Alloxan inhibits ligand binding to adrenoceptors of vascular smooth muscle microsomes.

We have examined the effects of alloxan on the binding of [3H]prazosin and [125I]monoiodocyanopindolol (ICYP) to plasma-membrane-enriched microsomes isolated from dog aortas and dog mesenteric arteries respectively. Preincubation of the vascular smooth muscle membranes with alloxan reduced the number of binding sites of the alpha- and beta-adrenoceptors in a concentration-dependent manner, whereas the affinity of the radioligands for the adrenoceptors was not affected by alloxan. Streptozotocin, which is also a diabetogenic agent like alloxan, had no effect on the radioligand binding to these adrenoceptors under similar experimental conditions. The inhibitory effects of alloxan on binding to beta-adrenoceptors were found to be highly pH-dependent. These results indicate that alloxan exerts adverse effects on cell membrane adrenoceptors in addition to those on the ion-transport function of vascular smooth muscle cell [Kwan (1988) Biochem. J. 254, 293-296], and also suggest that the primary site of action of alloxan is the plasma membrane.     

Prejunctional alpha 2 adrenoceptors inhibit contraction of tracheal smooth muscle by inhibiting cholinergic neurotransmission

Ring preparations obtained from the guinea pig trachea contracted on short trains of electrical field stimulation. These contractions were mediated by activation of cholinergic nerves since they were abolished by atropine or tetrodotoxin. In the presence of beta blocking drugs noradrenaline and adrenaline dose-dependently inhibited contractions induced by field stimulation. By contrast, contractions on exogenous acetylcholine were left completely unaffected. It is concluded that the adrenergic agonists inhibited cholinergic neurotransmission by a prejunctional action. In order to characterize the noradrenaline receptor the effects of alpha₁ and alpha₂ blockers were evaluated using the Schild plot. For comparison experiments were also conducted on the guinea pig aorta and electrically stimulated guinea pig ileum. The results indicate that in guinea pig trachea and ileum noradrenaline inhibits cholinergic neurotransmission by acting on prejunctional alpha₂ receptors whereas in guinea pig aorta it induces contraction by stimulating alpha₁ receptors.     

Characteristics of postsynaptic alpha 1 and alpha 2 adrenergic receptors in canine vascular smooth muscle

A previous study suggested the existence of two distinct postsynaptic alpha adrenergic receptors in canine intralobar pulmonary arteries (IPA) and veins. The present study, performed using rings of canine IPA and dorsal metatarsal vein (DMV), was designed to characterize the factors affecting the postsynaptic alpha 1 and alpha 2 receptors of these blood vessels. The responses of IPA and DMV to norepinephrine (NE), transmural nerve stimulation, phenylephrine (PE), guanabenz, and clonidine were obtained in the presence and absence of alterations in pH, extracellular calcium ion, sulfhydryl bond reduction and oxidation, and destruction of adrenergic nerves with 6-hydroxydopamine. The data demonstrate that: (i) alpha 2-receptors are inactivated by changes in pH above or below pH 7.4, contain a labile disulfide group, are susceptible to modulation by increases and decreases in calcium ion, and appear to be decreased by destruction of adrenergic nerves; (ii) the NE and PE sensitive alpha 1-receptors are insensitive to alterations in pH, refractory to disulfide reduction by dithiothreitol, slightly susceptible to modulation by calcium ion, and increased by destruction of adrenergic nerves. These data support the conclusion that the two subtypes of postsynaptic alpha adrenergic receptors differ in their properties and susceptibility to modification by alteration of the physiological environment.     

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.     

Renal alpha adrenoceptors

The alpha adrenoceptors modulate renal blood flow, electrolyte balance, and metabolism. In most species both alpha 1 and alpha 2 subtypes are present. The alpha 1 adrenoceptors control blood flow and increase sodium reabsorption and gluconeogenesis. Radioligand studies indicate that the majority of alpha 1 adrenoceptors are located on proximal tubules. The alpha 2 adrenoceptors inhibit adenylate cyclase, and their density varies among species as do the molecular characteristics. In rat but not guinea pig some alpha 2 adrenoceptors are associated with glomeruli, but in both species most receptors are found on proximal tubules.     

Regulation of alpha7-integrin expression in vascular smooth muscle by injury-induced atherosclerosis

Injury of vascular smooth muscle cells (VSMCs) by allylamine (AAM) leads to phenotypic changes associated with atherogenic progression including increased proliferation, migration, and alterations in cell adhesion. In the present study, the relationship between AAM-induced vascular injury and expression of the alpha(7)-integrin subunit was investigated. The alpha(7)-mRNA and protein expression were examined using real-time RT-PCR, fluorescence-activated cell sorting analysis (FACS), immunohistochemistry, and immunoblotting. In cultured VSMCs from aortas of AAM-treated rats (70 mg/kg for 20 days), alpha(7)-mRNA levels were increased more than twofold compared with control cells. No change was seen in beta(1)-integrin expression. FACS analysis revealed increased cell surface expression of alpha(7)-protein (25 +/- 9%; *P < 0.05). AAM treatment of naive VSMCs enhanced alpha(7)-mRNA expression (2.4 +/- 0.7-fold, mean +/- SE; *P < 0.05). The increased alpha(7)-mRNA expression was attenuated by the amine oxidase inhibitor semicarbazide and the antioxidant pyrrolidine dithiocarbamate, which confirms a role for oxidative stress in modulating alpha(7)-expression. In vivo alpha(7)-mRNA and protein expression were enhanced in the aortas of AAM-treated rats. In addition, increased alpha(7)-integrin expression facilitated AAM VSMC adhesion to laminin more efficiently compared with control (51 +/- 2%; *P < 0.05). Chemical injury induced by AAM significantly enhances alpha(7)-integrin expression in VSMCs. These findings implicate for the first time the expression of alpha(7)-integrin during the response of VSMCs to vascular injury.     

Tumor necrosis factor alpha inhibits insulin-induced mitogenic signaling in vascular smooth muscle cells

Tumor necrosis factor alpha (TNFalpha) interferes with insulin signaling in adipose tissue and may promote insulin resistance. Insulin binding to the insulin receptor (IR) triggers its autophosphorylation, resulting in phosphorylation of Shc and the downstream activation of p42/p44 extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2), which mediates insulin-induced proliferation in vascular smooth muscle cells (VSMC). Since insulin resistance is a risk factor for vascular disease, we examined the effects of TNFalpha on mitogenic signaling by insulin. In rat aortic VSMC, insulin induced rapid phosphorylation of the IR and Shc and caused a 5.3-fold increase in activated, phosphorylated ERK1/2 at 10 min. Insulin induced a biphasic ERK1/2 activation with a transient peak at 10 min and a sustained late phase after 2 h. Preincubation (30-120 min) with TNFalpha had no effect on insulin-induced IR phosphorylation. In contrast, TNFalpha transiently suppressed insulin-induced ERK1/2 activation. Insulin-induced phosphorylation of Shc was inhibited by TNFalpha in a similar pattern. Since mitogenic signaling by insulin in VSMC requires ERK1/2 activation, we examined the effect of TNFalpha on insulin-induced proliferation. Insulin alone induced a 3.4-fold increase in DNA synthesis, which TNFalpha inhibited by 48%. TNFalpha alone was not mitogenic. Inhibition of ERK1/2 activation with PD98059 also inhibited insulin-stimulated DNA synthesis by 57%. TNFalpha did not inhibit platelet-derived growth factor-induced ERK1/2 activation or DNA synthesis in VSMC. Thus, TNFalpha selectively interferes with insulin-induced mitogenic signaling by inhibiting the phosphorylation of Shc and the downstream activation of ERK1/2.     

Regulation of alpha(2)-adrenoceptors in human vascular smooth muscle cells

This study analyzed the regulation of alpha2-adrenoceptors (alpha2-ARs) in human vascular smooth muscle cells (VSMs). Saphenous veins and dermal arterioles or VSMs cultured from them expressed high levels of alpha2-ARs (alpha2C > alpha2A, via RNase protection assay) and responded to alpha2-AR stimulation [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304, 1 microM)] with constriction or calcium mobilization. In contrast, VSMs cultured from aorta did not express alpha2-ARs and neither cultured cells nor intact aorta responded to UK-14,304. Although alpha2-ARs (alpha2C > alpha2A) were detected in aortas, alpha2C-ARs were localized by immunohistochemistry to VSMs of adventitial arterioles and not aortic media. In contrast with aortas, aortic arterioles constricted in response to alpha2-AR stimulation. Reporter constructs demonstrated higher activities for alpha2A- and alpha2C-AR gene promoters in arteriolar compared with aortic VSMs. In arteriolar VSMs, serum increased expression of alpha2C-AR mRNA and protein but decreased expression of alpha2A-ARs. Serum induction of alpha2C-ARs was reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) with 2 microM SB-202190 or dominant-negative p38 MAPK. UK-14,304 (1 microM) caused calcium mobilization in control and serum-stimulated cells: in control VSMs, the response was inhibited by the alpha2A-AR antagonist BRL-44408 (100 nM) but not by the alpha2C-AR antagonist MK-912 (1 nM), whereas after serum stimulation, MK-912 (1 nM) but not BRL-44408 (100 nM) inhibited the response. These results demonstrate site-specific expression of alpha2-ARs in human VSMs that reflects differential activity of alpha2-AR gene promoters; namely, high expression and function in venous and arteriolar VSMs but no detectable expression or function in aortic VSMs. We found that alpha2C-ARs can be dramatically and selectively induced via a p38 MAPK-dependent pathway. Therefore, altered expression of alpha2C-ARs may contribute to pathological changes in vascular function.     

Nature of alpha 1 and postjunctional alpha 2 adrenoceptors in the pulmonary vascular bed

The subtypes of postjunctional alpha adrenoceptors in the feline pulmonary vascular bed were studied by using selective alpha-adrenoceptor agonists and antagonists. Under conditions of controlled pulmonary blood flow and constant left atrial pressure, intralobar injections of the alpha 1 agonists phenylephrine and methoxamine, and the alpha 2 agonists UK 14,304 and B-HT 933, increased lobar arterial pressure in a dose-related manner. Prazosin, an alpha 1-adrenoceptor antagonist, reduced responses to phenylephrine and methoxamine to a greater extent than responses to UK 14,304 and B-HT 933. Yohimbine, an alpha 2 blocker, decreased responses to UK 14,304 and B-HT 933 without altering responses to phenylephrine or methoxamine. The same pattern of blockade was observed in animals pretreated with 6-hydroxydopamine, an adrenergic neuronal blocking agent. However, in propranolol-treated animals, prazosin antagonized responses to phenylephrine and methoxamine without altering responses to UK 14,304 or B-HT 933, and the selectivity of the blocking effects of yohimbine were preserved. Responses to intralobar injections of norepinephrine (NE) were markedly decreased by prazosin, whereas yohimbine had only a small effect. These data suggest the presence of both postjunctional alpha 1 and alpha 2 adrenoceptors mediating vasoconstriction in the pulmonary vascular bed. These results also indicate that the vasoconstrictor responses to injected NE in the cat pulmonary vascular bed result mainly from activation of alpha 1 adrenoceptors.     

Actions of prostaglandins E1 and F2alpha on isolated intrapulmonary vascular smooth muscle.

The effects of PGE1 and PGF2alpha were studied on isolated strips of intrapulmonary arteries and veins from dog, sheep, swine and man. PGF2alpha contracted human arterial strips in a dose-dependent fashion, relaxed slightly sheep arteries and had no effect on dog arteries. Canine, sheep and human venous strips were contracted by PGF2alpha. PGE1 relaxed slightly both veins and arteries from dog and sheep. Human arteries usually contracted slightly and human veins usually relaxed slightly to PGE1. In a limited number of experiments, swine arteries and veins failed to respond to PGF2alpha or PGE1. All the vascular strips contracted well when exposed to NE. These results suggest that the responses of intrapulmonary vessels to PGF2alpha and PGE1 are species-dependent. PGF2alpha generally exhibits a contractile action, especially on veins. PGE1 usually relaxes intrapulmonary vessels. With regard to vessels from man, PGF2alpha is a powerful stimulant while PGE1 produces only small, variable effects.     

The alpha adrenoceptors on endothelial cells

Endothelial cells release a powerful factor (endothelium-derived relaxing factor [EDRF]) that relaxes smooth muscle cells in response to some vasodilating agents such as acetylcholine. Contraction curves to norepinephrine (NE) in greyhound, mongrel dog, and pig coronary artery rings were studied in vitro in the presence of propranolol. Removal of endothelium increased the sensitivity and maximum contraction in response to NE. In other experiments pig coronary rings were precontracted with a thromboxane mimetic U 46619 in the presence of propranolol. NE relaxed these arteries only if endothelium was present. Methoxamine was without effect but the relaxation response to NE was antagonized by phentolamine, idazoxan, and yohimbine, which suggests that there are alpha 2 adrenoceptors on endothelial cells that mediate the release of EDRF. Greyhound and mongrel dog large coronary arteries relaxed to NE only if prazosin was present, which suggests that alpha 1-adrenoceptor stimulation on the vascular smooth muscle can override the relaxation response to EDRF. Comparison of NE responses in carotid, mesenteric, renal, and femoral large arteries of the pig, greyhound, and mongrel dog indicate the nonuniformity of distribution of alpha 2 adrenoceptors on endothelium and alpha 1 and alpha 2 adrenoceptors on vascular smooth muscle. The integrity of the endothelium must now be considered in interpreting the vascular responses to alpha-adrenoceptor agonists.     

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).     

Lysolecithin actions on vascular smooth muscle cells.

Oxidation of low density lipoprotein increases its atherogenic potential. During oxidation there is an extensive conversion of lecithin to lysolecithin. In rat aortic smooth muscle cells, 2-25 micrograms/ml lysolecithin elevated cytosolic calcium concentration up to 560%. Lysolecithin (10-20 micrograms/ml) increased [3H]thymidine incorporation from 15 cpm/mg cell protein (controls) up to 189 cpm/mg cell protein. Lysolecithin (10 micrograms/ml) potentiated the PDGF-induced (50 ng/ml) [3H]thymidine incorporation up to 6.3 times. The results indicate that lysolecithin could induce mechanisms, by which oxidized low density lipoproteins could promote cell growth and thus contribute to atherosclerosis.     

TRAIL expression in vascular smooth muscle

TRAIL is a cell-associated tumor necrosis factor-related apoptosis-inducing ligand originally identified in immune cells. The ligand has the capacity to induce apoptosis after binding to cell surface receptors. To examine TRAIL expression in murine vascular tissue, we employed in situ hybridization and immunohistochemistry. In these studies, we found that TRAIL mRNA and protein were specifically localized throughout the medial smooth muscle cell layer of the pulmonary artery. Notably, a similar pattern of expression was observed in the mouse aorta. Consistent with these findings, we found that cultures of primary human aorta and pulmonary artery smooth muscle cells express abundant TRAIL mRNA and protein. We also found that these cells and endothelial cells undergo cell lysis in response to exogenous addition of TRAIL. Last, we confirmed that TRAIL specifically activated a death program by confirming poly(ADP ribose) polymerase cleavage. Overall, we believe that these findings are relevant to understanding the factors that regulate cell turnover in the vessel wall.     

Thick filaments in vascular smooth muscle

Two sets of myofilaments were demonstrated after incubation of strips of rabbit portal-anterior mesenteric vein under moderate stretch in a physiological salt solution. Thick filaments had a mean diameter of 18 nm and reached a maximum length of 1.4 µm with a mean length of 0.61 µm. In transverse sections, 2.5–5 nm particles were resolved as subunits of the thick filaments. Thin filaments had an average diameter of 8.4 nm and generally conformed to the structure believed to represent actin filaments in smooth and striated muscles. In the areas of maximum concentration there were 160–328 thick filaments/µm² and the lowest ratio of thin to thick filaments was 12:1. Thick filaments were present in approximately equal numbers in vascular smooth muscle relaxed by theophylline, in Ca⁺⁺-free solution, or contracted by norepinephrine. The same preparatory procedures used with vascular smooth muscle also enabled us to visualize thick filaments in guinea pig and rabbit taenia coli and vas deferens.