Pharmacological profile of binedaline, a new antidepressant drug

The interactions of binedaline (binodaline), a new antidepressant drug, and its main metabolites with neurotransmitter receptors and monoamine uptake sites were studied. In receptor binding assays, binedaline was compared to amitriptyline, imipramine, maprotiline and mianserin. Unlike these drugs binedaline did not show any significant affinity for an alpha adrenergic, muscarinic cholinergic, histamine H1 or serotonin2 (5-HT2) receptors. Binedaline and desmethylbinedaline were potent inhibitors of the uptake of norepinephrine in synaptosomes from rat cerebral cortex (Ki = 25 and 29 nM, respectively). Binedaline also inhibited 5-HT uptake with a weak affinity (Ki = 847 nM) but was inactive as an inhibitor of dopamine uptake in synaptosomes from rat striatum (Ki greater than 2 microM). No specific binding was found using [3H]binedaline. After 2 weeks of daily administration of binedaline (20 mg/kg i.p.), the number of beta adrenergic recognition sites labeled with [3H]CGP 12177 remained constant in rat forebrain, as did 5-HT2 receptors and benzodiazepine receptors. In contrast a prolonged treatment with maprotiline (20 mg/kg i.p.) increased the apparent Kd value of [3H]CGP 12177 by 43% and the apparent maximal binding value of [3H]RO 15-1788 by 20% as compared to control. Our results indicate that binedaline is comparable to a tricyclic antidepressant drug in inhibiting the norepinephrine uptake but, however, it is devoid of affinity for neurotransmitter receptors. This probably explains why this drug does not induce the classical side effects of tricyclic antidepressant drugs. These results also suggest that a reduction in beta adrenergic, 5-HT2 or benzodiazepine receptors is not always related to an antidepressant chronic treatment.     

Preclinical evaluation of McN-5707 as a potential antidepressant

Based on its activity in a variety of tests in vivo and in vitro McN-5707 [trans-6-(2-chlorophenyl)-1,2,3,5,6,10b-hexahydropyrrolo- (2,1-a)isoquinoline] is a novel potential antidepressant. McN-5707 blocked tetrabenazine-induced sedation and ptosis in mice and rats, and potently inhibited the uptake of norepinephrine by synaptosomes from rat hypothalamus (Ki approximately 2 nM), and the uptake of serotonin by synaptosomes from rat cerebral cortex (Ki approximately 10 nM). McN-5707 also inhibited the uptake of dopamine by synaptosomes from rat striatum (Ki approximately 40 nM); however, the stereotypic behavior often caused by dopamine uptake inhibitors was not evident in rats at doses of 300 mg/kg (p.o.) or less. In receptor binding assays, McN-5707 potently inhibited ketanserin binding to serotonin 5-HT2 receptors in synaptic membranes from rat cerebral cortex (apparent Ki approximately 8 nM). In mice, McN-5707 antagonized 5-hydroxytryptophan-induced head twitches. Spiperone binding to dopamine D2 receptors in synaptic membranes from rat striatum was weakly inhibited by McN-5707 (apparent Ki approximately 400 nM), as was the binding of WB4101 to alpha-1 adrenergic receptors (apparent Ki approximately 150 nM). McN-5707 was essentially inactive as an inhibitor of [3H]clonidine binding to alpha-2 adrenergic receptors and of [3H]quinuclidinyl benzilate binding to muscarinic receptors. In experiments with guinea pig ileum, McN-5707 weakly antagonized histamine-induced contractions and exhibited virtually no cholinergic or anticholinergic activity. Our observations indicate McN-5707 possesses attributes of both tricyclic and newer atypical antidepressants because it inhibits the uptake of both norepinephrine and serotonin, and blocks 5-HT2 receptors, but lacks some of the anticholinergic and behavioral properties often associated with them.     

Effects of clenbuterol and antidepressant drugs on beta adrenergic receptor/N-protein coupling in the cerebral cortex of the rat

Repeated administration of the centrally acting beta adrenergic agonist clenbuterol to rats reduced the ability of isoproterenol to increase levels of cyclic AMP in slices of cerebral cortex. This lessened response to isoproterenol was not due to a reduction in beta receptor density but appeared to be due to diminished receptor/N-protein coupling. This was determined by measuring the ability of isoproterenol to inhibit the binding of the beta adrenergic antagonist [125I]iodopindolol to membranes prepared from cerebral cortex. Using membranes prepared from vehicle-treated rats, isoproterenol, in the absence of GTP, inhibited the binding of [125I]iodopindolol with an IC50 value of 85 nM and a Hill coefficient of 0.65. GTP (250 microM) increased the IC50 value to 290 nM and the Hill coefficient to 0.98. After repeated administration of 10mg/kg of clenbuterol to rats for 8 days, isoproterenol inhibited the binding of [125I]iodopindolol with an IC50 value of 125 nM and a Hill coefficient of 0.90; GTP increased the IC50 value to 170 nM and the Hill coefficient to 0.98. It was inferred from the results of modeling of the isoproterenol competition curves that the repeated administration of clenbuterol reduced or eliminated the high-affinity component of isoproterenol binding. These effects of clenbuterol were found to depend on dose and duration of treatment and were reversible. Repeated administration of the antidepressant drugs desipramine, imipramine, phenelzine, zimelidine and mianserin twice daily for 21 days, by contrast, did not affect receptor/N-protein coupling.(ABSTRACT TRUNCATED AT 250 WORDS)     

The positive chronotropic effect induced by BRL 37344 and CGP 12177, two beta-3 adrenergic agonists, does not involve cardiac beta adrenoceptors but baroreflex mechanisms.

The presence of a beta-3 adrenoceptor (in addition to the classical beta-1 and beta-2 adrenoceptors) and its involvement in the control of heart rate was investigated in the dog. Experiments were carried out in conscious normal and sinoaortic denervated dogs (i.e. animals deprived of baroreceptor pathways). In normal dogs, infusion of isoproterenol, BRL 37344 (4-[-[(2-hydroxy-(3-chlorophenyl) ethyl)-amino]propyl]phenoxyacetate) (a beta-3 adrenergic agonist) or CGP 12177 (4-[3-t-butylamino-2-hydroxypropoxy]benzimidazol-2- one) (a beta-1 beta-2 adrenergic antagonist reported to act as an agonist for the beta-3 adrenergic receptor) increased heart rate with an order of potency: BRL 37344 > isoproterenol > CGP 12177. [125I]Cyanopindolol binding (2-2000 pM) was saturable and Scatchard analysis indicated the presence of an homogenous population of binding sites. KD was 12.8 +/- 18.5 pM and maximum binding was 94.2 +/- 12.5 fmol/mg of protein. Competition binding studies on dog heart membranes using 150 pM [125I] cyanopindolol indicated an order of potency (CGP 12177 > isoproterenol > BRL 37344) different from that observed in cardiovascular studies. Isoproterenol stimulated adenylate cyclase activity in heart membranes from normal dogs, whereas CGP 12177 and BRL 37344 were without any stimulating action. The positive chronotropic effects of isoproterenol, BRL 37344 and CGP 12177 were accompanied with a reduction in arterial blood pressure. In sinoaortic denervated animals, isoproterenol infusion provoked tachycardia and hypotension. BRL 37344 and CGP 12177 were without any significant effect on heart rate but induced a rapid and dramatic hypotension.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-1 receptor is the predominant beta-adrenoreceptor on rat brown adipose tissue

A new in vitro radioligand binding assay is described for brown adipose tissue using the beta adrenergic antagonist [3H]CGP 12177 (4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H]benzimidazol-2-one). Binding was saturable and stereoselectively inhibited by propranolol. There was 60 to 80% specific binding using either 30 microM l-isoproterenol or 10 microM l-propranolol to define nonsaturable binding. [3H]CGP 12177 was bound to partially purified membranes from collagenase-separated brown adipocytes with a Kd of 0.84 nM, as determined from kinetic studies, and 1.24 +/- 0.13 nM as found by equilibrium binding studies; maximum binding was 14.2 +/- 0.9 fmol/mg of protein. Membranes from whole-pad homogenates had a similar Kd of 1.17 +/- 0.14 nM but twice the maximum number of binding sites (28.5 +/- 4.4 fmol/mg of protein). Intact brown adipocytes had a Kd of 0.55 nM and a maximum binding of 29.4 +/- 1.5 fmol X 10(-6)/cell or 17,700 sites per cell. Competitive binding studies showed about 80% of the binding sites to be of the beta-1 and 20% of the beta-2 subtype. The pA2 values derived from inhibition of isoproterenol-stimulated in vitro oxygen consumption in intact brown adipocytes by the beta-1 selective antagonist metoprolol and beta-2 selective lCl 118551 were in close agreement with their respective K1 values at the beta-1 receptor as derived from competitive binding studies. These data strongly suggest that the beta-1 adrenoreceptor on brown adipose tissue is primarily responsible for the initiation of thermogenesis in this tissue.     

Multiple beta adrenergic receptor subclasses mediate the l-isoproterenol-induced lipolytic response in rat adipocytes.

l-Isoproterenol has been proposed to stimulate lipolysis in rat epididymal adipocytes via atypical beta adrenergic receptors, whereas radioligand binding studies only revealed the presence of beta 1 adrenergic receptors on adipocyte membranes. We have made use of the unique properties of CGP12177 to evidence that both the beta 1 and the atypical beta adrenergic receptor subtypes are mediating the lipolytic response of rat epididymal adipocytes to l-isoproterenol. CGP12177, an antagonist with high affinity for beta 1 receptors, triggers lipolysis by specifically stimulating the atypical receptors. For this response, CGP12177 displays low potency (EC50 = 68 nM), but high intrinsic activity (94% relative to l-isoproterenol). At low concentrations (3 nM), CGP12177 inhibits the lipolytic response to 10 nM l-isoproterenol by 43%, indicating that at least this fraction of the response is beta 1 receptor-mediated. The response to BRL37344, which is a selective agonist for the atypical receptors, is not inhibited by CGP12177. The pA2 values of the beta adrenergic antagonists propranolol, metoprolol and atenolol were calculated from the rightward shifts that they impose on dose-response curves of both l-isoproterenol and CGP12177. With l-isoproterenol, these values (6.54, 5.83 and 5.07, respectively) are lower than those expected for beta 1 and beta 2 receptors, indicating that atypical receptors are also involved in the lipolytic response to this agonist. With CGP12177, the pA2 values of propranolol, metoprolol and atenolol are even lower (5.80, 5.03 and 4.06, respectively), and are likely to be a more accurate reflection of their affinities for the atypical receptors.     

Binding and functional characteristics of beta adrenergic receptors in the intact neutrophil

Beta adrenergic receptors have been previously characterized in human neutrophil sonicates. In the present study the intact neutrophil has been assessed for the number and affinity of beta adrenergic binding sites by using the antagonist DNA. Agonist and antagonist potencies, characterized by their effect on DHA binding and cyclic AMP accumulation, are compared with agonist inhibition of lysosomal enzyme (beta glucuronidase) release. Criteria for beta adrenergic receptor identification were successfully demonstrated. At 30 degrees C, beta adrenergic binding was rapid (t 1/2 2 min) and reversible (t 1/2 9 min). Receptor binding was saturable, revealing approximately 900 high-affinity receptors per neutrophil with DHA concentrations of 0.1 to 10 nM. By utilizing both equilibrium and kinetic techniques, the KD was determined to be approximately 0.6 nM. Agonists and antagonists competed for DHA binding in a manner consistent with their effect on cyclic AMP generation. Rank order potency was suggestive of a beta-2 receptor: isoproterenol greater than epinephrine greater than norepinephrine. Stereoselectivity was shown by the greater potency of L-propranolol compared to the D isomer. A high degree of receptor-adenylate cyclase coupling efficiency was suggested by the observation that with only 1% receptor occupancy isoproterenol stimulated 50% maximal cyclic AMP generation. Finally, there was an excellent correlation between the isoproterenol concentration which resulted in 50% of maximal inhibition of beta glucuronidase release (Ki) and that causing 50% maximal cyclic AMP stimulation (Kact), suggestive of a close relationship between beta adrenergic-induced adenylate cyclase activation and beta adrenergic regulation of neutrophil lysosomal enzyme release. The data presented suggest that the use of the intact neutrophil for study of the beta adrenergic receptor is feasible and may provide information which is considerably more closely related to modulation of physiological function by neurohormones than is possible with disrupted cell preparations.     

Characterization of alpha-1 adrenergic receptors linked to [3H]inositol metabolism in rat cerebral cortex

The properties of alpha-1 adrenergic receptors in rat cerebral cortex were examined by measuring increases in [3H]inositol metabolism in brain slices. Slices of rat cerebral cortex were incubated in the presence of 0.23 microM [3H]inositol in Krebs-Ringer-bicarbonate buffer containing 10 mM lithium chloride, and the production of water-soluble [3H]inositol phosphates was monitored after extraction and anion-exchange chromatography. Norepinephrine caused a 4- to 6-fold increase in [3H]inositol metabolism in cerebral cortical slices, and this response was blocked much more potently by the alpha-1-selective antagonist prazosin than by the alpha-2-selective antagonist yohimbine. Epinephrine and norepinephrine were both full agonists and stimulated [3H]inositol metabolism to the same extent in this system. The synthetic drugs phenylephrine and methoxamine were both partial agonists at these receptors, with intrinsic activities only 56 to 58% of epinephrine and norepinephrine. A variety of imidazoline and other partial agonists caused no measurable stimulation of [3H]inositol metabolism in this preparation. The response to norepinephrine was completely blocked by alpha adrenergic receptor antagonists with the potency order prazosin greater than BE 2254 greater than indoramin = phentolamine greater than azapetine greater than piperoxan greater than yohimbine. In the absence of calcium, basal [3H]inositol metabolism was increased, but norepinephrine caused the same 5-fold stimulation as in the presence of 2.5 mM CaCl2. The potencies of both antagonists and agonists in inhibiting or activating [3H]inositol metabolism in slices of rat cerebral cortex were highly correlated with their ability to displace the alpha-1 adrenergic receptor selective radioligand [125I]BE 2254 from specific binding sites in membrane preparations of rat cerebral cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of beta adrenergic receptors on rat mononuclear leukocytes by stress: receptor redistribution and down-regulation are altered with aging

In vitro incubation of mononuclear leukocytes (MNL) with catecholamines desensitizes beta adrenergic receptors, meaning isoproterenol-stimulated cyclic AMP accumulation decreases. This desensitization is accompanied by two patterns of receptor changes: first, reduction of surface receptors (defined as binding of [3H]dihydroalprenolol inhibited by 1 microM CGP 12177 [4-3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole- 2-on-hydrochloride]) without any change in the total number of [3H] dihydroalprenolol binding sites inhibited by 1 microM propranolol (receptor redistribution); then reduction of the total number of receptors (receptor down-regulation). In the present study we investigated receptor redistribution and down-regulation under physiological conditions by raising endogenous catecholamines in the rat by stress. In young rats a single immobilization stress induced MNL beta adrenergic receptor redistribution: the number of surface receptors was reduced by about 50% but the total number remained the same. Receptor redistribution was prevented completely in rats pretreated with beta-blocking nadolol. Repeated stress down-regulated the MNL beta adrenergic receptors as shown by a reduction in the total number of sites. We also investigated the regulation of beta adrenergic receptors in three age-groups. After 60 min of immobilization stress the number of MNL surface receptors was reduced in young (4-month-old) rats but not in mature (12-month-old) or aged (26-month-old) rats. Using an alternative stress procedure, after single or repeated open-field sessions, we found receptor redistribution and down-regulation, respectively, in young rats. None of these adaptive receptor response was observed in 26-month-old rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of chick and frog beta adrenergic receptors in primary cultures of myocardial cells.

In membrane preparations derived from primary cultures of chick myocardial cells, beta adrenergic receptors modeled for a single low-affinity site for both betaxolol (beta-1-selective) and ICI 118551 (beta-2-selective) displacement of [125I]iodocyanopindolol (ICYP), indicating that the chick beta receptor is pharmacologically distinct from both mammalian beta-1 and beta-2 adrenergic receptors with respect to these antagonists. However, the highly beta-1-selective compound CGP 20712A was able to distinguish two binding sites on ICYP competition curves, a high-affinity "beta-1 site" (75%) and a low-affinity "beta-2 site" (25%). Also, in chick heart cell membranes the relative ability of agonists to displace ICYP produced a profile typical of beta-1 adrenergic receptors with a rank order of potency or efficacy of: isoproterenol greater than epinephrine = norephinephrine. When agonist-mediated adenylyl cyclase stimulation was assessed the order of potency was slightly different, isoproterenol greater than epinephrine greater than or equal to norepinephrine. Additionally, antagonism of isoproterenol stimulation of adenylyl cyclase by CGP 20712A yielded a Kb value (1.16 +/- 0.35 x 10(-7) M) intermediate between the high and low-affinity binding sites of CGP 20712A, suggesting that the low-affinity site is coupled to adenylyl cyclase. In membrane preparations of frog myocardial cells, ICYP/antagonist competition curves modeled for a mixed population of receptors, with subtype percentages varying from 50:50 beta-1:beta-2 to 100% beta-2 depending on the specific antagonist used and the individual cell preparation. For ICYP/agonist competition binding experiments the relative ability to displace ICYP was isoproterenol greater than epinephrine = norepinephrine, a profile typical of beta-1 adrenergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical profile of risperidone, a new antipsychotic

Risperidone was compared to the 5-hydroxytryptamine2 antagonist ritanserin and to the dopamine-D2 antagonist haloperidol. The in vitro receptor binding (neurotransmitter-, peptide- and ion channel binding sites) and neurotransmitter uptake profile were investigated. Risperidone revealed, like ritanserin, a very high binding affinity for 5-hydroxytryptamine2 receptors (Ki = 0.16 and 0.30 nM, respectively) and a slow dissociation (half-time, 31 and 160 min). In accordance, risperidone (IC50 = 0.5 nM) and ritanserin (IC50 = 1.8 nM) potently blocked serotonin-induced 32P-phosphatidic acid formation in human blood platelets. Risperidone showed, like haloperidol, high binding affinity for dopamine-D2 receptors (Ki = 3.13 and 1.55 nM, respectively) and rapid dissociation (half-time, 2.7 and 5.8 min). Risperidone displayed higher binding affinity than ritanserin and haloperidol for alpha-1 adrenergic (Ki = 0.8 nM), histamine-H1 (Ki = 2.23 nM) and alpha-2 adrenergic receptors (Ki = 7.54 nM). In in vitro superfusion experiments, risperidone and haloperidol reversed at nanomolar concentrations the inhibition by LY 171555 (a dopamine-D2 agonist) and by amphetamine of potassium and electrically evoked release of [3H]acetylcholine from striatal slices (postsynaptic dopamine-D2 effects). Both drugs reversed with similar potency the inhibition by LY 171555 of electrically evoked release of [3H]dopamine (a presynaptic dopamine-D2 effect). Risperidone did not affect the activation by amphetamine of [3H]dopamine efflux from rat striatal slices. Risperidone enhanced at nanomolar concentrations the stimulated [3H]norepinephrine efflux from cortical slices and it similarly reversed the inhibition by clonidine, at concentrations corresponding to its binding affinity for alpha-2 adrenergic receptors. The in vitro biochemical properties of risperidone are in agreement with the reported in vivo pharmacological profile, the relation to clinical findings is discussed.     

Characterization of a bromoacetylated derivative of pindolol as a high affinity, irreversible beta adrenergic antagonist in cultured cells

We have utilized cultured cell lines to test the utility of N8-(bromoacetyl)-N1-[3-(4-indolyoxy)-2-hydroxypropyl]-(Z)-1,8-diam ino- p-menthane, BIM, a recently synthesized, irreversible beta adrenergic antagonist. Previously available irreversible antagonists of beta adrenergic receptors have generally exhibited low affinity (typical IC50 values greater than or equal to 1 microM). By contrast, S49 lymphoma cells incubated with 10 nM BIM for 120 min and then washed extensively showed a 70% loss in beta adrenergic receptors, as measured by [125I]iodocyanopindolol binding. This loss, which could be prevented by propranolol, represented a decrease in receptor number without a change in affinity of the remaining receptors for [125I]iodocyanopindolol. The BIM-induced decrease in binding sites was persistent in membranes incubated for several hours after BIM treatment. BIM did not inactivate alpha-1 adrenergic receptors on Madin Darby canine kidney cells, alpha-2 adrenergic receptors on human erythroleukemia cells, nor did BIM treatment alter guanyl-5'-yl-imidodiphosphate-mediated regulation of agonist binding to the beta adrenergic receptors in S49 cell membranes. BIM treatment decreased cyclic AMP (cAMP) accumulation in S49 cells in response to the beta adrenergic agonist isoproterenol, but increased prostaglandin E1-stimulated cAMP accumulation (P = .09) without altering cAMP production in response to forskolin. The inactivation of beta receptors in S49 cells by BIM (IC50 = 0.30 nM) correlated closely with the loss in beta adrenergic receptor-mediated cAMP accumulation in these cells (IC50 = 0.59 nM), implying the absence of substantial receptor reserve for this response. We conclude that BIM is a potent, irreversible, selective beta adrenergic antagonist for the study of beta adrenergic receptors in cultured cells.     

Agonist interactions with beta adrenergic receptors in rat brain

Agonist interactions with beta adrenergic receptors on membranes prepared from rat brain were examined by measuring agonist inhibition of [125I]iodopindolol binding in the absence or presence of GTP. When rat cerebral cortical membranes were prepared with 1 mM EDTA in the homogenization medium and 2.5 mM MgCl2 was included in the binding reaction, then 250 microM GTP increased the Hill coefficient for isoproterenol from 0.77 to 0.99 and increased the IC50 from 88 to 213 nM. By contrast, I-propranolol competition curves were steep (Hill coefficient = 0.98) and were not affected by GTP. It was inferred from the results of computer-modeling that, in the absence of GTP, isoproterenol bound to two states of the receptor; GTP converted isoproterenol binding to a single low-affinity state. I-Propranolol bound to a single state in the absence or presence of GTP. The effect of GTP on I-epinephrine inhibition of [125I]iodopindolol binding was essentially identical to its effect on isoproterenol inhibition. GTP and GDP were the most potent of all the nucleotides tested. Guanylylimidodiphosphate (1 mM) produced only partial shifts in the isoproterenol competition curves and GMP and ATP were inactive. In membranes prepared from rat hippocampus and hypothalamus, isoproterenol competition curves and GTP effects were qualitatively similar to those observed in cerebral cortex. However, GTP produced only partial shifts of I-isoproterenol competition curves in cerebellum and neostratium. It appears that agonists, but not antagonists, can stabilize a high-affinity ternary complex with the beta adrenergic receptor and the guanine nucleotide binding regulatory protein in membranes prepared from various regions of the rat brain.     

Pineal beta adrenergic receptor: correlation of binding of 3H-l-alprenolol with stimulation of adenylate cyclase.

3H-l-Alprenolol, a potent competitive beta adrenergic antagonist, binds to sites in rat pineal gland membranes. The properties of these binding sites were compared to those of the receptors which mediate the beta adrenergic activation of pineal adenylate cyclase. Both sites are highly stereospecific. The l-stereoisomers of alprenolol and propranolol were at least two orders of magnitude more potent than the d-stereoisomers in inhibiting isoproterenol-stimulated adenylate cyclase or 3H-l-alprenolol binding. The dissociation constants (Kd) of the l-stereoisomers of both alprenolol and propranolol were 10 to 22 nM as determined by competition for binding sites or by inhibition of isoproternol-stimulated adenylate cyclase. Beta adrenergic agonists which stimulated adenylate cyclase also competitively inhibited the binding of 3H-l-alprenolol. They showed the same order of potency (isoproterenol greater than norepinephrine greater than or equal to epinephrine) and the same individual affinities in the two systems. Alpha adrenergic blockers were ineffective in inhibiting either adenylate cyclase stimulation or 3H-l-alprenolol binding. Isoproternol stimulation of adenylate cyclase acrivity, and 3H-l-alprenolol binding, were rapid and rapidly reversible. The 3H-l-alprenolol binding sites were saturable and bound 0.6 pmol of ligand per mg of added protein. The data suggest that the binding of 3H-l-alprenolol occurs at sites indistinguishable from the pineal beta adrenergic receptor.     

Impaired beta adrenergic receptor binding and function in cystic fibrosis neutrophils.

Cystic fibrosis (CF), a genetic disease characterized by abnormalities of exocrine gland and mucociliary function, has recently been shown to be associated with abnormal adrenergic and cholinergic physiologic responses in addition to decreased beta adrenergic-induced cyclic AMP generation in human leukocytes. In this study we have attempted to elucidate the nature of this hyporesponsiveness by assessing beta adrenergic receptor number and affinity (KD) in the intact neutrophil using the antagonist ligand [3H] dihydroalprenolol and cyclic AMP responses to isoproterenol in addition to histamine, and prostaglandin E1 in CF subjects, CF obligate heterozygotes (CFH), and normal control subjects. CF patients had significantly less (p less than 0.025) cyclic AMP stimulation above basals levels with isoproterenol (0.1 microM to 0.1 mM), compared with control values, but no consistent differences between groups were noted with histamine or PGE1. CF neutrophils had significantly fewer (p less than 0.005) beta adrenergic receptors per neutrophil (398.0 +/- 54.2 vs. 819.4 +/- 67.2) compared with control neutrophils, but the KD (0.740 +/- 0.11 vs. 0.630 +/- 0.05 nM) did not differ significantly (p greater than 0.05). There was no correlation between clinical severity and either cyclic AMP generation or dihydroalprenolol binding (r = 0.27 and 0.24, respectively, p greater than 0.05). The CFH group had approximately 50% of the cyclic AMP stimulation compared with controls, but the number (909.8 +/- 89.3) and KD (0.710 +/- 0.09 nM) of their beta adrenergic receptors were indistinguishable from control subjects. These findings suggest "down regulation" of the beta receptor in the CF patient. The cause of this remains unknown. Although the etiology of the decreased cyclic AMP responses in CFH was not due to decreased beta adrenergic receptors as assessed by antagonist ligand binding, further studies inthe CFH group to include agonist binding, receptor-adenylate cyclase coupling, intrinsic adenylate cyclase activity, and catecholamine metabolism may help determine the basic cause of beta adrenergic hyperesposiveness in both CFH and CF.     

Muscarinic cholinergic ligand binding to intact mouse pituitary tumor cells (AtT-20/D16-16) coupling with two biochemical effectors: adenylate cyclase and phosphatidylinositol turnover

(-)-[3H]Quinuclidinyl benzilate (QNB) binding to muscarinic receptors on intact mouse pituitary tumor cells (AtT-20/D16-16) was characterized in an attempt to correlate radioligand binding properties with receptor-coupled biochemical responses. Performing rinse time studies for 2 hr produced a remarkably improved ratio of specific/total (+)-[3H]QNB binding (85%). Kinetic experiments yielded association (k+1) and dissociation (k-1) rate constants of 2.2 X 10(8) M-1 min-1 and 6.8 X 10(-3) min-1, respectively. Receptor occupancy curves demonstrated a uniform population of specific, saturable (-)-[3H]QNB binding sites with a Hill coefficient equal to 1.0 and an apparent dissociation constant (Kd) equal to 34 pM under our conditions. Stereoselectivity was observed with the enantiomers (dexetimide and levetimide) of benzetimide (a factor of 4300). Concentrations of carbachol that produced a half-maximal inhibition of cyclic AMP formation and a concentration of carbachol for producing half-maximal stimulation of phosphatidylinositol turnover in the intact cells were 0.45 and 170 microM, respectively. Schild analysis revealed that pirenzepine, a nonclassical muscarinic antagonist, had a 40-fold greater affinity for reversing carbachol-stimulated phosphatidylinositol turnover (inhibition constant or Ki = 7 nM), compared to its antagonism of the carbachol-mediated inhibition of isoproterenol-stimulated cyclic AMP formation (Ki = 280 nM). Interestingly, pirenzepine inhibited (-)-[3H]QNB binding with a Ki value of 72 nM. In contrast, atropine was nearly equipotent (Ki = 0.3-0.5 nM) in binding studies and in both effector systems.     

Effect of imipramine and adrenocorticotropin administration on the rat brain norepinephrine-coupled cyclic nucleotide generating system: alterations in alpha and beta adrenergic components

Continuous treatment (1-3 weeks) with imipramine or adrenocorticotropin (ACTH) decreases the responsiveness of the norepinephrine-coupled cyclic nucleotide generating system in rat brain cerebral cortex. Experiments were undertaken to determine which component of the second messenger system is influenced by the hormone and antidepressant. Neither treatment modified the amount or function of extractable stimulatory guanine nucleotide binding protein or the activities of adenylate cyclase or phosphodiesterase. While both imipramine and ACTH treatment decreased the cyclic AMP response to norepinephrine, only imipramine administration influenced the response to isoproterenol. ACTH treatment was found to reduce the alpha adrenergic potentiation of isoproterenol- and 2-chloroadenosine-stimulated cyclic AMP production, as well as reduce the sensitivity of the norepinephrine response to prazosin. These findings indicate that imipramine and ACTH treatments decrease the responsiveness of the rat brain norepinephrine-stimulated cyclic AMP generating system through actions on the alpha and beta adrenergic receptor components. The results suggest that noradrenergic receptor activity may be under the control of adrenal and/or pituitary hormones.     

Effects of clenbuterol on central beta-1 and beta-2 adrenergic receptors of the rat

Repeated administration of the centrally acting beta adrenoceptor agonist, clenbuterol, to rats reduced the ability of isoproterenol to increase the concentration of cyclic AMP (cAMP) in slices of cerebellum. This reduced responsiveness to isoproterenol was accompanied by a marked reduction in the density of beta adrenoceptors as measured by the binding of the beta adrenoceptor antagonist [125I]iodopindolol. In addition, the agonist-binding properties of remaining cerebellar beta adrenoceptors were altered after clenbuterol treatment. The clenbuterol-induced reduction in the density of beta adrenoceptors in the cerebellum is in marked contrast to its inability to do this in cerebral cortex. Comparison of the ability of clenbuterol to that of isoproterenol to increase levels of cAMP in slices of cerebral cortex or cerebellum showed that clenbuterol is a weakly potent agonist in both brain regions. The increase in cAMP induced by isoproterenol in the cortex was significantly reduced in the presence of the selective beta-1 adrenoceptor antagonist, ICI 89,406. In contrast, the clenbuterol-induced increase in cortical cAMP was unchanged by ICI 89,406 but was reduced significantly by the beta-2 adrenoceptor antagonist, ICI 118,551. In cerebellum, both isoproterenol- and clenbuterol-stimulated accumulation of cAMP were antagonized much more potently by ICI 118,551 than by ICI 89,406. Furthermore, clenbuterol antagonized the cAMP response induced by isoproterenol in the presence of ICI 118,551 in a concentration-dependent manner. In terms of measurement of cAMP in brain slices, clenbuterol is weakly potent as an agonist at beta-2 adrenoceptors and has antagonist properties at beta-1 adrenoceptors.     

Role of alpha-adrenergic receptors in the effect of the beta-adrenergic receptor ligands, CGP 12177, bupranolol, and SR 59230A, on the contraction of rat intrapulmonary artery

This study investigates the effect of the aryloxypropanolamines 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one (CGP 12177), bupranolol, and 3-(2-ethylphenoxy)-1[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-(2S)-2-propanol oxalate (SR 59230A) [commonly used as beta(3)- and/or atypical beta-adrenergic receptors (beta-AR) ligands] on the contractile function of rat intralobar pulmonary artery. Affinities of beta-AR ligands for alpha(1)-adrenergic receptors (alpha(1)-AR) were also evaluated using [(3)H]prazosin binding competition experiments performed in rat cortical membranes. In intralobar pulmonary artery, CGP 12177 did not modify the basal tone, but antagonized the contraction induced by the alpha(1)-AR agonist phenylephrine (PHE). In arteries precontracted with PHE, CGP 12177 elicited relaxation, whereas in those precontracted with prostaglandin F(2alpha) (PGF(2alpha)), it further enhanced contraction. CGP 12177 induced an increase in intracellular calcium concentration in pressurized arteries loaded with Fura PE-3 and precontracted with PGF(2alpha). In PGF(2alpha) precontracted arteries, phentolamine (an alpha-AR antagonist) and phenoxybenzamine (an irreversible alpha-AR antagonist) antagonized the contractile responses to PHE and CGP 12177. Both responses were also decreased by bupranolol and SR 59230A. Specific [(3)H]prazosin binding was displaced by CGP 12177, bupranolol, and SR 59230A with pK(i) values of 5.2, 5.7, and 6.6, respectively. In contrast, (+/-)-(R*,R*)-[4-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]phenoxy]acetic acid sodium (BRL 37344) and disodium 5-[(2R)-2-([(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino)propyl]-1,3-benzodioxole-2,2-dicarboxylate (CL 316243) (nonaryloxypropanolamines beta(3)-AR agonists) displayed very low affinity for [(3)H]prazosin binding sites (pK(i) values below 4). These data suggest that CGP 12177 exhibits partial agonist properties for alpha(1)-AR in rat pulmonary artery. They also show that bupranolol and SR 59230A exert an alpha(1)-AR antagonist effect. As a consequence, these aryloxypropanolamine compounds should be used with caution when investigating the role of beta(3)- and atypical beta-AR in the regulation of vascular tone.     

N-n-alkylnicotinium analogs,a novel class of nicotinic receptor antagonists: interaction with alpha4beta2* and alpha7* neuronal nicotinic receptors

The current study demonstrates that N-n-alkylnicotinium analogs with increasing n-alkyl chain lengths from 1 to 12 carbons have varying affinity (Ki = 90 nM-20 microM) for S-(-)-[3H]nicotine binding sites in rat striatal membranes. A linear relationship was observed such that increasing n-alkyl chain length provided increased affinity for the alpha4beta2* nicotinic acetylcholine receptor (nAChR) subtype, with the exception of N-n-octylnicotinium iodide (NONI). The most potent analog was N-n-decylnicotinium iodide (NDNI; Ki = 90 nM). In contrast, none of the analogs in this series exhibited high affinity for the [3H]methyllycaconitine binding site, thus indicating low affinity for the alpha7* nAChR. The C8 analog, NONI, had low affinity for S-(-)-[3H]nicotine binding sites but was a potent inhibitor of S-(-)-nicotine-evoked [3H]dopamine (DA) overflow from superfused striatal slices (IC50 = 0.62 microM), thereby demonstrating selectivity for the nAChR subtype mediating S-(-)-nicotine-evoked [3H]DA overflow (alpha3alpha6beta2* nAChRs). Importantly, the N-n-alkylnicotinium analog with highest affinity for the alpha4beta2* subtype, NDNI, lacked the ability to inhibit S-(-)-nicotine-evoked [3H]DA overflow and, thus, appears to be selective for alpha4beta2* nAChRs. Furthermore, the present study demonstrates that the interaction of these analogs with the alpha4beta2* subtype is via a competitive mechanism. Thus, selectivity for the alpha4beta2* subtype combined with competitive interaction with the S-(-)-nicotine binding site indicates that NDNI is an excellent candidate for studying the structural topography of alpha4beta2* agonist recognition binding sites, for identifying the antagonist pharmacophore on the alpha4beta2* nAChR, and for defining the role of this subtype in physiological function and pathological disease states.