Vascular Smooth Muscle Rings Research Articles

Transduction of recombinant heat shock protein 27 increases contractility of vascular smooth muscle

Heat shock protein 27 (HSP27) is a stress induced cytoprotective protein, which has been shown be involved in contraction, stress fiber formation, and reorganization of actin cytoskeleton. In this study, the role of HSP27 on contractility in rat aortic vascular smooth muscle (VSM) tissue was examined by using a transducible recombinant HSP27 (rPTDHSP27). Human HSP27 cDNA, that was fused with a protein transduction domain and 6x Histidine tag at the N terminal end, was expressed in Escherichia coli and purified using a metal chelating affinity column. The purified protein was verified through Western blot and HPLC ESI MS/MS. VSM rings were equilibrated in a muscle bath, and contracted with norepinephrine (NE) (1–1000 nM) with or without 3.8 μM rPTDHSP27 and force generated was measured. Norepinephrine induced a dose dependent contraction, which was enhanced with the pretreatment of rPTDHSP27 (84.1% compared to 16.9% in the control with 16 nM NE). The concentration needed to produce maximal contraction was 10 fold lower when the tissue was pretreated with rPTDHSP27 (88% at 666 nM compared to 96% at 66 nM, for control and rPTDHSP27 treated rings respectively). NIH RO1 70715

Vascular Smooth Muscle Contraction Evoked by Cell Volume Modulation: Role of the Cytoskeleton Network

Previously, we reported that hyposmotic swelling evoked transient vascular smooth muscle cell (SMC) contraction that was completely abolished by L-type Ca²⁺ channel blockers. In contrast, sustained contraction revealed in hyper- and isoosmotically-shrunken SMCs was insensitive to L-type channel blockers and was diminished in Ca²⁺-free medium by only 30-50%. Several research groups reported cell volume-dependent cytoskeleton network rearrangements. This study examines the role of cytoskeleton proteins in cell volume-dependent contraction of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat thoracic aorta. Hyperosmotic shrinkage and hyposmotic swelling were triggered by modulation of medium osmolality; isosmotic shrinkage was induced by VSMR transfer from hypo- to isosmotic medium. The relative content of globular (G) and fibrillar (F) actin was estimated by fluorescence microscopy. Hyperosmotic shrinkage and hyposmotic swelling led to elevation of the F-actin/G-actin ratio by 2.5- and 1.8-fold respectively. Contraction of shrunken and swollen VSMR was insensitive to modulators of microtubules such as vinblastine, colchicine and docetaxel. Microfilament disassembly by cytochalasin B resulted in dramatic attenuation of the maximal amplitude of contraction of hyperosmotically-shrunken and hyposmotically-swollen VSMR, and almost completely abolished the contraction triggered by isosmotic shrinkage. These data suggest that both L-type Ca²⁺ channel-mediated contraction of swollen vascular SMC and Ca²⁺_o-insensitive contractions of shrunken cells are triggered by reorganization of the microfilament network caused by elevation of the F-actin/G-actin ratio.

Important role of Abelson tyrosine kinase (Abl) in regulating vascular smooth muscle contraction

There is emerging evidence that the actin cytoskeleton is involved in diverse array of pathways leading to vascular smooth muscle (VSM) contraction. Among the protein kinases participating in actin rearrangement, Abl is known for its ability to bind directly to F-actin. However, Abl implication in VSM excitation-contraction coupling remains unclear. This study elucidates the role of Abl in the contractions of vascular smooth muscle rings (VSMR) from the rat mesenteric artery. VSMR stimulation with norepinephrine (NE) initiated phosphorylation of Abl at Tyr-245 and Tyr-412 (indication of Abl activation) and the putative Abl downstream molecule p130 Crk-associated substrate (CAS) phosphorylation on Tyr-410. To silence gene expression, Abl shRNA was introduced into VSMR by reversible permeabilization plus liposomes and the rings were incubated for two days allowing Abl to degrade. Immunoblot showed lower Abl expression in shRNA-treated rings. The treatment with shRNA attenuated NE-induced contractions and the increase in F-actin/G-actin ratios. Abl downexpression affected NE-induced phosphorylation of CAS known to regulate active force generation in smooth muscle. Our study shows for the first time that Abl plays an important role in the regulation of VSM contraction. CAS phosphorylation may be a downstream mechanism of the Abl-associated pathway. Supported by NIH HL-75388

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl- cotransport, intracellular Cl- and L-type Ca2+ channels.

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca2+ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca2+ o. Thirty minutes preincubation with bumetanide, a potent Na+, K+, CI- cotransport (NKCC) inhibitor, decreased Cl(-)i content, nifedipine-sensitive 45Ca uptake and contractions triggered by modest depolarization ([K+]o = 36 mM). Elevation of [K+]o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl(-)i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.

ETB-type receptors mediate endothelin-stimulated contraction in the aortic vascular smooth muscle of the spiny dogfish shark, Squalus acanthias.

Using various agonists, and the specific antagonist BQ-123, we have examined the sensitivity to endothelin of the vascular smooth muscle of the ventral aorta of the spiny dogfish shark, Squalus acanthias. Human endothelin-1 produced significant contraction of isolated vascular smooth muscle rings, with an EC50 of 10 nmol.l-1. The presence of an intact endothelium did not alter this response but the magnitude of the contraction was greater in rings with an intact endothelium. The response to 0.2 mumol.l-1 endothelin-1 was equivalent to that of 0.1 mmol.l-1 acetylcholine, and significantly greater than that to 80 mmol.l-1 KCl, suggesting high sensitivity even to the heterologous, mammalian peptide. The Hill plot of the contractile response was a straight line with a slope of 1.12, indicating that a single receptor was mediating the response. Endothelin-1, endothelin-3, and sarafotoxin S6c produced similar concentration-response curves, and the response to endothelin-1 was insensitive to the ETA-specific inhibitor BQ-123. Our data are consistent with the hypothesis that the receptor involved in the contractile response to endothelin of shark aortic vascular smooth muscle is of the ETB-rather than the ETA-type.

Production of [Asn1, Val5] angiotensin II and [Asp1, Val5] angiotensin II in kallikrein-treated trout plasma (T60K).

Incubation of heat-denatured plasma from the rainbow trout Oncorhynchus mykiss with porcine pancreatic kallikrein generates, in addition to bradykinin-related peptides, previously uncharacterized peptides that contract mammalian and amphibian vascular smooth muscle. Using rings of vascular smooth muscle from the bullfrog systemic arch as bioassay, we have isolated two myotropic peptides whose primary structures were established as: Asn-Arg-Val-Tyr-Val-His-Pro-Phe ([Asn1, Val5]angiotensin II) and Asp-Arg-Val-Tyr-Val-His-Pro-Phe ([Asp1, Val5]angiotensin II). These peptides are the same as those generated in salmon plasma by an extract of kidney. The data raise the possibility that activation of the kallikrein-kinin system in trout generates both bradykinin-related and angiotensin II-related peptides that may act synergistically in the regulation of blood pressure.

Cardiovascular Actions of Frog Urotensin II in the Frog, Rana catesbeiana

The effects of synthetic frog urotensin II on cardiac output and arterial blood pressure and on the motility of isolated vascular smooth muscle were investigated in the bullfrog, Rana catesbeiana. Bolus injections of frog urotensin II (100 nmol/kg) into the left systemic arch produced a rapid and sustained fall in blood flow through the right branch of the truncus arteriosus (to 62 ± 5% of preinjection values; n = 8). The response exhibited strong tachyphylaxis. There were no significant effects on heart rate and central arterial blood pressure but the fact that a fall in cardiac output was not accompanied by a fall in pressure suggests that the peptide produced an increase in peripheral vascular resistance. Rings of vascular smooth muscle from the proximal and distal regions of the left and right systemic arches responded to urotensin II with sustained and concentration-dependent contractions. The tissues from the different regions did not significantly differ in their maximum response and sensitivity to the peptide (EC 50 values from 4.6 × 10 -9 to 6.5 × 10 -9 M; n = 6). Acetylcholine (3 × 10 -8 to 3 × 10 -6 M) significantly ( P < 0.05) relaxed the rings in an endothelium-dependent manner but urotensin II did not produce relaxation at any concentration tested. The contractile effect of urotensin II (10 -7 M) was not affected by preincubation of the rings with atropine, tetrodotoxin, and somatostatin-14 but indomethacin produced a significant ( P < 0.05) decrease in the amplitude of the contractions of the systemic arch (to 18 ± 2% of control values; n = 6). Arachidonic acid (10 -5 M) also produced a sustained contraction of the rings. The data suggest an involvement of prostaglandin synthesis in the contractile action of urotensin II on isolated frog vascular smooth muscle but, unlike in mammals, urotensin II does not stimulate nitric oxide synthesis in this tissue.

Effects of Melatonin on Isolated Pulmonary Artery and Vein: Role of the Vascular Endothelium

Summary: The influence of melatonin on tension of isolated pulmonary vascular smooth muscle rings was examined in these experiments. Melatonin caused a dose-dependent relaxation of precontracted (30 mM KCl) pulmonary artery and pulmonary vein. Removal of the endothelium did not alter the relaxant response to melatonin in pulmonary artery. In pulmonary artery with endothelium intact, preincubation with indomethacin, but not L-NG-monomethyl-L-arginine (L-NMMA) or deferoxamine, antagonized the relaxant response to melatonin. In pulmonary artery with endothelium removed, preincubation with deferoxamine, but not indomethacin, phorbol esters, or lithium chloride, antagonized the relaxant response to melatonin. In pulmonary vein, removal of the endothelium significantly increased the maximum relaxation attained in response to melatonin. In pulmonary vein with endothelium intact, preincubation with indomethacin, but not L-NMMA or deferoxamine, antagonized the relaxant response to melatonin. On the other hand, preincubation of de-endothelialized pulmonary vein with lithium chloride significantly increased the relaxation response to melatonin. These experiments demonstrate that both pulmonary artery and vein relax in response to melatonin and the mechanisms are apparently different.

Evidence for the presence of A1 and A2 adenosine receptors in the ventral aorta of the dogfish shark, Squalus acanthias.

Isolated, endothelium-free rings of vascular smooth muscle (VSM) from the ventral aorta of the dogfish shark, Squalus acanthias, were used to examine the vasoactive effects of various adenosine agonists. Cumulative addition of 2-chloroadenosine (2 Cl-ADO) over the concentration range 10 nM-1 mM resulted in a biphasic response, with a significant increase in tension at 1 microM and a more significant decline in tension at 100 microM and 1 mM, suggesting that this tissue may possess both A1 and A2 adenosine receptors. N6-Cyclopentyladenosine (N-6 CPA) and N6-(2-phenylisopropyl)adenosine, R(-)isomer (R-PIA), generally considered to be more A1 specific, also produced slight, but significant increases in tension, but only at relatively high concentrations. The more specific A1 agonist, N6-(25)-[2-endo-norbonyl] adenosine [(S)-ENBA] produced a significant increase in tension at 1 pM, reaching 28% above control at 10 nM. The response to (S)-ENBA was also biphasic, with a fall in tension at 10 microM. The relatively non-specific agonist 5'-N-ethylcarboxamidoadenosine (NECA) produced a small, but significant, increase in tension at 1 microM, with no subsequent decline in tension at higher concentrations. These results allow us to assign a tentative structure-activity relationship (SAR) for an increase in tension of (S)-ENBA much much greater than R-PIA greater than or equal to 2-Cl ADO = N-6 CPA = NECA; for the decrease, the SAR is (S)-ENBA greater than 2-Cl ADO greater than R-PIA greater than N-6 CPA = NECA.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen-dependent tension in vascular smooth muscle. Does the endothelium play a role?

We investigated a hypothesis that an oxygen sensor involved in hypoxia-induced relaxation of vascular smooth muscle may reside in endothelial cells. We also determined the oxygen dependence of hypoxia-induced decreases in cyclic guanosine 3',5'-monophosphate concentrations in vascular smooth muscle rings. Rings of canine femoral artery, rabbit thoracic aorta, and lamb ductus arteriosus, either with an intact endothelium or with damaged or absent endothelium, were studied using organ baths that allowed changes in PO2 without a change in pH. Hypoxia-induced relaxations of rabbit thoracic aorta, lamb ductus arteriosus, and canine femoral artery were not dependent on an intact endothelium. The magnitude of hypoxia-induced relaxations was unchanged in rings of canine femoral artery without intact endothelium compared to rings with endothelium. Quasi-steady state organ bath PO2-mechanical tension relationships were unchanged in rings of canine femoral artery without endothelium over an organ PO2 range of 200-20 mm Hg. With rabbit thoracic aorta, magnitudes of hypoxia-induced relaxations were significantly smaller in rings without endothelium. Quasi-steady state plots, where mechanical tension was given as percentage of maximal relaxation, were similar in rings either with or without intact endothelium. Cyclic guanosine 3',5'-monophosphate concentrations were shown to be oxygen-sensitive, decreasing during hypoxia-induced relaxations with a threshold PO2 of 80-100 mm Hg with canine femoral artery, and 60-80 mm Hg with rabbit thoracic aorta rings, but this finding seems unrelated to the mechanism of hypoxia-induced relaxation.

Vascular reactivity in the spontaneously hypertensive rat. Effect of high pressure stress and extracellular calcium.

The role of extracellular calcium and high blood pressure stress in altered vascular adrenergic responsiveness in rings of femoral artery from spontaneously hypertensive rats (SHR) was investigated. A model in which partial ligation of the external iliac artery prevents the increase in blood pressure to the ipsilateral femoral artery was used to assess the effect of the increase in pressure stress on these alterations. Age-matched (5-week-old) male Wistar-Kyoto rats (WKY) and SHR were used in the study. Partial ligation was performed before a substantial increase in blood pressure occurred (6 weeks of age), and studies on vascular reactivity were carried out at 10 to 12 weeks of age when the SHR were considered hypertensive (indirect systolic blood pressure greater than 150 mm Hg). Maximal contractility of rings of unprotected femoral artery from the SHR in response to KCl in either a normal (2.5 mM) or low (0.25 mM) calcium Krebs solution was significantly greater (p less than 0.05) than was that of protected vessels from the SHR or protected and unprotected vessels from the WKY; however, no difference in the sensitivity to KCl was observed. Isoproterenol-induced relaxation was significantly attenuated in rings of vascular smooth muscle from unprotected femoral arteries of the SHR (p less than 0.05), while the responses of protected vessels from the SHR were similar to controls. Equilibration of vascular smooth muscle in a low calcium Krebs solution resulted in an increase in beta-adrenergic mediated relaxation in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)