Effects of alpha 1 and alpha 2 activation of adrenergic receptors on aqueous humor dynamics

Effects of phenylephrine (alpha 1-adrenergic agonist), prazosin (alpha 1-adrenergic antagonist), clonidine (alpha 2-adrenergic agonist), and yohimbine (alpha 2-adrenergic antagonist) on aqueous humor (AH) dynamics were studied with a cat eye model. Phenylephrine (130 microgram/ml) inhibited AH outflow (67% at 90 min. period) more than AH formation (26% at the same period) indicating the intraocular pressure (IOP) might be raised by the administration of phenylephrine. Prazosin (0.1 microgram/ml) produced effects opposite to those of phenylephrine (55% reduction of AH formation and 25% reduction of AH outflow at 3 hr. period) suggesting the alpha 1-adrenergic receptor is responsible for increases rather than decreases of IOP. Both clonidine (10 microgram/ml) and yohimbine (0.1-1.0 microgram/ml) inhibited AH formation (60% inhibition) more than AH outflow (no inhibition for clonidine and 40% inhibition for yohimbine) to lower IOP. The conventional theory of receptor antagonism does not seem to function at alpha 2-receptor sites.     

The interaction of the enantiomers of carvedilol with alpha 1- and beta 1-adrenoceptors

The stereoselectivity of carvedilol, a novel beta-adrenoceptor antagonist and vasodilator with one asymmetric carbon atom, was examined at alpha 1- and beta 1-adrenoceptors in vitro and in vivo. (-)-(S)-Carvedilol is a potent, competitive antagonist of the beta 1-adrenoceptor-mediated positive chronotropic response to isoproterenol in guinea pig atrium, with a dissociation constant (KB) of 0.4 nM. (+)-(R)-Carvedilol was more than 100-fold less potent than the (-)-S-enantiomer as an antagonist of beta 1-andrenoceptors, having a KB of approximately 45 nM. Consistent with these findings (-)-(S)-carvedilol (0.1 mg/kg, i.v.) produced a 25-fold rightward shift in the beta 1-adrenoceptor-mediated positive chronotropic response to isoproterenol in pithed rats, whereas the (+)-R-enantiomer had no beta 1-adrenoceptor blocking activity in vivo at this dose. In contrast to the marked degree of stereoselectivity observed at beta 1-adrenoceptors, both (-)-(S)- and (+)-(R)-carvedilol produced equal antagonism of the alpha 1-adrenoceptor-mediated vasoconstrictor response to norepinephrine in rabbit aorta, with KB values of 14 and 16 nM, respectively. Furthermore, in the pithed rat, the alpha 1-adrenoceptor-mediated pressor dose-response curve to cirazoline was shifted approximately 6-fold to the right by both the (+)-R- and (-)-S-enantiomers of carvedilol at a dose of 1 mg/kg, i.v. In anesthetized spontaneously hypertensive rats, (-)-(S)-carvedilol was 6-fold more potent as an antihypertensive than (+)-(R)-carvedilol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta 2-adrenergic stimulation of androgen production by cultured mouse testicular interstitial cells

The present studies characterized the beta-receptor subtype involved in androgen production by cultured mouse testicular interstitial cells and explored the possible stimulation of androgen release by alpha-adrenergic agonists. During a 3-hour incubation period, LH and a non-specific beta-adrenergic agonist, L-isoproterenol steadily increased androgen production with a similar time-course. Isoproterenol, epinephrine, norepinephrine and a specific beta 2-receptor agonist, salbutamol stimulated androgen release in a concentration-dependent manner. The concentrations of the agonists required for half-maximum stimulation (EC50) were approximately 1 nM (isoproterenol), 8 nM (epinephrine), 9 nM (salbutamol) and 2 microM (norepinephrine) giving an order of potency of isoproterenol greater than epinephrine = salbutamol much greater than norepinephrine. L- but not the D-isomer of isoproterenol induced androgen production. A non-selective beta-receptor antagonist, propranolol, abolished androgen production induced by isoproterenol. A selective beta 2-receptor antagonist ICI 118,551 inhibited the isoproterenol effect in a concentration-dependent manner with half-maximum inhibition (IC50) at approximately 23 nM. The beta 1-receptor antagonists, metoprolol and atenolol had no effect on isoproterenol-induced androgen release. The stimulatory effect of norepinephrine (an alpha- and beta-agonist) was completely (100%) abolished by propranolol, unaffected by the alpha-antagonist phentolamine and only partially (35%) inhibited by phenoxybenzamine. Phenoxybenzamine and the alpha 2-agonist, clonidine reduced basal androgen production. These studies indicate that androgen production by primary cultures of mouse testicular interstitial cells occurs exclusively via the beta 2-receptor subtype and that alpha-receptor agonists do not stimulate androgen release by these cells.     

Rapid inverse changes in alpha 1B- and beta 2-adrenergic receptors and gene transcripts in acutely isolated rat liver cells.

In vitro incubation of hepatocytes acutely isolated from adult male rats leads to a rapid conversion of the adrenergic activation of glycogenolysis from an alpha 1-receptor (alpha 1AR) to a beta 2-receptor (beta 2AR) mediated response within 4 h. In order to understand the underlying mechanism, we examined time-dependent changes in alpha 1- and beta 2-adrenergic activation of glycogenolysis and second messenger systems, the cellular density and affinity of alpha 1AR and beta 2AR, and the steady state levels of alpha 1BAR and beta 2AR mRNAs. Incubation of hepatocytes for 4 h resulted in a decrease in phosphorylase activation and inositol 1,4,5 trisphosphate accumulation in response to phenylephrine, a 40% decrease in alpha 1AR density, and a 70% decrease in alpha 1BAR mRNA levels. Incubation of hepatocytes for 4 h also resulted in the emergence of a phosphorylase response to isoproterenol, an increase in isoproterenol-induced but not in glucagon- or forskolin-induced cAMP accumulation, no significant change in beta 2AR density, and a twofold increase in beta 2AR mRNA levels. Exposure of cells to cycloheximide, 2 microM throughout the 4 h incubation, prevented the emergence of the phosphorylase response to isoproterenol and reduced beta 2AR densities, while the decrease in alpha 1AR density was not affected and the decrease in phosphorylase activation by phenylephrine was attenuated. The results indicate that dissociation of rat liver cells triggers a rapidly developing decrease in alpha 1BAR mRNA and increase in beta 2AR mRNA levels and corresponding inverse changes in the synthesis of alpha 1BAR and beta 2AR which account, at least in part, for the rapid conversion from alpha 1- to beta 2-adrenergic glycogenolysis.     

Cloning, sequence analysis, and permanent expression of a human alpha 2-adrenergic receptor in Chinese hamster ovary cells. Evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation

The gene encoding a human alpha 2-adrenergic receptor was isolated from a human genomic DNA library using a 367-base pair fragment of Drosophila genomic DNA that exhibited 54% identity with the human beta 2-adrenergic receptor and 57% identity with the human alpha 2-adrenergic receptor. The nucleotide sequence of a fragment containing the human alpha 2-receptor gene and 2.076 kilobases of untranslated 5' sequence was determined, and potential upstream regulatory regions were identified. This gene encodes a protein of 450 amino acids and was identified as an alpha 2-adrenergic receptor by homology with published sequences and by pharmacological characterization of the protein expressed in cultured cells. Permanent expression of the alpha 2-receptor was achieved by transfecting Chinese hamster ovary (CHO) cells which lack adrenergic receptors with a 1.5-kilobase NcoI-HindIII fragment of the genomic clone containing the coding region of the gene. The alpha 2-receptor expressed in CHO cells displayed pharmacology characteristic of an alpha 2 A-receptor subtype with a high affinity for yohimbine (Ki = 1 nM) and a low affinity for prazosin (Ki = 10,000 nM). Agonists displayed a rank order of potency in radioligand binding assays of para-aminoclonidine greater than or equal to UK-14304 greater than (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol, consistent with the identification of this protein as an alpha 2-receptor. The role of the alpha 2-receptor in modulating intracellular cyclic AMP concentrations was investigated in three transfected cell lines expressing 50, 200, and 1200 fmol of receptor/mg membrane protein. At low concentrations (1-100 nM), (-)-epinephrine attenuated forskolin-stimulated cyclic AMP accumulation by up to 60% in a receptor density-dependent manner. At epinephrine concentrations above 100 nM, cyclic AMP levels were increased up to 140% of the forskolin-stimulated level. Pertussis toxin pretreatment of cells eliminated alpha 2-receptor-mediated attenuation of forskolin-stimulated cyclic AMP levels and enhanced the receptor density-dependent potentiation of forskolin-stimulated cyclic AMP concentrations from 3 to 8-fold. Potentiation of forskolin-stimulated cyclic AMP levels was also elicited by the alpha 2-adrenergic agonists, UK-14304 and para-aminoclonidine, and blocked by the alpha 2-adrenergic antagonist yohimbine, but not by the alpha 1-adrenergic antagonist prazosin or the beta-adrenergic antagonist propranolol.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Different accessibility from the artery and the portal vein of alpha- and beta-receptors involved in the sympathetic nerve action on glycogenolysis and hemodynamics in perfused rat liver

In perfused rat liver perivascular nerve stimulation (7.5 Hz, 20 V, 2 ms, 5 min) at the liver hilus caused an increase in glucose and lactate output and a decrease in flow. The influence of the alpha 1-receptor blocker prazosine and the beta-blocker propranolol on these nerve effects was studied in the isolated rat liver perfused classically via the portal vein only and, as developed recently, via both the hepatic artery and the portal vein. 1) In livers perfused via the portal vein only the nerve stimulation-dependent metabolic alterations were nearly completely inhibited by prazosine (5 microM), but not influenced by propranolol (10 microM). The hemodynamic changes were lowered to only 33% by prazosine and not altered by propranolol either. 2) In livers perfused via the hepatic artery (100 mm Hg, 20-40% of flow) and the portal vein (10 mm Hg, 80-60% of flow)--similar to portal perfusions--the nerve stimulation--dependent metabolic alterations were almost completely blocked by arterial, portal or simultaneously applied arterial and portal prazosine. However--in contrast to portal perfusions--the metabolic alterations were reduced to about 20% (glucose) and 50% (lactate) also by propranolol independently of its site of application. The decrease in flow was reduced by prazosine to about 60%, 50% and 30% when applied via the artery, the portal vein or via both vessels, respectively. The hemodynamic alterations were not influenced by propranolol. These results allow the following conclusions: A subpopulation of beta-receptors can play a permissive role in the alpha 1-receptor-mediated sympathetic nerve action on glucose and lactate metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential control of glycogenolysis and flow by arterial and portal acetylcholine in perfused rat liver.

The effects of acetylcholine on glucose and lactate balance and on perfusion flow were studied in isolated rat livers perfused simultaneously via the hepatic artery (100 mmHg, 25-35% of flow) and the portal vein (10 mmHg, 75-65% of flow) with a Krebs-Henseleit bicarbonate buffer containing 5 mM-glucose, 2 mM-lactate and 0.2 mM-pyruvate. Arterial acetylcholine (10 microM sinusoidal concentration) caused an increase in glucose and lactate output and a slight decrease in arterial and portal flow. These effects were accompanied by an output of noradrenaline and adrenaline into the hepatic vein. Portal acetylcholine elicited only minor increases in glucose and lactate output, a slight decrease in portal flow and a small increase in arterial flow, and no noradrenaline and adrenaline release. The metabolic and haemodynamic effects of arterial acetylcholine and the output of noradrenaline and adrenaline were strongly inhibited by the muscarinic antagonist atropine (10 microM). The acetylcholine-dependent alterations of metabolism and the output of noradrenaline were not influenced by the alpha 1-blocker prazosin (5 microM), whereas the output of adrenaline was increased. The acetylcholine-dependent metabolic alterations were not inhibited by the beta 2-antagonist butoxamine (10 microM), although the overflow of noradrenaline was nearly completely blocked and the output of adrenaline was slightly decreased. These results allow the conclusion that arterial, but not portal, acetylcholine caused sympathomimetic metabolic effects, without noradrenaline or adrenaline being involved in signal transduction.     

Mechanism of action of cysteinyl leukotrienes on glucose and lactate balance and on flow in perfused rat liver. Comparison with the effects of sympathetic nerve stimulation and noradrenaline

Rat livers were perfused at constant pressure via the portal vein with media containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. Leukotrienes C4 and D4 enhanced glucose and lactate output and reduced perfusion flow to the same extent and with essentially identical kinetics. They both caused half-maximal alterations (area under the curve) of carbohydrate metabolism at a concentration of about 1 nM and of flow at about 5 nM. The leukotriene-C4/D4 antagonist CGP 35949 B inhibited the metabolic and hemodynamic effects of 5 nM leukotrienes C4 and D4 with the same efficiency, causing 50% inhibition at about 0.1 microM. 2. Leukotriene C4 elicited the same metabolic and hemodynamic alterations with the same kinetics as leukotriene D4 in livers from rats pretreated with the gamma-glutamyltransferase inhibitor, acivicin. 3. The calcium antagonist, nifedipine, at a concentration of 50 microM did not affect the metabolic and hemodynamic changes caused by 5 nM leukotriene D4. The smooth-muscle relaxant, nitroprussiate, at a concentration of 10 microM reduced flow changes, without significantly affecting the metabolic alterations. 4. Leukotriene D4 not only reduced flow; it also caused an intrahepatic redistribution of flow, restricting some areas from perfusion. Thus, leukotrienes increased glucose and lactate output directly in the accessible parenchyma and, in addition, indirectly by washout from restricted areas during their reopening upon termination of application. 5. The phospholipase A2 inhibitor, bromophenacyl bromide, but not the cyclooxygenase inhibitor, indomethacin, at a concentration of 20 microM reduced the metabolic and hemodynamic effects of 5 mM leukotriene D4. 6. Stimulation of the sympathetic hepatic nerves with 2-ms rectangular pulses at 20 Hz and infusion of 1 microM noradrenaline increased glucose and lactate output and decreased flow, similar to 10 nM leukotrienes C4 and D4. The kinetics of the metabolic and hemodynamic changes caused by the leukotrienes differed, however, from those due to nerve stimulation and noradrenaline. 7. The leukotriene-C4/D4 antagonist, CGP 35949 B, even at very high concentrations (20 microM) inhibited the metabolic and hemodynamic alterations caused by nerve stimulation or noradrenaline infusion only slightly and unspecifically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Signal propagation via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves.

Cell-to-cell communication via gap junctions has been proposed to be involved in the metabolic actions of sympathetic liver nerves in the rat. The effects of hepatic nerve stimulation and noradrenaline-, PGF2 alpha- and glucagon infusion on glucose metabolism and perfusion flow were studied in perfused rat liver in the absence and presence of the gap junctional inhibitors, heptanol, carbenoxolone and (4 beta)phorbol 12-myristate 13-acetate (4 beta PMA). (i) Stimulation of the hepatic nerve plexus increased glucose output, decreased flow and caused an overflow of noradrenaline into the hepatic vein. (ii) Heptanol completely inhibited not only the nerve stimulation-dependent metabolic and hemodynamic alterations but also the noradrenaline overflow. Thus the heptanol-dependent inhibitions were caused primarily by a strong impairment of transmitter release. (iii) Carbenoxolone inhibited the effects of neurostimulation on glucose metabolism partially by about 50%, whereas it left perfusion flow and noradrenaline overflow essentially unaltered. (iv) 4 beta PMA reduced the nerve stimulation-dependent enhancement of glucose release by about 80% but the noradrenaline-dependent increase in glucose output only by about 30%; the increase in glucose release by PGF2 alpha and by glucagon remained essentially unaltered. 4 beta PMA reduced the nerve stimulation-dependent decrease in portal flow by about 35% but did not affect the noradrenaline-and PGF2 alpha-elicited alterations, nor did it alter noradrenaline overflow. The results allow the conclusion that gap junctional communication plays a major role in the regulation of hepatic carbohydrate metabolism by sympathetic liver nerves, but not by circulating noradrenaline, PGF2 alpha or glucagon.     

Noradrenergic stimulation of BDNF synthesis in astrocytes: mediation via alpha1- and beta1/beta2-adrenergic receptors

Brain-derived neurotrophic factor (BDNF) synthesis in astrocytes induced by noradrenaline (NA) is a receptor-mediated process utilizing two parallel adrenergic pathways: beta1/beta2-adrenergic/cAMP and the novel alpha1-adrenergic/PKC pathway. BDNF is produced by astrocytes, in addition to neurons, and the noradrenergic system plays a role in controlling BDNF synthesis. Since astrocytes express various subtypes of alpha- and beta-adrenergic receptors that have the potential to be activated by synaptically released NA, we focused our present study on the mediatory role of adrenergic receptors in the noradrenergic up-regulation of BDNF synthesis in cultured neonatal rat cortical astrocytes. NA (1 microM) elevates BDNF levels by four-fold after 6 h of incubation. Its stimulation was partly inhibited by either the beta1-adrenergic antagonist atenolol, the beta2-adrenergic antagonist ICI 118,551, or by the alpha1-adrenergic antagonist prazosin, while the alpha2-adrenergic antagonist yohimbine showed no effect. BDNF levels in astrocytes were increased by the specific beta1-adrenergic agonist dobutamine and the beta2-adrenergic agonist salbutamol, as well as by adenylate cyclase activation (by forskolin) and PKA activation (by dBcAMP). However, none of the tested agonists or mediators of the intracellular beta-adrenergic pathways were able to reach the level of NA's stimulatory effect. BDNF cellular levels were also elevated by the alpha1-adrenergic agonist methoxamine, but not by the alpha2-adrenergic agonist clonidine. The increase in intracellular Ca2+ by ionophore A23187 showed no effect, whereas PKC activation by phorbol 12-myristate 13-acetate (TPA) potently stimulated BDNF levels in the cells. The methoxamine-stimulated BDNF synthesis was inhibited by desensitizing pretreatment with TPA, indicating that the alpha1-stimulation was mediated via PKC activation. In conclusion, the synthesis of astrocytic BDNF stimulated by noradrenergic neuronal activity is an adaptable process using multiple types (alpha1 and beta1/beta2) of adrenergic receptor activation.     

Human apolipoprotein A-II: nucleotide sequence of a cloned cDNA, and localization of its structural gene on human chromosome 1

[3H]yohimbine, a potent and selective alpha 2-adrenergic antagonist was used to label alpha-adrenoceptors in intact human lymphocytes. Binding of [3H]yohimbine was rapid (t1/2 1.5 -2.0 min) and readily reversed by 10 microM phentolamine (t1/2 = 5 - 6 min) and of high affinity (Kd = 3.7 +/- 0.86 nM). At saturation, the total number of binding sites was 19.9 +/- 5.3 fmol/10(7) lymphocytes. Adrenergic agonists competed for [3H]yohimbine binding sites with an order of potency: clonidine greater than (-) epinephrine greater than (-) norepinephrine greater than (+) epinephrine much greater than (-) isoproterenol; adrenergic antagonists with a potency order of yohimbine greater than phentolamine greater than prazosin. These results indicate the presence of alpha 2-adrenoceptors in human lymphocytes.     

Role of extracellular calcium in the metabolic and hemodynamic actions of sympathetic nerve stimulation, noradrenaline and prostaglandin F2 alpha in perfused rat liver. Differential inhibition by nifedipine and verapamil

In perfused rat liver hepatic nerve stimulation (10 Hz, 2 ms) caused an increase in glucose and lactate output, a decrease in flow and an overflow of noradrenaline into the hepatic vein. Noradrenaline (1 microM) (NA) and prostaglandin F2 alpha (5 microM) (PGF2 alpha), which are implicated as mediators of nerve action, elicited similar effects. 1) All actions of nerve stimulation and the hemodynamic but not the metabolic effects of noradrenaline and PGF2 alpha were largely dependent on extracellular calcium. 2) The dihydropyridine type calcium antagonist nifedipine (5 microM) inhibited the hemodynamic but not the metabolic actions of nerve stimulation, NA and PGF2 alpha, while the phenylalkylamine type calcium antagonist verapamil (5 microM) had no effect. These findings allow the following conclusions: Calcium influx into I nerve endings, necessary for the release of neurotransmitter, II parenchymal cells, for the display of metabolic effects induced by nerve stimulation, and III the actions of NA and PGF2 alpha, do not appear to be mediated by the normal affinity nifedipine- or the verapamil-sensitive channels. Calcium influx into vascular smooth muscle and/or endothelial cells for the display of hemodynamic action induced by nerve stimulation and the NA and PGF2 alpha effects, appear to occur through nifedipine-sensitive but verapamil-insensitive channels.     

The mechanism of SR95531 inhibition at GABA receptors examined in human alpha1beta1 and alpha1beta1gamma2S receptors

We examined the interaction of GABA and the competitive inhibitor SR95531 at human alpha1beta1gamma2S and alpha1beta1 GABA(A) receptors expressed in Sf9 cells. The efficacy and potency of inhibition depended on the relative timing of the GABA and SR95531 applications. In saturating (10 mM) GABA, the half-inhibitory concentrations of SR95531 (IC50) when coapplied with GABA to alpha1beta1gamma2S or alpha1beta1 receptors were 49 and 210 microM for the peak and 18 and 130 microM for the plateau current, respectively. Our data are explained by an inhibition mechanism in which SR95531 and GABA bind to two sites on the receptor where the binding of GABA allows channel opening but SR95531 does not. The SR95531 affinity for both receptor types was approximately 200 nM and the binding rate was found to be 10-fold faster than that for GABA. The dual binding-site model gives insights into the differential effects of GABA and SR95531 on the peak and plateau currents. The model predicts the effect of SR95531 on GABA currents in the synapse (GABA concentration approximately mM) and at extrasynaptic (GABA concentration < or = microM) sites. The IC50 (50-100 nM) for the synaptic response to SR95531 was insensitive to the GABA affinity of the receptors whereas the IC50 (50-800 nM) for extrasynaptic inhibition correlated with the GABA affinity.     

Multiple Adrenergic Receptor Subtypes Controlling Cyclic AMP Formation: Comparison of Brain Slices and Primary Neuronal and Glial Cultures

The adrenergic receptor subtypes involved in cyclic AMP responses to norepinephrine (NE) were compared between slices of rat cerebral cortex and primary neuronal and glial cultures from rat brain. In neuronal cultures, NE and the beta-adrenergic receptor agonist isoproterenol (ISO) caused similar increases in cyclic AMP, which were not altered by the alpha-adrenergic receptor antagonist phentolamine. In glial cultures, NE caused a much smaller cyclic AMP response than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists (phentolamine greater than yohimbine greater than prazosin). alpha 2-Adrenergic receptor agonists partially inhibited the ISO response in glial cultures to a level similar to that observed with NE alone (clonidine = UK 14,304 greater than NE greater than 6-fluoro-NE greater than epinephrine). In slices from cerebral cortex, NE caused a much larger increase in cyclic AMP than did ISO, and this difference was reversed by alpha-adrenergic receptor antagonists with a different order of potency (prazosin greater than phentolamine greater than yohimbine). alpha 1-Adrenergic receptor agonists potentiated the response to ISO to a level similar to that observed with NE alone (epinephrine = NE greater than phenylephrine greater than 6-fluoro-NE greater than methoxamine). In all three tissue preparations, large responses to both alpha 1-receptor activation (increases in inositol phosphate accumulation) and alpha 2-receptor activation (decreases in forskolin-stimulated cyclic AMP accumulation) were observed. These data indicate that all of the major adrenergic receptor subtypes (beta, alpha 1, alpha 2) are present in each tissue preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adrenergic regulation of glucose metabolism in rat heart. A calcium-dependent mechanism mediated by both alpha- and beta-adrenergic receptors

Epinephrine treatment of the perfused rat heart led to an increase in glucose uptake, detritiation of [5-3H] glucose, glycogenolysis, and the formation of lactate. The change in the rate of formation of 3H2O from [5-3H]glucose was slower to develop (commencing at approximately 30 s) than changes in cyclic AMP concentration, hexose-6-P concentration, and the phosphorylase a/(a + b) ratio which were maximal at 24 s. Epinephrine plus propranolol (alpha-adrenergic combination) treatment of the perfused heart also led to increases in glucose uptake, detritiation of [5-3H]glucose, and the formation of lactate, but these occurred without significant changes in cyclic AMP concentration, hexose-6-P concentration, or the phosphorylase a/(a + b) ratio. Half-maximal stimulation of glucose uptake occurred at 0.2 microM epinephrine, 1.5 microM methoxamine, and 1 microM isoproterenol. The increase in glucose uptake mediated by 1 microM epinephrine was blocked by 10 microM prazosin but unaffected by 10 microM propranolol. The increase in glucose uptake mediated by 10 microM epinephrine plus 10 microM propranolol was partly blocked by yohimbine and completely blocked by prazosin. A role for Ca2+ in the adrenergic regulation of glucose uptake was indicated by the sensitivity of the epinephrine dose curve to Ca2+ and the dependence of epinephrine on Ca2+. In addition the increases in glucose uptake mediated by 1 microM epinephrine, 1 microM epinephrine plus 10 microM propranolol, 1 microM isoproterenol, and by 10 mM CaCl2 were each blocked by the Ca2+ channel blocker nifedipine (1 microM). It is concluded that Ca2+-dependent alpha- and beta-adrenergic receptor mechanisms are present in rat heart for controlling glucose uptake. At submicromolar levels of epinephrine the predominant receptors utilized appear to be alpha 1.     

Protection against alloxan-induced diabetes in mice by stimulation of alpha 2-adrenergic receptors

A single intravenous injection of alloxan in mice induced hyperglycemia in a dose dependent fashion. This diabetogenic action of alloxan was prevented by a single intraperitoneal injection of the alpha 2-adrenergic agonists, i.e. oxymetazoline, clonidine or epinephrine 40 min prior to the injection of alloxan. The alpha 1-adrenergic agonists, i.e. methoxamine and phenylephrine, and a beta-adrenergic agonist, isoproterenol, failed to prevent the diabetogenic action of alloxan. The inhibitory effect of clonidine on alloxan-induced diabetes was antagonized by yohimbine or phentolamine, but not by prazosin. Although alpha 2-adrenergic agonists caused a transient hyperglycemia at the time of alloxan administration (40 min after the administration of alpha 2-adrenergic agonists), the plasma glucose level at the time of alloxan injection did not correlate with the anti-diabetogenic effect of alpha 2-adrenergic agents. These results clearly demonstrate that the alpha 2-adrenergic mechanism which inhibits insulin release from pancreatic B cells prevented the diabetogenic action of alloxan in mice.     

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.     

Identification of alpha1-adrenergic receptors and their involvement in phosphoinositide hydrolysis in the frog heart

The aim of this study was to characterize alpha(1)-adrenergic receptors in frog heart and to examine their related signal transduction pathway. alpha(1)-Adrenergic binding sites were studied in purified heart membranes using the specific alpha(1)-adrenergic antagonist [(3)H]prazosin. Analysis of the binding data indicated one class of binding sites displaying a K(d) of 4.19 +/- 0.56 nM and a B(max) of 14.66 +/- 1.61 fmol/mg original wet weight. Adrenaline, noradrenaline, or phenylephrine, in the presence of propranolol, competed with [(3)H]prazosin binding with a similar potency and a K(i) value of about 10 microM. The kinetics of adrenaline binding was closely related to its biological effect. Adrenaline concentration dependently increased the production of inositol phosphates in the heart in the presence or absence of propranolol. Maximal stimulation was about 8.5-fold, and the half-maximum effective concentration was 30 and 21 microM in the absence and presence of propranolol, respectively. These data clearly show that alpha(1)-adrenergic receptors are coupled to the phosphoinositide hydrolysis in frog heart. To our knowledge, this is the first direct evidence supporting the presence of functional alpha(1)-adrenergic receptors in the frog heart.     

Dual regulation of adenylate cyclase and guanylate cyclase: alpha 2-adrenergic signal transduction in adrenocortical carcinoma cells

Isolated adrenocortical carcinoma cells of rat contain alpha 2- and beta-adrenergic receptors. When these cells are incubated with alpha 2-adrenergic agonists, there is a concentration-dependent increase of cyclic GMP that is blocked by the alpha 2-adrenergic antagonist yohimbine but not by the beta-antagonist propranolol. Concomitantly, both p-aminoclonidine (20 microM) and clonidine (100 microM), the alpha 2-adrenergic agonists, stimulate membrane guanylate cyclase activity. In calcium free medium there is no alpha 2-agonist-dependent increase in cyclic GMP. Isoproterenol, a beta-agonist, and forskolin cause an increase in cyclic AMP but not cyclic GMP. The cyclic AMP increase induced by isoproterenol is blocked by propranolol but not by yohimbine. Isoproterenol- and forskolin-dependent increases in cyclic AMP are inhibited by p-aminoclonidine and the inhibition is relieved by yohimbine. These results indicate a dual regulation of guanylate cyclase and adenylate cyclase by the alpha 2-receptor signal: guanylate cyclase is coupled to the receptor in a positive fashion, whereas adenylate cyclase is coupled in a negative fashion. Calcium is obligatory in the cyclic GMP-mediated response.