The role of cyclic AMP in the positive inotropic effect mediated by β1- and β2-adrenoceptors in isolated human right atrium

Summary The time course of the effects of isoprenaline (3 × 10^–7 mol/l) on contractile force and on the cyclic AMP level was studied in the electrically driven isolated muscle strip of the human right atrium. Isoprenaline produced a rise in cyclic AMP content (maximum increase after 60 s) preceding the increase in contractile force. The effects of isoprenaline on contractile force and on the intracellular level of cyclic AMP were enhanced in the presence of the phosphodiesterase inhibitor papaverine (10^–5 mol/l). On the other hand, the -adrenoceptor antagonist propranolol (10^–7 mol/l) suppressed isoprenaline-induced cyclic AMP increases, but reduced the increase in force of contraction by only 35%. In addition, both the ₁-selective antagonist bisoprolol (3 × 10^–9 × 10^–8 mol/l) and the ₂-selective antagonist ICI 118,551 (3 × 10^–9 × 10^–8 mol/l) inhibited the isoprenaline-induced cyclic AMP increase concentration-dependently; ICI 118,551 produced more pronounced inhibition than bisoprolol. It is concluded that cyclic AMP is involved in the positive inotropic action of isoprenaline evoked by ₁- and ₂-adrenoceptor stimulation in isolated human right atrium; however, an additional cyclic AMP independent mechanism cannot be ruled out. Send offprint requests to O.-E. Brodde at the above address     

Role of β2-adrenoceptor blockade and circulating adrenaline level for the pressor responses to β-adrenoceptor blocking drugs in rats

Summary The present experiments were designed to elucidate what mechanism(s) would be responsible for -adrenoceptor blocking drugs (-blockers)-induced pressor responses in rats. In urethane-anaesthetized rats, 6 -blockers at i.v. doses ranging from 0.3 to 300 g/kg evoked the pressor response in a dose-dependent manner. The relative potency in causing the pressor action was correlated not to their ₁-blocking activities (r=0.374, P>0.05) but to their ₂-blocking ones (r=0.856, P<0.05). In pithed or adrenalectomized rats with low levels of plasma catecholamines, however, propranolol failed to exert its sustained pressor action. Propranolol (300 g/kg i.v.) distinctly potentiated the pressor responses not to noradrenaline but to adrenaline and to electrical stimulation of the sympathetic outflow (E.S.) in pithed rats. On the contrary, there was not any potentiation of pressor response to E.S. in pithed, adrenalectomized rats treated with propranolol (300 g/kg i.v.). In rats treated with phenoxybenzamine (5 mg/kg i.v.), adrenaline was shown to have much more potent vasodilating action resulting from ₂-stimulation than noradrenaline, the dose difference for causing the diastolic blood pressure decrease by a 25 mm Hg being almost 80 times. In pithed rats, infusion of adrenaline at the rate of 0.02 g/min caused a significant increase in plasma adrenaline level from 0.02±0.01 to 0.45±0.048 ng/ml, being close to basal level obtained in urethane-anaesthetized rats. Under this situation, propranolol (1–100 g/kg i.v.) showed a distinct pressor response in a dose-dependent fashion as observed in adrenal intact rats anaesthetized with urethane. These results strongly suggest that the pressor responses to -blockers are largely due to both their inhibitory actions on the vasodilatory ₂-adrenoceptors and to their potentiating actions on the vascular response to circulating adrenaline.     

Comparison of the densities of 5-HT4 receptors, β1- and β2-adrenoceptors in human atrium: functional implications

We measured in human atrium the density of 5-HT₄ receptors, labelled with [¹²⁵I]-SB 207710 (1-butyl-4-piperidinyl) methyl 8-amino-7-iodo-1, (4-benzodioxan-5-carboxylate), and compared it with the density of ₁- and ₂-adrenoceptors, labelled with (–)-[¹²⁵I]-cyanopindolol. [¹²⁵I]-SB 207710 (5–1200 pmol/l) labelled a small population of saturable binding sites (B _max 4 fmol/mg protein) with a pK_D of 9.7 and with 5-HT₄ receptor characteristics, as assessed with competing ligands. The density of atrial binding sites with 5-HT₄ receptor characteristics was 10 and 5 times lower, respectively, than the density of ₁- and ₂-adrenoceptors. We suggest that the small 5-HT₄ receptor population may in part explain why the positive inotropic effects of 5-HT are smaller than those of catecholamines mediated through ₁- and ₂-adrenoceptors.     

5-Hydroxytryptamine causes rate-dependent arrhythmias through 5-HT4 receptors in human atrium: facilitation by chronic β-adrenoceptor blockade

We have investigated the ability of 5-hydroxytryptamine (5-HT) to elicit arrhythmic contractions in isolated human atrial strips as a function of pacing rate (0.1–2 Hz) using a method recently introduced by us (Kaumann and Sanders, this journal, 1993b) and examined the nature of the 5-HT receptors involved. Right atrial appendage tissue was obtained from 14 patients undergoing cardiac surgery. None of the patients had advanced heart failure. 5-HT (0.6–20 mol/l) induced arrhythmic contractions during pacing in 4/11 atrial strips from 3/4 patients who had not received blockers and in 21/27 atrial strips from 9/10 patients who had been chronically treated with blockers (primarily ₁-selective). The incidence of arrhythmic contractions evoked by 5-HT did not reach statistical significance in the atrial tissue from the non- blocked patients but was highly significant in the atrial tissue from the chronically blocked patients. The arrhythmic contractions usually occurred more frequently at low than at high pacing rates and were observed at the physiological frequency of 1 Hz in 1/4 atrial strips from 1/4 of the non- blocked patients and 6/11 strips from 5/10 of the blocked patients. The 5-HT-evoked arrhythmic contractions were observed during blockade of ₁-adrenoceptors, ₂-adrenoceptors and 5-HT₃ receptors, ruling out the participation of these receptors. The 5-HT-evoked arrhythmic contractions were totally inhibited within 30 min by the selective 5-HT₄ receptor antagonist SB 203186 ((1-piperidinyl)ethyl 1H-indole 3-carboxylate) 100 nmol/l whereas they persisted in time-matched controls. The blockade of 5-HT-evoked arrhythmic contractions by SB 203186 was surmounted by high concentrations (400–1800 mol/l) of 5-HT. Our results demonstrate that 5-HT elicits rate-dependent arrhythmic contractions in isolated human atrium through the 5-HT₄ receptor and that they are facilitated in atrial tissue from patients treated with blockers. Our results suggest that endogenous, platelet-derived 5-HT may cause atrial arrhythmias and that exogenous 5-HT₄ agonists/partial agonists may be arrhythmogenic. Correspondence to: A. J. Kaumann at the above address     

The pacemaker current If in single human atrial myocytes and the effect of β-adrenoceptor and A1-adenosine receptor stimulation

1. We used single human atrial myocytes to study I_f occurrence, properties and pharmacological modulation. Cells were obtained by chunk enzymatic digestion from samples of right atrial appendages of patients undergoing corrective cardiac surgery.
2. Patch-clamped cells in the whole-cell configuration were superfused with a modified Tyrode solution to reduce contamination by interfering currents and to amplify I_f. The average cell membrane capacitance was 85.06±2.41 pF (n=531). Data were consistent with the geometrical dimensions of the cells (length 94.2±1.89 μm, width 17.9±0.42 μm, n=126).
3. When hyperpolarizing to −120 mV from a holding potential of −40 mV, 252 of 306 tested cells (82%) expressed a hyperpolarization-activated inward current (I_f density =3.77±0.25 pA pF⁻¹); the current was considered to be present in a given cell if its density at −120 mV was larger than 0.5 pA pF⁻¹.
4. Current activation was sigmoidal and fitted a Boltzmann model; the average activation curve (n=25) showed a maximum current amplitude of 205.97±19.94 pA, corresponding to 3.87±0.63 pA pF⁻¹, voltage of half-maximal activation (V_1/2) at −86.68±2.19 mV and a slope of −11.39±0.69 mV. The reversal potential of I_f measured by tail-current analysis was −13.07±1.92 mV (n=6). The addition of CsCl (5 mM) fully and reversibly blocked the current.
5. In the presence of the β-adrenoceptor agonist isoprenaline (Iso, 1 μM), V_1/2 was significantly shifted toward less negative potentials by 6.06±1.96 mV (n=16, P=0.0039). The selective A₁-adenosine receptor agonist cyclopentyladenosine (CPA, 1 μM) caused a statistically significant shift of V_1/2 toward more negative potentials with respect to the control curve, both in the absence (−7.37±1.83 mV, P=0.0005, n=11) and in the presence of 1 μM Iso (−4.97±1.78, P=0.031, n=6).
6. These results demonstrate that a current with the properties of I_f described in cardiac primary and secondary pacemakers occurs in the majority of human atrial cells. While the pathophysiological relevance of I_f in human atrial tissue remains to be defined, our data clearly show that it is modulated through stimulation of β-adrenoceptors and A₁-adenosine receptors.

     

Both β1- and β2-adrenoceptors mediate catecholamine-evoked arrhythmias in isolated human right atrium

Summary The involvement of ₁- and ₂-adrenoceptors in catecholamine-evoked arrhythmias was investigated in isolated human right atrial appendages obtained from 22 patients chronically treated with blockers (usually ₁-selective) and 9 patients not treated with blockers. A simple experimental model that assesses the incidence of arrhythmic contractions as a function of heart rate (pacing) is introduced. ₁-adrenoceptors were activated by (–)-noradrenaline during ₂-adrenoceptor blockade with 50 nmol/l ICI 118551. ₂-adrenoceptors were activated by (–)-adrenaline during ₁-adrenoceptor blockade with 300 nmol/l CGP 20712A. Both (–)noradrenaline and (–)-adrenaline caused arrhythmic contractions whose incidence was greater at low than at high pacing rates. CGP 20712A (300 nmol/l) blocked the (–)-noradrenaline-evoked contractions in 1/1 atrial strip from 1/1 patient not treated with a blocker and 17/17 atrial strips from 15/15 patients chronically treated with blockers. ICI 118551 (50 nmol/l) blocked the (–)-adrenaline-evoked contractions in 3/4 atrial strips from 3/4 patients not treated with blockers and 17/20 atrial strips from 15/18 patients chronically treated with blockers. The incidence of arrhythmic contractions evoked by both (–)-noradrenaline and (–)-adrenaline was higher in chronically blocked patients than in non blocked patients. We conclude that both ₁- and ₂-adrenoceptors mediate atrial arrhythmias and that the generation of these arrhythmias is facilitated by chronic ₁-adrenoceptor blockade. Correspondence to: A. J. Kaumann at the Clinical Pharmacology Unit, University of Cambridge, as above     

Diuretic-related hypokalaemia: the role of diuretics,potassium supplements,glucocorticoids and β2-adrenoceptor agonists

All 5,047 consecutive inpatients admitted to the Internal Medicine Division of a teaching hospital (Zieglerspital, Berne) between 1982 and 1985 were registered in accordance with the CHDM (Comprehensive Hospital Drug Monitoring) questionnaire of adverse drug reactions (ADRs). Of them, 2,439 were treated with at least one potassium losing diuretic. The hospital records of the patients were reviewed with particular regard to serum potassium levels, and on the basis of this evaluation, the patients were assigned to four different diuretic treatment groups, and the incidence of hypokalaemia related to diuretic treatment was estimated. The overall rate of occurrence of hypokalaemia was 21.1% at a serum potassium level <3.5 mmol·1^–1, and 3.8% <3.0 mmol·1^–1. Hypokalaemia of less than 3.5 mml·1^–1 developed 24.9% (217/870) of patients treated with potassium losing diuretics alone; in 19.7% (101/513) treated with potassium losing diuretics in conjunction with potassium substitution, in 15.1% (66/438) treated with a combination of diuretics (potassium losing with potassium sparing), and in 20.0% (12/60) treated with combined diuretics and potassium substitution. Only the differences between the first and the two subsequent groups were statistically significant. The overall incidence of hypokalaemia below 3.0+mmol·1^–1 was significantly lower in the patients on combined diuretics without potassium substitution than in the patients on potassium losing diuretics with potassium substitution.Oral or parenteral administration of glucocorticoids (prednisone 5 to 2,000 mg/d) was a significant risk factor for hypokalaemic events. ₂-Adrenoceptor agonists had not effect. The patient's age, sex, renal function and numbers of drugs received were evaluated in a multivariate analysis, in order to take into account their influence on the risk of developing hypokalaemia. The number of drugs above 12 (and, less importantly, female sex) was the main risk factor for this ADR.The comparison between hypokalaemia and hyperkalaemia in this group of inpatients showed the significance of reduced renal function in the occurrence of hyperkalaemia.     

Agonist effects of zinterol at the mouse and human β3-adrenoceptor

The present study investigates the action of zinterol at β₃-adrenoceptors. We used mouse primary brown adipocytes and Chinese hamster ovary (CHO-K1) cells expressing the mouse or human β₃-adrenoceptor. Zinterol was a full agonist at increasing cyclic AMP levels in primary brown adipocytes (which express β₁- and β₃-adrenoceptors but not β₂-adrenoceptors), and this effect was almost totally abolished in adipocytes derived from β₃-adrenoceptor knock-out (KO) mice. Zinterol was also a full agonist at increasing another biological end-point, glucose uptake in brown adipocytes. This effect was reduced in adipocytes derived from β₃-adrenoceptor KO mice, with the remaining response sensitive to β₁-adrenoceptor antagonism. To determine whether the effect of zinterol on β₃-adrenoceptors in primary brown adipocytes can be replicated in a recombinant system, we used CHO-K1 cells expressing the mouse or human β₃-adrenoceptor. Zinterol was a full agonist at mouse and human receptors with respect to increasing cyclic AMP levels, with pEC₅₀ values similar to that of the selective β₃-adrenoceptor agonist (R, R)-5-[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]-amino]-propyl]1,3-benzodioxole-2,2-dicarboxylate (CL316243) at the mouse receptor. At the human receptor, zinterol was more potent at increasing cyclic AMP levels than CL316243. In cytosensor microphysiometer studies, zinterol was a full agonist for increases in extracellular acidification rates at the mouse and human β₃-adrenoceptor. Therefore, we have shown that zinterol is a potent, high-efficacy β₃-adrenoceptor agonist at the endogenous mouse β₃-adrenoceptor in primary brown adipocytes and at the cloned mouse and human β₃-adrenoceptor expressed in CHO-K1 cells. Zinterol is therefore one of few β-adrenoceptor agonists with high potency and efficacy at the human β₃-adrenoceptor.     

Regional distribution of β1- and β2-adrenoceptors in the failing and nonfailing human heart

Summary Total -adrenoceptor density and ₁- and ₂-subtype distribution in right and left atria and in different ventricular regions from 14 failing and seven nonfailing human hearts have been compared. End-stage heart failure was due to idiopathic dilated cardiomyopathy (n=8) or ischaemic cardiomyopathy (n=6).In nonfailing hearts the total -adrenoceptor density was similar in the right and left atria and in all the ventricular regions studied (about 70 to 80 fmol/mg protein). The ₁:₂-adrenoceptor ratio in both nonfailing atria was similar (about 70:30%) and was significantly smaller than in the different regions of both ventricles (about 80:20%). The ₁-subtype density was similar in nonfailing atria and ventricles (about 55 fmol/mg protein). The ₂-subtype density was significantly higher in the right and left atrium (about 25 fmol/mg protein) than in both ventricles (about 15 fmol/mg protein).In patients with end-stage heart failure due to idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy the total -adrenoceptor density was reduced by 50–60% in all regions. On the other hand, the ₁- and ₂-subtype distribution differed with the cause of heart failure. In patients with idiopathic dilated cardiomyopathy, the ₁-adrenoceptor density was lower in all regions, but the ₂-adrenoceptor density was not significantly reduced. In patients with ischaemic cardiomyopathy both ₁- and ₂-adrenoceptors were reduced in all regions.It is concluded that downregulation of -adrenoceptors in patients with end-stage idiopathic dilated cardiomyopathy or ischaemic cardiomyopathy occurs uniformly throughout the heart. The results support the hypothesis that changes in -adrenoceptor subtypes may be related to the cause of heart failure.     

The effects of chronic dosing on the β1 and β2-adrenoceptor antagonism of betaxolol and atenolol

Summary Six normal subjects were given once daily treatment for 15 days with placebo (PL), betaxolol 10 mg (B10), 40 mg (B40); atenolol 100 mg (A100); and nadolol 40 mg (N40). Measurements of ₁-adrenoceptorblockade (reduction of exercise heart rate) and of ₂-adrenoceptor-blockade (attenuation of isoprenaline induced finger tremor) were made after the first, eighth and fifteenth doses of each drug.Plasma concentrations showed dose related increases between 10 mg and 40 mg doses of betaxolol, and there was significant drug accumulation at steady state compared with after single dosing. The reduction in exercise heart rate (EHR) with B10 was less in comparison with all other treatments.There were no significant differences in effects between single and chronic-dosing for any of the treatments (% reduction EHR compared with placebo, on days 1 and 15): B10 (18.2, 19.0), B40 (28.6, 26.5); A100 (22.7, 23.1); N40 (26.6, 23.8). Dose-ratios for attenuation of isoprenaline-induced finger tremor (IT₁₀₀) were significantly greater with B40 compared with B10 or A100 (no dose-ratio for finger tremor could be calculated for N40).There were no differences between single and chronic-dosing (IT₁₀₀ dose-ratios on days 1 and 15): B10 (3.0, 2.5), B40 (4.4, 5.3); A100 (3.0, 3.0). The attenuation of isoprenaline-induced chronotropic response (IH₂₅) by N40 was significantly greater in comparison with all other treatments. IH₂₅ dose-ratios (on days 1 and 15) were as follows: B10 (2.8, 3.6), B40 (5.1, 5.8); A100 (3.6, 3.6); N40 (19.0, 17.4).Thus, despite drug accumulation after chronic-dosing, there was no evidence of any increase in either ₁ or ₂-adrenoceptor antagonism at steady-state in comparison with after single-dosing. The apparent dissociation between plasma concentration and -adrenoceptor antagonism after chronic-dosing my be a consequence of -adrenoceptor up-regulation, resulting in partial attenuation of -blockade.     

Influence of calcium entry blockade on α 1 - and α 2-adrenoceptor mediated vasoconstriction in the forearm of hypertensive patients

Summary The influence of treatment with the calcium entry blockers PY 108-068 (PY) and PN 200-110 (PN) on ₁- and ₂-adrenoceptor mediated vasoconstriction has been investigated in the forearms of hypertensive patients. Changes in forearm vascular resistance (FVR) in response to the intra-arterial infusion of drugs were determined at the end of a placebo period and after 2–4 weeks of treatment with PY or PN. The drugs used were the selective agonists methoxamine (₁) and B-HT 933 (₂). During placebo, basal FVR was dose-dependently increased by methoxamine and B-HT 933. Basal blood pressure was lowered during PN but not during PY. Treatment with the calcium entry blockers did not influence the effect of methoxamine, but the vasoconstriction induced by B-HT933 was attenuated by both of the calcium entry blockers. These results confirm the findings in animal studies that calcium entry blockers preferentially inhibit the ₂-adrenoceptor mediated vasoconstriction induced by selective agonists.     

Comparison of three radioligands for the labelling of human β-adrenoceptor subtypes

We have compared the ability of three radioligands, [¹²⁵I]-cyanopindolol, [³H]-CGP 12,177 and [³H]-dihydroalprenolol, to label the three human β-adrenoceptor subtypes. Saturation and competition binding experiments were performed using membrane preparations from Chinese hamster ovary cells stably transfected with the three subtypes. While [³H]-CGP 12,177 had very similar affinity for β₁- and β₂-adrenoceptors (about 40 pM), [¹²⁵I]-cyanopindolol and [³H]-dihydroalprenolol had 4- to 6-fold higher affinity for β₂- as compared to β₁-adrenoceptors (10 vs 45 and 187 vs 1,021 pM, respectively). The affinity of [¹²⁵I]-cyanopindolol at β₃-adrenoceptors was considerably lower (440 pM) than at the other two subtypes. The β₃-adrenoceptor affinity of [³H]-CGP 12,177 and [³H]-dihydroalprenolol was so low that it could not be estimated within the tested range of radioligand concentrations (up to 4,000 pM and 30,000 pM for [³H]-CGP 12,177 and [³H]-dihydroalprenolol, respectively). We conclude that all three radioligands are ill-suited to label β₃-adrenoceptors, particularly in preparations co-expressing multiple subtypes. In the absence of alternatives, [¹²⁵I]-cyanopindolol appears the least unsuitable to label β₃-adrenoceptors. There is a need for high-affinity radioligands which are either selective for β₃-adrenoceptors or reasonably non-selective among all three β-adrenoceptor subtypes.     

Muscarinic receptor interaction with full and partial β-adrenoceptor agonists in the rat colon strip

Summary -Adrenoceptor agonists inhibit contractile activity in isolated colon strips. In order to demonstrate that -adrenoceptors are located at different functional levels within the colon wall, increasing concentrations of muscarinic agonists were used to interact functionally with the -adrenoceptor-mediated inhibition of spontaneous colon activity. The effects of the full agonists isoprenaline and terbutaline and of the partial agonist prenalterol were functionally antagonized by carbachol (0.03 and 0.3 mol/l) and bethanechol (1,3 and 30 ol/l). This functional antagonism was parallelled by an increase in baseline tension and spontaneous contractile activity of the isolated colon strip. At lower concentrations of carbachol (0.003 and 0.01 ol/l) or bethanechol (0.03 and 0.31 mol/l) no effect on the contractile status of the smooth muscle or on the pD₂-values of the full agonists was seen. However, at these lower concentrations of muscarinic agonists a marked decrease in the maximal inhibitory response to the partial -adrenoceptor agonist prenalterol was demonstrated. The inhibitory response to prenalterol showed a biphasic concentration-response curve. The muscarinic antagonist atropine produced an increase in the maximal response of the high potency component of the concentration-response curve for prenalterol and an increase in the sensitivity to isoprenaline. These results demonstrate the presence of a high cholinergic tone in the colon preparation of a magnitude that clearly reduces the sensitivity to -adrenoceptor agonists. The different responses to full and partial -adrenoceptor agonists in the presence of increasing concentrations of muscarinic agonists may indicate that -adrenoceptors are located on two different functional units within the colon wall.     

Chronic treatment in vivo with β-adrenoceptor agonists induces dysfunction of airway β(2) -adrenoceptors and exacerbates lung inflammation in mice

BACKGROUND AND PURPOSE

Inhalation of a β-adrenoceptor agonist (β-agonist) is first-line asthma therapy, used for both prophylaxis against, and acute relief of, bronchoconstriction. However, repeated clinical use of β-agonists leads to impaired bronchoprotection and, in some cases, adverse patient outcomes. Mechanisms underlying this β₂-adrenoceptor dysfunction are not well understood, due largely to the lack of a comprehensive animal model and the uncertainty as to whether or not bronchorelaxation in mice is mediated by β₂-adrenoceptors. Thus, we aimed to develop a mouse model that demonstrated functional β-agonist-induced β₂-adrenoceptor desensitization in the context of allergic inflammatory airway disease.

EXPERIMENTAL APPROACH

We combined chronic allergen exposure with repeated β-agonist inhalation in allergen-treated BALB/C mice and examined the contribution of β₂-adrenoceptors to albuterol-induced bronchoprotection using FVB/NJ mice with genetic deletion of β₂-adrenoceptors (KO). Associated inflammatory changes – cytokines (ELISA), cells in bronchoalevolar lavage and airway remodelling (histology) and β₂-adrenoceptor density (radioligand binding) – were also measured.

KEY RESULTS

β₂-Adrenoceptors mediated albuterol-induced bronchoprotection in mice. Chronic treatment with albuterol induced loss of bronchoprotection, associated with exacerbation of the inflammatory components of the asthma phenotype.

CONCLUSIONS AND IMPLICATIONS

This animal model reproduced salient features of human asthma and linked loss of bronchoprotection with airway pathobiology. Accordingly, the model offers an advanced tool for understanding the mechanisms of the effects of chronic β- agonist treatment on β-adrenoceptor function in asthma. Such information may guide the clinical use of β-agonists and provide insight into development of novel β-adrenoceptor ligands for the treatment of asthma.