Extracellular calcium sensing in rat aortic vascular smooth muscle cells

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.     

ATP-induced calcium transient in cultured rat aortic smooth muscle cells

To characterize the excitatory purinoceptors in vascular smooth muscle cells and the biochemical mechanisms of their actions, the effects of ATP and other nucleotides on Ca2+ mobilization in cultured smooth muscle cells mainly from rat aorta were investigated. ATP induced a transient and dose-dependent increase in the cytosolic Ca2+ concentration. ATP also induced a rapid production of inositol trisphosphate (IP3). The agonist form of ATP was metal-free ATP and its half-maximal effect was obtained at about 0.1 microM. 4-beta-Phorbol 12-myristate 13-acetate (PMA) or 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both Ca2+ response and IP3 production. In addition, TMB-8 but not PMA, significantly decreased the amount of releasable Ca2+ presumably in the sarcoplasmic reticulum. Pertussis toxin also inhibited the Ca2+ response. Based on the dose-dependent effects of various nucleotides and adenosine on the Ca2+ response, it was concluded that the P2 subclass of purinoceptor is involved in the observed ATP effects. In addition, the observed absence or very weak effect of alpha, beta-methylene ATP relative to the effect of ATP suggests that the excitatory P2-purinoceptors in vascular smooth muscle cells do not form a homogeneous group, because the opposite order of potency for these two nucleotides was reported previously for the P2 purinoceptors involved in contraction of some isolated blood vessels.     

Differential proliferation of rat aortic and mesenteric smooth muscle cells in culture.

Smooth muscle cells (SMC) from various arterial origins have been successfully maintained in culture. The present study evaluates the proliferative activity of aortic and mesenteric SMC in culture. Aortic and mesenteric SMC were obtained from male Wistar rats by explant and enzyme digestion techniques, respectively. Vascular SMC obtained by either method exhibited a characteristic hill-and-valley growth pattern in culture after confluence and were positively labelled with either anti-smooth muscle actin or myosin by an indirect immunofluorescent method. The rate of incorporation of thymidine into DNA and cell number counting were used as indices of proliferation in vitro. Vascular SMC from passages 4-33 were first synchronized with either Dullbecco's Modified Eagle's Medium (DME) or Ham's F-12 medium, supplemented with insulin-transferring-selenium (ITS), for 72 hours. SMC were then stimulated with 10% bovine serum for either 24 or 72 hours with the former processed for scintillation counting, the latter for cell number determination. The incorporation of tritiated thymidine into DNA following a 2 hour incubation was determined by scintillation counting after perchloric acid extraction. In terms of cell numbers, proliferative responses to bovine serum were determined by Coulter counting. Autoradiography was also carried out in some cultures to determine both thymidine and mitotic labelling indices. The rate of thymidine incorporation in aortic cells was 2-3 fold higher than in mesenteric cells. Aortic and mesenteric SMC lines exhibited similar cell cycle intervals in terms of total duration and individuals cycle parameters. However, the total thymidine index was higher in the aortic than mesenteric SMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual effect of thapsigargin on cell death in porcine aortic smooth muscle cells

A sustained increase in the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) can cause cell death. In this study, we found that, in cultured porcine aortic smooth muscle cells, endoplasmic reticulum (ER) stress, triggered by depletion of Ca²⁺ stores by thapsigargin (TG), induced an increase in the [Ca²⁺]_i and cell death. However, the TG-induced death was not related to the [Ca²⁺]_i increase but was mediated by targeting of activated Bax to mitochondria and the opening of mitochondrial permeability transition pores (PTPs). Once the mitochondrial PTPs had opened, several events, including collapse of the mitochondrial membrane potential, cytochrome c release, and caspase-3 activation, occurred and the cells died. TG-induced cell death was completely inhibited by the pan-caspase inhibitor Z-VAD-fmk and was enhanced by the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting the existence of a Ca²⁺-dependent anti-apoptotic mechanism. After TG treatment, Ca²⁺-sensitive mitogen-activated protein kinase (MAPK) activation was induced and acted as a downstream effector of phosphatidylinositol 3-kinase (PI 3-kinase). The protective effect of Z-VAD-fmk on TG-induced cell death was reversed by BAPTA, PD-098059 (an MAPK kinase inhibitor), or LY-294002 (a PI 3-kinase inhibitor). Taken together, our data indicate that ER stress simultaneously activate two pathways, the mitochondrial caspase-dependent death cascade and the Ca²⁺-dependent PI 3-kinase/MAPK anti-apoptotic machinery. The Bax activation and translocation, but not the [Ca²⁺]_i increase, may activate mitochondrial PTPs, which, in turn, causes activation of caspases and cell death, whereas Ca²⁺-dependent MAPK activation counteracts death signaling; removal of Ca²⁺ activated a second caspase-independent death pathway. sarco(endo)plasmic reticulum calcium ion adenosine triphosphatase; cytosolic calcium ion concentration; mitogen-activated protein kinase     

Homocysteine potentiates calcification of cultured rat aortic smooth muscle cells

Aortic calcification was demonstrated in experimental animal models of hyperhomocysteinemia. Mild hyperhomocysteinemia was associated with aortic calcification, suggesting a relationship between homocysteine (HCY) and the pathogenesis of aortic calcification. In the present study, the effect of HCY on vascular calcification was examined in calcifying and non-calcifying vascular smooth muscle cells (VSMCs). Cell calcification was induced by incubation of VSMCs with beta-glycerophosphate. Proliferation of VSMCs was studied by cell counting, 3H-thymidine (3H-TdR) and 3H-leucine (3H-Leu) incorporation. 45Ca accumulation, cell calcium content, and alkaline phosphatase (ALP) activity were measured as indices of calcification. The results showed that the proliferation of calcifying VSMCs, which was indicated by cell counting, 3H-TdR and 3H-Leu incorporation in calcifying VSMCs, was enhanced as compared with that of non-calcifying VSMCs. HCY promoted increases in cell number, 3H-TdR and 3H-Leu incorporation in both calcifying and non-calcifying VSMCs, but with more prominent effect in calcifying VSMCs. The stimulating effects of HCY on the three parameters in calcifying VSMCs were antagonized by PD98059, a specific inhibitor of mitogen activated protein kinase kinase (MAPKK). The ALP activity, 45Ca uptake, and calcium deposition in the calcifying VSMCs were greater than those in non-calcifying VSMCs. PD98059 had no effect on ALP activity, 45Ca uptake, and calcium deposition in calcifying VSMCs. HCY caused marked increases in 45Ca uptake and calcium deposition both in calcifying and non-calcifying VSMCs. HCY, however, enhanced ALP activity in the calcified VSMCs but not in the non-calcifying VSMCs. The non-calcifying VSMCs treated with HCY showed the same low ALP activity, as did the control VSMCs. In calcifying VSMCs, the HCY-induced increases in 45Ca uptake, calcium deposition, and ALP activity were also attenuated by PD98059. The results demonstrated that HCY potentiated VSMC calcification probably through the mechanisms by which HCY promotes atherosclerosis.     

Phenotype modulation in primary cultures of rat aortic smooth muscle cells

The transition of adult rat aortic smooth muscle cells from a contractile to a synthetic phenotype during the first week of primary culture on a substrate of fibronectin in serum-free medium was studied by light and electron microscopy. The weak base chloroquine and the carboxylic ionophore monensin were both found to inhibit the spreading of the cells and the accompanying changes in cellular fine structure. The exchange of myofilament bundles for a prominent rough endoplasmic reticulum and Golgi complex was delayed and vacuoles filled with incompetely degraded material accumulated in the cytoplasm. The microtubule-disruptive drugs colchicine and nocodazole likewise opposed the spreading and fine structural reorganization of the cells. Most typically, the Golgi stacks were small and widely dispersed. In addition, vacuoles of the type mentioned above increased in number. On the other hand, there was surprisingly little effect of cytochalasin B, a drug that is supposed to interfere with the assembly of actin filaments. The observations suggest that the phenotypic modulation of arterial smooth muscle cells is dependent on: (a) lysosomal degradation of discarded cellular constituents, (b) active vesicular transport along the exocytic pathway to provide the expanding cell surface with new membrane, and (c) a normal microtubular cytoskeleton to ensure the establishment of a new and functionally efficient intracellular organization.     

Vanadate increases cytosolic free calcium in rat aortic smooth muscle cells

Although several studies have shown that vanadate evokes vasoconstriction whether it elevates cytosolic free calcium, [Ca²⁺]_i, in vascular smooth muscle (VSM) cells has not been investigated. The present study shows that acute additions of low concentrations of vanadate (10–200) to cultured aortic smooth muscle cells (ASMC) produced a rapid and a concentrationdependent increase in [Ca²⁺]_i with an EC₅₀ (mean ± SEM) value of 42 ± 11 μM. Inclusion of vanadate (200 μM) led to a significant increase (p < 0.05) in the peak [Ca²⁺]_i level to 190 ± 23 nM from a basal level of 102 ± 2 nM. At concentrations > 200 μM, vanadate caused quenching of fura-2 fluorescence. For example, addition of 1 mM vanadate led to an apparent decrease in fluorescence by about 50 % (due to a quenching effect), followed by a transient rise. H₂O₂, which is used in the preparation of peroxide forms of vanadate, pervanadate (PV), also produced a rise in [Ca²⁺]_i. These data suggest that vanadate promotes vascular tone by elevating [Ca²⁺]_i in ASMC. However, [Ca²⁺]_i measurements made with higher concentrations of vanadate and PV, using the fura-2 method, must be interpreted with caution.     

Nitroalkenes induce rat aortic smooth muscle cell apoptosis via activation of caspase-dependent pathways

Nitroalkene derivatives of nitro-linoleic acid (LNO₂) and nitro-oleic acid (OA-NO₂) are nitrated unsaturated fatty acids that can be detected in healthy human plasma, red blood cells and urine. It has been shown that nitroalkenes have potent anti-inflammatory properties in multiple disease models. In the present study, we are the first to investigate the apoptotic effects of nitroalkenes in rat aortic smooth muscle cells (RASMCs). We observed that nitroalkenes induce RASMCs apoptosis in a dose-dependent manner. In addition, nitroalkenes stimulate extrinsic caspase-8 and intrinsic caspase-9 activity to trigger the caspase-3 apoptotic signaling cascade, resulting in RASMCs death. Furthermore, the pro-apoptotic protein, Bad was upregulated and antiapoptotic protein, Bcl-xl was downregulated during nitroalkene-induced apoptosis. These results demonstrate that nitroalkenes can induce RASMCs apoptosis via stimulation of caspase activity and the regulation of apoptotic protein expression levels.     

Cyclosporine A -induced contraction of isolated rat aortic smooth muscle cells

The mechanisms by which the immunosuppressive drug cyclosporine A (CsA) induces hypertension and nephrotoxicity are still not fully understood. Although smooth muscle cell (SMC) contraction is probably the mechanism of vasoconstriction, the direct contractive effect of CsA on SMCs has not yet been demonstrated. Thus, it was the purpose of this study to evaluate the direct effects of CsA in cultured SMCs through interactive image analysis. In aortic SMCs, CsA at the concentrations of 0.01, 0.1 and 1 μM, caused a concentration-dependent decrease of the planar cross-sectional area (PCSA) after 30 min and 60 min of treatment. The PCSA decreases were statistically significantly different from control at all concentrations. No cytotoxicity was observed under these conditions. Ten minutes preincubation of SMCs with a monoclonal antibody against endothelin-1 (ET-1) significantly prevented the CsA effects at 1 μM. When the same antibody was heat inactivated or an unspecific antibody (anti-desmin immunoglobulin G) was applied, the CsA-induced contractions were not affected. These data suggest that CsA can cause a direct contractive effect on vascular SMCs. This effect is partly mediated by ET-1.     

Cell cycle versus density dependence of smooth muscle alpha actin expression in cultured rat aortic smooth muscle cells

Cultured smooth muscle cells (SMC) undergo induction of smooth muscle (SM) alpha actin at confluency. Since confluent cells exhibit contact inhibition of growth, this finding suggests that induction of SM alpha actin may be associated with cell cycle withdrawal. This issue was further examined in the present study using fluorescence-activated cell sorting of SMC undergoing induction at confluency and by examination of the effects of FBS and platelet-derived growth factor (PDGF) on SM alpha actin expression in postconfluent SMC cultures that had already undergone induction. Cell sorting was based on DNA content or differential incorporation of bromodeoxyuridine (Budr). The fractional synthesis of SM alpha actin in confluent cells was increased two- to threefold compared with subconfluent log phase cells, but no differences were observed between confluent cycling (Budr+) and noncycling (Budr-) cells. In cultures not exposed to Budr, confluent cycling S + G2 cells exhibited similar induction. These data indicate that cell cycle withdrawal is not a prerequisite for the induction of SM alpha actin synthesis in SMC at confluency. Growth stimulation of postconfluent cultures with either FBS or PDGF resulted in marked repression of SM alpha actin synthesis but the level of repression was not directly related to entry into S phase in that PDGF was a more potent repressor of SM alpha actin synthesis than was FBS despite a lesser mitogenic effect. This differential effect of FBS versus PDGF did not appear to be due to transforming growth factor-beta present in FBS since addition of transforming growth factor-beta had no effect on PDGF-induced repression. Likewise, FBS (0.1-10.0%) failed to inhibit PDGF-induced repression. Taken together these data demonstrate that factors other than replicative frequency govern differentiation of cultured SMC and suggest that an important function of potent growth factors such as PDGF may be the repression of muscle-specific characteristics.     

PDGF-BB and IGF-I use different signaling pathways to induce fibronectin synthesis in cultured rat thoracic aortic smooth muscle cells

Fibronectin, an extracellular matrix protein, acts as an early signal in initiating cell proliferation. Results have indicated that platelet-derived growth factor BB (PDGF-BB) and insulin-like growth factor-I (IGF-I) both enhance fibronectin gene expression. Genistein inhibits PDGF-BB-induced fibronectin levels without inhibiting IGF-I-induced fibronectin levels. It indicates that PDGF-BB and IGF-I utilize separate signaling pathways to induce the synthesis of fibronectin.     

PDGF-BB and IGF-I use different signaling pathways to induce NaK-ATPase subunits in cultured rat thoracic aortic smooth muscle cells

(Na(+)+K(+))-adenosine triphosphatase (NaK-ATPase), an ubiquitous membrane transport protein consisting of alpha and beta subunits, regulates Na(+)/K(+)fluxes and maintains many vital physiological functions, including cell growth. Results have indicated that platelet-derived growth factor (PDGF) and insulin-like growth factor-I (IGF-I) both enhance NaK-ATPase subunits. Genistein, an inhibitor of tyrosine phosphorylation, inhibits serum- and PDGF-BB-induced NaK-ATPase alpha(1)subunit protein levels without inhibiting IGF-I-induced NaK-ATPase alpha(1)subunit protein levels. These results indicate that PDGF-BB and IGF-I utilize separate signaling pathways to induce the synthesis of NaK-ATPase alpha(1)subunits. In addition, genistein failed to inhibit PDGF-BB-stimulated NaK-ATPase beta(1)subunit levels, suggesting that two separate pathways are involved to induce the synthesis of the NaK-ATPase alpha(1)and beta(1)subunits, respectively.     

Mechanisms related to NO-induced motility in differentiated rat aortic smooth muscle cells

Nitric oxide (NO) is thought to play an important role as an inhibitor of vascular cell proliferation, motility, and neointima formation. This effect is mediated, in part, via the upregulation of protein tyrosine phosphatase (PTP)1B. Conversely, studies have reported that in presumably hyperinsulinemic mice fed a high-fat diet, NO enhances vascular remodeling, whereas a deficit of NO attenuates vascular remodeling. We have reported that in differentiated cultured smooth muscle cells treated with insulin, NO induces a motogenic effect that is dependent on Src homology-2 domain PTP 2 (SHP2) upregulation. In the present study, we describe novel mechanisms relevant to the motogenic effect of NO. Treatment of cultured cells with the selective angiontensin type 1 receptor antagonist losartan, but not with the selective angiotensin type 2 receptor antagonist PD-123319, blocked the comotogenic capacity of NO and insulin. Insulin and NO increased the secretion of ANG II into the culture media by 2- and 2.5-fold (P < 0.05), respectively, whereas treatment of cells with ANG II uncovered the motogenic effect of NO (1.4-fold above control, P < 0.05) and decreased the levels of PTP1B to 45% of control (P < 0.05). Suppression of PTP1B function was sufficient to uncover the motogenic effect of NO. The capacity of insulin to suppress PTP1B activity was blocked by losartan, implicating ANG II function in mediating this effect. Both insulin and ANG II induced the upregulation of phosphatidyl inositol 3-kinase (PI3K)-δ by two- to threefold (P < 0.05), and this effect was both necessary and sufficient to uncover NO-induced motogenesis. Finally, suppression of PTP1B function potentiated, whereas overexpression of PTP1B inhibited, SHP2-induced motogenesis. These results support the hypothesis that the comotogenic effect of insulin and NO occurs via an ANG II-mediated effect involving the suppression of PTP1B and upregulation of PI3K-δ and SHP2.     

Induction of fatty acid cyclooxygenase in rat aortic smooth muscle cells by estradiol

We have previously reported that estradiol stimulates prostacyclin biosynthesis in cultured rat aortic smooth muscle cells by increasing fatty acid cyclooxygenase activity, and induction of new protein biosynthesis is involved since the effect of estradiol is blocked by cycloheximide treatment. In order to see whether estradiol stimulates de novo biosynthesis of fatty acid cyclooxygenase in cells, a specific radioimmunoassay of fatty acid cyclooxygenase was used to measure the enzyme. Estradiol significantly increased immunoreactive fatty acid cyclooxygenase in cells. Consequently, increase in cyclooxygenase activity results in the production of more prostaglandin endoperoxides for final increased synthesis of prostacyclin in these cells.     

TIMP-1 stimulates proliferation of human aortic smooth muscle cells and Ras effector pathways

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein, which is found in most tissues and body fluids. Here, we demonstrated that recombinant TIMP-1 but not the synthetic matrix metalloproteinase inhibitor, GM6001, stimulated proliferation of human aortic smooth muscle cells (AoSMC) in a dose-dependent manner. The mitogenic effect was associated with activation of Ras, increased phosphorylation of ERK, and stimulation of cyclin D1 expression. The phosphatidylinositol 3-kinase (PI3K) signaling pathway was also involved since the PI3K inhibitor, LY294002, abolished the TIMP-1-mediated growth stimulation. These data suggest that TIMP-1 activates Ras, which then turns on the ERK and PI3K signaling pathways to promote cell cycle progression of the AoSMC.     

20-Hydroxyeicosatetraenoic acid (20-HETE) stimulates migration of vascular smooth muscle cells.

AIM: We tested the hypothesis that 20-HETE production contributes to platelet derived growth factor (PDGF)-BB stimulated migration of VSMC in a cell culture model. METHODS: Studies were performed with A10 cells which are a rat vascular smooth muscle derived cell line. Migration was determined using a Boyden chamber chemotactic assay. RESULTS: Pre-treatment of cells with two doses of 20-HETE (100 and 500 nM) significantly increased PDGF-BB stimulated VSMC migration by 34-58% of control; whereas, prior incubation of cells with inhibitors of 20-HETE production, 17-ODYA (1-25 M) or HET0016 (100 nM), significantly decreased PDGF-BB stimulated migration by 40-90%. 20-HETE mediated increase in PDGF-BB migration was completely prevented by the 20-HETE antagonist, WIT-002. In order to determine what second messenger pathways are involved in the 20-HETE mediated stimulation of VSM migration, experiments were performed with specific inhibitors of tyrosine kinase (tyrphostin 25, 10 microM), mitogen-activated extracellular signal-regulated kinase (MEK, PD98059, 20 microM and U0126, 10 microM), protein kinase C (Myr-PKC, 50 microM), and phosphoinositide 3-kinases (PI3Ks) (wortmannin, 50 nM). Blockade of MEK and PI3K all abolished the increase in 20-HETE mediated migration. CONCLUSION: 20-HETE stimulates PDGF-mediated VSM migration acting through pathways that involve MEK and PI3K.