Alberto J. Kaumann

Is there a third heart β-adrenoceptor?

Abstract A series of partial agonists with high affinity for myocardial β 1 - and β 2 -adrenoceptors cause stimulant effects in heart that are resistant to blockade of β 1 - and -adrenoceplors. The concentrations of partial agonist that cause stimulant effects greatly exceed those that cause blockade. Alberto Kaumann suggests that such non-conventional partial agonists, often analogues of pindolol, may act through a third heart β 2 -adrenoceptor, which resembles the β 3 -adrenoceptor of white adipocytes and smooth muscle of airways and ileum

Modulation of human cardiac function through 4 beta-adrenoceptor populations

Abstract In human heart there is now evidence for the involvement of four β-adrenoceptor populations, three identical to the recombinant β1-, b2- and β3-adrenoceptors, and a fourth as yet uncloned putative β-adrenoceptor population, which we designate provisionally as the cardiac putative β4-adrenoceptor. This review described novel features of β-adrenoceptors as modulators of cardiac systolic and diastolic function. We also discuss evidence for modulation by unoccupied β1- and β2-adrenoceptors. Human cardiac and recombinant β1- and β2-adrenoceptors are both mainly coupled to adenylyl cyclase through Gs protein, the latter more tightly than the former. Activation of both human β1- and β2-adrenoceptors not only increases cardiac force during systole but also hastens relaxation through cyclic AMP-dependent phosphorylation of phospholamban and troponin I, thereby facilitating diastolic function. Furthermore, both β1 and β2-adrenoceptors can mediate experimental arrhythmias in human cardiac preparations elicited by noradrenaline and adrenaline. Human ventricular β3-adrenoceptors appear to be coupled to a pertussis toxin-sensitive protein (Gi?). β3-Adrenoceptor-selective agonists shorten the action potential and cause cardiodepression, suggesting direct coupling of a Gi protein to a K+ channel. In a variety of species, including man, cardiac putative β4-adrenoceptors mediate cardiostimulant effects of non-conventional partial agonists, i.e. high affinity β1- and β2-adrenoceptor blockers that cause agonist effects at concentrations considerably higher than those that block these receptors. Putative β4-adrenoceptors appear to be coupled positively to a cyclic AMP-dependent cascade and can undergo some desensitisation.

A 5‐hydroxytryptamine receptor in human atrium

1 The effects of 5‐hydroxytryptamine (5‐HT) were investigated on right atrial appendages obtained from patients treated with β‐adrenoceptor blocking agents who were undergoing open heart surgery. Atrial strips were paced under isometric conditions. 2 5‐HT increased contractile force to approximately one half of the force produced by a saturating concentration of (−)−isoprenaline. Both 5‐HT and (−)−isoprenaline accelerated the onset of relaxation, as indicated by an abbreviation of time to peak force. 3 The effects of 5‐HT were resistant to blockade by 0.4 μm (±)‐propranolol, 1μm (−)−pindolol, 0.4 μm methiothepin, 4 μm yohimbine, 0.4 μm ketanserin, 10 μm phenoxybenzamine, 1μm methysergide, 2 μm MDL 72222 and 20 μm granisetron. 4 Cocaine 6 μm potentiated the effects of 5‐HT, increasing the pEC50 from 6.6 to 7.4. The inotropic potency of 5‐HT is five times greater than that of (−)−noradrenaline. 5 ICS 205930 antagonized competitively the effects of 5‐HT with a pKB of 6.7. 6 In the presence of 0.4 μm (±)‐propranolol, 10 μm 5‐HT increased both adenosine 3′:5′ cyclicmonophosphate (cyclic AMP) levels and cyclic AMP‐dependent protein kinase activity by approximately one half and two thirds respectively, of the corresponding effects of 200 μm (−)−isoprenaline. 7 Both the increase in cyclic AMP levels and the stimulation of protein kinase activity are consistent with the inotropic effects of 5‐HT being mediated by cyclic AMP‐dependent phosphorylation of Ca2+ channels and of proteins involved in contraction and relaxation. 8 The human atrial 5‐HT receptor resembles the neuronal ‘so called’ 5‐HT4 receptor of rodents both in increasing cyclic AMP levels and in its affinity for ICS 205930.

Autoradiographical localization of binding sites for porcine [125I]endothelin-1 in humans, pigs, and rats: functional relevance in humans.

Summary Endothelin-1 (ET-1) is a recently discovered 21 amino acid peptide with potent vasoconstrictor properties. So far, its expression has been found only in porcine aorta, whereas its putative role as the endothelium-derived constricting factor (EDCF) would require it to be expressed and active in most vascular beds. We have used quantitative receptor autoradiography on pig, rat, and some human tissues to determine the distribution and localization of specific binding sites for ET-1. In some cases where binding sites were found, studies were performed to determine whether these are likely to be functional receptors. Binding sites for ET-1 have been found in heart (nerves > atria > ventricle > coronary arteries), kidney (glomeruli > papilla), adrenal (zona glomerulosa > medulla), cerebellum, spinal cord, gut, spleen, and lung. The binding of [125I]ET-1 was displaced at all these sites by unlabeled ET-1 but not by nitrendipine, apamin, and other vasoconstrictor peptides. ET-1 contracted strips of human coronary artery at an EC50 of 15 nM, with a maximal contraction 130% that of K+. A positive inotropic effect was found in strips of human atria (EC50 = 1 nM), which was not blocked by α-or β-blockade. The widespread distribution of its binding sites suggests a more extensive role than control of vascular tone.

Differences between the third cardiac β‐adrenoceptor and the colonic β3‐adrenoceptor in the rat

1 The heart of several species including man contains atypical β‐adrenoceptors, in addition to coexisting β1‐ and β2‐adrenoceptors. We now asked the question whether or not the third cardiac β‐adrenoceptor is identical to the putative β3‐adrenoceptor. We compared the properties of the third cardiac β‐adrenoceptor with those of β3‐adrenoceptors in isolated tissues of the rat. To study the third cardiac β‐adrenoceptor we used spontaneously beating right atria, paced left atria and paced left ventricular papillary muscles. As a likely model for putative β3‐adrenoceptors we studied atypical β‐adrenoceptors of the colonic longitudinal muscle precontracted with 30 mM KCl. We used β3‐adrenoceptor‐selective agonists, antagonists and non‐conventional partial agonists (ie high‐affinity blockers of both β1‐ and β2‐adrenoceptors known to exert also stimulant effects through β3‐adrenoceptors). 2 The non‐conventional partial agonist (−)−CGP 12177 caused positive chronotropic effects in right atria (pD2 = 7.3) and positive inotropic effects in left atria (pD2 = 7.5). The stimulant effects of (−)−CGP 12177 were resistant to blockade by 200 nM‐2μm (−)−propranolol and 3 μm ICI 118551 (a β2‐selective antagonist) but antagonized by 1 μm (−)−bupranolol (pKB = 6.4–6.8), 3 μm CGP 20712A (a β1‐ selective antagonist) (pKB = 6.3–6.4) and 6.6 μm SR 59230A (a β3‐selective antagonist, pKB = 5.1–5.4). 3 The non‐conventional partial agonist cyanopindolol caused positive chronotropic effects in right atria (pD2 = 7.7) and positive inotropic effects in left atria (pD2 = 7.1). The stimulant effects of cyanopindolol were resistant to blockade by 200 nM (−)−propranolol but antagonized by 1 μm (−)−bupranolol (pKB = 6.8‐7.1). 4 Neither (−)−CGP 12177 nor cyanopindolol caused stimulant effects in papillary muscles at concentrations between 0.2 nM and 20 μm. 5 In the presence of 200 nM (−)−propranolol the β3‐adrenoceptor‐selective agonists BRL 37344 (6 μm), SR 58611A (6 μm), ZD 2079 (60 μm) and CL 316243 (60 μm) did not cause stimulant effects or modify the potency and efficacy of the effects of (−)−CGP 12177 in right and left atria. The combination of 2 μm (−)−propranolol and 2 μm (−)−noradrenaline did not modify the chronotropic potency and efficacy of (−)−CGP 12177 compared to the potency and efficacy in the presence of 2 μm (−)−propranolol alone. 6 (−)−CGP 12177 relaxed the colon with a pD2 of 6.9 and a maximum effect of 55% compared to (−)−isoprenaline. The relaxant effects of (−)−CGP 12177 were resistant to blockade by 200 nM (−)−propranolol, 3 μm CGP 20712A, 3 μm ICI 118551 but blocked by 2 μm (−)−propranolol (pKB = 6.0), 1 μm (−)−bupranolol (pKB = 6.4) and 3 μm SR 59230A (pKB = 6.3). In the presence of 200 nM (−)−propranolol, (−)−CGP 12177 (20 μm) antagonized surmountably the relaxant effects of BRL 37344 (pKP = 7.3), (−)−noradrenaline (pKP = 7.0); and CL 316243 (pKP = 7.0). 7 Cyanopindolol in the presence of 200 nM (−)−propranolol relaxed the colon with a pD2 of 7.0 and a maximum effect of 40% compared to (−)−isoprenaline. As expected from a partial agonist, cyanopindolol antagonized the relaxant effects of both BRL 37344 and CL 316243 with a pKP = 7.6 and (−)−noradrenaline with a pKP = 7.4. 8 The following β3‐adrenoceptor‐selective agonists were potent colonic relaxants (pD2 values between parentheses): BRL 37344 (9.1), ZD 2079 (7.0), CL 316243 (9.0) and SR 58611A (8.2). The relaxant effects of these agonists were only marginally affected by 200 nM (−)−propranolol, not blocked by 3 μm CGP 20712A or 3 μm ICI 118551, and blocked by SR 59230A 3 μm (pKB = 6.9‐7.5), 1 μm (−)−bupranolol (pKB = 6.2–6.4) and 2 μm (−)−propranolol (pKB = 6.3–6.5). 9 The colonic relaxation caused by the nanomolar concentrations of the β3‐adrenoceptor‐selective agonists and the non‐conventional partial agonists (−)−CGP 12177 and cyanopindolol and their relative resistance to blockade by antagonists with high affinity for β1‐ and β2‐adrenoceptors but blockade by the β3‐adrenoceptor selective SR 59230A agree with the hypothesis that the receptors involved are β3‐adrenoceptors. On the other hand, the failure of micromolar concentrations of β3‐adrenoceptor‐selective agonists to produce cardiac stimulation or affect the cardiostimulant effects of (−)−CGP 12177 is inconsistent with the hypothesis that the third cardiac β‐adrenoceptor is β3. Additionally, the selective blockade of the colonic putative β3‐adrenoceptor compared to the third cardiac β‐adrenoceptor by SR 59230A, as well as the blockade of cardiac but not colonic receptors by CGP 20712A is also inconsistent with an identical putative β3‐adrenoceptor in colon and heart. We conclude that in the rat the third cardiac β‐adrenoceptor is different from the colonic β3‐adrenoceptor.

Variable participation of 5-HT1-like receptors and 5-HT2 receptors in serotonin-induced contraction of human isolated coronary arteries. 5-HT1-like receptors resemble cloned 5-HT1D beta receptors.

BackgroundSerotonin may contract human large coronary arteries through two 5-hydroxtryptamine (5-HT) receptors, 5-HT1-like and 5 -HT2. These 5-HT1-like receptors resemble both cloned 5-HT1D receptor subtypes, 5-HT1Dα and 5 -HT1Dβ Although these subtypes have similar pharmacology, 5-HT1Dαreceptors appear to have lower affinity for ketanserin than 5-HT1Dα receptors. We assessed the relative participation of 5-HT1-like and 5-HT2 receptors and attempted to identify whether vasoconstrictor 5-HT1-like receptors are 5 1HT1Dα or 5-.HT1Dβ. Methods and ResultsEpicardial coronary arteries were dissected from the hearts of 29 patients (including 1 healthy donor) undergoing heart transplant operation. Endothelium-denuded strips were set up to contract at 37°C. To assess the relative contributions of 5-HT2-like and 5-HT2 receptors, we blocked the latter with ketanserin (0.1 to 1.0 μmol/L) and ketanserin-resistant receptors with methiothepin (0.1 μmol/ L). Concentration-effect curves for 5-HT, in the absence and presence of ketanserin, were analyzed by using a model for two receptor subtypes. The fractional contributions of 5-HT1-like and 5 -HT2 receptors to the maximum effect of 5 -HT, f1 and f2, were estimated in arteries from 28 patients: f1 (0.71±0.20, mean±SD) was significantly larger than f2 (0.29±0.20) (P<.0001). Using [3H]-serotonin to label transfected and ex-pressed receptors, we verified that ketanserin has lower affinity for 5-HT1Dβ (pK1-log Ki, mol/L] less than 5.0) than for 5-HTlDα (pKi=7.1±0.1) receptors. A concentration of ketanserin (1 μmol/L) that would occupy more than 90% of 5-HT1Dα receptors failed to block 5-HT-induced contractions (4 patients). The 5-HT1-like receptor stimulant sumatriptan evoked maximal contractions that matched f1 and was equipotent with 5-HT through 5-HT1-like receptors (8 patients). No systematic influence of disease, atheroma, or therapy on f1 and f2 was detected. ConclusionsCoronary artery contractile 5-HT1-like receptors resemble cloned 5-HT1Dβ receptors and predominate over 5-HT2 receptors in mediating serotonin-evoked contractions. Sumatriptan contracts coronary arteries as a full agonist through 5-HT1-like receptors.

Do human atrial 5-HT4 receptors mediate arrhythmias?

Since their first description five years ago, knowledge about human atrial 5-HT4 receptors has increased considerably. Progress has been facilitated by the advent of selective antagonists with high affinity for human atrial 5-HT4 receptors. The receptors have been detected in both right and left atrium where they mediate increases in contractile force. Human sinoatrial 5-HT4 receptors may mediate the tachycardia caused by 5-HT and cisapride, and 5-HT elicits arrhythmias via 5-HT4 receptors in human atrium. In this article, Alberto Kaumann suggests that 5-HT may be involved in the genesis of atrial fibrillation and associated thromboembolic stroke and that both the arrhythmia and stroke could be prevented by inhibiting 5-HT4 receptors.

Activation of β2-Adrenergic Receptors Hastens Relaxation and Mediates Phosphorylation of Phospholamban, Troponin I, and C-Protein in Ventricular Myocardium From Patients With Terminal Heart Failure

BACKGROUND Catecholamines hasten cardiac relaxation through beta-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of beta1- and beta2-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. METHODS AND RESULTS Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 micromol/L) or zinterol (10 micromol/L), mediated through beta2-adrenergic receptors, and of norepinephrine (10 micromol/L), mediated through beta1-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17. Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15+/-3%, 5+/-2%, and 20+/-3%, respectively, and reduced the time to half-relaxation by 26+/-3%, 21+/-3%, and 37+/-3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14+/-3, 12+/-4, and 12+/-3; troponin I 40+/-7, 33+/-7, and 31+/-6; and C-protein 7.2+/-1.9, 9.3+/-1.4, and 7.5+/-2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both beta1- and beta2-adrenergic receptors. CONCLUSIONS Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both beta1- and beta2-adrenergic receptors, thereby potentially improving diastolic function.

(−)-CGP 12177-induced increase of human atrial contraction through a putative third β-adrenoceptor

1 The inotropic effects of (−)−4‐(3‐t‐butylamino‐2‐hydroxypropoxy)benzimidazol‐2‐one ((−)−CGP 12177), an antagonist for β1‐ and β2‐adrenoceptors as well as an agonist for β3‐adrenoceptors, were investigated on paced preparations of isolated right atrial appendages obtained from patients without advanced heart failure undergoing open heart surgery. 2 In the presence of (−)−propranolol (200 nM), (−)−CGP 12177 increased contractile force with a ‐log EC50, M, of 7.3. The maximum effects of (−)−CGP 12177 amounted to 15% and 11% of the effects of (−)−isoprenaline (400 μm) and of CaCl2 (6.75 mM) respectively. 3 (—)‐Bupranolol 1 μm, an antagonist with a pKB of ∼7.5 for β3‐adrenoceptors, antagonized surmountably the positive inotropic effects of (—)‐CGP 12177 (in the presence of 200 nM (−)−propranolol) with an apparent pKB of 7.3. 4 The potent positive inotropic effects of (−)−CGP 12177 and their resistance to blockade by (−)−propranolol but antagonism by (−)−bupranolol are consistent with the existence in human atrial myocardium of a minor third β‐adrenoceptor population, possibly related to β3‐adrenoceptors.

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

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