immobility time across all groups, consistent with an antidepressant-like response. However, there were no significant differences in forced swim test immobility time between HCC and CUS exposed animals. Taken together, these data point to a role of LTCCs in the regulation of mood disorder-related behavioral phenotype responses to chronic stress exposure.

Nimodipine inhibits spreading depolarization, ischemic injury, and neuroinflammation in mouse live brain slice preparations.

Nimodipine is used to prevent delayed ischemic deficit in patients with aneurysmal subarachnoid hemorrhage (aSAH). Spreading depolarization (SD) is recognized as a factor in the pathomechanism of aSAH and other acute brain injuries. Although nimodipine is primarily known as a cerebral vasodilator, it may have a more complex mechanism of action due to the expression of its target, the L-type voltage-gated calcium channels (LVGCCs) in various cells in neural tissue. This study was designed to investigate the direct effect of nimodipine on SD, ischemic tissue injury, and neuroinflammation. SD in control or nimodipine-treated live mouse brain slices was induced under physiological conditions using electrical stimulation, or by subjecting the slices to hypo-osmotic stress or mild oxygen-glucose deprivation (mOGD). SD was recorded applying local field potential recording or intrinsic optical signal imaging. Histological analysis was used to estimate tissue injury, the number of reactive astrocytes, and the degree of microglia activation. Nimodipine did not prevent SD occurrence in mOGD, but it did reduce the rate of SD propagation and the cortical area affected by SD. In contrast, nimodipine blocked SD occurrence in hypo-osmotic stress, but had no effect on SD propagation. Furthermore, nimodipine prevented ischemic injury associated with SD in mOGD. Nimodipine also exhibited anti-inflammatory effects in mOGD by reducing reactive astrogliosis and microglial activation. The results demonstrate that nimodipine directly inhibits SD, independent of nimodipine's vascular effects. Therefore, the use of nimodipine may be extended to treat acute brain injuries where SD plays a central role in injury progression.

Pharmacological Research Progress of Novel Antihypertensive Drugs.

Cardiovascular disease stands as the leading cause of death globally, with hypertension emerging as an independent risk factor for its development. The worldwide prevalence of hypertension hovers around 30%, encompassing a staggering 1.2 billion patients, and continues to escalate annually. Medication plays a pivotal role in managing hypertension, not only effectively regulating blood pressure (BP) but also substantially mitigating the occurrence of cardiovascular and cerebrovascular diseases. This review comprehensively outlines the categories, mechanisms, clinical applications, and drawbacks of conventional antihypertensive drugs. It delves into the five primary pharmacological classifications, namely ß-receptor blockers, calcium channel blockers (CCBs), angiotensin-converting enzyme inhibitors (ACEIs), angiotensin receptor blockers (ARBs), and diuretics. The emphasis is placed on elucidating the mechanisms, advantages, and research progress of novel antihypertensive drugs targeting emerging areas. These include mineralocorticoid receptor antagonists (MRAs), atrial natriuretic peptides (ANPs), neutral endopeptidase inhibitors (NEPIs), sodium-dependent glucose transporter 2 inhibitors (SGLT-2Is), glucagon-like peptide-1 receptor agonists (GLP-1RAs), endothelin receptor antagonists (ERAs), soluble guanylate cyclase (sGC) agonists, brain aminopeptidase A inhibitors (APAIs), and small interfering ribonucleic acids (siRNAs) targeting hepatic angiotensinogen. Compared to conventional antihypertensive drugs, these novel alternatives exhibit favorable antihypertensive effects with minimal adverse reactions. This review serves as a valuable reference for future research and the clinical application of antihypertensive drugs.

Chamaecyparis lawsoniana and Its Active Compound Quercetin as Ca2+ Inhibitors in the Contraction of Airway Smooth Muscle.

The Cupressaceae family includes species considered to be medicinal. Their essential oil is used for headaches, colds, cough, and bronchitis. Cedar trees like Chamaecyparis lawsoniana (C. lawsoniana) are commonly found in urban areas. We investigated whether C. lawsoniana exerts some of its effects by modifying airway smooth muscle (ASM) contractility. The leaves of C. lawsoniana (363 g) were pulverized mechanically, and extracts were obtained by successive maceration 1:10 (w:w) with methanol/CHCl3. Guinea pig tracheal rings were contracted with KCl, tetraethylammonium (TEA), histamine (HIS), or carbachol (Cch) in organ baths. In the Cch experiments, tissues were pre-incubated with D-600, an antagonist of L-type voltage-dependent Ca2+ channels (L-VDCC) before the addition of C. lawsoniana. Interestingly, at different concentrations, C. lawsoniana diminished the tracheal contractions induced by KCl, TEA, HIS, and Cch. In ASM cells, C. lawsoniana significantly diminished L-type Ca2+ currents. ASM cells stimulated with Cch produced a transient Ca2+ peak followed by a sustained plateau maintained by L-VDCC and store-operated Ca2+ channels (SOCC). C. lawsoniana almost abolished this last response. These results show that C. lawsoniana, and its active metabolite quercetin, relax the ASM by inhibiting the L-VDCC and SOCC; further studies must be performed to obtain the complete set of metabolites of the extract and study at length their pharmacological properties.

Inhibitory mechanisms of docosahexaenoic acid on carbachol-, angiotensin II-, and bradykinin-induced contractions in guinea pig gastric fundus smooth muscle.

We studied the inhibitory actions of docosahexaenoic acid (DHA) on the contractions induced by carbachol (CCh), angiotensin II (Ang II), and bradykinin (BK) in guinea pig (GP) gastric fundus smooth muscle (GFSM), particularly focusing on the possible inhibition of store-operated Ca2+ channels (SOCCs). DHA significantly suppressed the contractions induced by CCh, Ang II, and BK; the inhibition of BK-induced contractions was the strongest. Although all contractions were greatly dependent on external Ca2+, more than 80% of BK-induced contractions remained even in the presence of verapamil, a voltage-dependent Ca2+ channel inhibitor. BK-induced contractions in the presence of verapamil were not suppressed by LOE-908 (a receptor-operated Ca2+ channel (ROCC) inhibitor) but were suppressed by SKF-96365 (an SOCC and ROCC inhibitor). BK-induced contractions in the presence of verapamil plus LOE-908 were strongly inhibited by DHA. Furthermore, DHA inhibited GFSM contractions induced by cyclopiazonic acid (CPA) in the presence of verapamil plus LOE-908 and inhibited the intracellular Ca2+ increase due to Ca2+ addition in CPA-treated 293T cells. These findings indicate that Ca2+ influx through SOCCs plays a crucial role in BK-induced contraction in GP GFSM and that this inhibition by DHA is a new mechanism by which this fatty acid inhibits GFSM contractions.

Novel Scorpion Toxin ω-Buthitoxin-Hf1a Selectively Inhibits Calcium Influx via CaV3.3 and CaV3.2 and Alleviates Allodynia in a Mouse Model of Acute Postsurgical Pain.

Venom peptides have evolved to target a wide range of membrane proteins through diverse mechanisms of action and structures, providing promising therapeutic leads for diseases, including pain, epilepsy, and cancer, as well as unique probes of ion channel structure-function. In this work, a high-throughput FLIPR window current screening assay on T-type CaV3.2 guided the isolation of a novel peptide named ω-Buthitoxin-Hf1a from scorpion Hottentotta franzwerneri crude venom. At only 10 amino acid residues with one disulfide bond, it is not only the smallest venom peptide known to target T-type CaVs but also the smallest structured scorpion venom peptide yet discovered. Synthetic Hf1a peptides were prepared with C-terminal amidation (Hf1a-NH2) or a free C-terminus (Hf1a-OH). Electrophysiological characterization revealed Hf1a-NH2 to be a concentration-dependent partial inhibitor of CaV3.2 (IC50 = 1.18 µM) and CaV3.3 (IC50 = 0.49 µM) depolarized currents but was ineffective at CaV3.1. Hf1a-OH did not show activity against any of the three T-type subtypes. Additionally, neither form showed activity against N-type CaV2.2 or L-type calcium channels. The three-dimensional structure of Hf1a-NH2 was determined using NMR spectroscopy and used in docking studies to predict its binding site at CaV3.2 and CaV3.3. As both CaV3.2 and CaV3.3 have been implicated in peripheral pain signaling, the analgesic potential of Hf1a-NH2 was explored in vivo in a mouse model of incision-induced acute post-surgical pain. Consistent with this role, Hf1a-NH2 produced antiallodynia in both mechanical and thermal pain.

Novel protocol for multiple-dose oral administration of the L-type Ca2+ channel blocker isradipine in mice: A dose-finding pharmacokinetic study.

Studies in genetically modified animals and human genetics have recently provided new insight into the role of voltage-gated L-type Ca2+ channels in human disease. Therefore, the inhibition of L-type Ca2+ channels in vivo in wildtype and mutant mice by potent dihydropyridine (DHP) Ca2+ channel blockers serves as an important pharmacological tool. These drugs have a short plasma half-life in humans and especially in rodents and show high first-pass metabolism upon oral application. In the vast majority of in vivo studies, they have therefore been delivered through parenteral routes, mostly subcutaneously or intraperitoneally. High peak plasma concentrations of DHPs cause side effects, evident as DHP-induced aversive behaviors confounding the interpretation of behavioral readouts. Nevertheless, pharmacokinetic data measuring the exposure achieved with these applications are sparse. Moreover, parenteral injections require animal handling and can be associated with pain, discomfort and stress which could influence a variety of physiological processes, behavioral and other functional readouts. Here, we describe a noninvasive oral application of the DHP isradipine by training mice to quickly consume small volumes of flavored yogurt that can serve as drug vehicle. This procedure does not require animal handling, allows repeated drug application over several days and reproducibly achieves peak plasma concentrations over a wide range previously shown to be well-tolerated in humans. This protocol should facilitate ongoing nonclinical studies in mice exploring new indications for DHP Ca2+ channel blockers.

Calcium channel inhibitor and extracellular calcium improve aminoglycoside-induced hair cell loss in zebrafish.

Aminoglycosides are commonly used antibiotics for treatment of gram-negative bacterial infections, however, they might act on inner ear, leading to hair-cell death and hearing loss. Currently, there is no targeted therapy for aminoglycoside ototoxicity, since the underlying mechanisms of aminoglycoside-induced hearing impairments are not fully defined. This study aimed to investigate whether the calcium channel blocker verapamil and changes in intracellular & extracellular calcium could ameliorate aminoglycoside-induced ototoxicity in zebrafish. The present findings showed that a significant decreased number of neuromasts in the lateral lines of zebrafish larvae at 5 days' post fertilization after neomycin (20 µM) and gentamicin (20 mg/mL) exposure, which was prevented by verapamil. Moreover, verapamil (10-100 µM) attenuated aminoglycoside-induced toxic response in different external calcium concentrations (33-3300 µM). The increasing extracellular calcium reduced hair cell loss from aminoglycoside exposure, while lower calcium facilitated hair cell death. In contrast, calcium channel activator Bay K8644 (20 µM) enhanced aminoglycoside-induced ototoxicity and reversed the protective action of higher external calcium on hair cell loss. However, neomycin-elicited hair cell death was not altered by caffeine, ryanodine receptor (RyR) agonist, and RyR antagonists, including thapsigargin, ryanodine, and ruthenium red. The uptake of neomycin into hair cells was attenuated by verapamil and under high external calcium concentration. Consistently, the production of reactive oxygen species (ROS) in neuromasts exposed to neomycin was also reduced by verapamil and high external calcium. Significantly, zebrafish larvae when exposed to neomycin exhibited decreased swimming distances in reaction to droplet stimulus when compared to the control group. Verapamil and elevated external calcium effectively protected the impaired swimming ability of zebrafish larvae induced by neomycin. These data imply that prevention of hair cell damage correlated with swimming behavior against aminoglycoside ototoxicity by verapamil and higher external calcium might be associated with inhibition of excessive ROS production and aminoglycoside uptake through cation channels. These findings indicate that calcium channel blocker and higher external calcium could be applied to protect aminoglycoside-induced listening impairments.

Development and therapeutic implications of small molecular inhibitors that target calcium-related channels in tumor treatment.

Calcium ion dysregulation exerts profound effects on various physiological activities such as tumor proliferation, migration, and drug resistance. Calcium-related channels play a regulatory role in maintaining calcium ion homeostasis, with most channels being highly expressed in tumor cells. Additionally, these channels serve as potential drug targets for the development of antitumor medications. In this review, we first discuss the current research status of these pathways, examining how they modulate various tumor functions such as epithelial-mesenchymal transition (EMT), metabolism, and drug resistance. Simultaneously, we summarize the recent progress in the study of novel small-molecule drugs over the past 5 years and their current status.

Brain-derived neurotrophic factor scales presynaptic calcium transients to modulate excitatory neurotransmission.

Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic physiology, as well as mechanisms underlying various neuropsychiatric diseases and their treatment. Despite its clear physiological role and disease relevance, BDNF's function at the presynaptic terminal, a fundamental unit of neurotransmission, remains poorly understood. In this study, we evaluated single synapse dynamics using optical imaging techniques in hippocampal cell cultures. We find that exogenous BDNF selectively increases evoked excitatory neurotransmission without affecting spontaneous neurotransmission. However, acutely blocking endogenous BDNF has no effect on evoked or spontaneous release, demonstrating that different approaches to studying BDNF may yield different results. When we suppressed BDNF-Tropomyosin receptor kinase B (TrkB) activity chronically over a period of days to weeks using a mouse line enabling conditional knockout of TrkB, we found that evoked glutamate release was significantly decreased while spontaneous release remained unchanged. Moreover, chronic blockade of BDNF-TrkB activity selectively downscales evoked calcium transients without affecting spontaneous calcium events. Via pharmacological blockade by voltage-gated calcium channel (VGCC) selective blockers, we found that the changes in evoked calcium transients are mediated by the P/Q subtype of VGCCs. These results suggest that BDNF-TrkB activity increases presynaptic VGCC activity to selectively increase evoked glutamate release.

Discovery and Structure-Activity Relationship of a Ryanodine Receptor 2 Inhibitor.

Ryanodine receptor 2 (RyR2) is a large Ca2+-release channel in the sarcoplasmic reticulum (SR) of cardiac muscle cells. It serves to release Ca2+ from the SR into the cytosol to initiate muscle contraction. RyR2 overactivation is associated with arrhythmogenic cardiac disease, but few specific inhibitors have been reported so far. Here, we identified an RyR2-selective inhibitor 1 from the chemical compound library and synthesized it from glycolic acid. Synthesis of various derivatives to investigate the structure-activity relationship of each substructure afforded another two RyR2-selective inhibitors 6 and 7, among which 6 was the most potent. Notably, compound 6 also inhibited Ca2+ release in cells expressing the RyR2 mutants R2474S, R4497C and K4750Q, which are associated with cardiac arrhythmias such as catecholaminergic polymorphic ventricular tachycardia (CPVT). This inhibitor is expected to be a useful tool for research on the structure and dynamics of RyR2, as well as a lead compound for the development of drug candidates to treat RyR2-related cardiac disease.

Amlodipine inhibits Synaptotagmin-4's oncogenic activity on gastric cancer proliferation by targeting calcium signaling.

BACKGROUND: Gastric cancer (GC) remains a leading cause of cancer mortality globally. Synaptotagmin-4 (SYT4), a calcium-sensing synaptic vesicle protein, has been implicated in the oncogenesis of diverse malignancies. PURPOSE: This study delineates the role of SYT4 in modulating clinical outcomes and biological behaviors in GC. METHODS: We evaluated SYT4 expression in GC specimens using bioinformatics analyses and immunohistochemistry. Functional assays included CCK8 proliferation tests, apoptosis assays via flow cytometry, confocal calcium imaging, and xenograft models. Western blotting elucidated MAPK pathway involvement. Additionally, we investigated the impact of the calcium channel blocker amlodipine on cellular dynamics and MAPK pathway activity. RESULTS: SYT4 was higher in GC tissues, and the elevated SYT4 was significantly correlated with adverse prognosis. Both univariate and multivariate analyses confirmed SYT4 as an independent prognostic indicator for GC. Functionally, SYT4 promoted tumorigenesis by fostering cellular proliferation, inhibiting apoptosis, and enhancing intracellular Ca2+ influx, predominantly via MAPK pathway activation. Amlodipine pre-treatment attenuated SYT4-driven cell growth and potentiated apoptosis, corroborated by in vivo xenograft assessments. These effects were attributed to MAPK pathway suppression by amlodipine. CONCLUSION: SYT4 emerges as a potential prognostic biomarker and a pro-oncogenic mediator in GC through a Ca2+-dependent MAPK mechanism. Amlodipine demonstrates significant antitumor effects against SYT4-driven GC, positing its therapeutic promise. This study underscores the imperative of targeting calcium signaling in GC treatment strategies.

Structural basis for human Cav3.2 inhibition by selective antagonists.

The Cav3.2 subtype of T-type calcium channels has been targeted for developing analgesics and anti-epileptics for its role in pain and epilepsy. Here we present the cryo-EM structures of Cav3.2 alone and in complex with four T-type calcium channel selective antagonists with overall resolutions ranging from 2.8 Å to 3.2 Å. The four compounds display two binding poses. ACT-709478 and TTA-A2 both place their cyclopropylphenyl-containing ends in the central cavity to directly obstruct ion flow, meanwhile extending their polar tails into the IV-I fenestration. TTA-P2 and ML218 project their 3,5-dichlorobenzamide groups into the II-III fenestration and place their hydrophobic tails in the cavity to impede ion permeation. The fenestration-penetrating mode immediately affords an explanation for the state-dependent activities of these antagonists. Structure-guided mutational analysis identifies several key residues that determine the T-type preference of these drugs. The structures also suggest the role of an endogenous lipid in stabilizing drug binding in the central cavity.

Lead exerts a depression of neurotransmitter release through a blockade of voltage dependent calcium channels in chromaffin cells.

The present work, using chromaffin cells of bovine adrenal medullae (BCCs), aims to describe what type of ionic current alterations induced by lead (Pb2+) underlies its effects reported on synaptic transmission. We observed that the acute application of Pb2+ lead to a drastic depression of neurotransmitters release in a concentration-dependent manner when the cells were stimulated with both K+ or acetylcholine, with an IC50 of 119,57 µM and of 5,19 µM, respectively. This effect was fully recovered after washout. Pb2+ also blocked calcium channels of BCCs in a time- and concentration-dependent manner with an IC50 of 6,87 µM. This blockade was partially reversed upon washout. This compound inhibited the calcium current at all test potentials and shows a shift of the I-V curve to more negative values of about 8 mV. The sodium current was not blocked by acute application of high Pb2+ concentrations. Voltage-dependent potassium current was also shortly affected by high Pb2+. Nevertheless, the calcium- and voltage-dependent potassium current was drastically depressed in a dose-dependent manner, with an IC50 of 24,49 µM. This blockade was related to the prevention of Ca2+ influx through voltage-dependent calcium channels coupled to Ca2+-activated K+-channels (BK) instead a direct linking to these channels. Under current-clamp conditions, BCCs exhibit a resting potential of -52.7 mV, firing spontaneous APs (1-2 spikes/s) generated by the opening of Na+ and Ca2+-channels, and terminated by the activation of K+ channels. In spite of the effect on ionic channels exerted by Pb2+, we found that Pb2+ didn't alter cellular excitability, no modification of the membrane potential, and no effect on action potential firing. Taken together, these results point to a neurotoxic action evoked by Pb2+ that is associated with changes in neurotransmitter release by blocking the ionic currents responsible for the calcium influx.

Do calcium channel blockers applied to cardiomyocytes cause increased channel expression resulting in reduced efficacy?

In the initial hours following the application of the calcium channel blocker (CCB) nifedipine to microtissues consisting of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we observe notable variations in the drug's efficacy. Here, we investigate the possibility that these temporal changes in CCB effects are associated with adaptations in the expression of calcium ion channels in cardiomyocyte membranes. To explore this, we employ a recently developed mathematical model that delineates the regulation of calcium ion channel expression by intracellular calcium concentrations. According to the model, a decline in intracellular calcium levels below a certain target level triggers an upregulation of calcium ion channels. Such an upregulation, if instigated by a CCB, would then counteract the drug's inhibitory effect on calcium currents. We assess this hypothesis using time-dependent measurements of hiPSC-CMs dynamics and by refining an existing mathematical model of myocyte action potentials incorporating the dynamic nature of the number of calcium ion channels. The revised model forecasts that the CCB-induced reduction in intracellular calcium concentrations leads to a subsequent increase in calcium ion channel expression, thereby attenuating the drug's overall efficacy. The data and fit models suggest that dynamic changes in cardiac cells in the presence of CCBs may be explainable by induced changes in protein expression, and that this may lead to challenges in understanding calcium based drug effects on the heart unless timings of applications are carefully considered.

T- and L-Type Calcium Channels Maintain Calcium Oscillations in the Murine Zona Glomerulosa.

BACKGROUND: The zona glomerulosa of the adrenal gland is responsible for the synthesis and release of the mineralocorticoid aldosterone. This steroid hormone regulates salt reabsorption in the kidney and blood pressure. The most important stimuli of aldosterone synthesis are the serum concentrations of angiotensin II and potassium. In response to these stimuli, voltage and intracellular calcium levels in the zona glomerulosa oscillate, providing the signal for aldosterone synthesis. It was proposed that the voltage-gated T-type calcium channel CaV3.2 is necessary for the generation of these oscillations. However, Cacna1h knock-out mice have normal plasma aldosterone levels, suggesting additional calcium entry pathways. METHODS: We used a combination of calcium imaging, patch clamp, and RNA sequencing to investigate calcium influx pathways in the murine zona glomerulosa. RESULTS: Cacna1h-/- glomerulosa cells still showed calcium oscillations with similar concentrations as wild-type mice. No calcium channels or transporters were upregulated to compensate for the loss of CaV3.2. The calcium oscillations observed were instead dependent on L-type voltage-gated calcium channels. Furthermore, we found that L-type channels can also partially compensate for an acute inhibition of CaV3.2 in wild-type mice. Only inhibition of both T- and L-type calcium channels abolished the increase of intracellular calcium caused by angiotensin II in wild-type. CONCLUSIONS: Our study demonstrates that T-type calcium channels are not strictly required to maintain glomerulosa calcium oscillations and aldosterone production. Pharmacological inhibition of T-type channels alone will likely not significantly impact aldosterone production in the long term.

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 8 weeks, they were given 40 mg/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 ± 0.45 l/min, P = 0.006) and leg vascular conductance (Δ 5.38 ± 5.67 ml/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 8 weeks' 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.