Temporal relation between long-lasting aftercontractions and action potentials in cat papillary muscles.

Sotalol, an adrenergic-blocking and antiarrhythmic agent, increases markedly and simultaneously the duration of both action potentials and contractions in papillary muscles. The active tension is manifested as a main twitch contraction followed by a maintained low level of residual tension (aftercontraction) which persists until the terminal phase of rapid repolarization. The Strength of the aftercontraction is augmented when the extracellular concentration of calcium is increased.

Phosphodiesterase-4 blunts inotropism and arrhythmias but not sinoatrial tachycardia of (-)-adrenaline mediated through mouse cardiac beta(1)-adrenoceptors.

BACKGROUND AND PURPOSE: beta(1) and beta(2)-adrenoceptors coexist in murine heart but beta(2)-adrenoceptor-mediated effects have not been detected in atrial and ventricular tissues, possibly due to marked phosphodiesterase (PDE) activity. We investigated the influence of the PDE3 inhibitor cilostamide and PDE4 inhibitor rolipram on the effects of (-)-adrenaline in three regions of murine heart. EXPERIMENTAL APPROACH: (-)-Adrenaline-evoked cardiostimulation was compared on sinoatrial beating rate, left atrial and right ventricular contractile force in isolated tissues from 129SvxC57B1/6 cross mice. Ventricular arrhythmic contractions were also assessed. KEY RESULTS: Both rolipram (1 microM) and cilostamide (300 nM) caused transient sinoatrial tachycardia but neither enhanced the chronotropic potency of (-)-adrenaline. Rolipram potentiated 19-fold (left atrium) and 7-fold (right ventricle) the inotropic effects of (-)-adrenaline. (-)-Adrenaline elicited concentration-dependent ventricular arrhythmias that were potentiated by rolipram. All effects of (-)-adrenaline were antagonized by the beta(1)-adrenoceptor-selective antagonist CGP20712A (300 nM). Cilostamide (300 nM) did not increase the chronotropic and inotropic potencies of (-)-adrenaline, but administered jointly with rolipram in the presence of CGP20712A, uncovered left atrial inotropic effects of (-)-adrenaline that were prevented by the beta(2)-adrenoceptor-selective antagonist ICI118551. CONCLUSIONS AND IMPLICATIONS: PDE4 blunts the beta(1)-adrenoceptor-mediated effects of (-)-adrenaline in left atrium and right ventricle but not in sinoatrial node. Both PDE3 and PDE4 reduce basal sinoatrial rate in a compartment distinct from the beta(1)-adrenoceptor compartment. PDE3 and PDE4, acting in concert, prevent left atrial beta(2)-adrenoceptor-mediated inotropy. PDE4 partially protects the right ventricle against (-)-adrenaline-evoked arrhythmias.

The function of alpha- and beta-adrenoceptors of the saphenous artery in caveolin-1 knockout and wild-type mice.

BACKGROUND AND PURPOSE: Adrenoceptors can associate with cardiac caveolae. To investigate the function of vascular caveolae, adrenoceptor-mediated effects were compared in the saphenous artery of caveolin-1 knockout (cav-1KO) and wild-type (WT) mice. EXPERIMENTAL APPROACH: Electronmicroscopy was used to detect caveolae. Real-Time quantitative PCR was used for adrenoceptor subtypes. Catecholamine-evoked contractions and relaxations were studied in arterial segments. KEY RESULTS: Caveolae were found in arterial smooth muscle from WT but not from cav-1KO mice. Arterial mRNA levels for the adrenoceptors alpha1A, alpha1B, alpha1D, beta1, beta2 and beta3 were similar in cav-1KO and WT. (-)-Noradrenaline contracted cav-1KO (-log EC50M=7.1) and WT (-log EC50M=7.3) arteries through prazosin-sensitive receptors. Maximum (-)-noradrenaline-evoked contractions were greater in cav-1KO than WT arteries. (-)-Isoprenaline relaxed WT arteries (-log EC50M=7.3) more potently than cav-1KO arteries (-log EC50M=6.8); the effects were antagonized partially and similarly by the beta2-selective antagonist ICI118551 (50 nM). The (-)-isoprenaline-evoked relaxation was partially antagonized by the beta1-adrenoceptor-selective antagonist CGP20712 (300 nM) in WT but not cav-1KO arteries. The beta3-adrenoceptor-selective antagonist L748337 (100 nM) partially antagonized the relaxant effects of (-)-isoprenaline in cav-1KO but not in WT arteries. BRL37344 partially relaxed arteries through beta3-adrenoceptors in cav-1KO but not WT. The relaxant effects of BRL37344 were decreased by the NO synthase inhibitor OmegaL-nitroarginine. CONCLUSIONS AND IMPLICATIONS: The function of arterial alpha1- and beta2-adrenoceptors is similar in cav-1KO and WT mice. beta1-adrenoceptor-mediated relaxation in WT is lost in cav-1KO and replaced by the appearance of beta3-adrenoceptors.

Cilostamide potentiates more the positive inotropic effects of (-)-adrenaline through beta(2)-adrenoceptors than the effects of (-)-noradrenaline through beta (1)-adrenoceptors in human atrial myocardium.

Activation of both beta(1)- and beta(2)-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 beta-adrenoceptor subtypes. We have compared the positive inotropic effects of (-)-noradrenaline and (-)-adrenaline, mediated through beta(1)- and beta(2)-adrenoceptors, respectively, in the absence and presence of the PDE3 inhibitor cilostamide (300 nM) or PDE4 inhibitor rolipram (1 muM) on human atrial trabeculae from non-failing hearts. Cilostamide, but not rolipram, potentiated the effects of both (-)-noradrenaline and (-)-adrenaline. Cilostamide increased the -logEC(50)M of (-)-adrenaline more than of (-)-noradrenaline (P < 0.05), regardless of whether or not the patients had been chronically treated with beta-blockers. The results are consistent with a greater PDE3-catalysed hydrolysis of inotropically relevant cyclic AMP produced through beta(2)-adrenoceptors than beta(1)-adrenoceptors in human atrium.

Is there a third heart beta-adrenoceptor?

A series of partial agonists with high affinity for myocardial beta 1- and beta 2-adrenoceptors cause stimulant effects in heart that are resistant to blockade of beta 1- and beta 2-adrenoceptors. 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 beta-adrenoceptor, which resembles the beta 3-adrenoceptor of white adipocytes and smooth muscle of airways and ileum.

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.

L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac beta 2-adrenoceptor.

OBJECTIVES: The reported increase in basal activity of hearts from transgenic mice (TG4) overexpressing the human beta 2-adrenoceptor (beta 2-AR) was explained by spontaneously active beta 2-ARs that stimulate the beta-adrenergic cascade in the absence of an agonist. In order to examine altered myocardial function on a cellular level, we have investigated L-type calcium current (ICa,L) and cell shortening in ventricular myocytes from TG4 hearts. Myocytes from littermates (LM) and wild type animals (WT) served as controls. METHODS: Cardiac beta-AR density was measured by [125I]-iodocyanopindolol binding to ventricular membranes. ICa,L was assessed by standard whole-cell voltage clamp technique. Contractility was measured as cell shortening in ventricular myocytes and as force of contraction in electrically stimulated left atria. RESULTS: Overexpression of beta 2-ARs was confirmed by an almost 400-fold increase in beta-AR density. The beta 1:beta 2-AR ratio in WT mice was 71:29. Myocytes from TG4 and LM mice were similar in size as judged by membrane capacitance and two dimensional cell area. ICa,L amplitude was significantly lower in TG4 than in LM myocytes (with 2 mM [Ca2+]o -4.82 +/- 0.48 vs. -6.56 +/- 0.38 pA/pF, respectively). In TG4 myocytes, the ICa,L response to isoproterenol (1 microM) was almost abolished. Cell shortening was not different in physiological [Ca2+]o, but smaller in maximum [Ca2+]o when comparing TG4 to control myocytes. Basal force of contraction in left atria did not differ between TG4 and LM at any age investigated. In TG4 left atria the inotropic response to isoproterenol was also absent, whereas responses to high [Ca2+]o or dibutyryl-cAMP (1 mM) were present but reduced. The rate of spontaneous beating of right atria was elevated in TG4 mice. CONCLUSIONS: Since only spontaneous beating rate but neither basal ICa,L amplitude nor basal contractile activity were elevated, our data fail to reveal evidence for spontaneously active, stimulating beta 2-ARs in left atrium and ventricle. A contractile deficit unrelated to the beta-adrenoceptor pathway is evident in TG4 myocytes and left atria.

Molecular cloning of a human gene (S31) encoding a novel serotonin receptor mediating inhibition of adenylyl cyclase.

We report the molecular cloning of human gene (S31) containing an open reading frame of 1095 nucleotides, which encodes a protein of 365 amino acids. The encoded protein contains seven hydrophobic putative transmembrane domains considered the hallmark of G protein-coupled receptors. The amino acid sequence shows highest homology to receptors for serotonin (5-hydroxytryptamine). Expression of this receptor in murine Ltk- cells conferred upon these cells the ability to respond to serotonin by inhibition of adenylyl cyclase. No response was observed to isoproterenol, epinephrine, histamine, dopamine or melatonin in the transfected cells. We propose that the human gene S31 encodes a novel serotonin receptor.

Blockade of human atrial 5-HT4 receptors by GR 113808.

1. The mode of antagonism of 5-hydroxytryptamine (5-HT)-induced positive inotropic effects by the highly selective 5-HT4 receptor antagonist GR 113808 ([1-[2-methylsulphonylamino ethyl]-4-piperidinyl]methyl 1-methyl-1H-indole-3-carboxylate) was investigated on isolated preparations of human right atrium. 2. GR 113808 caused concentration-dependent (2-100 nM) surmountable antagonism of the effects of 5-HT with a pKB (M) of 8.8. 3. The affinity of GR 113808 for human atrial 5-HT4 receptors, together with its high selectivity for 5-HT4 receptors comprise useful properties for investigating the question of 5-HT4 receptor subtypes.

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

1. The inotropic effects of (-)-4-(3-t-butylamino-2-hydroxypropoxy)benzimidazol-2-one ((-)-CGP 12177), an antagonist for beta 1- and beta 2-adrenoceptors as well as an agonist for beta 3-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 microM) and of CaCl2 (6.75 mM) respectively. 3. (-)-Bupranolol 1 microM, an antagonist with a pKB of approximately 7.5 for beta 3-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 beta-adrenoceptor population, possibly related to beta 3-adrenoceptors.

Potentiation of responses to sympathetic nerve stimulation and vasoconstrictor agents by SK&F 103829 in the feline mesenteric circulation.

1. The amplification of vasoconstrictor effects of several agonists and sympathetic nerve stimulation, caused by 5-HT2 receptor activation, was studied in the autoperfused mesenteric circulation of anaesthetized cats. To produce long lasting and selective 5-HT2 receptor stimulation we used SK&F 103829 (2,3,4,5 tetrahydro-8[methyl-sulphonyl]-1H3-benzazepin-7-ol methensulphonate). We assessed that SK&F 103829 was a strong contractile partial agonist in isolated preparations of rat tail artery and calf pulmonary artery. 2. The intrinsic activity of SK&F 103829 with respect to 5-hydroxytryptamine (5-HT) was 0.8 in rat tail artery and 0.6 in calf pulmonary artery. SK&F 103829-induced contractile responses were surmountably antagonized by ketanserin with a potency expected from its affinity for 5-HT2 receptors. SK&F 103829 surmountably antagonized the effects of 5-HT in rat tail artery with a pKp of 5.8. 3. Concentrations of SK&F 103829 causing greater than threshold constrictions enhanced vasoconstrictor responses of sympathetic nerve stimulation, noradrenaline, angiotensin II, methoxamine and alpha, beta-methylene ATP in the mesenteric arterial bed. Increases in mesenteric arterial pressure by noradrenaline, observed in the presence of prazosin, were also potentiated by SK&F 103829. 4. Ketanserin prevented both the constrictor effect of SK&F 103829 and the SK&F 103829-evoked potentiation of the responses to noradrenaline and angiotensin II in the mesenteric arterial bed. Ketanserin, however, failed to abolish (once established) the SK&F 103829-evoked potentiation of the constrictor effects caused by both noradrenaline and angiotensin II. 5. Short lasting constrictor effects of 5-HT were reversed to dilator effects by SK&F 103829 in both the mesenteric arterial and venous bed, thereby revealing the existence of vasodilator 5-HT receptors.6. The main finding is consistent with a sensitization of the mesenteric arterial bed to vasoconstrictor responses mediated through alpha 1- and alpha2-adrenoceptors, angiotensin II receptors and purinoceptors by SK&F 103829-evoked activation of 5-HT2 receptors. This property, together with the direct constrictor effect of the mesenteric arterial bed suggest that SK&F 103829 can reduce portal venous flow thereby being a useful therapeutic principle for the treatment of portal hypertension.

Mode of action of (-)-pindolol on feline and human myocardium.

(-)-Pindolol antagonized competitively and to a similar extent the positive inotropic effects of both (-)-noradrenaline and (-)-adrenaline in human ventricular preparations. An equilibrium dissociation constant KD (-log mol 1(-1) = pKD) of 9.2-9.3 was estimated regardless of disease present or agonist used. (-)-Pindolol antagonized competitively the positive inotropic effects of (-)-adrenaline more than those of (-)-noradrenaline in human atrial preparations. pKD values of (-)-pindolol were 9.6 against (-)-adrenaline and 9.1 against (-)-noradrenaline. The results are consistent with a moderate selectivity of (-)-pindolol for beta 2-compared to beta 1-adrenoceptors in human atrium. (-)-Pindolol competed with [3H]-(-)-bupranolol with a pKD of 9.4 for beta-adrenoceptors of human ventricle. Positive inotropic effects of (-)-pindolol were not detected on human atrium or ventricle in a concentration range of 1-1000 nmol 1(-1). The affinity of (-)-pindolol estimated for human myocardial beta-adrenoceptors, its moderate beta 2-selectivity and its lack of intrinsic activity for contractile force agreed with similar characteristics in other species. (-)-Pindolol caused marked positive chronotropic effects in kitten right atria with an intrinsic activity of 0.5 with respect to catecholamines. On kitten left atria it caused only weak positive inotropic effects with an intrinsic activity of 0.1. (-)-Pindolol (0.6-6000 nmol-1) did not cause positive inotropic effects in kitten papillary muscle. The concentration-effect curve for (-)-pindolol on kitten right and left atria was biphasic. Its positive chronotropic and inotropic effects were not blocked by methysergide, suggesting that 5-hydroxytryptamine (5-HT)-receptors were not involved. Low concentrations of antagonists selective for beta 1- and beta 2-adrenoceptors blocked the high sensitivity component but not the low sensitivity component of the positive chronotropic and inotropic effects. The biphasic nature of the positive chronotropic effects of (-)-pindolol in kitten agreed with previous observations made on guinea-pig right atria and support the concept that 3 receptors in the sinoatrial pacemaker contribute to these chronotropic effects: beta 1, beta 2 and a low-affinity receptor for (-)-pindolol which is neither beta 1 nor beta 2. The partial agonistic activity of (-)-pindolol in the heart appears to be mainly (kitten) or completely (man) restricted to the sinoatrial pacemaker.

Relationship between beta-adrenoceptors and calcium channels in human ventricular myocardium.

The stoichiometric relationship between adrenoceptors and saturable binding sites for 1,4-dihydropyridines in calcium channels was investigated in human ventricular myocardium. Membrane particles were prepared from heart specimens of patients undergoing open heart surgery. The patients suffered from hypertrophic obstructive cardiomyopathy (HOCM) or mitral valve disease. Using [3H]-prazosin and [125I]-2-beta-hydroxy-3-iodiphenyl-ethyl-aminoethyl tetralone ([125I]-HEAT) as labels we detected only a marginal density of alpha 1-adrenoceptors, regardless of disease. No alpha 2-adrenoceptors were detected with [3H]-rauwolscine. In HOCM patients we estimated 72 +/- 10 fmol mg-1 (n = 12) beta-adrenoceptors labelled with [3H]-(-)-dihydroalprenolol and 74 +/- 5 fmol mg-1 (n = 2) beta-adrenoceptors labelled with [125I]-(-)-iodocyanopindolol; the equilibrium dissociation constants KD, were 1.2 +/- 0.2 nmol l-1 for [3H]-(-)-dihydroalprenolol and 7 +/- 1 pmol l-1 for [125I]-(-)-iodocyanopindolol. In patients with mitral valve disease we estimated 84 +/- 11 fmol mg-1 (n = 3) labelled with [3H]-(-)-dihydroalprenolol and 66 +/- 13 fmol mg-1 (n = 2) labelled with [125I]-(-)-iodocyanopindolol. The KD values were 1.8 +/- 0.6 nmol l-1 for [3H]-(-)-dihydroalprenolol and 8 +/- 2 pmol l-1 for [125I]-(-)-iodocyanopindolol. In 14 HOCM patients we estimated 107 +/- 12 fmol mg-1 calcium channel sites labelled with [3H]-nimodipine with a KD of 280 +/- 4 pmol l-1. In 5 patients with mitral valve disease the density of calcium channel sites labelled with [3H]-nimodipine was 78 +/- 5 fmol mg-1 with a KD of 290 +/- 20 pmol l-1, In HOCM patients the density of calcium channel sites labelled with the benzoxadiazol 1,4-dihydropyridine ([3H]-(+)-PN 200-110) was 1.6 fold of that labelled with [3H]-nimodipine with a KD of 84 +/- 11 pmol l-1. In a group of 4 HOCM patients in which calcium channels were labelled with [125I]-iodipine, the density of sites was 1.37 +/- 0.07 fold the density of sites labelled by [3H]-(+)-PN 200-11-. The KD value of [125I]-iodipine was 246 +/- 16 pmol-1. (+)-PN 200-110 was approximately 100 fold more potent than (-)-PN 200-110 as a competitor of [125I]-iodipine binding. For the HOCM group a significant correlation was found between beta-adrenoceptor density and calcium channel density, whereas in the mitral valve group no such correlation was found. This does not prove that there is causal interaction leading to a relationship between the density of beta-adrenoceptors and calcium channels. However, because positive inotropic effects of catecholamines mediated by beta-adrenoceptors are associated with opening of calcium channels, this suggests that the density of both beta-adrenoceptors and calcium channels could be co-regulated.

Characterization of the 5-HT4 receptor mediating tachycardia in piglet isolated right atrium.

1. In order to explore whether 5-HT4 receptor subtypes exist, we have characterized further the 5-HT4 receptor that mediates tachycardia in the piglet isolated right atrium. All experiments were carried out in the presence of propranolol (400 nM) and cocaine (6 microM). We used tryptamine derivatives, substituted benzamides and benzimidazolone derivatives as pharmacological tools. 2. Tachycardia responses to 5-hydroxytryptamine (5-HT) were mimicked by other tryptamine derivatives with the following order of potency: 5-HT > 5-methoxytryptamine alpha-methyl-5-HT = bufotenine bufotenine > 5-carboxamidotryptamine = tryptamine (after treatment with pargyline) > 5-methoxy-N,N-dimethyltryptamine > 2-methyl-5-HT. 3. The substituted benzamides were all partial agonists relative to 5-HT except (-)-zacopride which was a full agonist. The stimulant potency order was renzapride > cisapride = (-)-zacopride > metoclopramide > (+)-zacopride. 4. The benzimidazolone derivatives had contrasting effects. BIMU 8 (endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-(1-methyl(eth yl- 2-oxo-1H-benzimidazole-1-carboxamide hydrochloride) was a full agonist relative to 5-HT whilst BIMU 1 (endo-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dihydro-3-ethyl-2-oxo - 1H-benzimidazole-1-carboxamide hydrochloride) was a partial agonist with low intrinsic activity compared to 5-HT but had similar potency. We estimated a pKB of 7.9 for BIMU 1 antagonism of 5-HT-induced tachycardia. DAU 6215 (N-endo-8-methyl-8-azabicyclo[3.2.1]-oct-3-yl)-2,3-dihydro-2-oxo-lH-benzimidazole-l-carboxamide, hydrochloride) had no chronotropic activity and was found to be a simple competitive antagonist with a pKB of 7.15.SB 203186 (1-piperidinyl)ethyl lH-indole 3-carboxylate) was a potent antagonist with a pKB of 8.3.The affinity of SB 203186 was approximately 20 times higher than that of tropisetron (ICS 205-930;pKB= 6.9) and DAU 6215 (pKB= 7.0). GR1 13808 (([1-[2-[methylsulphonyl amino]ethyl]-4-piperidinyl]methyl 1-methyl-1H-indole-3-carboxylate) and SDZ 205-557 ((2-diethylaminoethyl)2-methoxy-4-amino-5-chloro-benzoate) also antagonized 5-HT-induced tachycardia but not by simple competitive blockade.6. The sinoatrial 5-HT4 receptor in the piglet has a pharmacological profile that correlates well with 5-HT4 receptors characterized in rat oesophagus, guinea-pig ileum and colon, mouse embryonic colliculi neurones and human atrium.

Stimulation of cyclic AMP-dependent protein kinase in rat atria by (-)-CGP 12177 through an atypical beta-adrenoceptor.

Mammalian hearts possess an atypical beta-adrenoceptor (non-beta 1, non-beta 2, non-beta 3) through which (-)-4-(3-t-butylamino-2 -hydroxypropoxy)benzimidazol-2-one ((-)-CGP 12177) causes cardiostimulant effects. Here we showed that (-)-CGP 12177 increased the activity of adenosine 3':5'-cyclic monophosphate (cyclic AMP)-dependent protein kinase in the presence of 200 nM (-)-propranolol in rat atria at a concentration (10 microM) that elicits maximum positive chronotropic and inotropic effects. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) potentiated the positive chronotropic and inotropic effects of (-)-CGP 12177. We suggest that the atypical beta-adrenoceptor is coupled positively to the Gs protein-adenylyl cyclase system.

Differences between the third cardiac beta-adrenoceptor and the colonic beta 3-adrenoceptor in the rat.

1. The heart of several species including man contains atypical beta-adrenoceptors, in addition to coexisting beta 1- and beta 2-adrenoceptors. We now asked the question whether or not the third cardiac beta-adrenoceptor is identical to the putative beta 3-adrenoceptor. We compared the properties of the third cardiac beta-adrenoceptor with those of beta 3-adrenoceptors in isolated tissues of the rat. To study the third cardiac beta-adrenoceptor we used spontaneously beating right atria, paced left atria and paced left ventricular papillary muscles. As a likely model for putative beta 3-adrenoceptors we studied atypical beta-adrenoceptors of the colonic longitudinal muscle precontracted with 30 mM KCl. We used beta 3-adrenoceptor-selective agonists, antagonists and non-conventional partial agonists (ie high-affinity blockers of both beta 1- and beta 2-adrenoceptors know to exert also stimulant effects through beta 3-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-2 microM (-)-propranolol and 3 microM ICI 118551 (a beta 2-selective antagonist) but antagonized by 1 microM (-)-bupranolol (pKB = 6.4-6.8), 3 microM CGP 20712A (a beta 1-selective antagonist) (pKB = 6.3-6.4) and 6.6 microM SR 59230A (a beta 3-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 microM (-)-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 microM. 5. In the presence of 200 nM (-)-propranolol the beta 3-adrenoceptor-selective agonists BRL 37344 (6 microM), SR 58611A (6 microM), ZD 2079 (60 microM) and CL 316243 (60 microM) 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 microM (-)-propranolol and 2 microM (-)-noradrenaline did not modify the chronotropic potency and efficacy of (-)-CGP 12177 compared to the potency and efficacy in the presence of 2 microM (-)-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 microM CGP 20712A, 3 microM ICI 118551 but blocked by 2 microM (-)-propranolol (pKB = 6.0), 1 microM (-)-bupranolol (pKB = 6.4) and 3 microM SR 59230A (pKB = 6.3). In the presence of 200 nM (-)-propranolol, (-)-CGP 12177 (20 microM) 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 beta 3-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 microM CGP 20712A or 3 microM ICI 118551, and blocked by SR 59230A 3 microM (pKB = 6.9-7.5), 1 microM (-)-bupranolol (pKB = 6.2-6.4) and 2 microM (-)-propranolol (pKB = 6.3-6.5). 9...

Beta 1- and beta 2-adrenoceptor-mediated relaxation in human internal mammary artery and saphenous vein: unchanged beta- and alpha-adrenoceptor responsiveness after chronic beta 1-adrenoceptor blockade.

1. We have recently reported that patients taking beta 1-adrenoceptor-selective antagonists exhibit marked sensitization of beta 2-adrenoceptor responses but unaltered beta 1-adrenoceptor responses in the heart, both in vitro and in vivo. We therefore investigated beta 1- and beta 2-adrenoceptor-mediated relaxant responses in rings of human internal mammary artery and saphenous vein without endothelium, taken from beta 1-blocked and non-beta-blocked patients undergoing coronary artery bypass graft surgery, for comparison. We also examined alpha 1-adrenoceptor-mediated contraction in these vessels, to determine whether beta 1-blockade had any cross-regulatory effect. 2. Following alpha-blockade with 10 microM phenoxybenzamine, both noradrenaline adrenaline produced concentration-dependent relaxations in both blood vessels, their effects being mediated predominantly through beta 2-adrenoceptors; a lesser beta 1-adrenoceptor component to relaxation was also found in internal mammary artery and a minor beta 1-adrenoceptor component was present in saphenous vein. No differences were found in beta 1- or in beta 2-adrenoceptor-mediated vasorelaxation between beta 1-blocked and non-beta-blocked patients. 3. Methoxamine produced concentration-dependent contractions in both blood vessels, and the potency and efficacy were not significantly different between vessels from beta 1-blocked and from non-beta-blocked patients. 4. These findings indicate that, in these tissues, which possess a relatively minor beta 1-adrenoceptor component in contrast to myocardial tissue, chronic beta 1-blocker treatment does not alter either beta 1- or beta 2-adrenoceptor responses. Likewise, in such tissues, alpha 1-adrenoceptor responses are unaffected by prior beta 1-blockade.

Contribution of beta 1- and beta 2-adrenoceptors of human atrium and ventricle to the effects of noradrenaline and adrenaline as assessed with (-)-atenolol.

1. (-)-Atenolol was used as a tool to assess the function of beta 1- and beta 2-adrenoceptors in human heart. Right atrial and left ventricular preparations from patients undergoing open heart surgery were set up to contract isometrically. Membrane particles were prepared for beta-adrenoceptor labelling with [3H]-(-)-bupranolol and adenylate cyclase assays. 2. The positive inotropic effects of (-)-noradrenaline were antagonized to a similar extent by (-)-atenolol in atrial and ventricular preparations. (-)-Atenolol consistently antagonized the effects of (-)-adrenaline to a lesser extent than those of (-)-noradrenaline in atrial preparations. In ventricular preparations (-)-atenolol antagonized the effects of low concentrations of (-)-adrenaline to a lesser extent than those of high concentrations. 3. pKB values (M) of (-)-atenolol, estimated with non-linear analysis from the blockade of the positive inotropic effects of the catecholamines, were 7.4 for beta 1-adrenoceptors and 6.0 for beta 2-adrenoceptors. 4. (-)-Atenolol inhibited the binding of [3H]-(-)-bupranolol to ventricular beta 1-adrenoceptors with a pKD (M) of 5.9 and to ventricular beta 2-adrenoceptors with a pKD of 4.6. 5. (-)-Atenolol inhibited the catecholamine-induced adenylate cyclase stimulation in the atrium and ventricle with pKB values of 5.8-6.4 for beta 1- and pKB values of 4.7-5.7 for beta 2-adrenoceptors. The binding and cyclase assays suggest a partial affinity loss for (-)-atenolol inherent to membrane preparations. 6. beta 1-Adrenoceptors mediate the maximum positive inotropic effects of (-)-noradrenaline in both the atrium and ventricle of man. beta 2-Adrenoceptors appear to be capable of mediating maximal positive inotropic effects of (-)-adrenaline in atrium. In contrast, ventricular beta 2-adrenoceptors mediated only submaximal effects of (-)-adrenaline.

Validity of (-)-[3H]-CGP 12177A as a radioligand for the 'putative beta4-adrenoceptor' in rat atrium.

1. We have recently suggested the existence in the heart of a 'putative beta4-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta1- and beta2-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[3H]-CGP 12177A ([-]-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[3H]-CGP 12177A for this purpose for two reasons, because it is a non-conventional partial agonist and also because it is a hydrophilic radioligand. 2. Increasing concentrations of (-)-[3H]-CGP 12177A, in the absence or presence of 20 microM (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta1- and beta2-adrenoceptors (pKD 9.4+/-0.1, Bmax 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta4-adrenoceptor' (pKD 7.5+/-0.1, Bmax 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta1- and beta2-adrenoceptors, (-)-[3H]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pKD 7.6+/-0.1, Bmax 50.8+/-7.4 fmol mg(-1) protein). 3. The non-conventional partial agonists (-)-CGP 12177A (pKi 7.3+/-0.2), (+/-)-cyanopindolol (pKi 7.6+/-0.2), [-]-pindolol (pK1 6.6+/-0.1) and (+/-)-carazolol (pKi 7.2+/-0.2) and the antagonist (-)-bupranolol (pKi 6.6+/-0.2), all competed for (-)-[3H]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta4-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4. The catecholamines competed with (-)-[3H]-CGP 12177A at the 'putative beta 4-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pKiH 6.3+/-0.3, pKiL 3.5+/-0.1), (-)-adrenaline (pKiH 6.5+/-0.2, pKiL 2.9+/-0.1), (-)-isoprenaline (pKiH 6.2+/-0.5, pKiL 3.4+/-0.1), (+)-isoprenaline (pKi< 1.7), (-)-RO363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy++ +)-2-propranol)oxalate, pKi 5.5+/-0.1). 5. The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta4-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[3H]-CGP 12177A for one receptor state in the absence (pKi 7.3+/-0.2) or presence of GTP (pKi 7.3+/-0.2). (-)-Isoprenaline competed with (-)-[3H]-CGP 12177A for two states in the absence (pKiH 6.6+/-0.3, pKiL 3.5+/-0.1; % H 25+/-7) or presence of GTP (pKiH 6.2+/-0.5, pKiL 3.4+/-0.1; % H 37+/-6). In contrast, at beta1-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6. The specificity of binding to the 'putative beta 4-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta 3-adrenoceptor agonists, BRL 37344 ((RR+SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy-ethyl]amino]propyl]phenoxy]acetic acid, 6 microM), SR 58611A (ethyl{(7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl2-yloxy} acetate hydrochloride, 6 microM), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)-ethan-1-ol, 60 microM), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 microM) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 microM) caused less than 22% inhibition of (-)[3H]-CGP 12177A binding in the presence of 500 nM (-)propranolol. Histamine (1mM), atropine (1 microM), phentolamine (10 microM), 5-HT (100 microM) and the 5-HT4 receptor antagonist SB 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1,4-benzodioxan-5-carboxylate, 10nM) caused less than 26% inhibition of binding. 7.Non-conventional partial agonists, the antagonist (-)bupranolol and catecholamines all competed for (-)[3H]-CGP 12177A binding in the absence of (-)propranolol at beta1-adrenoceptors, with affinities (pKi) ranging from 1.6-3.6 log orders greater than at the 'putative beta 4-adrenoceptor'. 8.We have established and validated a radioligand binding assay in rat atrium for the 'putative beta 4-adrenoceptor' which is distinct from beta1-, beta2- and beta 3-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta 4-adrenoceptor'.

Relationship between the stereoselective negative inotropic effects of verapamil enantiomers and their binding to putative calcium channels in human heart.

Ventricular preparations from patients with mitral disease and hypertrophic obstructive cardiomyopathy (HOCM) were set up to contract isometrically. Ventricular membrane particles were also prepared and putative calcium channels were labelled with [3H]-nimodipine. Positive staircase was induced by varying the rate of stimulation of isolated strips from 6 min-1 to 120 min-1 in the presence of 6-60 microM (-)-adrenaline or (-)-noradrenaline. (-)-Verapamil 3-5 microM or (+)-verapamil 20-30 microM reversed the force-frequency relationship (i.e. caused negative staircase) in preparations from patients with mitral disease or HOCM. In subendocardial strips of ventricular septum from 5 patients with HOCM paced at 60 min-1, both (-)-verapamil and (+)-verapamil caused cardiodepression. Half-maximal cardiodepression was observed with 0.4 microM (-)-verapamil and with 3 microM (+)-verapamil. [3H]-nimodipine bound to ventricular membrane particles in a saturable, reversible fashion to a high affinity site with an equilibrium dissociation constant of 0.23 nM. The density of these sites was 95 fmol mg-1 of membrane protein. Binding of the tritiated 1,4-dihydropyridine was stereoselectively inhibited by 1,4-dihydropyridine enantiomers and nifedipine. (-)-Verapamil and (+)-verapamil inhibited high affinity [3H]-nimodipine binding in a negative heterotropic allosteric manner with (-)-verapamil being 5 times more potent than (+)-verapamil on an IC50 basis. At a given [3H]-nimodipine concentration, (+)-verapamil inhibited a greater fraction of specific [3H]-nimodipine binding. The allosteric mode of (+)-verapamil inhibition of [3H]-nimodipine binding was confirmed by kinetic studies. (-)-Verapamil shifted (+)-verapamil-binding inhibition curves to the right in an apparently competitive fashion. The inversion of staircase caused by both verapamil enantiomers suggests that they cause a use-dependent channel blockade. The similar potency ratios for binding and for cardiodepression are indicative of a common locus of action for both verapamil enantiomers within the calcium channel.