Identification of a-Adrenergic Receptors in Human Platelets by [3H]Dihydroergocryptine Binding KURT D. NEWMAN, LEWIS T. WILLIAMS, N. HAHR BISHOPRIC, and ROBERT J. LEFKOWITZ, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 A B ST RA CT Binding of [3H]dihydroergocryptine to INTRODUCTION platelet lysates appears to have all the characteristics of binding to a-adrenergic receptors. At 25°C binding The endogenous catecholamines epinephrine and nor- reaches equilibrium within 20 min and is reversible epinephrine exert a variety of regulatory functions on upon addition of excess phentolamine. Binding is satu- human physiological processes. The initial sites of ac- rable with 183+22 fmol of [3H]dihydroergocryptine tion of catecholamines seem to be at membrane adren- bound per mg of protein at saturation, corresponding ergic receptors. Based on the relative order of potencies to 220+26 sites per platelet. Kinetic and equilibrium of a series of adrenergic agonists, Ahlquist proposed studies indicate the dissociation constant of [3H]dihy- that the effects of catecholamines were mediated by droergocryptine for the receptors is 1-3 nM. The spec- two distinct types of adrenergic receptors, termed a- ificity of the binding sites is typical of an a-adrenergic and ,3-adrenergic receptors (1). /-Adrenergic responses receptor. Catecholamine agonists compete for occu- (isoproterenol> epinephrine> norepinephrine) which pancy of the [3H]dihydroergocryptine binding sites are specifically antagonized by propranolol are typified with an order of potency (-)epinephrine> (-)norepi- by relaxation of smooth muscle and stimulation of car- nephrine> (-)isoproterenol. Stereospecificity was dem- diac contractility. a-Adrenergic responses (epineph- onstrated inasmuch as the (+)isomers of epinephrine rine> norepinephrine> isoproterenol) are potently an- and norepinephrine were 10-20-fold less potent than tagonized by phentolamine and are typified by con- the (-)isomers. The potent a-adrenergic antagonists traction of vascular and uterine smooth muscle. phentolamine, phenoxybenzamine, and yohimbine Recent studies have shown that epinephrine and competed potently for the sites, whereas 8-antagonists norepinephrine induce human platelet aggregation such as propranolol and dichlorisoproterenol were whereas isoproterenol does not (2-4). This aggregation quite weak. Dopamine and serotonin competed only at is blocked by the a-antagonist phentolamine (2-4). high concentrations (0.1 mM). Epinephrine and norepinephrine also cause a rapid The [3H]dihydroergocryptine binding sites could decrease in prostaglandin E,-(PGE,)1- enhanced cyclic also be demonstrated in intact platelets where they AMP (cAMP) levels in platelets and produce immedi- displayed comparable specificity, stereospecificity, ate decreases in platelet adenylate cyclase activity (5- and saturability. Saturation binding studies with the 11). These responses are also blocked by the a-adrener- intact platelets indicated 220+±45 receptors per plate- gic blocker phentolamine. Thus, there is evidence that let, in good agreement with the value derived from these adrenergic effects on platelets are mediated by studies with platelet lysates. Ability of a-adrenergic a-adrenergic receptors. agonists to inhibit adenylate cyclase and of a-adrener- The development of radioligand binding methods gic antagonists to antagonize this inhibitory effect di- for the direct study of hormone and drug receptors over rectly paralleled ability to interact with the [3H]dihy- the past few years has facilitated the investigation of droergocryptine binding sites. These data demonstrate the properties and regulation of these receptors. Ap- the feasibility of directly studying a-adrenergic recep- plied to human cell types, these methods provide an tor binding sites in human platelets with [3H]dihydro- approach to the direct study of abnormalities of recep- ergocryptine. tor function in various disease states (12, 13). Recently, Williams et al. described a ligand, [3H] Dr. Lefkowitz is an Investigator of the Howard Hughes dihydroergocryptine, which could be used to identify Medical Institute. Received for publication 4 April 1977 and in revised 1Abbreviations used in this paper: cAMP, cyclic AMP; form 29 September 1977. PGE, prostaglandin E,. The Journal of Clinical Investigation Volume 61 February 1978 395-402 395 a-adrenergic receptors in uterine smooth muscle (14). resuspended in "incubation buffer" (Tris-HCl 0.05 M, pH 7.5, In the present studies we report the successful identifi- EDTA 0.005 M) for use in the binding assay. Whole platelet and lysate preparations were examined by cation and characterization of a-adrenergic receptors phase-contrast and interference-contrast microscopy at x 1000 in human platelets with [3H ]dihydroergocryptine. magnification as well as by electron microscopy. The platelet Binding of [3H]dihydroergocryptine to human platelet preparations appeared intact. The lysate preparations show lysates has all the characteristics of specificity, stereo- lysis of some platelets with areas of cell debris including selectivity, saturability, and reversibility which would scattered cytoplasmic granules and granule-free membrane fragments. Some apparently unlysed platelets were also seen, be expected of binding to a physiological a-adrenergic but these were markedly swollen. receptor. a-Receptor binding is also demonstrated in [3H]Dihydroergocryptine binding assay. [3H]Dihydroer- intact platelets. The specificity of the binding data is gocryptine (5 nM unless otherwise specified) and platelet ly- also correlated with an a-adrenergic response in the sates (2.0 mg/ml unless otherwise specified) or intact platelets (1-2 x 109/ml) were incubated at 25°C with shaking for 18 min platelet lysates inhibition of PGE,-stimulated adenyl- in a total volume of 150 ,ul of incubation buffer for platelet ate cyclase. lysates, or buffer I for intact platelets. In competition experi- ments, varying concentrations of agonists or antagonists were added to the incubation as indicated. Incubations were ter- METHODS minated by rapidly diluting the total volume with 2 ml of incu- bation buffer or buffer I at 250C followed by rapid filtration Pharmacological agents. [3H] Dihydroergocryptine was through Whatman GFC glass fiber filters (Whatman, Inc., used as the a-adrenergic radioligand for this study. It has a Clifton, N. J.). Filters were rapidly washed with 20 ml of incu- specific activity of 23.0 Ci/mmol and has biologic activity and bation buffer. After drying, filters were counted in a Triton- chromatographic properties identical to native dihydroer- toluene scintillation mixture at an efficiency of 40-50%. gocryptine (14). Other compounds used in this study were "Nonspecific" binding was defined as binding which was from the following sources: Sigma Chemical Co., St. Louis, not displaced by a high concentration of 10 ,uM phentolamine, Mo.-(-)epinephrine bitartrate, (-)norepinephrine bitar- a potent a-adrenergic antagonist, or 1 mM epinephrine, an trate, (- )isoproterenol bitartrate, dopamine hydrochloride, a-adrenergic agonist. In platelet lysates, 1 mM epinephrine dihydroxyphenylalanine, dihydroxymandelic acid, (+)norme- and 10 ,uM phentolamine inhibited binding to the same extent, tanephrine, .dihydroergotamine, ergotamine tartrate, ergocryp- and higher concentration of phentolamine did not inhibit tine, (-)phenylephrine hydrochloride, and (+)propranolol; binding further. In intact platelets, 10 ,M phentolamine in- Winthrop Laboratories, New York-phenylephrine hydro- hibited binding slightly, but consistently to a greater extent chloride (used only in certain experiments); Sandoz Pharma- (- 20%), than 1 mM epinephrine. Accordingly in studies with ceuticals, E. Hanover, N. J.-dihydroergocryptine methane intact platelets we have used 1 mM epinephrine to determine sulfonate; Sterling Drug, Inc., New York-(+)epinephrine nonspecific binding, assuming that all true a-adrenergic re- bitartrate and (+)norepinephrine bitartrate; Ayerst Labora- ceptors should be accessible to epinephrine as well as phento- tories, New York-practolol hydrochloride and (+)butacla- lamine. "Specific" binding, defined as total binding minus mol; Eli Lilly and Co., Indianapolis, Ind. -(+)dichlorisopro- nonspecific binding is shown in all figures and tables. Specific terenol; CIBA Pharmaceutical Co., Div. of CIBA-Geigy Corp., binding was generally 55-70% of the [3H]dihydroergocryp- Summit, N. J. -phentolamine hydrochloride; McNeil Labora- tine bound in the platelet lysates and 30-40% in the intact tories, Inc., Fort Washington, Pa.-dibozane and haloperidol; platelets. In competition experiments with [3H]dihydroergo- ICN Nutritional Biochemicals Div., Cleveland, Ohio-yo- cryptine present at -5 nM, specific binding was generally himbine hydrochloride; Boehringer Ingelheim Ltd., Elms- -110 fmol/mg protein in the platelet lysates. ford, N. Y.-clonidine hydrochloride; Burroughs Wellcome To assess the integrity of the [3H]dihydroergocryptine the Co., Research Triangle Park, N. C.-methoxamine hydro- following experiment was performed. Binding incubations chloride; and Smith Kline & French Laboratories, Div. of were performed in the usual manner. The membranes were Smith Kline Corp., Philadelphia, Pa.-phenoxy-benzamine then collected by centrifugation. Radioactivity from the super- hydrochloride. nate and that extracted from the pellet with ethanol was then Preparation of platelets and platelet lysates. 100-200 cm3 chromatographed