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.     

Regulation of beta-adrenergic receptors and calcium channel agonist binding sites in cultured human embryonal smooth muscle cells

Recent electrophysiological studies with cell membrane patches of cardiac myocytes and an electrically excitable cell line derived from rat pituitary tumor suggested that voltage activated calcium channels must be phosphorylated to respond to membrane depolarization (Armstrong and Eckert 1986; Trautwein and Kameyama 1986). In view of the "phosphorylation hypothesis" we investigated the adenylate-cyclase activity, the characteristics of beta-adrenergic and calcium channel agonist binding sites in control and desensitized (exposure to isoproterenol) human embryonal cells (HEC), and in fragmented membrane preparations of canine coronary smooth muscle. Our results suggest that down-regulation of the membrane-bound beta-adrenergic receptors, induced by isoproterenol in human embryonal cells and also in adult canine vascular tissue, results in physical translocation of beta-adrenergic binding sites into the light membrane fraction. This phenomenon is accompanied with an increased intracellular concentration of cAMP in and an increased binding of the calcium channel agonist (3H) BAYK 8644 to both HEC and canine smooth muscle membrane preparations. It could be concluded that phosphorylation of beta-adrenergic receptors regulates not only the beta subcellular distribution of the beta receptors but also the availability of calcium channel agonist binding sites in the cellular membrane.     

Lipoproteins increase growth of mitogen-stimulated arterial smooth muscle cells

The relationship between lipoproteins and growth of aortic smooth muscle cells has been a matter of controversy. We therefore reexamined this issue using serum-free defined media methodology. By themselves, LDL or HDL (50-500 micrograms/ml) from normolipemic human or bovine plasma produced little or no growth of homologous aortic smooth muscle cells incubated in serum-free medium that was supplemented with insulin and transferrin to maintain cell viability. In fact, LDL prepared in the absence of an antioxidant (BHT) was toxic to these cells. However, in the presence of maximally effective concentrations of platelet-derived growth factor (PDGF), LDL or HDL consistently increased the growth of homologous smooth muscle cells (up to twofold increased in DNA accumulation in 48 hr). Lipoproteins also augmented the growth response of arterial smooth muscle cells to fibroblast growth factor or epidermal growth factor. The mechanism of this effect was investigated further with HDL, because, in contrast to LDL, HDL apoproteins are water-soluble. Neither HDL delipidated by solvent extraction (apoHDL), purified bovine apoA-I, nor cholesterol added in the form of phospholipid vesicles appreciably increased PDGF-induced growth of bovine smooth muscle cells. However, HDL-like particles reconstituted by sonication of apoHDL with cholesterol and phospholipids did increase the growth of cultures of bovine smooth muscle cells treated with PDGF. Uptake of tritiated thymidine by cultures incubated with partially purified PDGF alone (10 micrograms/ml) was 5,693 +/- 235 dpm/24 hr compared to 10,381 +/- 645 dpm/24 hr (p less than 0.01) in the presence of both PDGF and reconstituted HDL-like particles (250 micrograms protein/ml). Thus both the lipid and protein components of HDL may be necessary for optimal potentiation of growth of mitogen-stimulated cells. These results indicate that lipoproteins from normolipemic sera are not bona fide growth factors but can potentiate the growth of mitogen-stimulated cells, perhaps by supplying exogenous cholesterol required for membrane biogenesis. This finding might be important in arterial injury when the release of PDGF and exposure to plasma lipoproteins could act in concert to stimulate the proliferation of smooth muscle cells.     

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.     

Beta-adrenergic receptor binding characteristics and responsiveness in cultured Wistar-Kyoto rat arterial smooth muscle cells

The tone of arterial blood vessels is regulated by the catecholamines through their receptors on arterial smooth muscle cells (ASMC). beta 2-adrenergic receptors of ASMC mediate vasodilation through agonist mediated c-AMP production. Previous reports have described these receptors on freshly isolated blood vessels. This study demonstrates the presence of beta 2-adrenergic receptors on cultured rat ASMC and that these receptors are functional. beta-adrenergic receptor binding was measured using [3H]-dihydroalprenolol (DHA) binding to the membrane of cultured ASMC from normotensive Wistar-Kyoto rats. The ASMC beta-adrenergic receptors have a Kd of 0.56 +/- 0.16 nM and a Bmax of 57.2 +/- 21.7 fmol/mg protein. Competition binding studies revealed a much greater affinity of these receptors for epinephrine than norepinephrine, indicating the preponderance of a beta 2-adrenergic receptor subtype. Isoproterenol stimulation of cultured ASMC resulted in a 14 +/- 7 fold increase in intracellular c-AMP content of these cells indicating these receptors are functional. beta-adrenergic receptors of cultured ASMC provide an excellent system in which the association between hypertension and observed beta-adrenergic receptor differences can be further explored.     

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.     

Functional angiotensin II receptors in cultured vascular smooth muscle cells

To study cellular mechanisms influencing vascular reactivity, vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the rat mesenteric artery, a highly reactive, resistance-type blood vessel, and established in primary culture. Cellular binding sites for the vasoconstrictor hormone angiotensin II (AII) were identified and characterized using the radioligand 125I-angiotensin II. Freshly isolated VSMC, and VSMC maintained in primary culture for up to 3 wk, exhibited rapid, saturable, and specific 125I-AII binding similar to that seen with homogenates of the intact rat mesenteric artery. In 7-d primary cultures, Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant (Kd) of 2.8 +/- 0.2 nM and a total binding capacity of 81.5 +/- 5.0 fmol/mg protein (equivalent to 4.5 x 10(4) sites per cell). Angiotensin analogues and antagonists inhibited 125I-AII binding to cultured VSMC in a potency series similar to that observed for the vascular AII receptor in vivo. Nanomolar concentrations of native AII elicited a rapid, reversible, contractile response, in a variable proportion of cells, that was inhibited by pretreatment with the competitive antagonist Sar1,Ile8-AII. Transmission electron microscopy showed an apparent loss of thick (12-18 nm Diam) myofilaments and increased synthetic activity, but these manifestations of phenotypic modulation were not correlated with loss of 125I-AII binding sites or hormonal responsiveness. Primary cultures of enzymatically dissociated rat mesenteric artery VSMC thus may provide a useful in vitro system to study cellular mechanisms involved in receptor activation-response coupling, receptor regulation, and the maintenance of differentiation in vascular smooth muscle.     

Characterization of epidermal growth factor receptors in cultured vascular smooth muscle cells of rat aorta

Characteristics of specific receptors for epidermal growth factor (EGF) and its effect on cellular proliferation and synthesis of DNA and protein were studied in cultured vascular smooth muscle cells (VSMC) from rat aorta. Binding studies using 125I-EGF revealed the presence of high affinity binding sites for EGF on VSMC in culture: the apparent dissociation constant was approximately 2.5 X 10(-10)M and the maximal binding capacity was approximately 67,000 sites/cell. EGF stimulated cellular proliferation and incorporation of [3H]thymidine and [3H]leucine into the cells in a dose-dependent fashion; the approximate half-maximal stimulation was induced with 1.5 X 10(-10)M. Platelet-derived growth factor (PDGF) had an additive effect with EGF on DNA synthesis by VSMC. Preincubation of VSMC with unlabeled EGF resulted in a substantial reduction in the number of receptors without changing the affinity, suggesting receptor "down-regulation" mechanism. These data indicate that rat aortic VSMCs have specific receptors for EGF, and suggest that EGF, in addition to PDGF, is also involved in the cell growth of VSMC.     

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.     

Endogenous arachidonic acid metabolism by cultured arterial smooth muscle cells

Cultured aortic smooth muscle cells originated from healthy and atherosclerotic rabbits produce prostaglandins (namely prostacyclin) at a basal state. Prostaglandin secretion is dramatically reduced in atherosclerotic cells. This impairment was not correlated with any alteration of acyl hydrolase activities and probably involved a decrease of cyclooxygenase activities.     

Lipoprotein uptake by cultured human arterial smooth muscle cells.

Human arterial smooth muscle cells growing in tissue culture, in contrast to rat cells, preferentially bind and take up large, lipid-rich lipoproteins (125I-labeled low density and very low density lipoproteins) in comparison to the known difference in the propensity of these two species to develop atherosclerosis.     

Functional receptors for vasoactive intestinal peptide in cultured vascular smooth muscle cells from rat aorta

Specific binding sites for vasoactive intestinal peptide (VIP), a potent vasodilatory polypeptide, and its effect on formation of intracellular cyclic AMP levels were studied in cultured vascular smooth muscle cells (VSMC) from rat aorta. Specific binding of ¹²⁵I-labeled-VIP to cultured VSMCs was time- and temperature-dependent. Scatchard analysis of binding studies suggested the presence of two classes of high and low affinity binding sites for VIP; the apparent K_d and the number of maximal binding capacity were ∼8×10⁻⁹ M and 60,000 sites/cell (high-affinity sites) and ∼4×10⁻⁸ M and 140,000 sites/cells (low-affinity sites), respectively. Unlabeled VIP competitively inhibited the binding of ¹²⁵I-labeled-VIP to its binding sites, whereas neither peptides structurally related to VIP, nor other vasoactive substances affected the binding. VIP stimulated formation of intracellular cyclic AMP in cultured VSMCs in a dose-dependent manner; the stimulatory effect of VIP on cyclic AMP formation was not blocked by propranolol and was additive with isoproterenol. The present study first demonstrates the presence of specific receptors for VIP in VSMCs functionally coupled to adenylate cyclase system. It is suggested that VIP exerts its vasodilatory effect through its specific receptors distinct from β-adrenergic receptors.     

Dimethyl sulfoxide induces microtubule formation in cultured arterial smooth muscle cells

Prolonged exposure of cultured arterial smooth muscle cells to 1% dimethyl sulfoxide (DMSO) resulted in a remarkable increase in cytoplasmic microtubules and an appearance of bundle-like aggregates of microtubules associated with ribosomes (microtubule-ribosome-complex). In this complex, fine filaments of 8 to 16 nm diameter were interspersed, some of which displayed continuity with the microtubules and were studded with a single array of ribosomes. The microtubule-ribosome-complex may represent an unusual aggregate of microtubules containing incompletely polymerized forms of newly synthesized tubulin induced by prolonged effect of DMSO.     

Growth-mediated, density-dependent inhibition of endocytosis in cultured arterial smooth muscle cells

The effects of cell density and growth upon fluid phase endocytosis were investigated in quiescent and growing cultures of monkey arterial smooth muscle cells. Cells were maintained in a quiescent state of growth in 5% plasma-derived serum. Subsequent exposure of subconfluent cultures to the specific mitogens, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), or to whole blood serum, resulted in up to 4-fold increases in the rate of fluid endocytosis/cell. The changes began several hours after entry into G1 phase of the cell cycle and continued through S. The fraction of cells entering the growth cycle was variable (PDGF=FGF>EGF) and a close correlation existed between the rate of endocytosis and the fraction of [³H]thymidine-labelled cells (r = 0.929, p<0.01). At a range of cell densities, the rate of fluid endocytosis/cell was similar in sparse, confluent and post-confluent cultures of quiescent cells; in contrast, in growing cells there was density-dependent inhibition of endocytosis. Furthermore, when quiescent cells were in contact with each other and were then exposed to mitogens, the growth response was diminished and there was only a 25–50% increase in the rate of endocytosis, even in the presence of high concentrations of growth factors.These studies indicate that the influence of cell density upon fluid endocytosis in arterial smooth muscle cells is indirect in that it represents a secondary effect of decreased mitogenic response to specific growth factors.     

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.     

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.     

Processing of soluble elastin in cultured neonatal rat smooth muscle cells

The synthesis and extracellular deposition of elastin by cultured neonatal rat aorta smooth muscle cells has been followed. The addition of beta-aminopropionitrile to the culture medium promotes accumulation of soluble precursors of elastin. Under such conditions, a protein possessing characteristics of a soluble elastin precursor with an apparent molecular weight of 77,000 was detected and partially purified. Pulse-chase studies suggested that this 77-kDa protein undergoes an extracellular, enzymatically catalyzed process to a 71-kDa protein. This 71-kDa protein is strikingly similar to tropoelastins isolated from other tissue systems, in which no evidence for higher molecular weight soluble precursors is at present available. Data presented in this communication suggest that the 77-kDa protein, which we have designated protropoelastin, represents a precursor to the tropoelastin moiety produced in the neonatal rat smooth muscle cell culture.