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The characteristics of the beta-receptor in turkey erythrocyte adenylate cyclase were studied using both kinetics of enzyme activation and direct binding measurement of the beta-agonists and antagonists to the beta-receptor. The regulatory ligands Gpp(NH)p and Ca2+ do not have any direct effect on the beta-receptor, but modulate the enzyme activity through the interaction with specific regulatory sites.

Several new clonidine analogs were synthesized and their ability to inhibit [3H]phentolamine binding to human platelet alpha 2-adrenergic receptors was tested. The order of potency and calculated dissociation constants for clonidine and its analogs were as follows: clonidine (0.020 +/- 0.005 microM) greater than p-aminoclonidine (0.100 +/- 0.010 microM) greater than hydroxy-phenacetyl-aminoclonidine (0.20 +/- 0.03 microM) greater than p-dansyl clonidine (1.00 +/- 0.20 microM) greater than t-boc-tyrosine clonidine (1.80 +/- 0.60 microM). Thus, p-amino substitution reduces alpha 2-adrenergic affinity in the platelet system. The effects of clonidine and its p-amino analogs on platelet adenylate cyclase were also evaluated. This enzyme is inhibited by epinephrine acting via alpha 2-adrenergic receptors. Both clonidine and p-aminoclonidine cause slight inhibition of basal adenylate cyclase and reverse the inhibition induced by epinephrine. These observations indicate that clonidine is a partial agonist for platelet alpha 2-adrenergic receptors.

The relative influences of the in vivo administration of phenoxybenzamine on in vitro binding to alpha 1-adrenergic receptors and alpha 1-receptor-mediated responses were studied. Phenoxybenzamine treatment reduced maximal specific binding of the alpha 1-selective antagonist [3H]prazosin to liver cell membranes. This response was rapid (less than 90 min) and half-maximal following a phenoxybenzamine dose of approx. 10 mg/kg. A similar decrease in the ability of phenylephrine to stimulate glucose release and 45Ca2+ efflux from liver slices was also noted after phenoxybenzamine treatment. During the recovery period following administration of 30 mg/kg phenoxybenzamine, [3H]prazosin specific binding and phenylephrine-stimulated glucose release and 45Ca2+ efflux returned to their respective control levels with t 1/2 values of 42, 49 and 38 h, respectively. At all times studied during the recovery period, alpha 1-binding and both of the alpha 1-responses were similar fractions of their respective control values. These observations indicate that a close relationship exists between the density of [3H]prazosin binding sites and the ability of rat liver to respond to alpha 1-stimulation. We suggest that the binding sites identified in studies using the antagonist [3H]prazosin and those through which the agonist phenylephrine stimulates glucose release and 45Ca2+ efflux are either identical or in equilibrium with each other.

1 The synthesis of several butyrophenone analogues of haloperidol is described. 2 The effects of these compounds on alpha-adrenoceptors were evaluated by examining their ability to reduce alpha 1-stimulated K+ release from rat parotid slices and to displace [3H]-phentolamine from human platelet membrane alpha 2-adrenoceptors. 3 The affinity of haloperidol and its analogues for alpha 1-receptors was found to be 1 to 2 orders of magnitude greater than that for alpha 2-adrenoceptors. These observations suggest that most of the alpha-adrenoceptor activity of butyrophenones results from their interaction with alpha 1-adrenoceptors. 4 The relatively high affinity of the butyrophenones for alpha 1-adrenoceptors suggests that they may be useful as probes in studies of alpha 1-adrenoceptors in these and other tissues.

Exposure of intact human platelets or platelet membranes to the clonidine analog clonidine p-isothiocyanate (clonidine-NCS), followed by extensive washing, results in the loss of [3H]yohimbine binding to platelet alpha 2-receptors. In addition, exposure of intact platelets to clonidine-NCS, followed by extensive washing, results in the loss of of epinephrine-induced inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in frozen--thawed platelets and in purified platelet membranes. This effect is dependent on time and concentration (t 1/2 at 30 degrees C is less than 15 min; half-maximal effect occurs with clonidine-NCS at less than 10 microM). Clonidine-NCS appears to interact by irreversibly blocking the platelet alpha 2-receptors because (i) it abolishes alpha 2-receptor effects of adenylate cyclase activity (i.e., epinephrine-induced inhibition of basal and prostaglandin E1-stimulated activity) while not altering other cyclase activity (basal, prostaglandin E1-stimulated, and NaF-stimulated) and (ii) its effect on both [3H]yohimbine binding and epinephrine-induced inhibition of adenylate cyclase can be specifically prevented by alpha-agonists [(-)-epinephrine and clonidine] and alpha-antagonists (yohimbine and phentolamine). These observations indicate that clonidine-NCS is an effective affinity label for platelet alpha 2-receptors.

The radioiodinated pindolol analogs 125I-labeled cyanopindolol ([125I]CYP) and 125I-labeled hydroxybenzylpindolol ([125I]HBP) have been used to study binding to human platelet beta-adrenergic receptors. [125I]CYP binds to a saturable class of binding sites on platelet membranes with a dissociation constant (Kd) of 14 +/- 3 pM and maximal binding capacity (Bmax) of 18 +/- 4 fmol/mg protein. Binding of [125I]CYP is reversible and is characterized by forward and reverse rate constants of 1.8 . 10(7) s-1 . M-1 and 3.8 . 10(-4) s-1, respectively. [125I]HBP binds to a saturable class of platelet membrane sites with a Kd of 50 +/- 10 pM and Bmax of 32 +/- 6 fmol/mg protein. [125I]HBP also binds to a saturable class of sites on intact platelets with a Kd of 58 +/- 14 pM and Bmax of 24 +/- 4 molecules per platelet. Binding of [125I]CYP and [125I]HBP is stereospecifically inhibited by propranolol and epinephrine; the (-) stereoisomers are at least 50-times more potent than the (+) stereoisomers. Binding of both radioligands is inhibited by adrenergic ligands with a potency order of propranolol much greater than isoproterenol greater than epinephrine greater than practolol greater than norepinephrine greater than phenylephrine. These observations indicate that [125I]CYP and [125I]HBP bind to platelet sites which have the pharmacological characteristics of beta-adrenergic receptors but which are not typical of either the beta 1 or beta 2 sub-type.

The stereospecificity of the beta-adrenergic receptor of turkey erythrocyte ghosts was studied. Binding of the specific beta-adrenergic antagonist, propranolol, was found to occur exclusively with the l-stereoisomer. The stereospecificity of catecholamine binding was determined by assaying the ability of the catecholamines to displace [(3)H]propranolol. Binding of the catecholamines was also found to be stereospecific for the l-stereoisomers. Furthermore, the d-stereoisomers do not compete with the l form for binding. Using this displacement technique, we were able to calculate the dissociation constants for l-epinephrine, l-norepinephrine, and l-isoproterenol from the beta-receptor and compare these values to the apparent dissociation constants obtained from the direct activation of adenylate cyclase (EC 4.6.1.1) by these catecholamines.

Turkey erythrocyte ghosts (empty membranes) possess a class of receptors that can bind both L-[(3)H]isoproterenol and DL-[(3)H]propranolol. The binding of [(3)H]isoproterenol to these receptors occurs with a dissociation constant of 0.15 muM and can be fully inhibited by 1 muM propranolol. The binding of [(3)H]propranolol occurs with a dissociation constant of 2.5 nM and can be fully inhibited by 0.2 mM DL-isoproterenol. Ligand binding is sensitive to sonication, boiling, and 8 M urea. The cells possess 500 to 1000 beta-adrenergic receptors per cell. Binding of propranolol to the beta-receptor was found to be stereospecific for the L stereoisomer. If one assumed a 1:1 relationship between beta-adrenergic receptors and adenylate cyclase, the turnover number of this adenylate cyclase would be close to 100 min(-1).

The compound N-[2-hydroxy-3-(1-naphthoxy)-propyl]-N'-bromoacetylethylenediamine (NHNP-NBE) was found to label covalently the beta-adrenergic receptor in turkey erythrocytes. The compound inhibits irreversibly 1-epinephrine-dependent adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in the whole turkey erythrocyte as well as in the erythrocyte membranes possessing the beta-receptor. The affinity label blocks, also irreversibly, the specific [3H] propranolol binding, whereas other bromoacetyl compounds tested have no effect on binding, even at high concentrations, which cause enzyme inactivation. 1-Epinephrine and propranolol offer protection against the affinity label in whole turkey erythrocytes as well as in membranes prepared from these cells. The potential usefulness of an irreversible beta-antagonist is discussed.