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.     

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.     

α2-Adrenergic Receptors in Neuronal and Glial Cultures: Characterization and Comparison

Membranes prepared from either neuronal or glial cultures contain alpha 2-adrenergic receptors as determined by the characteristics of [3H]yohimbine [( 3H]YOH) binding. The binding was rapid, reversible, saturable, dependent on the protein concentration used, and reached equilibrium by 5 min in membranes from both neuronal and glial cultures. Scatchard analyses of saturation isotherms revealed similar KD values of 13.7 +/- 1.35 nM (n = 10) for neuronal cultures and 18.42 +/- 2.34 nM (n = 10) for glial cultures. Glial cultures contained many more binding sites for [3H]YOH than neuronal cultures, having a Bmax of 1.6 +/- 0.33 pmol/mg protein (n = 10) compared with 0.143 +/- 0.018 pmol/mg protein (n = 10) in neurons. Drugs selective for alpha 2-adrenergic receptors were the most effective displacers of [3H]YOH binding in both neuronal and glial cultures, i.e., the alpha 2-adrenergic antagonists rauwolscine and yohimbine were better displacers than the other catecholamine antagonists prazosin, corynanthine, or propranolol. The agonists showed the same pattern with the alpha 2-selective drugs clonidine and naphazoline being the most effective competitors for the [3H]YOH site. GTP and its nonhydrolyzable analog. 5'-guanylyl-imidodiphosphate, were able to lower the affinity of the alpha 2-receptors for agonists but not antagonists in membranes from both neuronal and glial cultures, suggesting that the receptors are linked to a G protein in both cell types. The presence of alpha 2-adrenergic receptors in neuronal cultures was also substantiated by light microscopic autoradiography of [3H]YOH binding. In summary, we have demonstrated that both neuronal and glial cultures contain alpha 2-adrenoceptors.     

Transient high-affinity binding of agonists to alpha 1-adrenergic receptors of intact liver cells

At alpha 1-adrenergic receptors in isolated rat liver parenchymal cells, (-)-epinephrine is potent in eliciting a maximal increase in glycogenolysis (Kact = 24 nM). This contrasts with a 100-fold lower affinity for the agonist at alpha 1-adrenergic receptors of intact hepatocytes determined from equilibrium competition assays with the alpha 1-adrenergic antagonist [3H]prazosin. We demonstrate here that agonists bind to alpha 1-adrenergic receptors of intact liver cells initially with a markedly higher affinity than under equilibrium conditions. When incubations are performed for 15 s at 37 degrees C, the affinity is more than 100-fold higher than that obtained in equilibrium (45 min) assays (IC50 = 28 +/- 3 vs 5300 +/- 400 nM for (-)-epinephrine and 32 +/- 3 vs 6100 +/- 500 nM for (-)-norepinephrine). When incubations are performed at 4 degrees C (150 min), high-affinity binding similar to that obtained in short-term incubations can also be demonstrated. In contrast, antagonist compete with similar affinities in 15 s and 45 min assays, and their dissociation constants are not affected by changes in the incubation temperature. These results indicate that agonists bind to native alpha 1-adrenergic receptors transiently with high affinity. The conversion of receptors to a state of predominantly low affinity for agonists, which occurs rapidly and irreversibly with increasing incubation at 37 degrees C, is inhibited at low incubation temperatures. It is suggested that the high-affinity configuration of the alpha 1-adrenergic receptor for agonists observed in nonequilibrium experiments or at reduced incubation temperatures represents the physiologically relevant state of the alpha 1-adrenergic receptor.     

Identification of α2-Adrenergic Receptors in Chicken Pineal Gland Using [3H]Rauwolscine

The norepinephrine-induced inhibition of avian pineal N-acetyltransferase activity appears to be mediated by alpha 2-adrenergic receptors. In this study, alpha 2-adrenergic receptors in the chicken pineal gland were directly identified by radioligand binding. Membrane preparations of pineal glands from chickens from 1 to 6 weeks of age were examined using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, to characterize the binding sites. The results indicate no ontological change in either the affinity (KD) or density of receptor binding sites (Bmax) during the time span examined. The binding was saturable and of high affinity with a mean KD of 0.27 +/- 0.01 nM and a mean Bmax of 242 +/- 12 fmol/mg protein. Further characterization of these binding sites indicated that the alpha 2-adrenergic receptor is of the alpha 2A subtype, since prazosin and ARC-239 bound with low affinities and oxymetazoline bound with high affinity.     

Involvement of alpha 1- and alpha 2-adrenergic receptors in the hypothalamo-pituitary-ovarian axis of the domestic fowl, Gallus domesticus

The alpha 1-adrenergic receptor ligand, 3H-WB4101, and the alpha 2-adrenergic receptor ligand, 3H-para-aminoclonidine, were utilized at a 1.0 nM incubation concentration to determine relative alpha 1-and alpha 2-adrenergic receptor binding by cell membranes from selected tissues within the brain, ovary and oviduct of the domestic fowl. Significant specific alpha 1-adrenergic binding was observed in the hypothalamus, anterior pituitary, pineal, cerebrum and cerebellum but only the cerebrum had significant alpha 2-receptor binding. Significant levels of alpha 1-adrenergic binding were observed in the granulosa cells of the three largest ovarian follicles and in the postovulatory follicle. Significant specific alpha 2-adrenergic binding was measured in the infundibulum, magnum, isthmus and shell gland of the oviduct. The physiological implications of alpha-adrenergic receptors in these tissues are discussed.     

Purification of the alpha 2-adrenergic receptor from porcine brain using a yohimbine-agarose affinity matrix

A procedure has been developed for purification of the porcine brain alpha 2-adrenergic receptor to homogeneity. alpha 2-Adrenergic receptors were solubilized from porcine brain particulate preparations using sequential extraction into sodium cholate- and digitonin-containing buffers. The alpha 2-adrenergic receptors in the digitonin extract were identified using the alpha 2-adrenergic selective antagonist, [3H]yohimbine, and demonstrated the same specificity for interaction with adrenergic ligands as did the receptors in particulate preparations. Extraction into digitonin-containing buffers eliminated the modulation of receptor-agonist interactions by guanine nucleotides, but not by monovalent cations. A novel affinity resin, yohimbine-agarose, was synthesized and used for purification of alpha 2-adrenergic receptors. Using two sequential yohimbine-agarose affinity chromatography steps, digitonin-solubilized alpha 2-adrenergic receptors from porcine brain cortex were purified to homogeneity as assessed by radioiodination and silver stain analysis of these preparations on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified alpha 2-adrenergic receptor has an approximate Mr = 65,000, as determined by photolabeling of the adrenergic ligand-binding subunit. The yohimbine-agarose affinity resin should be useful for purifying quantities of receptor sufficient for studies of receptor structure and function.     

An affinity label for alpha 2-adrenergic receptors in rat brain

Clonidine, a potent and highly selective alpha 2-adrenergic agonist of the central nervous system, was modified. Insertion of the strong alkylating isothiocyanate group (NCS) group, at its aromatic residue, makes clonidine a potential affinity label of the alpha 2-adrenergic receptors. In displacement of [3H]clonidine and p-[3H]aminoclonidine from rat brain membrane preparations, clonidine-NCS demonstrates high affinity for the alpha 2-adrenergic receptors (Kd = 50 mM). The covalent labelling of the central alpha 2-receptors requires higher concentrations of the irreversible ligand (1-70 microM), thus indicating possible non-productive interactions at the environment of the receptor site. Only partial protection of the receptors is observed with a reversible alpha 2-agonist. The new clonidine analog appears to be a general ligand for the alpha 2-adrenergic receptors and might serve as a potential affinity probe for these receptors.     

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)     

Biochemical characterization of alpha-adrenergic receptors in human brain and changes in Alzheimer-type dementia

Using ligand binding techniques, we studied alpha-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the alpha-adrenergic antagonists [3H]prazosin and [3H]yohimbine to membranes of human brains exhibited characteristics compatible with alpha 1- and alpha 2-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [3H]prazosin and 5.5 nM for [3H]yohimbine. [3H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [3H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [3H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [3H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that alpha 1- and alpha 2-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

Attenuation of inositol trisphosphate generation and cytosolic Ca2+ elevation in dispersed rat parotid acini stimulated simultaneously at muscarinic and alpha 1-adrenergic receptors

We have examined intracellular signalling events, peak cytosolic [Ca2+] and inositol trisphosphate levels, in rat parotid acini simultaneously stimulated with two Ca2+ mobilizing agonists, carbachol (muscarinic-cholinergic) and epinephrine (alpha 1-adrenergic). When the agonists were added together, either at sub-maximal (200 nM each, i.e. 400 nM total agonist concentration) or maximal (10 uM each, i.e. 20 uM total) stimulatory concentrations, the resulting elevations in both cytosolic [Ca2+] and inositol trisphosphate levels were not greater than those achieved when each agonist was added individually. However, with 400 nM carbachol these responses were significantly greater than those seen with either 200 nM carbachol or 200 nM carbachol + 200 nM epinephrine. The data indicate that when muscarinic and alpha 1-adrenergic receptors of rat parotid acini are simultaneously stimulated a novel regulatory mechanism is induced, which attenuates inositol trisphosphate generation and, consequently, intracellular Ca2+ release.     

Alpha 2-adrenergic receptors on the circular smooth muscle of canine small intestine.

We have studied the distribution and properties of alpha 2-adrenergic receptors in the circular muscle layer (containing deep muscular plexus) of canine small intestine. Using radioactivity labelled rauwolscine, we located the binding sites to the neuronal membranes supporting the prejunctional action of alpha 2-adrenergic agents in the gut. Moreover, although the functional data to suggest the existence of postjunctional alpha 2-adrenergic receptors coupled to contraction are not available so far, we measured a substantial number of rauwolscine binding sites on the smooth muscle plasma membranes. Scatchard and Hill analyses of the saturation data were indicative of the presence of a single high affinity site (Hill coefficient 0.996) with a KD value of 8.8 nM and the maximum number of binding sites (Bmax) of 313 fmol/mg of protein. Competition studies suggested the presence of multiple subtypes of alpha 2-adrenoceptors.     

Binding of antipsychotic drugs to human brain receptors focus on newer generation compounds

Using radioligand binding assays and post-mortem normal human brain tissue, we obtained equilibrium dissociation constants (K(d)s) for nine new antipsychotic drugs (iloperidone, melperone, olanzapine, ORG 5222, quetiapine, risperidone, sertindole, ziprasidone, and zotepine), one metabolite of a new drug (9-OH-risperidone), and three older antipsychotics (clozapine, haloperidol, and pimozide) at nine different receptors (alpha1-adrenergic, alpha2-adrenergic, dopamine D2, histamine H1, muscarinic, and serotonin 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2C receptors). Iloperidone was the most potent drug at the two adrenergic receptors. ORG 5222 was the most potent drug at dopamine D2 and 5-HT2c receptors, while ziprasidone was the most potent compound at three serotonergic receptors (5-HT1A, 5-HT1D, and 5-HT2A). At the remaining two receptors, olanzapine was the most potent drug at the histamine H1 receptor (Kd=0.087 nM); clozapine at the muscarinic receptor (Kd=9 nM). Certain therapeutic and adverse effects, as well as certain drug interactions can be predicted from a drug's potency for blocking a specific receptor. These data can provide guidelines for the clinician in the choice of antipsychotic drug.     

Isolation of an endogenous clonidine-displacing substance from rat brain

An endogenous substance which specifically displaces clonidine, yohimbine and rauwolscine from rat brain alpha 2-adrenergic receptors, has been isolated. The new compound, designed clonidine-displacing-substance (CDS), has been partially purified by ion exchange chromatography, zone electrophoresis and high performance liquid chromatography (HPLC). CDS binds specifically to alpha 2-adrenergic receptors by competing with either alpha 2-adrenergic agonists or alpha 2-antagonists, but has no effect on the specific binding of [3H]prazosin to alpha 1-adrenergic receptors in rat brain membranes. In the course of isolation, CDS was shown to be neither the endogenous neurotransmitter (-)norepinephrine (NE) nor the guanyl nucleotide GTP which lowers the specific binding of alpha 2-agonists to the alpha 2-adrenergic receptors.     

Photoaffinity labeling of the alpha 1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin

alpha 1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquin azoline (CP63,155), an arylamine analogue of the selective alpha 1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP63,789), bind reversibly and with high affinity (KD = 1 nM and 0.6 nM, respectively) to rat hepatic membrane alpha 1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP65,526), which prior to photolysis binds competitively and with high affinity (KD = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar alpha 1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the alpha 1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the alpha 1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alpha 1-adrenergic receptors in the liver parenchyma of children: the changes in cirrhosis]

In the present study alpha 1-adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of prehepatic portal hypertension children without parenchymal affection (control group, n = 7) and the resection of children in parenchymal affection (group of cirrhosis, n = 8). It has been shown, that the binding of alpha 1-adrenergic antagonist 3H-prasozin (3H-PRZ) in liver parenchyma membranes of both control and cirrhosis groups is saturable and shows a high affinity. The Scatchard analysis of the binding data indicated that the binding site is characterized by Kd and Bmax of 0.6 +/- 0.12 nM, 92.8 +/- 8.0 fmol/mg, respectively, for the control group; and 1.5 +/- 0.4 nM, 254.1 +/- 28.4 fmol/mg, respectively, for the group of cirrhosis; (mean +/- SEM). It has been found that the number of binding sites of 3H-PRZ significantly increases in cirrhosis liver parenchyma in comparison with the control group. The results obtained suggest that alpha 1-adrenergic receptors play an important role in cirrhosis formation in children, showing liver parenchyma affection severity and its regenerative properties.     

Cloning, sequencing, and expression of the gene encoding the porcine alpha 2-adrenergic receptor. Allosteric modulation by Na+, H+, and amiloride analogs

The gene for an alpha 2-adrenergic receptor has been cloned from a porcine genomic library, using as a probe a 0.95-kilobase Pst fragment of the gene for the human platelet alpha 2-adrenergic receptor. The identity of the cloned porcine gene was confirmed initially on the basis of partial amino acid sequence information obtained following cyanogen bromide digestion of homogeneous preparations of porcine brain alpha 2-adrenergic receptors. The deduced amino acid sequence for the porcine receptor, when compared to other members of the family of guanine nucleotide-binding protein-coupled receptors, shares the same overall structural characteristics and most closely resembles the human platelet C10 alpha 2-adrenergic receptor (greater than 93% homology). The putative porcine alpha 2-receptor gene was expressed in the COS-M6 cell line. Transfected cells display saturable [3H]yohimbine binding. The KD for [3H]yohimbine, determined in digitonin-solubilized preparations, is 5.8 nM. The selectivity of agonists and antagonists in competing for [3H]yohimbine binding to membranes prepared from the transfected cells is characteristic of the alpha 2A subtype of adrenergic receptors. The porcine alpha 2-receptor also was expressed permanently in LLC-PK1 porcine kidney cells at a level of 100 pmol/mg protein. The alpha 2-agonist UK14304 is able to attenuate forskolin or vasopressin-stimulated cAMP accumulation by at least 50% in these cells. Allosteric modulation of [3H] yohimbine binding by Na+, H+, and 5-amino-substituted analogs of amiloride also was demonstrated for the alpha 2-receptor expressed in COS-M6 cells. Moreover, these modulatory effects were quantitatively similar to those observed for homogeneous preparations of the alpha 2-receptor purified from porcine brain cortex. Retention of the effects of cations and amiloride analogs in transiently expressed alpha 2-receptors supports the interpretation that the allosteric sites for these agents reside in the alpha 2-receptor molecule itself.     

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.     

Alpha 2-adrenergic receptor stimulation mobilizes intracellular Ca2+ in human erythroleukemia cells

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.