CONSIDERABLE evidence exists that the tricyclic antidepressant drugs are selective inhibitors of monoamine uptake into monoaminergic nerve terminals1,2. These drugs also potentiate pharmacological responses to the monoamines in both the peripheral and central nervous systems3–6. These effects do not explain the discrepancy between the time-course of the biochemical and pharmacological responses which are elicited by these antidepressant drugs within minutes or hours and their clinical antidepressant action which requires treatment for weeks for efficacy to be reached. Furthermore, desipramine, an antidepressant drug, can potentiate neuronal responses to noradrenaline and dopamine in the caudate nucleus7, although it does not block the catecholamine uptake in this area of the brain1,2. Finally the antidepressant iprindole does not influence noradrenaline turnover8 or metabolism9,10 or its uptake into catecholaminergic neurones11, but is effective in potentiating the responses of single cortical and caudate neurones to monoamines12. Vetulani and his associates13 suggested that the therapeutic action of tricyclic antidepressants may be related to postsynaptic adaptive changes in the sensitivity of the noradrenergic adenylate cyclase receptor system rather than to acute presynaptic events. The purpose of the experiments described here was to explore the molecular basis for antidepressant-induced noradrenergic subsensitivity by examining the kinetic properties of the β-adrenergic receptors in the microsomal suspension of rat brain by using a potent β-adrenergic receptor antagonist 3H-dihydroalprenolol as a radiolabelled ligand14–19. Our results indicate that the main mechanism is a reduction in the number of receptors.

Development of β-adrenergic receptor subsensitivity by antidepressants

We systematically modified isoproterenol's chemical structure to reduce chronotropic, arrhythmogenic, and vascular side effects. Experiments on dogs showed that the resulting drug, dobutamine, had an inotropic efficacy as great as that of epinephrine due to a direct action on beta1 cardiac receptors. However, unlike epinephrine, dobutamine's effect on alpha and beta2 vascular receptors was slight. At equivalent inotropic doses, dobutamine had less than a fourth of the chronotropic effect of isoproterenol. Desmethylimipramine (DMI), which blocks the sympathetic nerve fiber uptake mechanism, had no effect on dobutamine's actions. In contrast, DMI antagonized dopamine's inotropic effect, and marked chronotropic and pressor responses occurred when we used doses of dopamine large enough to elicit a direct inotropic effect. Dobutamine increased the contractility of isolated cat papillary muscles more but the automaticity less than did isoproterenol. In ischemic dog hearts, dobutamine lacked significant arrhythmic activity, whereas dopamine, norepinephrine, and isoproterenol caused severe ectopic activity. In dogs with experimentally induced low cardiac contractility, low cardiac output, and hypotension, dobutamine produced dose-related increases in cardiac contractility and output, restored arterial blood pressure, and reduced total peripheral resistance slightly. In contrast, isoproterenol failed to restore blood pressure, had only a meager effect on cardiac contractility and output, cuased extreme tachycardia, and lowered peripheral resistance more than did dobutamine. Norepinephrine, which did not increase cardiac contractility or output as much as dobutamine, excessively elevated peripheral resistance and arterial blood pressure.

Dobutamine: development of a new catecholamine to selectively increase cardiac contractility.

Receptors for peptide hormones and neurotransmitters are integral components of the plasma membrane of cells which serve to couple the external milieu to the intracellular regulators of metabolism. These macromolecules are usually high molecular weight glycoproteins, and in many cases appear to have more than one subunit capable of binding the hormone. The interaction of the hormone or neurotransmitter with its receptor is rapid, reversible, and of high affinity and specificity. Many receptors exhibit cooperative properties in hormone binding or biological function. The concentration of receptors on the membrane is a function of continued synthesis and degradation, and may be altered by a variety of factors including the hormone itself. The fluid mosaic nature of the membrane may allow hormone receptors and effectors to exist in free floating states. Further investigations of the hormone-receptor interaction will no doubt yield new insights into both the mechanism of hormone action and membrane structure and function.

/pdf/membrane-receptors-for-hormones-and-neurotransmitters-1mqu6naxr2.pdf

Membrane receptors for hormones and neurotransmitters.

Publisher Summary Studies of receptor binding generally employ highly radioactive ligand derivatives; however, knowledge of the biological activity of the radioactive derivative is essential for a complete interpretation of the binding data. This chapter describes the relation between biological activity and binding, physicochemical detection and assay of receptors, solubilization of membrane receptors, provides examples of membrane-localized receptors, affinity techniques, polyvalent hormone derivatives, and receptor studies, and theoretical mechanisms of hormone–receptor binding and action. The biological response toward many neurotransmitters and hormones, such as insulin, acetylcholine, and epinephrine is rapid both in onset and in the termination of action on removal of these agents; therefore, it may be anticipated that the initial hormone–receptor interaction will also be reversible. The chapter shows that progress has been made toward understanding the way by which hormones, via interactions with cell–surface receptors, modulate overall cellular function. This interaction is only the first in a presumably complicated series of protein–protein interactions within the plane of the cell membrane; the mechanism, whereby the ultimate signal is transmitted to the internal cellular milieu is undoubtedly an equally intricate process. The chapter is considered to be useful in delineating the challenge that the elucidation of these processes presents and in providing a basis and incentive for more discoveries in this field.

Membrane receptors and hormone action.

Injection of frogs with beta-adrenergic catecholamines for 1-24 hr produces marked subsensitivity of the erythrocyte membrane adenylate cyclase [ATP pyrophosphate-lyase (cyclizing); EC 4.6.1.1.] to in vitro stimulation by isoproterenol. The subsensitization is specific for catecholamine stimulation, since basal and fluoride-stimulated enzyme activity are unaffected. Maximum isoproterenol-stimulated adenylate cyclase activity declines by 75% in the isoproterenol-treated animals (P less than 0.001). The concentration of isoproterenol causing one-half maximal activation of adenylate cyclase, however, is unaltered. (-)[3H]Alprenolol, a potent competitive beta-adrenergic antagonist, was used to study directly the beta-adrenergic receptor binding sites in the erythrocyte membranes from control and subsensitized animals. A highly significant (P less than 0.005) 60% fall in the number of the beta-adrenergic receptor binding sites ("specific"(-)[3H]alprenolol binding sites) in the treated animals was found. The binding affinity of the sites was not markedly altered. These data suggest that beta-adrenergic catecholamines are able to regulate catecholamine sensitivity of tissues in vivo, by regulating the properties of the beta-adrenergic receptor binding sites.

Catecholamine-induced subsensitivity of adenylate cyclase associated with loss of beta-adrenergic receptor binding sites

Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase.

SUMMARY ( -)-Alprenolol, a potent, competitive @-adrenergic antagonist labeled to high specific activity with tritium (17 Ci per mmol), has been used to identify binding sites in frog erythrocyte membranes having many of the characteristics to be expected of the P-adrenergic receptors which are linked to adenylate cyclase in these membranes. The chromatographic behavior and biological activity of the labeled and native drug were essentially identical. ( -)-Alprenolol and ( -)-[3H]alprenolol both competitively antagonize isoproterenol stimulation of frog erythrocyte membrane adenylate cyclase with a KD of 5 to 10 KIM. ( -)-[3H]Alprenolol binding to sites in the frog erythrocyte membranes was studied by a centrifugal assay. At 37”, equilibrium binding was established within 5 min and the half-time for dissociation of bound ( -)-[3H]alprenolol was approximately 30 s. This rapid onset and dissociation of ( -)-[3H]alprenolol binding was in good agreement with the rapid onset of action of /3-adrenergic agonists and antagonists on the frog erythrocyte adenylate cyclase. ( -)-[3H]Alprenolol binding was saturable. There were 0.25 to 0.35 pmol of ( -)-[3H]alprenolol binding sites per mg of protein corresponding to 1300 to 1800 binding sites per intact frog erythrocyte. The binding sites showed halfmaximal saturation at 5.0 to 10 no ( -)-[3H]alprenolol, which is in good agreement with the K. for alprenolol antagonism of isoproterenol stimulation of adenylate cyclase. The ( -)[3H]alprenolol binding sites exhibited strict stereospecificity. ( -)-Stereoisomers of /3-adrenergic antagonists or agonists were approximately 2 orders of magnitude more potent than the (+)-stereoisomers in competing for the binding sites. Comparable stereospecificity was apparent when agonists and antagonists were tested for their ability to interact with the adenylate cyclase-coupled P-adrenergic receptors in the membranes. Potency series of 11 agonists and 13 antagonists for inhibition of binding and interaction with adenylate cyclase were identical and were characteristic of a Pz-adrenergic receptor. A variety of nonphysiologically active compounds containing a catechol moiety as well as several metabolites and

Michael Coverstone

Papers

Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase.

Identification of Adenylate Cyclase-coupled fi -Adrenergic Receptors in Frog Erythrocytes with (-) -( 311) Alprenolol"