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Rat arterial smooth muscle devoid of ryanodine receptor function: effects on cellular Ca(2+) handling.

ABSTRACT: The roles of intracellular Ca(2+) stores and ryanodine (Ry) receptors for vascular Ca(2+) homeostasis and viability were investigated in rat tail arterial segments kept in organ culture with Ry (10 - 100 microM) for up to 4 days. Acute exposure to Ry or the non-deactivating ryanodine analogue C(10)-O(eq) glycyl ryanodine (10 microM) eliminated Ca(2+) release responses to caffeine (20 mM) and noradrenaline (NA, 10 microM), whereas responses to NA, but not caffeine, gradually returned to normal within 4 days of exposure to RY: Ry receptor protein was detected on Western blots in arteries cultured either with or without RY: Brief Ca(2+) release events (sparks) were absent after culture with Ry, whereas Ca(2+) waves still occurred. The propagation velocity of waves was equal ( approximately 19 microm s(-1)) in tissue cultured either with or without RY: Inhibition of Ca(2+) accumulation into the sarcoplasmic reticulum (SR) by culture with caffeine (5 mM), cyclopiazonic acid or thapsigargin (both 10 microM) decreased contractility due to Ca(2+)-induced cell damage. In contrast, culture with Ry did not affect contractility. Removal of Ca(2+) from the cytosol following a Ca(2+) load was retarded after Ry culture. Thapsigargin reduced the rate of Ca(2+) removal in control cultured rings, but had no effect after Ry culture. It is concluded that intracellular Ca(2+) stores recover during chronic Ry treatment, while Ry receptors remain non-functional. Ry receptor activity is required for Ca(2+) sparks and for SR-dependent recovery from a Ca(2+) load, but not for Ca(2+) waves or basal Ca(2+) homeostasis.

SUBMITTER: Dreja K

PROVIDER: S-EPMC1572725 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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Dihydropyridine Ca(2+) channel blockers increase cytosolic [Ca(2+)] by activating Ca(2+)-sensing receptors in pulmonary arterial smooth muscle cells.

Project description:An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and pulmonary vascular remodeling. The dihydropyridine Ca(2+) channel blockers, such as nifedipine, have been used for treatment of idiopathic pulmonary arterial hypertension (IPAH).Our previous study demonstrated that the Ca(2+)-sensing receptor (CaSR) was upregulated and the extracellular Ca(2+)-induced increase in [Ca(2+)](cyt) was enhanced in PASMC from patients with IPAH and animals with experimental pulmonary hypertension. Here, we report that the dihydropyridines (eg, nifedipine) increase [Ca(2+)](cyt) by activating CaSR in PASMC from IPAH patients (in which CaSR is upregulated), but not in normal PASMC.The nifedipine-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC was concentration dependent with a half maximal effective concentration of 0.20 µmol/L. Knockdown of CaSR with siRNA in IPAH-PASMC significantly inhibited the nifedipine-induced increase in [Ca(2+)](cyt), whereas overexpression of CaSR in normal PASMC conferred the nifedipine-induced rise in [Ca(2+)](cyt). Other dihydropyridines, nicardipine and Bay K8644, had similar augmenting effects on the CaSR-mediated increase in [Ca(2+)](cyt) in IPAH-PASMC; however, the nondihydropyridine blockers, such as diltiazem and verapamil, had no effect on the CaSR-mediated rise in [Ca(2+)](cyt).The dihydropyridine derivatives increase [Ca(2+)](cyt) by potentiating the activity of CaSR in PASMC independently of their blocking (or activating) effect on Ca(2+) channels; therefore, it is possible that the use of dihydropyridine Ca(2+) channel blockers (eg, nifedipine) to treat IPAH patients with upregulated CaSR in PASMC may exacerbate pulmonary hypertension.

| S-EPMC3642037 | biostudies-literature

The properties of ryanodine-sensitive Ca(2+) release in mouse gastric smooth muscle cells.

Project description:1. Under voltage-clamped conditions, gastric smooth muscle cells of BALB/c mice developed spontaneous (STOCs) and caffeine- (I(CAF)) and carbachol-induced (I(CCh)) transient outward currents. 2. In fura-2 microscopic measurements of intracellular Ca(2+) concentration ([Ca(2+)](i)), caffeine and carbachol (CCh) provoked similar transient [Ca(2+)](i) elevations. 3. Both I(CCh) and CCh-induced [Ca(2+)](i) elevation of single smooth muscle cells occurred in an 'all-or-nothing' fashion in contrast to the reproducible caffeine responses. 4. On the basis of the suppression of STOCs and I(CAF) by nicardipine, tetraethylammonium and iberiotoxin, but not by charybdotoxin nor apamin, it was suggested that both currents were generated by large conductance type Ca(2+)-activated K(+) channels. 5. In measurements of isometric tension, caffeine produced relaxation of gastric smooth muscle strips in a concentration-dependent manner (0.1 -- 3 mM). The concentration-dependent relaxation with caffeine was mimicked by dibutyryl cyclic AMP which produced potentiation of contraction triggered by 50 mM KCL. 6. At caffeine concentrations >3 mM, a transient contraction followed by relaxation was provoked as the quasi maximal response to caffeine. In the quasi maximal response, caffeine acted as a potent relaxant in smooth muscle strips precontracted with 50 mM KCl or 3 microM CCh. 7. The relaxation with caffeine was significantly accelerated in those strips precontracted with KCl or CCh. All these results suggest that ryanodine-sensitive Ca(2+) release, which is triggered by caffeine, is an important modifier of Ca(2+) homeostasis in the cytoplasm and the contractility of gastric smooth muscle cells of mice.

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Kv2.1 channels play opposing roles in regulating membrane potential, Ca2+ channel function, and myogenic tone in arterial smooth muscle.

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| S-EPMC7035623 | biostudies-literature

CD38 mediates angiotensin II-induced intracellular Ca(2+) release in rat pulmonary arterial smooth muscle cells.

Project description:CD38 is a multifunctional enzyme that catalyzes the formation of the endogenous Ca(2+)-mobilizing messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenosine dinucleotide phosphate (NAADP) for the activation of ryanodine receptors (RyRs) of sarcoplasmic reticulum and NAADP-sensitive Ca(2+) release channels in endolysosomes, respectively. It plays important roles in systemic vascular functions, but there is little information on CD38 in pulmonary arterial smooth muscle cells (PASMCs). Earlier studies suggested a redox-sensing role of CD38 in hypoxic pulmonary vasoconstriction. This study sought to characterize its roles in angiotensin II (Ang II)-induced Ca(2+) release (AICR) in PASMCs. Examination of CD38 expression in various rat arteries found high levels of CD38 mRNA and protein in pulmonary arteries. The Ang II-elicited Ca(2+) response consisted of extracellular Ca(2+) influx and intracellular Ca(2+) release in PASMCs. AICR activated in the absence of extracellular Ca(2+) was reduced by pharmacological or siRNA inhibition of CD38, by the cADPR antagonist 8-bromo-cADPR or ryanodine, and by the NAADP antagonist Ned-19 or disruption of endolysosomal Ca(2+) stores with the vacuolar H(+)-ATPase inhibitor bafilomycin A1. Suppression of AICR by the inhibitions of cADPR- and NAADP-dependent pathways were nonadditive, indicating interdependence of RyR- and NAADP-gated Ca(2+) release. Furthermore, AICR was inhibited by the protein kinase C inhibitor staurosporine, the nonspecific NADPH oxidase (NOX) inhibitors apocynin and diphenyleneiodonium, the NOX2-specific inhibitor gp91ds-tat, and the scavenger of reactive oxygen species (ROS) tempol. These results provide the first evidence that Ang II activates CD38-dependent Ca(2+) release via the NOX2-ROS pathway in PASMCs.

| S-EPMC4370261 | biostudies-literature

Effects of Hyperglycemia on Vascular Smooth Muscle Ca2+ Signaling.

Project description:Diabetes is a complex disease that is characterized with hyperglycemia, dyslipidemia, and insulin resistance. These pathologies are associated with significant cardiovascular implications that affect both the macro- and microvasculature. It is therefore important to understand the effects of various pathologies associated with diabetes on the vasculature. Here we directly test the effects of hyperglycemia on vascular smooth muscle (VSM) Ca2+ signaling in an isolated in vitro system using the A7r5 rat aortic cell line as a model. We find that prolonged exposure of A7r5 cells to hyperglycemia (weeks) is associated with changes to Ca2+ signaling, including most prominently an inhibition of the passive ER Ca2+ leak and the sarcoplasmic reticulum Ca2+-ATPase (SERCA). To translate these findings to the in vivo condition, we used primary VSM cells from normal and diabetic subjects and find that only the inhibition of the ER Ca2+ leaks replicates in cells from diabetic donors. These results show that prolonged hyperglycemia in isolation alters the Ca2+ signaling machinery in VSM cells. However, these alterations are not readily translatable to the whole organism situation where alterations to the Ca2+ signaling machinery are different.

| S-EPMC5497615 | biostudies-literature

Cannabidiol attenuates pulmonary arterial hypertension by improving vascular smooth muscle cells mitochondrial function.

Project description:Rationale: Pulmonary arterial hypertension (PAH) is a chronic disease associated with enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) and dysfunctional mitochondria, and the clinical therapeutic option for PAH is very limited. Recent studies showed that cannabidiol (CBD), a non-psychoactive constituent of cannabinoids, possessed antioxidant effect towards several cardiovascular diseases, whereas the mechanistic effect of CBD in PAH is unknown. Methods: In this study, the effects of CBD in PAH were determined by analyzing its preventive and therapeutic actions in PAH rodent models in vivo and PASMCs' proliferation test in vitro. Additionally, CBD's roles in mitochondrial function and oxidant stress were also assessed in PASMCs. Results: We found that CBD reversed the pathological changes observed in both Sugen-hypoxia and monocrotaline-induced PAH rodent models in a cannabinoid receptors-independent manner. Our results also demonstrated that CBD significantly inhibited the PASMCs' proliferation in PAH mice with less inflammation and reactive oxygen species levels. Moreover, CBD alleviated rodent PAH by recovering mitochondrial energy metabolism, normalizing the hypoxia-induced oxidant stress, reducing the lactate overaccumulation and abnormal glycolysis. Conclusions: Taken together, these findings confirm an important role for CBD in PAH pathobiology.

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Selective targeting of the α5-subunit of GABAA receptors relaxes airway smooth muscle and inhibits cellular calcium handling.

Project description:The clinical need for novel bronchodilators for the treatment of bronchoconstrictive diseases remains a major medical issue. Modulation of airway smooth muscle (ASM) chloride via GABAA receptor activation to achieve relaxation of precontracted ASM represents a potentially beneficial therapeutic option. Since human ASM GABAA receptors express only the α4- and α5-subunits, there is an opportunity to selectively target ASM GABAA receptors to improve drug efficacy and minimize side effects. Recently, a novel compound (R)-ethyl8-ethynyl-6-(2-fluorophenyl)-4-methyl-4H-benzo[f]imidazo[1,5-a][1,4] diazepine-3-carboxylate (SH-053-2'F-R-CH3) with allosteric selectivity for α5-subunit containing GABAA receptors has become available. We questioned whether this novel GABAA α5-selective ligand relaxes ASM and affects intracellular calcium concentration ([Ca(2+)]i) regulation. Immunohistochemical staining localized the GABAA α5-subunit to human ASM. The selective GABAA α5 ligand SH-053-2'F-R-CH3 relaxes precontracted intact ASM; increases GABA-activated chloride currents in human ASM cells in voltage-clamp electrophysiology studies; and attenuates bradykinin-induced increases in [Ca(2+)]i, store-operated Ca(2+) entry, and methacholine-induced Ca(2+) oscillations in peripheral murine lung slices. In conclusion, selective subunit targeting of endogenous α5-subunit containing GABAA receptors on ASM may represent a novel therapeutic option to treat severe bronchospasm.

| S-EPMC4421780 | biostudies-literature

A junctophilin-caveolin interaction enables efficient coupling between ryanodine receptors and BKCa channels in the Ca2+ microdomain of vascular smooth muscle.

Project description:Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (<400 nm) from fluorescently labeled JP2 or Cav1 particles, when they were moderately expressed in primary SMCs from mouse mesenteric artery. The removal of caveolae by genetic Cav1 ablation or methyl-β-cyclodextrin treatments significantly reduced coupling efficiency between Ca2+ sparks and BKCa channel activity in SMCs, an effect also observed after JP2 knockdown in SMCs. A 20-amino acid-long region in JP2 appeared to be essential for the observed JP2-Cav1 interaction, and we also observed an interaction between JP2 and the BKCa channel. It can be concluded that the JP2-Cav1 interaction provides a structural and functional basis for the Ca2+ microdomain at PM-SR junctions and mediates cross-talk between RyRs and BKCa channels, converts local Ca2+ sparks into membrane hyperpolarization, and contributes to stabilizing resting tone in SMCs.

| S-EPMC6721949 | biostudies-literature

Bone morphogenetic protein 2 decreases TRPC expression, store-operated Ca(2+) entry, and basal [Ca(2+)]i in rat distal pulmonary arterial smooth muscle cells.

Project description:Recent studies indicate that multiple bone morphogenetic protein (BMP) family ligands and receptors are involved in the development of pulmonary arterial hypertension, yet the underlying mechanisms are incompletely understood. Although BMP2 and BMP4 share high homology in amino acid sequence, they appear to exert divergent effects on chronic hypoxic pulmonary hypertension (CHPH). While BMP4 promotes vascular remodeling, BMP2 prevents CHPH. We previously demonstrated that BMP4 upregulates the expression of canonical transient receptor potential channel (TRPC) proteins and, thereby, enhances store-operated Ca(2+) entry (SOCE) and elevates intracellular Ca(2+) concentration ([Ca(2+)]i) in pulmonary arterial smooth muscle cells (PASMCs). In this study, we investigated the effects of BMP2 on these variables in rat distal PASMCs. We found that treatment with BMP2 (50 ng/ml, 60 h) inhibited TRPC1, TRPC4, and TRPC6 mRNA and protein expression. Moreover, BMP2 treatment led to reduced SOCE and decreased basal [Ca(2+)]i in PASMCs. These alterations were associated with decreased PASMC proliferation and migration. Conversely, knockdown of BMP2 with specific small interference RNA resulted in increased cellular levels of TRPC1, TRPC4, and TRPC6 mRNA and protein, enhanced SOCE, elevated basal [Ca(2+)]i, and increased proliferation and migration of PASMCs. Together, these results indicate that BMP2 participates in regulating Ca(2+) signaling in PASMCs by inhibiting TRPC1, TRPC4, and TRPC6 expression, thus leading to reduced SOCE and basal [Ca(2+)]i and inhibition of cell proliferation and migration.

| S-EPMC3651608 | biostudies-literature

Reactive Oxygen Species Mediate the Suppression of Arterial Smooth Muscle T-type Ca2+ Channels by Angiotensin II.

Project description:Vascular T-type Ca2+ channels (CaV3.1 and CaV3.2) play a key role in arterial tone development. This study investigated whether this conductance is a regulatory target of angiotensin II (Ang II), a vasoactive peptide that circulates and which is locally produced within the arterial wall. Patch clamp electrophysiology performed on rat cerebral arterial smooth muscle cells reveals that Ang II (100 nM) inhibited T-type currents through AT1 receptor activation. Blocking protein kinase C failed to eliminate channel suppression, a finding consistent with unique signaling proteins enabling this response. In this regard, inhibiting NADPH oxidase (Nox) with apocynin or ML171 (Nox1 selective) abolished channel suppression highlighting a role for reactive oxygen species (ROS). In the presence of Ni2+ (50 µM), Ang II failed to modulate the residual T-type current, an observation consistent with this peptide targeting CaV3.2. Selective channel suppression by Ang II impaired the ability of CaV3.2 to alter spontaneous transient outward currents or vessel diameter. Proximity ligation assay confirmed Nox1 colocalization with CaV3.2. In closing, Ang II targets CaV3.2 channels via a signaling pathway involving Nox1 and the generation of ROS. This unique regulatory mechanism alters BKCa mediated feedback giving rise to a "constrictive" phenotype often observed with cerebrovascular disease.

| S-EPMC5823855 | biostudies-literature

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