Venous smooth muscle contains vasoconstrictor ETB-like receptors.

Two endothelin (ET) receptor subtypes have been identified to date: the ETA receptor which preferentially binds ET-1 over ET-3, and the ETB receptor which is non-selective. This study characterized the ET receptor subtypes present in several vascular smooth muscle preparations using standard in vitro techniques. In all but one of the arteries tested, ET-3 was significantly less potent than ET-1. In contrast, the potency of ET-3 was very similar to that of ET-1 in all of the veins. The selective ETA receptor antagonist BQ-123 blunted the ET-1 contractions in rabbit carotid artery, but not in saphenous vein. The selective ETB receptor ligand sarafotoxin S6c contracted the rabbit saphenous vein, but not the carotid artery. These data suggest that vascular smooth muscle cells express ETA and ETB receptors. Stimulation of either receptor subtype can result in force development.     

Staurosporine inhibition of zipper-interacting protein kinase contractile effects in gastrointestinal smooth muscle.

Zipper-interacting protein kinase (ZIPK) is a serine-threonine kinase that has been implicated in Ca2+-independent myosin II phosphorylation and contractile force generation in vascular smooth muscle. However, relatively little is known about the contribution of this kinase to gastrointestinal smooth muscle contraction. The addition of a recombinant version of ZIPK that lacked the leucine zipper domain to permeabilized ileal strips evoked a Ca2+-independent contraction and resulted in myosin regulatory light chain diphosphorylation at Ser19 and Thr18. Neither Ca2+-independent force development nor myosin regulatory light chain phosphorylation was elicited by the addition of kinase-dead ZIPK to the ileal strips. The sensitivity of ZIPK-induced contraction to various kinase inhibitors was similar to the in vitro sensitivity of purified ZIPK to these inhibitors. Staurosporine was the most effective ZIPK inhibitor, with a Ki value calculated to be 2.6 +/- 0.3 micromol/L. Through the use of specific kinase inhibitors, we determined that Rho-associated protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C) do not mitigate ZIPK-induced contraction in ileum. Our findings support a role for ZIPK in Ca2+-independent contractile force generation in gastrointestinal smooth muscle.     

Binding and receptor down-regulation of a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells

Binding of a novel endothelium-derived vasoconstrictor endothelin (ET) and the regulation of its receptor were studied in cultured rat vascular smooth muscle cells. 125I-labeled-ET bound to the cells was resistant to acid extraction and the majority of the acid-resistant compartment was extractable with chloroform/methanol with minimal degradation. Autoradiographic studies using electron microscopy revealed that the grains were predominantly localized in the plasma membranes, but some were adjacent to and within the lysosome. Pretreatment with ET resulted in a substantial reduction of ET receptor number without changing its binding affinity. ET-induced increase in cytosolic free Ca2+ levels [( Ca2+]i] was absent or attenuated in the ET-pretreated cells. These data suggest that tight association of ET with its receptor is due to a strong interaction of its hydrophobic domain with the membrane lipids and/or its internalization within cells and that down-regulation of ET receptor is functionally linked to decreased ET-induced [Ca2+]i response.     

Energetic effects of adenosine on vascular smooth muscle

Adenosine (Ado) is a naturally occurring compound that has several important cardiovascular actions, including activation of ATP-sensitive K(+) channels in vascular smooth muscle, vasorelaxation, and an effect to alter glucose metabolism of cardiac muscle. The metabolic effects of Ado on vascular smooth muscle have not been defined and were examined in this study. Porcine carotid artery strips were incubated in the presence and absence of 0.5 mM Ado. Compared with the control, Ado had no effect on glucose uptake, glucose oxidation, or fatty acid (octanoate) oxidation. Ado suppressed glycolysis but enhanced glycogen synthesis. Relative to the rate of glycolysis, Ado increased lactate production. Ado stimulated O(2) consumption by 52 +/- 10%, altered the activities of the tricarboxylic acid cycle and malate-aspartate shuttle, and increased the content of ATP, ADP, AMP, and phosphocreatine. Alteration in the metabolic variables by Ado could not be attributed to diminished energy requirements of reduced resting muscle tone of the arterial strips. Relaxation of the arterial strips in response to Ado were abolished in arteries incubated under hypoxic conditions (95% N(2)-5% CO(2)). Hypoxia was associated with increased ADP content. It is concluded that Ado affected glucose metabolism indirectly. The metabolic and energetic effects of 0.5 mM Ado are mediated by alterations in the concentrations of AMP, ATP, and phosphorylation potential (ATP/ADP).     

Endothelin: differential effects in vascular and nonvascular smooth muscle

Endothelin, a potent vasoconstrictor, produced concentration-dependent contractions in aorta, trachea and bladder body obtained from rat and rabbit. Contractions developed slowly, reaching maxiMum within 15-20 min. Although, in both rat and rabbit tissues, endothelin was 3- to 10-fold more potent in contracting vascular (approximate EC50, 1 nM) than nonvascular smooth muscle, rat trachea and rabbit bladder did contract in response to endothelin. Rat bladder body and rabbit trachea were the least sensitive tissues with only modest contractile responses to endothelin. To determine the role of calcium in these endothelin-induced contractions, the effects of diltiazem and nitrendipine were examined. Although diltiazem (5 x 10-5) M) or nitrendipine (10(-6) M) markedly attenuated contractions produced by KCl, neither agent significantly affected concentration response curves produced by endothelin in rabbit aorta or rat trachea. In rat aorta, nitrendipine had no effect on endothelin responses, whereas diltiazem modestly decreased the maximal contraction to endothelin. However, in rabbit bladder, both calcium channel blockers significantly decreased the maximum response to endothelin with no change in EC50. These results indicate that smooth muscle sensitivity to the contractile effects of endothelin may be both species and tissue specific.     

The effects of ruthenium tetraammine compounds on vascular smooth muscle.

The time course of the relaxation effect induced by a single dose (3 x 10(-6) mol/L) of trans-[Ru(NH3)4L(NO)]3+ (L=nic, 4-pic, py, imN, P(OEt)3, SO(3)(2-), NH3, and pz) species and sodium nitroprusside (4 x 10(-9) mol/L) was studied in aortic rings without endothelium and pre-contracted with noradrenaline (1 x 10(-6) mol/L). All the compounds induced a relaxing effect in the aortic rings, but the intensity and time of relaxation were different. Only the species where L=py, 4-pic, and P(OEt)3 were able to induce 100% (99-100%) of the relaxing effect during the assay. trans-[Ru(NH3)4(L)(NO)]3+ (L=SO(3)(2-) and NH3) showed the lowest relaxing effect (36 and 37%, respectively) when compared with the other compounds. Relationship was observed between the time corresponding to half of the maximum relaxation intensity observed and, respectively, k-NO, E0'[Ru(NO)]3+/[Ru(NO)]2+ in trans-[Ru(NH3)4(L)(NO)]3+ species and E0'Ru(III)/Ru(II) in trans-[Ru(NH3)4(L)(H2O)]3+ ions. These relationships strongly suggested that the NO liberation from the reduced nitrosyl complexes was responsible for the observed relaxation.     

Phorbol ester-induced expression of the common, low-affinity binding site for primary prostanoids in vascular smooth muscle cells

We showed in an earlier study (Hanasaki, K., and Arita, H. (1989) Biochim. Biophys. Acta 1013, 28-35) that there is a common, low-affinity binding site for primary prostanoids in cultured vascular smooth muscle cells (VSMC). This site, called the "primary prostaglandin (PG) site," can be evaluated by radioreceptor assay using [3H]PGF2 alpha and [3H]PGE1. Comparison of the capacity of several PGF2 alpha analogs to displace both radioligand bindings indicated strict requirements of the 15-hydroxy group as well as the 13,14-double bond in the omega-side chain of prostaglandins for recognition of this site. Treatment of VSMC with phorbol 12-myristate 13-acetate (PMA), a known protein kinase C activator, led to concentration- and time-dependent increases in the binding activities of [3H] PGF2 alpha as well as [3H]PGE1, which could be completely suppressed by the addition of protein kinase C inhibitor, H-7. The PMA effects could be mimicked by phorbol 12,13-dibutylate, but not by inactive phorbol ester. Scatchard analyses revealed an approximately 8-fold increase in the binding density with unaltered binding affinity after PMA treatment. This expression of the primary PG site was blocked by the addition of cycloheximide and actinomycin D. In contrast, PMA did not affect the binding activity for the thromboxane A2/prostaglandin H2 receptor in VSMC. These results suggest that the expression of the primary PG site is regulated by a protein kinase C-dependent mechanism in VSMC.     

Cellular mechanism of action by a novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells

Specific binding sites for synthetic porcine endothelin (pET), a novel potent vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells, and its effects on cytosolic free Ca2+ concentrations ([Ca2+]i) and phosphatidylinositol (PI) response were studied in cultured rat aortic vascular smooth muscle cells (VSMC). Binding of 125I-labeled-pET to rat VSMC was time- and temperature-dependent and the cell-bound 125I-labeled-pET was resistant to dissociate. Scatchard analysis of binding studies indicated the presence of a single class of high-affinity binding sites: the apparent Kd was 2-4 X 10(-10) M and the maximal binding capacity was 11,000-13,000 sites/cell. The binding was highly specific for pET because neither well-recognized vasoconstrictors, peptide neurotoxins, nor Ca2+-channel blockers affected the binding. pET dose-dependently (10(-9)-10(-7) M) induced a transient and sustained increase in [Ca2+]i in fura-2-loaded cells of which effect was largely dependent on extracellular Ca2+, whereas it had no significant effect on PI response in 3H-myoinositol-prelabeled cells. The present data clearly demonstrates the presence of specific receptors for pET distinct from those of the well-recognized vasoconstrictors and voltage-dependent Ca2+-channels in cultured rat VSMC, and suggest that pET-induced increase in [Ca2+]i is involved in the mechanism of its vasoconstriction.     

Flow effects on cultured vascular endothelial and smooth muscle cell functions.

Cultured vascular endothelial cells were exposed to fluid shear stress by means of a rotary-disc shear-loading device, and the physiological effects of the conditioned medium (CM) and the homogenate (HM) of the cells on migration, adhesion and growth of endothelial cells (EC) or smooth muscle cells (SMC) were studied. Effects of shear stress on the production and secretion of collagen, one of the extracellular matrices of EC, were also studied. CM stimulated the adhesion and growth of SMC, but not of EC themselves. The ability to stimulate SMC adhesion and growth was similar in CM obtained from the static and shear-loaded cells. HM of the shear-loaded EC stimulated SMC migration. Further, HM of the shear-loaded EC contained increased amounts of collagen compared with the static EC. These results suggest that: 1) EC produce and secrete accelerators for the adhesion and growth of SMC, 2) EC react to the physical stimulus of fluid shear stress to produce stimulators of SMC migration, and 3) EC produce collagen, the production of which is enhanced by fluid shear stress.     

Ca2+-sensitivity and cGMP-independent effects of NO in vascular smooth muscle.

The effects of NO on Ca2+-sensitivity of vascular smooth muscle (VSM) myofilaments have been the focus of this study. Simultaneous measurements of [Ca2+]i and force were carried out in rat tail artery segments. NO, 10(-7) M, evoked a transient decrease in [Ca2+]i accompanied by sustained relaxation (45.3+/-6.3 vs. 69.45+/-7.2%, P<0.05, respectively) of VSM precontracted with K+ (70 mM), suggesting a decrease in Ca2+-sensitivity of VSM. This decrease in Ca2+-sensitivity was completely abolished by preincubation of VSM with ODQ (10(-6) M) (63.9+/-7.8% for [Ca2+]i vs. 20.5+/-8.4% for relaxation, P<0.05). Ca2+-presensitization of VSM myofilaments with PE (10(-6) M) decreased the efficacy of NO to relax VSM (44.25+/-6.9% vs. 69.45+/-7.2%, P<0.05), but increased its ability to lower [Ca2+]i (70.5+/-6.8% vs. 45.3+/-6.3%, P<0.05). Application of DTT (10(-3) M) together with ODQ (10(-6) M) to subtract possible cGMP-independent effects revealed the total suppression of both the relaxant responses and [Ca2+]i of VSM under high-K+ preactivation of VSM. The data indicate that NO not only relaxes VSM and lowers [Ca2+]i in K+-preactivated VSM, but also decreases Ca2+-sensitivity of VSM myofilaments and these effects are strongly cGMP-dependent. In PE-induced contractions of VSM, NO relaxed VSM of rat tail artery and lowered [Ca2+]i, but failed to reverse Ca2+-presensitized myofilaments. We suggest that alternative cGMP-independent effects of NO are primarily manifested via activation of K+-channels and inhibition of Ca2+ current rather than to affect relaxation. An importance of reduced SH-groups within VSM myoplasm for both relaxation and [Ca2+]i disposal evoked by NO is evident whatever Ca2+-mobilization pathways are involved.     

Effects of altering carbohydrate metabolism on energy status and contractile function of vascular smooth muscle

Substrate-dependent changes in vascular smooth muscle energy metabolism and contractile function were investigated in isolated porcine carotid arteries. In media containing glucose glycogen catabolism accounted for all the estimated high-energy phosphate turnover that occurred in conjunction with contraction induced by 80 mM KCl. However, in glucose-free media glycogen catabolism accounted for only a portion of the estimated ATP utilization in resting and contracting arteries, even though glycogen stores were not depleted. The glycogenolysis and lactate production that ordinarily accompanies contraction was completely inhibited by 5 mM 2-deoxyglucose (2-DG). However, there was no decrease in the high-energy phosphate levels when compared to control resting arteries similarly treated with 2-DG. The results suggest that an endogenous non-carbohydrate source may be an important substrate for energy metabolism. Treatment of arteries with 50 microM iodoacetate (IA) in media containing glucose resulted in a marked reduction of high energy phosphate levels and an accumulation of phosphorylated glycolytic intermediates, as demonstrated by 31P-NMR spectroscopy. In glucose-free media, 50 microM IA had only a slight effect on high-energy phosphate levels, while glycogenolysis proceeded unhindered. With 1 mM IA in glucose-free media, the oxidative metabolism of glycogen was inhibited as evidenced by the depletion of high-energy phosphates and the appearance of sugar phosphates in the 31P-NMR spectra. Thus, the titration of enzyme systems with IA reveals a structural partitioning of carbohydrate metabolism, as suggested by previous studies.     

Catecholamine-mediated constrictor effects of aldosterone on vascular smooth muscle

The possibility that the corticosteroid hormone, aldosterone, might possess direct vasoconstrictor properties was examined in the isolated central ear artery of the New Zealand white rabbit. Aldosterone alone produced only minimal contractile effects in the arterial segments; but following pretreatment of the tissue with desipramine (10^?7M), a blocker of neuronal uptake of norepinephrine, aldosterone concentrations of 10^?6M, 10^?5M, and 10^?4M produced stepwise contractile responses of 0.16 ± 0.03 (SE)g, 0.48 ± .04g, and 1.31 ± 0.06g. The possible involvement of norepinephrine in this action of aldosterone was tested in a series of tissues stored for 2 days at 2°C in Krebsbicarbonate medium so as to deplete endogenous catecholamine stores. Treatment with desipramine followed by aldosterone (10^?4M) now produced an average contraction of only 0.1 ± 0.06g; but if the labile neuronal tissue stores of norepinephrine in these tissues were then replenished by exposure to norepinephrine 10^?7M, contractions of 1.2 ± 0.3g now occurred when desipramine and aldosterone were added. To examine whether aldosterone's action might be due to blockade of extraneuronal norepinephrine uptake (uptake-2), ³H-norepinephrine was added to ear artery tissues exposed to desipramine with or without aldosterone: a significant (P<0.005) decrease of 25% in ³H-norepinephrine uptake occurred in the tissues treated with aldosterone. In further studies, the contractile effects of aldosterone could be prevented by pretreatment with prazosin (10^?7M) or phentolamine (10^?7M); if added after the aldosterone, each of these alpha-blockers completely reversed the contractile responses. Although the physiological relevance of these findings is yet to be fully defined, these studies indicate that the $\text{in}$$\text{vitro}$ contractile effects of aldosterone are dependent upon its inhibition of extraneuronal uptake of endogenous norepinephrine; it is likely that the resulting increase in extracellular norepinephrine concentration then produces vasoconstriction by stimulation of post-synaptic alpha-adrenergic receptors.     

Assembly of contractile and cytoskeletal elements in developing smooth muscle cells.

Specific developmental changes in smooth muscle were studied in gizzards obtained from 6-, 8-, 10-, 12-, 14-, 16-, 18-, and 20-day chick embryos and from 1- and 7-day posthatch chicks. Myoblasts were actively replicating in tissue from 6-day embryos. Cytoplasmic dense bodies (CDBs) first appeared at Embryonic Day 8 (E8) and were recognized as patches of increased electron density that consisted of actin filaments (AFs), intermediate filaments (IFs), and cross-connecting filaments (CCFs). Although the assembly of CDBs was not synchronized within a cell, the number, size, and electron density of CDBs increased as age increased. Membrane-associated dense bodies (MADBs) also could be recognized at E8. The number and size of MADBs increased as age increased, especially after E16. Filaments with the diameter of thick filaments first appeared at E12. Smooth muscle cells were able to divide as late as E20. The axial intermediate filament bundle (IFB) could first be identified in 1-day posthatch cells and became larger and more prominent in 7-day posthatch cells. Immunogold labeling of 1- and 7-day posthatch cells with anti-desmin showed that the IFB contained desmin IFs. The developmental events during this 23-day period were classified into seven stages, based primarily on the appearance and the growth of contractile and cytoskeletal elements. These stages are myoblast proliferation, dense body appearance, thick filament appearance, dense body growth, muscle cell replication, IFB appearance, and appearance of adult type cells. Smooth muscle cells in each stage express similar developmental characteristics. The mechanism of assembly of myofilaments and cytoskeletal elements in smooth muscle in vivo indicates that myofilaments (AFs and thick filaments) and filament attachment sites (CDBs and MADBs) are assembled before the axial IFB, a major cytoskeletal element.     

Impact of alpha1-adrenoceptor expression on contractile properties of vascular smooth muscle cells

Low-frequency blood pressure oscillations (Mayer waves) are discussed as a marker for sympathetic modulation of vascular tone. However, the factors that determine the frequency response of the vasculature to sympathetic stimuli are not fully understood. Possible mechanisms include functions related to alpha(1)-adrenergic receptors (alpha(1)-AR) and postreceptor processes involved in the vascular contractile response. The purpose of the present study was to examine the hypothesis that expression levels of alpha(1)-AR and their subtype distribution determine velocity and magnitude of alpha(1)-AR-mediated vascular smooth muscle cell (VSMC) contraction. alpha(1A)-, alpha(1B)-, and alpha(1D)-AR subtypes were transfected into VSMCs from rat aorta and characterized immunocytochemically via confocal microscopy. Functional studies in isolated cells were performed using video microscopy. The alpha(1)-AR agonist phenylephrine produced dose-dependent contractions of VSMCs. All transfected groups were more sensitive to phenylephrine compared with controls. Maximal contraction velocity almost doubled in transfected cells. However, no differences in observed parameters were found between the three transfected groups. Contractile properties in response to membrane depolarization with KCl were similar in all groups. In conclusion, alpha(1)-AR density determines velocity and sensitivity of alpha(1)-AR-mediated contraction in VSMCs. alpha(1)-AR subtype distribution does not appear to influence vasoconstriction to sympathetic stimuli.     

The contractile response of vascular smooth muscle to thrombin and its inhibition by thrombin inhibitors

Thrombin (0.1-30 NIH-U/ml) caused a contractile response of rabbit aortic rings. The vasocontraction was independent upon intact endothelium, however in deendothelialized vessels the contractile effect was more pronounced. The thrombin-induced vasocontraction was diminished in calcium-free medium; the same effect was attained by the calcium channel blocker verapamil at high concentrations. In human femoral and saphenous vein strips thrombin produced a contractile effect, too, which was very low and inconsistent in femoral arterial strips. To inhibit the contractile response of vascular smooth muscle to thrombin, higher concentrations of both the specific tight-binding inhibitors hirudin and beta Nas-Gly-(pAM)Phe-Pip were required than for the inhibition of fibrinogen clotting.