Arrhythmias and alpha 1-adrenoceptor binding characteristics of the guinea-pig perfused heart during ischaemia and reperfusion.

Guinea-pig isolated hearts were perfused by the Langendorff method. Low flow (10%) global ischaemia for 30 min induced ventricular tachycardia (VT) and fibrillation (VF) in 87.5 and 37.5% respectively of the hearts. The onset times for VT and VF were 15.7 +/- 1.0 and 23.5 +/- 1.6 min respectively. On reperfusion the incidences of VT and VF were 81.3 and 75.0% and occurred after 16.0 +/- 1.5 and 35.0 +/- 4.9 s of reperfusion. In those hearts exhibiting arrhythmias, [3H]-prazosin binding to alpha 1-adrenoceptors of ventricular membrane fractions was measured and compared with normally perfused time-matched controls. There was no significant change in dissociation constant (KD) or density Bmax, of binding throughout the control perfusion period. Similarly, the KD (0.38 +/- 0.06 nM) and Bmax (15.5 +/- 1.4 fmol/mg protein) values obtained during ischaemia did not differ significantly from the corresponding control values (0.48 +/- 0.05 nM and 15.8 +/- 1.5 fmol/mg protein). Also, at 1 min of reperfusion the KD (0.42 +/- 0.04 nM) and Bmax (19.3 +/- 2.0 fmol/mg protein) values were not significantly different from the time-matched controls (0.38 +/- 0.09 nM and 20.1 +/- 2.6 fmol/mg protein). The same result was obtained if the crude membrane fraction pelleted by the initial slow spin was used. Thus, although cardiac arrhythmias are induced by ischaemia and reperfusion of the guinea-pig isolated perfused heart and previous studies have shown these to be susceptible to alpha-adrenoceptor blockade, they are not accompanied by an increase in alpha 1-adrenoceptor affinity or density.

Whether phenylephrine exerts inotropic effects through alpha- or beta-adrenoceptors depends upon the relative receptor populations.

Phenylephrine produced concentration-related positive inotropic responses in isolated left atria and papillary muscles of guinea-pigs and rats. In rat tissues, these responses were unaffected by propranolol but antagonized by prazosin and therefore mediated via alpha 1-adrenoceptors. The alpha 1-adrenoceptor agonist methoxamine also exerted positive inotropic effects in these rat tissues. The maximum alpha-adrenoceptor-mediated effect of methoxamine (relative to the isoprenaline maximum) was greater than that of phenylephrine in left atria (in the presence of propranolol), whereas in papillary muscles phenylephrine exerted the greater maximum. In guinea-pig papillary muscles, the response to phenylephrine was unaffected by prazosin but was antagonized by propranolol and therefore caused by stimulation of beta-adrenoceptors. Methoxamine had no effect in guinea-pig papillary muscles. Guinea-pig left atria produced biphasic concentration-response curves for phenylephrine, the lower portion being antagonized by phentolamine and was therefore alpha-adrenoceptor-mediated, while the upper portion was antagonized by propranolol and therefore beta-adrenoceptor-mediated. Methoxamine exerted a small inotropic response, the maximum of which was similar to that of the first component of the phenylephrine response. Phenylephrine was a partial agonist for the cardiac beta-adrenoceptor. The density of rat ventricular alpha-adrenoceptors was 4 times greater than beta-adrenoceptor density, as measured by [3H]-prazosin and [3H]-dihydroalprenolol binding. This explains why the responses of rat papillary muscles were alpha-adrenoceptor-mediated. In contrast, the density of beta-adrenoceptor binding sites in guinea-pig ventricles was 6 times greater than the alpha-adrenoceptor density. This explains why the phenylephrine responses were beta-adrenoceptor-mediated in guinea-pig papillary muscles. In the left atria of guinea-pigs, which displayed both alpha- and beta-adrenoceptor-mediated responses, the densities of alpha- and beta-adrenoceptor binding sites were similar. Thus, phenylephrine exerts positive inotropic effects through alpha- or beta-adrenoceptors depending upon their relative densities.

Cardiac alpha- and beta-adrenoceptor sensitivity and binding characteristics after chronic reserpine pretreatment.

Cardiac alpha- and beta-adrenoceptor sensitivities were examined after chronic pretreatment of rats with reserpine. Increases in sensitivity would indicate that the receptor is under the influence of the sympathetic innervation, removal by catecholamine depletion with reserpine of the tonic effect of neurotransmitter release would permit receptor upregulation. The positive inotropic responses of paced left atria and papillary muscles and the positive chronotropic responses of spontaneously beating right atria were recorded. A concentration-response curve to isoprenaline (beta-adrenoceptor-mediated) was followed, in the presence of beta-blockade, by one to methoxamine (alpha-adrenoceptor-mediated). Methoxamine exerted positive inotropy of left atria and papillary muscles, the maxima being 43.2 +/- 2.7 and 26.8 +/- 4.4% of the isoprenaline maxima. A small positive chronotropy (16.5 +/- 5.6% maximum) of right atria occurred. After pretreatment with reserpine (1.0 mg kg-1 i.p. daily) for 7 days, the three preparations displayed supersensitivity to isoprenaline, revealed as a significant displacement (P less than 0.05) of the concentration-response curves to the left of those for control rats. Reserpine pretreatment, however, had no effect on the sensitivity to methoxamine. The increase in beta-adrenoceptor sensitivity to isoprenaline after reserpine pretreatment was accompanied by a significant 41.3% increase (P less than 0.05) in the number of [3H]-dihydroalprenolol [( 3H]-DHA) binding sites (Bmax) in ventricular membranes, although the dissociation constant (KD) was unaffected. There were more alpha-adrenoceptor [3H]-prazosin binding sites in ventricular than atrial membranes. However, there was no difference in KD or Bmax between reserpine-pretreated and control tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Examination of cardiac alpha-adrenoceptors from pharmacological responses and radioligand binding. Comparison of rat and guinea pig tissues.

The object of this study was to determine suitable experimental conditions for the pharmacological evaluation of cardiac alpha-adrenoceptors. Atrial and ventricular preparations of the guinea pig and rat were employed, and the alpha-adrenoceptor responsiveness was compared with the binding of the alpha-adrenoceptor radioligand [3H]prazosin in membranes prepared from these cardiac regions. The experimental variables examined were the pacing frequency, bath temperature, choice of agonist, and cardiac region. In guinea pig atria the optimum alpha-adrenoceptor-mediated positive inotropic response to phenylephrine was at 32 degrees C and a pacing frequency of 1 Hz. A comparison of phenylephrine with methoxamine showed that the former yielded biphasic concentration-response curves in guinea pig left atria; the lower portion was alpha-adrenoceptor mediated and the upper, more substantial portion, was beta mediated. Methoxamine produced monophasic curves due entirely to alpha-adrenoceptor stimulation and was therefore used for comparisons between rat and guinea pig tissues. Of the guinea pig tissues, only the left atrium produced positive inotropic responses. Negative chronotropy was obtained with right atria and negative inotropy with ventricular strips and papillary muscles. The rat tissues all responded with positive responses, the largest maximum being in the left atrium. Binding data showed a larger number of alpha-adrenoceptors in the rat tissues, of which the ventricles had the greatest number. The lack of response of guinea pig ventricular tissues was therefore reflected in the low binding. From this study, the most appropriate model for characterizing cardiac alpha-adrenoceptors is therefore the rat left atria at 32 degrees C and paced at 1 Hz with methoxamine as the agonist.

Cardiac post-junctional alpha 1- and beta-adrenoceptors: effects of chronic chemical sympathectomy with 6-hydroxydopamine.

Alpha 1- and beta-adrenoceptor responsiveness and binding have been examined in cardiac tissues removed from guinea-pigs pretreated with 6-hydroxydopamine (6-OHDA) for 3 weeks. Results were compared with control tissues from sham-injected animals. Chemical sympathectomy with 6-OHDA resulted in an increase in the sensitivity of postjunctional beta-adrenoceptor-mediated responses to isoprenaline. No such increase was observed for the alpha 1-adrenoceptor-mediated responses to methoxamine. The increase in beta-adrenergic responsiveness was accompanied by a significant (P less than 0.05) 52% increase in the number of [3H]-dihydroalprenolol binding sites with no change in binding affinity. [3H]-Prazosin binding was not affected by pretreatment with 6-OHDA. The results suggest that cardiac beta- but not alpha 1-adrenergic responsiveness is regulated by the sympathetic innervation.

Calculated and actual changes in beta-adrenoceptor number associated with increases in rat and guinea-pig cardiac sensitivity.

Chronic catecholamine depletion induced by reserpine pretreatment of rats, or 6-hydroxydopamine pretreatment of guinea-pigs, resulted in an enhanced sensitivity of isolated papillary muscles to isoprenaline. This hypersensitivity was accompanied by 1.41-(rats) and 1.52-fold (guinea-pigs) increases in the number of [3H]dihydroalprenolol binding sites, without changes in binding affinity. An equation was derived for calculation of increases in receptor number. Application of this showed that substantially greater increases in receptor number were required (2.32- to 4.04-fold) to account for the degree of supersensitivity observed.

A physiological basis for subclassifying beta-adrenoceptors examined by chemical sympathectomy of guinea-pigs.

Chemical sympathectomy of guinea-pigs was induced by chronic pretreatment with 6-hydroxydopamine over a 20 day period. Control animals were sham injected with vehicle at the same times. Isolated tissues were removed from the animals and beta-adrenoceptor sensitivity assessed from cumulative concentration-response curves for isoprenaline, followed after wash-out by a partial agonist (salbutamol, ritodrine or prenalterol). The following responses were measured: increases in force and rate of contraction of left and right atria respectively, inhibition of carbachol-induced ileal contractions, relaxation of intrinsic tone of lung strips and tracheal spirals, inhibition of contractions of vas deferens and soleus muscle induced by field stimulation. Left and right atria and ileum from 6-hydroxydopamine-pretreated guinea-pigs exhibited supersensitivity to beta-adrenoceptor stimulation. This was measured as a leftwards shift of the concentration-response curve for isoprenaline and as an elevation of the partial agonist maximum response (relative to isoprenaline), when compared with tissues from sham-injected controls. The supersensitivity was assumed to be due to the loss of endogenous neurotransmitter release by chemical sympathectomy and specific for the beta-adrenoceptor. In contrast, lung strips, vas deferens and soleus muscle were not supersensitive. The responses of these tissues are thought to be mediated via beta 2-adrenoceptors whereas cardiac and ileal responses are beta 1-adrenoceptor mediated. The latter receptor subtype would therefore appear to be under the influence of sympathetic innervation, but since no supersensitivity occurred at beta 2-adrenoceptors these were presumed to be non-innervated but stimulated by circulating adrenaline. These results obtained by use of chemical sympathectomy with 6-hydroxydopamine support the contention that the physiological basis of beta-adrenoceptor subclassification is that the beta 1-subtype are innervated whereas the beta 2-subtype are non-innervated.

The use of forskolin to investigate the site of cardiac beta-adrenoreceptor supersensitivity.

The positive inotropic responses of left atria and papillary muscles and the positive chronotropic responses of right atria of guinea-pigs to isoprenaline and forskolin were examined. An increase in sensitivity of the three preparations to isoprenaline was observed by lowering the bath temperature from 38 to 30 degrees C as demonstrated by a leftwards shift of the concentration-response curves. A similar degree of supersensitivity was observed for forskolin. Since forskolin is reputed to stimulate adenylate cyclase directly, whereas isoprenaline stimulates via the regulatory nucleotide Ns protein, this would suggest a common site for the supersensitivity at adenylate cyclase. However, the possibility that forskolin also stimulates via the Ns protein in producing cardiac stimulation and that this is the site of hypothermia-induced supersensitivity is discussed. Supersensitivity to isoprenaline was also observed in left atria and papillary muscles from guinea-pigs chronically pretreated with reserpine for 3 days (5 mg/kg at 72 h, 3 mg/kg at 48 and 24 h) or 7 days (0.1 mg/kg daily). In the same tissues, there was no change in the sensitivity to forskolin. The site of the supersensitivity can therefore be concluded to occur before the level of adenylate cyclase activation either directly or via the regulatory Ns protein; possibly at the beta-adrenoreceptor itself.

Cardiac postjunctional supersensitivity to beta-agonists after chronic chemical sympathectomy with 6-hydroxydopamine.

The sensitivity to sympathomimetic amines of isolated atria removed from sham-injected and 6-hydroxydopamine-treated (6-OHDA) guinea-pigs was examined in the presence of an extraneuronal uptake blocker and an alpha-adrenoceptor antagonist. Three weeks of pretreatment with 6-OHDA resulted in leftwards shifts of the dose-response curves for the positive chronotropic and inotropic responses of right and left atria to isoprenaline. The responses to the partial agonist salbutamol were also potentiated after 6-OHDA pretreatment, revealed as an increase in the maximum response relative to isoprenaline. The supersensitivity was post-synaptic in origin and independent of changes in disposition or metabolism, since it was observed with agonists immune to neuronal uptake and O-methylation, and in the presence of extraneuronal uptake inhibition by metanephrine. It was also specific for the beta-adrenoceptor, no supersensitivity to histamine being found. In the right atria, the supersensitivity was partially masked by an opposing depressant effect after 6-OHDA pretreatment which was observed with histamine. Dissociation constants (KA) for the left atrial inotropic responses to orciprenaline were determined by use of the antagonist Ro 03-7894. Atria from 6-OHDA-pretreated animals were supersensitive to orciprenaline, but the KA value did not differ from that after sham injection. It could therefore be concluded that the increase in sensitivity was not due to an increase in affinity for the beta-adrenoceptor.

Temperature dependence of beta 1-adrenoceptor-mediated responses examined by use of partial agonists.

The inotropic and chronotropic responses of guinea pig atria, and the relaxation responses of guinea pig intestine, trachea, lung, uterus and vas deferens to catecholamines have been examined at bath temperatures of 38 degrees C and 30 degrees C. Hypothermia resulted in a supersensitivity of cardiac tissues with a decrease in isoprenaline EC50 and an increase in the maximum response to the partial agonist, salbutamol. Ileum responses to isoprenaline were potentiated at 30 degrees C but no partial agonist could be found on this tissue. Responses of the lung and vas deferens to partial agonists were not affected by temperature, while uterine responses were inhibited by hypothermia. The trachea was supersensitive to isoprenaline at 30 degrees C, however this was not due to a change in beta-adrenoceptor sensitivity but an inhibition of COMT. Partial agonist responses of trachea were similar at both temperatures. beta-Adrenoceptor supersensitivity was therefore observed only where responses are mediated primarily by beta 1-adrenoceptors and supports the concept that beta 1- but not beta 2-adrenoceptors exhibit hypothermia-induced supersensitivity.

A fundamental temperature-dependent difference between beta-adrenoceptor agonists and antagonists.

Positive inotropic and chronotropic responses of guinea-pig isolated left and right atria to sympathomimetic amines were examined at bath temperatures of 38, 30 or 25 degrees C. The concentration-response curves to isoproterenol and orciprenaline were displaced to the left by cooling, indicating hypothermia-induced supersensitivity. The affinities of isoproterenol and orciprenaline were determined as their dissociation constants (pKA) from antagonism of their responses by either the functional antagonist carbachol or Ro 03-7894 which is reported to be an irreversible beta-adrenoceptor antagonist. By both methods of calculation, the affinities of isoproterenol and orciprenaline for the beta-adrenoceptors mediating inotropic and chronotropic responses were increased by lowering the temperature. In contrast, the affinity of practolol, measured as the pA2 for competitive antagonism of the isoproterenol- and orciprenaline-induced inotropic and chronotropic responses, did not increase with cooling. Thus hypothermia-induced supersensitivity is associated with an increase in agonist affinity only, which indicates a fundamental temperature-dependent difference between agonist and antagonist interactions with the beta-adrenoceptor.

Ex vivo examination of beta-adrenoceptor characteristics after propranolol withdrawal.

Propranolol (3.6 mg kg-1 day-1) was administered to guinea pigs for 14 days via subcutaneously implanted "Alzet" osmotic minipumps. Blockade of cardiac and vascular beta-adrenoceptors was confirmed by a depression of heart rate and blood pressure responses to isoprenaline in anaesthetized animals. After 14 days of propranolol treatment, minipumps were removed and 48 h later beta-adrenoceptor sensitivity was determined in cardiac and lung tissues. Left atrial inotropic responses were unaltered following propranolol withdrawal. However, right atrial chronotropic responses to sympathomimetic amines were supersensitive when compared with tissues from animals implanted with empty minipumps. Relaxation responses of lung strip preparations to beta-adrenoceptor agonists after withdrawal of propranolol were similar to controls. (3H)Dihydroalprenolol binding to ventricular and lung tissue indicated that there was no change in either the dissociation constant (KD) or the maximum number of (3H)DHA binding sites (Bm) in these tissues. These results suggest that following withdrawal from beta-adrenoceptor antagonist treatment there is a selective increase in the chronotropic sensitivity of the heart to sympathomimetic amines.