Molecular pharmacology
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Molecular pharmacology · Nov 1991
Species differences in the toxicity and cytochrome P450 IIIA-dependent metabolism of digitoxin.
In rats, cytochrome P450 (P450) IIIA enzymes are an important determinant of digitoxin toxicity. Induction of these liver microsomal enzymes decreases the toxicity of digitoxin by increasing its oxidative cleavage to digitoxigenin bis- and monodigitoxoside (dt2 and dt1). The present study shows that the susceptibility of different mammalian species to digitoxin toxicity is inversely related to liver microsomal P450 IIIA activity (measured as testosterone 6 beta-hydroxylase activity). ⋯ The rate of dt2 formation varied approximately 41-fold among 22 samples of human liver microsomes, which was highly correlated (r = 0.841) with the rate of testosterone 6 beta-hydroxylation. Antibody against rat P450 IIIA1 inhibited the high rate of dt2 formation by rat liver microsomes and the low rate catalyzed by mouse, guinea pig, dog, monkey, and human liver microsomes. In contrast, anti-P450 IIIA1 did not inhibit the 12-, 16-, or 17-hydroxylation of digitoxin (or the formation of the unknown metabolite), despite the fact that anti-P450 IIIA1 strongly inhibited (greater than 70%) the 6 beta-hydroxylation of testosterone by liver microsomes from each of the species examined (except rabbit liver microsomes, which were inhibited only approximately 30%).(ABSTRACT TRUNCATED AT 400 WORDS)
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Molecular pharmacology · May 1990
gamma-Aminobutyric acidA receptor regulation in culture: altered allosteric interactions following prolonged exposure to benzodiazepines, barbiturates, and methylxanthines.
In previous reports, we have described the use of primary neuronal cultures derived from chick brain to study the regulation of the gamma-aminobutyric acidA (GABAA) receptor complex. Chronic exposure of cultures to GABA, benzodiazepines, or methylxanthines results in decreased enhancement of [3H]flunitrazepam binding by GABA, consistent with an allosteric uncoupling of GABA and benzodiazepine recognition sites of the GABAA receptor. In the present communication, we extend our studies of the pharmacology of benzodiazepine- and methylxanthine-induced uncoupling of GABA/benzodiazepine recognition site interactions and present evidence to show that certain barbiturates (barbital and pentobarbital) also induce uncoupling. ⋯ The onset of flurazepam-induced uncoupling (EC50 approximately 1 microM) exhibits a t 1/2 of about 18 hr, in general agreement with the half-time for receptor turnover. Uncoupling is reversible following washout and recovery at 37 degrees. These results are discussed in terms of mechanisms of GABAA receptor regulation in response to chronic exposure to functionally homologous or heterologous ligands.
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Molecular pharmacology · May 1988
Tachykinin-induced phosphoinositide breakdown in airway smooth muscle and epithelium: relationship to contraction.
We have studied the contractile response and phosphoinositide hydrolysis induced by substance P (SP), neurokinin A (NKA), neurokinin B (NKB), and Alp-Phe-Phe(R)-Gly[ANC-2]-Leu-Met-NH2 (L 363851), a selective NK2-receptor agonist, in guinea pig tracheal smooth muscle. The four tachykinins elicited a concentration-dependent contraction in tracheal smooth muscle devoid of epithelium, with the following order of potency: NKA greater than L 363851 greater than NKB greater than SP, (EC50 1.0 x 10(-9) M, 3.2 x 10(-9) M, 7.5 x 10(-9) M and 1.2 x 10(-7) M, respectively), which suggests that NK2 receptors predominate in airway smooth muscle. In the presence of epithelium, the sensitivity of airway smooth muscle to tachykinins was decreased, and the concentration response curves to tachykinins were shifted rightward by 30-fold for SP, 9-fold for NKA, and 5-fold for NKB. ⋯ In airway smooth muscle, we found that tachykinins elicited phosphoinositide breakdown with an order of potency similar to that for contractile response (EC50 2.2 x 10(-5) M, 3.6 x 10(-5) M, 4.4 x 10(-5) M, and 5.9 x 10(-5) M). In epithelium, SP alone elicited a significant phosphoinositide breakdown, suggesting that epithelial receptors to tachykinins may be of the NK1 subtype. Since it is established that phosphoinositide derivatives can elicit mobilization of intracellular calcium, our results suggest that phosphoinositide breakdown is the coupling mechanism for tachykinin-induced contraction of airway smooth muscle.
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Molecular pharmacology · Dec 1986
Ni-coupled receptors in cultured neural hybrid cells: cell specificity for dibutyryl cyclic AMP-induced down-regulation but not morphological differentiation.
Opiate, muscarinic, and alpha 2-adrenergic receptors and the Ni-coupled response of adenylate cyclase (AC) inhibition were examined in neuroblastoma X glioma NG108-15 (108 CC15) and neuroblastoma X Chinese hamster brain NCB-20 clonal hybrid cells, induced to differentiate with 1.0 mM dibutyryl cAMP (dBcAMP). Scatchard analysis of binding of the opiate agonist 3H-(D-Ala2,D-Leu5)enkephalin (DADLE) and the antagonist [3H] diprenorphine to dBcAMP-treated NCB-20 cell membranes indicated an 80% reduction in opiate receptor density relative to untreated cells (Bmax = 47 +/- 11 fmol/mg of protein versus 220 +/- 48 fmol/mg of protein), with no change in ligand affinities. Binding of the muscarinic cholinergic antagonist [3H]quinuclidinyl benzilate and the alpha 2-adrenergic agonist [3H]-p-aminoclonidine to dBcAMP-treated NCB-20 membranes was also reduced by 50% and 28%, respectively. ⋯ Prostaglandin E1-stimulated AC was maximally inhibited by 1 microM DADLE in membranes from undifferentiated cells to different degrees (30% in NCB-20 and 54% in NG108-15). dBcAMP treatment had no effect on opiate inhibition of AC in NG108-15 cells but reduced by 50% the maximum opiate inhibition of AC in NCB-20 cells. These data indicate that the signal for receptor down-regulation which was triggered by dBcAMP in the NCB-20 cell comes uniquely from the Chinese hamster brain cell NCB-20 parent. The differences between NCB-20 and NG108-15 cells in the regulation of Ni-coupled receptors provides an example of dBcAMP-induced heterologous down-regulation with unique cell specificity, which is unrelated to the morphological differentiation process triggered by dBcAMP, which is common to both cells.
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Molecular pharmacology · Jan 1985
Receptors for beta-adrenergic agonists in cultured chick ventricular cells. Relationship between agonist binding and physiologic effect.
To determine if the two-state, guanine nucleotide-modulated beta-adrenergic receptor model elucidated in erythrocyte membranes accurately describes hormone binding to intact heart cells, and to determine the relationship of agonist binding to physiologic contractile response, we studied beta-adrenergic antagonist and agonist binding to intact cultured heart cells and homogenates of these cells from embryonic chick ventricle and related the binding observations to alterations in amplitude of contraction of intact cells under identical conditions. The levo isomer of the beta-adrenergic antagonist pindolol was radioiodinated, purified, and utilized to characterize the beta-adrenergic receptor in intact, beating heart cells under physiologic conditions. Computer analysis of iodopindolol-binding isotherms revealed a KD = 22 +/- 3 pM with Bmax = 10.3 fmol/mg of protein in intact cells; in homogenates of cells, the KD was 39 +/- 12 pM in the absence of exogenous guanine nucleotides and 19 +/- 7 pM in their presence. ⋯ At the isoproterenol concentration causing 50% maximal inotropic response, 67% occupancy of high affinity receptors occurs. Thus, there is a close relationship between high affinity receptor occupancy and augmentation of contractility in intact cells. These findings support the view that agonist interaction with the guanine nucleotide-sensitive, high affinity receptor state initiates the physiologic response of myocardial tissue to beta-adrenergic agonists.