Involvement of phospholipase D in store-operated calcium influx in vascular smooth muscle cells

In non-excitable cells, sustained intracellular Ca2+ increase critically depends on influx of extracellular Ca2+. Such Ca2+ influx is thought to occur by a 'store-operated' mechanism, i.e. the signal for Ca2+ entry is believed to result from the initial release of Ca2+ from inositol 1,4,5-trisphosphate-sensitive intracellular stores. Here we show that the depletion of cellular Ca2+ stores by thapsigargin or bradykinin is functionally linked to a phosphoinositide-specific phospholipase D (PLD) activity in cultured vascular smooth muscle cells (VSMC), and that phosphatidic acid formed via PLD enhances sustained calcium entry in this cell type. These results suggest a regulatory role for PLD in store-operated Ca2+ entry in VSMC.     

Calcium channels in smooth muscle cells

In smooth muscle cells, the electrophysiological properties of potential-dependent calcium channels are similar to those described in other excitable cells. The calcium current is dependent on the extracellular calcium concentration; it is insensitive to external sodium removal and tetrodotoxin application. Other ions (Ba2+, Sr2+, Na+) can flow through the calcium channel. This channel is blocked by Mn2+, Co2+, Cd2+ and by organic inhibitors. The inactivation mechanism is mediated by both the membrane potential and the calcium influx. Ca2+ ions can also penetrate into the cell through receptor-operated channels. These channels show a low ionic selectivity and are generally less sensitive to organic Ca-blockers than the potential-dependent calcium channels. The finding of specific channel inhibitors as well as the study of the biochemical pathways between receptor activation and channel opening are prerequisites to further characterization of receptor-operated channels.     

S1P activates store-operated calcium entry via receptor- and non-receptor-mediated pathways in vascular smooth muscle cells

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.     

Protein kinases modulate store-operated channels in pulmonary artery smooth muscle cells

Background  

This study investigates whether protein kinase G (PKG), protein kinase A (PKA) and protein kinase C (PKC) are involved in the regulatory mechanisms of store-operated channel (SOC) in pulmonary arteries.     

Store-operated Ca-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca²⁺ store in smooth muscle triggered a Ca²⁺ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca²⁺ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca²⁺ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca²⁺ ions. Two activation mechanisms have been identified which involve Ca²⁺ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of α-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

Evidence that Orai1 does not contribute to store-operated TRPC1 channels in vascular smooth muscle cells

Ca²⁺-permeable store-operated channels (SOCs) mediate Ca²⁺ entry pathways which are involved in many cellular functions such as contraction, growth, and proliferation. Prototypical SOCs are formed of Orai1 proteins and are activated by the endo/sarcoplasmic reticulum Ca²⁺ sensor stromal interaction molecule 1 (STIM1). There is considerable debate about whether canonical transient receptor potential 1 (TRPC1) proteins also form store-operated channels (SOCs), and if they do, is Orai1 involved. We recently showed that stimulation of TRPC1-based SOCs involves store depletion inducing STIM1-evoked Gαq/PLCβ1 activity in contractile vascular smooth muscle cells (VSMCs). Therefore the present work investigates the role of Orai1 in activation of TRPC1-based SOCs in freshly isolated mesenteric artery VSMCs from wild-type (WT) and Orai1^?/? mice. Store-operated whole-cell and single channel currents recorded from WT and Orai1^?/? VSMCs had similar properties, with relatively linear current-voltage relationships, reversal potentials of about +20mV, unitary conductances of about 2pS, and inhibition by anti-TRPC1 and anti-STIM1 antibodies. In Orai1^?/? VSMCs, store depletion induced PLCβ1 activity measured with the fluorescent phosphatidylinositol 4,5-bisphosphate/inositol 1,4,5-trisphosphate biosensor GFP-PLCδ1-PH, which was prevented by knockdown of STIM1. In addition, in Orai1^?/? VSMCs, store depletion induced translocation of STIM1 from within the cell to the plasma membrane where it formed STIM1-TRPC1 interactions at discrete puncta-like sites. These findings indicate that activation of TRPC1-based SOCs through a STIM1-activated PLCβ1 pathway are likely to occur independently of Orai1 proteins, providing evidence that TRPC1 channels form genuine SOCs in VSMCs with a contractile phenotype.     

A store-operated nonselective cation channel in lymphocytes is activated directly by Ca(2+) influx factor and diacylglycerol

Agonist-receptor interactions at the plasma membrane often lead to activation of store-operated channels (SOCs) in the plasma membrane, allowing for sustained Ca(2+) influx. While Ca(2+) influx is important for many biological processes, little is known about the types of SOCs, the nature of the depletion signal, or how the SOCs are activated. We recently showed that in addition to the Ca(2+) release-activated Ca(2+) (CRAC) channel, both Jurkat T cells and human peripheral blood mononuclear cells express novel store-operated nonselective cation channels that we termed Ca(2+) release-activated nonselective cation (CRANC) channels. Here we demonstrate that activation of both CRAC and CRANC channels is accelerated by a soluble Ca(2+) influx factor (CIF). In addition, CRANC channels in inside-out plasma membrane patches are directly activated upon exposure of their cytoplasmic side to highly purified CIF preparations. Furthermore, CRANC channels are also directly activated by diacylglycerol. These results strongly suggest that the Ca(2+) store-depletion signal is a diffusible molecule and that at least some SOCs may have dual activation mechanisms.     

Platelet-Derived growth factor activates phospholipase D and chemotactic responses in vascular smooth muscle cells

Summary Platelet-derived growth factor (BB dimer; PDGF-BB) stimulates a mitogenic response in A-10 vascular smooth muscle cells. In addition, PDGF-BB stimulates phospholipase D activity against phosphatidylcholine in A-10 cells. This response was observed as a rapid metabolism of phosphatidylcholine to phosphatidate and choline; a subsequent metabolism generates sustained levels of diacylglycerol. The accumulation of phosphatidylethanol, a transphosphatidylation product of phospholipase D, was obvious in PDGF-treated cells. PDGF-BB also stimulates a chemotactic response in A-10 cells. The concentrations of PDGF-BB required to stimulate mitogenesis, phospholipase D activity and chemotaxis are similar. This finding shows that PDGF induces a variety of cellular responses and suggests that these responses may share common metabolic pathways. That conception was tested by investigating the activity of the different PDGF dimers. PDGF-AA had little or no activity in A-10 cells for any of the responses measured. PDGF-AB and PDGF-BB were equally potent in stimulating mitogenic responses. However, the AB heterodimer was only half as active as PDGF-BB with respect to activation of phospholipase D and chemotactic responses. These results demonstrate that PDGF stimulates phospholipase D in vascular smooth muscle cells. In addition, the data indicate that different PDGF dimers can transduce varying signals and suggest a link between the mechanisms by which PDGF-BB activates phospholipase D and the chemotactic response. Partial support for this project was obtained through a grant to C. J. W. from the American Heart Association (#88-034G) and from the W. Alton Jones Foundation.     

Parathyroid hypertensive factor inhibits voltage-gated K+ channels in vascular smooth muscle cells

Parathyroid hypertensive factor (PHF) has been implicated in regulation of vascular smooth muscle tone and pathogenesis of several forms of hypertension. Earlier studies have suggested that PHF enhances the actions of other vasoconstrictors, while it has no in vitro vasoconstrictor property of its own. PHF was previously found to enhance the L-type Ca channel currents and intracellular Ca responses to depolarization in vascular smooth muscle cells (VSMCs). The present study examined whether PHF might act on K channels in the plasma membrane of VSMCs. Primary cultured VSMCs from rat tail artery were used. The whole-cell version of the patch-clamp technique was used under conditions in which there was no contribution of Ca-activated K channels to the outward current. Both purified and semipurified PHF inhibited the delayed rectifier type potassium current in a dose-dependent manner. The effect was time dependent and was first significantly different from the control current after 30 min. The inhibition of the delayed rectifier K channel was associated with a time-dependent decrease in the resting membrane potential. Therefore, PHF may alter VSMC cellular Ca responses by reducing the membrane potential to a level closer to the activation potential of Ca channels.     

IL-22 activates oxidant signaling in pulmonary vascular smooth muscle cells

Reactive oxygen species (ROS) mediate cell-signaling processes in response to various ligands and play important roles in the pathogenesis of cardiovascular diseases. The present study reports that interleukin-22 (IL-22) elicits signal transduction in vascular smooth muscle cells (SMCs) through a ROS-dependent mechanism. We find that pulmonary artery SMCs express IL-22 receptor alpha 1 and that IL-22 activates STAT3 through this receptor. IL-22-induced signaling is found to be mediated by NADPH oxidase, as indicated by the observations that the inhibition and siRNA knock-down of this enzyme inhibit IL-22 signaling. IL-22 triggers the oxidative modifications of proteins through protein carbonylation and protein glutathionylation. Mass spectrometry identified some proteins that are carbonylated in response to IL-22 stimulation, including α-enolase, heat shock cognate 71 kDa protein, mitochondrial 60 kDa heat shock protein, and cytoplasmic 2 actin and determined that α-tubulin is glutathionylated. Protein glutathionylation and STAT3 phosphorylation are enhanced by the siRNA knock-down of glutaredoxin, while IL-22-mediated STAT3 phosphorylation is suppressed by knocking down thioredoxin interacting protein, an inhibitor of thioredoxin. IL-22 is also found to promote the growth of SMCs via NADPH oxidase. In rats, pulmonary hypertension is found to be associated with increased smooth muscle IL-22 expression. These results show that IL-22 promotes the growth of pulmonary vascular SMCs via a signaling mechanism that involves NADPH oxidase-dependent oxidation.     

Bradykinin activates calcium-dependent potassium channels in cultured human airway smooth muscle cells

Bradykinin (BK) is an inflammatory mediator that can cause bronchoconstriction. In this study, we investigated the membrane currents induced by BK in cultured human airway smooth muscle (ASM) cells. Depolarization of the cells induced outward currents, which were inhibited by tetraethylammonium (TEA) in a concentration-dependent manner with an IC50 of 0.33 microM. The currents were increased by elevating intracellular free Ca2+ concentration, suggesting they are calcium-activated potassium channels [I(K(Ca))]. Preexposure to inhibitor of I(K(Ca)) of large conductance (BKCa), iberiotoxin, and small conductance (SKCa), apamin, inhibited the increase of outward current induced by BK. The relative contribution of BKCa was greatest in early passage cells. Both nickel and SKF-96365 (10 microM) inhibited the increase of the I(K(Ca)) induced by BK; however, the l-type Ca2+ channel blocker, nifedipine, had no effect. Activation of the BK-induced current was inhibited by heparin, indicating dependence on intact inositol 1,4,5-triphosphate (IP3)-sensitive intracellular Ca2+ stores. BK also increased inositol phosphate accumulation and induced a transient Ca2+-activated chloride current (CACC) and a sustained nonselective cation current (I(CAT)). In summary, BK activates BKCa, SKCa, CACC, and I(CAT) via IP3-sensitive stores in human ASM.     

Store-operated Ca2+-permeable non-selective cation channels in smooth muscle cells

Over twenty years ago it was shown that depletion of the intracellular Ca2+ store in smooth muscle triggered a Ca2+ influx mechanism. The purpose of this review it to describe recent electrophysiological data which indicate that Ca2+ influx occurs through discrete ion channels in the plasmalemma of smooth muscle cells. The effect of external Ca2+ on the amplitude and reversal potential of whole-cell and single channel currents suggests that there are at least two, and probably more, distinct store-operated channels (SOCs) which have markedly different permeabilities to Ca2+ ions. Two activation mechanisms have been identified which involve Ca2+ influx factor and protein kinase C (PKC) activation via diacylglycerol. In addition, in rabbit portal vein cells there is evidence that stimulation of alpha-adrenoceptors can stimulate SOC opening via PKC in a store-independent manner. There is at present little knowledge on the molecular identity of SOCs but it has been proposed that TRPC1 may be a component of the functional channel. We also summarise the data showing that SOCs may be involved in contraction and cell proliferation of smooth muscle. Finally, we highlight the similarities and differences of SOCs and receptor-operated cation channels that are present in native rabbit portal vein myocytes.     

Calcium homeostasis and nerve growth factor secretion from vascular and bladder smooth muscle cells

Bladder and vascular smooth muscle cells cultured from four rat strains (WKY, SHR, WKHA, WKHT) differing in rates of nerve growth factor (NGF) production were used to determine whether a relationship exists between intracellular calcium and NGF secretion. Basal cytosolic calcium was related to basal NGF secretion rates in bladder and vascular smooth muscle cells from all four strains with the exception of WKHT bladder muscle cells. Thrombin is a calcium-mobilizing agent and increases NGF production from vascular but not bladder smooth muscle cells. Strain differences were found in the magnitude of the calcium peak induced by thrombin in vascular smooth muscle cells, but these differences did not correlate with NGF secretion. Thrombin caused a calcium response in bladder smooth muscle cells without influencing NGF production. Quenching the calcium transient with a calcium chelator had no effect on thrombin-inducted NGF secretion rates in vascular smooth muscle cells. Thus, basal intracellular calcium may establish a set point for NGF secretion from smooth muscle. In addition, transient elevations in cytosolic calcium were unrelated to the induction of NGF output.     

Cyclic nucleotide regulation of store-operated Ca2+ influx in airway smooth muscle

Sarcoplasmic reticulum (SR) Ca2+ release and plasma membrane Ca2+ influx are key to intracellular Ca2+ ([Ca2+]i) regulation in airway smooth muscle (ASM). SR Ca2+ depletion triggers influx via store-operated Ca2+ channels (SOCC) for SR replenishment. Several clinically relevant bronchodilators mediate their effect via cyclic nucleotides (cAMP, cGMP). We examined the effect of cyclic nucleotides on SOCC-mediated Ca2+ influx in enzymatically dissociated porcine ASM cells. SR Ca2+ was depleted by 1 microM cyclopiazonic acid in 0 extracellular Ca2+ ([Ca2+]o), nifedipine, and KCl (preventing Ca2+ influx through L-type and SOCC channels). SOCC was then activated by reintroduction of [Ca2+]o and characterized by several techniques. We examined cAMP effects on SOCC by activating SOCC in the presence of 1 microM isoproterenol or 100 microM dibutryl cAMP (cell-permeant cAMP analog), whereas we examined cGMP effects using 1 microM (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO nitric oxide donor) or 100 microM 8-bromoguanosine 3',5'-cyclic monophosphate (cell-permeant cGMP analog). The role of protein kinases A and G was examined by preexposure to 100 nM KT-5720 and 500 nM KT-5823, respectively. SOCC-mediated Ca2+ influx was dependent on the extent of SR Ca2+ depletion, sensitive to Ni2+ and La3+, but not inhibitors of voltage-gated influx channels. cAMP as well as cGMP potently inhibited Ca2+ influx, predominantly via their respective protein kinases. Additionally, cAMP cross-activation of protein kinase G contributed to SOCC inhibition. These data demonstrate that a Ni2+/La3+-sensitive Ca2+ influx in ASM triggered by SR Ca2+ depletion is inhibited by cAMP and cGMP via a protein kinase mechanism. Such inhibition may play a role in the bronchodilatory response of ASM to clinically relevant drugs (e.g., beta-agonists vs. nitric oxide).     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.