The effect of isoprenaline on plasma concentrations of immunoreactive β-endorphin and β-lipotropin in the conscious rat

Summary The effect of the -adrenoceptor agonist isoprenaline on the plasma concentrations of -endorphin (-E) and -lipotropin (-LPH) was investigated in conscious rats. Isoprenaline (i.m.) elevated plasma -endorphin-like immunoreactivity (-EI) as measured by radioimmunoassay of unextracted plasma, with peak values 24 min after drug administration. This effect was dose-dependent. The lowest effective dose of isoprenaline was 15 g kg^–1; 240 g kg^–1 exerted a maximum effect, raising plasma -EI about ten-fold above control values. Plasma vasopressin concentrations also increased in response to isoprenaline following a timecourse identical to that of plasma -EI. (±)-Propranolol (1 mg kg^–1) but not phentolamine (10 mg kg^–1) rendered isoprenaline (240 g kg^–1) injections almost ineffective. Because of the cross-reactivity of -LPH in the radioimmunoassay used, plasma was extracted by means of a cation exchange resin and subjected to gel chromatography on a Sephadex G-50 column, avoiding artefactual degradation of the peptides. In isoprenaline-treated rats about 50% of the -EI behaved similar to human -LPH, whereas 45% co-migrated with human -E; immunoreactivity corresponding to -LPH or -E comprised about 70% or 30%, respectively, in the plasma extract of vehicle-treated rats. Dexamethasone pretreatment reduced the isoprenaline-induced increase in plasma -EI by 87%, but left the simultaneous elevation of plasma vasopressin concentrations unchanged.These data demonstrate that isoprenaline stimulates -LPH and -E release in vivo. The possibility of an interrelationship between vasopressin and -E release is discussed.     

Vasopressin stimulates release of β-lipotropin and β-endorphin in conscious rats as measured by radioimmunoassay of unextracted plasma

Summary Using a newly developed radioimmunoassay to determine the -endorphin-like immunoreactivity (-EI) in unextracted plasma, the effect of vasopressin injections on plasma -EI was investigated in conscious rats. Arginine vasopressin caused a dose-dependent increase of plasma -EI from 34.5±7.8 fmol ml^–1 (n=6) in vehicle-treated animals to 205.0±36.1 fmol ml^–1 (n=7) after injection of the highest vasopressin dose employed (486 ng/100 g b.w.). In view of the appreciable cross-reactivity of -lipotropin (-LPH) in the radioimmunoassay used, plasma was extracted and subjected to gel chromatography on a Sephadex G-50 column. On average, about 70% of the -EI co-eluted with human -LPH and about 30% with human -endorphin in plasma extracts obtained from both control and vasopressin-treated rats. No peripheral conversion of human -LPH occurred under the experimental conditions, since after i.v. bolus injection of human -LPH 97% of the -EI comigrated with human -LPH during gel filtration. A similar blood pressure increase to that induced by the vasopressin injections, when elicited by noradrenaline or angiotensin II i.v., was not followed by an elevation of plasma -EI.These data indicate that vasopressin stimulates -lipotropin and -endorphin release into the systemic circulation in vivo.     

Esmolol,an ultrashort-acting,selective β1-adrenoceptor antagonist: pharmacodynamic and pharmacokinetic properties

The effects of esmolol at different rates of infusion (100, 250 and 500 g·kg^–1 BW·min^–1) were compared with -adrenoceptor occupancy (₁ and ₂, estimated by a subtype selective radioreceptor assay) and plasma concentrations of esmolol and its acid metabolite were measured by HPLC. Up to a rate of infusion of esmolol of 500 g·kg^–1 BW·min^–1 there was a maximal ₁-receptor occupancy of 84.7% while ₂-receptor occupancy was below the detection limit; confirming the ₁ selectivity of esmolol. Exercise-induced increases in heart rate and systolic blood pressure were reduced by esmolol in a dose-dependent manner. The estimated EC₅₀ values of rate of infusion for the reduction in heart rate and systolic blood pressure during exercise were 113 and 134 g·kg^–1 BW · min^–1, respectively. Additionally, heart rate and systolic blood pressure were reduced moderately at rest. Because of the short elimination half-life of esmolol caused by the rapid hydrolysis to its acid metabolite, 45 min after end of infusion high plasma concentrations of the metabolite (maximally 80 g·ml^–1) but no esmolol were detectable. Since no in vivo effects have been observed, despite the presence of high plasma concentrations of the metabolite, the metabolite did not participate in the observed effects up to an infusion rate of esmolol of 500 g·kg^–1 BW·min^–1. The plasma concentrations of antagonist detected by radioreceptor assay and plasma concentrations of esmolol detected by HPLC showed a good correlation (r=0.97). Since the cardiovascular effects, determined before and 45 min after termination of infusion of esmolol were similar, it can be concluded that the observed effects on heart rate and systolic blood pressure are exclusively mediated by esmolol.Dedicated to Dr. P.Rajagopal, Kuantan Specialist Hospital, Kuantan, Malaysia     

The selective P2X purinoceptor agonist, β,γ-methylene-L-adenosine 5′-triphosphate,discriminates between smooth muscle and neuronal P2X purinoceptors

The effects of the putative selective P_2X purinoceptor agonist, ,-methylene-l-adenosine 5-triphosphate (me-l-ATP), were determined at rat neuronal and smooth muscle P_2X purinoceptors.Me-l-ATP had no effect on the extracellularly recorded membrane potential of the rat isolated vagus nerve preparation at concentrations up to 300 M. In contrast, the archetypal P_2X purinoceptor agonist, , methylene ATP (meATP;1–100 M), produced concentration-related depolarisation responses with a mean EC₅₀ value of 10.8 M. The depolarising effects of meATP were not attenuated by me-l-ATP (100 M). In voltage clamp experiments on single nodose ganglion neurones, ATP (100 M), but not me-l.-ATP (1–300 M), evoked rapid ( < 20 ms onset) inward currents when applied using a concentration-clamp method. In receptor binding studies to rat brain membranes, me-d-ATP and meATP competed with high affinity for [³H]Lx meATP binding sites, with mean pIC₅₀ values of 7.7 and 8.3, respectively. However, me-l-ATP possessed low affinity for these sites and competed only at concentrations in excess of 10 M (mean pIC₅₀ value 4.1).In prostatic segments of the rat vas deferens, me-l-ATP (1–100 M) and meATP (0.3–100 M) each produced concentration-related contractile responses with mean EC₅₀ values of 17.1 and 3.6 M, respectively. Me-l-ATP (1–10 M) evoked fast inward currents in freshly dispersed vas deferens smooth muscle cells, indicative of an action at ligand-gated ion channels. Binding sites in vas deferens membranes labelled using 1 nM [³H]meATP exhibited high affinity for me-l-ATP, meATP and me-d-ATP with mean PIC₅₀ values of 7.7, 8.4 and 7.3, respectively.These results indicate that me-l-ATP exhibits neither agonist nor antagonist properties at P_2X purinoceptors on rat vagal neurones and possesses only very low affinity for [³H]meATP binding sites in rat brain. In contrast, me-l-ATP is a potent, high affinity agonist at smooth muscle P_2X purinoceptors of the rat vas deferens. This selective agonist action of me-l-ATP suggests that P_2X purinoceptors in smooth muscle and neurones are different and represent distinct P_2X purinoceptor subtypes.     

Neuronally released (−)-noradrenaline relaxes smooth muscle of calf trachea mainly through β1-adrenoceptors : comparison with (−)-adrenaline and relation to adenylate cyclase stimulation

Summary The nature of the receptors that mediate the relaxation of smooth muscle by field stimulation, (–)-noradrenaline and (–)-adrenaline was investigated in calf tracheal smooth muscle. The relation between relaxation, stimulation of the adenylate cyclase and density of -adrenoceptor subtypes was studied with the help of antagonists of ₁- and ₂-adrenoceptors. The question of the existence of catecholamine-containing nerves was also investigated. (1) Nerves with varicosities exhibiting catecholaminergic fluorescence were observed between bundles of smooth muscle cells. (2) Consistent with the existence of adrenergic nerves (–)-noradrenaline was also found. The content of (–)-noradrenaline (1 g · g^–1 w.w.) was the same in smooth muscle strips from the sublaryngeal region and from the region close to the bifurcation of the calf trachea. (–)-Adrenaline was not detected. (3) Smooth muscle relaxation by low (–)-noradrenaline concentration (0.6–2 nmol/l) was mediated through ₁-adrenoceptors. Low concentrations of (–)-adrenaline (0.06–1 nmol/l) relaxed through ₂-adrenoceptors. High concentrations of (–)-noradrenaline and (–)adrenaline also caused relaxation through ₂- and ₁-adrenoceptors respectively. (4) Field stimulation caused relaxation which was half maximal at 0.2–0.8 Hz. Blockade of ₁-adrenoceptors strongly attenuated the relaxant response to field stimulation and shifted the frequency-relaxation curves to 4 times higher frequencies. These results are consistent with a ₁-adrenoceptor-mediated relaxation caused by (–)-noradrenaline released from sympathetic nerve endings at low stimulation frequencies. (5) Blockade of ₂-adrenoceptors failed to reduce smooth muscle relaxation caused by field stimulation at low stimulation frequencies (0.1–1 Hz). However, after ₁-adrenoceptor blockade, additional blockade of ₂-adrenoceptors reduced the relaxant effects observed at high frequencies (2–400 Hz). The results suggest that high concentrations of endogenous (–)-noradrenaline cause relaxation through ₂-adrenoceptors. (6) Binding experiments with ³H-(–)-bupranolol and ³H-ICI 118,551 revealed between 10,000 and 20,000 -adrenoceptors per smooth muscle cell of which 3/4 were ₂ and 1/4 ₁. The equilibrium dissociation constant of (–)-adrenaline for both ₁- and ₂-adrenoceptors and of (–)-noradrenaline for ₁-adrenoceptors was 1 mol/l. The affinity of (–)noradrenaline for ₂-adrenoceptors was 10 to 20 times lower than for ₁-adrenoceptors. (7) The adenylate cyclase of smooth muscle cells was stimulated more through ₂-adrenoceptors by both (–)-noradrenaline (77%) and adrenaline (83%) than through ₁-adrenoceptors. Half maximum stimulation of the cyclase was observed at 2 to 4 times lower catecholamine concentrations than the concentrations that half saturate ₁- or ₂-adrenoceptors. (8) Both the low affinity and low cyclase stimulating potency of the catecholamines suggest considerable amplification of the receptor signals. Half maximum relaxation of tracheal muscle by (–)-noradrenaline is associated with the activation of less than 50 ₁-adrenoceptors per cell producing less than 250 molecules cyclic AMP per second.     

Effect of captopril on myocardial β-adrenoceptor density and Giα-proteins in patients with mild to moderate heart failure due to dilated cardiomyopathy

In end-stage heart failure due to idiopathic dilated cardiomyopathy ₁-adrenoceptors are downregulated and G₁-proteins are upregulated. The aim of the present study was to investigate the influence of the angiotensin-converting enzyme inhibitor captopril on -adrenoceptor density and G_i-proteins in sequential endomyocardial biopsies. Nineteen patients with mild to moderate congestive heart failure due to idiopathic dilated cardiomyopathy (NYHA Class II–III) were studied before and after 8–11 weeks of therapy. Patients were randomised into a captopril and a control group; 9 patients received captopril 12.5–50 mg per day, (divided in 2–3 doses) p.o. in addition to conventional therapy with digoxin and diuretics, and 10 controls received conventional therapy only. Echocardiography, spiroergometry, right heart catheterisation and endomyocardial biopsies were performed before (baseline) and after treatment. Compared to baseline, captopril increased total -adrenoceptor density by selectively increasing ₁-adrenoceptors (31.6 vs 41.2 fmol·mg^–1; p<0.05) but had no significant effect on G_i-proteins. The results indicate that treatment with angiotensin-converting enzyme inhibitors partly restores myocardial ₁-adrenoceptor density, and this action effect may contribute to the clinical improvement of patients with idiopathic dilated cardiomyopathy treated in this way.     

Selective inhibition of aldosterone synthesis by 11-hydroxylated spirolactone in rat adrenals

Summary The effect of the spirolactone SC5233 [3-(3-oxo-17-hydroxy-4-androstene-17-yl)propionic acid--lactone] and of the 11-hydroxylated SC5233 on the synthesis of corticosteroids was examined in rat adrenal tissue. SC5233 markedly inhibited the production of both corticosterone and aldosterone. 11-Hydroxy-SC5233 also inhibited markedly the production of aldosterone but only slightly that of corticosterone. 50% inhibition of aldosterone production by 11-Hydroxy-SC5233 was obtained at a concentration of 7×10^–5 M when the synthesis of corticosteroids was stimulated by dibutyryl cyclic AMP, while 50% inhibition of corticosterone production was obtained only at 4×10^–4 M. In incorporation experiments a highly significant inhibition by SC5233 of the incorporation of ¹⁴C-progesterone into corticosterone, 18-hydroxycorticosterone and aldosterone was detectable in connexion with an accumulation of labelled deoxycorticosterone. In experiments with 11-Hydroxy-SC5233 a significant inhibition of the incorporation of ¹⁴C-progesterone was shown for only 18-hydroxycorticosterone and aldosterone with a moderate accumulation of labelled deoxycorticosterone. Incorporation of ¹⁴C-progesterone into corticosterone was not affected by 11-Hydroxy-SC5233. The results show that 11-hydroxylated SC5233 induced a largely selective inhibition of aldosterone production; in contrast, SC5233 had an unselective inhibitory effect on the synthesis of both corticosteroids, aldosterone and corticosterone.     

Vergleich der enteralen Resorbierbarkeit vonβ-Acetyldigoxin und Digoxin am Meerschweinchen

Zusammenfassung Vergleichende Untersuchungen anwachen Meerschweinchen über die enterale Resorption von Digoxin und -Acetyldigoxin (-AD) ergaben, daß -AD nach peroraler Gabe der LD 50 (2,62 mg/kg) innerhalb von 45–90 min zum Tod der Versuchstiere führt. Für Digoxin liegt die perorale LD 50 wesentlich höher (13,3 mg/kg), und die Zeitspanne bis zum Eintritt des Todes variiert von 1 Std bis zu 4 Tagen.Versuche annarkotisierten Meerschweinchen wiesen darauf hin, daß -Acetyldigoxin im Ileum am besten resorbiert wird; aus der Zeit bis zum Wirkungseintritt sowie den Angaben über die i.v. Toxicität wird geschlossen, daß -Acetyldigoxin enteral rascher und vollständiger resorbiert wird als Digoxin.

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Summary Comparative studies on enteral resorption of digoxine and -acetic digoxine (-AD) were carried out in not narcotized guinea-pigs. -AD, after application of the oral LD 50 (2,62 mg/kg), proved to be lethal within 45–90 min. For digoxine the oral LD 50 is considerably higher (13,3 mg/kg), times of death were found between 1 hour and 4 days.Studies with narcotized guinea-pigs showed, that -AD is optimally resorbed in the ileum. From the time between application and response and from their intravenous toxicity it can be concluded, that the enteral resorption of -acetic digoxine takes place more quickly and more completely than of digoxine.

     

Different steric characteristics of β1- and β2-adrenoceptors

Summary -Adrenoceptors of lung (75% ₂) and heart (95% ₁) of calf were labelled with ³H-(–)-propranolol. The stereoisomers of 10 ligands were used to inhibit the binding of ³H-(–)-propranolol to membrane particles. The affinity ratio of sereoisomers is consistently greater for ₁-adrenoceptors than for ₂-adrenoceptors, regardless of whether the ligands are agonists, partial agonists or antagonists. The ₁-adrenoceptor appears to possess stricter steric requirements than the ₂-adrenoceptor. This property may prove helpful in differentiating the -adrenoceptor subtypes during receptor solubilization and purification.     

α-Adrenoceptor-mediated inhibitory effect of the sympathetic nervous system on the isoprenaline-induced increase in plasma renin concentration

Summary The importance of the sympatho-adrenal system for the isoprenaline-induced increase in plasma renin concentration was investigated in conscious rats. Ganglionic blockade by trimethidinium (10 mg kg^–1) increased the dose-dependent elevation of plasma renin concentration induced by isoprenaline (0.03–0.48 g kg^–1 min^–1). Also treatment of the rats with guanethidine (6 mg kg^–1) or reserpine (2.5 mg kg^–1, given 16 and 7 h prior to the experiments) further increased the effect of isoprenaline (0.5 g kg^–1 min^–1) on plasma renin concentration. Unilateral renal denervation combined with contralateral nephrectomy doubled the effect of the -sympathomimetic amine on renin release. The -adrenoceptor antagonist phenoxybenzamine (3 mg kg^–1) also enhanced the effect of isoprenaline on this parameter.It is concluded that apart from a stimulation of renin release via -adrenoceptors the sympathetic nervous system may inhibit renin release via stimulation of -adrenoceptors.Supported by DFG Me 541/1Partial preliminary communication: Meyer et al. (1976)     

Receptor binding assays in analysing the bioavailability and pharmacodynamic bioequivalence of active drug moieties

The bioavailability and pharmacodynamic bioequivalence of a conventional and an experimental sustained-release formulation of 100 mg metoprolol tartrate were studied in a randomised cross-over study in seven healthy volunteers by assessing over 24 h the plasma kinetics of R,S-metoprolol, its ₁-adrenoceptor binding component, and by determining the extent to which the active drug moiety in plasma occupied rabbit lung ₁-and rat reticulocyte ₂-adrenoceptors.The formulations differed markedly in their kinetic characteristics: the peak plasma concentration (C_max) of R,S-metoprolol after administration of the conventional formulation was 140 ng·ml^–1, (n=7) and it was approximately one-third of that after the sustained-release formulation, 49 ng·ml^–1, (n=6); the AUC_{0–24 h}-values for the formulations were 700 and 310 ng·h·ml^–1, respectively. The C_max for the ₁-adrenoceptor binding component of metoprolol was 180 ng·ml^–1 (n=7) after administration of the conventional, and 74 ng·ml^–1 after administration of the sustained-release formulation. The corresponding AUC_{0–24 h}-values for the receptor binding component were 920 and 470 ng·h·ml^–1 (n=7).Thus, the kinetic differences between R,S-metoprolol and the ₁-receptor binding component were considerable and they were affected by the type of formulation. In general, after administration of the sustained-release formulation, the percentage ₁- and ₂-adrenoceptor occupancy of metoprolol in plasma was 5–15% less than after administration of the conventional formulation. At 0.5–1.5 h after drug intake the average ₁-adrenoceptor occupancy of the conventional formulation varied between 80–90% and that of the sustained release formulation between 20–76%. At these times the differences in receptor occupancy were significant; at 0.5–2 h after drug intake the average ₂-adrenoceptor occupancy of the conventional formulation varied from 20–30%, and that of the sustained-release formulation was 2–17%. At other times the difference in receptor occupancy between the formulations was not significant.The results demonstrate that plasma concentration-kinetics were more discriminating than -adrenoceptor-binding in analysing bioequivalence. It was possible to determine the bioavailability of the active ingredient of metoprolol and to study pharmacodynamic bioequivalence by using receptor binding assays.     

β-Endorphin-induced release of 5-hydroxytryptamine from human platelets

Platelets of healthy human subjects were incubated with 5-hydroxytryptamine (5-HT) followed by a second incubation with either -endorphin (-end) or the combination of -end and naloxone. -End (300 pg/ml) reduced the levels of 5-HT to 50% of initial values within 15–40 min. After 40–80 min incubation, the levels of 5-HT decreased to approximately zero. Addition of increasing amounts of -end (up to 300 pg/ml) produced increasing releases of 5-HT with increasing doses of -end. The response was dose-related, however variable, across subjects. Addition of either 28.7 or 57.4 pg naloxone to 300 pg -end did not antagonize the effects of -end on 5-HT.     

Changes in plasma and urinary 11-dehydrothromboxane B2 in healthy subjects produced by oral CS-518, a novel thromboxane synthase inhibitor

Summary In 18 healthy volunteers, in a double-blind placebo-controlled study, we investigated of whether 14 days treatment with a therapeutic dose of ibopamine (3×100 mg/day p.o.), respectively its active metabolite epinine, would desensitize lymphocyte ₂- or platelet ₂-adrenoceptors, or ₁- and -adrenoceptor mediated (phenylephrine-and isoprenaline infusions, respectively), changes in systolic and diastolic blood pressure and heart rate.Ibopamine-treatment, which resulted in peak plasma epinine concentrations of 4–5 nmol·l^–1, neither affected resting heart rate or blood pressure, nor any of the - or -adrenoceptor parameters measured. Since in man in general long-term administration of - and -adrenoceptor agonists desensitizes - and -adrenoceptors, the lack of any - and -adrenoceptor desensitizing effect of ibopamine suggests that, in the dose employed (3×100 mg per day), ibopamine does not exert - or -adrenoceptor agonistic effect in humans.     

The β2-adrenoceptor antagonist CGP 20712 A unmasks β2-adrenoceptors activated by (−)-adrenaline in rat sinoatrial node

Summary The role of sinoatrial ₁- and ₂-adrenoceptors mediating positive chronotropic effects of (–)-adrenaline and (–)-noradrenaline was investigated in rat right atria. Concentration effect curves for (–)-adrenaline, but not for (–)-noradrenaline, became biphasic in the presence of the ₁-adrenoceptors antagonist CGP 20712 A. The curves for (–)-adrenaline in the presence of 300 nmol/l CGP 20712 A (equivalent to 1,000 times its K _B, K _B=0.3 nmol/l for ₁-adrenoceptors) comprise a high-sensitivity component that saturates at 1/4 of maximum effect, and a low sensitivity component. The high-sensitivity component is blocked by the ₂-adrenoceptor-selective antagonist ICI 118,551. These results are consistent with an involvement in the rat of both ₁-adrenoceptors (to a major extent) and ₂-adrenoceptors [only at high (–)-adrenaline concentrations] in the positive chronotropic effects of (–)-adrenaline. (–)-Noradrenaline appears to activate mostly rat sinoatrial ₁-adrenoceptors.     

Kinetics of the Aspartyl Transpeptidation of Daptomycin,a Novel Lipopeptide Antibiotic

Two degradation products of the lipopeptide antibiotic, daptomycin, were identified and the reaction pathway and kinetics were delineated in aqueous solution at 60°C, pH range 3 to 8 and ionic strength 0.01. The degradation products were 1) a succinimido intermediate (anhydro-daptomycin) formed by attack of side-chain carbonyl on the peptide-bond nitrogen in the asp–gly sequence and 2) a -asp daptomycin isomer formed by rehydration of the anhydrodaptomycin succinimide. This aspartyl transpeptidation pathway was found to be reversible. Formation of the anhydrodaptomycin from either daptomycin or -asp daptomycin was pH dependent but the pH–rate profiles for anhydrodaptomycin formation were not mechanistically interpretable. The pH–rate profiles for the formation of daptomycin or -asp daptomycin from the anhydrodaptomycin were linear with slopes = 1, which is consistent with nucleophilic hydroxide ion attack of the succinimido intermediate at either the -carbonyl, giving rise to the -asp daptomycin, or the -carbonyl, giving rise to daptomycin.     

Aspartate138 is required for the high-affinity ligand binding site but not for the low-affinity binding site of the β<Subscript>1</Subscript>-adrenoceptor

The ₁-adrenoceptor two-site ligand binding hypothesis was investigated by comparing the pharmacological activities of the receptor with an Asp to Glu mutation of amino acid 138 after transient transfection into CHO cells. The high-affinity binding of (–)-[³H]-CGP12177 (pK_D=9.4) and binding inhibition by (–)-isoprenaline (pK_i=6.2), observed with Asp138-₁-adrenoceptors, were absent at Glu138-₁-adrenoceptors. (–)-[³H]-CGP12177 bound with a pK_D=7.6 to Glu138-₁-adrenoceptors and (–)-isoprenaline enhanced binding, probably allosterically. Glu138-₁-adrenoceptors compared with Asp138-₁-adrenoceptors showed a 500,000-fold decrease in cyclic AMP-enhancing potency by (–)-isoprenaline and antagonism by (–)-bupranolol (1 M) was abolished. At Glu138-₁-adrenoceptors, the agonist potency of (–)-CGP12177, compared with (–)-isoprenaline was reduced five-fold, but the antagonism by (–)-bupranolol (pK_B=7.1) was not significantly changed, compared with Asp138-₁-adrenoceptor. Thus, Asp138 of the ₁-adrenoceptor is essential for the binding of (–)-isoprenaline, (–)-bupranolol and (–)-CGP12177 to a high-affinity site, but not for the binding of (–)-CGP12177 and (–)-bupranolol to a low-affinity site.     

(±)[125Iodo]cyanopindolol,a new ligand for β-adrenoceptors: Identification and quantitation of subclasses of β-adrenoceptors in guinea pig

Summary (±)[¹²⁵Iodo]cyanopindolol (ICYP) is a radioligand which binds with an extraordinarily high affinity and specificity to -adrenoceptors. In contrast to (±)[¹²⁵Iodo]-hydroxybenzylpindolol (IHYP), the new ligand has neither affinity to - nor to 5-HT-receptors. The dissociation constants of ICYP for -adrenoceptors in various tissues range from 27 to 40 pM, thereby exceeding the affinity of IHYP by a factor of 3.ICYP does not discriminate between ₁– and ₂–. Therefore, the densities of the two receptor subtypes can be determined from competition curves of ICYP by drugs previously found to show in vitro selectivity for ₁–adrenoceptors.The guinea pig left ventricle contains only ₁– adrenoceptors, whereas in the lung tissue, the ratio of ₁– to ₂–adrenoceptors is 1 to 4. The calculated affinities of five ₁– selective antagonists for ₁–adrenoceptors were nearly identical in the ventricle and the lung.Kinetic studies of ICYP binding to guinea pig lung membranes indicated that the dissociation reaction consists of two components, a fast process (t _1/2=9 min) and a slower process (t _1/2=8.8 h). A mathematical treatment revealed two possibilities of interpretation: 1. Two forms of the receptor exist which are interconvertible. 2. The (+)- and (–)-enantiomers of ICYP dissociate with different rate constants.The low dissociation constant of ICYP in combination with its high specific radioactivity (2175 Ci mmole^–1) allows binding studies to be carried out with small protein and ligand concentrations, e.g. 3 g protein per assay in guinea pig lung membranes.Abbreviations CYP (±)cyanopindolol, [(±)4-(3-tert-butylamino-2-hydroxypropoxy)-1H-indole-2-carbonitrile] - ICYP (±)-3-[¹²⁵iodo]-cyanopindolol - HYP (±)hydroxybenzylpindolol; IHYP, (±)[¹²⁵iodo]-hydroxybenzylpindolol - ³H-DHA (–)-[³H]dihydroalprenolol Part of this work has been presented in Mainz, March 1980, at the Spring Session of the German Pharmacological Society, and in Brussels, June 1980, at the 4th International Conference on Cyclic Nucleotides     

Some 4-methylesculetins substituted byβ-dialkylaminoethyl radicals

Conclusions The 3-(-dialkylaminoethyl)-6,7-dimethoxy-4-methylcoumarins, 6-(-dialkylaminoethoxy)-7-methoxy-and 6-methoxy-7-(-dialkylaminoethoxy)-4-methylcoumarins were synthesized by reacting the -bromo-ethyl-4-methylesculetins with secondary amines.Translated from Khimiko-Farmatsevticheskii Zhurnal, No. 9, pp. 32–35, September, 1969.     

Differentiation of cardiac chronotropic and inotropic effects of β-adrenoceptor agonists

Summary The relative inotropic and chronotropic activity of -adrenoceptor agonists was studied in the noradrenaline-depleted, anaesthetized cat. Terbutaline, a selective ₂-adrenoceptor agonist, gave at a certain dose a more pronounced chronotropic than inotropic response, while a new ₁-selective adrenoceptor agonist (–)-H 80/62 produced the same degree of chronotropic and inotropic stimulation. The results indicate that there is some difference between the -adrenoceptors in the sinus node mediating chronotropic stimulation and -adrenoceptors in the ventricular myocardium mediating stimulation of the contractile force. It has been shown that there are both ₁- and ₂-adrenoceptors in the heart (Carlsson et al., 1972). In the light of this finding it is hypothetized that there are differences in the relative distribution of ₁- and ₂-adrenoceptors in the sinus node and in the myocardium. Although ₁ is the predominant type of -adrenoceptor in both regions, the ₁: ₂ concentration ratio seems to be higher in the myo-cardium, than in the sinus node.     

Bedeutung der N- und α-Methylierung für die Affinität von Brenzcatechinaminen zu den adrenergischen Receptoren

Summary 1. N-methylation of dopamine yielding epinine means potentiation of the -adrenergic pressor action in the cat. It is only N-methylation which renders dopamine to a direct acting -sympathomimetic amine: the positive inotropic effect of epinine on the isolated electrically driven guinea pig auricle remained uninfluenced by pretreatment of the animals with reserpine, whereas the dose-response curve of dopamine was shifted to the right.The blood pressure lowering effect of epinine after phenoxybenzamine was—in contrast to that of dopamine—abolished by pronethalol.—N-methylation of noradrenaline doubled the affinity for the -receptors of the heart.2. Also by -methylation dopamine gains affinity to the adrenergic -receptors (heart and vessels): d--methyldopamine, an exclusively directly acting catecholamine, had a 40 times stronger inotropic action than dopamine on reserpinized auricles; the action of l--methyldopamine, a mainly indirectly acting amine, was only 4 times stronger than dopamine.-methylnoradrenaline, although having the same inotropic activity as noradrenaline, had a 10 times stronger depressor action than noradrenaline in the cat pretreated with phenoxybenzamine.By -methylation, the -adrenergic pressor effect of dopamine as well as that of noradrenaline was enhanced only in the range of lower doses since the dose-response curves of the -methylated compounds were generally less steep than those of the non-methylated catecholamines, indicating a lower intrinsic activity of the -methylated derivatives.3. The N- and -methylated catecholamines -methylepinine and -methyladrenaline resp. were blood pressure lowering agents per se, because both methylgroups additively enhanced the affinity to the vascular -receptors. -methylepinine was the most potent -sympathomimetic on the heart in the dopamine series (dopamine < d--methyldopamine epinine < dl--methylepinine). However, in the noradrenaline series the twofold methylated compound -methyladrenaline had the lowest positive inotropic action (d(–)-adrenaline > d(–)-noradrenaline (–)erythro--methylnoradrenaline > (–) erythro--methyladrenaline).4. From the results the following conclusions are drawn: The N- as well as the -methyl-group exerts and +I-effect on the ammonium group. Thereby, protonation will be increased which leads to an enhanced affinity of the resp. catecholamine to the adrenergic - and -receptor. Since the -CH₃ group—dependant upon its steric configuration—also causes a steric hindrance at the receptor site, the increase in affinity due to the +I-effect is partially neutralized. By that the lower intrinsic affinity of the -methylated compounds as indicated by the different slope of their pressor dose-response curves, is also readily explained. Furtheron it is intelligible why -methylation enhanced the -adrenergic activity in the less potent dopamine series (preponderance of the +I-effect), whereas it lowered the affinity to the cardiac -receptors in the noradrenaline series (preponderance of the steric hindrance).5. Although -methyladrenaline was the least potent -sympathomimetic of the noradrenaline series in the guinea pig heart, it was the most potent compound in lowering the cat's blood pressure. Therefore, it seems to be questionable whether the cardiac and the vascular -receptors and/or the mechanisms by which they induce the pharmacodynamic actions are identical.

Über einen Teil der Ergebnisse wurde auf der 7. Frühjahrstagung der Deutschen Pharmakologischen Gesellschaft in Mainz, 24.–27. April 1966, berichtet (Palm, Langeneckert u. Holtz, 1966).