Evidence for receptor-mediated bivalent-cation entry in A10 vascular smooth-muscle cells.
Ontology highlight
ABSTRACT:
SUBMITTER: Simpson AW
PROVIDER: S-EPMC1131279 | biostudies-other | 1990 Apr
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Sphingosine-1-phosphate (S1P) has been shown to modulate intracellular Ca(2+) through both G protein-coupled receptors and intracellular second messenger pathways. The precise mechanism by which S1P activates store-operated calcium entry (SOCE) in vascular smooth muscle cells (VSMCs) has not been fully characterized. Because sphingolipids and Ca(2+) modulate proliferation and constriction in VSMCs, characterizing the connection between S1P and SOCE may provide novel therapeutic targets for vascular diseases. We found that S1P triggered STIM1 puncta formation and SOCE in VSMCs. S1P-activated SOCE was inhibited by 2-aminoethoxydiphenyl borate (2-APB), diethylstilbestrol (DES), and gadolinium (Gd(3+)). SOCE was observed in VSMCs lacking either S1P(2) or S1P(3) receptors, suggesting that S1P acts via multiple signaling pathways. Indeed, both extracellular and intracellular S1P application increased the total internal reflection fluorescence signal in VSMCs cells transfected with STIM1-yellow fluorescent protein in a 2-APB-sensitive manner. These data, and the fact that 2-APB, DES, and Gd(3+) all inhibited S1P-induced cerebral artery constriction, suggest that SOCE modulates S1P-induced vasoconstriction in vivo. Finally, S1P-induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P(1), and S1P(3) receptor mRNA. These data demonstrate that S1P can act through both receptors and a novel intracellular pathway to activate SOCE. Because S1P-induced SOCE contributes to vessel constriction and is increased in proliferative VSMCs, it is likely that S1P/SOCE signaling in proliferative VSMCs may play a role in vascular dysfunction such as atherosclerosis and diabetes.
| S-EPMC3074633 | biostudies-literature
Project description:Differentiation of BC3H1 cells leads to expression of a variety of proteins characteristic of smooth muscle and to changes in the behaviour of intracellular Ca2+ stores. Treatment of both differentiated and undifferentiated cells with thapsigargin (2 microM) emptied their intracellular Ca2+ stores, and in the presence of extracellular Ca2+ caused an increase in cytosolic [Ca2+] that rapidly reversed after its removal. The amplitudes of these capacitative Ca2+ entry signals were 101 +/- 8 nM (n = 42) in differentiated cells and 188 +/- 16 nM (n = 35) in undifferentiated cells. Mn2+ entry in thapsigargin-treated cells, measured by recording the quenching of cytosolic fura 2 fluorescence, was 374 +/- 26% (n = 34) and 154 +/- 7% (n = 41) of control rates in differentiated and undifferentiated cells, respectively. Empty stores caused Ba2+ entry to increase to 282 +/- 20% (n = 8) of its basal rate in differentiated cells and to 187 +/- 20% (n = 8) in undifferentiated cells. Rates of Ca2+ extrusion, measured after rapid removal of extracellular Ca2+ from cells in which capacitative Ca2+ entry had been activated, were similar in differentiated (t1/2 = 23 +/- 2 s, n = 7) and undifferentiated (23 +/- 1 s, n = 6) cells. The different relationships between capacitative Ca2+ and Mn2+ signals are not, therefore, a consequence of more active Ca2+ extrusion mechanisms in differentiated cells, nor are they a consequence of different fura 2 loadings in the two cell types. We conclude that during differentiation of BC3Hl cells, the cation selectivity of the capacitative pathway changes, becoming relatively more permeable to Mn2+ and Ba2+. The change may result either from expression of a different capacitative pathway or from modification of the permeation properties of a single pathway.
| S-EPMC1217415 | biostudies-other
Project description:In non-excitable cells, activation of G-protein-coupled phospholipase C (PLC)-linked receptors causes the release of Ca(2+) from intracellular stores, which is followed by transmembrane Ca(2+) entry. This Ca(2+) entry underlies a small and sustained phase of the cellular [Ca(2+)](i) increases and is important for several cellular functions including gene expression, secretion and cell proliferation. This form of transmembrane Ca(2+) entry is supported by agonist-activated Ca(2+)-permeable ion channels that are activated by store depletion and is referred to as store-operated Ca(2+) entry (SOCE) and represents a major pathway for agonist-induced Ca(2+) entry. In excitable cells such as smooth muscle cells, Ca(2+) entry mechanisms responsible for sustained cellular activation are normally considered to be mediated via either voltage-operated or receptor-operated Ca(2+) channels. Although SOCE occurs following agonist activation of smooth muscle, this was thought to be more important in replenishing Ca(2+) stores rather than acting as a source of activator Ca(2+) for the contractile process. This review summarizes our current knowledge of SOCE as a regulator of vascular smooth muscle tone and discusses its possible role in the cardiovascular function and disease. We propose a possible hypothesis for its activation and suggest that SOCE may represent a novel target for pharmacological therapeutic intervention.
| S-EPMC2267267 | biostudies-other
Project description:ObjectiveTransient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR-TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice.ConclusionsWe show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.
| S-EPMC8299307 | biostudies-literature
Project description:Acid-sensing ion channels (ASIC) are voltage-insensitive, cationic channels that have recently been identified in vascular smooth muscle (VSM). It is possible that ASIC contribute to vascular reactivity via Na(+) and Ca(2+) conductance; however, their function in VSM is largely unknown. In pulmonary VSM, store-operated Ca(2+) entry (SOCE) plays a significant role in vasoregulatory mechanisms such as hypoxic pulmonary vasoconstriction and receptor-mediated arterial constriction. Therefore, we hypothesized that ASIC contribute to SOCE in pulmonary VSM. We examined SOCE resulting from depletion of intracellular Ca(2+) stores with cyclopiazonic acid in isolated small pulmonary arteries and primary cultured pulmonary arterial smooth muscle cells by measuring 1) changes in VSM [Ca(2+)](i) using fura-2 indicator dye, 2) Mn(2+) quenching of fura-2 fluorescence, and 3) store-operated Ca(2+) and Na(+) currents using conventional whole cell patch-clamp configuration in voltage-clamp mode. The role of ASIC was assessed by the use of the ASIC inhibitors, amiloride, benzamil, and psalmotoxin 1, or siRNA directed towards ASIC1, ASIC2, or ASIC3 isoforms. We found that store-operated VSM [Ca(2+)](i) responses, Mn(2+) influx, and inward cationic currents were attenuated by either pharmacological ASIC inhibition or treatment with ASIC1 siRNA. These data establish a unique role for ASIC1 in mediating SOCE in pulmonary VSM and provide new insight into mechanisms of VSM Ca(2+) entry and pulmonary vasoregulation.
| S-EPMC2742795 | biostudies-literature
Project description:Atrial natriuretic peptide (ANP) mediates a variety of physiological effects through its interaction with ANP-A, ANP-B or ANP-C receptors. However, controversies exist regarding the involvement of ANP-C receptor and adenylyl cyclase/cAMP signal-transduction systems to which these receptors are coupled in mediating these responses. In the present studies, we have employed an antisense approach to eliminate the ANP-C receptor and to examine the effect of this elimination on adenylyl cyclase inhibition. An 18-mer antisense phosphorothioate oligodeoxynucleotide (OH-2) targeted at the initiation codon of the ANP-C receptor was used to examine its effects on the expression of the ANP-C receptor and ANP-C-receptor-mediated inhibition of adenylyl cyclase in vascular smooth-muscle cells (A10). Treatment of the cells with antisense oligonucleotide resulted in complete attenuation of C-ANP(4-23) [des(Gln(18), Ser(19), Gln(20), Leu(21), Gly(22))ANP(4-23)-NH(2)]-mediated inhibition of adenylyl cyclase, whereas sense and missense oligomers did not affect the inhibition of adenylyl cyclase by C-ANP(4-23). In addition, the stimulatory effects of guanine nucleotides, isoproterenol, sodium fluoride and forskolin as well as the inhibitory effects of angiotensin II on adenylyl cyclase were not affected by antisense-oligonucleotide treatment. The attenuation of C-ANP(4-23)-mediated inhibition of adenylyl cyclase by antisense oligonucleotide was dose- and time-dependent. A complete attenuation of ANP-C-receptor-mediated inhibition of adenylyl cyclase was observed at 2.5 microM. In addition, treatment of the cells with antisense oligonucleotide and not with sense or missense oligomers resulted in the inhibition of the levels of ANP-C-receptor protein and mRNA as determined by immunoblotting and Northern blotting using antisera against the ANP-C receptor and a cDNA probe of the ANP-C receptor respectively. On the other hand, ANP-A/B-receptor-mediated increases in cGMP levels were not inhibited by antisense-oligonucleotide treatment. Our results demonstrate conclusively that the elimination of ANP-C receptor by antisense oligonucleotide attenuates ANP-induced inhibition of adenylyl cyclase and provide evidence that antisense oligonucleotide of the ANP-C receptor may serve as a useful pharmacological tool to elucidate the physiological functions of the ANP-C receptor.
| S-EPMC1220855 | biostudies-other
Project description:The preferential accumulation of vascular smooth muscle cells (vSMCs) on arteries versus veins during early development is a well-described phenomenon, but the molecular pathways underlying this polarization are not well understood. In zebrafish, the cxcr4a receptor (mammalian CXCR4) and its ligand cxcl12b (mammalian CXCL12) are both preferentially expressed on arteries at time points consistent with the arrival and differentiation of the first vSMCs during vascular development. We show that autocrine cxcl12b/cxcr4 activity leads to increased production of the vSMC chemoattractant ligand pdgfb by endothelial cells in vitro and increased expression of pdgfb by arteries of zebrafish and mice in vivo. Additionally, we demonstrate that expression of the blood flow-regulated transcription factor klf2a in primitive veins negatively regulates cxcr4/cxcl12 and pdgfb expression, restricting vSMC recruitment to the arterial vasculature. Together, this signalling axis leads to the differential acquisition of vSMCs at sites where klf2a expression is low and both cxcr4a and pdgfb are co-expressed, i.e. arteries during early development.
| S-EPMC7719186 | biostudies-literature
Project description:Hypoxia augments PAF receptor (PAFr) binding and PAFr protein expression in venous SMC (SMC-PV). We compared effect of acute and prolonged hypoxia (pO(2)<40 torr) on PAFr-mediated responses in arterial SMC (SMC-PA) and SMC-PV. Cells were studied for 30 min (acute) or for 48 h (prolonged) hypoxia and compared to normoxic (pO(2) ~100 torr) conditions. PAF binding was quantified in fmol/10(6) cells (mean ± SEM). PAF binding in normoxia were SMC-PA, 5.2 ± 0.2 and in SMC-PV, 19.3 ± 1.1; values in acute hypoxia were SMC-PA, 7.7 ± 0.4 and in SMC-PV, 27.8 ± 1.7. Prolonged hypoxia produced 6-fold increase in binding in SMC-PA, but only 2-fold increase in SMC-PV, but binding in SMC-PV was still higher. Acute hypoxia augmented inositol phosphate release by 50% and 40% in SMC-PA and SMC-PV, respectively. During normoxia, PAFr mRNA expression by both cell types was similar, but expression in hypoxia by SMC-PA was greater. In SMC-PA, hypoxia and PAF augmented intracellular calcium flux. Re-exposure of cells to 30 min normoxia after 48 h hypoxia decreased binding by 45-60%, suggesting immediate down-regulation of hypoxia-induced PAFr-mediated effects. We speculate that re-oxygenation immediately reverses hypoxia effect probably due to oxygen tension-dependent reversibility of PAFr activation and suggest that exposure of the neonate to prolonged state of hypoxia will vilify oxygen exchange capacity of the neonatal lungs.
| S-EPMC2991540 | biostudies-literature
Project description:An indazole derivative, YC-1, was identified in this study to be capable of reversibly and effectively inhibiting proliferation of rat A10 vascular smooth-muscle cells (VSMCs) in vitro. YC-1 (1-100 microM) dose-dependently inhibited [3H]thymidine incorporation into DNA in rat A10 VSMCs that were synchronized by serum depletion and then restimulated by addition of 10% foetal calf serum (FCS), whereas FCS-induced [3H]thymidine incorporation into rat synchronized endothelial cells was unaffected by this agent. The dose of YC-1 required to cause inhibition of FCS-induced proliferation was similar to that necessary for the formation of cellular cyclic GMP (cGMP). Guanylate cyclase activity in soluble fractions of VSMCs was activated by YC-1 (1-100 microM), whereas cGMP-specific phosphodiesterase activity was unaffected by this compound. The anti-proliferative effect of YC-1 was mimicked by 8-bromo-cGMP, a membrane-permeable cGMP analogue, and was antagonized by KT 5823 (0.2 microM), a selective inhibitor of protein kinase G. The anti-proliferative effect of YC-1 was also antagonized by Methylene Blue (50 microM), a guanylate cyclase inhibitor, and was potentiated by 3-isobutyl-1-methylxanthine (500 microM), a phosphodiesterase inhibitor. These results verified that YC-1 is a direct soluble guanylate cyclase activator in A10 VSMCs, and the anti-proliferative effect of YC-1 is mediated by cGMP. YC-1 still inhibited FCS-induced DNA synthesis even when added 10-18 h after restimulation of the serum-deprived A10 VSMCs with 10% FCS. Flow cytometry in synchronized populations revealed an acute blockage of FCS-inducible cell-cycle progression at a point in the G1/S-phase in YC-1 (100 microM)-treated cells. The inhibition of proliferation by YC-1 was demonstrated to be independent of cell damage, as documented by several criteria of cell viability. In conclusion, YC-1 reversibly and effectively inhibited the proliferation of VSMCs, suggesting that it has potential as a therapeutic agent in the prevention of vascular diseases.
| S-EPMC1136590 | biostudies-other
Project description:Stimulation of Jurkat E6 cells with anti-CD3 antibody results in a characteristic rise in [Ca2+]i which is due to both the release of Ca2+ from intracellular stores and the entry of external Ca2+. Individual components of the [Ca2+]i increase were investigated by measuring intracellular Ca2+ release in the absence of external Ca2+ and determining influx of bivalent cations by following the entry of Mn2+. The increase in [Ca2+]i induced by anti-CD3 antibody in the presence or absence of extracellular Ca2+ could be inhibited by the non-selective kinase inhibitor staurosporine, which also inhibits anti-CD3-stimulated phospholipase C activity. Staurosporine also inhibits the influx of bivalent cations induced by anti-CD3 antibody, but not that induced by depletion of intracellular Ca2+ stores using thapsigargin. The effect of staurosporine was compared with that of Ro 31-8425, a potent and selective inhibitor of protein kinase C (PKC). Ro 31-8425, at concentrations up to 10 microM, has no inhibitory effect on the anti-CD3 antibody-induced [Ca2+]i increase or phospholipase C activity. These studies are consistent with the concept that augmentation of [Ca2+]i by stimulated T-cell receptors requires activation of a kinase, probably a tyrosine kinase such as p56lck, ZAP-70 or p59fyn, and is independent of PKC. Phorbol esters inhibit the anti-CD3-stimulated [Ca2+]i increase and phospholipase C activity, showing that this can be negatively regulated by PKC. A small potentiation of the anti-CD3 antibody-induced [Ca2+]i rise in the presence of extracellular Ca2+ was detected in the presence of Ro 31-8425; this suggests that T-cell-receptor ligation can also limit the increase in [Ca2+]i via PKC activation.
| S-EPMC1137379 | biostudies-other