Iodocyanopindolol Binding Research Articles

Decreased myometrial β-adrenoceptors in women receiving β2-adrenergic tocolytic therapy: Correlation with lymphocyte β-adrenoceptors

We have determined simultaneously the density of beta-adrenoceptors in human myometria (by (-)-[125I]iodopindolol binding) derived from 36 women undergoing cesarean section and in the corresponding circulating lymphocytes (by (-)-[125I]iodocyanopindolol binding). In myometrial membranes about 80% to 85% of the beta-adrenoceptors were of the beta 2-subtype. The density of myometrial and lymphocyte beta-adrenoceptors in women treated with the beta 2-adrenoceptor agonist hexoprenaline to prevent preterm labor was about 65% to 70% lower than that in nontreated women. Concomitantly, in hexoprenaline-treated women the 10 mumol/L isoproterenol-evoked increase in lymphocyte cyclic adenosine monophosphate content (as index for lymphocyte beta-adrenoceptor responsiveness) was diminished to a similar extent. Combining all data resulted in a significant positive correlation between myometrial and lymphocyte beta-adrenoceptor densities (r = 0.7303; n = 36; p less than 0.001). It is possible that determination of beta-adrenoceptor function in circulating lymphocytes may be a useful model to monitor myometrial beta-adrenoceptor changes during tocolytic therapy.

Lung tissue binding of iodobenzyl-propanediamine: Involvement of beta-adrenergic receptors

The basic compound N-N-N′-trimethyl- N′-(2-hydroxy-3-methyl-5-iodobenzyl)-1,3-propanediamine (HIPDM) accumulates in human and rabbit lungs, where it forms a slowly effluxable pool. In isolated perfused rat lung, HIPDM is taken up by a saturable, energy-independent mechanism, which is competitively inhibited by imipramine, chlorpromazine and propranolol. To ascertain whether beta-adrenergic receptors are involved in the binding process of HIPDM to lung tissue, the ability of unlabelled HIPDM to displace the beta-adrenergic receptor ligand [ 125I]iodocyano-pindolol (ICYP) from rabbit lung beta-receptors was examined. Lung microsomal membrane fractions (75 μg ml −1) were incubated at 37°C for 3 h with 68 pM ICYP (with or without 1 μM of (±)-propranolol) in the presence of HIPDM (10 −10−10 −3 M). Bound and free radioactivity were separated through glass-fibre filters and the retained radioactivity was counted in a gamma-spectrometer. HIPDM competed with ICYP for beta-adrenoceptors (13% displacement at 10 −5 M, 50% at 5 × 10 −5 M, and 90% at 2 × 10 −4 M). The inhibition curve of ICYP binding by HIPDM was similar to that observed for (−)-noradrenaline. Although the results of the in vitro studies cannot be extrapolated to in vivo conditions, they suggest that beta-adrenergic receptors may be involved in the observed lung uptake of the basic amine HIPDM.

Regulation of adrenergic receptor number following chronic noradrenaline infusion in the rabbit.

In order to study noradrenaline-induced regulation of alpha- and beta-adrenoceptors, groups of male New Zealand White rabbits (n = 8) were treated with intravenous noradrenaline (0.09 mumol/kg x h) or ascorbate (0.1%) for 10 days via osmotic minipumps implanted in the femoral vein, and the number of cardiac, lung and lymphocyte beta-adrenoceptors as well as renal and platelet alpha 2-adrenoceptors were determined. 1. The mean arterial blood pressure, heart rate and catecholamine levels were measured before commencing, and after 24 h and 10 days infusion. Circulating noradrenaline concentrations were elevated approximately 6-fold at 24 h and were sustained at these levels after 10 days administration of noradrenaline. There were no significant alterations in the blood pressure while a significant decrease in the heart rate was observed at 24 h. 2. Alpha 2-adrenoceptor density was assessed using [3H]-yohimbine. A significant decrease in the number of alpha 2-adrenoceptors in the kidney was observed following the 10 days infusion with noradrenaline. This down-regulation was in marked contrast to the lack of alteration in platelet alpha 2-adrenoceptor number and the platelet alpha 2-adrenoceptor mediated aggregatory response. 3. The density of beta-adrenoceptors in lymphocytes, heart and lung were quantified using (-)[125I]iodocyanopindolol (ICYP). The noradrenaline infusions caused significant reductions in beta-adrenoceptor number in the heart and lung (containing predominantly beta 1-adrenoceptors) but not in lymphocytes (possessing mainly beta 2-adrenoceptors). The KD-values (pM) for ICYP binding to heart and lung were also significantly decreased in the present studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular mechanism of β‐adrenergic receptor blockers with intrinsic sympathomimetic activity

beta-Adrenergic receptor (beta AR) blocking agents with intrinsic sympathomimetic activity (ISA) can induce modest increases in beta AR-stimulated activity, such as rate and force of contraction in cardiac tissue. The molecular basis for this activity has been elusive. Previous studies have suggested that these compounds do not stimulate cyclic AMP (cAMP) formation even though activation of adenylate cyclase is the generally accepted mechanism for beta AR promotion of target cell response. In the current studies, we show that several beta AR antagonists with ISA (dichloroisoproterenol, pindolol, and celiprolol) stimulate cAMP accumulation five-, two-, and threefold, respectively, in S49 lymphoma cells, but only if cells are simultaneously incubated with the diterpene forskolin. The KI values observed for inhibition of isoproterenol-stimulated cAMP accumulation or of beta AR [( 125I]iodocyanopindolol) binding for each of the beta blockers with ISA were comparable in magnitude to their respective EC50 values for forskolin-potentiated cAMP accumulation. The forskolin-potentiated responses of these compounds were abolished by the beta AR-antagonist propranolol. These results indicate that the ISA of beta-blocking drugs most likely results from a modest beta AR-mediated stimulation of adenylate cyclase activity. The results further suggest that treatment of target cells with forskolin provides a means to define partial agonism at receptors that are linked to stimulation of adenylate cyclase.

The role of zinc status in altered cardiac adenylate cyclase activity in diabetic rats.

Zinc deficiency and altered myocardial adenylate cyclase activity commonly occur in diabetes. To determine whether the zinc intake of the animal can account for the altered beta-adrenergic receptor activity in the diabetic heart, we determined the beta-adrenergic receptor number and isoproterenol-, NaF- and forskolin-stimulated adenylate cyclase activity in diabetic and control rats maintained on low, normal and high zinc diets for 3 weeks. Scatchard analysis of [125I]iodocyanopindolol binding to control heart membrane preparations revealed a binding capacity of 17.3 +/- 1.3 fmol/mg protein with a Kd of 35 +/- 1.0 pmol/l. Neither the diabetic state nor the zinc status altered these binding parameters. The isoproterenol-stimulated adenylate cyclase activity was significantly lower in diabetic rats on low zinc diets compared with controls. The NaF- (65.1 +/- 5.4 vs 60.8 +/- 6.4 pmol cAMP.mg protein-1.min-1) and forskolin-stimulated adenylate cyclase activities (161 +/- 9.3 vs 154 +/- 21.2 pmol cAMP.mg protein-1. min-1) were not significantly altered in diabetic rats. Low dietary zinc intake compared with high zinc diet significantly increased NaF- and forskolin-stimulated adenylate cyclase activity both in diabetic rats and controls. The effect of dietary zinc content on isoproterenol-stimulated adenylate cyclase was significant in control rats only. Thus zinc intake appears to be an important determinant of cardiac adenylate cyclase activity level. Additional factors peculiar to the diabetic state are involved in the modulation of beta-adrenergic responsiveness of the diabetic heart.

Selective regulation of beta 1- and beta 2-adrenoceptors in the human heart by chronic beta-adrenoceptor antagonist treatment.

1. In 44 patients undergoing coronary artery bypass grafting, the effect of chronic administration of the beta-adrenoceptor antagonists sotalol, propranolol, pindolol, metoprolol and atenolol on beta-adrenoceptor density in right atria (containing 70% beta 1- and 30% beta 2-adrenoceptors) and in lymphocytes (having only beta 2-adrenoceptors) was studied. 2. beta-Adrenoceptor density in right atrial membranes and in intact lymphocytes was assessed by (-)-[125I]-iodocyanopindolol (ICYP) binding; the relative amount of right atrial beta 1- and beta 2-adrenoceptors was determined by inhibition of ICYP binding by the selective beta 2-adrenoceptor antagonist ICI 118,551 and analysis of the resulting competition curves by the iterative curve fitting programme LIGAND. 3. With the exception of pindolol, all beta-adrenoceptor antagonists increased right atrial beta-adrenoceptor density compared to that observed in atria from patients not treated with beta-adrenoceptor antagonists. 4. All beta-adrenoceptor antagonists increased right atrial beta 1-adrenoceptor density; on the other hand, only sotalol and propranolol also increased right atrial beta 2-adrenoceptor density, whereas metoprolol and atenolol did not affect it and pindolol decreased it. 5. Similarly, in corresponding lymphocytes, only sotalol or propranolol increased beta 2-adrenoceptor density, while metoprolol and atenolol did not affect it and pindolol decreased it. 6. It is concluded that beta-adrenoceptor antagonists subtype-selectively regulate cardiac and lymphocyte beta-adrenoceptor subtypes. The selective increase in cardiac beta 1-adrenoceptor density evoked by metoprolol and atenolol may be one of the reasons for the beneficial effects observed in patients with end-stage congestive cardiomyopathy following intermittent treatment with low doses of selective beta 1-adrenoceptor antagonists.

Effect of thyroid status on ?-adrenoceptors and calcium channels in rat cardiac and vascular tissue

To determine the influence of thyroid hormone on beta-adrenoceptors and Ca2+ channels, rats were treated with thyroxine (75 micrograms/100 g sc daily for 5 days) or propylthiouracil (0.05% in drinking water for 30 days). beta-Adrenoceptor density in ventricular tissue, measured by [125I]iodocyanopindolol binding, was significantly increased and decreased respectively, following thyroxine or propylthiouracil treatment to 124.7 +/- 7.11 fmol/mg protein and 71.98 +/- 5.37 fmol/mg protein from euthyroid (control) levels of 93.7 +/- 4.58 fmol/mg protein. Ca2+ channel density, measured by [3H]nitrendipine binding, was altered in the opposite direction; it was significantly decreased and increased to 324 +/- 24 fmol/mg protein and 691 +/- 31 fmol/mg protein from 562 +/- 35 fmol/mg protein after thyroxine or propylthiouracil treatment, respectively. No changes in affinity of either ligand were observed. Responses of isolated papillary muscles from propylthiouracil-treated animals accorded with changes seen in the binding studies. The geometric mean EC50 of isoproterenol increased from 9.5 x 10(-9) mol/l to 5.5 x 10(-8) mol/l, and the EC50 for calcium decreased from 3.16 x 10(-3) mol/l to 1.36 x 10(-3) mol/l; moreover, the responsiveness to the Ca2+ channel activator Bay K 8644 was increased. The corresponding responses in thyroxine-treated animals could not be examined because of prominent arrhythmic activity. As with papillary muscles the sensitivity of left atria to isoproterenol was decreased after treatment with propylthiouracil, with geometric mean EC50 values increasing from 3.21 x 10(-9) mol/l to 89.4 x 10(-9) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic influences on agonist binding to cardiac β-receptors

Regulation of β-adrenoceptor-agonist function in the Maudsley Reactive (MR/Har) and the Maudsley Non-Reactive (MNRA/Har) rat strains was assessed by comparison of isoproterenol competition for [ 125I]iodocyanopindolol (ICYP) binding sites in crude left ventricular homogenate preparations. Non-linear, least-squares analysis of isoproterenol competition for ICYP binding in the absence of guanine nucleotide revealed different proportions of high- and low-affinity receptors in the two strains; MR/Har rats (59±3.3%) had a significantly greater proportion of receptors in the high-affinity state than the MNRA/Har rats (41±4.5%). Addition of the non-hydrolyzable guanine nucleotide analog guanylylimidodiphosphate (Gpp(NH)p) converted receptors to the low-affinity state. Analysis of Gpp(NH)p concentration-response curves in left ventricular homogenates of the two strains revealed that the MR/Har strain had a significantly (P<0.02) lower EC 50 for guanyl nucleotide inhibition of isoproterenol competition for ICYP binding than the MNRA/Har. Confirming previous experimental results, a significantly (P<0.04) greater density of ventricular β-receptors was found in MR/Har rats (13.16±0.92 fmol/mg protein) than in MNRA/Har rats (10.81±0.63 fmol/mg protein). Left ventricular catecholamine levels were found to be correlated inversely with β-adrenoceptor density in the two strains; norepinephrine (NE) and epinephrine (EPI) concentrations (ng/mg protein) in left ventricle were 12.19±0.94 for NE and 0.165±0.038 for EPI in MNRA/Har, and 8.73±0.95 and 0.018±0.018, respectively, in MR/Har. All other parameters of agonist interactions with the cardiac β-adrenoceptor for the MR/Har and MNRA/Har rat strains were similar [the IC 50 for displacement of ICYP binding by isoproterenol, the accompanying Hill coefficients in the Gpp(NH)p present and absent condition, the K d of the high- and low-affinity states in the absence of Gpp(NH)p, and the K d of the uniform low-affinity state in the presence of Gpp(NH)p]. We hypothesize that the strain-dependent differences in high-affinity state formation reported here may account for some of the in vivo differences in cardiovascular function previously demonstrated in the Maudsley rats.

β-Adrenergic receptors and stimulatory effects of (−) isoproterenol on testosterone production in fetal mouse Leydig cells

Beta-adrenergic receptors were characterized in freshly excised fetal mouse testis using the radioiodinated antagonist iodocyanopindolol (ICYP). [125I]-CYP bound to a single class of high affinity sites with a KD value of 42.2 +/- 7.0 pM. Adrenergic agonists competed for ICYP binding sites with the following order of potency: (-)isoproterenol greater than (-)epinephrine much greater than (-)norepinephrine which is typical for a beta 2-adrenergic receptor. A selective beta 2-receptor antagonist ICI 118-551 showed an approximately 200 fold higher affinity than the beta 1-selective compound, betaxolol. The beta-adrenergic agonist (-)isoproterenol did not or slightly affect testosterone production by freshly isolated fetal Leydig cells. The ability of fetal Leydig cells to respond to (-)isoproterenol increased during culture. This change in responsiveness was not accompanied either by modification of the number of binding sites or by change in the binding affinity. Taken together these data suggest that i) the stimulatory effect of (-)isoproterenol on testosterone production by cultured fetal Leydig cells is mediated through beta 2-adrenergic receptors ii), the inability of freshly Leydig cells to respond to catecholamines is probably due to post receptor events.

The effect of hydrogen peroxide on beta-adrenoceptor function in the heart.

A membrane preparation of calf heart left ventricle has been used to study the effect of radical stress on the beta-adrenoceptor complex. To this end the membranes were incubated for 30 minutes with several concentrations of hydrogen peroxide. This resulted in a dose dependent peroxidation of the membrane lipids. Preincubation with hydrogen peroxide in the concentration range 10(-7)-10(-3) M caused an increase in specific (-)-[125I]-Iodocyanopindolol binding, possibly due to a decrease in membrane fluidity as a result of lipid peroxidation, thus making the receptor protein more accessible. Higher concentrations H2O2 reduced the specific (-)-[125I]-Iodocyanopindolol binding, which is most likely the effect of deterioration of the receptor protein by the more pronounced radical stress induced by these higher concentrations. Also adenylate cyclase activity was affected by radical stress. Basal cyclic-AMP production and cyclic-AMP production induced by NaF (10(-2) M) or guanylylimidodiphosphate (10(-4) M), was suppressed after pretreatment with concentrations of H2O2 above 10(-4) M. This indicates a higher sensitivity of the adenylate cyclase toward radical stress when compared to the receptor protein. Our results show that radical stress can perturb beta-adrenoceptor function considerably in the heart.

Effects of stress and chronic propranolol infusion on cardiac beta-receptor function in the maudsley reactive and non-reactive rat strains

The effects of β-adrenoceptor blockade on left ventricular β-receptor characteristics were evaluated in the Maudsley Reactive (MR/Har) and Non-Reactive (MNRA/Har) rat strains. After propranolol infusion for one week administered by subcutaneously implanted osmotic minipumps, differences in β-adrenoceptor-agonist interactions between strains were assessed by comparison of isoproterenol competition for [ 125I]iodocyanopindolol (ICYP) binding sites in crude left ventricular homogenate preparations from both strains. Propranolol exposure had no significant effect on the binding parameters investigated (B max, K d, % receptors in high affinity state, IC 50). In contrast to previous observations in naive animals, these experiments revealed no strain differences in the sham-implanted Maudsleys in any of the left ventricular β-adrenoceptor parameters investigated. We hypothesize that the surgical procedures and/or presence of the plastic inserts acted as stressors in the Maudsley strains and thus altered β-adrenoceptor function. This hypothesis is supported by the observation that exposure to another stress (footshock) also eliminated strain differences in ventricular β-receptor binding variables.

Post-natal evolution of rat cardiac beta-adrenoceptors

Cardiac β-adrenoceptors (βAR) were studied using membranes prepared at birth (day 0) and at days 7, 10, 15, 21, 30, 45 and 60. Saturation experiments using the antagonist ligand ( 125I)-iodocyanopindolol (ICYP) allowed the determination of βAR number (Bmax) and ICYP dissociation constant (K d), while (-) isoproterenol competition curves of ICYP binding, performed in the absence or presence of Gpp(NH)p (10 −4M), were used to measure the relative proportions of high and low affinity states of the βAR for the agonist and to assess the ability of βAR to couple with the GTP-binding protein. Rat cardiac βAR evolved at 3 distinct periods: during the first period (days 0–10), the receptor density and ICYP K d were half that of adults, and βAR were present only in an homogenous high affinity state. The second period (days 15–21) was characterized by a progressive increase in βAR number and ICYP K d, while analysis of (-)isoproterenol competition curves indicated that βAR were poorly coupled to the GTP-binding protein. In the third period (days 30–60), ICYP Bmax and K d were respectively 53.9±1.2 fmoles/mg proteinand 106.4±2.9 pM, while analysis of (-)isoproterenol competition curves showed the existence of high and low affinity binding states in equal proportions in the absence Gpp(NH)p, and of one homologous low affinity state of the receptor in its presence. These data indicate that βAR follow a post-natal evolution marked by an increase in βAR density concomittant with a decrease in affinity toward the antagonist ligand ICYP, accompanied by the progressive appearance of a poorly-coupled βAR. However, the number of efficiently coupled receptors was found to be similar in adult and newborn rats.

Adrenergic receptors in bovine retinal microvessels: Presence ofα 2- and β- but notα 1-receptors

In bovine retinal microvessels,α 1-, α 2- and β-adrenergic receptors were characterized by binding assay, using [ 3H]prazosin, [ 3H]para-aminoclonidine and [ 125I]iodocyanopindolol as radioligands, respectively. The microvessels were purified from bovine eyes by differential centrifugation through a high concentration of bovine serum albumin followed by use of a glass bead filtration technique. In the preparation, specific binding sites for [ 3H]para-aminoclonidine and [ 125I]iodocyanopindolol were observed, whereas [ 3H]prazosin binding was not detected. The [ 3H]para-aminoclonidine binding sites localized to the microvessels were characterized by high affinity and saturability ( K D: 173 ± 9pM; B max: 394 ± 11fmol/mg protein) as well as the [ 125I]iodocyanopindolol binding sites ( K D: 20 ± 3pM; B max: 43 ± 4fmol/mg protein). Furthermore, the specificity of both binding sites was pharmacologically evaluated by measuring the inhibitory effects of various adrenergic reagents on binding. The existence ofα 2- and β-adrenergic receptors which were characterized by high affinity, saturability and stereospecificity, leads to the hypothesis that the retinal microcirculation is under neuronal control.

The effect of ischemia and recirculation, hypoxia and recovery on anti-oxidant factors and β-adrenoceptor density: Is the damage in the erythrocytes a reflection of brain damage caused by complete cerebral ischemia and by hypoxia?

This investigation was focussed on the gravity of tissue injury caused by complete ischemia (for five min) and hypoxia (for three weeks) in the cerebral cortex (homogenate) and the erythrocyte lysate or the erythrocyte membrane of the rat in order to investigate if the changes that occur in brain tissue are reflected in the erythrocyte. To this end, glutathione (GSH), superoxide dismutase (SOD) and catalase were measured, also alterations in β-adrenoceptor density under these two conditions were examined. It was found that in ischemia partial parallellism in changes that occur in the central nervous system (cerebral cortex) and the erythrocyte exists. The SOD activity became higher and the β-adrenoceptor density (measured as specific (−)-[ 125I] iodocyanopindolol binding) was decreased in both tissues. However after the hypoxic condition we established a decrease in the number of β-adrenoceptors in the cerebral cortex but an increase in β-adrenoceptor density in the erythrocyte.

The effects of halothane on the human beta-adrenergic receptor of lymphocyte membranes.

The effects of halothane on beta-adrenergic receptor antagonist interaction were studied using the membranes of human lymphocytes as a model. Membrane preparations of lymphocytes were obtained from blood samples withdrawn from seven healthy young volunteers. Beta-receptor studies were performed using (-)125I iodocyanopindolol (125ICP) binding. Non-specific binding was determined in the presence of (-)isoproterenol. Beta-receptor density (Bmax) and the dissociation constant (KD) for 125ICP were determined from saturation curves. Beta-receptor affinity for agonists evaluated by the IC50 (the concentration of isoproterenol required to inhibit 50% of specific 125ICP binding) and the dissociation constant (KL) for isoproterenol was established from competition curves. The effect of halothane 1%, in an air oxygen mixture (oxygen fraction: 0.3) administered by tonometry during ligand membrane incubation, on beta-adrenergic receptor, was compared to that of control experiments not exposed to halothane. Halothane produced a moderate but significant decrease of Bmax (-10%) and a significant increase in non-specific binding (+30%), while KD, IC50, and KL were unchanged. The authors conclude that halothane, in vitro, decreases beta-adrenergic receptor density. This effect could be mediated by an alteration of the receptor in the membrane due to action of halothane on the lipid phase of the membrane.

Effects of Ischemia on the Canine Myocardial β-Adrenoceptor-Linked Adenylate Cyclase System

Several studies indicate that myocardial ischemia causes a redistribution of beta-adrenergic receptors from a presumably intracellular compartment to the cell surface. However, a decreased adenylate cyclase and contractile responsiveness to beta-adrenergic stimuli has also been reported. The aim of the present study was to investigate possible ischemia-induced changes in myocardial beta-adrenoceptor coupling to adenylate cyclase. Myocardial ischemia was induced by hydraulic occlusion of the LAD in mongrel dogs anesthetized with isoflurane. After 90 min of ischemia, tissue samples were removed from the ischemic and nonischemic regions for tissue catecholamine determinations and for the preparation of particulate fractions from tissue homogenates. Saturation experiments on microsomal fractions obtained from the ischemic and control areas did not reveal any significant changes in the calculated dissociation constant for (-)[125I]iodocyanopindolol binding nor in the calculated receptor density. Likewise, the relative numbers of beta 2-adrenergic receptors were comparable in both preparations (approximately 20%). On the other hand, the proportion of beta-adrenoceptors stabilized in the high-affinity state by (-)isoproterenol was significantly reduced in the ischemic region when compared with the control myocardium (17 +/- 5 vs. 41 +/- 4%). This change was accompanied by a significant decrease in the intrinsic activity of (-)isoproterenol in stimulating adenylate cyclase activity. We propose that the initial uncoupling of the beta-adrenoceptor from its effector is a physiologically important, protective mechanism which guards the ischemic myocardium against the deleterious effect of excessive sympathetic stimulation.

Alpha- and beta-adrenergic receptors in proximal tubules of rat kidney.

Proximal tubules were isolated from the rat kidney by collagenase digestion of the cortical tissue followed by Percoll gradient centrifugation. Microscopic and hormone-stimulated adenylate cyclase activity studies proved the purity of the preparation. [3H]Prazosin, [3H]rauwolscine, and [125I]iodocyanopindolol were used to identify and quantitate respectively the alpha 1-, alpha 2- and beta-adrenergic receptors. Proximal tubular (F4) particulate fraction was compared against other cortical nephron segment (F1, F2) fractions and the total collagenase-digested cortex particulate suspension (Ft). Proximal tubules were enriched in alpha 1- and alpha 2-adrenergic receptors compared with Ft (alpha 1-receptor, 100.4 +/- 4.5 vs. 87.4 +/- 4.9; alpha 2-receptor, 250 +/- 16.2 vs. 185.1 +/- 12 fmol/mg protein). The fractions enriched in glomeruli and distal tubular segments (F1, F2) had relatively low concentrations of alpha 1- and alpha 2-adrenergic receptors. In contrast, beta-adrenergic receptor concentration in the proximal tubules was approximately 25% of that in the Ft fraction and approximately 10% of that in the F1 fraction. Isoproterenol-stimulated adenylate cyclase activities in the different fractions corroborated well with the pattern suggested by the [125I]iodocyanopindolol binding studies. Our results suggest that whole-cortex preparation radioligand binding studies may reflect proximal tubular alpha 1- and alpha 2-adrenergic receptor changes quite well. They may, however, miss or give erroneous impressions about beta-adrenergic receptor changes occurring in different cortical nephron segments.

Ischemia- and agonist-induced changes in alpha- and beta-adrenergic receptor traffic in guinea pig hearts.

We have used radioligand binding techniques and subcellular fractionation to assess whether changes in expression of myocardial alpha 1- and beta-adrenergic receptors are mediated by a redistribution of receptors between various membrane fractions. Three fractions were prepared from the left ventricles of guinea pigs that underwent either 1 h of ischemia or injection of epinephrine (0.25 mg/kg ip): a crude membrane, a purified sarcolemma, and a light vesicle fraction. In control animals alpha 1-adrenergic receptors ([3H]prazosin binding) in light vesicles was only 25% of the total alpha 1-receptor density found in sarcolemmal and light vesicle fractions as compared with 50% for beta-adrenergic receptors ([125I]iodocyanopindolol binding sites). Although ischemia was associated with a 53% decrease in the number of light vesicle beta-adrenergic receptors and a 42% increase in the number of sarcolemma beta-receptors (P less than 0.05), there was no change in the number of light vesicle alpha 1-receptors, even though the number of sarcolemmal alpha 1-receptors increased 34%. Epinephrine treatment promoted internalization of beta-adrenergic receptors; sarcolemma beta-receptors decreased 37% and light vesicle beta-receptors increased 28% (P less than 0.025). For alpha 1-receptors, epinephrine treatment decreased the number of sarcolemmal receptors 41% (P less than 0.025) but failed to increase the number of receptors in the light vesicle fraction. The changes in receptor binding to beta-adrenergic receptors in sarcolemmal fractions were mirrored by parallel changes in isoproterenol-stimulated adenylate cyclase activity. These results indicate that alpha 1- and beta-adrenergic receptors may undergo a different cellular itinerary in guinea pig myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha-adrenoceptor antagonistic action of amiloride

1. In isolated perfused rat liver, the effects of alpha-adrenergic stimulation by phenylephrine (2 μM), such as an increase of portal pressure, glucose output, Ca 2+ release into the perfusate and the characteristic K + flux changes across the hepatocyte plasma membrane were almost completely abolished in the presence of amiloride (0.5 mM). 2. When the phenylephrine concentration was raised to about 100 μM, the effects of the alphaadrenergic agonist on hepatic metabolism, Ca 2+ and K + fluxes, but not on the portal venous pressure, were restored, suggesting a competitive antagonism by amiloride. 3. Amiloride antagonized in a concentration-dependent manner noradrenaline-induced isometric contractions of strips of the rabbit pulmonary artery. The concentration-response curve of noradrenaline was shifted to the right, and the maximal response obtained was also depressed, suggesting a mixed competitive and non-competitive antagonism. The estimated amiloride-adrenoceptor-dissociation constant was 8 μM. 4. The affinity of amiloride to the alpha- and β-adrenoceptor subtypes was determined by radioligand binding assays using [ 125I]BE 2254 binding to rat liver plasma membranes (alpha 1-subtype), [ 3H]yohimbine binding to human platelet membranes (alpha 2-subtype), (−)-[ 125I]iodocyanopindolol (ICYP) binding to rabbit lung membranes in presence of the β 2-adrenoceptor antagonist ICI 118, 551 (β 1-subtype) and ICYP binding to rat lung membranes in presence of the β 1-blocker atenolol (β 2- subtype). In all systems, amiloride inhibited specific ligand binding concentration-dependently, the K i values for amiloride were about 25, 52, 148 and 161 μM for alpha 1- alpha 2-, beta 1- and beta 2- adrenoceptor subtypes, respectively. 5. It is concluded that amiloride in concentrations below those required for inhibition of the Na +/H + exchanger is a potent antagonist of alpha- and beta-adrenoceptors in a variety of experimental systems. Whether the adrenergic antagonism of amiloride is important for antihypertensive therapy, remains to be elucidated.

Inhibition of (-) [125I]-iodocyanopindolol binding to rat lung beta adrenoceptors by uremic plasma ultrafiltrates.

Numerous data have suggested that beta-adrenoceptor-mediated responses were decreased in uremia and that parathormone could be implicated in this phenomenon. In a previous paper we have shown that the beta2 receptor density of mononuclear cells of uremic patients is significantly increased despite a significant increase in plasma epinephrine, suggesting that an endogenous substance could interfere and disregulate the beta 2 receptor density. In order to further evaluate this phenomenon we have firstly studied the influence of one non uremic and five uremic plasma ultrafiltrates on the binding of (-)-[125I]iodocyanopindolol using rat lung beta adrenoceptors. The results show that uremic plasma ultrafiltrates induce a decrease in the Bmax value without any variation on the Kd value. In a second step we have assessed the ability of human synthetic 1-34 and 53-84 parathormone to interact directly with beta-adrenoceptors. No variation in the (-)-[125I]iodocyanopindolol binding parameters was observed. These results suggest that an uremic endogenous substance might interfere on the beta adrenergic receptors and that the alteration in the beta-adrenergic response in uremia is probably not due to a direct action of parathormone on the beta-adrenoceptors.