Inhibition Of Vascular Smooth Muscle Research Articles

DiGeorge Syndrome, Tbx1, and Retinoic Acid Signaling Come Full Circle

See related article, pages 686-694 Among the most well-characterized cardiac congenital malformations are those associated with DiGeorge syndrome (DGS)/velocardiofacial syndrome. DGS is mainly caused by heterozygous deletion of a region of chromosome 22q11.2 and is characterized by cardiac conotruncal malformations, aortic arch anomalies, dysmorphic face and hypoplasia of the thymus and parathyroid.1 This spectrum of anomalies is attributed to defects in neural crest-derived structures and associated tissues. The T-box-containing transcription factor TBX1 has been identified as responsible for cardiovascular, thymic, and parathyroid phenotypes of DGS.2 Alterations in retinoic acid (RA) metabolism are similarly associated with cardiac conotruncal, aortic arch, and pharyngeal structural abnormalities similar to DGS.3 In this issue of Circulation Research , Ryckebusch et al4 establish a new functional link between Tbx1 and RA signaling in the regulation of aortic arch anomalies in a mouse model of DGS. A characteristic feature of DGS is malformation of the pharyngeal arch arteries (PAAs), particularly of the fourth PAA, which leads to interrupted aortic arch type B (IAA-B). Mice heterozygous for Tbx1 exhibit defects in the development in the fourth PAA related to aortic arch anomalies including IAA.5,6 Ryckebusch et al define molecular and cellular mechanisms underlying aortic arch anomalies in Tbx1 +/− mice.4 The fourth PAA in embryonic day 10.5 embryos is derived from neural crest cells and is absent or hypomorphic in Tbx1 +/− embryos. This PAA anomaly is accompanied by neural crest migration defects as well as inhibition of vascular smooth muscle (VSM) differentiation. Both neural crest migration and VSM differentiation related to PAA defects are controlled by a precise balance of RA signaling …

Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances alpha1D-adrenergic receptor constriction.

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.

Heteromultimeric Kv1 Channels Contribute to Myogenic Control of Arterial Diameter

Inhibition of vascular smooth muscle (VSM) delayed rectifier K+ channels (K(DR)) by 4-aminopyridine (4-AP; 200 micromol/L) or correolide (1 micromol/L), a selective inhibitor of Kv1 channels, enhanced myogenic contraction of rat mesenteric arteries (RMAs) in response to increases in intraluminal pressure. The molecular identity of K(DR) of RMA myocytes was characterized using RT-PCR, real-time PCR, and immunocytochemistry. Transcripts encoding the pore-forming Kvalpha subunits, Kv1.2, Kv1.4, Kv1.5, and Kv1.6, were identified and confirmed at the protein level with subunit-specific antibodies. Kvbeta transcript (beta1.1, beta1.2, beta1.3, and beta2.1) expression was also identified. Kv1.5 message was approximately 2-fold more abundant than that for Kv1.2 and Kv1.6. Transcripts encoding these three Kv1alpha subunits were approximately 2-fold more abundant in 1st/2nd order conduit compared with 4th order resistance RMAs, and Kvbeta1 was 8-fold higher than Kvbeta2 message. RMA K(DR) activated positive to -50 mV, exhibited incomplete inactivation, and were inhibited by 4-AP and correolide. However, neither alpha-dendrotoxin or kappa-dendrotoxin affected RMA K(DR), implicating the presence of Kv1.5 in all channels and the absence of Kv1.1, respectively. Currents mediated by channels because of coexpression of Kv1.2, Kv1.5, Kv1.6, and Kvbeta1.2 in human embryonic kidney 293 cells had biophysical and pharmacological properties similar to those of RMA K(DR). It is concluded that K(DR) channels composed of heteromultimers of Kv1 subunits play a critical role in myogenic control of arterial diameter.

Loss of function of vascular smooth muscle cells by nitric oxide‐dependent and ‐independent interactions with tumorigenic cells

Little is known about the interaction of tumor cells with host vascular smooth muscle cells. In reconstitution experiments, tumorigenic cell lines (including the rat hepatocarcinoma Morris 7777 and human melanoma M-21) were cultured for 17 hr in the presence of rat aortic rings, subsequently evaluated in contractility assays (response to phenylephrine and KCl). An agonist-independent loss of contractility was observed in rings pre-incubated with either tumorigenic cell lines or their conditioned medium (CM). The depressing effect of Morris cells depends largely on the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and was reversed by an inhibitor of this enzyme; iNOS immunoreactivity was verified in some muscular vessels at the periphery of tumors formed by the Morris cell line in rats. The M-21 melanoma produces cytotoxicity in rat aortic rings (presence of single stranded DNA, cleavage of PARP, in differentiated smooth muscle only), accounting for the irreversible loss of contractility. The cytotoxicity produced by M-21 CM is not dependent on NO. Gel filtration of CM suggests that both the iNOS- and cytotoxicity-inducing substances from Morris hepatoma cells or M-21 cells, respectively, are mainly of low molecular weight (1 kDa or less). Other cell lines derived from rat or human tumors produce minimal effects on the rat aorta smooth muscle (H4-II-E-C3) or an irreversible anergy (RBL, MDA-MB-231, HEP-3B, HEP G2). The results emphasize that inhibition of vascular smooth muscle is relevant to tumor biology both by modulation of tumoral hemodynamics and by influencing the state of vessel maturation.

Smooth muscle membrane potential modulates endothelium-dependent relaxation of rat basilar artery via myo-endothelial gap junctions.

The release of endothelium-derived relaxing factors, such as nitric oxide (NO), is dependent on an increase in intracellular calcium levels ([Ca(2+)](i)) within endothelial cells. Endothelial cell membrane potential plays a critical role in the regulation of [Ca(2+)](i) in that calcium influx from the extracellular space is dependent on membrane hyperpolarization. In this study, the effect of inhibition of vascular smooth muscle delayed rectifier K(+) (K(DR)) channels by 4-aminopyridine (4-AP) on endothelium-dependent relaxation of rat basilar artery to acetylcholine (ACh) was assessed. ACh-evoked endothelium-dependent relaxations were inhibited by N-(Omega)-nitro-L-arginine (L-NNA) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), confirming a role for NO and guanylyl cyclase. 4-AP (300 microM) also suppressed ACh-induced relaxation, with the maximal response reduced from approximately 92 to approximately 33 % (n = 11; P < 0.01). However, relaxations in response to exogenous NO, applied in the form of authentic NO, sodium nitroprusside or diethylamineNONOate (DEANONOate), were not affected by 4-AP treatment (n = 3-11). These data are not consistent with the view that 4-AP-sensitive K(DR) channels are mediators of vascular hyperpolarization and relaxation in response to endothelium-derived NO. Inhibition of ACh-evoked relaxation by 4-AP was reversed by pinacidil (0.5-1 microM; n = 5) or 18beta-glycyrrhetinic acid (18betaGA; 5 microM; n = 5), indicating that depolarization and electrical coupling of the smooth muscle to the endothelium were involved. 4-AP caused depolarization of both endothelial and vascular smooth muscle cells of isolated segments of basilar artery (mean change 11 +/- 1 and 9 +/- 2 mV, respectively; n = 15). Significantly, 18betaGA almost completely prevented the depolarization of endothelial cells (n = 6), but not smooth muscle cells (n = 6) by 4-AP. ACh-induced hyperpolarization of endothelium and smooth muscle cells was also reduced by 4-AP, but this inhibition was not observed in the combined presence of 4-AP and 18betaGA. These data indicate that 4-AP can induce an indirect inhibition of endothelium-dependent relaxation in the rat basilar artery by electrical coupling of smooth muscle membrane depolarization to the endothelium via myo-endothelial gap junctions.

Inhibition of vascular smooth muscle and endothelial cell migration by apolipoprotein J

Sivamurthy, Nayan MD; Rohan, Darren I MD; Stone, David H MD; Quist, William C MD, PhD; LoGerfo, Frank W MD Author Information

Gbetagamma-Mediated Signaling: New Therapeutic Target for Proliferative Vascular Disease

Summary Proliferation of vascular smooth muscle (VSM) severely affects theoutcomeofcoronaryarterybypassandangioplastyprocedures, causing the failure of venous grafts or restenosis of the reopened vessel. Investigation intothemechanisms underlying theprocess of VSMcellularproliferationhasprovidedevidencethatintracellular signaling mechanisms triggered by extracellular hormonal factors acting through G protein‐coupled receptors, can mediate and sustain this pathological process. Inhibition of common pathways of G protein‐coupled receptor signaling has recently proven effective in preventing VSM cellular activation and proliferation. In particular, inhibition of the G¯°-mediated mitogen-activated protein (MAP) kinase signaling pathway results in the inhibition of VSM proliferationinvitro.Moreover,use ofadenoviralvectors todeliver a peptide inhibitor of G¯° signaling in vivo has resulted in inhibition of intimal hyperplasia in experimental models of vein-graft failure and restenosis. IUBMBLife, 48: 257‐261, 1999

NOS gene transfer inhibits expression of cell cycle regulatory molecules in vascular smooth muscle cells.

The mechanisms of nitric oxide (NO)-mediated inhibition of vascular smooth muscle (VSM) cell proliferation are still obscure. Cyclins A and E in association with cyclin-dependent kinase 2 (cdk2) serve as positive regulators for mammalian cell cycle progression through the G1/S checkpoint of the cell cycle and subsequent cell proliferation. Therefore, we have tested the effect of adenovirus-mediated transfection of the endothelial nitric oxide synthase (eNOS) gene into guinea pig coronary VSM cells on platelet-derived growth factor (BB homodimer) (PDGF-BB)-stimulated cell proliferation and the expression of cell cycle regulatory molecules. Transfection of the eNOS gene (eNOS) into VSM cells significantly inhibited (P < 0.05) [3H]thymidine incorporation into the DNA in response to PDGF-BB stimulation compared with lacZ-transfected control cells. The eNOS transfer significantly inhibited (P < 0.05) PDGF-BB-induced proliferating cell nuclear antigen (PCNA) and cyclin A expression in VSM cells compared with cells transfected with the control vector. The time course of cyclin E expression in response to PDGF-BB stimulation was delayed in eNOS-transfected cells. Levels of cyclin-dependent kinase inhibitors p21 and p27 were not significantly affected by eNOS transfer. eNOS transfer did not decrease PDGF-beta receptor number, affinity, and autophosphorylation measured by radioreceptor assay and Western analysis. These results suggest that inhibition of PDGF-stimulated expression of cyclin A, cyclin E, and PCNA is the target of NO action. These findings could explain, at least in part, NO-mediated inhibition of VSM cell proliferation.

Inhibition of mitosis and microtubule function through direct tubulin binding by a novel antiproliferative naphthopyran LY290181.

The mechanism of action of a novel antiproliferative compound LY290181 [2-amino-4-(3-pyridyl)-4H-naphtho(1,2-b)pyran-3-carbonitrile] was characterized. LY290181 is a potent inhibitor of cell proliferation, producing 50% inhibition of vascular smooth muscle, endothelial, Chinese hamster ovary, HeLa, and human erythroleukemia cells at concentrations of 8-40 nM. Cell cycle analysis showed that LY290181 caused accumulation of smooth muscle cells at the G2/M phase and induced mitotic arrest in Chinese hamster ovary cells and HeLa cells. At low concentrations (3-30 nM), LY290181 blocked transition of cells from metaphase to anaphase and disrupted mitotic spindle organization. At high concentrations (>/=100 nM), LY290181 produced a concentration-dependent loss of cytoplasmic and spindle microtubules. LY290181 inhibited the polymerization of purified bovine brain microtubule protein into microtubules, and it depolymerized preformed microtubules. Using tubulin-1-anilino-8-naphthalene sulfonate complex fluorescence, we have shown that LY290181 directly interacted with tubulin in a unique manner. These studies show that LY290181 induces cell growth arrest in prometaphase/metaphase, and tubulin appears to be its molecular target.

Characterization of endothelium-dependent relaxations resistant to nitro-L-arginine in the porcine coronary artery.

1. Previous studies, demonstrated that endothelium-dependent relaxations which are resistant to nitro-L-arginine (an inhibitor of nitric oxide synthase) are accompanied by membrane hyperpolarization in the porcine coronary artery. The present experiments were designed to characterize further this type of endothelium-dependent relaxation in response to bradykinin by measuring isometric force in isolated rings of that artery. The experiments were performed in the presence of indomethacin to rule out vasoactive prostanoids. 2. Bradykinin induced comparable endothelium-dependent relaxations of proximal and distal rings of porcine coronary arteries contracted with prostaglandin F2 alpha in the presence of nitro-L-arginine. 3. Bradykinin and SIN 1 (a donor of nitric oxide) reduced contractions induced by prostaglandin F2 alpha in an additive fashion in the presence of nitro-L-arginine. 4. Bradykinin (in the presence of nitro-L-arginine) relaxed the tissues contracted with tetraethylammonium, prostaglandin F2 alpha, phorbol 12, 13-diacetate or endothelin, with similar pD2 values. 5. The time course of the relaxations induced by bradykinin (in the presence of nitro-L-arginine) and UK14304 (an alpha 2-adrenoceptor agonist, in the absence of the inhibitor of nitric oxide synthase) were comparable. 6. These results suggest that, in the porcine coronary artery, nitro-L-arginine-resistant relaxations (a) are distributed similarly in the proximal and distal parts of the artery, (b) contribute to inhibition of vascular smooth muscle with nitric oxide in an additive fashion, (c) occur during contractions induced by various contractile agents and (d) do not precede those mediated by nitric oxide.

Effects of Lead and a Low-Molecular-Weight Endogenous Plasma Inhibitor on the Kinetics of Sodium—Potassium-Activated Adenosine Triphosphatase and Potassium-Activated p-Nitrophenylphosphatase

1. Lead, ouabain and an endogenous plasma inhibitor were all found to be potent inhibitors of purified hog cerebral cortex sodium-potassium-activated adenosine triphosphatase and potassium-stimulated p-nitrophenyl-phosphatase. 2. The kinetic characteristics of inhibition of both enzymes by lead and the endogenous plasma inhibitor differed in several respects. For sodium-potassium-activated adenosine triphosphatase, lead and the endogenous plasma inhibitor were non-competitive inhibitors with respect to potassium; lead was competitive with respect to sodium, whereas the endogenous plasma inhibitor had no effect; lead was competitive with respect to magnesium adenosine triphosphate, whereas the endogenous plasma inhibitor was uncompetitive. For potassium-activated p-nitrophenylphosphatase, both lead and the endogenous plasma inhibitor were competitive with respect to potassium; lead showed a mixed type of inhibition with respect to p-nitrophenylphosphate, whereas the endogenous plasma inhibitor was non-competitive. 3. Lead and the endogenous plasma inhibitor exhibited synergistic inhibitory activity on sodium-potassium-activated adenosine triphosphatase. 4. These results suggest that lead could play a contributory role in the pathogenesis of essential hypertension via an additive inhibition of vascular smooth muscle sodium-potassium-activated adenosine triphosphatase.

Effects of lead and natriuretic hormone on kinetics of sodium-potassium-activated adenosine triphosphatase: possible relevance to hypertension.

Inhibition of vascular smooth muscle sodium-potassium-activated adenosine triphosphatase (Na-K-ATPase) has been postulated as a central mechanism in enhancing vascular contractility. In the present study, kinetics of inhibition of Na-K-ATPase by lead, ouabain, and natriuretic hormone (NH) was studied in a purified hog cerebral cortex enzyme preparation. Determination of I50 values for lead, ouabain, and NH revealed that NH is the most potent inhibitor of the enzyme system (0.8 x 10(-6) M ouabain equivalents). Kinetic analyses indicated that lead and NH exhibited different inhibitory mechanisms. The inhibition by lead was noncompetitive with respect to potassium and competitive with respect to sodium and MgATP. Natriuretic hormone was noncompetitive with respect to potassium, uncompetitive with respect to MgATP, and exhibited no inhibitory effect with respect to sodium. Synergism between lead and NH in the inhibition of Na-K-ATPase raises the possibility that lead may be a contributory factor in hypertension via this mechanism.

Serotonin and the vascular wall

Serotonin possesses both vasoconstrictor and vasodilator properties. The constrictor action of the monoamine can be due to: (a) direct activation of vascular smooth muscle; in most blood vessels, this is mediated by S 2-serotonergic receptors; (b) augmentation (amplification) of the action of other endogenous vasoconstrictors such as catecholamines, angiotensin II and the prostanoids; and (c) release of norepinephrine from adrenergic nerves. The dilator action of serotonin can be due to: (a) activation of endothelial cells which release endothelium-derived relaxing factor(s); this response appears to be mediated by S 1-serotonergic receptors; (b) direct inhibition of vascular smooth muscle; (c) inhibition of adrenergic neurotransmission by an action on S 1-serotonergic prejunctional receptors; and (d) release of other endogenous mediators. The net effect of serotonin on the blood vessel wall depends on: (a) the integrity of the endothelium; (b) the degree of activation of the vascular smooth muscle; (c) the level of sympathetic tone; and (d) local (e.g. P O 2 , temperature) and chronic (e.g. blood pressure) modulating factors. S 2-serotonergic antagonists prevent the constrictor action of serotonin, and often unmask its dilator potential.

VIP inhibition of vascular smooth muscle: complementary to beta 2-adrenoceptor mediated relaxation in the isolated rat portal vein.

Exogenous VIP caused a concentration dependent inhibition of the spontaneous mechanical activity in the isolated rat mesenteric-portal vein preparation via a mechanism which was completely independent of the propranolol-blocked beta-adrenoceptor, of high K+ in the medium and of exogenous bovine pancreatic polypeptide, neurotensin and opioids. The potency of VIP (pD2 = 7.52 +/- 0.18, n = 6) was about 30 times higher than that of isoprenaline in the atropine and phentolamine-blocked preparation. The isoprenaline inhibition was mediated via a beta 2-type of adrenoceptor with low apparent affinity for noradrenaline (intrinsic activity (alpha) = 0.27 +/- 0.01, n = 8). Opposite effects of exogenous VIP and noradrenaline were on the other hand observed in the atropinized and beta-blocked preparation. These results suggest that in the rat portal vein neuronal VIP and circulating adrenaline may be complementary in their antagonism of the alpha-adrenoceptor mediated increase in contractility.

Pharmacological studies of snake ( Bitis arietans) venom

Venom from Bitis arietans was fractionated on Sephadex G-100 into five main fractions, of which only fraction 1 was lethal to mice. The crude venom and fractions 1 and 2 produced a gradual and precipitous fall in the blood pressure of the cat. This fall was not antagonized by pretreatment with hexamethonium, cyproheptadine, propranolol, or atropine. Also, it was not blocked by indomethacin or the kinin inhibitor, trasylol. Both the crude venom and fraction 1 produced a depression of the isolated heart which was not prevented by atropine or hexamethonium. Similarly, the crude venom and fractions 1 and 2 increased the capillary permeability in rabbits. The crude venom and fractions 1 and 3 produced an inhibition of a variety of smooth muscle preparations such as the rabbit intestine, guinea-pig ileum and the rat uterus. However, the spontaneously contracting rat uterus was stimulated by the crude venom and fraction 1. The venom produced hyperglycaemia in the rabbit and a decrease in the potassium level of the cat blood. It is suggested that the circulatory shock produced by the venom is due to a direct inhibition of vascular smooth muscle, together with a direct depression of the myocardium and an increased capillary permeability.

The effect of guancydine on blood pressure, vascular reactivity, and norepinephrine uptake in essential hypertension.

Guancydine was administered to 10 patients for a period of 3 weeks to 6 months. The drug produced a decrease in both supine and standing blood pressures as well as in digital vascular reactivity but caused no change in tritiated norepinephrine uptake. Its action appears to be direct inhibition of vascular smooth muscle. Side effects were minimal and transitory.