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.     

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.     

Specific receptors for thromboxane A2 in cultured vascular smooth muscle cells of rat aorta

The specific binding site for thromboxane A2 (TXA2) was studied in cultured vascular smooth muscle cells (VSMC) of the rat aorta. [3H]SQ29,548, a potent and selective TXA2 receptor antagonist, displayed high-affinity and specificity, as well as saturable and displaceable binding to rat VSMC in culture. Scatchard analysis of equilibrium binding at 24 degrees C revealed a single class of binding sites with a Kd of 1.7 nM and a Bmax of 8.0 fmol/10(6) cells. A series of TXA2 receptor antagonists completely suppressed [3H]SQ29,548 binding to rat VSMC, and the rank order of their inhibitory potencies (Ki) correlated well with the potencies for suppression of the U46619-induced contraction of rat thoracic aorta. These results suggest that specific binding sites for [3H]SQ29,548 represent the TXA2 receptor in rat VSMC.     

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.     

Agonist-induced desensitization of 5-HT2 receptors on cultured calf aortic smooth muscle cells

[³²P]Phosphatidic acid (PA)-formation was quantified in calf aortic smooth muscle cultures for measuring the activation of the signal transducing system coupled to the 5-hydroxytryptamine₂-(5-HT₂) receptor. [³²P]PA-formation was increased upon stimulation of smooth muscle cells with serotonin (5-HT) and 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM), but not with the 5-HT₁ agonists N,N-dipropyl-8-hydroxy-2-aminotetralin and RU 24969. The potency of drugs to inhibit the 5-HT induced [³²P]PA-formation closely corresponded to their binding affinity for 5-HT₂ receptors. 24-Hour treatment of smooth muscle cultures with 5-HT or DOM resulted in a substantial decrease of 5-HT induced [³²P]PA-formation. In contrast to the anomalous 5-HT₂ receptor regulation in vivo, 5-HT₂ receptors on smooth muscle cells appeared to be desensitized by agonist treatment.     

Specific receptors for adrenomedullin in cultured rat vascular smooth muscle cells

The effects of synthetic rat adrenomedullin (rAM), a novel vasorelaxant peptide originally isolated from human pheochromocytoma, on receptor binding and cAMP generation were studied in cultured rat vascular smooth muscle cells (VSMC). A binding study using [¹²⁵I]rAM revealed the presence of a single class of high-affinity (K_d1.3 × 10⁻⁸ M) binding sites for rAM in VSMC. The apparent K_i of rat calcitonin gene-related peptide (rCGRP) was 3 × 10⁻⁷ M. Affinity labeling of VSMC membranes with [¹²⁵I]rAM revealed two distinct labeled bands with apparent molecular weights of 120 and 70 kDa, both of which were abolished by excess unlabeled rAM or rCGRP. rAM stimulated cAMP formation with an approximate EC₅₀ of 10⁻⁸ M, the effect of which was additive with isoproterenol, but not with rCGRP. The rAM-induced cAMP response was unaffected by propranalol, indomethacin, or quinaerine, but inhibited by a CGRP receptor antagonist, human CGRP[8–37]. These data suggest that VSMC possesses specific AM receptors functionally coupled to adenylate cyclase with which CGRP interacts.     

Formation of epoxyeicosatrienoic acids from arachidonic acid by cultured rat aortic smooth muscle cell microsomes.

The vasodilatory effect of epoxyeicosatrienoic acids (EpETrE), especially 5(6)-EpETrE, has been reported recently and a role of P-450-dependent arachidonic acid monooxygenase metabolites was suggested in vasoregulation. Accordingly, the presence of P-450-dependent arachidonic acid monooxygenase was investigated in rat aortic smooth muscle cells. Incubation of the microsomes of rat cultured aortic smooth muscle cells with 14C-arachidonic acid in the presence of 1 mM NADPH resulted in the formation of oxygenated metabolites. The metabolites were separated and purified by reverse phase and straight phase high performance liquid chromatography and identified by gas chromatography-mass spectrometry. Identified metabolites were 5(6)-EpETrE, 5,6-dihydroxyeicosatrienoic acid (DiHETrE), and 14,15-DiHETrE. The formation of these metabolites was totally dependent on the presence of NADPH, and inhibitors of cytochrome P-450-dependent enzymes, SKF-525A and metyrapone, reduced the formation of these metabolites. This is the first report that cytochrome P-450-dependent arachidonic acid metabolites, especially 5(6)-EpETrE and 14(15)-EpETrE, can be produced in the microsomes of vascular smooth muscle cells of rats.     

Mineralocorticoid-induced increase in beta-adrenergic receptors of cultured rat arterial smooth muscle cells

We have investigated the effect of mineralocorticoids on beta-adrenergic receptors in cultured arterial smooth muscle cells. Mineralocorticoid (aldosterone) treatment resulted in a significant increase in beta-adrenergic receptors measured by [3H]dihydroalprenolol (DHA) binding. This effect required at least 20 hours of incubation with aldosterone and was completely blocked by cycloheximide (10 micrograms/ml), indicating protein synthesis was required for this response. Aldosterone at the concentration range of 10(-8)-10(-6) M increased [3H]DHA binding, but was ineffective at 10(-9) M. Scatchard analysis of [3H]DHA binding revealed that the observed significant increase in binding was due to an increased number of binding sites (P less than 0.05), and that the affinity was unchanged. The aldosterone (1 x 10(-8) M) effect was completely blocked by the combination of RU 38486 (10(-6) M) and spironolactone (10(-7) M), but not by the glucocorticoid antagonist RU 38486 alone. While basal c-AMP levels were not changed by aldosterone (10(-6) M) treatment, the isoproterenol (10(-6) M) stimulated level of c-AMP was significantly higher in cells treated with aldosterone (P less than 0.05). We conclude that aldosterone, acting through the mineralocorticoid receptor, has a direct effect on arterial smooth muscle cells mediated through modulation of beta-adrenergic receptors of these cells.     

Regulation of fibronectin by platelet-derived growth factors in cultured rat thoracic aortic smooth muscle cells

Induction of Fibronectin (FN) gene expression by platelet-derived growth factor (PDGF) isoforms in rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances FN levels in SMC cultures in a time- and concentration-response fashion. PDGF-AA and PDGF-AB show no effect on FN levels. The effects of insulin and insulin-like growth factor-I (IGF-I) on PDGF-BB-induced FN levels were examined. No additivity of FN levels is observed between PDGF-BB and insulin and/or IGF-I. Experiments also show that PDGF-BB enhances FN mRNA levels, implying that acquisition of additional FN mRNA units accounts for the increase in FN levels. Induction of FN and FN mRNA levels by PDGF-BB could be one of the initial events in vascular SMC proliferation and extracellular matrix expansion, leading to atherosclerosis and hypertension.     

Regulation of NaK-ATPase by platelet-derived growth factors in cultured rat thoracic aortic smooth muscle cells

Regulation of (Na⁺ + K⁺)-adenosine triphosphatase (NaK-ATPase) by platelet-derived growth factor (PDGF) in cultured rat thoracic aortic smooth muscle cells (SMC) was examined. PDGF-BB enhances SMC proliferation and NaK-ATPase activity. Ouabain, an inhibitor of NaK-ATPase activity, prevents PDGF-BB-induced SMC proliferation. As shown by Western blot and immunochemiluminescence analysis, PDGF-BB also enhances ₁, truncated ₁, and ₁ NaK-ATPase subunit levels. PDGF-AA and PDGF-AB show no effect on ₁ and truncated ₁ levels in slot blot analysis. Induction of NaK-ATPase subunit levels by PDGF-BB could be one of the initial events in vascular SMC proliferation.     

Metabolism of cytoplasmic triacylglycerol in cultured aortic smooth muscle cells

A turnover of cytoplasmic triacylglycerol was studied in cultured rat, rabbit, and bovine aortic smooth muscle cells. Cytoplasmic triacylglycerol was labeled with [3H]glycerol in the presence of oleic acid in the medium and its loss from the cell was studied in the presence of carrier glycerol. Multiple additions of Isuprel or dibutyryl cyclic AMP during the chase period did not enhance the loss of labeled triacylglycerol. The rate of hydrolysis of cellular triacylglycerol was unchanged in the absence or in the presence of 100 microM chloroquine. Modulation of cellular cholesterol content by addition of low density lipoprotein or high density apolipoprotein--sphingomyelin liposomes did not affect the residence time of the cellular triacylglycerol. We conclude that cytoplasmic triacylglycerol in cultured aortic smooth muscle cells is metabolized by an extralysosomal enzyme which is neither catecholamine responsive nor affected by modulation of cellular cholesterol.     

Superoxide anion impairs contractility in cultured aortic smooth muscle cells

We examined the effects of superoxide anion (O) generated by xanthine plus xanthine oxidase (X/XO) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and muscle contractility in cultured bovine aortic smooth muscle cells (BASMC). Cells were grown on collagen-coated dish for the measurement of [Ca(2+)](i). Pretreatment with X/XO inhibited ATP-induced Ca(2+) transient and Ca(2+) release-activated Ca(2+) entry (CRAC) after thapsigargin-induced store depletion, both of which were reversed by superoxide dismutase (SOD). In contrast, Ca(2+) transients induced by high-K(+) solution and Ca(2+) ionophore A-23187 were not affected by X/XO. BASMC-embedded collagen gel lattice, which was pretreated with xanthine alone, showed contraction in response to ATP, thapsigargin, high-K(+) solution, and A-23187. Pretreatment of the gel with X/XO impaired gel contraction not only by ATP and thapsigargin, but also by high-K(+) solution and A-23187. The X/XO-treated gel showed normal contraction; however, when SOD was present during the pretreatment period. These results indicate that O(2)(-) attenuates smooth muscle contraction by impairing CRAC, ATP-induced Ca(2+) transient, and Ca(2+) sensitivity in BASMC.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

Regulation of atrial natriuretic peptide receptors in cultured vascular smooth muscle cells of rat

To elucidate the regulation of vascular receptors for atrial natriuretic peptide (ANP), we have studied the binding capacity of 125I-labeled rat (r) ANP using cultured vascular smooth muscle cells from rat aorta. After preincubation with 3.2 X 10(-8) M rANP at 37 degrees C, the binding capacity decreased as a function of time; the maximal receptor loss (70-75%) occurred after 4 hrs and persisted for 24 hrs. Pretreatment with cycloheximide (20 micrograms/ml) and actinomycin D (2 micrograms/ml) similarly caused a dramatic reduction (approximately 80%) of the binding capacity after 24 hrs; the half-life (t1/2) of the receptor loss was approximately 7-8 hrs. Following removal of rANP, the "down-regulated" ANP receptors fully recovered in the presence of 10% fetal calf serum, but not in combination with either actinomycin D or cycloheximide. Concanavalin A dose-dependently inhibited the binding. The binding capacity also decreased with time in the presence of tunicamycin (1 microgram/ml) with t1/2 of approximately 30 hrs. These data indicate that protein and carbohydrate moieties are essential for the functional integrity of the vascular receptor binding sites for ANP, and suggest that the recovery of the receptor loss by "down-regulation" requires concomitant RNA and protein synthesis.     

Intercellular calcium waves in primary cultured rat mesenteric smooth muscle cells are mediated by Connexin43

Abstract

Intercellular Ca^{2 +} wave propagation between vascular smooth muscle cells (SMCs) is associated with the propagation of contraction along the vessel. Here, we characterize the involvement of gap junctions (GJs) in Ca^{2 +} wave propagation between SMCs at the cellular level. Gap junctional communication was assessed by the propagation of intercellular Ca^{2 +} waves and the transfer of Lucifer Yellow in A7r5 cells, primary rat mesenteric SMCs (pSMCs), and 6B5N cells, a clone of A7r5 cells expressing higher connexin43 (Cx43) to Cx40 ratio. Mechanical stimulation induced an intracellular Ca^{2 +} wave in pSMC and 6B5N cells that propagated to neighboring cells, whereas Ca^{2 +} waves in A7r5 cells failed to progress to neighboring cells. We demonstrate that Cx43 forms the functional GJs that are involved in mediating intercellular Ca^{2 +} waves and that co-expression of Cx40 with Cx43, depending on their expression ratio, may interfere with Cx43 GJ formation, thus altering junctional communication.