Pharmacological subclassification of alpha 1-adrenoceptors in vascular smooth muscle

1. We examined whether alpha 1-adrenoceptors in various blood vessels can be divided into subtypes by antagonist affinity or by susceptibility to chloroethylclonidine or nifedipine. 2. Noradrenaline or phenylephrine produced concentration-dependent contractions in all the tissues tested, which were competitively inhibited by phentolamine, yohimbine, prazosin, WB4101 and HV723. However, there were large differences between the tissues in the pA2 values for all the antagonists except phentolamine. 3. The blood vessels could be classified into three groups (I, II and III) on the basis of their affinity variation. In group I (dog mesenteric artery and vein, saphenous vein), the pA2 values for HV723 were greater than 9, and those for HV723 and WB4101 were approximately 1 log unit higher than for prazosin. This rank order of affinity reversed in group II (dog carotid artery and rat thoracic aorta), where prazosin was more potent (pA2 values greater than 9.5) than HV723 or WB4101. In group III (rabbit mesenteric artery, thoracic aorta and carotid artery and guinea-pig thoracic aorta), on the other hand, prazosin, HV723 and WB4101 inhibited the noradrenaline response with a similar affinity (pA2 values ranging from 8 to 9). 4. Yohimbine inhibited the responses to noradrenaline and phenylephrine with a lower affinity than prazosin, HV723 or WB4101. The pA2 values for yohimbine were similar in groups I and II (the values greater than 6.5), which were greater than those in group III (values less than 6.4).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ketanserin blocks alpha 1-adrenoceptors of porcine vascular smooth muscle cells

The effect of ketanserin on alpha-adrenoceptors was studied in membrane preparations of the porcine aorta using [3H]prazosin and [3H]yohimbine binding assays to identify alpha 1- and alpha 2-adrenoceptors. Ketanserin bound to alpha-adrenoceptors and the Ki value of ketanserin for alpha 1-adrenoceptors was 8.3 nM, a value practically equal to that of phentolamine (Ki = 7.2 nM). The Ki value of ketanserin for alpha 2-adrenoceptors was 3.3 microM. Thus, at the doses prescribed clinically, ketanserin blocks alpha 1- but not alpha 2-adrenoceptors of porcine vascular smooth muscle.     

Selective blockade of alpha 1-adrenoceptors of porcine vascular smooth muscle cells by bunazosin

The characteristics of bunazosin binding to alpha-adrenoceptors in the porcine aortic membranes were investigated using [3H]prazosin and [3H]yohimbine binding assays to identify alpha 1- and alpha 2-adrenoceptors, respectively. The extent of the inhibition (Ki values) of [3H]prazosin binding to alpha 1-adrenoceptors induced by bunazosin was 0.29 nmol/l and about the same as that induced by prazosin (Ki = 0.10 nmol/l). The Ki value of bunazosin inhibition of [3H]yohimbine binding to alpha 2-adrenoceptors was 350 nmol/l. There was over a 1000-fold difference in Ki value for bunazosin between alpha 1-and alpha 2-adrenoceptors. Thus, bunazosin is a highly alpha 1 selective agent in vascular smooth muscle.     

Competitive interaction of amiloride and verapamil with alpha 1-adrenoceptors in vascular smooth muscle

We have characterized [3H]prazosin binding to purified plasma membranes isolated from bovine carotid arteries and studied the effects of Na+ and Ca2+ channel blockers on [3H]prazosin binding to alpha 1-adrenoceptors. Amiloride and verapamil competitively inhibited the specific binding of [3H]prazosin to purified plasma membranes isolated from bovine carotid artery in a dose-dependent manner. The Ki values of verapamil and amiloride for alpha 1-adrenergic receptor were 1.04 +/- 0.037 microM and 32.6 +/- 0.59 microM, respectively. Verapamil (10 microM) and amiloride (100 microM) caused a 6-fold and 2.7-fold decrease in affinity of [3H]prazosin binding, respectively, with no change in the number of binding sites. The inhibition of [3H]prazosin binding by amiloride and verapamil could be reversed after the membranes were washed. Another Ca2+-channel blocker, nifedipine, and a Na+-channel blocker, furosemide, did not significantly inhibit [3H]prazosin binding up to 0.1 mM concentrations. Our results suggest that amiloride and verapamil may produce vascular smooth muscle relaxation by modulating alpha 1-adrenoceptor affinity in addition to blocking Na+ and Ca2+ channels, respectively.     

Postsynaptic alpha 2-adrenoceptors predominate over alpha 1-adrenoceptors in canine tracheal smooth muscle and mediate neuronal and hormonal alpha-adrenergic contraction

Alpha-adrenoceptor subtypes in canine tracheal smooth muscle were investigated by radioligand binding and by in vitro responses of muscle strips to electrical field stimulation and exogenous alpha-agonists. [3H]Yohimbine identified a high density of alpha 2-receptors (51.4 +/- 4.9 fmoles/mg of protein; n = 5) in tracheal smooth muscle membranes, whereas [3H]prazosin revealed a low density of alpha 1-receptors (11.1 +/- 2.9 fmoles/mg of protein; n = 5). In peripheral lung membranes, however, alpha 1-receptors predominated (46.8 +/- 7.7 fmoles/mg of protein; n = 4) over alpha 2-receptors (4.1 +/- 1.5 fmoles/mg of protein; n = 4). After pretreatment with atropine and propranolol and precontraction with serotonin or histamine, the contractile response of tracheal smooth muscle to electrical field stimulation was partially inhibited by 0.3 microM prazosin (16%), potently inhibited by 0.3 microM yohimbine (89%), and abolished by a combination of the two drugs. The response to neuronally released norepinephrine is therefore mediated predominantly by alpha 2-receptors. The rank order of potency for adrenergic agonists was clonidine greater than norepinephrine greater than phenylephrine in both competition studies with [3H]yohimbine and in contraction studies, signifying a predominance of postsynaptic alpha 2-receptors. The contractile responses to exogenous norepinephrine, clonidine, and phenylephrine were only weakly inhibited by 0.3 microM prazosin but markedly inhibited by 0.3 microM yohimbine, with a Kb of 1.2 nM, which was similar to the Kd of [3H]yohimbine binding to airway smooth muscle membranes (2.7 nM).     

Location of vascular alpha 2-adrenoceptors in man.

To determine whether vascular postsynaptic alpha 2-adrenoceptors are innervated by sympathetic nerves in man, normal volunteers were infused for 90 min with angiotensin II (A II) and the alpha 2-adrenoceptor agonist alpha-methylnoradrenaline (MNA) in a double-blind cross-over study. Whilst systolic blood pressure returned to baseline within 5 min of terminating the A II infusion it remained elevated 40 min after stopping MNA. The prolongation of the pressor response to MNA, a substrate for neuronal uptake, was probably due to activation of alpha 2-adrenoceptors by MNA re-released from contiguous sympathetic nerve endings. The proximity of alpha 2-adrenoceptors to sympathetic nerve terminals suggests that they could contribute to blood pressure regulation in man.     

Glibenclamide relaxes vascular smooth muscle constriction produced by prostaglandin F2 alpha

The present study has demonstrated: (1) glibenclamide can reduce resting tension in canine cerebral arteries but has no effect on resting tension in the rat aorta; (2) glibenclamide can relax prostaglandin F2 alpha-induced contractions in the rat aorta, and in canine femoral, mesenteric, renal, coronary, basilar and middle cerebral arteries; (3) the relaxation produced by glibenclamide in rat aorta is comparable to that of glyceryl trinitrate and stronger than that of papaverine; (4) canine femoral arteries are less sensitive to glibenclamide than the other arteries; (5) in cerebral arteries glibenclamide was as effective as papaverine, but less effective than glyceryl trinitrate; (6) the actions of glibenclamide on cerebral arteries are not mediated by cGMP as they were not blocked by methylene blue, an inhibitor of guanylate cyclase; (7) the effects of glibenclamide are not endothelium-dependent. The mechanism by which glibenclamide produces relaxation is not clear; while the drug is known to block ATP-dependent potassium channels, in vascular smooth muscle this would cause contraction, not dilation. The action of glibenclamide may be at the level of the receptor or the signal transduction process.     

血管平滑肌钙动员和钙敏感机制在高血压中的改变

血管张力受血管平滑肌(vascular smooth muscle,VSM)收缩水平的调控,是调节外周血管阻力和血压的重要因素。血管激动剂通过细胞内信号转导通路影响肌球蛋白轻链(myosin light chain20,MLC20)磷酸化水平,从而调节VSM的收缩。而MLC20磷酸化水平主要受肌球蛋白轻链激酶(myosin light chain kinase,MLCK)和肌球蛋白轻链磷酸酶(myosin light chain phosphatase,MLCP)的双向调节。前者与细胞内Ca2+浓度有关,后者取决于肌丝对Ca2+敏感性。原发性高血压中Ca2+动员和肌丝Ca2+敏感性增强,导致VSM过度收缩,是高血压重要的病理生理改变之一。所以,对血管平滑肌细胞(vascular smooth muscle cell,VSMC)钙动员和钙敏感机制在高血压中的改变进行研究可为原发性高血压的治疗提供新思路和新靶点。该文就VSMC钙动员和钙敏感机制在原发性高血压中的改变及其发病机制进行阐述。     

Dopamine preferentially stimulates postsynaptic alpha 2-adrenoceptors in the femoral vascular bed, but alpha 1-adrenoceptors in the renal vascular bed of the anaesthetised dog

The decrease in blood flow in response to dopamine (DA) injected intraarterially (i.a.) into the femoral or renal vascular beds was examined in the anaesthetised dog. DA or noradrenaline (NA) were 10 times more potent as vasoconstrictor agents in the femoral than in the renal vasculature. In the femoral bed, the DA induced vasoconstriction was completely resistant to antagonism by prazosin (30-300 micrograms/kg i.v.), but was dose-dependently blocked by the alpha 2-receptor antagonist idazoxan (30-300 micrograms/kg i.v.). In the renal bed the vasoconstrictor effects of DA were resistant to blockade by idazoxan, but were prazosin sensitive indicating that alpha 1-adrenoceptors were involved in this response. The alpha-receptor agonist profile for DA was not modified in the femoral bed after blockade of dilatory D1-receptors with SCH 23390 (0.5 mg/kg i.v. and 0.1 mg/kg per h i.v.). However, this antagonist significantly increased the vasoconstrictor potency for DA in the renal bed. The decrease in femoral blood flow induced by an injection of DA, appears to be mediated by alpha 2-adrenoceptors. In the renal vascular bed where the predominant alpha-adrenoceptor corresponds to the alpha 1-subtype and there are few postsynaptic alpha 2-receptors subserving vasoconstriction, DA can stimulate alpha 1-receptors but this action requires higher doses of agonist than those needed for alpha 2-adrenoceptor stimulation.     

Alpha 1- and alpha 2-adrenoceptors in the smooth muscle of male and female rabbit urethra

[3H]Prazosin and [3H]rauwolscine, specific alpha 1- and alpha 2-antagonists respectively, were used to label alpha-adrenoceptors in membranes from male and female rabbit urethra. In the male rabbit, [3H]prazosin bound with high affinity (Kd = 0.56 nM) to a single population of sites with a capacity of 73 fmol/mg protein. [3H]Rauwolscine bound with a lower affinity (Kd = 2.24 nM) to another single class of sites with a capacity of 41 fmol/mg protein. The order of potencies of various adrenergic compounds in inhibiting radioligand binding suggested that [3H]prazosin and [3H]rauwolscine interacted in the urethra with sites having the characteristics of alpha 1- and alpha 2-adrenoceptors, respectively. In addition, studies on the female rabbit urethra showed that alpha 2-adrenoceptor density and affinity were respectively 6 times higher and 2 times lower than in the male. No significant sex difference was observed for urethral alpha 1-adrenoceptors.     

Changes in platelet alpha2-adrenoceptors in human phaeochromocytoma

Summary In a 44 year-old male with a surgically proven phaeochromocytoma platelet ₂-adrenoceptor density, determined by³H-yohimbine binding, was only 50% of that in an age-matched control group, and plasma catecholamines were elevated. Two weeks after removal of the tumour, platelet ₂-adrenoceptor density and plasma catecholamines had become normal and were not significantly different from the controls. It is concluded that endogenous catecholamines may play an important role in regulation of ₂-adrenoceptor density and hence tissue sensitivity to -adrenergic stimulation in the human being.     

Crk-associated substrate, vascular smooth muscle and hypertension

Hypertension is characterized by vascular smooth muscle constriction and vascular remodeling involving cell migration, hypertrophy and growth. Crk-associated substrate (CAS), the first discovered member of the adapter protein CAS family, has been shown to be a critical cellular component that regulates various smooth muscle functions. In this review, the molecular structure and protein interactions of the CAS family members are summarized. Evidence for the role of CAS in the regulation of vascular smooth muscle contractility is presented. Contraction stimulation induces CAS phosphorylation on Tyr-410 in arterial smooth muscle, creating the binding site for the Src homology (SH) 2/SH3 protein CrkII, which activates neuronal Wiskott-Aldrich syndrome protein (N-WASP)-mediated actin assembly and force development. The functions of CAS in cell migration, hypertrophy and growth are also summarized. Abelson tyrosine kinase (Abl), c-Src, focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (PYK2), protein tyrosine phosphatase-proline, glutamate, serine and threonine sequence protein (PTP-PEST) and SHP-2 have been documented to coordinate the phosphorylation and dephosphorylation of CAS. The downstream signaling partners of CAS in the context of cell motility, hypertrophy, survival and growth are also discussed. These new findings establish the important role of CAS in the modulation of vascular smooth muscle functions. Furthermore, the upstream regulators of CAS may be new biologic targets for the development of more effective and specific treatment of cardiovascular diseases such as hypertension.     

Characterization of postsynaptic alpha1-adrenoceptors in the rabbit iris dilator smooth muscle

Summary Interactions of several alpha-adrenoceptor agonists and antagonists with their receptors were studied in rabbit and guinea pig iris dilator smooth muscle and rabbit aortic strips using pharmacological procedures.In rabbit iris dilators and aortic strips, noradrenaline acted as a full agonist, while oxymetazoline, clonidine and tizanidine acted as partial agonists. The dissociation constants of full and partial agonists in the dilators, calculated after irreversible blockade of a proportion of the active receptors with phenoxybenzamine, were similar to those in the aortic strips. Furthermore, the relative intrinsic efficacies of partial agonists were practically equal in the two tissues, suggesting that these drugs act on the same alpha-adrenoceptors. Since the alpha₂-agonists clonidine and tizanidine had low affinity in the rabbit dilators, the alpha-adrenoceptors in this tissue appear to be of alpha₁-type. These results were further supported by the fact that the pA₂-value of prazosin, an alpha₁-antagonist, was approximately 2 log units higher than that of yohimbine, an alpha₂-antagonist.However, pA₂-values of four quinazolines (prazosin, bunazosin, SM911 and SM2470) and two yohimbine alkaloids (yohimbine and corynanthine) were significantly lower in the rabbit dilator muscle than in rabbit aortic strips. Two imidazoline antagonists (phentolamine and tolazoline) and a phenethanolamine (labetalol) acted on the alpha₁-adrenoceptors in the two tissues nonselectively. These results suggest that alpha₁-adrenoceptors in the rabbit dilator muscle and aortic strips may not be identical and that both selective and nonselective antagonists which act on these receptor sites exist. The pA₂-values of prazosin, SM911 and SM2470 against noradrenaline in guinea pig iris dilator smooth muscle were also significantly lower than those in the rabbit aortic strips, but practically equal to those in the rabbit iris dilators, suggesting that the alpha₁-adrenoceptors in the dilators are the same in these two species.     

Nebivolol induces a hyperpolarizing effect on smooth muscle cells in the mouse renal artery by activation of beta-2-adrenoceptors

Nebivolol is a highly selective beta(1)-adrenoceptor antagonist with vasodilator properties involving the vascular endothelium, but its effect on the smooth muscle cells (SMC) is still unclear. In this paper, we tested the effect of nebivolol on renal artery smooth muscle cells and investigated the cellular mechanism involved. To this purpose, the denuded renal arteries isolated from mice were studied in vitro using the myograph and the nitric oxide (NO) sensor techniques, while the SMC in culture were analyzed by the patch-clamp technique. The myograph technique was used to assay the vasodilator effect of nebivolol on the arterial muscular layer, and to establish the optimal dose of the drug to be tested on single SMC by the patch-clamp technique. Using both the myograph and the patch-clamp techniques, we examined the potential contribution of beta(2)-adrenoceptors and Ca(2+)-activated K(+) channels to the nebivolol-induced effects, by exposing the denuded arteries and SMC cultures to specific inhibitors such as butoxamine (100 micromol/l), tetraethylammonium (TEA, 1 mmol/l), and iberiotoxin (100 nmol/l). The direct measurement of NO using the NO sensor enabled us to evaluate if nebivolol induces/or not the release of NO in denuded renal arteries. The results of this study show that nebivolol exerts vasodilator effects on the SMC in the denuded renal arteries and the maximal response is achieved at a concentration of 50 micromol/l. Nebivolol effects involve binding to the beta(2)-adrenoceptors and the subsequent activation of Ca(2+)-activated K(+) channels in SMC, with no contribution of NO. Taken together, the study brings new insights into the mechanism underlying the nebivolol-induced arterial vasodilation.     

Influence of Bay K 8644 on vascular responses mediated by alpha 1- and alpha 2-adrenoceptors

1. The calcium channel activator Bay K 8644 increased the potency of noradrenaline in cat middle cerebral (alpha 2-adrenoceptors) and mesenteric (atypical or mixed alpha 1- and alpha 2-adrenoceptor population) arteries, but not in rat middle cerebral and mesenteric arteries (alpha 1-adrenoceptors). 2. In cat arteries, exposure to 15 mM K+ solution shifted the noradrenaline concentration-response curve to the left in an almost identical manner as did Bay K 8644. 3. Bay K 8644 completely reversed the relaxation produced by nifedipine in K+-contracted cat middle cerebral arteries, whereas the relaxation induced by verapamil, diltiazem or flunarizine was only partially reversed. This suggests a specific interaction between Bay K 8644 and the dihydropyridine receptors on the calcium channels. 4. It is concluded that the degree to which noradrenaline promotes calcium influx through membrane channels is at least partly related to the alpha-adrenoceptor subtype mediating the response.     

Alpha 1- and alpha 2-adrenoceptors in the smooth muscle of isolated rabbit urinary bladder and urethra

The present experiment was undertaken to determine the existence of alpha 1- and alpha 2-adrenoceptors in the smooth muscle of the rabbit bladder dome, trigone and proximal urethra. In the dome pretreated with propranolol (10(-6) M), phenylephrine (10(-5) M-10(-3) M) and norepinephrine (10(-7) M-10(-5) M) caused only a small contraction but norepinephrine, only at high concentrations (10(-4) M-10(-3) M), produced a small relaxation. Clonidine, however, had no effect on the dome. In both trigone and urethra, phenylephrine, clonidine and norepinephrine caused dose-dependent contractions. The contractile response to phenylephrine or norepinephrine was significantly greater than that to clonidine in the trigone but no such difference was observed in the urethra. Prazosin (10(-8) M-10(-6) M, alpha 1-adrenoceptor antagonist) produced a rightward shift of the phenylephrine and clonidine dose-response curve in both the trigone and urethra. Yohimbine (10(-8) M-10(-6) M, alpha 2-adrenoceptor antagonist) inhibited the response to clonidine without significantly affecting the responses to phenylephrine. These studies indicated that alpha 1- and alpha 2-adrenoceptors are present in both the trigone and proximal urethra. In the dome, only alpha 1-adrenoceptors are sparsely distributed.     

Role of Ca2+ in vascular smooth muscle contractions induced by Phoneutria nigriventer spider venom.

Phoneutria nigriventer venom (PNV) contracts vascular tissues and increases arterial blood pressure. This study aimed to investigate the mechanisms involved on PNV-induced contractions of rabbit mesenteric and celiac arteries. Strips of mesenteric and celiac arteries were suspended in a cascade system and superfused with warmed and oxygenated Krebs solution. PNV was dialyzed in order to exclude the participation of biogenic amines in the contractions elicited by the venom. Noradrenaline (NA, 30-300 pmol), PNV (1-10 microg), Bay K-8644 (0.3-3 nmol) and KCl (10-100 micromol) dose-dependently contracted the preparations. Ca(2+)-free solution reduced by 38 and 83% the PNV-induced contractions of mesenteric and celiac arteries, respectively. Subsequent infusion of EGTA (0.2 mM) suppressed the residual contractions. Nifedipine (1 microM) and verapamil (10 microM) abolished PNV- and Bay K-8644-evoked contractions, whereas those induced by NA were reduced to a lesser extent. Lanthanum chloride (0.2 mM) inhibited by 75-90% the mesenteric and celiac contractions mediated by PNV. Caffeine (2 mM) fully blocked contractions induced by NA (95% mean inhibition), but only partly reduced those induced by PNV (35% mean inhibition). Ryanodine (10 microM) inhibited by 50% the contractions evoked by NA, but had no effect on the PNV-induced contractions in both tissues. Our findings indicate that PNV contracts vascular smooth muscle mainly due to increased influx of Ca(2+) from extracellular sources.     

Catecholamines act as alpha 2-adrenoceptors to cause contraction of circular smooth muscle of guinea-pig stomach

Circular smooth muscle strips taken from the body region of the guinea-pig stomach responded to dopamine and noradrenaline with contraction at lower concentrations followed by relaxation at higher concentrations. A beta-adrenoceptor-mediated relaxation response was excluded by propranolol treatment and this allowed the remaining alpha-adrenoceptor involvement with relaxation and contraction to be incisively differentiated in terms of two distinct alpha-adrenoceptor mechanisms. Thus, the relaxation responses to the catecholamines were mimicked by phenylephrine and antagonized by prazosin, phentolamine but not by yohimbine or rauwolscine. In contrast, the catecholamine-induced contractions were mimicked by clonidine and antagonized by yohimbine and phentolamine but not by prazosin. It is therefore concluded that the alpha mechanisms via which dopamine and noradrenaline are able to relax and contract the circular smooth muscle from the body region of guinea-pig stomach are of the alpha 1- and alpha 2-type respectively.