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     

Preponderance of β- over α-adrenoceptors in mediating the positive inotropic effect of phenylephrine in the ferret ventricular myocardium

Summary [³H]prazosin bound to the membrane fraction derived from the ferret ventricular muscle with high affinity in a saturable manner (K _d = 0.25 nmol/l and B _max = 27 fmol/mg protein in the right ventricle). [³H]CGP-12177, a -adrenoceptor ligand, bound to the membrane fraction with a _{K d} value of 0.29 nmol/l and a B _max of 42 fmol/mg protein. In the isolated ferret papillary muscle driven at 1 Hz at 37°C, phenylephrine elicited a concentration-dependent positive intropic effect. The maximal effect of phenylephrine was comparable to that of isoprenaline. Prazosin (0.3 ol/l) shifted the concentration-response curve for phenylephrine slightly but significantly to the right, the maximal response being unaffected. In contrast, bupranolol (0.3 gmol/l) shifted the curve for phenylephrine markedly downwards: the maximal response was depressed significantly to 40% and the curve became less steep. In the presence of prazosin and bupranolol the curve was shifted to the right, being essentially parallel to the control curve. These results indicate that in the ferret ventricular myocardium both - and -adrenoceptors mediate the positive inotropic effect of phenylephrine. The extent of contribution of the two classes of adrenoceptor is quite different from that in other mammalian species. In the ferret heart, -adrenoceptors predominate over -adrenoceptors in mediating the positive inotropic effect of phenylephrine, although the number of -adrenoceptors is not especially high when compared with other species. Send offprint requests to M. Endoh at the above address     

Cilostamide potentiates more the positive inotropic effects of (−)-adrenaline through β2-adrenoceptors than the effects of (−)-noradrenaline through β1-adrenoceptors in human atrial myocardium

Activation of both β₁- and β₂-adrenoceptors increases the contractility of human atrial myocardium through cyclic AMP-dependent pathways. Cyclic AMP is hydrolised by phosphodiesterases, but little is known about which isoenzymes catalyse inotropically relevant cyclic AMP accumulated upon stimulation of β-adrenoceptor subtypes. We have compared the positive inotropic effects of (−)-noradrenaline and (−)-adrenaline, mediated through β₁- and β₂-adrenoceptors, respectively, in the absence and presence of the PDE3 inhibitor cilostamide (300 nM) or PDE4 inhibitor rolipram (1 μM) on human atrial trabeculae from non-failing hearts. Cilostamide, but not rolipram, potentiated the effects of both (−)-noradrenaline and (−)-adrenaline. Cilostamide increased the −logEC₅₀M of (−)-adrenaline more than of (−)-noradrenaline (P < 0.05), regardless of whether or not the patients had been chronically treated with β-blockers. The results are consistent with a greater PDE3-catalysed hydrolysis of inotropically relevant cyclic AMP produced through β₂-adrenoceptors than β₁-adrenoceptors in human atrium.     

Chronic β1-adrenoceptor blockade sensitises the H1 and H2 receptor systems in human atrium: role of cyclic nucleotides

We have reported that chronic treatment of patients with ₁-adrenoceptor blockers sensitises isolated atrial preparations to adrenaline, noradrenaline and 5-HT. We have now examined the effect of chronic treatment with -adrenoceptor blockers on responses to histamine of human right atrial appendages. We compared the effects of histamine on contractile force, cyclic AMP and cyclic GMP levels as well as cyclic AMP-dependent protein kinase (PKA) activity and explored the arrhythmogenic effects of histamine in preparations obtained from patients chronically treated or not treated with -adrenoceptor blockers.Histamine increased contractile force in paced preparations; the effects were blocked by the H₂ receptor antagonist famotidine (0.1–30 mol/1). The maximum inotropic response to histamine was doubled and the inotropic potency of histamine 0.4 log units greater in atria from -adrenoceptor blocker-treated compared to non -adrenoceptor blocker-treated patients. Histamine elicited frequency-dependent arrhythmias that were blocked by famotidine (30 mol/1) but not by mepyramine (1 mol/1). The incidence of arrhythmias was higher in atria from -adrenoceptor blocker-treated compared to untreated patients. Histamine increased both cyclic AMP and cyclic GMP levels, as well as PKA activity, significantly more in atria from -adrenoceptor blocker-treated compared to those from untreated patients. Mepyramine 1 mol/l prevented the histamine-evoked increase in cyclic GMP levels, reduced the inotropic hyperresponsiveness and abolished the hyperresponsiveness in cyclic AMP levels and PKA activity observed in patients chronically treated with blockers. Sodium nitroprusside 10 mol/l caused smaller increases of cyclic GMP levels than histamine and restored the contracile force depressed by mepyramine to its original level in atria from -adrenoceptor blocker-treated patients.The evidence is consistent with sensitisation of both the histamine H₁ and histamine H₂ receptor systems by chronic ₁-adrenoceptor blockade. H₁ receptor-mediated increases in cyclic GMP, enhanced through an as yet unknown mechanism by chronic ₁-adrenoceptor blockade, may inhibit phosphodiesterase 3 activity, thereby causing enhanced histamine-evoked increases in cyclic AMP levels and PKA activity, and accounting partially for the increased inotropic responses to histamine through H₂ receptors.     

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

Summary 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 (K _D ) 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 K _D fall in the range 0.5–1.4 nmol/l. The stereo-selectivity ratio (K _D (+)-isomer/K _D (–)-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.     

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.     

α-adrenoceptors mediating the positive inotropic effect of phenylephrine in the right ventricular muscle of the monkey (Macaca fuscata)

Summary The property of adrenoceptors mediating the positive inotropic effect (PIE) in the ventricular muscle of the Japanese monkey (Macaca fuscata) was investigated by the use of phenylephrine (PE) and adrenoceptor antagonists. The intrinsic activity (0.6) and the pD₂-value (5.41) for PE were comparable to those in other mammalian species. The -adrenoceptor antagonist, pindolol (3×10^–8 mol/l) shifted only the upper part of the concentration-response curve (CRC) for PE to the right; the pD₂-value for PE was not significantly affected by pindolol. On the other hand, phentolamine (10^–6 mol/l) shifted the lower part of the CRC for PE more than the upper part. In the presence of both pindolol and phentolamine the curve was shifted to the right in a parallel manner. The time required for twitch relaxation was negatively correlated to the degree of PIE of PE in the presence of phentolamine but not pindolol. These results indicate that both - and -adrenoceptors mediate the positive inotropic action in the ventricular muscle of the Japanese monkey and that in contrast to the action via -adrenoceptors the action via -adrenoceptors is not accompanied by the relaxant effect.     

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.     

β2-Adrenoceptor-mediated positive inotropic effect of adrenaline in human ventricular myocardium

Summary Experiments were designed to unravel the relative contribution of ₁- and 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 ₁- and ₂-adrenoceptors, estimated from binding assays, and stimulation of adenylate cyclase and contractile force by adrenaline and noradrenaline. 1) Selective blockade of ₂-adrenoceptors by erythro(±)-(-methyl-indan-4-yloxy)-3-isopropylaminobutan-2-ol (ICI 118,551) attenuated the increase of contractile force caused by adrenaline but not by noradrenaline, suggesting some involvement of ₂-adrenoceptors. Selective blockade of ₂-adrenoceptors without affecting ₁-adrenoceptors still enabled both adrenaline and noradrenaline to cause maximum possible increases of contractile force through ₁-adrenoceptors. 2) A direct involvement of ₂-adrenoceptors became manifest by selectively antagonizing 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 ₂-adrenoceptor. 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. 3) -adrenoceptors were labelled in membrane particles with ³H-(–)-bupranolol in the absence (₁ & ₂) and presence of 500 nmol/l CGP 20712 A (₂). 71 % of the -adrenoceptors Were ₁ and 29% ₂. Binding inhibition experiments with CGP 20712 A and ICI 118,551 yielded 74% ₁ and 26% ₂-4) With the help of ICI 118,551 and CGP 20712 A it was found that, in membrane particles, 33–36% and 64–67% of maximum stimulation of the adenylate cyclase by noradrenaline was mediated through ₁- and ₂-adrenoceptors, respectively. Adrenaline caused 11–25% and 75–89% of maximum cyclase stimulation through ₁- and ₂-adrenoceptors, respectively. 5) The contribution of ₁- and ₂-adrenoceptors to the positive inotropic effects of adrenaline and noradrenaline cannot be inferred straightforwardly from biochemical estimates of receptor fractions and fractional adenylate cyclase stimulation. Send offprint requests to A. J. Kaumann, ICI Pharmaceutical Division, Mereside Alderley Park, Macclesfield, Cheshire SK10 4TG, UK     

The preferential inhibition of α1- over β-adrenoceptor-mediated positive inotropic effect by organic calcium antagonists in the rabbit papillary muscle

Summary Experiments were carried out to elucidate the mechanism that the positive inotropic effect mediated by ₁-adrenoceptors is more susceptible to organic calcium antagonists than the -adrenoceptor-mediated effect. Verapamil and diltiazem displaced the specific binding of [³H]prazosin to the membrane fraction derived from the rabbit ventricular myocardium, verapamil being about 70 times more potent than diltiazem. Nifedipine did not displace the binding. While these compounds suppressed the positive inotropic effect mediated via _l-adrenoceptors in ₁- concentration-dependent manner, there was no correlation between the potency of the compounds to displace the [³H]prazosin binding and to inhibit the -mediated positive inotropic effect. The relative potency of three calcium antagonists to decrease the basal force of contraction and the al-mediated effect (of the same extent as compared to basal force of contraction) was consistent to each other. The positive inotropic effect mediated by -adrenoceptors was inhibited much less, and was enhanced by low concentrations of organic calcium antagonists. The differential action of calcium antagonists on the - and -mediated positive inotropic effect was mimicked by lowering the extracellular calcium concentration to 1/2, 1/4 and 1/8 of that in normal Krebs-Henseleit solution (2.5 mmol/l). These results indicate that the ₁-adrenoceptor blocking activity does not play an essential role for the preferential inhibition of -mediated positive inotropic effect by organic calcium antagonists. Difference in the subcellular mechanism involved in mobilization of intracellular Ca²⁺ subsequent to ₁-and -adrenoceptor activation may be responsible for the differential inhibitory action of calcium antagonists in the rabbit heart. Send offprint requests to M. Endoh at the above address     

The effects of both noradrenaline and CGP12177, mediated through human β1-adrenoceptors, are reduced by PDE3 in human atrium but PDE4 in CHO cells

(-)-Noradrenaline and (-)-CGP12177 activate β₁-adrenoceptors through a high (H)- and low-affinity (L) site, respectively. The positive inotropic effects of (-)-noradrenaline are blunted by phosphodiesterase4 (PDE4) but not PDE3, while both PDE isoenzymes, acting in concert, prevent the effects of (-)-CGP12177 through β₁-adrenoceptors in rat ventricle. We sought to unravel the role of PDE3 and PDE4 on signals through the H and L sites in human myocardium. The kinetics of matching positive inotropic effects of (-)-noradrenaline (20 nM) and (-)-CGP12177 (100 nM) were investigated on human atrial trabeculae in the absence and presence of the PDE3 inhibitor cilostamide (300 nM), PDE4 inhibitor rolipram (1 μM) or both. The influence of cilostamide and rolipram on agonist-evoked cyclic adenosine monophosphate (cAMP) increases were also compared in Chinese hamster ovary (CHO) cells expressing recombinant human β₁-adrenoceptors. (-)-Noradrenaline and (-)-CGP12177 caused matching inotropic responses that faded during a 60-min time course. Cilostamide, but not rolipram, increased the positive inotropic effects and abolished the time dependent fade of both agonists. In CHO cells, rolipram, but not cilostamide, enhanced the cAMP signals caused by both (-)-noradrenaline and (-)-CGP12177. PDE3, but not PDE4, blunts the positive inotropic effects of both (-)-noradrenaline and (-)-CGP12177 through H and L sites, respectively, of human atrial β₁-adrenoceptors. However, in CHO cells, PDE4 blunts the cAMP signals of both (-)-noradrenaline and (-)-CGP12177. Neither CHO cells nor the rat ventricle are appropriate models for the β₁-adrenoceptor-evoked signalling to PDE3 observed in human atrium.     

Distinct down-regulation of cardiac β1- and β2-adrenoceptors in different human heart diseases

Summary Cardiac -adrenoceptor density and ₁- and ₂-subtype distribution were examined in human left ventricular myocardium from transplant donors serving as controls and from patients with mitral valve stenosis, aortic valve stenosis, idiopathic dilated cardiomyopathy, and ischaemic cardiomyopathy respectively. The total -adrenoceptor density was similar in transplant donors and patients with moderate heart failure (NYHA II–III) due to mitral valve stenosis, but was markedly reduced in all forms of severe heart failure (NYHA III–IV) studied. A reduction of both ₁- and ₂-adrenoceptors was found in patients with severe heart failure due to mitral valve stenosis or ischaemic cardiomyopathy. In contrast, a selective down-regulation of ₁-adrenoceptors with unchanged ₂-adrenoceptors and hence a relative increase in the latter was observed in idiopathic dilated cardiomyopathy and aortic valve stenosis. It is concluded that the extent of total -adrenoceptor down-regulation is related to the degree of heart failure. Selective loss of ₁-adrenoceptors is not specific for idiopathic dilated cardiomyopathy but also occurs in aortic valve stenosis. Changes in ₁- and ₂-subtype distribution are rather related to the aetiology than to the clinical degree of heart failure. Send offprint requests to M. Steinfath at the above address     

Stimulation of cyclic AMP production in human alveolar macrophages induced by inflammatory mediators and β-sympathicomimetics

We have investigated the effects of inflammatory mediators and β-adrenoceptor agonists on the adenylyl cyclase responsiveness in alveolar macrophages from control subjects, patients suffering from chronic obstructive pulmonary disease (COPD) and asthmatics. Basal cyclic AMP (cAMP) levels in alveolar macrophages from COPD patients were significantly elevated (plus 42%) as compared to controls. In addition, the adenylyl cyclase responsiveness to prostaglandin E₂, histamine and the β-adrenoceptor agonist salbutamol was significantly impaired in alveolar macrophages from COPD patients and asthmatics. The lipid mediator platelet activating factor showed no effect on cAMP production in all three alveolar macrophage populations. Furthermore, the cAMP-enhancing effects of isoprenaline, salbutamol and histamine appeared to be mediated via β₂-adrenoceptors and histamine H₂-receptor subtypes respectively. Taken together, these data suggest an intrinsic desensitization phenomenon in alveolar macrophages from COPD patients and asthmatics.     

Contribution of both α- and β-adrenoceptors to the inotropic effects of catecholamines in the rabbit heart

Summary The functional role of -adrenoceptors was investigated in different parts of the rabbit heart. Phenylephrine (PE) caused a marked increase in force of contraction (F_c) and a prolongation of the action potential (AP) in preparations from the left atrium and the right ventricle. The response was less pronounced in the right atrium and in the left ventricle, whereas APs of spontaneously beating sinoatrial preparations remained completely unchanged. Phentolamine as well as the diesters phorbol 12,13 dibutyrate (PDBu) or 12-O-tetradecanoylphorbol-13-acetate (TPA) eliminated the effects of PE. The contribution of a-adrenoceptors to the effects of adrenaline (Adr) and noradrenaline (NA) on F_c was determined in preparations from the right ventricle. Phentolamine and the phorbol diesters reduced the effects of Adr and NA by about 30 to 60%; the remaining response was abolished by propranolol. It can be derived from our experiments that, in some parts of the rabbit heart, a considerable amount of the effects of Adr and NA is due to the stimulation of a-adrenoceptors. The present findings therefore support the view that, in the rabbit heart, the maximally effective drive of the heart requires the stimulation of both - and -adrenoceptors. The inhibitory effects of phorbol diesters on the -adrenoceptor-mediated response indicate that the activation of protein kinase C (PKC) specifically uncouples -adrenoceptors from the effector system, whereas the response to -adrenoceptor stimulation remains unchanged.Correspondence to: H. Nawrath at the above address     

Age-related alterations in α1- and β-adrenoceptors mediated responsiveness of rat aorta

Summary We have examined the responsiveness of - and -adrenoceptors in aortae from 1.5, 3, 6 and 24 month old rats. The isometric contraction to phenylephrine was antagonised competitively by prazosin with a pA₂ value of 9.45, suggesting that the receptor is an ₁-adrenoceptor. The potency of phenylephrine was significantly reduced in 24 months old as compared with all younger rats combined. The maximum contraction to phenylephrine was unaltered in 24 month old rats. The maximum contraction to potassium chloride was significantly less than that to phenylephrine only in 1.5 months old rats. In tissues contracted by potassium chloride, isoprenaline produced a marked relaxation in 1.5 months old animals, but there was a progressive loss with increasing age of the -adrenoceptor-mediated relaxation which was markedly reduced by 6 months and abolished in 24 months old. It is concluded that, in the rat aorta, there is a decrease in ₁-adrenoceptor responsiveness in senescence, and a loss of -adrenoceptor-mediated responses in maturation. Send offprint requests to J. R. Docherty at the above address     

α2-Adrenoceptor mediated inhibition of forskolin-stimulated cyclic AMP accumulation in isolated porcine palmar lateral veins

The aim of this study was to use a ³H-adenine pre-labelling technique to characterise the effect of ₂-adrenoceptor activation on forskolin-stimulated cyclic AMP accumulation in the isolated porcine palmar lateral vein. Forskolin (10^–7–10^–4 M) stimulated ³H-cyclic AMP accumulation in the isolated porcine palmar lateral vein in a biphasic and concentration-dependent manner. In the absence of the cyclic AMP-selective phosphodiesterase inhibitor rolipram, forskolin stimulated ³H-cyclic AMP accumulation approximately 7–8 fold. The response reached a peak after 5 min. In the presence of rolipram (10^–5 M), basal ³H-cyclic AMP levels were approximately 70% higher than in its absence (basal: 1823 ± 57 dpm; rolipram: 3088 ± 229, n \2 = 3) and forskolin (3 × 10^–5 M) stimulated ³H-cyclic AMP accumulation approximately 8 fold. The latter response reached a plateau 10 min after the addition of forskolin. In all subsequent studies, the tissues were incubated with forskolin (3 × 10^–5 M) for 5 min in the absence of rolipram. Noradrenaline (NA; 10^–9–10^–4 M) and UK14304 (10^–9–10^–4 M) inhibited forskolin-stimulated ³H-cyclic AMP accumulation in a concentration-dependent manner with mean pIC₅₀ values of 7.61 ± 0.37 (n \s = 4) and 7.76 ± 0.23 (n \s = 5), respectively. With either NA or UK14304, the maximal inhibition of the forskolin response obtained was approximately 75%. Neither NA (10^–4 M) nor UK14304 (10^–4 M) altered basal ³H-cyclic AMP levels. Phenylephrine (10^–4 M) had no effect on basal ³H-cyclic AMP levels and produced a 25.4 ± 7.1% inhibition of the forskolin-stimulated response, an effect that was reversed by 10^–6 M rauwolscine. Rauwolscine (10^–9–10^–6 M) produced a concentration-dependent reversal of the inhibitory effect of UK14304 10^–6 M on forskolin-stimulated ³H-cyclic AMP accumulation with a mean pK _i of 8.35 ± 0.39 (n = 3), but had no effect on basal or on forskolin-stimulated ³H-cyclic AMP levels. Similarly, prazosin (3 × 10^–8–3 × 10^–5 M) or imiloxan (3 × 10^–8––3 × 10^–5 M) produced a concentration-dependent reversal of the UK14304 (10^–7 M)-induced inhibition of forskolin-stimulated ³H-cyclic AMP accumulation, with mean pK _i values of 6.32 ± 0.22 (n = 4) and 6.01 ± 0.30 (n = 3), respectively; neither drug had any effect on basal or on forskolin-stimulated ³H-cyclic AMP levels. This suggests that the receptor is of the _2A-adrenoceptor subtype. It can be seen from these studies that it is possible to measure changes in cyclic AMP accumulation in porcine vascular smooth muscle using a pre-labelling technique, and it has been possible to demonstrate the presence of functional ₂-adrenoceptors, stimulation of which results in inhibition of forskolin-stimulated cyclic AMP formation.     

Blockade of putative β4- and β1-adrenoceptors by carvedilol in ferret myocardium

The putative β₄-adrenoceptor mediates positive inotropic effects, action potential shortening and arrhythmias in ferret ventricle. Here we compared the affinity of carvedilol at the putative β₄-adrenoceptor and β₁-adrenoceptor, activated by (±)-CGP 12177 and (–)-isoprenaline, respectively. In paced right ventricular preparations, carvedilol (0.01–10 μmol/l) was a simple competitive antagonist of the positive inotropic effects of (±)-CGP 12177 (slope of Schild-plot = 1.02, pK _B = 6.8) and (–)-isoprenaline (slope of Schild-plot = 0.98, pK _B = 8.1). Carvedilol also blocked putative β₄- and β₁-adrenoceptors of left ventricle, left atrium and sino-atrial pacemaker. Carvedilol therefore interacts with the putative β₄-adrenoceptor according to the law of mass action and may provide a lead in the development of putative β₄-adrenoceptor-selective antagonists. Received: 25 January 1999 / Accepted: 2 March 1999     

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.     

Functional effects of polyamines via activation of human β1- and β2-adrenoceptors stably expressed in CHO cells

Polyamines mediate acute metabolic effects and cardiac hypertrophy associated with β-adrenoceptor stimulation. They may also modulate β-adrenoceptors, causing functional responses in rat atria and tracheal smooth muscle. The aim of this study was to determine whether polyamines interact with human β(1)- and β(2)-adrenoceptors and the functional consequences of such an interaction. Chinese hamster ovary (CHO) cells stably transfected with human β(1)- and β(2)-adrenoceptors were used to evaluate the effect of polyamines binding to β-adrenoceptors, cAMP production and morphological changes, which were pharmacologically validated by investigating the effects of the β-adrenoceptor agonists, isoproterenol and salbutamol. Polyamines interacted with human β(1)- and β(2)-adrenoceptors, as shown by the displacement of [(125)I]iodocyanopindolol in the binding assay. Putrescine showed higher affinity to β(1)- than β(2)-adrenoceptors. Spermidine and spermine produced partial displacement (approximately 50%) and, at the highest concentration, the effect was reversed. Putrescine and spermine acutely increased cAMP and, in a serum-free medium, induced a stellate-like form in cells, which was inhibited by propranolol, a β-blocker. A 10 to 15 h incubation with putrescine produced a spindle-like form and spatial organization via β-adrenoceptor activation, evidenced by the antagonizing effect by propranolol and lack of effect in wild-type CHO cells. Additionally, it decreased cell proliferation independently of β-adrenoceptor activation. Spermine caused cell death via fetal bovine serum-dependent and -independent mechanisms. The results suggest that putrescine may act as a non-selective and low affinity agonist of human β(1)- and β(2)-adrenoceptors, eliciting morphological changes. These findings may be of importance in physiology and in diseases involving β-adrenoceptor functionality.