Sympathetic nerve stimulation induces local endothelial Ca2+ signals to oppose vasoconstriction of mouse mesenteric arteries

It is generally accepted that the endothelium regulates vascular tone independent of the activity of the sympathetic nervous system. Here, we tested the hypothesis that the activation of sympathetic nerves engages the endothelium to oppose vasoconstriction. Local inositol 1,4,5-trisphosphate (IP(3))-mediated Ca(2+) signals ("pulsars") in or near endothelial projections to vascular smooth muscle (VSM) were measured in an en face mouse mesenteric artery preparation. Electrical field stimulation of sympathetic nerves induced an increase in endothelial cell (EC) Ca(2+) pulsars, recruiting new pulsar sites without affecting activity at existing sites. This increase in Ca(2+) pulsars was blocked by bath application of the α-adrenergic receptor antagonist prazosin or by TTX but was unaffected by directly picospritzing the α-adrenergic receptor agonist phenylephrine onto the vascular endothelium, indicating that nerve-derived norepinephrine acted through α-adrenergic receptors on smooth muscle cells. Moreover, EC Ca(2+) signaling was not blocked by inhibitors of purinergic receptors, ryanodine receptors, or voltage-dependent Ca(2+) channels, suggesting a role for IP(3), rather than Ca(2+), in VSM-to-endothelium communication. Block of intermediate-conductance Ca(2+)-sensitive K(+) channels, which have been shown to colocalize with IP(3) receptors in endothelial projections to VSM, enhanced nerve-evoked constriction. Collectively, our results support the concept of a transcellular negative feedback module whereby sympathetic nerve stimulation elevates EC Ca(2+) signals to oppose vasoconstriction.     

Dopaminergic Receptors Linked to Adenylate Cyclase in Human Cerebromicrovascular Endothelium

Cultured endothelium derived from three fractions of human cerebral microvessels was used to characterize dopamine (DA) receptors linked to adenylate cyclase activity. DA or D1 agonist, (+/-)-SKF-82958 hydrobromide, stimulated endothelial cyclic AMP formation in a dose-dependent manner. The selective D1 antagonist, (+/-)SCH-23390, inhibited in a dose-dependent manner the production of cyclic AMP induced by DA. The affinity for the D1 receptor appeared to be greater in endothelium derived from large and small microvessels than from capillaries. Cholera toxin ADP-ribosylation of Gs proteins abolished the DA stimulatory effect on endothelial adenylate cyclase, whereas pertussis toxin ADP-ribosylation enhanced the DA-inducible formation, indicating the presence of both D1 and D2 receptors. Agonists of alpha 1-adrenergic receptors (phenylephrine, 6-fluoronorepinephrine) or serotonin (5-HT), which stimulated the production of cyclic AMP, had no additive effect on DA-stimulated cyclic AMP formation. Incubation of these agents with DA produced the same or lower levels of cyclic AMP as compared to that formed by DA alone. The effect of alpha 1-adrenergic agonists or 5-HT on DA production of cyclic AMP was partially prevented by the D2 antagonist, S(-)-sulpiride, or ketanserin (5-HT2 greater than alpha 1 greater than H1 antagonists), respectively. These findings represent the first demonstration of D1- (stimulatory) and D2- (inhibitory) receptors linked to adenylate cyclase in microvascular endothelium derived from human brain. The data also indicate that dopaminergic receptors can interact with either alpha 1-adrenergic or or 5-HT receptors in endothelium on the adenylate cyclase level.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presynaptic alpha 2 -adrenergic stimulation leads to growth hormone release in the dog

In previous studies we have shown that the alpha 2 -adrenergic receptor agonist clonidine (CLON) releases growth hormone (GH) in conscious dogs, an effect abolished by the selective alpha 2-receptor antagonist yohimbine (YOH) and by reserpine, but not by the alpha 1-receptor antagonist prazosin (1). In the present work intravenous (iv) administration of CLON in conscious dogs evoked a dose-related rise in plasma GH at doses of 2-8 /micrograms/Kg, but not at 16 and 32 /micrograms/Kg. Acute pretreatment with the selective inhibitor of norepinephrine (NE) synthesis, DU-18288, or with a potent antagonist of presynaptic alpha 2-receptors, mianserin abolished the GH rise induced by CLON (4 /micrograms/Kg iv). In contrast, a 10-day-pretreatment with YOH greatly enhanced the GH-releasing effect of CLON (2 /micrograms/Kg iv). In all these data indicate that in the dog: 1) CLON induces GH release via activation of alpha 2-adrenergic receptors; 2) these receptors are likely located on presynaptic sites [experiments with reserpine (1), DU-18288, mianserin, dose-response curve with CLON 2-32/micrograms/kg iv]; 3) the adrenergic receptors involved in GH release exhibit supersensitivity upon (YOH-induced) chronic pharmacologic denervation. In view of the inhibitory action of presynaptic alpha 2-adrenergic receptors (autoreceptors) on NE function, it may be envisioned that in the dog noradrenergic activation is inhibitory and not stimulatory to GH release.     

Norepinephrine- and phorbol ester-induced phosphorylation of alpha(1a)-adrenergic receptors. Functional aspects

Maximal adrenergic responses in Rat-1 fibroblasts expressing alpha(1a)-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of alpha(1b)-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on alpha(1a)-adrenoreceptor phosphorylation and coupling to G proteins were studied. Both stimuli lead to dose-dependent receptor phosphorylation. Interestingly, protein kinase C activation affected to a much lesser extent the actions of alpha(1a)-adrenergic receptors than those of the alpha(1b) subtype (norepinephrine elicited increases in calcium in whole cells and [(35)S]GTPgammaS binding to membranes). Basal phosphorylation of alpha(1a)-adrenergic receptors was much less than that observed with the alpha(1b) subtype. The carboxyl terminus seems to be the main domain for receptor phosphorylation. Therefore, chimeric receptors, where the carboxyl-terminal tails of alpha(1a) and alpha(1b) adrenergic receptors were exchanged, were constructed and expressed. alpha(1a)-Adrenoreceptors wearing the carboxyl tail of the alpha(1b) subtype had a high basal phosphorylation and displayed a strong phosphorylation in response to norepinephrine and phorbol esters. Our results demonstrate that stimulation of alpha(1a)-adrenergic receptor, or activation of protein kinase C, leads to alpha(1a)-adrenergic receptor phosphorylation. alpha(1a)-Adrenoreceptors are affected to a much lesser extent than alpha(1b)-adrenoreceptors by protein kinase C activation.     

Vipoxin. A protein from Russell's viper venom with high affinity for biogenic amine receptors

We have purified a small, basic protein with high affinity and selectivity for biogenic amine receptors to apparent homogeneity from the venom of Russell's viper (Vipera russelli). This protein, which we designate "vipoxin," has Mr = 13,000, and appears to exist in solution as a single polypeptide chain. It may contain 2 atypical amino acids. Vipoxin inhibits in a dose-dependent manner the binding of 3H-ligands to biogenic amine receptors, with apparent Ki values of 3 nM at alpha 1-adrenergic receptors, 5 nM at alpha 2-adrenergic receptors, 15 nM at dopamine receptors, and 32 nM at serotonin receptors. At concentrations up to 1 microM, vipoxin is inactive at beta-adrenergic, histamine, nicotinic cholinergic, muscarinic cholinergic, adenosine, gamma-aminobutyric acid, benzodiazepine, or opiate receptor binding sites. The effect of vipoxin is essentially irreversible over 20 h at alpha 1- and alpha 2-adrenergic receptors and serotonin receptors and is only slightly reversible at dopamine receptors. Norepinephrine protects alpha-adrenergic receptors from inhibition by vipoxin, while dopamine does not. Vipoxin has no protease activity but does have phospholipase A2 activity, which cannot account for its action on receptors, since receptor binding is assayed in the presence of 1 mM CoSO4 which completely and selectively inhibits the phospholipase activity. Other phospholipases A2 in the same venom lack vipoxin's action on receptors. In physiologic experiments, vipoxin behaves as an agonist at alpha 2-adrenergic receptors in the rat vas deferens and is over an order of magnitude more potent than norepinephrine itself. At alpha 1-adrenergic receptors, it is neither a simple agonist nor an antagonist, but selectively potentiates norepinephrine. Vipoxin may be a useful tool for biogenic amine receptor characterization.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

Prostacyclin synthesis elicited by cholinergic agonists is linked to activation of M2 alpha and M2 beta muscarinic receptors in the rabbit aorta

This study was conducted to investigate the subtypes of muscarinic receptors involved in the action of cholinergic agents on prostacyclin synthesis in the rabbit aorta. Prostacyclin production measured as 6-keto-PGF1 alpha was assessed after exposing the aortic rings to different cholinergic agents. Acetylcholine (ACh) (M1 and M2 agonist) (1-10 microM) and arecaidine proparagyl ester (APE) (M2 selective agonist) (1-10 microM) enhanced 6-keto-PGF1 alpha output in a concentration-dependent manner. A selective M1 receptor agonist, McN-A-343, at 1 microM-1 mM did not alter 6-keto-PGF1 alpha output. ACh- and APE induced increases in 6-keto-PGF1 alpha output were attenuated by the M1/M2 antagonist atropine (0.1 microM), M2 alpha antagonist (AF-DX 116), (0.1-1.0 microM), and by selective M2 beta antagonist, hexahydro-sila-difendiol (HHSiD) (0.1-1.0 microM), but not by the M1 antagonist pirenzepine (1.0 microM). 6-Keto-PGF1 alpha output elicited by ACh- or APE was not altered by the adrenergic receptor antagonists phentolamine and propranolol or by the nicotinic receptor blocker hexamethonium. Similarly, the arachidonic acid- or norepinephrine induced 6-keto-PGF1 alpha accumulation was not altered by these muscarinic receptor antagonists. Indomethacin, a cyclooxygenase inhibitor, prevented arachidonic acid, ACh- or APE induced 6-keto-PGF1 alpha output. Removal of the endothelium abolished the production of 6-keto-PGF1 alpha elicited by ACh, APE, bradykinin, and calcium ionophore A 23187, but not that induced by angiotensin II, K+ or norepinephrine. These data suggest that vascular prostaglandin generation elicited by cholinergic agonists is mediated via activation of M2 alpha and M2 beta but not M1 muscarinic receptors, which are most likely located on the endothelium.     

α2-Adrenergic stimulation enhances growth hormone secretion in the dog: A presynaptic mechanism?

Intravenous administration of clonidine (CLO), (2,4 and 8/micrograms/Kg), a predominantly alpha 2-adrenergic receptor agonist, induced in unanesthetized dogs clear-cut and dose-related rises in plasma GH (cGH) levels. Pretreatment with the selective antagonist of alpha 1-adrenergic receptors prazosin (0.1 mg/Kg iv) left unaltered the cGH rise induced by 4/micrograms/Kg of CLO whilst blockade of alpha 2-adrenergic receptors by yohimbine (2.5 mg/Kg iv) completely prevented it. In dogs treated 24 h previously, with reserpine (0.5 mg/Kg iv), a depletor of brain catecholamine stores, CLO was ineffective to stimulate cGH release. These data indicate that in the dog the GH-releasing effect of CLO occurs via stimulation of alpha 2-adrenergic receptors and suggest that the latter are located presynaptically in relation to norepinephrine neurons.     

Studies on the mechanism of inhibition of glucose-stimulated insulin secretion by noradrenaline in rat islets of Langerhans.

Noradrenaline (norepinephrine) was shown to be a potent inhibitor of glucose-induced insulin release from rat pancreatic islets, with half-maximal inhibition of the secretory response to 20 mM-glucose occurring at approx. 0.3 microM, and complete suppression of the response occurring at 4 microM-noradrenaline. Inhibition of insulin secretion by noradrenaline was antagonized by the alpha 2-adrenergic antagonist yohimbine (half maximally effective dose approximately 1 microM), but was largely unaffected by the alpha 1-adrenergic antagonist prazosin at concentrations up to 50 microM, suggesting that the response was mediated by alpha 2-adrenergic receptors. Noradrenaline significantly reduced the extent of 45Ca2+ accumulation in glucose-stimulated islets, although as much as 5 microM-noradrenaline was required for 50% inhibition of this response. The ability of noradrenaline to inhibit islet-cell 45Ca2+ uptake was totally abolished in media containing 1 mM-dibutyryl cyclic AMP, suggesting that the response may have been secondary to lowering of islet cyclic AMP. Under these conditions, however, noradrenaline was still able to inhibit insulin secretion maximally. The data suggest that the site(s) at which noradrenaline acts to mediate inhibition of insulin secretion in rat islets lies distal to both islet-cell cyclic AMP accumulation and Ca2+ uptake.     

Extracellular ATP induces Ca2+ transients in cardiac myocytes which are potentiated by norepinephrine

Isolated rat ventricular cardiac myocytes loaded with the fluorescent calcium indicator fura2 showed significant changes in intracellular calcium concentrations upon exposure to greater than 1 microM ATP (EC50 = 7.4 +/- 1.3 microM, n = 4, SE), suggesting that extracellular ATP may have an important influence on myocardial contractility. The response was found to be highly ATP specific and required extracellular calcium. Furthermore, 30 s pretreatment of the cells with 0.2-1 microM norepinephrine decreased the concentration of ATP required for the Ca2+ transient, shifting the EC50 for ATP to 1.7 +/- 0.1 microM (n = 3, SE). beta-Propranolol (a beta 1-receptor antagonist) prevented potentiation, whereas phentolamine (an alpha 1-receptor antagonist) did not, indicating that regulation is through the beta 1-adrenergic receptor. ATP and norepinephrine released locally from sympathetic neurons may act in concert through the ATP and beta 1-adrenergic receptors to regulate myocardial calcium homeostasis.     

Identification of alpha 2-adrenergic receptor sites in human retinoblastoma (Y-79) and neuroblastoma (SH-SY5Y) cells

The existence of specific alpha 2-adrenergic receptor sites has been shown in human retinoblastoma (Y-79) and neuroblastoma (SH-SH5Y) cells using direct radioligand binding. [3H]Rauwolscine, a selective alpha 2-adrenergic receptor antagonist, exhibited high affinity, saturable binding to both Y-79 and SH-SY5Y cell membranes. The binding of alpha 1 specific antagonist, [3H]Prazocine, was not detectable in either cell type. Competition studies with antagonists yielded pharmacological characteristics typical of alpha 2-adrenergic receptors: rauwolscine greater than yohimbine greater than phentolamine greater than prazocine. Based on the affinity constants of prazocine and oxymetazoline, it appears that Y-79 cells contain alpha 2A receptor, whereas SH-SY5Y cells probably represent a mixture of alpha 2A and alpha 2B receptors. alpha 2-agonists clonidine and (-)epinephrine inhibition curves yielded high and low affinity states of the receptor in SH-SY5Y cells. Gpp(NH)p and sodium ions reduced the proportion of high affinity sites of alpha 2 receptors. These two neuronal cell lines of human origin would prove useful in elucidating the action and regulation of human alpha 2-adrenergic receptors and their interaction with other receptor systems.     

Neuropeptide Y Inhibits β-Adrenergic Agonist– and Vasoactive Intestinal Peptide–Induced Cyclic AMP Accumulation in Rat Pinealocytes Through Pertussis Toxin–Sensitive G Protein

The effects of neuropeptide Y (NPY) on pineal gland cyclic AMP (cAMP) accumulation were investigated using dispersed pinealocytes from rats. NPY inhibited the intracellular cAMP accumulation stimulated by isoproterenol and norepinephrine in a dose-dependent manner during a 10-min incubation of pinealocytes. NPY (1 x 10(-7) M) also inhibited vasoactive intestinal peptide (VIP)- and cholera toxin-induced cAMP accumulation. The inhibitory effect of NPY on isoproterenol-induced cAMP accumulation was completely abolished by a 5-h pretreatment of pinealocytes with 1 microgram/ml of pertussis toxin (PT). These results suggest that NPY participates in modulation of cAMP production in the rat pineal gland through PT-sensitive G protein. Yohimbine, an alpha 2-adrenergic antagonist, blocked NPY inhibition of isoproterenol-stimulated cAMP accumulation. On the other hand, the alpha 2-adrenergic agonist clonidine by itself did not affect cAMP accumulation stimulated by isoproterenol but significantly potentiated NPY action. The present study demonstrates that NPY inhibits beta-adrenergic or VIPergic stimulation of the pineal gland cAMP accumulation. The inhibitory effect of NPY is mediated through PT-sensitive G protein. Our results also suggest that NPY exerts its action to affect alpha 2-adrenoceptor function.     

Participation of endothelial cells in the protein C-protein S anticoagulant pathway: the synthesis and release of protein S

The protein C-protein S anticoagulant pathway is closely linked to the endothelium. In this paper the synthesis and release of the vitamin K-dependent coagulation factor protein S is demonstrated. Western blotting, after SDS PAGE of Triton X-100 extracts of bovine aortic endothelial cells grown in serum-free medium, demonstrated the presence of protein S. A single major band was observed at Mr approximately 75,000, closely migrating with protein S purified from plasma absent from cells treated with cycloheximide. Metabolic labeling of endothelial cells with [35S]methionine confirmed de novo synthesis of protein S. Using a radioimmunoassay, endothelium was found to release 180 fmol/10(5) cells per 24 h and contain 44 fmol/10(5) cells of protein S antigen. Protein S released from endothelium was functionally active and could promote activated protein C-mediated factor Va inactivation on the endothelial cell surface. Warfarin decreased secretion of protein S antigen by greater than 90% and increased intracellular accumulation by almost twofold. Morphological studies demonstrated intracellular protein S was in the Golgi complex, concentrated at the trans face, rough endoplasmic reticulum, lysosomes, and in vesicles at the periphery. In contrast, protein S was not found in vascular fibroblasts or smooth muscle cells. A pool of intracellular protein S could be released rapidly by the calcium ionophore A23187 (5 microM). This effect was dependent on the presence of calcium in the culture medium and could be blocked by LaCl3, which suggests that cytosolic calcium flux may be responsible for protein S release. These results demonstrate that endothelial cells, but not the subendothelial cells of the vessel wall, can synthesize and release protein S, which indicates a new mechanism by which the inner lining of the vessel wall can contribute to the prevention of thrombotic events.     

Hippocampal astrocytes exhibit Ca2+-elevating muscarinic cholinergic and histaminergic receptors in situ

Recent findings suggest that astrocytes respond to neuronally released neurotransmitters with Ca2+ elevations. These Ca2+ elevations may trigger astrocytes to release glutamate, affecting neuronal activity. Neuronal activity is also affected by modulatory neurotransmitters that stimulate G protein-coupled receptors. These neurotransmitters, including acetylcholine and histamine, might affect neuronal activity by triggering Ca2+-dependent release of neurotransmitters from astrocytes. However, there is no physiological evidence for histaminergic or cholinergic receptors on astrocytes in situ. We asked whether astrocytes have these receptors by imaging Ca2+-sensitive dyes sequestered by astrocytes in hippocampal slices. Our results show that immunocytochemically identified astrocytes respond to carbachol and histamine with increases in intracellular free Ca2+ concentration. The H1 histamine receptor antagonist chlorpheniramine inhibited responses to histamine. Similarly, atropine and the M1-selective muscarinic receptor antagonist pirenzepine inhibited carbachol-elicited responses. Astrocyte responses to histamine and carbachol were compared with responses elicited by alpha1-adrenergic and metabotropic glutamate receptor agonists. Individual astrocytes responded to different subsets of receptor agonists. Ca2+ oscillations were the prevalent response pattern only with metabotropic glutamate receptor stimulation. Finally, functional alpha1-adrenergic receptors and muscarinic receptors were not detected before postnatal day 8. Our data show that astrocytes have acetylcholine and histamine receptors coupled to Ca2+. Given that Ca2+ elevations in astrocytes trigger neurotransmitter release, it is possible that these astrocyte receptors modulate neuronal activity.     

Subcellular distribution of alpha 1-adrenergic receptors in rat liver

The distribution of alpha 1-adrenergic receptors in rat liver subcellular fractions was studied using the alpha 1-adrenergic receptor ligand [3H]prazosin. The highest number of [3H]prazosin binding sites was found in a plasma membrane fraction followed by 2 Golgi and a residual microsomal fraction, the numbers of binding sites were 1145, 845, 629 and 223 fmol/mg protein, respectively. When the binding in these fractions was compared with the activity of plasma membrane 'marker' enzymes in the same fractions a relative enrichment of [3H]prazosin binding sites was found in the residual microsomes and one of the Golgi fractions. Photoaffinity labelling with 125I-arylazidoprazosin in combination with SDS-polyacrylamide gel electrophoresis revealed the specific binding to 40 and 23 kDa entities in a Golgi fraction, while in plasma membranes the binders had an apparent molecular mass of 36 and 23 kDa. When [3H]prazosin was injected in vivo into rat portal blood followed by subcellular fractionation of liver, a pattern of an initial rapid decline and thereafter a slow decline of radioactivity was noted in all fractions. Additionally, in the two Golgi fractions a transient accumulation of radioactivity occurred between 5 and 10 min after the injection. The ED50 values for displacement of [3H]prazosin with adrenaline was lowest in the plasma membrane fraction, followed by the residual microsomes and Golgi fractions, the values were 10(-6), 10(-5) and 10(-4) mol/l, respectively. On the basis of lack of correlation between distribution of alpha 1-adrenergic antagonist binding and adenylate cyclase activity, differences in the molecular mass of alpha 1-adrenergic antagonist binders, differences in the kinetics of in vivo binding and accumulation of [3H]prazosin and also differences in agonist affinity between plasma membrane and Golgi fractions, it is concluded that alpha 1-adrenergic receptors are localized to low-density intracellular membranes involved in receptor biosynthesis and endocytosis.     

Regulation of adrenergic receptor function by phosphorylation. I. Agonist-promoted desensitization and phosphorylation of alpha 1-adrenergic receptors coupled to inositol phospholipid metabolism in DDT1 MF-2 smooth muscle cells

Continuous exposure of DDT1 MF-2 smooth muscle cells to 10-100 microM norepinephrine results in a dramatic attenuation of the ability of norepinephrine to stimulate inositol phospholipid hydrolysis via alpha 1-adrenergic receptors (alpha 1-AR). In addition to the functional desensitization, norepinephrine exposure also reduces the number of accessible cell surface alpha 1-AR as assayed by [3H]prazosin binding at 4 degrees C. Desensitization of the cells with norepinephrine results in an increase in the phosphorylation of the Mr 80,000 alpha 1-AR ligand binding peptide (2.4 +/- 0.2 mol of 32P per mol of alpha 1-AR; n = 5) when compared to control cells (1.1 +/- 0.1 mol of 32P per mol of alpha 1-AR; n = 5). The time courses of these three processes are all comparable being half-maximal within 1-2 min. These norepinephrine-promoted effects can be prevented by the alpha 1-AR receptor antagonist phentolamine indicating that they are mediated via the alpha 1-AR. Treatment of cells with the vasoactive peptide bradykinin (10 microM) induces desensitization of alpha 1-AR function similar to that induced by tumor-promoting phorbol ester treatment (Leeb-Lundberg, L. M. F., Cotecchia, S., Lomasney, J. W., DeBernardis, J. F., Lefkowitz, R. J., and Caron, M. G. (1985) Proc. Natl. Acad. Sci. USA 82, 5651-5655). Both treatments also result in phosphorylation of the alpha 1-AR, with stoichiometries of 1.7 +/- 0.1 (bradykinin; n = 5) and 3.6 +/- 0.1 (PMA; n = 5) mol of 32P/mol of alpha 1-AR. However, neither phorbol esters nor bradykinin reduce the number of accessible cell surface alpha 1-AR. Similar phosphopeptide maps are obtained from tryptic phosphopeptides generated from phosphorylated alpha 1-AR derived from cells treated with norepinephrine, phorbol 12-myristate 13-acetate, and bradykinin. Phosphoamino acid analysis reveals that the various agents induce phosphorylation on both serine and threonine residues. Thus, phosphorylation of receptors linked to the inositol phospholipid/Ca2+ signaling pathway may represent an important mechanism of regulation of receptor responsiveness.     

Clonidine-induced coronary vasodilatation in isolated guinea pig heart is not mediated by endothelial alpha2 adrenoceptors.

Functional role of endothelial alpha(2)-adrenoceptor in coronary circulation remains unclear. Clonidine, an agonist of alpha(2)-adrenoceptors, was reported to induce coronary vasodilatation via stimulation of endothelial alpha(2)-adrenoceptors or coronary vasoconstriction involving vascular smooth muscle alpha(2)-adrenoceptors. Moreover, H(2) receptor-dependent responses to clonidine were described. Here, we reassess the contribution of endothelial alpha(2)-adrenoceptor and H(2) receptors to coronary flow and contractility responses induced by clonidine in the isolated guinea pig heart. We found that clonidine (10(-9) - 10(-6) M) produced concentration-dependent coronary vasoconstriction without a significant change in contractility. This response was inhibited by the alpha(1)/alpha(2)-adrenoceptor antagonist - phentolamine (10(-5) M) and the selective alpha(2)-adrenoceptor antagonist yohimbine (10(-6) M), but it was not changed by the selective alpha(1)-adrenoceptor antagonist prazosin (10(-6) M). In the presence of nitric oxide synthase inhibitor, L-NAME (10(-4) M) the clonidine-induced vasoconstriction was potentiated. Clonidine at high concentrations of 10(-5) - 3 x 10(-5) M produced coronary vasodilatation, and an increase in myocardial contractility. These responses were abolished by a selective H(2)-receptor antagonist, ranitidine (10(-5) M), but not by phentolamine (10(-5) M). We conclude that in the isolated guinea pig heart, clonidine-induced vasoconstriction is mediated by activation of smooth muscle alpha(2)-adrenoceptors whereas clonidine-induced coronary vasodilatation is mediated by activation of vascular H(2) histaminergic receptors. Accordingly, endothelial alpha(2)-adrenoceptors does not seem to play a major role in coronary flow response induced by clonidine.     

Guanine nucleotides mediate stimulatory and inhibitory effects on cerebral-cortical membrane phospholipase C activity.

alpha 2-Adrenergic receptors on NG 108 15 cell membranes were identified by [3H]rauwolscine binding: Bmax. = 661 +/- 81 fmol/mg of protein, Kd = 6.9 +/- 2.5 nM (mean +/- S.E.M., n = 6). On intact cells, stimulation of these receptors by (-)-adrenaline inhibited the prostaglandin-E1-stimulated adenylate cyclase activity by about 60%. The effect of (-)-adrenaline was pertussis-toxin-sensitive, indicating the involvement of an inhibitory G protein. (-)-Adrenaline/[3H]rauwolscine competition-binding experiments revealed that only 50% of the alpha 2 receptors were coupled to G proteins (i.e. displayed high agonist affinity). Pre-treatment of the cells with 20 microM-(-)-adrenaline provoked homologous desensitization of the alpha 2 receptors. The alpha 2-adrenergic response decreased after a time lag of about 2 h, to reach a minimum after 12 h. The bradykinin and muscarinic responses were not affected. The alpha 2-receptor concentration decreased without time lag. The high-agonist-affinity sites disappeared more rapidly (t1/2 = 42 min) than did the low-affinity uncoupled sites (t1/2 approx. 20 h). In contrast, pertussis-toxin-mediated [32P]ADP-ribosylation of inhibitory G proteins was unaffected by the pre-treatment. Pretreatment of intact NG 108 15 cells with 1 microM-phorbol 12-myristate 13-acetate (PMA) provoked a rapid decrease of the alpha 2-adrenergic response. The effect was nearly complete after 40 min. PMA also decreased the bradykinin response, suggesting a heterologous desensitization process. The alpha 2-receptor concentration, the (-)-adrenaline competition-binding curves and the pertussis- and cholera-toxin-mediated [32P]ADP-ribosylation of their respective G proteins were not affected.     

Pre- and postjunctional alpha(2)-adrenergic receptors in fetal and adult ovine cerebral arteries

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered alpha(2)-adrenergic receptor (alpha(2)-AR) density and/or affinity. In fetal (approximately 140 days) and adult sheep, we measured alpha(2)-AR density and affinity with the antagonist [(3)H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the alpha(2)-AR antagonists yohimbine and idazoxan and contractile responses to the alpha(2)-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, alpha(2)-AR density was 201 +/- 18 and 52 +/- 6 fmol/mg protein, respectively (P < 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, 10(-7) M yohimbine significantly decreased the pD(2) for NE-induced tension in the presence of 3 x 10(-5) M cocaine, 10(-5) M deoxycorticosterone, and 10(-6) M tetrodotoxin. In fetal, but not adult, MCA, UK-14304 induced a significant decrease in pD(2) for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of 10(-6) M idazoxan to block alpha(2)-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, alpha(2)-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional alpha(2)-AR.