Beta-adrenoceptor subtypes in human, rat, guinea pig, and rabbit atria

Selective beta-adrenoceptor agonists and antagonists were used to characterize the beta-adrenoceptor subtypes in human right atrium. The activity order of selective agonists for rat, guinea pig, and rabbit atrial inotropic and chronotropic responses was consistent with the hypothesis that only beta 1-adrenoceptors mediate these responses. In human right atrium, however, the beta 2-selective agonists fenoterol and salbutamol were relatively much more active. The beta 1-selective antagonist practolol preferentially antagonized the human atrial response to noradrenaline (beta 1-selective) as opposed to fenoterol (beta 2-selective), whereas the beta 2-selective antagonist ICI 118,551 preferentially antagonized the response to fenoterol. The data indicate that the response of human right atrium to beta-adrenoceptor agonists is mediated by a mixed beta 1-plus beta 2-adrenoceptor population. The implications of these results with respect to the therapeutic uses of beta-adrenoceptor agonists are discussed.     

Beta-adrenoceptor blocking and electrophysiological effects of bufetolol in the guinea pig atria.

The potency of blockade of bufetolol, a beta-adrenoceptor blocking drug, and effects of bufetolol on the action potential, contractile force and various electrophysiological properties of the atrium were investigated in comparison with propranolol and quinidine. Bufetolol had a pA2 of 8.65 against the positive chronotropic action of isoproternol on the guinea pig sinus node. Bufetolol, 10(-7) g/ml, did not affect the action potential of the atrial muscle, while the drug, 3 X 10(-5) g/ml, significantly decreased the overshoot potential, the amplitude and the maximum rate of rise of the action potential and prolonged the times for 50% and 90% repolarization. The contractile force was reduced by bufetol, 3 X 10(-5) g/ml. The maximum responsive frequency to the driving stimulus was decreased by bufetol, 3 X 10(-5) g/ml. The excitability of the muscle membrane was suppressed by bufetolo, 3 X 10(-5) g/ml, as indicated by changes in membrane responsiveness, membrane reactivation and strength-duration curve. Propranolol, 10(-5) g/ml and quinidine, 10(-5) g/ml showed similar effects on excitability. The authors suggest that these effects of bufetolol are responsible for its antidyshythmic effects.     

Beta-adrenoceptor blocking effects of two bopindolol metabolites in isolated guinea-pig atria.

The beta-adrenoceptor blocking effects of the bopindolol (Wandonorm, CAS 62658-63-3) metabolites 18-502 (indole-2,4- methylaminopropoxy(N-tert.butyl)-tartrate) and 20-785 (indole-2,4-carboxyaminopropoxy(N-tert.butyl] were studied in electrically stimulated guinea-pig left atria and in spontaneously beating guinea-pig atria in vitro. Both compounds shifted the concentration-response curve of isoprenaline to the right, but did not reduce the maximum effect of this drug. For compound 20-785, pA2 values of 7.44 (left atrium, inotropic effect) and 7.58 (right atrium, chronotropic effect) were calculated. The metabolite 18-502 had a much greater beta-adrenoceptor blocking potency, as judged from its pA2 values of 9.53 and 9.48, resp., and in the concentration of 10(-8) mol/l it caused a significant flattening of the concentration-response curve of isoprenaline. From these results, compound 20-785 can be classified as a competitive beta-adrenoceptor antagonist, while for higher concentrations of the metabolite 18-502 additional noncompetitive mechanisms of action cannot be excluded.     

Synthesis, receptor binding affinities and alpha-MSH releasing activities of TRH analogues

New syntheses of three thyrotropin releasing hormone (TRH) analogues ([Dopa2]THR, [Nic1]TRH, and [Tyr(30NO2)2]TRH) have been reported (Dopa stands for L-3,4-dihydroxyphenylalanine, Nic--for nicotinic acid and Tyr(3-NO2)--for L-3-nitrotyrosine). These three TRH analogues and five already known ones ([Aad1Tca3]TRH, [D-His2]TRH, [D-Pro3]TRH, [Pro-NH-NH2(3)]TRH and [Tyr2]TRH), were studied in vitro for their binding activity to rat pituitary TRH receptors and a-MSH releasing activity in the neuro-intermediate lobe of frogs. Competition of analogues for 3H-TRH binding to rat anterior pituitary membrane fraction was used. One of ten tested analogues ([Aad1, Tca]3 TRH) was as potent as TRH in competing for high-affinity binding sites (Kd = 8.5 nM). The binding activity of diastereoisomers ([D-His2]TRH and [D-Pro3]TRH) was reduced as well as that of analogue [Pro-NH-NH2(3)]TRH. The rest of the analogues were inactive. The binding activities were in good accordance with alpha-MSH releasing activities.     

Beta-adrenoceptor subtypes in young and old rat ventricular myocytes: a combined patch-clamp and binding study.

1. We used electrophysiological and binding techniques to assess the presence of beta 1- and beta 2-adrenoceptors (beta 1AR and beta 2AR) in rat cardiac myocytes and to determine their ratio during aging. Experiments were performed in left ventricular myocytes enzymatically dissociated from the heart of 3-(young) or 22-month-old (old) Wistar Kyoto rats. 2. In patch-clamp experiments, myocytes from old rats showed a prolonged action potential duration (at -20 mV: 41.7 +/- 3.6 vs 26.2 +/- 3.1 ms; at -60 mV: 154.4 +/- 17.7 vs 87.1 +/- 6.9 ms, P < 0.05) and an augmented membrane capacitance (an index of cell size) (271.7 +/- 20.2 vs 164.3 +/- 14.6 pF, P < 0.05) compared to young rats. beta 2AR stimulation, achieved by superfusing myocytes with the selective beta 2AR agonist, zinterol (10 microM) or with (-)-isoprenaline (1 microM) in the presence of the selective beta 1AR antagonist, CGP 20712A (0.1 microM), significantly increased L-type calcium current (ICa,L) in rat ventricular myocytes. The percentage increase was similar in both young and old rats, either with zinterol (26.9 +/- 3.6% and 24.2 +/- 2.8%, respectively) or isoprenaline plus CGP 20712A (30.4 +/- 3.7% and 22.4 +/- 4.1%, respectively). Isoprenaline alone (beta 1AR and beta 2AR stimulation) caused a much smaller increase in ICa,L in old rats (58.4 +/- 12.1%) than in younger ones (95.3 +/- 8.1%) (P = 0.067).(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-adrenoceptor stimulants and mesovarian leiomyomas in the rat

The experiment reported here shows that mesovarian leiomyomas may be induced in rats by the administration of 2 chemically distinct adrenergic stimulants, salbutamol or terbutaline. That the induction of these benign tumours is a function of adrenergic stimulation is shown by the fact that the concurrent administration of the adrenergic blocker propranolol prevented their development.     

Relationship between receptor-binding affinities and TSH-releasing activities of novel TRH analogs in the rat

Specific bindings of 3H-TRH to rat pituitary homogenate and apparent affinities of TRH analogs for binding sites were studied. The dissociation constant for 3H-TRH binding to rat pituitary homogenate was 30 nM at 0 degrees C, and the number of the binding sites was 120 fmoles/mg protein. The apparent affinities of TRH analogs for the binding sites, which were estimated from the ability to displace 3H-TRH from those binding sites, were found to correlate well with their TSH releasing activities. These findings support the idea that the TSH releasing activities of TRH analogs depend almost entirely upon their binding abilities to the TRH receptor in the pituitary.     

Beta-adrenoceptor desensitization by clenbuterol in rat uterus

• 1.1. The relaxant response and cAMP production mediated by stimulation of isoproterenol is reduced in uterine rings from clenbuterol treated rats (0.25 mg kg⁻¹ s.c. 24 hr before experiments) precontracted with 50 mM KCl.
• 2.2. Forskolin, in contrast, showed similar relaxant responses in untreated or clenbuterol treated rats.
• 3.3. Isoproterenol produces a biphasic response that is composed of a rapid relaxation followed by a slower regaining of tension, which is considered as desensitization.
• 4.4. The kinetic study demonstrates marked changes in the desensitization process of β-adrenoceptors after clenbuterol administration.

     

Protein binding of sotalol enantiomers in young and elderly human and rat serum using ultrafiltration

The protein binding of sotalol (STL) enantiomers was evaluated using an ultrafiltration technique with serum from young (32±2 years, n=5) and elderly (73±6 years, n=5) male and female humans, and young (8 weeks, n=4) and elderly (60 weeks, n=3) male Sprague—Dawley rats. Serum samples were collected and immediately frozen at ?20°C. Within 1 week, the serum samples were thawed at room temperature, and adjusted to pH 7.4 using 0.05 M phosphate buffer, pH 5.0. Aliquots were spiked with 250 ng mL^?1 and 500 ng mL^?1 of each STL enantiomer, placed in ultrafiltration sets (Microsep, 30K molecular weight cut-off), capped, equilibrated to 37°C, and centrifuged at 1850g for 1.5h at 37°C. Aliquots of ultrafiltrate and unspun serum were analysed for STL enantiomer concentration using a stereospecific HPLC assay. In all groups, bound fraction was less than 7% for both STL enantiomers. There were no significant differences in bound fraction between groups, or between enantiomers. Adsorption of STL enantiomers to the ultrafiltration device and membrane, evaporative loss of serum samples during centrifugation, and protein concentration in each ultrafiltrate sample were all negligible. It is concluded that the binding of STL in human and rat serum at therapeutic concentrations and physiological temperature and pH is negligible and non-stereoselective.     

Species-dependent binding of disopyramide enantiomers

Serum protein binding of the basic enantiomers of disopyramide were studied in several animal species. (S)-(+)-Disopyramide was more highly bound than the (R)-(-)-enantiomer to serum protein in the man, gorilla, and pig. The reverse was true in cow serum, and in serum and albumin from sheep. Enantioselective differences in binding were due to differences in association constants. No enantioselective differences in binding were observed in serum protein from horse and goat, or in albumin from cow and pig. Disopyramide was highly bound to two sites on horse albumin. The association constant characterizing the binding of disopyramide to the first (major) site on horse albumin was 1.3 x 10(7) M-1. At predialysis concentrations of 10(-7) M, tris-(2-butoxyethyl)phosphate displaced disopyramide from sites on horse albumin and from sites on serum protein from the horse, man, gorilla, cow, and pig. At predialysis concentrations of 10(-5) M, warfarin and diazepam had no effect on disopyramide binding in these animal species. It is concluded that the enantioselective binding of disopyramide is species dependent, the site that is responsible for the moderate to high binding of disopyramide enantiomers is probably located on alpha 1-acid glycoprotein, and the sites that bind disopyramide in the horse are located on albumin and may be unique.     

Sodium channel binding and anticonvulsant activities for the enantiomers of a bicyclic 2,4-oxazolidinedione and monocyclic models

Racemic 7-phenyl-9,10-dioxo-1-aza-8-oxabicyclo[5.2.1]decane (1), a bicyclic 2,4-oxazolidinedione that we previously reported was a possible sodium channel anticonvulsant, was resolved into its enantiomeric forms, the absolute configurations were determined, and the stereoisomers were evaluated for relative sodium channel binding and whole animal anticonvulsant activities. Similar studies were carried out with two monocyclic models, 5-ethyl-5-phenyl-2,4-oxazolidinedione (2) and 5-ethyl-3-methyl-5-phenyl-2,4-oxazolidinedione (3). None of these isomers exhibited stereoselective effects in the sodium channel assay, and only modest enantioselectivities were observed for 2 and 3 in the anticonvulsant assays. (R)-(-)-1 was, however, 4 times more toxic than (S)-(+)-1 in the rotorod test, and due to its larger protective index, (S)-(+)-1 exhibited greater therapeutic potential than either (R)-(-)-1 or racemic 1.     

Beta-adrenoceptor blocking drugs and calcium transport in isolated rat mast cells

The beta-adrenoceptor blocking (BAB) drugs exaprolol (EXA), metipranolol (MET) and propranolol (PRO) inhibited histamine liberation and degranulation from isolated rat mast cells stimulated with the calcium ionophore A23187. MET was the most and EXA the least active. Atenolol (ATE) had no effect. Inhibition by BAB drugs of secretion induced with A23187 was not accompanied by any change in 45Ca uptake. On the other hand, EXA, MET and PRO significantly decreased 45Ca uptake by mast cells stimulated with 48/80. The effect of BAB drugs on inhibition of A23187-induced secretion from isolated mast cells was dependent on the lipid solubility of the studied drugs.     

Beta-adrenoceptor antagonist activity of bivalent ligands. 1. Diamide analogues of practolol

Two series of bivalent ligands (P-X-P) containing the (R,S)-3-[(4-aminoaryl)oxy]-1-(isopropylamino)propan-2-ol pharmacophore and a connecting alpha,omega-dicarbonylpoly(methylene) [X = -OC(CH2)nCO-] or alpha,omega-N,N'-bis(carbonylmethylene) polymethylenediamine [X = -OCCH2NH(CH2)nNHCH2CO-] spanner were synthesized and evaluated for beta-adrenoceptor antagonist activity in rat heart and lung membrane preparations. The target compounds were obtained as a mixture of stereoisomers in modest yields by using a three to four step sequence beginning with N-benzylpractolol. The results from the competitive binding studies indicated that binding affinity increased by a factor of up to 160 by increasing the length of the group spanning the pharmacophore moieties. Modest increases in cardioselectivity were also obtained. The data suggest that further increases in spanner length and lipophilicity and optical resolution may improve the potential of a labeled bivalent beta 1-adrenoceptor antagonist to function as a myocardial imaging agent.     

Beta-adrenoceptor stimulating effects of phenylephrine and noradrenaline in the rat pulmonary vascular bed

The effects of phenylephrine and noradrenaline have been investigated on the perfusion pressure of the rat isolated lung. Both drugs (0.3-30 micrograms) produced a dose-dependent decrease in perfusion pressure elevated by 20 mM KCl, which was reversed to a dose-dependent increase after addition of propranolol (1 x 10(-7) M) to the perfusion fluid. Increments due to both agonists in the presence of propranolol were antagonized by prazosin (1 x 10(-6) M). Propranolol, but not prazosin, elevated the basal perfusion pressure. The results indicate that phenylephrine and noradrenaline are more effective in stimulating beta-adrenoceptors than alpha-adrenoceptors in the rat pulmonary vascular bed and that beta-adrenoceptors may regulate the vascular tone of the rat pulmonary circulation.     

Characteristics of sodium-induced increase in opiate antagonist binding sites in rat brain membranes

Sodium decreases agonist binding and increases antagonist binding to opiate receptor sites in brain membranes. This study characterizes in detail the 20-40% increase in [3H]naloxone binding caused by sodium. This increase in binding was specific to sodium (not mimicked by potassium or lithium) and was maximal at 10 mM NaCl. The sodium effect was reversible: washing membranes free of sodium restored binding to normal. Sodium increased Bmax, not KD, of [3H]naloxone binding. The sodium-induced increased binding was not inhibited by either N-ethylmaleimide (NEM) or phospholipase A2 at concentrations which inhibit 50-85% of normal [3H]naloxone binding. In NEM-treated membranes, the effect of sodium on increasing naloxone binding was actually increased. The regional distribution of these sodium-dependent sites were different from normal naloxone sites. These results suggest that physiological concentrations of sodium expose naloxone sites which differ in biochemical properties from naloxone sites assayed in the absence of sodium.     

Dopaminergic and adrenergic binding affinities in rabbit retinal synaptosomes

1. Dopamine binding to amacrine membrane vesicles isolated as synaptosomal fractions P1 and P2 from rabbit retinas showed saturation within less than a minute. 2. Dopamine binding to retinal synaptosomal membranes (RSM) in P1 and P2 is a two-component system: the first saturated at 1.00 microM 14C-dopamine in P1 and 1.25 microM in P2, and the second saturated at 2.00 microM in both pellets. 3. The affinity of RSM receptors to dopamine in P1 was equal to that in P2 (Km = 2.00 microM), whereas the calculated Vmax of dopamine binding was increased in P2 (1.25 pmol/micrograms protein) as compared to P1 (0.625 pmol/micrograms protein). 4. Dopamine binding to the beta-adrenergic sites showed a lower affinity (Km = 10 microM) in P2 relative to P1 (Km = 4.0 microM), whereas Vmax in P2 (5.0 pmol/micrograms protein) was 4-fold higher than P1 (1.25 pmol/micrograms protein). 5. The P1 and P2 fractions of rabbit RSM contain dopaminergic and beta-adrenergic binding sites with higher concentration of dopaminergic receptors and lower concentration of beta-adrenergic receptors in P2 relative to P1.