Characterization Of Adrenergic Receptors Research Articles

Characterization of adrenergic receptors in the accessory lacrimal glands of the upper eyelid.

To identify the histologic location as well as the exact subtype of adrenergic receptors in the accessory lacrimal glands of the upper eyelid. Upper eyelid specimens were collected from 19 patients undergoing routine blepharoptosis correction via a posterior tarsoconjunctival mullerectomy. Immunohistochemical staining using polyclonal antibodies against human alpha 1, alpha 2, beta 1, and beta 2 receptors was performed on all specimens. beta 1 receptors were the predominant adrenergic receptor subtype in the glands of Wolfring. The presence of beta 1 receptors in the accessory lacrimal glands of the upper eyelid may suggest a possible role for selective beta 1 agonists in the treatment of keratitis sicca.

Characterization of adrenergic receptors and related transduction pathways in the liver of the rainbow trout

Epinephrine (EPI) is thought to act by stimulating adenylyl cyclase (ACase) and cAMP production through β-adrenoceptors in the liver of more primitive vertebrates. Recent observations, however, point to an involvement of α 1-adrenoceptors in EPI action, at least in some fish species. The role of the α 1- and β-adrenergic transduction pathways has been investigated in rainbow trout (Oncorhynchus mykiss) hepatic tissue. Radioligand-binding assays with the β-adrenergic antagonist 3H-CGP-12177 using hepatic membranes purified on a discontinuous sucrose gradient confirmed the presence of β-adrenoceptors ( K d0.36 nM, B max 8.61 fmol · mg −1 protein). We provide the first demonstration of α 1-adrenoceptors in these same membranes; analysis of binding data with the α 1-adrenergic antagonist 3H-prazosin demonstrated a single class of binding sites with a K dof 15.4 nM and a B max of 75.2 fmol · mg −1 protein. There is a straight correlation between β-adrenoceptor occupancy, ACase activation and cAMP production. On the contrary, the role of inositol 1,4,5-trisphosphate (IP 3) has to be elucidated; in fact, despite the presence of specific microsomal binding sites for IP 3 ( K d 6.03 nM, B max 90.2 fmol · mg −1 protein), its cytosolic concentration was not modulated by EPI. On the other hand, we have previously shown in American eel and bullhead hepatocytes that α 1-adrenergic agonists are able to increase intracellular concentrations of IP 3 and Ca 2+ and to activate glycogenolysis. These data suggest a marked variation in the liver of different fish both in terms of α 1-binding sites affinity and of α 1-adrenoceptor/IP 3/Ca 2+ transduction systems.

β-Adrenoreceptors in the Trout ( Oncorhynchus mykiss) Heart: Characterization, Quantification, and Effects of Repeated Catecholamine Exposure

Specific binding of the hydrophilic radioligand [ 3H]CGP-12177 to cell surface (functional) β-adrenoreceptors was quantified in ventricular micropunches (2 mm diameter, 350 μm thickness) from seawater-acclimated rainbow trout held at 7-9°. Binding was stereospecific, saturable, of high affinity, and displaceable by appropriate agonists and antagonists. Phentolamine failed to displace [ 3H]CGP at concentrations up to 10 -4 M, indicating an absence of [ 3H]CGP binding to α-adrenergic receptors. Trout ventricular β-adrenoreceptors are exclusively of the β 2 type. This conclusion is based on (1) the IC 50 value for the β 2-antagonist ICI 118551 (2.9 × 10 -6 M); (2) the inability of the β 1-antagonist atenolol to displace [ 3H]CGP from β-adrenoreceptors; and (3) the order of agonist-binding affinity (isoproterenol > epinephrine ⪢ norepinephrine). The B max and K d values for [ 3H]CGP binding to myocardial tissue were approximately 0.04 fmol μg protein -1 and 0.25 n M, respectively. The B max value indicates that the density of cell surface (functional) β-adrenoreceptors in the ventricle was 12,000 sites per cell or 3.38 sites per μm 2 of sarcolemma. The K d and B max values for [ 3H]CGP binding to ventricular β-adrenoreceptors were unaffected by the in vivo administration of five bolus catecholamine injections (4.0 μg kg -1 epinephrine, 2.0 μg kg -1 norepinephrine). This suggests that stress-induced increases in plasma catecholamines are unlikely to cause the down-regulation of heart β-adrenoreceptors in fish. The method described here represents a simple but powerful technique for the quantification and characterization of adrenergic receptors in the fish heart.

Characterization of Adrenergic Receptors in Membranes from the Rat Seminal Vesicle

Characterization of adrenergic receptors in membranes from the rat seminal vesicle was studied by radioligand binding assay. Seminal vesicle membranes contained saturable and high affinity binding sites for the β-adrenergic receptor antagonist 3H-dihydroalprenolol (3H-DHA) and for the α-adrenergic antagonist 3H-prazosin. The observed order of potency for adrenergic agonists in competing for the 3H-DHA binding sites: isoproterenol > epinephrine—salbutamol≃ norepinephrine indicates that these membrane receptors have the properties of β2-adrenergic receptors. α1-Adrenergic receptors were defined mainly as α1A subtypes by demonstrating their insensitivity to pretreatment with chlorethylclonidine and the different rank orders of antagonist affinities. No significant binding sites for the α2-adrenergic receptor agonist 3H-clonidine were observed. The GTP-induced reduction in the affinity of α,-adrenergic receptors for epinephrine was significantly reversed by the muscarinic cholinergic agonist carbachol. Atropine effectively antagonized this effect of carbachol on the competitive inhibition of 3H-prazosin binding by epinephrine in the presence of GTP, which suggests that muscarinic cholinergic receptors regulate the affinity of α,-adrenergic receptors by modulating the effect of guanine nucleotides. The effect of GTP on decreasing the affinity of β2-adrenergic receptors was not influenced by the addition of carbachol.

Adrenergic receptors: structure and function

Adrenergic receptors are cell surface glycoproteins that recognize and selectively bind the catecholamines, norepinephrine and epinephrine, which are released from sympathetic nerve endings and the adrenal medulla. By transducing the external catecholamine stimulus into an intracellular signal, these receptors mediate the actions of the sympathetic nervous system, including a variety of responses such as arteriolar smooth muscle contraction and cardiac contraction, while are critically involved in cardiac function and blood pressure homeostasis. Activation or blockade of these receptors is, thus, a major therapeutic approach for the management of a number of cardiovascular disorders, including hypertension, angina pectoris, and cardiac arrhythmias. Additionally, alterations in these receptors or in their coupled intracellular effectors may contribute to or underlie the pathogenesis of cardiovascular diseases such as cardiac hypertrophy, congestive heart failure, and hypertension. Over the past few years, major insights have been gained into the physiology and pharmacology of these receptors, particularly in relationship to the molecular mechanisms involved in their ability to mediate "transmembrane signalling." These insights, which will be reviewed here, have resulted from the isolation and characterization of adrenergic receptors, from the determination of their deduced primary and secondary structures, and from structure-function studies using classical biochemistry and pharmacology as well as recombinant DNA techniques.

Characterization of Adrenergic Receptors of the Cat Iris and Nictitating Membrane

Graded pupillary dilations and nictitating membrane (NM) contractions were elicited in anesthetized cats by electrical stimulation of the preganglionic sympathetic nerve or by i.a. administration of norepinephrine (NE) or phenylephrine into the carotid artery. Pupil and NM responses were measured simultaneously from the same side. Alpha-adrenoceptor antagonists were administered intravenously. All of the alpha 1-adrenoceptor blockers tested produced a dose-related reduction of NM responses to both neural and agonist activation; the potency rank order was prazosin greater than WB-4101 greater than phentolamine greater than phenoxybenzamine (PBZ). In contrast, responses of the iris dilator were antagonized only by WB-4101 and PBZ. The iris was almost totally refractory to doses of prazosin and phentolamine that blocked NM responses by more than 75% of control. Neither alpha 2- nor beta-adrenoceptor antagonism produced significant inhibition of neural or agonist activation of either organ (with the exception of high doses of yohimbine on the NM). These results suggest that the postjunctional adrenoceptors of the NM are exclusively of the alpha 1-adrenoceptor subtype. In contrast, those of the iris dilator muscle cannot be easily classified pharmacologically as either alpha 1 or alpha 2-adrenoceptors.

Characterization of adrenergic receptors in pial arteries of the bovine brain

Isolated pial arteries contract by the administration of adrenergic agonists in a dose dependent manner,and the contraction is blocked by alpha adrenergic antagonists. This response is mediated by alpha adrenergic receptors. We attempted to characterize the nature of adrenergic receptors in bovine pial arteries, performing radioligand binding assay. We found that the specific binding of 3H-yohimbine, a selective alpha 2 antagonist, was saturable, reversible and of high affinity (KD= 18 nM, Bmax = 690 fmol/mg protein) (N=4). Scatchard and Hill plot analyses of 3H-yohimbine binding indicated one class of binding site. On the contrary, there was no specific binding of ^H-prazosin, a selective alpha 1 antagonist, in these tissues. These results indicate that alpha adrenergic receptors in the bovine pial arteries are classified as the alpha 2 subtype and that the contraction is mediated by alpha 2 subtype receptors. Using 3H-dihydroalprenolol (3 H-DHA), a beta antagonist, we detected beta adrenergic receptors in the same tissues. 3H-DHA binding sites were revealed to have relatively high values of KD(83 nM) and Bmax (3,200 fmol/mg protein) (N=3). Scatchard and Hill analyses also indicated one class of binding site.These values may explain the low responsiveness of the pial arteries to stimulation mediated by beta adrenergic receptors.

Localization and characterization of adrenergic receptors on frog skin melanophores.

The functional location of adrenergic binding sites was studied in frog skin melanophores by injecting norepinephrine (NE) outside and inside a melanophore. In 49 groups of cells (75% of the fields tested) iontophoretic injection of NE outside the cell caused melanosome aggregation in the target cell and/or in the field. In six cells in which a resting membrane potential was measured before and after intracellular injection (10-90 nA), NE elicited no change in melanosome configuration. Once the receptors were localized, the effect of temperature on these receptors was determined by measuring the reflectance of skins (an indication of melanosome aggregation or dispersion) in two populations of frogs treated with NE, Rana pipiens pipiens (with dominant alpha-receptors) and Rana berlandieri forreri (with dominant beta-receptors). NE (0.1 mM) caused melanosome aggregation in the former and dispersion in the latter tested at 12, 22, and 40 degrees C. The iontophoretic and reflectance results suggest that the binding site of the adrenergic receptor is located on the outer surface of the plasma membrane of melanophores and that alpha- and beta-receptors evoke aggregation and dispersion, respectively, within the temperature range of these experiments.

The hepatic adrenergic receptors.

The presence of both alpha- and beta-adrenergic receptors in liver designated the hepatic plasma membrane as a useful tool for the elucidation of the mechanisms by which the hormonal signal is transferred through the membrane via a coupling system to an amplifying entity. This process is well documented for the beta-adrenergic receptor which is linked to adenylate cyclase, whereby it modulates the cyclic AMP level. Much less is known about the alpha-adrenergic receptor. Recently, two factors have contributed to a renewed interest in alpha- and beta-adrenergic receptors in liver: i) The fact that activation of glycogenolysis in isolated liver parenchymal cells by epinephrine may be mediated by either alpha- or beta-adrenergic receptors, depending on the species or on the state of nutrition, and not only by beta-adrenergic receptors as previously thought. ii) The existence of specific adrenergic agonists and antagonists radiolabeled to a high specific activity which has permitted the characterization of adrenergic receptors in terms of nature, number, affinity and regulation. The present review will be devoted to the recent progress made in the physiological, pharmacological and biochemical characterization of alpha- and beta-adrenergic receptors in the liver.

In vitro characterization of adrenergic receptors mediating extrusion of preformed sebum from preputial gland of rat.

Extrusion of preformed sebum from the preputial glands of rat after phenylephrine and adrenaline treatment, and its inhibition by alpha-receptor blocking agents under in vitro conditions, shows that secretory response of the glands is influenced by alpha-adrenergic receptors, and isoproterenol--a beta-agonist--is not effective in elicitation of exudation of secretion from the preputial gland.

Identification and characterization of adrenergic receptors and catecholamine-stimulated adenylate cyclase in hog pial membranes

In order to define the adrenergic receptors in small pial blood vessels, we studied [ 3H]dihydroalprenolol (DHA) binding, [ 3H]dihydroergocryptine (DHE) binding, and catecholamine-stimulated adenylate cyclase in membranes prepared from the hog pia. [ 3H]DHA bound to a saturable number of sites (0.09 pmol/mg protein) with a high affinity (10 nM). The ability of a small series of adrenergic agents to compete for [ 3H]DHA binding was as expected for binding to β-adrenergic membrane receptors. The receptor binding appeared to be composed largely ofβ 2-adrenergic sites but some sites withβ 1 properties were detected. The β-adrenergic agonist, isoproterenol, stimulated adenylate cyclase activity by 20% in pial membranes in the presence of GTP. The potency of catecholamines in stimulating adenylate cyclase correlated well with their ability to compete for [ 3H]DHA binding. [ 3H]DHE also bound to a saturable number of sites (0.39 pmol/mg) on these membranes. α-Adrenergic agents were potent competitors for [ 3H]DHE binding but dopamine, serotonin and histamine had effects only at high concentrations. These data demonstrate the presence of α- and β-adrenergic membrane receptors and β-adrenergically stimulated adenylate cyclase activity in small pial blood vessels from the hog.

Characterization of adrenergic receptors, with examples from some sympathetically innervated structures.

Characterization of adrenergic receptors, with examples from some sympathetically innervated structures.