KATP channel openers of the benzopyran type reach their binding site via the cytosol.

BACKGROUND AND PURPOSE: ATP-sensitive K+ (KATP) channels are composed of pore-forming subunits (Kir6.x) and of sulphonylurea receptors (SUR). Both sulphonylureas and K(ATP) channel openers act by binding to SUR. Sulphonylureas reach their binding site from the cytosol but it remains unknown whether this holds for openers too. EXPERIMENTAL APPROACH: A poorly membrane-permeant sulphonic acid derivative of the benzopyran-type opener, bimakalim, was synthesized, descyano-bimakalim-6-sulphonic acid (BMSA). Binding of BMSA and bimakalim was compared in membranes and intact cells expressing the Kir6.2/SUR2B channel and channel opening was compared in inside-out patches and whole cells. KEY RESULTS: In membranes, bimakalim and BMSA bound to Kir6.2/SUR2B with Ki values of 61 nM and 4.3 microM, showing that the negative charge decreased affinity 69-fold. In intact cells, however, binding of BMSA was much weaker than in membranes (75-fold) whereas that of bimakalim was unchanged. The Ki value of BMSA decreased with increasing incubation time. In inside-out patches, bimakalim (1 microM) and BMSA (100 microM) opened the Kir6.2/SUR2B channel closed by MgATP to a similar degree whereas in whole-cell experiments, only bimakalim was effective. CONCLUSIONS AND IMPLICATIONS: Despite its negative charge, BMSA is an effective channel opener. The fact that BMSA binds and acts more effectively when applied to the inner side of the cell membrane shows that benzopyran openers reach their binding site at SUR from the cytosol. This suggests that the binding pocket of SUR is only open on the cytoplasmic side.

[Does the paper speed in fetal heart monitoring during labour influence the variability in the interpretation by professionals?] / La vitesse de déroulement du rythme cardiaque fœtal en cours de travail a-t-elle un impact sur la variabilité d'interprétation par les professionnels ?

OBJECTIVE: Assessing inter- and intra- observer agreement in the reading of fetal heart rate (FHR) between two different paper speeds (1 and 2cm/min) using FIGO classification. MATERIAL AND METHODS: Single-centre experimental study consisting in reading 60minutes FHR tracings by six readers (3 midwives and 3 obstetricians) during 1cm and 2cm/min sessions within a period of three weeks. The reading guideline was based on FIGO classification. Inter- and intra-observer agreement was assessed thanks to Kappa coefficient (K) and percentage of agreement (PA) using the classification of FHR tracings drawn up by readers. RESULTS: Intra-observer agreement reached 60% between the two paper speeds, and PA ranged from 48 to 67%. Inter-observer agreement was poor to moderate (K=0.42 for 1cm/min sessions and K=0.38 for 2cm/min sessions). Inter-observer agreement was significantly higher for normal tracings (PA ranged from 55.2% for 2cm/min sessions to 57.4% for 1cm/min sessions). The preterminal category had the lowest concordance rates (PA=19% for 1cm/min sessions and 20, 7% for 2cm/min sessions). CONCLUSION: This study did not highlight significant differences in intra- and inter-observer variability between the two FHR paper speeds. The 1cm/min paper speed, which is commonly used in France, is more economical and gives a better bedside overview of FHR. Therefore, it should be recommended.

Regulation of endothelial permeability by beta-adrenoceptor agonists: contribution of beta 1- and beta 2-adrenoceptors.

The barrier function of cultured, macrovascular endothelial cells derived from bovine aorta was analyzed using confluent monolayers of cells and measuring the exchange of fluorescein dextrans of different molecular masses. The effects of beta-adrenoceptor agonists with different selectivity for beta 1- and beta 2-adrenoceptors (AR) were investigated. Formoterol, a novel high-affinity agonist for beta 2-AR recently introduced in the treatment of bronchial asthma, showed a significant reduction of cell permeability with subnanomolar concentrations, whereas the catecholamines (-)-isoproterenol and (-)-norepinephrine only showed significant effects with micromolar concentrations. In order to elucidate if this difference in potential to regulate cell permeability is related to appropriate changes in the selectivity and affinity of the agonists for beta 2 AR, we investigated the beta AR-coupled adenylate cyclase (AC) in membranes from endothelial cells and compared AC stimulation with the binding of agonists to the receptors using [125I](-)-iodopindolol as radioligand. beta-Adrenoceptors revealed to be closely coupled to AC as assessed by a similar magnitude of effects by receptor agonists in comparison to GTP analogues and direct stimulants of AC activity. AC activity was increased by formoterol in parallel to its receptor occupancy of beta 2AR with nanomolar concentrations which were 50-fold higher than those used for the regulation of cell permeability indicating the existence of spare receptors. In contrast to formoterol, the catecholamines (-)-isoproterenol and (-)-norepinephrine stimulated AC activity through both beta 1AR and beta 2AR. From the overproportional high contribution of beta 1AR to AC stimulation (42%) in comparison to its low fraction (13%) in receptor binding we calculated that beta 1AR is 3-4-fold more effectively coupled to AC than beta 2 AR.

6-Substituted benzopyrans as potassium channel activators: synthesis, vasodilator properties, and multivariate analysis.

During the last 10 years compounds have been discovered which can activate or block KATP channels. In particular, K channel activators (KCA) have been found to be smooth muscle relaxants with their main utility in hypertension and bronchodilation. In this paper we describe the synthesis of new KCA of the benzopyran type with a fixed 4-substituent and a systematic variation in the 6-position. The relaxant potency in rat aorta and trachea was used for biological characterization of the benzopyrans. In both biological test systems, they exhibit potency ranges of more than 3 log units. Structure-activity relationships are investigated by principal component analysis (PCA) and partial least-squares (PLS) analysis. Most striking outliers in an initial PLS analysis of the entire database were the unsubstituted 6-H compound 13 as well as 34 and 35. For the remaining set of 31 compounds, a 3-component PLS model explains the variance in biological activity to 81% in the aortic and to 82% in the tracheal test system. 6-Substituents influence affinity by a direct (presumably dipolar) interaction with the receptor site. According to the 2D-plot of the partial PLS weights, a strong electronegativity as well as high values for the integy moment and for the heat of formation in water dominate the first component; low values for substituent size (as defined by globularity or surface) are in addition favorable for high potency. High lipophilicity and low minimum energies of interaction dominate the second component. Chemical descriptors for the biological potency of the test set in rat aorta and rat trachea are very similar according to the almost identical projection of the Y-variables onto the X-component space.

Verapamil analogues with restricted molecular flexibility: synthesis and pharmacological evaluation of the four isomers of alpha-[1-[3-[N-[1- [2-(3,4-dimethoxyphenyl)ethyl]]-N-methylamino]cyclohexyl]]-alpha- isopropyl-3,4-dimethoxybenzene-acetonitrile.

The synthesis and pharmacological activities of the four isomeric racemates of alpha-[1-[3-[N-[1-[2-(3,4-dimethoxyphenyl)ethyl]]-N- methylamino]cyclohexyl]]-alpha-isopropyl-3,4-dimethoxybenzene-acetoni trile are reported (2a-d). The compounds are verapamil analogues with restricted molecular flexibility designed to gather information on the active conformation(s) of the parent drug. The relative stereochemistry of the four racemates was established by X-ray crystallography and by 1H NMR spectroscopy; conformational analysis was supported by theoretical calculations. Negative inotropic and chronotropic activities were evaluated on guinea pig atria, while vasodilatory activity on smooth muscle was tested on guinea pig aortic strips. Binding studies on cat ventricles were performed using (-)-[N-methyl-3H]desmethoxyverapamil (D888) as a reference ligand. The results seem to support the hypothesis that cardiac depressant and vasorelaxant activities are due to different conformations of the verapamil molecule.

Contribution of beta 1- and beta 2-adrenoceptors of human atrium and ventricle to the effects of noradrenaline and adrenaline as assessed with (-)-atenolol.

1. (-)-Atenolol was used as a tool to assess the function of beta 1- and beta 2-adrenoceptors in human heart. Right atrial and left ventricular preparations from patients undergoing open heart surgery were set up to contract isometrically. Membrane particles were prepared for beta-adrenoceptor labelling with [3H]-(-)-bupranolol and adenylate cyclase assays. 2. The positive inotropic effects of (-)-noradrenaline were antagonized to a similar extent by (-)-atenolol in atrial and ventricular preparations. (-)-Atenolol consistently antagonized the effects of (-)-adrenaline to a lesser extent than those of (-)-noradrenaline in atrial preparations. In ventricular preparations (-)-atenolol antagonized the effects of low concentrations of (-)-adrenaline to a lesser extent than those of high concentrations. 3. pKB values (M) of (-)-atenolol, estimated with non-linear analysis from the blockade of the positive inotropic effects of the catecholamines, were 7.4 for beta 1-adrenoceptors and 6.0 for beta 2-adrenoceptors. 4. (-)-Atenolol inhibited the binding of [3H]-(-)-bupranolol to ventricular beta 1-adrenoceptors with a pKD (M) of 5.9 and to ventricular beta 2-adrenoceptors with a pKD of 4.6. 5. (-)-Atenolol inhibited the catecholamine-induced adenylate cyclase stimulation in the atrium and ventricle with pKB values of 5.8-6.4 for beta 1- and pKB values of 4.7-5.7 for beta 2-adrenoceptors. The binding and cyclase assays suggest a partial affinity loss for (-)-atenolol inherent to membrane preparations. 6. beta 1-Adrenoceptors mediate the maximum positive inotropic effects of (-)-noradrenaline in both the atrium and ventricle of man. beta 2-Adrenoceptors appear to be capable of mediating maximal positive inotropic effects of (-)-adrenaline in atrium. In contrast, ventricular beta 2-adrenoceptors mediated only submaximal effects of (-)-adrenaline.

Structural dependence of the allosteric interaction of semi-rigid verapamil analogues with dihydropyridine-binding in kitten heart.

Structural determinants of the allosteric interaction of semi-rigid verapamil analogues with dihydropyridine binding were investigated in kitten heart using [3H](+)-isradipine as radioligand. Chemical variations were performed in the alkyl chain of verapamil and include introduction of unsaturation (double or triple bonds) or the insertion of cyclohexyl moieties. Introduction of unsaturation generally reduces the allosteric interaction in the case of 'double bond'-and abolishes it in the case of 'triple bond'-derivatives. Also the introduction of cyclohexyl moieties diminishes the potency of allosteric interaction: derivatives with the phenylethylamino side chain in an equatorial position exhibit the allosteric interaction, while it is lacking in derivatives with the basic side chain in axial position. Thus, the reduced conformational flexibility of the new verapamil congeners reduces or abolishes their ability to allosterically interfere with dihydropyridine binding. A molecular interpretation was approached by molecular modelling studies. The strategy was to find low energy conformations common to the active congeners, but not shared by the inactive ones. Structural features discriminating allosterically active and inactive congeners comprise: 1) the position of the nitrogen, 2) the volume occupied by the N-methyl groups, 3) the direction of the N-H bond and 4) the position of the phenyl ring in the basic side chain.

Relaxant activity in rat aorta and trachea, conversion to a muscarinic receptor antagonist and structure-activity relationships of new K(ATP) activating 6-varied benzopyrans.

To characterize ATP-sensitive channels (K(ATP) channels) benzopyrans with different substituents at position 6 were synthesized as new K(ATP)-activators. Their relaxant potencies were determined in rat aorta and trachea. In aorta, pEC50-values (-log, M) ranged from 7.37 to 5.43; in trachea, pEC50-values were 0.3 to 0.8 log units lower. Functional data were compared with binding data obtained in calf tracheal cells using the cyanoguanidine [3H]P1075 (N-cyano-N'-1,1-dimethyl[2,3(n)-3H]propyl)-N11-(3-pyridinyl)guanidine) as radioligand. A high correlation (r = 0.96) between pEC50- and pKD-values indicated that tracheal relaxation produced by benzopyrans is mediated via K(ATP) channels without signal amplification. The permanently charged trimethylammonium derivative designed as a probe for the membrane site of action completely lost its affinity for K(ATP) channels, but converted to an antagonist for muscarinic acetylcholine receptors (pK(B) = 6.12+/-0.10), as confirmed in radioligand binding studies (pK(D) = 5.77+/-0.04). Structure-activity analyses revealed that the 6-substituent influences biological activity by a direct receptor interaction of its own and not indirectly by withdrawing electrons from the benzopyran nucleus. The variance of the biological activity is primarily determined by electrostatic properties, but desolvation energies additionally contribute.

Allosteric properties of 5-HT2 receptors in tracheal smooth muscle.

5-Hydroxytryptamine (5-HT)-induced contractions were investigated in isolated tracheal smooth muscle of guinea pig and calf. In guinea-pig tracheae, ketanserin reduced to 60% the maximum response to 5-HT, but also shifted the concentration-effect curve for 5-HT to higher 5-HT concentrations, as expected from its affinity for 5-HT2 receptors [pKB = -log KB = 9.6, KB in mol/l]. The concentration effect-data for the depressant effect of ketanserin are closely associated with the curve for occupancy of 5-HT2-receptors by ketanserin. In calf tracheae, ketanserin caused surmountable antagonism of the effects of 5-HT with a pKB of 9.5. Methysergide reduced to 25% the maximum response to 5-HT in guinea-pig tracheae and to 20% in calf tracheae. The methysergide-depressed response to 5-HT was restored by ketanserin to 60% of maximum in guinea-pig tracheae, and to 100% in calf tracheae. The results support for tracheal smooth muscle a model of an allosteric regulation of 5-HT2-receptors which was proposed for arterial smooth muscle by Kaumann and Frenken (this journal 328:295-300, 1985). The model requires that: the 5-HT2 receptor exists in two interconvertible states R in equilibrium R'; 5-HT causes its effects through R; methysergide, by acting on an allosteric site near or on the 5-HT2 receptor, shifts the equilibrium into the inactive state R'; ketanserin competes with 5-HT for the 5-HT2 receptor and with methysergide for the allosteric site, thereby restoring the active state R of the 5-HT2-receptor. All four requirements were experimentally verified in calf trachea.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta 2-adrenoceptor-mediated positive inotropic effect of adrenaline in human ventricular myocardium. Quantitative discrepancies with binding and adenylate cyclase stimulation.

Experiments were designed to unravel the relative contribution of beta 1- and beta 2-adrenoceptors to the positive inotropic effects of adrenaline and noradrenaline in isolated tissues of left ventricular myocardium of man. We also analyzed relationships between the fractions of human left ventricular beta 1- and beta 2-adrenoceptors, estimated from binding assays, and stimulation of adenylate cyclase and contractile force by adrenaline and noradrenaline. Selective blockade of beta 2-adrenoceptors by erythro-(+/-)-(alpha-methyl-indan-4-yloxy)-3-isopropylaminobuta n-2-ol (ICI 118,551) attenuated the increase of contractile force caused by adrenaline but not by noradrenaline, suggesting some involvement of beta 2-adrenoceptors. Selective blockade of beta 2-adrenoceptors without affecting beta 1-adrenoceptors still enabled both adrenaline and noradrenaline to cause maximum possible increases of contractile force through beta 1-adrenoceptors. A direct involvement of beta 2-adrenoceptors became manifest by selectively antagonizing beta 1-adrenoceptors by 1-[2[3-carbamoyl-4-hydroxy)phenoxy)ethylamino]- 3-[4(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol (CGP 20712 A) without affecting beta 2-adrenoceptor. beta 2-adrenoceptors can mediate half of the maximum increase of contractile force elicited by low concentrations of adrenaline and also contribute to the increase of contractile force caused by high concentrations of noradrenaline. beta-adrenoceptors were labelled in membrane particles with 3H-(-)-bupranolol in the absence (beta 1 & beta 2) and presence of 500 nmol/l CGP 20712 A (beta 2). 71% of the beta-adrenoceptors were beta 1 and 29% beta 2. Binding inhibition experiments with CGP 20712 A and ICI 118,551 yielded 74% beta 1 and 26% beta 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct labelling of myocardial beta 1-adrenoceptors. Comparison of binding affinity of 3H-(-)-bisoprolol with its blocking potency.

A radioligand that selectively labels beta 1-adrenoceptors, 3H-(-)-bisoprolol (3H-BIS), is introduced. The binding properties of 3H-BIS to membrane particles of kitten heart are compared with the blocking properties of (-)-bisoprolol against stimulant effects of (-)-adrenaline and (-)-noradrenaline in heart preparations of kitten and guinea pig. 1. On kitten heart tissues (-)-bisoprolol antagonized the positive chronotropic and inotropic effects of catecholamines competitively. The effects of (-)-adrenaline were antagonized considerably less by (-)-bisoprolol than the corresponding effects of (-)-noradrenaline on sinoatrial pacemakers. The antagonism was nearly the same against both (-)-adrenaline and (-)-noradrenaline in left atria and papillary muscles. The data were analyzed with a model for 2-receptor subtypes by non-linear regression. Equilibrium dissociation constants KB (mol/l; -log KB = pKB) for a high-affinity beta 1-adrenoceptor of 8.8 and for a low-affinity beta 2-adrenoceptor of 7.0 were estimated in the three classes of tissues. In kitten sinoatrial pacemaker beta 1-adrenoceptors contribute 76% to the stimulus induced by (-)-adrenaline and 97% to the stimulus induced by (-)-noradrenaline. In ventricle and left atrium beta 1-adrenoceptors contribute 97-99% and 100% to the stimulus caused by (-)-adrenaline and (-)-noradrenaline, respectively. 2. Both 3H-BIS and unlabelled (-)-bisoprolol caused competitive blockade of the positive chronotropic effects of (-)-noradrenaline in guinea-pig right atria. pKB-values of 8.7 were estimated for both unlabelled and tritiated (-)-bisoprolol. The positive chronotropic effects of (-)-adrenaline were antagonized considerably less by (-)-bisoprolol than those of (-)-noradrenaline in guinea-pig atria. In the presence of low concentrations of beta 2-selective ICI 118,551, which did not antagonize beta 1-adrenoceptor mediated effects, (-)-bisoprolol antagonized positive chronotropic effects of (-)-adrenaline to the same extent as those of (-)-noradrenaline. The results are consistent with the concept of a significant role of sinoatrial beta 2-adrenoceptors of guinea pig for the effects of (-)-adrenaline but not for those of (-)-noradrenaline. 3. 3H-BIS associated and dissociated quickly with and from ventricular beta 1-adrenoceptors. A koff of 1.0 min-1 was estimated. An equilibrium dissociation constant pKL* of 8.2 for 3H-BIS was estimated from saturation binding.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel analysis of concentration-dependence of partial agonism Ring-demethylation of bupranolol results in a high affinity partial agonist (K 105) for myocardial and tracheal beta-adrenoceptors.

1. Ring-demethylation of the pure antagonist bupranolol results in a ligand (K 105) which induces conformational beta-adrenoceptor changes leading to partial agonistic effects in heart and trachea. However, these conformational receptor changes are not accompanied by changes in receptor affinity, because the affinity estimates for K 105 and bupranolol did not differ for a variety of myocardial tissues (including ventricular beta-adrenoceptors labelled with 3H-(-)-propranolol] and trachea, not even for tracheal receptor subtypes. 2. For the analysis of the concentration-dependence of the blocking actions of a partial agonist a double log-plot was derived, which includes the classical Schild-plot as a special case. The plot is based on the statistical analysis of the action of partial agonists by Marano and Kaumann (1976). They defined a slope m for the weighted regression of equieffective concentrations of agonist in the absence and presence of a concentration [P] of partial agonist P. We derived the dependence of m on [P], which is suitably expressed as: log (1/m-1) = log [P]-log Kp. For the case of a single class of non-interacting receptors the slope of the double log-regression should be unity. Our plot has incorporated information from complete concentration-effect curves, instead of a single concentration-ratio as in the Schild-plot. Analysis of data of K 105 with the new plot (intrinsic activity greater than 0) and the Schild-plot (intrinsic activity = 0) yielded slopes near unity, consistent with simple competition.

Regional differences of beta 1- and beta 2-adrenoceptor-mediated functions in feline heart. A beta 2-adrenoceptor-mediated positive inotropic effect possibly unrelated to cyclic AMP.

The effects of (-)-adrenaline and (-)-noradrenaline were studied on isolated preparations of kitten heart. To define the contribution of beta 1-adrenoceptors (beta 1AR) and beta 2-adrenoceptors (beta 2AR) we used as tools the highly beta 1AR-selective antagonist CGP 20,712 A and non-linear analysis of antagonism. The beta 2AR-mediated responses to the catecholamines, disclosed by CGP 20,712 A, were verified by blockade with the beta 2AR-selective ICI 118,551. The relative density and contribution of beta 1AR and beta 2AR to (-)-adrenaline- and (-)-noradrenaline-induced adenylyl cyclase stimulation was also estimated in right ventricular membranes. 1. In the sinoatrial pacemaker (-)-adrenaline caused positive chronotropic effects through both beta 1AR and beta 2AR while (-)-noradrenaline does so predominantly through beta 1AR. During beta 1AR blockade (-)-adrenaline did cause the same maximum effects through beta 2AR as (-)-noradrenaline did through beta 1AR. 2. In left atria (-)-adrenaline caused positive inotropic effects predominantly through beta 1AR. CGP 20,712 A also uncovered a beta 2AR component at high (-)-adrenaline concentrations comprising one third of the maximum beta 1AR-mediated response. 3. Receptor binding assays revealed that 80% of right ventricular beta AR were beta 1AR and 20% beta 2AR. Consistent with this finding, around 80% of the adenylyl cyclase stimulation by both (-)-noradrenaline and (-)-adrenaline was mediated through beta 1AR, around 20% through beta 2AR. The positive inotropic effects of (-)-noradrenaline appeared to be nearly exclusively mediated through beta 1AR in right ventricular papillary muscles. 4. The positive inotropic effects of (-)-adrenaline were quite variable with regard to beta 1AR and beta 2AR in right ventricular papillary muscles. Although beta 1AR-mediated effects are predominant in many muscles with only a small contribution of beta 2AR, in some muscles beta 2AR mediated around 50% of the maximum effect elicited through beta 1AR. In 3 out of 17 muscles beta 2AR mediated the same maximum effect of (-)-adrenaline as beta 1AR. 5. On occasion, we found marked beta AR heterogeneity amongst two muscles from the same right ventricle. One muscle only exhibited beta 1AR-mediated effects of (-)-adrenaline whereas in the other muscle maximal effects could be elicited through beta 2AR.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulant and blocking effects of optical isomers of pindolol on the sinoatrial node and trachea of guinea pig. Role of beta-adrenoceptor subtypes in the dissociation between blockade and stimulation.

The blocking and stimulant potencies of (-)-pindolol and (+)-pindolol were estimated on right atria and tracheae of guinea pig. Blocking affinities were estimated for beta-adrenoceptor subtypes by using several agonists. Binding affinities of (-)-pindolol and (+)-pindolol were also estimated for beta-adrenoceptors labelled with 3H-(-)-bupranolol in membranes of ventricular myocardium and lung of guinea pig. Both (-)-pindolol and (+)-pindolol caused tracheal relaxation with intrinsic activities of 0.3. The concentration-effect curve for (-)-pindolol exhibits a high-sensitivity and a low-sensitivity relaxant component; the curve for (+)-pindolol was nearly monophasic. The EC50's were (-log mol/l) 9.2 and 6.1 for (-)-pindolol and 7.6 for (+)-pindolol. Using subtype-selective blockers it was found that the relaxant effects of (+)-pindolol and those of the high-sensitivity component of (-)-pindolol are mediated through beta 2-adrenoceptors. The low-sensitivity component of relaxation of (-)-pindolol was antagonized by beta-blockers less than expected from their affinities for beta-adrenoceptors. Both (-)-pindolol and (+)-pindolol caused an increase of atrial beating rate with an intrinsic activity of 0.2. The concentration-effect curve of (-)-pindolol was biphasic; the curve of (+)-pindolol was monophasic. The EC50's were (-log mol/l) 9.1 and 7.0 for (-)-pindolol and 7.5 for (+)-pindolol. From the use of subtype-selective antagonists we conclude that the positive chronotropic effects of (+)-pindolol are mediated predominantly by beta 2-adrenoceptors. On the other hand, the high-sensitivity component of the positive chronotropic effects of (-)-pindolol appears to be mediated predominantly through beta 1-adrenoceptors, although beta 2-adrenoceptors may also participate. The low-sensitivity component of the positive chronotropic effects of (-)-pindolol is resistant to blockade by subtype-selective antagonists at concentrations causing at least 98% beta-adrenoceptor occupancy. Only high but non-depressant concentrations of non-selective (-)-bupranolol antagonized the low-sensitivity component of (-)-pindolol. (-)-Pindolol antagonized the effects of several agonists to similar extent in both trachea and right atrium. (+)-Pindolol was less potent as antagonist of the relaxant effects of (-)-noradrenaline on trachea than against those of (-)-adrenaline, (-)-isoprenaline and (+/-)-salbutamol.(ABSTRACT TRUNCATED AT 400 WORDS)

Direct labelling of beta 2-adrenoceptors. Comparison of binding potency of 3H-ICI 118,551 and blocking potency of ICI 118,551.

A radioligand that selectively labels beta 2-adrenoceptors, 3H-ICI 118,551 (3H-ICI), is introduced. Experiments were performed on guinea-pig tissues. The binding characteristics of 3H-ICI on lung membrane particles are compared with the blocking characteristics of ICI 118,551 against the tracheo-relaxant effects of (-)-noradrenaline, (-)-adrenaline and (+/-)-fenoterol. Binding to both beta 1- and beta 2-adrenoceptors were also performed with 3H-(-)-bupranolol on lung and ventricular myocardium. The binding inhibition characteristics of unlabelled ICI 118,551 on ventricle were compared with its characteristics as antagonist of the positive chronotropic effects of (-)-noradrenaline, (-)-adrenaline and (+/-)-fenoterol in spontaneously beating right atria. 1. ICI 118,551 blocked more the relaxant effects of (+/-)-fenoterol and (-)-adrenaline than those of (-)-noradrenaline on trachea. The positive chronotropic effects of (+/-)-fenoterol on sinoatrial node were blocked more than those of both (-)-adrenaline and (-)-noradrenaline. A non-linear regression analysis of blocking data with ICI 118,551 according to the model of Lemoine and Kaumann (1983) revelas that both beta 1- and beta 2-adrenoceptors contribute to the tracheo-relaxant and positive chronotropic effects of agonists. The estimated equilibrium dissociation constants pKB (-log KB = pKB; mol/l) were 7.1 and 9.6 for beta 1- and beta 2-adrenoceptors, respectively. Tracheal beta 2-adrenoceptors contribute 99%, 97% and 7%, sinoatrial beta 2-adrenoceptors contribute 76%, 3% and 0% to the fractional stimuli induced by (+/-)-fenoterol, (-)-adrenaline and (-)-noradrenaline, respectively. 2. 3H-ICI associated to beta 2-adrenoceptors of lung membranes with a kon of 0.521 X nmol-1 X min-1 and dissociated with a koff of 0.19 min-1. 3H-ICI bound to lung beta 2-adrenoceptors with an equilibrium dissociation constant pKL* of 9.2. Unlabelled ICI 118,551, (-)-bupranolol, (+)-bupranolol, (-)-adrenaline, (-)-noradrenaline and (+/-)-fenoterol competed with 3H-ICI for lung beta 2-adrenoceptors with pKL-values of 9.0, 9.4, 8.1, 5.9, 4.9 and 6.4, respectively. 3. 3H-(-)-bupranolol associated to beta-adrenoceptors of lung membranes with a kon 1.21 X nmol-1 X min-1 and dissociated with a koff of 0.26 min-1. 3H-(-)-bupranolol bound to lung beta 2-adrenoceptors and to heart beta 1-adrenoceptors with a pKL of 9.6 and a pKL of 8.8, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Improved evaluation of binding of ligands to membranes containing several receptor-subtypes.

In order to evaluate accurately affinity characteristics and relative size of populations of receptor-subtypes in one system we analysed three relevant problems encountered in binding assays. Binding to receptors caused a decrease in the free ligand concentration (i.e. "depletion"). The neglect of depletion may lead to significant distortions of the estimates of affinity and size of receptor-subtype population when the concentrations of both receptor and ligand are of similar magnitude. The distortion is particularly marked when the affinity of a competing ligand is higher than the affinity of the radioligand. We present a formula that describes binding inhibition in a system with receptor-subtypes under conditions of depletion. Binding data usually exhibit heteroscedasticity (i.e. heterogeneous variance), which can not be neglected especially in a system with receptor heterogeneity. Assuming a log normal distribution of experimental errors and a Poisson distribution for errors due to radioactivity counting we derived a function for the transformation of binding data. Transformed data show homoscedasticity, as illustrated with experiments on membranes of guinea-pig lung using ICI 118,551 as inhibitor of 3H-(-)-bupranolol binding to beta 1- and beta 2-adrenoceptors. The hypothesis that affinity characteristics of receptor subtypes are independent of the tissue class can not be tested accurately by the use of standard methods because of interferences of errors between experiments. We propose a method to account for differences between experiments. Assuming invariance of affinity characteristics one is able to perform common fits of data from different tissue classes.(ABSTRACT TRUNCATED AT 250 WORDS)

Relations between beta-adrenoceptor occupancy and increases of contractile force and adenylate cyclase activity induced by catecholamines in human ventricular myocardium. Acute desensitization and comparison with feline ventricle.

The function of beta-adrenoceptors was investigated in ventricular myocardium obtained from patients undergoing open heart surgery. 1. Dopamine increased contractile force up to 1/2 and 1/4 of the maximum increase caused by (-)-noradrenaline or (-)-adrenaline in right and left ventricular preparations, respectively. 2. beta-Adrenoceptors were labelled with 3H-(-)-bupranolol. For 3/4 of the receptors (beta 1) the affinity of (-)-noradrenaline was 20 times higher than for the remaining 1/4 (beta 2). (-)-Adrenaline and dopamine appeared to be non-selective for beta 1 and beta 2. 3. Dopamine was able to stimulate the adenylate cyclase only up to 1/3 of the maximum stimulation caused by (-)-noradrenaline and (-)-adrenaline. 4. Increases in contractile force by (-)-noradrenaline were closely associated with small increases of cyclase activity through beta 1-adrenoceptors, consistent with a common link. 5. The experiments on human myocardium were compared with similar experiments on feline myocardium. Feline ventricle exhibited a 20- to 30-fold higher sensitivity to catecholamines as activators of contractile force than did human ventricle. However, the binding affinities for catecholamines were similar in cat and man. 6. A 3 h exposure of human and feline ventricular myocardium to (-)-isoprenaline caused desensitization by uncoupling beta-adrenoceptors from the adenylate cyclase. Desensitization reduced the maximum contractile response to (-)-isoprenaline in human but not in feline ventricle. 7. The more efficient activation of contractile force by (-)-noradrenaline in cat, compared to man, appears to be related to a 2-fold higher density of beta 1-adrenoceptors, a 6-fold higher production of cyclic AMP per beta 1-adrenoceptor and possibly to a more effective use of cyclic AMP for contraction.

On minimum cyclic AMP formation rates associated with positive inotropic effects mediated through beta 1-adrenoceptors in kitten myocardium. Beta 1-specific and non-adrenergic stimulant effects of denopamine.

The agonist (-)-denopamine was used as a tool to study relationships between pharmacological effects and adenylate cyclase stimulation mediated through beta 1-adrenoceptors. 1. (-)-Denopamine was a full agonist in kitten papillary muscles (force), kitten left atria (force) and kitten and guinea-pig atria (sinoatrial frequency). (-)-Denopamine was a strong partial agonist in guinea-pig tracheae (relaxation). None of these effects was influenced by blockade of beta 2-adrenoceptors. beta 1-Adrenoceptors mediated all effects of (-)-denopamine in atria and effects of low (-)-denopamine concentrations in papillary muscles and tracheae, as assessed with beta 1-selective antagonists. 2. High (-)-denopamine concentrations caused positive inotropic effects in papillary muscles and tracheorelaxant effects that were resistant to blockade by beta 1-, beta 2- and alpha-adrenoceptor antagonists (non-adrenergic effects). 3. (-)-Denopamine stimulated the adenylate cyclase of membranes derived from kitten ventricle and calf tracheal cells with an intrinsic activity of 0.3 and 0.2, respectively, compared to catecholamines. The contribution of beta 1- and beta 2-adrenoceptors to cyclase stimulation was assessed by selective blockade. Cyclase stimulation through beta 2-adrenoceptors by (-)-denopamine was 12% in ventricle and 82% in trachea but is not associated with positive inotropic effects and tracheal relaxation. 4. (-)-Denopamine exhibited only a 2- to 5-fold selectivity for beta 1-adrenoceptors compared to beta 2-adrenoceptors, as estimated consistently from binding assays and blockade of cyclase stimulation in myocardial and tracheal cell membranes. 5. Desensitization of kitten ventricular tissues, caused by a 3 h exposure to 30 mumol/l (-)-isoprenaline followed by 5 h washout, reduced the inotropic sensitivity of papillary muscles without decreasing the maximum inotropic effects of (-)-denopamine. In desensitized tissues, the nonadrenergic effect contributed by 30% to the maximum inotropic effect of (-)-denopamine. 6. In membranes, derived from desensitized tissues, the maximum adenylate cyclase stimulation induced by (-)-isoprenaline, (-)-denopamine and xamoterol was reduced to 1/2 of the corresponding stimulations observed in membranes from sham desensitized tissues. The density of beta-adrenoceptors, assessed with 3H-(-)-CGP 12,177, was not changed by the desensitization procedure suggesting that part of the receptors was uncoupled from the adenylate cyclase. The partial inotropic agonist xamoterol, which has an intrinsic activity of 0.5 in non-desensitized tissues, failed to cause positive inotropic effects in desensitized papillary muscles suggesting that not all cyclic AMP possesses inotropic relevance.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence that (+)-bupranolol interacts directly with myocardial beta-adrenoceptors. Control of optical purity with differential thermal analysis.

Melting points measured with the capillary method were 150.5 degree C, 150.5 degree C and 224.0 degree C for hydrochlorides of (+)-bupranolol, (-)-bupranolol and (+/-)-bupranolol, respectively. The large difference in melting points of 73.5 degree C prompted us to determine possible contaminations of (+)-bupranolol with traces of (-)-bupranolol using differential scanning calorimetry. We detected as little as 0.001% (-)-bupranolol in a standard mixture of (+)-bupranolol and (-)-bupranolol. A batch of (+)-bupranolol not measurably contaminated with (-)-bupranolol (optically purity greater than 99.999%) was used in pharmacological and biochemical assays. The affinities of (-)-bupranolol and (+)-bupranolol were determined functionally by the blockade of isoprenaline stimulation of spontaneously beating rat right atria and electrically driven kitten papillary muscles; and directly by inhibition of binding of 3H-(-)-propranolol to kitten ventricle membrane particles. In all 3 systems the enantiomeric (-)/(+) affinity ratio was 50--120 for bupranolol. These experiments prove that (+)-bupranolol itself binds to the beta-adrenoceptors of mammalian myocardium.

An initial characterization of human heart beta-adrenoceptors and their mediation of the positive inotropic effects of catecholamines.

The positive inotropic effects of catecholamines were studied on samples of ventricular myocardium taken from patients undergoing open heart surgery. The adenylyl cyclase and binding of 3H-(-)-bupranolol were examined in membrane particles prepared from similarly obtained samples. The equilibrium dissociation constant (KD) for (-)-bupranolol was estimated in 4 ways: blockade of the positive inotropic effects of catecholamines, blockade of the stimulation of the adenylyl cyclase by catecholamines, saturation binding of 3H-(-)-bupranolol, inhibition of the binding of 3H-(-)-bupranolol by its unlabeled stereoisomers. The estimates of KD fall in the range 0.5-1.4 nmol/l. The stereo-selectivity ratio (KD (+)-isomer/KD (-)-isomer) is 73. Both values for bupranolol are very similar in cat and man. The inotropic potency of (-)-noradrenaline is nearly 2 orders of magnitude higher in cat heart tissues than in tissues from human hearts. The difference in inotropic potencies between species is only partially accounted for by the five-fold lower potency of (-)-noradrenaline for the human heart adenylyl cyclase as compared to the cat enzyme.