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PubMed1987

Effect of low extracellular calcium on shortening velocity in isolated single smooth muscle cells.

To understand how smooth muscle modulates its shortening velocity within the time course of a single contraction, the factors that govern crossbridge cycling in smooth muscle must be characterized. Since calcium plays an important role in regulation of the contractile apparatus, we studied the effect of lowering extracellular calcium on shortening velocity in single smooth muscle cells isolated enzymatically from the toad, Bufo marinus, stomach muscularis. Shortening velocity was estimated by three independent methods: (1) isotonic releases; (2) slack test; (3) video images of freely shortening cells. To determine the shortening velocity from isotonic releases and the slack test method, a single cell was attached at one end to an ultrasensitive force transducer and piezoelectric displacement device at its other end. At peak isometric force, in response to electrical stimulation, a series of isotonic releases to varying force levels were imposed and the resultant shortening responses used to construct force:velocity relationships in physiological saline containing low (0.18 mM) extracellular calcium. From force:velocity relationships and slack test data, an estimate of maximum shortening velocity (Vmax) was 0.19 cell lengths/s which was 3 times lower than in normal calcium (Warshaw 1987). Although Vmax was reduced in low extracellular calcium, the curvature of the force:velocity relationship estimated from the hyperbolic constant, a/Fmax, was unchanged (a/Fmax = 0.17). Additional evidence for an effect of extracellular calcium on shortening velocity was obtained from shortening responses of freely shortening cells attached at one end only. Data from cells exposed first to low (0.18 mM) and then normal (1.8 mM) calcium suggest at least a twofold decrease in free shortening velocity with lower extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Composition in situ and in vitro of vascular smooth muscle laminin in the rat.

Vascular smooth muscle cells are surrounded by a basal lamina containing an array of macromolecules: included among these are the laminins, a family of oligomeric glycoproteins composed of subunits encoded by different genes. In this study, we have used monoclonal antibodies to several of these subunits, including S-laminin, laminin B2, and laminin B1, to study these proteins in tail artery, superior mesenteric artery, and aorta of rats. In situ, immunostaining for the B2 and S chains was present in the basal lamina, between the smooth muscle cells, throughout the tunica media. In contrast, B1 chain immunostaining was concentrated around cells in the inner media. To investigate whether smooth muscle cells can produce S-laminin, laminin B2, and laminin B1, smooth muscle cells from the superior mesenteric artery were grown in culture and laminin subunit expression determined. In early culture (4 days), immunostaining showed abundant laminin B2 and less B1 synthesis and incorporation into the matrix. Staining for S-laminin was even less intense than for B1 and was localized to areas where cells were densely packed. The same pattern of S-laminin immunostaining was seen during early culture in cells grown on fibronectin, type IV collagen, or gelatin. Immunoblotting detected S-laminin in the conditioned medium from early cultured cells. In later culture (12 days), S-laminin incorporation into the matrix increased markedly compared to incorporation at 4 days. At this time, cells are much more densely packed and multilayered with extensive matrix accumulation. Cyclical stretching of cells in vitro did not increase immunostaining for S-laminin. Together these data show that S-laminin is a component of the arterial media in situ and that in vitro S-laminin is synthesized by smooth muscle cells. Increased incorporation of S-laminin into the matrix in later culture correlates with the presence of a more extensive matrix, suggesting that matrix organization may be critical to S-laminin incorporation.

Animals↗
PubMed1995

Regulation of sodium-hydrogen exchange in vascular smooth muscle.

The Na+/H+ exchanger in vascular smooth muscle cells represents a major mechanism for sodium influx and is also one of the principal mechanisms responsible for the regulation of intracellular pH (pHi). In this review, the relationship between pHi and vascular smooth muscle cell growth, the regulation of Na+/H+ exchange by vasoactive agents and growth factors, and the second messenger pathways that may be involved in activation of Na+/H+ exchange have been discussed. The exchanger appears to be important in vascular smooth muscle cell growth, based on results that (1) Na+/H+ exchange is stimulated by hypertrophic and hyperplastic agonists, (2) vascular smooth muscle cell proliferation is induced by cytoplasmic alkalinisation in the absence of mitogens, (3) vascular smooth muscle cell proliferation is dependent on extracellular sodium, and (4) inhibitors of Na+/H+ exchange block cell growth. Several pathways appear capable of activating the exchanger in vascular smooth muscle cells as there is evidence for both calcium and protein kinase C dependent and independent pathways. We speculate that the calcium and protein kinase C dependent pathways may play a role in the contractile response of differentiated vascular smooth muscle cells in the vessel wall, while the calcium and protein kinase C independent pathways may be involved in the proliferative response observed after arterial injury and in tissue culture.

Animals↗
PubMed2002

Upregulation of the cytoskeletal-associated protein Moesin in the neointima of coronary arteries after balloon angioplasty: a new marker of smooth muscle cell migration?

Migrating cells like coronary smooth muscle cells in restenosis change their cell shape and form cellular protrusions called filopodia. A prerequisite for filopodia formation is the rearrangement of the actin cytoskeleton. An essential role of the 78-kDa protein Moesin is described for Rho- and Rac-dependent assembly of actin filaments. In vivo Moesin is not observed in mature smooth muscle cells. The objective of this study was to demonstrate that Moesin is upregulated in migrating coronary smooth muscle cells during restenosis development. In vivo expression of Moesin was upregulated in neointimal coronary smooth muscle cells of dilated porcine coronary arteries compared to the undilated left circumflex coronary artery of the same swine. Concordant to these results Moesin expression was upregulated in migrating and invading human arterial smooth muscle cells in vitro analyzed by FACS, Western blotting and RT-PCR. In addition, the invasive potential of Moesin-positive Mel Im cells transfected with Moesin sense DNA increased by 28% as compared to mock-transfected control, whereas antisense transfected cells had a decreased invasive potential of 32%. Transfection of Moesin-negative HepG2 with Moesin sense cDNA increased the invasive potential by 43%. Finally, transfection of human arterial smooth muscle cells with Moesin sense cDNA caused an increased invasive potential of 30%. Transfection of haSMCs with antisense cDNA decreased the invasive potential by 37% in comparison to mock-transfected control. These results demonstrate for the first time an upregulation of Moesin expression in coronary smooth muscle cells of the neointima after arterial injury. The increased migrative and invasive potential of cells transfected with Moesin confirmed the functional role of Moesin in cell migration. This indicates an important role of Moesin during restenosis development.

Actins↗
PubMed2001

Regulation of cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle cells.

Nitric oxide and endogenous nitrovasodilators regulate smooth muscle tone by elevation of cGMP and activation of cyclic GMP-dependent protein kinase (PKG). The amplitude and duration of the cGMP signal in smooth muscle is regulated in large part by cGMP-specific cyclic nucleotide phosphodiesterase (PDE5). Previous in vitro data have suggested that both cAMP-dependent protein kinase and PKG can regulate the activity of PDE5. To test if this type of regulation is important in the intact cell, we have generated phospho-PDE5-specific antisera and have utilized isolated smooth muscle cells from mice having a disruption in the PKG I gene as well as cells from normal human smooth muscle. The data show that in human smooth muscle cells, activation of PKG by 8-Br-cGMP led to phosphorylation and activation of PDE5. In the same cells, 8-Br-cAMP had no significant effect on PDE5 phosphorylation. Treatment of wild-type mouse aortic smooth muscle cells with 8-Br-cGMP also induced the phosphorylation of PDE5, whereas no phosphorylation was seen in smooth muscle cells isolated from mice in which the gene for PKG I had been disrupted. As with the human cells, no phosphorylation was seen in the mouse cells in response to 8-Br-cAMP. These results strongly suggest that a major regulatory pathway for control of PDE5 phosphorylation and activity in intact smooth muscle is via PKG-dependent phosphorylation of PDE5. Finally, experiments with calyculin A and okadaic acid suggest that PP1 phosphatase, the catalytic subunit of myosin phosphatase, can regulate PDE5 dephosphorylation. Together, the data suggest that phosphorylation and activation of PDE5 by PKG I and its subsequent dephosphorylation by myosin phosphatase may be key steps in the regulation of relaxation/contraction cycles of smooth muscle.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2004

Modulation of cyclic AMP production by signal transduction pathways in preglomerular microvessels and microvascular smooth muscle cells.

Cyclic AMP affects microvascular smooth muscle contraction and growth. Therefore, it is important to elucidate mechanisms regulating cyclic AMP production in microvascular smooth muscle. In this study, we determined whether several signal transduction pathways regulate receptor-induced cyclic AMP in isolated preglomerular microvessels and microvascular smooth muscle cells. Preglomerular microvessels were incubated with isoproterenol (beta-adrenoceptor agonist) and with and without U73122 (phospholipase C inhibitor), GF109203X (protein kinase C inhibitor), 1-butanol (phospholipase D inhibitor), CGP77675 (c-src inhibitor), HA1077 (Rho kinase inhibitor), Y27632 (Rho kinase inhibitor), LY294002 (phosphatidylinositol-3-kinase inhibitor), dipenyleneiodonium (NADPH oxidase inhibitor), or Tempol (superoxide dismutase mimetic). Cultured preglomerular microvascular smooth muscle cells were incubated with isoproterenol or forskolin (direct activator of adenylyl cyclase) and with or without U73122, C(2)-ceramide (phospholipase D inhibitor), or PP1 [src family inhibitor, 1-(1,1-dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine]. All studies were conducted with 3-isobutyl-1-methylxanthine (broad-spectrum phosphodiesterase inhibitor) to eliminate changes in cyclic AMP degradation. In microvessels isoproterenol-induced cyclic AMP was not affected by Y27632, HA1007, LY294002, dipenylene-iodonium, or Tempol; was increased by U73122 and GF109203X; and was decreased by 1-butanol and CGP77675. In cells, U73122 increased and C(2)-ceramide and PP1 decreased isoproterenol-induced cyclic AMP. Forskolin-induced cyclic AMP was not altered. These results indicate that receptor-mediated activation of adenylyl cyclase is 1) not modulated by Rho kinase, phosphatidylinositol-3-kinase, NADPH oxidase, or superoxide; 2) is attenuated by phospholipase C and protein kinase C; and 3) is augmented by phospholipase D and src. Phospholipase C, phospholipase D, and src modulate receptor-induced cyclic AMP by affecting beta-adrenoreceptor/G protein/adenylyl cyclase coupling rather than by directly affecting adenylyl cyclase activity.

Adenylyl Cyclases↗
PubMed1991

[In vitro balloon treatment of coronary plaque material of the human: effect of dilation time on proliferation of smooth muscle cells].

The proliferation of smooth muscle cells (SMC) seems to be an important factor in the development of restenosing events after angioplasty. Primary stenosing plaque material was obtained from human coronary arteries by thrombendarterectomy. Specimens were cut in small pieces and dilated in a flexible tube with a balloon-catheter (Medtronic 18K2030E) for 1 min., 5 min. and 10 min. at 3 atm. For the isolation of plaque cells the expant technique was used. The majority of plaque cells was identified as smooth muscle cells by positive reaction with monoclonal antibodies against smooth muscle alpha-actin. For the cultivation of SMC a mixture of Waymouth's MB 752/1 and Ham F-12 (1:1) was used. After 8 weeks cell number was analysed by a cell counter (Casy I, Schärfe System). There was a clear increase of SMC-proliferation in dependence to the duration of angioplasty (1 min. = 100%, 5 min. = 112%, 10 min. = 153%). If these data are confirmed by clinical and experimental studies, the duration of angioplasty has to be considered as a risc factor for the development of restenosis.

Angioplasty, Balloon, Coronary↗
PubMed1992

Effect of conditioning of beta-migrating very low-density lipoprotein with macrophages on the accumulation of cholesteryl esters in smooth muscle cells.

The mechanism of cholesteryl ester accumulation in smooth muscle cells was investigated. Incubation of smooth muscle cells with beta-migrating very low-density lipoprotein (beta-VLDL, d less than 1.006) for 24 h did not result in accumulation of oil red O-stained particles in the cells. However, incubation of smooth muscle cells with beta-VLDL in the presence of rat peritoneal macrophages induced accumulation of oil red O-stained granules in smooth muscle cells. Medium containing [3H]-cholesteryl linoleate-labelled beta-VLDL ([3H]beta-VLDL) that was conditioned with rat peritoneal macrophages increased the incorporation of [3H]-cholesterol and the cholesteryl ester content in smooth muscle cells, whereas unconditioned [3H]beta-VLDL did not. On zonal ultracentrifugation of conditioned medium containing [3H]beta-VLDL with macrophages, radioactivity was found at two peaks of density 1.150 and less than 1.006. This new fraction with d = 1.150 (peak II) migrated at beta-positions, the same as that of low-density lipoprotein (LDL) on agarose gel electrophoresis, and contained cholesteryl esters (28%), free cholesterol (15%) and phospholipid (43%). When smooth muscle cells were incubated with the peak II fraction, the radioactivity incorporated into smooth muscle cells and the cholesterol ester content of the cells increased. These results show that aortic smooth muscle cells in cooperation with macrophages can accumulate cholesteryl ester from beta-VLDL.

Animals↗
PubMed1987

Catecholamine-induced polyploidization in vascular smooth muscle cells.

Polyploidy formation of vascular smooth muscle cells was observed in vivo by intravenous infusion of norepinephrine or epinephrine in Wistar-Crea rats for 12 days at the rate of 0.02 mumol/hr with an osmotic minipump and in vitro by exposing cultured vascular smooth muscle cells obtained from normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) to norepinephrine (10(-7)M to 10(-5)M) in the cultured medium. Infusion in vivo of norepinephrine but not epinephrine increased blood pressure slightly. However, both agents increased significantly the frequency of tetraploidy of smooth muscle cells up to 14.9 +/- 0.6% (mean +/- SE) and 9.2 +/- 0.5% compared with saline (6.5 +/- 0.7%). Exposure in vitro to norepinephrine (10(-6)M) increased the frequency of tetraploidy and octaploidy of smooth muscle cells from SHR up to 14.2% and 2.1%, respectively, compared with nonexposed cells (8.1% and 0.2%). These results appear to indicate that norepinephrine and epinephrine induce polyploidy of smooth muscle cells independently from blood pressure elevation.

Animals↗
PubMed1988

Effect of prostaglandin E2 on rabbit colonic smooth muscle cell contraction.

Isolated circular smooth muscle cells from the rabbit distal colon were used to study the effect of prostaglandin E2 (PGE2) on bethanechol-stimulated smooth muscle cell contraction. Contraction was expressed as the percentage decrease in mean cell length compared with unstimulated cells. Incubation with different concentrations of PGE2 (10(-10)-10(-6) M) did not cause contraction or relaxation of unstimulated smooth muscle cells. Bethanechol stimulated a dose-dependent contraction that was maximal at 30 s. The threshold for bethanechol-stimulated contraction was 10(-11) M; the ED50 was 10(-10) M; and the maximum contraction (23.0 +/- 1.8%) occurred at 10(-8) M. Preincubation with PGE2 reduced both the efficacy and potency of bethanechol-stimulated contraction. Preincubation with 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or dibutyryl-cAMP reduced the efficacy of bethanechol-stimulated contraction without affecting potency. Increasing concentrations of PGE2 stimulated a dose-dependent increase in the production of intracellular cAMP (P less than 0.05). These studies show that PGE2 inhibits bethanechol-stimulated contraction of isolated colonic circular myocytes and is associated with increased production of intracellular cAMP. There is also a cAMP-independent effect of PGE2 on the potency of bethanechol stimulation.

Animals↗
PubMed1999

A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells.

Coronary artery smooth muscle (SM) cells originate from proepicardial cells that migrate over the surface of the heart, undergo epithelial to mesenchymal transformation and invade the subepicardial and cardiac matrix. Prior to contact with the heart, proepicardial cells exhibit no expression of smooth muscle markers including SMalphaactin, SM22alpha, calponin, SMgammaactin or SM-myosin heavy chain detectable by RT-PCR or by immunostaining. To identify factors required for coronary smooth muscle differentiation, we excised proepicardial cells from Hamburger-Hamilton stage-17 quail embryos and examined them ex vivo. Proepicardial cells initially formed an epithelial colony that was uniformly positive for cytokeratin, an epicardial marker. Transcripts for flk-1, Nkx 2.5, GATA4 or smooth muscle markers were undetectable, indicating an absence of endothelial, myocardial or preformed smooth muscle cells. By 24 hours, cytokeratin-positive cells became SMalphaactin-positive. Moreover, serum response factor, undetectable in freshly isolated proepicardial cells, became strongly expressed in virtually all epicardial cells. By 72 hours, a subset of epicardial cells exhibited a rearrangement of cytoskeletal actin, focal adhesion formation and acquisition of a motile phenotype. Coordinately with mesenchymal transformation, calponin, SM22alpha and SMgammaactin became expressed. By 5-10 days, SM-myosin heavy chain mRNA was found, by which time nearly all cells had become mesenchymal. RT-PCR showed that large increases in serum response factor expression coincide with smooth muscle differentiation in vitro. Two different dominant-negative serum response factor constructs prevented the appearance of calponin-, SM22alpha- and SMgammaactin-positive cells. By contrast, dominant-negative serum response factor did not block mesenchymal transformation nor significantly reduce the number of cytokeratin-positive cells. These results indicate that the stepwise differentiation of coronary smooth muscle cells from proepicardial cells requires transcriptionally active serum response factor.

Animals↗
PubMed1989

Epidermal growth factor responsiveness in smooth muscle cells from hypertensive and normotensive rats.

Aortic smooth muscle cells from spontaneously hypertensive rats (SHR) exhibit inappropriate proliferation characteristics in culture that suggest a modified response to serum mitogens or growth factors. The present study compares vascular smooth muscle cells from SHR and normotensive Wistar-Kyoto (WKY) rats with respect to their proliferative and functional response to growth factors. Specific attention was focused on the interaction of these vascular smooth muscle cells with epidermal growth factor. An increased growth rate of vascular smooth muscle cells from SHR (vs. WKY rats) was observed when cells were cultured in the presence of serum (10% and 0.5%), but not under serum-free conditions. The additional presence of low serum concentrations (0.5%) was required for epidermal growth factor to elicit a proliferative response, whereupon smooth muscle cells from SHR displayed an increased (vs. WKY rats) growth rate. Saturation binding of [125I]epidermal growth factor to intact smooth muscle cells indicated a twofold increase in receptor density in SHR-derived cells (p less than 0.001 vs. WKY rats) without an alteration in affinity for the growth factor. Cells derived from SHR also exhibited greater functional responsiveness to epidermal growth factor when compared with smooth muscle cells from WKY rats as evidenced by amplifications of both S6 kinase activation, phosphoinositide catabolism, elevation of intracellular pH, and DNA synthesis (nuclear labeling). We conclude that increased responsiveness of SHR-derived smooth muscle cells to epidermal growth factor could contribute to alterations in vascular smooth muscle growth and tone that may be fundamental to the pathogenesis of hypertension and atherosclerosis.

Animals↗
PubMed1985

The effects of a calcium dependent protease on the ultrastructure and contractile mechanics of skinned uterine smooth muscle.

In situ substrates for a vascular smooth muscle calcium-dependent protease (CDP) were investigated using a chemically skinned uterine smooth muscle preparation. Treatment of skinned smooth muscles with CDP had no effect on the total content of actin and myosin. Electron microscopical observations demonstrated that membrane plaques, cytoplasmic dense bodies, and intermediate filaments were all degraded by CDP. In addition, CDP reduced both isometric force and isotonic shortening velocity of contracted muscles in a concentration and time-dependent manner. Treatment of contracting muscles with CDP resulted in a condensation of myofilaments away from the plasma membrane concurrent with the loss of contractility. The condensation of myofilaments was ATP-dependent and could be inhibited by removal of ATP prior to proteolysis. The effects of proteolysis on smooth muscle ultrastructure and contractility support previously proposed models which assign a role to cytoskeletal elements in coordinating the molecular interaction of actomyosin to produce muscle contraction. The loss of cytoskeletal structures following protease treatment suggests that one of the functions of CDP in smooth muscle may be the disassembly of the cell cytoskeleton.

Actins↗
PubMed1994

[Myocyte transformation in autologous transplantation of colonic smooth muscle in domestic pigs].

Autologous gut smooth muscle graft of two piglets was used for creating a ring in sigmoidostomy. The graft was investigated several times by histology and electron microscopy. A sort of myofibroblasts were observed the 7th day in the smooth muscle. Three months later the smooth muscle was completely transformed into a cellular connective tissue comprising myofibroblasts and fibroblasts. The experiment points to possible relation of smooth muscle cell, myofibroblast and fibroblast.

Animals↗
PubMed1991

Nerve growth factor synthesis in vascular smooth muscle.

Details of the interdependent, trophic relation between smooth muscle and its neural innervation are not well known despite suggestions that neural influences may contribute significantly to hypertensive and other cardiovascular disease. Vascular smooth muscle is a major target of innervation by neurons of the sympathetic nervous system. Sympathetic neurons depend on a constant supply of the potent neurotrophic peptide nerve growth factor. Nerve growth factor regulates an impressive list of neuronal and perhaps muscle properties, yet its source in vessels and the determinants of its synthesis are not known. We have taken advantage of the cytoarchitecture of the aorta to demonstrate that vascular smooth muscle cells synthesize nerve growth factor. The survival of cultured sympathetic neurons is supported in a nerve growth factor-dependent manner by co-culture with pure rat aortic vascular smooth muscle cells. Furthermore, pure smooth muscle cell cultures contain nerve growth factor-specific messenger RNA. Levels of messenger nucleic acid coding for nerve growth factor in smooth muscle are regulated by contractile agonists (angiotensin II, arginine vasopressin) and the adrenergic agonist phenylephrine. This suggests a link between muscle activity and growth factor production. Secretion of nerve growth factor protein by vascular smooth muscle was measured using a sensitive two-site immunoassay. Secretion is highest during muscle growth. Secretion is elevated by angiotensin II and arginine vasopressin but slightly inhibited by phenylephrine. These results suggest that cultured vascular smooth muscle can serve as a useful model in which to study the cellular regulation of trophic factor synthesis in health and disease.

Angiotensin II↗
PubMed1988

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells.

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.

Animals↗
PubMed1993

Up-regulation of the levels of androgen receptor and its mRNA by androgens in smooth-muscle cells from rat penis.

Smooth-muscle cells cultured from the penis of sexually immature (I-PSMC) and adult (A-PSMC) rats express similar high levels of the androgen receptor (AR) mRNA. This contrasts with the marked in vivo decline of both AR mRNA and androgen binding in the penile smooth muscle of adult rats, which appears to be responsible for the cessation of androgen-dependent penile growth upon sexual maturation. PSMC is therefore a good model to study putative down-regulators of AR expression as a function of cell proliferation in the smooth muscle of androgen-responsive vascular tissue. In order to determine whether AR protein levels in PSMC correlate with AR mRNA levels, the immunocytochemical detection of ARs and their androgen binding capacity were compared between I- and A-PSMC. The number of ARs and their protein half-lives suggested similar levels of translation of the AR mRNA in both cell lines. The effect of the synthetic analog methyltrienolone (R-1881) on androgen binding was studied in contact-inhibited androgen-deprived PSMC. In contrast to the postulated role of androgens as down-regulators of AR expression in rat penis, ARs were up-regulated in A-PSMC by R-1881. Contact inhibition of A-PSMC combined with serum depletion and androgen deprivation down-regulated AR mRNA levels, and dihydrotestosterone (DHT) counteracted this effect. These results suggest that the loss in A-PSMC of the age-dependent down-regulation of ARs observed in vivo in adult corpora cavernosa smooth muscle is related to the in vitro resumption of cell proliferation and that DHT acts directly on the penile smooth muscle as a positive modulator of AR levels.

Actin Cytoskeleton↗
PubMed1996

Cytoskeleton-dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells.

1. Vascular endothelial and smooth muscle cells generate nitric oxide (NO) via different nitric oxide synthase (NOS) isozymes. Activation of the endothelial constitutive NOS (ecNOS) contributes to the maintenance of cardiovascular homeostasis, whereas expression of the endotoxin- and cytokine-inducible pathway (iNOS) within the vascular smooth muscle is thought to be responsible for the cardiovascular collapse which occurs during septic shock and antitumour therapy with cytokines. Since the cytoskeleton is involved in the activation of certain genes and in some effects of endotoxin in macrophages, we investigated the role of microtubules and microfilaments in the activation of the NO pathway in cultured vascular cells. 2. Depolymerization of microtubules by either nocodazole or colchicine prevented lipopolysaccharide (LPS)- and interleukin-1 beta-induction of NO-dependent cyclic GMP accumulation. Steady state levels of iNOS mRNA, assessed by Northern blot and RT-PCR, and iNOS protein, assessed by Western blotting, were also decreased by either colchicine or nocodazole treatment. 3. Taxol enhanced microtubule polymerization alone, and prevented microtubule depolymerization elicited by nocodazole and colchicine. Associated with its effect on microtubule assembly, taxol prevented the inhibitory effects of nocodazole and colchicine on cyclic GMP accumulation and iNOS mRNA levels. 4. Disruption of microfilaments by cytochalasins had no inhibitory effect on the activation of the inducible NO pathway. 5. In contrast to cytokine-stimulated smooth muscle cells, modulation of either microtubule or microfilament assembly did not affect the constitutive NO pathway in endothelial cells, as endothelial cell- and NO-dependent cyclic GMP accumulation in endothelial-smooth muscle co-cultures remained unchanged. 6. Our findings demonstrate that microtubules play a prominent role in the activation of the inducible NO pathway in response to inflammatory mediators in smooth muscle cells but not of the constitutive synthesis of NO in endothelial cells.

Animals↗