Elisabeth Tuisl

A different desensitization pattern of cardiac β-adrenoceptor subtypes by prolonged in vivo infusion of isoprenaline

(–)Isoprenaline was continuously administered to rats at a rate of 0.4 mg/kg/h for 7 days via subcutaneously (s.c.) implanted osmotic minipumps. This treatment induced cardiac hypertrophy and a marked decrease in basal as well as catecholamine-stimulated adenylate cyclase activity in a ventricular plasma membrane fraction. The total number of (β-adrenoceptors was downregulated by one-half the amount of the receptor sites obtained in a control group. However, in the isoprenaline-treated group, the (β-adrenoceptors constituted a significantly smaller proportion of the total 4bT-adrenoceptor population (28%) than in the control group (50%). Transformation of these relative into absolute values indicates that prolonged isoprenaline treatment induced a significantly higher downregulation of β2- than of (β1-adrenoceptors. The fact of a different (β-adrenoceptor desensitization pattern in response to in vivo administration of nonselective β-adrenergic agonists therefore must be taken into consideration when desensitization is used as a method for determination of subtype selectivity of an agonist per se. However, we were unable to detect the “lost” (β-adrenoceptors in a light vesicular fraction. In our study, this fraction was not separable from plasma membranes, as substantiated by levels of plasma membrane markers as high as in the plasma membrane fraction and by a guanine nucleotide-dependent adenylate cyclase activity.

Adenosine receptor agonists: Binding and adenylate cyclase stimulation in rat liver plasma membranes

SummaryN6-Cyclohexyl[3H]adenosine([3H]CHA),[3H]adenosine, and 5′N-ethylcarboxamide[3H]adenosine ([3H]NECA), potent agonists in adenosine-responsive cellular systems, have been used to identify adenosine binding sites in rat liver plasma membranes. Endogenous ligands were removed by prior dialysis of the membranes. Specific binding of the ligands tested was characterized by rapid forward and reverse kinetics and heterogeneity as indicated by curvilinear Scatchard plots. The KD in the high affinity range was 80 nM for [3H]adenosine, 84 nM for [3H]NECA, and 168 nM for [3H]CHA; the respective binding capacities of 1.19, 1.03, and 1.05 pmol/mg protein were of virtually the same magnitude, suggesting labeling of identical sites. However, all ligands also displayed binding to large numbers of low affinity sites. This high level of apparently non-receptor binding markedly influenced the adenosine structure-activity profile of [3H]CHA displacement, which differs with pharmacological findings. — NECA and CHA stimulated hepatic adenylate cyclase with an apparent ED50 of 60 and 580 nM, respectively; adenosine was stimulatory at a concentration range from 0.1–2.0 μM, but inhibitory at higher concentrations. Hence, estimation of the true ED50 was not possible. Because the KD of high affinity binding and the ED50 of the biological effect of NECA and CHA are in the same range, it may be reasonable to assume that the high affinity sites represent adenosine receptors, recently classified as Ra-site receptors.

Glomeruli and microvessels of the rabbit kidney contain both A1- and A2-adenosine receptors.

SummaryRabbit renal cortices were fractionated by collagenase dispersion and glomeruli, microvessels and tubuli purified on a discontinuous sucrose gradient. Binding experiments with (−)[125I]N6-(4-hydroxyphenylisopropyl)-adenosine ([125I]HPIA) provided evidence for the presence of A1-adenosine receptors in the glomerular and microvascular fraction. With glomeruli, saturation isotherms for specific [125I]HPIA binding were mono-phasic with a KD of 1.3 nmol/l and a Bmaxof 7.7 fmol/mg protein. In kinetic experiments, an association rate constant of 4.9 × 105 (mol/ 1)−1 s−1 and a dissociation rate constant of 4.3 × 10−4 s−1 were obtained, yielding a KD of 0.9 nmol/l. Adenosine analogs displaced [125I]HPIA binding with a rank order of potency typical of A1-adenosine receptors; furthermore, binding was inhibited by methylxanthines and modulated by GTP. Saturation experiments with the microvessels revealed a KD of 1.9 nmol/l and a Bmax of 13.4 fmol/mg protein. However, no inhibition of glomerular and microvascular adenylate cyclase activity could be demonstrated, but instead both 5′-N-ethylcarboxamido-adenosine (NECA) and N6-(R-phenylisopropyl)-adenosine (R-PIA) stimulated enzyme activity, with EC50 values of 0.14 μmol/l and 1.5 μmol/l, respectively. The concentration-response curve for NECA was shifted to the right (factor 9) by 10 μmol/l 8-phenyltheophylline. On the other hand, computer simulation of biphasic curves (adenylate cyclase inhibition in the presence of activation via a stimulatory receptor) indicates that the failure to observe an A1-adenosine receptor-mediated inhibition of adenylate cyclase activity in the presence of stimulatory adenosine receptors may be attributable to methodological constraints. The results demonstrate that both A1- and A2-adenosine receptors are present in rabbit glomeruli and microvessels. It is suggested that both receptors are involved in the control of renin secretion.

P2-, but not P1-purinoceptors mediate formation of 1, 4, 5-inositol trisphosphate and its metabolites via a pertussis toxin-insensitive pathway in the rat renal cortex.

1 The adenosine receptor (P1‐purinoceptor) agonists N6‐cyclopentyladenosine and N‐5′‐ethyl‐carboxamidoadenosine at concentrations up to 10 μmol l−1 affected neither basal, nor noradrenaline‐ and angiotensin II‐stimulated formation of inositol‐1‐phosphate, inositol‐1,4‐bisphosphate, and inositol‐1,4,5‐trisphosphate in slices of rat renal cortex. 2 In contrast, adenine nucleotides (P2‐purinoceptor agonists) markedly stimulated inositol phosphate formation. The observed rank order of potency adenosine‐5′‐O‐(2‐thiodiphosphate) (EC50 39 μmol l−1) > adenosine‐5′‐O‐(3‐thio triphosphate) (587) ≥ 5′‐adenylylimidodiphosphate (App(NH)p, 899) > adenylyl‐(β,γ‐methylene)‐diphosphonate (4,181) was consistent with the interaction of the compounds with the P2y‐subtype of P2‐purinoceptors. AMP and the ADP analogue (α,β‐methylene)‐adenosine‐5′‐diphosphate were ineffective. ATP and ADP (≤ 10 mmol l−1) did not produce a consistent increase, owing to their hydrolytic degradation in the incubation medium. 3 Whereas the inositol phosphate response to App(NH)p was linear only up to 5 min incubation, the time‐dependent stimulation of noradrenaline declined at a slower rate. Following pre‐exposure of the renal cortical slices to App(NH)p, renewed addition of App(NH)p caused no further enhancement in the accumulation of inositol phosphates, whilst noradrenaline was still capable of eliciting a response. This suggests that the apparent loss of responsiveness to App(NH)p is not due to substrate depletion or enzymatic inactivation, but most likely attributable to homologous desensitization of the purinoceptor. 4 Pretreatment of the animals with pertussis toxin caused a substantial reduction of functional Gi‐protein, as indicated by the lack of [32P]‐NAD incorporation in a membrane preparation of the renal cortex. Nevertheless, the increase in inositol phosphate formation induced by noradrenaline, angiotensin II, and App(NH)p was not significantly impaired. 5 We conclude that P2y‐purinoceptors are present in the renal cortex; these receptors stimulate formation of inositol phosphates via a pertussis toxin‐insensitive pathway and undergo homologous desensitization. On the other hand, our results suggest that renal A1‐adenosine receptors do not use stimulation of phosphoinositide breakdown as a transmembrane signalling system.

Cardiac sarcolemmal purity is essential for the verification of adenylate cyclase inhibition via A1-adenosine receptors.

SummaryInhibition of cardiae adenylate cyclase by adenosine receptor agonists was reinvestigated in a more homogeneous sarcolemmal vesicular preparation than used in a previous study. Microsomal particles obtained by differential centrifugation were further fractionated on a shallow density gradient of Percoll. Two populations of plasma membrane vesicles were partially resolved. Identical peaks were identified for adenylate cyclase activity and [3H]ouabain binding, whereas 5′-nucleotidase activity and β-adrenoceptor binding displayed an additonal peak at higher density, where angiotensin converting enzyme, a marker for endothelial plasma membranes, was at maximal activity. Significant inhibition by N6-cyclohexyladenosine (CHA), as measured in each fractionation step following homogenization, was observed only at the activity peak of adenylate cyclase. Moreover, analysis of the degree and rank order of potency of several adenosine analogs was indicative for interaction with A1-adenosine receptors. Accordingly, the peak in adenosine receptor binding, using (-)[125I]iodo-N6-hydroxyphenyl-isopropyladenosine as the radioligand, coincided with CHA-inhibitable adenylate cyclase activity. By contrast, adenylate cyclase was slightly stimulated by CHA in the higher density range, an action suggested to be mediated via A2-adenosine receptors, which recently have been demonstrated to exist on guinea-pig coronary endothelium. It is concluded that the full extent of adenosine receptor-mediated adenylate cyclase inhibition in the heart is only to be demonstrated if contamination of the sarcolemmal preparation with endothelial membrane components is kept to a minimum.

Interaction of the antihypertensive drug urapidil with cardiac β-adrenoceptors in vitro

A possible interaction of the antihypertensive drug, urapidil with beta-adrenoceptors was investigated in a guinea-pig ventricular membrane preparation. Urapidil at concentrations above 1 microM antagonized the isoproterenol-induced stimulation of adenylate cyclase activity. Urapidil 10 microM shifted the concentration-response curve for isoproterenol in a parallel manner to the right by a factor of 16. Urapidil competed with specific beta-adrenoceptor binding of [125I]iodocyanopindolol with an IC50 of 12 microM. Since the affinity of urapidil to beta-adrenoceptors appeared rather low, any beta-adrenoceptor blocking property may be of relevance in vivo at high dosages only.

Alterations of the glomerular β-adrenoceptor-linked adenylate cyclase system in perinephritis hypertension

Summary: The present study was undertaken to investigate β-adrenoceptor–adenylate cyclase coupling in a myocardial ventricular membrane preparation and in isolated renal glomeruli of cellophane perinephritis hypertensive rats. Adenylate cyclase activity and [125I]iodocyanopindolol binding were differentially affected in these preparations. In isolated glomeruli of hypertensive rats a reduced intrinsic activity of isoproterenol was associated with an apparent loss of the guanine nucleotide-sensitive high-affinity state of the β-adrenoceptors, whereas their absolute number was unchanged when compared with the normotensive control rats. On the other hand, in the sarcolemmal preparation of hypertensive rats adenylate cyclase activity, the relative amount of high-affinity states, and the density of β-adrenoceptors were not different from the respective values in normotensive controls. Experiments performed on isolated glomeruli of rats with unilateral cellophane perinephritis that developed only moderate hypertension provide evidence that the apparent loss of the high-affinity state is a consequence of hypertension, since no difference was observed in glomeruli from the wrapped, as compared with the intact kidney.

Effects of the carboxylesterase inhibitor bis-(p-nitrophenyl)-phosphate on disposition and metabolism of hexobendine

Abstract The effect of pretreatment with the carboxylesterase inhibitor bis-( p -nitrophenyl)-phosphate (BNPP) on the disposition and metabolism of intravenously-administered [ 14 C]hexobendine was studied in rats. Inhibition of hexobendine ester cleavage by BNPP produced a marked diminution in the concentrations of the hexobendine hydrolysis products, 3,4,5-trimethoxybenzoic acid and N,N′-dimethyl-N-[3-(3′,4′,5′-trimethoxybenzoxy)-propyl]-N′-3-hydroxypropyl-ethylendiamine in plasma and in the liver and kidneys. A simultaneous elevation occurred in the levels of demethylated metabolites following BNPP pretreatment. The concentration-time relationship of non-metabolized [ 14 C]hexobendine was not significantly altered by BNPP pretreatment. The results suggest that BNPP pretreatment produced a shift towards the dealkylating pathway in hexobendine metabolism. Consequently, esterase inhibition did not significantly affect the hexobendine plasma and tissue levels.

Induction of drug metabolism in the rat by taglutimide, a sedative-hypnotic glutarimide derivative

SummaryPretreatment with taglutimide significantly decreased the plasma dicoumarol level and shortened the duration of hexobarbital-induced narcosis in rats. Furthermore, taglutimide pretreatment accelerated thein vitro metabolism of dicoumarol, hexobarbital, o-nitrophenyl acetate and procaine, but not of 3,4-benzpyrene, as assayed in the 10,000xg supernatant fraction of rat liver homogenate. No definite increase was observed in liver wet weight, nor in the amount of microsomal and total liver protein in comparison with the control values. No marked differences were found between the effects of short-(4-day) and long-term (17-day) pretreatment on any of the studied parameters. The changes in drug metabolism and liver protein observed after taglutimide pretreatment differed from those observed after pretreatment with either phenobarbital or 3,4-benzpyrene. Taglutimide, like other inducing agents, is lipophilic, but differs from them in not being a substrate of monooxygenases.

Effects of the carboxylesterase inhibitor bis-(p-nitrophenyl)-phosphate on elimination of hexobendine.

Abstract A study was conducted of the effect of pretreatment of rats with the carboxylesterase inhibitor, bis-( p -nitrophenyl)-phosphate (BNPP) on the biliary and urinary excretion of intravenously-administered 14 C-hexobendine (HB) and its metabolites. Inhibition of HB ester cleavage by BNPP produced a marked decrease in the urinary excretion of HB hydrolysis products: this effect was accompanied by a definite increase in the biliary excretion of conjugates of O -demethylated HB metabolites. The results indicate the participation of complementary metabolic and excretory pathways in the elimination of HB; this hypothesis is further supported by the finding that BNPP did not alter the half-life of the plasma 14 C level.