Intracellular pH as a determinant of vascular smooth muscle function

Intracellular pH (pHi) is a physiological parameter that is intimately linked to contractility, growth and proliferation of vascular smooth muscle (VSM). Regarding contractility, no general unifying concept of pHi regulation but a rather complex relation between pHi signals and vascular tone has been revealed so far. The modulation of vasotone by pHi depends on the type of blood vessel as well as on the pattern of regulatory input signals. In addition, changes in pHi have been recognized as an important cellular signal to determine the fate of cells in terms of proliferation or apoptosis. Cellular sensors for pHi include a variety of ion transport systems which control intracellular Ca2+ gradients and are likely to serve as a link between pHi and cell functions. Here we provide an overview on the potential targets and mechanisms that transduce pHi signals in VSM. The role of pHi-sensing signaling complexes and localized pHi signaling as the basis of diversity of pHi regulation of VSM function is discussed.     

Ca2+-transport ATPases of vascular smooth muscle

To characterize the Ca2+-transport properties of the plasma membrane and of the endoplasmic reticulum of bovine pulmonary artery, membrane vesicles are subfractionated by a procedure of density-gradient centrifugation that takes advantage of the selective effect of digitonin on the density of plasma-membrane vesicles. The obtained endoplasmic-reticulum fraction contains hardly any plasma-membrane vesicles, whereas the plasma-membrane fraction is still contaminated by a substantial amount of endoplasmic-reticulum vesicles. An adenosine 5'-triphosphate (ATP) energized Ca2+-transport system and a Ca2+-stimulated ATPase activity are present in both subcellular fractions. The Ca2+ transport by the plasma membrane is catalyzed by a (Ca2+,Mg2+)-ATPase of Mr 130,000. It binds calmodulin and it has a low steady-state phosphoprotein intermediate level. The endoplasmic-reticulum vesicles contain a Ca2+-transport ATPase of Mr 100,000 that is characterized by a high steady-state phosphointermediate level. It is antigenically related to the Ca2+-pump protein of cardiac sarcoplasmic reticulum. Phospholamban, the regulatory protein of the Ca2+-transport enzyme of cardiac sarcoplasmic reticulum, is also present in the endoplasmic reticulum of the pulmonary artery. A comparison of these fractions with the previously characterized fractions from porcine gastric smooth muscle reveals important differences in the basal Mg2-ATPase activity, in the ratio of the (Ca2+,Mg2+)-ATPase of the plasmalemma to that of the endoplasmic reticulum, and in the ratio of the (Na+,K+)-ATPase activity to the plasmalemmal (Ca2+,Mg2+)-ATPase activity. These differences can be ascribed in part to the species and in part to the tissue. These data suggest that in the bovine pulmonary artery the Ca2+ extrusion via the ATP-dependent Ca2+ pump may have a less predominant role, and that the Ca2+ uptake by the endoplasmic reticulum, and possibly also the Ca2+ extrusion via the Na+-Ca2+ exchanger could be more important in this tissue than in the porcine stomach.     

Protein kinase C as a modulator of response amplification in vascular smooth muscle

The amplification of alpha-adrenoceptor-mediated vasoconstriction by angiotensin II was studied in femoral artery rings from rabbits. Threshold concentrations of angiotensin II (0.1 nM) increased the maximal response to clonidine to 139 +/- 8% of control and produced a 3.2-fold increase in sensitivity. These effects of angiotensin II were reversed when tissues were pretreated with staurosporine (50 nM), an inhibitor of protein kinase C. The amplification of the alpha-adrenoceptor-mediated vasoconstrictor effects of thrombin and norepinephrine by angiotensin II were also reversed by pretreatment with staurosporine. Angiotensin II induced a response amplification in vascular smooth muscle known to be a nonspecific phenomenon, implying postreceptor interaction at intracellular transducer systems. Our findings suggest that upon activation of protein kinase C by angiotensin II, arterial responses to alpha-adrenoceptor agonists are amplified. This provides for nonspecific changes in vascular sensitivity by tonic alterations in postsynaptic modulation by enzyme systems known to regulate Ca2(+)-dependent phenomena, e.g. those related to vascular excitation-contraction mechanisms.     

Insulin attenuates vascular smooth muscle calcification but increases vascular smooth muscle cell phosphate transport

Medial artery vascular smooth muscle cell (VSMC) calcification increases the risk of cardiovascular mortality in type 2 diabetes. However, the influence of insulin on VSMC calcification is unclear. We explored the effects of insulin on rat VSMC calcification in vitro and found that in a dose-dependent fashion, insulin attenuates VSMC calcification induced by high phosphate conditions as quantified by the o-cresolphthalein calcium (OCPC) method. In an in vitro model of insulin resistance in which cells are exposed to elevated insulin concentrations and the PI 3-kinase pathway is selectively inhibited, increased VSMC calcification was observed, suggesting that the PI 3-kinase pathway is involved in this attenuating effect of insulin. We postulated that insulin may also have an effect on phosphate or calcium transport in VSMC. We found that insulin increases phosphate transport at 3 and 24 h. This effect was mediated by increased Vmax for phosphate transport but not Km. Because type III sodium-phosphate co-transporters Pit-1 and Pit-2 are found in VSMC, we examined their expression by Western blot and real-time RT-PCR. Insulin stimulates Pit-1 mRNA modestly (*p<0.01 versus control), an effect inhibited by PD98059 but not by wortmannin. Pit-1 protein expression is induced by insulin, an effect also inhibited by PD98059 (*p<0.001 versus insulin alone). Our results suggest a role for insulin in attenuating VSMC calcification which may be disrupted in selective insulin signaling impairment seen in insulin resistance. This effect of insulin contrasts with its effect to induce phosphate transport in VSMC.     

Activation of cell adhesion kinase beta by mechanical stretch in vascular smooth muscle cells

We have studied whether activation of cell adhesion kinase beta (CAKbeta) is involved in stretch-induced signaling pathway in cultured rat vascular smooth muscle cells. Cyclic stretch (1 Hz) induced a rapid (within 1 min) phosphorylation of CAKbeta, whose effect was time and strength dependent. Both Ca(2+) and Na(+) ionophores (A23187 and monensin) stimulated phosphorylation of CAKbeta in a similar fashion to mechanical stretch. The stretch-induced phosphorylation of CAKbeta was inhibited completely by an intracellular Ca(2+) chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester)] and largely by gadolinium, but only partially by an extracellular Ca(2+) chelator (EGTA). An angiotensin type 1 receptor antagonist (CV11974) abolished the phosphorylation of CAKbeta stimulated by angiotensin II, but not by mechanical stretch. Mechanical stretch rapidly (within 1 min) increased the association of CAKbeta with c-Src, but not pp125(focal adhesion kinase). Stretch-induced phosphorylation of ERK1/2 was inhibited by EGTA and an inhibitor of the Src kinase family [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine], but not by cytochalasin D, to disrupt actin polymerization. 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine or cytochalasin D did not affect stretch-induced phosphorylation of CAKbeta. These data suggest that mechanical stretch stimulates activation of CAKbeta, followed by its association with c-Src, which requires ion influx mainly via stretch-activated nonselective ion channels, thereby leading to activation of the p21(Ras)/ERK1/2 cascade in vascular smooth muscle cells.     

Inhibition of vascular smooth muscle cell proliferation by a novel fibroblast growth factor receptor antagonist.

OBJECTIVE: One of the key events in post-angioplasty restenosis is the migration and proliferation of medial smooth muscle cells leading to neo-intima formation. This phase is mediated by several growth factors, mainly platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF2/bFGF) and heparin-binding epidermal growth factor (HB-EGF). In this study, we have focused on the role of FGF2, which requires heparan sulfate proteoglycans (HSPG) as cofactors for binding and activation of its cell surface tyrosine kinase receptor. The aim of this study was to identify and explore the effect of novel FGF antagonists on vascular smooth muscle cell (VSMC) proliferation. METHODS: We have recently identified a novel class of small, positively charged molecules sharing a porphyrin core as inhibitors of FGF2 and vascular endothelial growth factor (VEGF) activity. Here we investigated the inhibitory effect of these compounds on VSMC proliferation and their effect on heparin-induced FGF receptor activity. RESULTS: We found that these molecules exert a marked inhibitory effect on FGF2-mediated smooth muscle cell (SMC) proliferation, manifested by reduced cell growth and DNA synthesis, which occurred in a dose-dependent manner with an IC(50) of approximately 1 microM of inhibitor. We demonstrate that the molecule, 5, 10, 15, 20-tetrakis (methyl-4-pyridyl)-21H, 23H-porphine tetra-p-tosylate salt (TMPP), inhibits binding of radiolabeled FGF2 to SMCs and to soluble FGF receptor 1 (FGFR1) in a manner that interferes with both ligand and receptor interactions with heparin, thereby blocking growth factor mediated SMC proliferation. CONCLUSION: We have identified an FGF antagonist, which may serve in clinical practice as a preventive measure of restenosis.     

AT2 receptor and vascular smooth muscle cell differentiation in vascular development.

The angiotensin II type 2 (AT2) receptor is transiently expressed at late gestation in the fetal vasculature, but its expression rapidly declines after birth. We have previously demonstrated that the expression of this receptor mediates decline in vascular DNA synthesis that occurs at this stage of vascular development. To examine further the role of the AT2 receptor in vasculogenesis, we have focused on the effect of the AT2 receptor on vascular smooth muscle cell (VSMC) differentiation. In this study, we examined the time-dependent expression of differentiation markers for VSMCs in the aorta of wild-type and AT2 receptor-null mice. alpha-Smooth muscle actin was expressed at the early stage of differentiation and exhibited unchanged expression before and after the peak of AT2 receptor expression, which was observed at embryonic day 20, neonatal day 1, and thereafter. No difference in alpha-smooth muscle actin expression was observed between the wild-type and AT2 receptor-null mice. In contrast, the mRNA levels for calponin, expressed in the late stage of VSMC differentiation, were significantly higher in the wild-type mouse aorta as compared with the AT2 receptor-null mice, which correlates with expression of the AT2 receptor. Moreover, the protein levels of calponin and high-molecular-weight caldesmon (h-caldesmon) showed lower expression in the aorta of AT2 receptor knockout mice at 2 and 4 weeks after birth. Taken together, our results suggest that the AT2 receptor promotes vascular differentiation and contributes to vasculogenesis.     

腺病毒介导的HCY2基因对血管平滑肌细胞凋亡的诱导作用

目的　观察高同型半胱氨酸诱导基因HCY2对于平滑肌细胞的作用。方法　以复制缺陷型腺病毒作为载体 ,将HCY2基因转移到平滑肌细胞中。提取平滑肌细胞的DNA ,进行凝胶电泳及ELISA ,行DNA片段化分析。用流式细胞术观察平滑肌细胞的亚二倍体。结果　转染HCY2后平滑肌细胞的DNA断裂成相差 2 0 0bp左右的片段 ,流式细胞术测定时发现 ,位于亚二倍体区的平滑肌细胞明显增多。结论　HCY2基因能引起平滑肌细胞的凋亡。     

Na-Ca exchange in vascular smooth muscle

The above section makes it clear that even if an Na-Ca exchange exists in vascular smooth muscles, the clear involvement of Na ions in the generation of tension can in many cases be explained by the operation of mechanisms other than Na-Ca exchange. Readers who have got this far may still be unclear whether we believe that smooth muscle cells do possess the exchange mechanism or not. In the next section we will briefly describe two more recent approaches which have convinced us that at least some smooth muscles do possess an Na-Ca exchange mechanism, one involving isolated smooth muscle membranes and the mechanisms they possess for transporting Ca, and one using ion-sensitive microelectrodes to follow in more detail changes in intracellular Na activity and membrane potential that occur when the extracellular ionic environment is manipulated.     

Smoothelin in vascular smooth muscle cells

Smoothelin-A and -B have only been found in fully differentiated contractile smooth muscle cells. They are increasingly used to monitor the smooth muscle cell differentiation process to a contractile or synthetic phenotype. Vascular-specific smoothelin-B is the first smooth muscle cell marker that disappears when vascular tissues are compromised, for example, in atherosclerosis or restenosis. Recently obtained data show that smoothelin deficiency results in a considerable loss of contractile potential and hence in impaired smooth muscle function and suggest that smoothelins are part of the contractile apparatus.     

Chemokine receptors in vascular smooth muscle

Atherosclerosis is considered to be an inflammatory disease. Chemokines are low-molecular-weight proteins that exert their effects, in part, through mediating leukocytic infiltration into the vessel wall. Recently, studies have determined that chemokines and their receptors are present, and function on other cellular components comprising the arterial wall, such as the endothelium and vascular smooth muscle. Smooth muscle cells (SMC) constitute the major cellular element of the arterial wall and are located predominantly in the arterial media. Recent studies have demonstrated that SMC possess a number of functional chemokine receptors, including CCR5, CXCR4, and a receptor for monocyte chemoattractant protein-1 (MCP-1). It is likely that SMC are increasingly recognized as potential targets for chemokines, and that these effects may influence a variety of normal and pathological processes involving SMC such as atherosclerosis and arterial injury.