Inhibition by prostacyclin and carbacyclins of endothelin-induced DNA synthesis in cultured vascular smooth muscle cells

Effects of prostacyclin and carbacyclins on endothelin-induced DNA synthesis were investigated in vascular smooth muscle cells. DNA synthesis was estimated by [3H]thymidine incorporation. Five carbacyclins used in this report were 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo [3.3.0]oct-2-en-3-yl) pentanoic acid (TEI-7165), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo[3.3.0]oct-2-en-3- yl]pentanoate (TEI-9090), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1-nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)penta noic acid (TEI-9063), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1- nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)pentanoate (TEI-1324), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-4-hydroxy-4-methyl-1- octenyl]bicyclo[3.3.0]oct-2-en-3-yl] pentanoic acid (TEI-3356). Prostacyclin and the carbacyclins inhibited the endothelin-induced DNA synthesis within the nanomolar range. These results suggest that prostacyclin and carbacyclins are possibly effective in inhibiting the proliferation of vascular smooth muscle cells under some situations in vivo.     

Effects of cholesterol oxidase on cultured vascular smooth muscle cells

Summary Cholesterol oxidase (3-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives. The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells. Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum. If fetal calf serum was absent, cells were unaffected by the treatment. The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed. After moderate treatment with cholesterol oxidase, cells excluded trypan blue. Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment. Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase. Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment. These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology. The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media. These changes occur prior to the cytotoxic effects of extensive oxidation. Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.     

Endothelin-mediated stimulation of DNA synthesis in vascular smooth muscle cells

Effects of endothelin on DNA synthesis were investigated in two clones of vascular smooth muscle cells, 1YB4 and A7r5. The peptide stimulated DNA synthesis in both clones with apparent EC50 of less than 1 ng/ml. More than 17 h was required before initiating endothelin-stimulated DNA synthesis. The platelet-derived growth factor at a concentration which had no effects by itself on DNA synthesis enhanced the effect of low concentrations of endothelin. A calcium antagonist, nifedipine, inhibited endothelin-induced DNA synthesis. These data suggest that endothelin stimulates DNA synthesis in vascular smooth muscle cells through nifedipine-sensitive mechanisms that can be modulated by platelet-derived growth factor.     

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.     

Effects of nicotine on phenotypic modulation and initiation of DNA synthesis in cultured arterial smooth muscle cells

During the early stages of atherogenesis, as well as during in vitro cultivation, smooth muscle cells modulate from a contractile to a synthetic phenotype. This process includes the loss of myofilaments and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex; it leads to decreased contractility and the commencement of cell growth and secretion of extracellular matrix components. In this paper, the effects of nicotine on adult rat arterial smooth muscle cells cultivated in vitro were studied by transmission electron microscopy and 3H-thymidine autoradiography. The results show that the drug speeded the initial rate of transition of the cells from contractile to synthetic phenotype in primary culture. Further, it stimulated the initiation of DNA synthesis in growth-arrested secondary cultures. Its effect was independent of other mitogens and additive to that of serum. The influences of nicotine, both on the modulation of the smooth muscle phenotype and the initiation of DNA synthesis, occurred at concentrations lower than those obtained in the blood after smoking and could contribute to the role of smoking as a risk factor for atherosclerosis.     

Angiotensin II effect on cytosolic pH in cultured rat vascular smooth muscle cells

This study investigated fluctuations of cytosolic pH (pHi) of cultured rat vascular smooth muscle cells (VSMCs) in reaction to metabolic alterations induced by angiotensin II (AII). Serially passed VSMCs from Wistar rat aortae were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. A biphasic reaction was seen after exposure of these cells to AII (1 nM to 1 microM); an initial and relatively brief phase of acidification was followed by sustained alkalinization. The rate of acidification and magnitude of alkalinization were dose-dependent. This biphasic effect of AII was also demonstrated in Ca2+-free medium and was mimicked by subjecting VSMCs to the calcium ionophore A23187 (5 microM) in Ca2+-containing medium but not in Ca2+-free medium. Verapamil (10 microM) almost entirely eliminated the AII-induced acidification, whereas amiloride analogues 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride (100 microM) as well as Na+-deficient medium abolished the subsequent (alkalinization) phase produced by the hormone. Activation of the Na+/H+ antiport by subjecting VSMCs to phorbol 12-myristate 13-acetate (100 nM) prevented a subsequent effect of AII on the pHi profile. This resistance to a further action of the hormone was not mediated via cytoplasmic alkalinization. AII produced a dramatic redistribution in the cellular compartments of 45Ca2+ associated with accelerated 45Ca2+ washout. These findings suggest that the AII-induced acidification phase may relate to activation of the Ca2+ pump (Ca2+/H+ exchange) and that this process can take place in the presence and absence of extracellular Ca2+. The alkalinization phase is the consequence of stimulation of the Na+/H+ antiport, which in cultured VSMCs can be activated by a rise in cytosolic free Ca2+ as well as other mechanisms.     

Glucocorticoids stimulate collagen and noncollagen protein synthesis in cultured vascular smooth muscle cells

The effect of glucocorticoids on collagen synthesis was examined in cultured bovine aortic smooth muscle (BASM) cells. BASM cells treated with 0.1 microM dexamethasone during their proliferative phase (11 d) were labeled with [3H]proline for 24 h, and the acid-precipitable material was incubated with bacterial collagenase. Dexamethasone produced an approximate twofold increase in the incorporation of proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) in the cell layer and medium. The stimulation was present in both primary mass cultures and cloned BASM. An increase in CDP and NCP was detected at 0.1 nM, while maximal stimulation occurred at 0.1 microM. Only cells exposed to dexamethasone during their log phase of growth (1-6 d after plating) showed the increase in CDP and NCP when labeled 11 d after plating. The stimulatory effect was observed in BASM cells treated with the natural bovine glucocorticoid, cortisol, dexamethasone, and testosterone, but was absent in cells treated with aldosterone, corticosterone, cholesterol, 17 beta-estradiol, and progesterone. The increase in CDP and NCP was absent in cells treated with the inactive glucocorticoid, epicortisol, and totally abolished by the antagonist, 17 alpha-hydroxyprogesterone, suggesting that the response was mediated by specific cytoplasmic glucocorticoid receptors. Dexamethasone-treated BASM cells showed a 4.5-fold increase in the specific activity of intracellular proline, which was the result of a twofold increase in the uptake of proline and depletion of the total proline pool. After normalizing for specific activity, dexamethasone produced a 2.4- and 2.8-fold increase in the rate of collagen and NCP synthesis, respectively. Cells treated with dexamethasone secreted 1.7- fold more collagen protein in 24 h compared to control cultures. The BASM cells secreted 70% Type I and 30% Type III collagen into the media as assessed by two-dimensional gel electrophoresis. The ratio of these two types was not altered by dexamethasone. The results of the present study demonstrate that glucocorticoids can act directly on vascular smooth muscle cells to increase the synthesis and secretion of collagen and NCP.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca2+ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca2+ as well as PG synthesis. The increase of cytosolic Ca2+ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca2+ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGTA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca2+, whereas TMB-8 (10 microM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca2+, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca2+. These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca2+ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca2+ and on the other hand, PG synthesis.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca²⁺ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca²⁺ as well as PG synthesis. The increase of cytosolic Ca²⁺ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca²⁺ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGIA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca²⁺, whereas TMB-8 (10 μM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca²⁺, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca²⁺.These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca²⁺ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca²⁺ and on the other hand, PG synthesis.     

Effects of cholesterol surface transfer on cholesterol and phosphatidylcholine synthesis in cultured rat arterial smooth muscle cells

The purpose of this study was to examine the effects of cholesterol surface transfer between lipid vesicles and rat arterial smooth muscle cells on endogenous synthesis of cholesterol and phosphatidylcholine. Lipid vesicles containing cholesterol and egg phosphatidylcholine in different proportions were used as the extracellular lipid source. The rate of cellular cholesterol and phosphatidylcholine synthesis was determined from the [14C]acetate incorporation into these lipid classes. [3H]Cholesterol in lipid vesicles, with a cholesterol/phospholipid (C/P) mole ratio of 1:1, was rapidly transferred into rat smooth muscle cells, with a half-time of about 3.6 hours in the absence of serum proteins. Incubation of cells for 5 hours with vesicles of a high C/P mole ratio (i.e. 1.5:1) at vesicle-cholesterol concentrations above 100 micrograms/ml resulted in a marked reduction of cellular cholesterol synthesis, whereas the rate of phosphatidylcholine synthesis was increased. Cells incubated with lipid vesicles of C/P 1:2 did not show any change in cellular cholesterol or phosphatidylcholine synthesis. Incubation of cells with egg phosphatidylcholine vesicles at concentrations above 300 micrograms/ml, on the other hand, stimulated endogenous synthesis of cholesterol without affecting cellular phosphatidylcholine synthesis. The main conclusion is that cholesterol surface transfer may influence cellular lipid metabolism in the absence of mediating serum lipoproteins in a model system with cultured cells and lipid vesicles.     

Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: II. Effects of calmodulin and calmodulin inhibitors.

The role of calmodulin (CaM) in modulating calcium (Ca) uptake by sarcoplasmic reticulum (SR) of vascular smooth muscle was studied in saponin skinned strips of rat caudal artery. Exogenous CaM concentrations ranging from 0.3-1.8 microM did not statistically change the steady state MgATP-dependent Ca content, the MgATP-independent Ca content, or the oxalate-stimulated Ca influx. Calmidazolium (CDZ), W-7, and trifluoperazine (TFP) were used to examine the potential effect of an endogenous CaM pool on inward Ca transport. The IC50 of these antagonists for inhibition of Ca-CaM-stimulated phosphodiesterase activity and Ca-activated superprecipitation of canine aortic actomyosin was measured and found to be in the low micromolar range with a rank order of potency for inhibition of CDZ greater than TFP greater than W-7. In skinned tissues, micromolar concentrations of antagonists that inhibited CaM-mediated reactions in isolated enzyme systems did not reduce Ca content or oxalate-stimulated Ca influx. At higher concentrations of 100-200 microM, the MgATP-dependent Ca content was significantly reduced by TFP and W-7 but not by CDZ. The order of potency for inhibition of Ca uptake was TFP greater than W-7 greater than CDZ. The MgATP-independent Ca content was significantly decreased only by 200 microM TFP. Although none of these inhibitors significantly altered Ca efflux at concentrations up to 100 microM, Ca release was significantly stimulated by all three at 200 microM. The TFP-stimulated Ca release was partially inhibited by ruthenium red. The results indicate that neither exogenous CaM nor an endogenous CaM pool directly modulates inward Ca transport by the SR of saponin skinned caudal artery. The inhibition of Ca uptake produced by hundred micromolar concentrations of CaM antagonists fails to correlate with the order of and with the potency of inhibition measured in isolated enzyme systems. This suggests that the inhibition of Ca uptake produced by high concentrations of these antagonists may be independent of a specific interaction with CaM. The activation of Ca release by high concentrations of CaM antagonists may involve a nonspecific increase in membrane permeability as well as modulation of a membrane Ca channel.     

Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells

The effect of arginine vasopressin (AVP) on Na+ kinetics was examined in cultured rat vascular smooth muscle cells (VSMC) and rat renal papillary collecting tubule cells (RPCT) by the direct measurement of intracellular sodium concentration [(Na+]i) using fluorescence dye; SBFI. AVP increased [Na+]i in a dose-dependent manner at a concentration of 10(-9) M or higher in rat VSMC but did not affect [Na+]i in rat RPCT. The calcium (Ca2+)-free solution completely blocked the increasing effect of AVP on [Na+]i in rat VSMC. A Ca2+ ionophore, ionomycin (1-2 x 10(-6) M) increased [Na+]i both in rat VSMC and RPCT. The Ca2(+)-free solution abolished the ionomycin-increased [Na+]i both in rat VSMC and RPCT. These results therefore indicate that after binding the V1 receptor AVP increases [Na+]i mediated through an increase in cellular Ca2+ uptake in VSMC.     

Effect of TPA on ion fluxes and DNA synthesis in vascular smooth muscle cells

Previous reports have suggested that phorbol esters can decrease the affinity of epidermal growth factor (EGF) for its cellular receptors. Investigations of the consequences of the interaction between phorbol esters and EGF, however, have been limited to EGF-stimulated Na/H exchange in A431 cells (Whitely, B., D. Cassel, Y.-X. Zuang, and L. Glaser, 1984, J. Cell Biol., 99:1162-1166). In the present study, the effect of the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) on EGF-stimulated ion transport and DNA synthesis was determined in cultured vascular smooth muscle cells (A7r5). It was found that TPA stimulated Na/H exchange when added alone (half-maximal stimulatory concentration, 25 nM). However, when cells were pretreated with TPA and then challenged with EGF, TPA significantly inhibited EGF-stimulated Na/H exchange (78%; half-maximal inhibition [Ki] at 2.5 nM). Subsequently the effects of TPA on Na/K/Cl co-transport were measured. TPA was observed to inhibit Na/K/Cl co-transport (half-maximal inhibitory concentration, 50 nM) and also to inhibit EGF-stimulated Na/K/Cl co-transport (100%; Ki at 5 nM). Finally, the effects of TPA on DNA synthesis were assessed. TPA had a modest stimulatory effect on DNA synthesis (half-maximal stimulatory concentration, 6 nM), but had a significant inhibitory effect on EGF-stimulated DNA synthesis (56%; Ki at 5 nM). These findings suggest that the inhibitory effect of TPA on EGF-receptor functions goes beyond previously reported effects on Na/H exchange in A431 cells and extends to EGF-stimulation of Na/K/Cl co-transport and DNA synthesis in vascular smooth muscle cells.     

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.     

The calcium antagonist nisoldipine and the calmodulin antagonist W-7 synergistically inhibit initiation of DNA synthesis in cultured arterial smooth muscle cells

Cultured arterial smooth muscle cells go through a transition from a contractile to a synthetic phenotype. Morphologically, the transition includes a reduction in size of the myofilament bundles and the formation of an extensive rough endoplasmic reticulum and a large Golgi complex. Functionally, it leads to loss of contractility, onset of cellular proliferation, and secretion of extracellular matrix components. This change in differentiated characteristics under in vitro conditions has attracted attention because of its resemblance to the modification of the smooth muscle cells that occurs in vivo during atherogenesis. Here, transmission electron microscopy and [3H]-thymidine autoradiography were used to study the role of calcium ions in the control of phenotypic properties and growth of cultivated rat aortic smooth muscle cells. The calcium antagonist nisoldipine was found to lack distinct effect on the structural reorganization of the cells, but showed a moderate prohibitory effect on the start of DNA synthesis early in primary culture. In growth-arrested secondary cultures, nisoldipine inhibited induction of DNA synthesis by serum or platelet-derived growth factor (PDGF). The agent's effect was inversely related to the concentration of calcium ions in the extracellular medium and was partially counteracted by the calcium agonist BAY K 8644. In contrast, W-7, an antagonist of the calcium-binding protein calmodulin, potentiated the effect of nisoldipine and, at higher concentrations, inhibited induction of DNA synthesis in itself. The results suggest that the mitogenic stimulation of arterial smooth muscle cells involves a flux of calcium ions through the plasma membrane and requires participation of calmodulin.     

Intercellular communication in cultured human vascular smooth muscle cells

Intercellular communication through gap junction channelsplays a fundamental role in regulating vascular myocyte tone. We investigated gap junction channel expression and activity in myocytes from the physiologically distinct vasculature of the human internal mammary artery (IMA, conduit vessel) and saphenous vein (SV,capacitance vessel). Northern and Western blots documented the presenceof connexin43 (Cx43) in frozen tissues and cultured cells from both vessels. Northern blots also confirmed the presence of Cx40 mRNA incultured IMA and SV myocytes. Dual whole cell patch-clamp experiments revealed that macroscopic junctional conductance was voltage dependent and characteristic of that observed for Cx43. In the majority ofrecords, in both vessels, single-channel activity was dominated by amain-state conductance of 120 pS, with subconducting events comprisingless than 10% of the amplitude histograms. However, some recordsshowed "atypical" unitary events that had a conductance similar toCx40 (~140-160 pS), but gating behavior like that of Cx43. Assuch, it is conceivable that the presence and coexpression of Cx40 andCx43 in IMA and SV myocytes may result in heteromeric channelformation. Nonetheless, in terms of gating, Cx43-like behavior clearly dominates.

     

Vasoconstrictor-induced protein-tyrosine phosphorylation in cultured vascular smooth muscle cells

In cultured rat aortic smooth muscle cells, angiotensin II induced tyrosine phosphorylation of at least 9 proteins with molecular masses of 190, 117, 105, 82, 79, 77, 73, 45 and 40 kDa in time- and dose-dependent manners. Other vasoconstrictors such as [Arg]vasopressin, 5-hydroxytryptamine and norepinephrine induced the tyrosine phosphorylation of the same set of proteins as angiotensin II. The tyrosine phosphorylation of these proteins was mimicked by the protein kinase C-activating phorbol ester, phorbol 12 myristate 13-acetate, and the Ca2+ ionophore, ionomycin. These results demonstrate that the vasoconstrictors stimulate the tyrosine phosphorylation of several proteins in vascular smooth muscle cells and suggest that the tyrosine phosphorylation reactions are the events distal to the activation of protein kinase C and Ca2+ mobilization in the intracellular signalling pathways of the vasoconstrictors.