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.     

Presence of specific bradykinin receptors in arterial and venous smooth muscle

The relationship between bradykinin action and its concentration was examined on isolated rings of the rabbit aorta, femoral artery, jugular vein and on isolated strips of the rat portal vein. The sensitivity of femoral artery and portal vein smooth muscles to bradykinin was disclosed. Venous smooth muscles were more sensitive to bradykinin as compared with arterial smooth muscles. Dissociation constants for the rabbit aorta, femoral artery, jugular vein and for the rat portal vein were 3.98 X 10(-6), 6.3 X 10(-6), 1.26 X 10(-7), and 7.6 X 10(-9)M, respectively. Effects of endogenous bradykinin in vivo might result from its primary action on the venous smooth muscle, action on the arterial smooth muscle and veno-arterial interactions.     

Evidence that unphosphorylated smooth muscle myosin supports smooth muscle contraction.

Unphosphorylated smooth muscle myosin filaments do not disassemble in MgATP, provided that the solution is supplemented either by 25% serum albumin or by 6% polyethylene glycol 6000. These filaments are able to support actomyosin retraction but their ATPase activity is not activated by tropomyosin-decorated F-actin.     

The smooth muscle cell. III. Elastin synthesis in arterial smooth muscle cell culture

Primate arterial smooth muscle cells and skin fibroblasts were examined for their ability to synthesize elastin in culture. In the presence of the lathyrogen beta-aminopropionitrile, the smooth muscle cells incorporate [3H]lysine into a lysyl oxidase substrate that was present in the medium and associated with the cell layer. A component having a mol wt of 72,000 and an electrophoretic mobility similar to that of authentic tropoelastin was isolated from the labeled smooth muscle cells by coacervation and fractionation with organic solvents. In the absence of beta-aminopropionitrile, long-term cultures of smooth muscle cells incorporated [14C]lysine into desmosine and isodesmosine, the cross-link amino acids unique to elastin. In contrast, no desmosine formation occurred in the fibroblast cultures. These characteristics demonstrate that arterial smooth muscle cells are capable of synthesizing both soluble and cross-lined elastin in culture.     

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.     

A specific gastrin receptor on plasma membranes of antral smooth muscle

Plasma membranes with a 17 fold enrichment in 5'-nucleotidase over homogenate were prepared from antral smooth muscle. A specific gastrin receptor on the plasma membranes has been demonstrated. By Scatchard analysis receptor has a Kaff of 2x10(9)M(-1) and a binding capacity of 5x10(-14) moles/mg of membrane protein.     

Tyrosine kinase receptor activation inhibits NPR-C in lung arterial smooth muscle cells

We have previously demonstrated that expression of the atrial natriuretic peptide (ANP) clearance receptor (NPR-C) is reduced selectively in the lung of rats and mice exposed to hypoxia but not in pulmonary arterial smooth muscle cells (PASMCs) cultured under hypoxic conditions. The current study tested the hypothesis that hypoxia-responsive growth factors, fibroblast growth factors (FGF-1 and FGF-2) and platelet-derived growth factor-BB (PDGF-BB), that activate tyrosine kinase receptors can reduce expression of NPR-C in PASMCs independent of environmental oxygen tension. Growth-arrested rat PASMCs were incubated under hypoxic conditions (1% O2) for 24 h; with FGF-1, FGF-2, or PDGF-BB (0.1-20 ng/ml for 1-24 h); or with ANG II (1-100 nM), endothelin-1 (ET-1, 0.1 microM), ANP (0.1 microM), sodium nitroprusside (SNP, 0.1 microM), or 8-bromo-cGMP (0.1 mM) for 24 h under normoxic conditions. Steady-state NPR-C mRNA levels were assessed by Northern blot analysis. FGF-1, FGF-2, and PDGF-BB induced dose- and time-dependent reduction of NPR-C mRNA expression within 1 h at a threshold concentration of 1 ng/ml; hypoxia, ANG II, ET-1, ANP, SNP, or cGMP did not decrease NPR-C mRNA levels in PASMCs under the above conditions. Downregulation of NPR-C expression by FGF-1, FGF-2, and PDGF-BB was inhibited by the selective FGF-1 receptor tyrosine kinase inhibitor PD-166866 and mitogen-activated protein/extracellular signal-regulated kinase inhibitors U-0126 and PD-98059. These results indicate that activation of tyrosine kinase receptors by hypoxia-responsive growth factors, but neither hypoxia per se nor activation of G protein-coupled receptors, inhibits NPR-C gene expression in PASMCs. These results suggest that FGF-1, FGF-2, and PDGF-BB play a role in the signal transduction pathway linking hypoxia to altered NPR-C expression in lung.     

Molecular cloning of the rat vascular smooth muscle thrombin receptor. Evidence for in vitro regulation by basic fibroblast growth factor.

To study thrombin's receptor-mediated effects on vascular cells, we cloned and characterized a cDNA encoding a rat smooth muscle cell thrombin receptor. A rat aortic smooth muscle (RASM) cell cDNA library was screened with a 500-base pair (bp) sequence from the human thrombin receptor, obtained by polymerase chain reaction (PCR) amplification of cDNA synthesized from human erythropoietic leukemia (HEL) cell mRNA with PCR primers based on the published human thrombin receptor sequence. Clone pRTHR17 contains a 3418-bp insert that includes 50 bp of the 5'-untranslated region and the entire coding and 3'-untranslated regions of the RASM cell thrombin receptor. The sequence of pRTHR17 is 85% similar, at the nucleotide level, and 78% similar, at the deduced amino acid level, to the human thrombin receptor. Although the putative thrombin cleavage and binding sites are present, there are significant differences between the rat and human receptors in their amino-terminal sequences. Detectable signals (consisting of a single band of 3.45 kb) are present by Northern analysis of mRNA from RASM cells, and rat lung, kidney, and testes, but not in aorta or other tissues probed. The results of Southern analysis of rat genomic DNA are consistent with the existence of a single copy of the gene encoding this receptor. The steady state thrombin receptor mRNA level is low in cultured growth-arrested RASM cells and not detectable in rat aorta. To determine whether regulation of the RASM cell thrombin receptor occurs under growth-stimulating conditions, growth-arrested RASM cells were treated with basic fibroblast growth factor (bFGF, recently proposed to be a major mitogen controlling vascular smooth muscle cell growth following injury (Lindner, V., and Reidy, M. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3739-3743)). There was a significant increase in thrombin receptor mRNA following the addition of bFGF. These data demonstrate that: 1) mRNA for a thrombin receptor similar to that reported from human megakaryocyte and hamster fibroblast cell lines is present in proliferating primary culture rat smooth muscle cells, 2) the most significant sequence differences are present in the amino-terminal tail of the thrombin receptor, and 3) the mRNA level for this receptor is regulated under growth-stimulating conditions in vitro.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Replication of cytomegalovirus in human arterial smooth muscle cells.

Cytomegalovirus (CMV) strain AD-169 replicated in smooth muscle cell (SMC) cultures derived from human umbilical arteries, producing enveloped infectious virions. However, unlike the effects of CMV on fully permissive human lung fibroblasts, the effects of strain AD-169 on SMC cultures were delayed and prolonged, resulting in extended survival of a fraction of the starting population. This period of survival did not exceed the life-span of the control SMC cultures. Infectious CMV continued to be isolated from the surviving SMC cultures after extinction of the original inoculum by dilution and after treatment of the cultures with CMV neutralizing antibody. The implications of these findings for the pathogenesis of atherosclerosis are discussed.     

Estimates of cellular mechanics in an arterial smooth muscle.

Estimates of force generation or shortening obtained from smooth muscle tissues are valid for individual cells only if each cell is contracting homogeneously and if cells anatomically arranged in series are mechanically coupled. These two assumptions were tested and shown to be valid for the pig carotid media under certain conditions. Homogeneity of cellular responses in carotid strips was estimated from the motion of markers on the tissue during K+ -induced isometric contractions. When tissues were stretched to L0 (the optimum length for force generation), there was little marker movement on stimulation. However, considerable marker movement was observed on stimulation at shorter muscle lengths, reflecting localized shortening or stretching. The mechanical coupling of the very small cells in the media was determined by measuring the dependence of cell length on tissue length. Tissues were fixed with glutaraldehyde during isometric contractions at various tissue lengths (0.4--1.1 x L0). The fixed tissues were macerated with acid and the lengths of the dispersed cells were measured. Cell lengths were broadly distributed at all muscle lengths. However, the direct proportionality between mean cell length and muscle length (as a fraction of L0) indicated that cells which are anatomically in series are coupled force-transmitting structures. We conclude that valid estimates of cellular mechanical function in this preparation can be obtained from tissue measurements at lengths greater than about 0.9L0.     

Evidence for presence of ATP-sensitive K+ channels in rat colonic smooth muscle cells.

Coexpression of sulfonylurea receptor (SUR) and inward-rectifying K+ channel (Kir6.1 or 6.2) subunit yields ATP-sensitive K+ (K(ATP)) channels. Three subtypes of SUR have been cloned: pancreatic (SUR1), cardiac (SUR2A), and vascular smooth muscle (SUR2B). The distinct responses to K+ channel openers (KCOs) produced in different tissues may depend on the SUR isoform of K(ATP) channel. Therefore, we investigated the effects of pinacidil and diazoxide, two KCOs, on K(ATP) currents in intestinal smooth muscle cells of the rat colon (circular layer) using whole-cell voltage clamp. Pinacidil stimulated a time-independent K+ current evoked by various test potentials from a holding potential of -70 mV. The reversal potential of the stimulated current was about -75 mV, which is close to the equilibrium potential for K+ (E(K)). Both pinacidil and diazoxide dose-dependently stimulated K+ currents (evoked by ramp pulses), with EC50 values of 1.3 and 34.2 microM, respectively. The stimulated current was completely reversed by glybenclamide (3 microM). Since the EC50 values are close to those reported for vascular smooth muscle (VSM) cells, the SUR subtype may be similar to that in VSM cells, and could form the functional K(ATP) channel in rat colonic smooth muscle cells.     

Crosstalk between protein kinase A and growth factor receptor signaling pathways in arterial smooth muscle.

Crosstalk between the cyclic AMP-dependent protein kinase (PKA) and growth factor receptor signaling is one of many emerging concepts of crosstalk in signal transduction. Understanding of PKA crosstalk may have important implications for studies of crosstalk between other, less well known, signaling pathways. This review focuses on PKA crosstalk in arterial smooth muscle. Proliferation and migration of arterial smooth muscle cells (SMCs) contribute to the thickening of the blood vessel wall that occurs in many types of cardiovascular disease. PKA potently inhibits SMC proliferation by antagonizing the major mitogenic signaling pathways induced by growth factors in SMCs. PKA also inhibits growth factor-induced SMC migration. An intricate crosstalk between PKA and the mitogen-activated protein kinase (MAPK/ERK) pathway, the p70 S6 kinase pathway and cyclin-dependent kinases has been described. Further, PKA regulates expression of growth regulatory molecules. The result of PKA activation in SMCs is the potent inhibition of cell cycle traverse and SMC migration. In this review, we discuss recent advances in our understanding of the crosstalk between PKA and signaling pathways induced by growth factor receptors in SMCs, and where relevant, in other cell types in which interesting examples of PKA crosstalk have been described.     

Response of arterial smooth muscle to length perturbation.

The ability of arterial smooth muscle to generate tension is influenced by muscle length. An unsettled question is whether the length-tension relationship is a simple reflection of the contractile filament overlap, as it is in skeletal muscle. There are several factors that could potentially affect tension generation in arterial smooth muscle; these include stretch-induced myogenic response and length-oscillation-induced disruption of the contractile filament organization. In this study, in which rabbit carotid arterial preparations were used, we found that different length-tension curves could be obtained at different times after a length change. In addition, length oscillation at a frequency of normal pulse rate and with small to moderate oscillation amplitude was found to potentiate tension generation but reduced tension at large amplitudes. The observed response could be attributed to adaptation of the muscle to length change over time and to myogenic potentiation associated with stretching of the muscle.     

Phosphorylation of caldesmon in arterial smooth muscle

We have isolated caldesmon (Mr = 145,000), by immunoprecipitation, from [32P]orthophosphate-loaded porcine carotid arteries. In resting muscles, caldesmon was phosphorylated to 0.45 mol of PO4/mol protein, while the 20,000-dalton myosin regulatory light chain (LC20) was phosphorylated to less than 0.05 mol/mol. After stimulation by KCl (110 mM) for 75 min and phorbol 12,13-dibutyrate (PDBu, 1 microM) for 60 min, caldesmon phosphorylation levels rose to 0.96 and 1.1 mol/mol, respectively. LC20 phosphorylation increased to 0.49 mol/mol at 1 min of stimulation by KCl and decreased to 0.17 mol/mol at 60 min. With PDBu, phosphate incorporation into LC20 rose only slightly, reaching 0.09 mol/mol after 90 min. Muscles contracted with histamine (10 microM) or ouabain (1 microM) also demonstrated elevated levels of phosphate incorporation into caldesmon. In these muscles, LC20 phosphorylation levels were less than 0.05 mol/mol. Three major phosphopeptides of indistinguishable mobility were identified on maps of caldesmon from resting, KCl-stimulated, and PDBu-stimulated muscles. There was, however, little similarity between the phosphopeptide maps of caldesmon phosphorylated in intact tissue and maps of purified caldesmon phosphorylated in vitro by protein kinase C (Ca2+/phospholipid-dependent enzyme) or Ca2+/calmodulin kinase II.     

Force-velocity relationships in hypertensive arterial smooth muscle

Increased total peripheral resistance is the cardinal haemodynamic disorder in essential hypertension. This could be secondary to alterations in the mechanical properties of vascular smooth muscle. Adequate study has not been made of the force-velocity (F-V) relationship in hypertensive arterial smooth muscle. Increased shortening in arterial smooth muscle would result in greater narrowing of arteries. The objectives of this investigation were to see if there is (i) increased shortening or increased maximum change in muscle length (delta Lmax where L stands for muscle length), (ii) an increased maximum velocity of shortening (Vmax) measured in l omicron per second where l omicron is the optimal muscle length for tension development, and (iii) a difference in maximum isometric tension (P omicron) developed in spontaneously hypertensive rat (SHR; N = 6) compared with normotensive Wistar Kyoto rat (WKY;N = 5) caudal artery strips. An electromagnetic muscle lever was employed in recording force-velocity data. Analysis of these data revealed the following: (a) the SHR mean P omicron of 6.21 +/- 1.01 N/cm2 was not different from the mean WKY P omicron of 6.97 +/- 1.64 N/cm2 (p greater than 0.05); (b) the SHR preparations showed greater shortening for all loads imposed; (c) the SHR Vmax of 0.016 l omicron/s was greater than the WKY Vmax of 0.013 l omicron/s (p less than 0.05). This study provides evidence that while hypertensive arterial smooth muscle is not able to produce more force than normotensive arterial smooth muscle, it is capable of faster and greater shortening. The latter could result in increased narrowing of hypertensive arteries and increased blood pressure.