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Thyroid hormone binds to specific nuclear receptors, regulating the expression of target genes, with major effects on cardiac function. Triiodothyronine (T3) increases the expression of key proteins related to calcium homeostasis, such as the sarcoplasmic reticulum calcium ATPase pump, but the detailed mechanism of gene regulation by T3 in cardiac voltage-gated calcium (Cav1.2) channels remains incompletely explored. Furthermore, the effects of T3 on Cav1.2 auxiliary subunits have not been investigated. We conducted quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence experiments in H9c2 cells derived from rat ventricular tissue, examining the effects of T3 on the expression of α1c, the principal subunit of Cav1.2 channels, and Cavβ4, an auxiliary Cav1.2 subunit that regulates gene expression. The translocation of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) by T3 was also examined. We found that T3 has opposite effects on these channel proteins, upregulating α1c and downregulating Cavβ4, and that it increases the nuclear translocation of pCREB while decreasing the translocation of Cavβ4. Finally, we found that overexpression of Cavβ4 represses the mRNA expression of α1c, suggesting that T3 upregulates the expression of the α1c subunit in response to a decrease in Cavβ4 subunit expression. [ABSTRACT FROM AUTHOR]

Abstract The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination.

Abstract Background The study aimed to identify transcripts of specific ion channels in rat ventricular cardiomyocytes and determine their potential role in the regulation of ionic currents in response to mechanical stimulation. The gene expression levels of various ion channels in freshly isolated rat ventricular cardiomyocytes were investigated using the RNA-seq technique. We also measured changes in current through CaV1.2 channels under cell stretching using the whole-cell patch-clamp method. Results Among channels that showed mechanosensitivity, significant amounts of TRPM7, TRPC1, and TRPM4 transcripts were found. We suppose that the recorded L-type Ca 2+ current is probably expressed through CaV1.2. Furthermore, stretching cells by 6, 8, and 10 μm, which increases I SAC through the TRPM7, TRPC1, and TRPM4 channels, also decreased I Ca,L through the CaV1.2 channels in K+ in/K+ out, Cs+ in/K+ out, K+ in/Cs+ out, and Cs+ in/Cs+ out solutions. The application of a nonspecific I SAC blocker, Gd3+, during cell stretching eliminated I SAC through nonselective cation channels and I Ca,L through CaV1.2 channels. Since the response to Gd3+ was maintained in Cs+ in/Cs+ out solutions, we suggest that voltage-gated CaV1.2 channels in the ventricular myocytes of adult rats also exhibit mechanosensitive properties. Conclusions Our findings suggest that TRPM7, TRPC1, and TRPM4 channels represent stretch-activated nonselective cation channels in rat ventricular myocytes. Probably the CaV1.2 channels in these cells exhibit mechanosensitive properties. Our results provide insight into the molecular mechanisms underlying stretch-induced responses in rat ventricular myocytes, which may have implications for understanding cardiac physiology and pathophysiology.

The experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis was used in combination with a Cav1.2 conditional knock-out mouse (Cav1.2KO) to study the role of astrocytic voltage-gated Ca++ channels in autoimmune CNS inflammation and demyelination. Cav1.2 channels were specifically ablated in Glast-1-positive astrocytes by means of the Cre-lox system before EAE induction. After immunization, motor activity was assessed daily, and a clinical score was given based on the severity of EAE symptoms. Cav1.2 deletion in astrocytes significantly reduced the severity of the disease. While no changes were found in the day of onset and peak disease severity, EAE mean clinical score was lower in Cav1.2KO animals during the chronic phase of the disease. This corresponded to better performance on the rotarod and increased motor activity in Cav1.2KO mice. Furthermore, decreased numbers of reactive astrocytes, activated microglia, and infiltrating lymphocytes were found in the lumbar section of the spinal cord of Cav1.2KO mice 40 days after immunization. The degree of myelin protein loss and size of demyelinated lesions were also attenuated in Cav1.2KO spinal cords. Similar results were found in EAE animals treated with nimodipine, a Cav1.2 Ca++ channel inhibitor with high affinity to the CNS. Mice injected with nimodipine during the acute and chronic phases of the disease exhibited lower numbers of reactive astrocytes, activated microglial, and infiltrating immune cells, as well as fewer demyelinated lesions in the spinal cord. These changes were correlated with improved clinical scores and motor performance. In summary, these data suggest that antagonizing Cav1.2 channels in astrocytes during EAE alleviates neuroinflammation and protects the spinal cord from autoimmune demyelination. [ABSTRACT FROM AUTHOR]

Background: The study aimed to identify transcripts of specific ion channels in rat ventricular cardiomyocytes and determine their potential role in the regulation of ionic currents in response to mechanical stimulation. The gene expression levels of various ion channels in freshly isolated rat ventricular cardiomyocytes were investigated using the RNA-seq technique. We also measured changes in current through CaV1.2 channels under cell stretching using the whole-cell patch-clamp method. Results: Among channels that showed mechanosensitivity, significant amounts of TRPM7, TRPC1, and TRPM4 transcripts were found. We suppose that the recorded L-type Ca2+ current is probably expressed through CaV1.2. Furthermore, stretching cells by 6, 8, and 10 μm, which increases ISAC through the TRPM7, TRPC1, and TRPM4 channels, also decreased ICa,L through the CaV1.2 channels in K+in/K+out, Cs+in/K+out, K+in/Cs+out, and Cs+in/Cs+out solutions. The application of a nonspecific ISAC blocker, Gd3+, during cell stretching eliminated ISAC through nonselective cation channels and ICa,L through CaV1.2 channels. Since the response to Gd3+ was maintained in Cs+in/Cs+out solutions, we suggest that voltage-gated CaV1.2 channels in the ventricular myocytes of adult rats also exhibit mechanosensitive properties. Conclusions: Our findings suggest that TRPM7, TRPC1, and TRPM4 channels represent stretch-activated nonselective cation channels in rat ventricular myocytes. Probably the CaV1.2 channels in these cells exhibit mechanosensitive properties. Our results provide insight into the molecular mechanisms underlying stretch-induced responses in rat ventricular myocytes, which may have implications for understanding cardiac physiology and pathophysiology. [ABSTRACT FROM AUTHOR]

Sesquiterpenes such as leucodin and the labdane-type diterpene manool are natural compounds endowed with remarkably in vitro vasorelaxant and in vivo hypotensive activities. Given their structural similarity with the sesquiterpene lactone (+)-sclareolide, this molecule was selected as a scaffold to develop novel vasoactive agents. Functional, electrophysiology, and molecular dynamics studies were performed. The opening of the five-member lactone ring in the (+)-sclareolide provided a series of labdane-based small molecules, promoting a significant in vitro vasorelaxant effect. Electrophysiology data identified 7 as a CaV1.2 channel blocker and a KCa1.1 channel stimulator. These activities were also confirmed in the intact vascular tissue. The significant antagonism caused by the CaV1.2 channel agonist Bay K 8644 suggested that 7 might interact with the dihydropyridine binding site. Docking and molecular dynamic simulations provided the molecular basis of the CaV1.2 channel blockade and KCa1.1 channel stimulation produced by 7. Finally, 7 reduced coronary perfusion pressure and heart rate, while prolonging conduction and refractoriness of the atrioventricular node, likely because of its Ca2+ antagonism. Taken together, these data indicate that the labdane scaffold represents a valuable starting point for the development of new vasorelaxant agents endowed with negative chronotropic properties and targeting key pathways involved in the pathophysiology of hypertension and ischemic cardiomyopathy. [ABSTRACT FROM AUTHOR]

Sesquiterpenes such as leucodin and the labdane-type diterpene manool are natural compounds endowed with remarkably in vitro vasorelaxant and in vivo hypotensive activities. Given their structural similarity with the sesquiterpene lactone (+)-sclareolide, this molecule was selected as a scaffold to develop novel vasoactive agents. Functional, electrophysiology, and molecular dynamics studies were performed. The opening of the five-member lactone ring in the (+)-sclareolide provided a series of labdane-based small molecules, promoting a significant in vitro vasorelaxant effect. Electrophysiology data identified 7 as a CaV1.2 channel blocker and a KCa1.1 channel stimulator. These activities were also confirmed in the intact vascular tissue. The significant antagonism caused by the CaV1.2 channel agonist Bay K 8644 suggested that 7 might interact with the dihydropyridine binding site. Docking and molecular dynamic simulations provided the molecular basis of the CaV1.2 channel blockade and KCa1.1 channel stimulation produced by 7. Finally, 7 reduced coronary perfusion pressure and heart rate, while prolonging conduction and refractoriness of the atrioventricular node, likely because of its Ca2+ antagonism. Taken together, these data indicate that the labdane scaffold represents a valuable starting point for the development of new vasorelaxant agents endowed with negative chronotropic properties and targeting key pathways involved in the pathophysiology of hypertension and ischemic cardiomyopathy.

In atherosclerosis, vascular smooth muscle cells (VSMC) migrate from the media toward the intima of the arteries in response to cytokines, such as platelet-derived growth factor (PDGF). However, molecular mechanism underlying the PDGF-induced migration of VSMCs remains unclear. The migration of rat aorta-derived synthetic VSMCs, A7r5, in response to PDGF was potently inhibited by a CaV1.2 channel inhibitor, nifedipine, and a Src family tyrosine kinase (SFK)/Abl inhibitor, bosutinib, in a less-than-additive manner. PDGF significantly increased CaV1.2 channel currents without altering CaV1.2 protein expression levels in A7r5 cells. This reaction was inhibited by C-terminal Src kinase, a selective inhibitor of SFKs. In contractile VSMCs, the C-terminus of CaV1.2 is proteolytically cleaved into proximal and distal C-termini (PCT and DCT, respectively). Clipped DCT is noncovalently reassociated with PCT to autoinhibit the channel activity. Conversely, in synthetic A7r5 cells, full-length CaV1.2 (CaV1.2FL) is expressed much more abundantly than truncated CaV1.2. In a heterologous expression system, c-Src activated CaV1.2 channels composed of CaV1.2FL but not truncated CaV1.2 (CaV1.2Δ1763) or CaV1.2Δ1763 plus clipped DCT. Further, c-Src enhanced the coupling efficiency between the voltage-sensing domain and activation gate of CaV1.2FL channels by phosphorylating Tyr1709 and Tyr1758 in PCT. Compared with CaV1.2Δ1763, c-Src could more efficiently bind to and phosphorylate CaV1.2FL irrespective of the presence or absence of clipped DCT. Therefore, in atherosclerotic lesions, phenotypic switching of VSMCs may facilitate pro-migratory effects of PDGF on VSMCs by suppressing posttranslational CaV1.2 modifications. [ABSTRACT FROM AUTHOR]

Lead ions (Pb) possess characteristics similar to Ca. Because of this and its redox capabilities, lead causes different toxic effects. The neurotoxic effects have been well documented; however, the toxic effects on cardiac tissues remain allusive. We utilized isolated guinea pig hearts and measured the effects of Pb on their contractility and excitability. Acute exposure to extracellular Pb had a negative inotropic effect and increased diastolic tension. The speed of contraction and relaxation were affected, though the effects were more dramatic on the speed of contraction. Excitability was also altered. Heart beat frequency increased and later diminished after lead ion exposure. Pro-arrhytmic events, such as early after-depolarization and a reduction of the action potential plateau, were also observed. In isolated cardiomyocytes and tsA 201 cells, extracellular lead blocked currents through Cav1.2 channels, diminished their activation, and enhanced their fast inactivation, negatively affecting their gating currents. Thus, Pb was cardiotoxic and reduced cardiac contractility, making the heart prone to arrhythmias. This was due, in part, to Pb effects on the Cav1.2 channels; however, other channels, transporters or pathways may also be involved. Acute cardiotoxic effects were observed at Pb concentrations achievable during acute lead poisoning. The results suggest how Cav1.2 gating can be affected by divalent cations, such as Pb, and also suggest a more thorough evaluation of heart function in individuals affected by lead poisoning. [ABSTRACT FROM AUTHOR]

Extensive research is currently underway, seeking better diagnostic methods and treatments and a better understanding of the molecular mechanisms involved in cancer, from the role of specific genetic mutations to the intricate biochemical and molecular pathways involved. Because of their role in regulating relevant physiological events such as cell proliferation, migration, and invasion, ion channels have recently been recognized as important elements in cancer initiation and progression. Moreover, it has been reported that pharmacological intervention in ion channel activity might provide protection against diverse types of cancer, and that ion channels could be used as targets to counteract tumor growth, prevent metastasis, and overcome the therapy resistance of tumor cells. In this context, Ca 2+ channels have been found to play a role in tumorigenesis and tumor progression. Specifically, L-type Ca 2+ channel inhibition may affect cell proliferation, differentiation, and apoptosis. This review aims to provide insights into the potential role of these channels in cancer cell lines, emphasizing their participation in cell proliferation, migration, and autophagy induction, as well as their potential as rational targets for new cancer therapeutics. [ABSTRACT FROM AUTHOR]

A role for mitochondrial fission in vascular contraction has been proposed based on the vasorelaxant activity of the dynamin (and mitochondrial fission) inhibitors mdivi-1 and dynasore. However, mdivi-1 is capable to inhibit Ba2+ currents through Ca V 1.2 channels (I Ba1.2), stimulate K Ca 1.1 channel currents (I KCa1.1), and modulate pathways key to the maintenance of vessel active tone in a dynamin-independent manner. Using a multidisciplinary approach, the present study demonstrates that dynasore, like mdivi-1, is a bi-functional vasodilator, blocking I Ba1.2 and stimulating I KCa1.1 in rat tail artery myocytes, as well as promoting relaxation of rat aorta rings pre-contracted by either high K+ or phenylephrine. Conversely, its analogue dyngo-4a, though inhibiting mitochondrial fission triggered by phenylephrine and stimulating I KCa1.1 , did not affect I Ba1.2 but potentiated both high K+- and phenylephrine-induced contractions. Docking and molecular dynamics simulations identified the molecular basis supporting the different activity of dynasore and dyngo-4a at Ca V 1.2 and K Ca 1.1 channels. Mito-tempol only partially counteracted the effects of dynasore and dyngo-4a on phenylephrine-induced tone. In conclusion, the present data, along with previous observations (Ahmed et al., 2022) rise caution for the use of dynasore, mdivi-1, and dyngo-4a as tools to investigate the role of mitochondrial fission in vascular contraction: to this end, a selective dynamin inhibitor and/or a different experimental approach are needed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Artificial intelligence accelerates the drug discovery. • O -substituted morin derivatives show interesting vasorelaxant activity. • Ca V 1.2 channels seem to be the main targets involved in the vasorelaxant activity. • The pentamethyl derivative 5a reveals a multi-target activity. Morin is a vasorelaxant flavonoid, whose activity is ascribable to Ca V 1.2 channel blockade that, however, is weak as compared to that of clinically used therapeutic agents. A conventional strategy to circumvent this drawback is to synthesize new derivatives differently decorated and, in this context, morin-derivatives able to interact with Ca V 1.2 channels were found by employing the potential of PLATO in target fishing and reverse screening. Three different derivatives (5a - c) were selected as promising tools, synthesized, and investigated in in vitro functional studies using rat aorta rings and rat tail artery myocytes. 5a - c were found more effective vasorelaxant agents than the naturally occurring parent compound and antagonized both electro- and pharmaco-mechanical coupling in an endothelium-independent manner. 5a , the series' most potent, reduced also Ca2+ mobilization from intracellular store sites. Furthermore, 5a ≈ 5c > 5b inhibited Ba2+ current through Ca V 1.2 channels. However, compound 5a caused also a concentration-dependent inhibition of K Ca 1.1 channel currents. [ABSTRACT FROM AUTHOR]

Aim The primary aim of this study was to identify the effects of hyperammonaemia on functional expression of Cav1.2 L-type Ca2+ channels in astroglia. Methods Primary cultures of mouse astrocytes were used to study effects of chronic treatment (1-5 days) with ammonium chloride, at 1, 3 and 5 m m on depolarization-induced increases in free cytosolic Ca2+ concentration ([Ca2+]i, measured with Fura-2 based microfluorimetry) in control conditions and following treatment with the L-type Ca2+ channel inhibitor, nifedipine, or with ryanodine receptor inhibitor, ryanodine. Expression of Cav1.2 mRNA was identified with RT- PCR, whereas protein content was determined by Western blotting. Sustained hyperammonaemia in vivo was induced by daily injections of urease (33 units kg body weight−1, i.p.) for 3 days. Results Depolarization-induced [Ca2+]i transients sensitive to nifedipine (peak of the response) and to ryanodine (plateau phase) were significantly increased in astrocytes chronically exposed to ammonium. The ammonium-induced increase in Ca2+ influx in astrocytes resulted from an upregulation of Cav1.2 channel's expression detected at mRNA and protein levels. Increase in Cav1.2 expression was prevented by ouabain antagonist canrenone. Similar upregulation of Cav1.2 gene expression was found in the brains of adult mice subjected to intraperitoneal injection of urease. In transgenic mice tagged with an astrocyte-specific or neurone-specific markers and treated with intraperitoneal injections of urease, the fluorescence-activated cell sorting of neurones and astrocytes demonstrated that Cav1.2 mRNA expression was upregulated in astrocytes, but not in neurones. Conclusions Ammonium-induced deregulation of astroglial Ca2+ signalling, is, in part, associated with upregulation of Cav1.2 L-type calcium channels. [ABSTRACT FROM AUTHOR]

[Display omitted] Mdivi-1 is widely used as a pharmacological tool to inhibit dynamin-related protein-1-mediated mitochondrial fission. Whether this compound may interact directly or indirectly with ion channels or cellular pathways fundamental for the regulation of vascular smooth muscle tone remains unknown. The present study aimed to assess the effect of mdivi-1 on Ca V 1.2 and K Ca 1.1 channels, both in vitro and in silico as well as on the mechanical activity of rat aorta rings. Mdivi-1 was an effective Ca V 1.2 channel blocker, docking in a Ca V 1.2 channel antagonist binding region, stimulated K Ca 1.1 channel current, binding to a sensing region common to other stimulators, and possibly inhibited the Rho-kinase pathway. These effects contributed to its vasorelaxant activity observed in rings stimulated with high KCl, phenylephrine, or NaF. Neither structurally different dynamin inhibitors nor a stimulator affected the Ca2+ antagonistic and vasorelaxant activities of the compound. However, mito-tempol reduced its vasorelaxant potency towards phenylephrine. Finally, mdivi-1 antagonized mitochondrial fission triggered by phenylephrine. In conclusion, mdivi-1 is an effective in vitro vasorelaxant agent at concentrations routinely employed to block dynamin-related protein-1. Ion channels and pathways key to the maintenance of vessel active tone are involved in this mechanism. These yet undiscovered off-target effects raise caution for the interpretation of mitochondrial fission signalling. [ABSTRACT FROM AUTHOR]

The article focuses on a study investigating the coordination of Cav1.2 or L-Type Ca2+ channels which are vital regulators for muscle and neural functions. It reports that couple gating of the channels represent a new mechanism of Ca2+ influx regulation and excitability in neurons, cardiac and arterial smooth muscle under physiological and pathological conditions. It mentions that Cav1.2 channels can open and close in tandem and increase in frequency in hypertensive smooth muscle cells.

Inhibition provided by local GABAergic interneurons (INs) activates ionotropic GABA A and metabotropic GABA B receptors (GABA B Rs). Despite GABA B Rs representing a major source of inhibition, little is known of their function in distinct IN subtypes. Here, we show that, while the archetypal dendritic-inhibitory somatostatin-expressing INs (SOM-INs) possess high levels of GABA B R on their somato-dendritic surface, they fail to produce significant postsynaptic inhibitory currents. Instead, GABA B Rs selectively inhibit dendritic Ca V 1.2 (L-type) Ca 2+ channels on SOM-IN dendrites, leading to reduced calcium influx and loss of long-term potentiation at excitatory input synapses onto these INs. These data provide a mechanism by which GABA B Rs can contribute to disinhibition and control the efficacy of extrinsic inputs to hippocampal networks., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)