Fraser D. Russell

Cardiostimulant effects of urotensin-II in human heart in vitro.

The effects of the recently identified human peptide urotensin‐II (hU‐II) were investigated on human cardiac muscle contractility and coronary artery tone. In right atrial trabeculae from non‐failing hearts, hU‐II caused a concentration‐dependent increase in contractile force (pEC50=9.5±0.1; Emax=31.3±4.8% compared to 9.25u2003mM Ca2+; n=9) with no change in contraction duration. In right ventricular trabeculae from explanted hearts, 20u2003nM hU‐II caused a small increase in contractile force (7.8±1.4% compared to 9.25u2003mM Ca2+; n=3/6 tissues from 2 out of 4 patients). The peptide caused arrhythmic contractions in 3/26 right atrial trabeculae from 3/9 patients in an experimental model of arrhythmia and therefore has less potential to cause arrhythmias than ET‐1. hU‐II (20u2003nM) increased tone (17.9% of the response to 90u2003mM KCI) in 7/7 tissues from 1 patient, with no response detected in 8/8 tissues from 2 patients. hU‐II is a potent cardiac stimulant with low efficacy.

The human heart endothelin system: ET-1 synthesis, storage, release and effect

Endothelin-1 (ET-1) is synthesized, stored and released in the human heart. On release, ET-1 has the potential to activate specific receptors in a paracrine manner to modify heart function directly. Two binding sites corresponding to endothelin ETA and ETB receptors are localized to human heart. Contractility is modified by ETA receptors in the left ventricle and by ETA and ETB receptors, and a receptor that is possibly a different conformation of ETA and/or ETB receptors, in the right atrium. The endothelin system might directly support the failing heart but might also contribute to its pathology, providing a basis for the therapeutic use of endothelin receptor antagonists.

(-)-CGP 12177 increases contractile force and hastens relaxation of human myocardial preparations through a propranolol-resistant state of the β1-adrenoceptor

Abstract. Two forms of the activated β1-adrenoceptor exist, one that is stabilized by (-)-noradrenaline and is sensitive to blockade by (-)-propranolol and another which is stabilized by partial agonists such as (-)-pindolol and (-)-CGP 12177 but is relatively insensitive to (-)-propranolol. We investigated the effects of stimulation of the propranolol-resistant β1-adrenoceptor in the human heart. Myocardium from non-failing and failing human hearts were set up to contract at 1xa0Hz. In right atrium from non-failing hearts in the presence of 200xa0nM (-)-propranolol, (-)-CGP 12177 caused concentration-dependent increases in contractile force (–logEC50[M] 7.3±0.1, Emax 23±1% relative to maximal (-)-isoprenaline stimulation of β1- and β2-adrenoceptors, n=86 patients), shortening of the time to reach peak force (–logEC50[M] 7.4±0.1, Emax 37±5%, n=61 patients) and shortening of the time to reach 50% relaxation (t50%, –logEC50[M] 7.3±0.1, Emax 33±2%, n=61 patients). The potency and maxima of the positive inotropic effects were independent of Ser49Gly- and Gly389Arg-β1-adrenoceptor polymorphisms but were potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (–logEC50[M] 7.7±0.1, Emax 68±6%, n=6 patients, P<0.0001). In the presence of (-)-propranolol and 3-isobutyl-1-methylxanthine, the potency (–logEC50[M] 7.4±0.1, P=0.0013, n=9 patients) but not the maximal effect of (-)-CGP 12177 was reduced in right atrium from failing hearts, which was associated with 64% and 52% reductions in the densities of low-affinity and high-affinity (-)-[3H]CGP 12177 binding sites. In the presence of (-)-propanolol and 3-isobutyl-1-methylxanthine, (-)-CGP 12177 increased atrial cyclic AMP levels and activated cyclic AMP-dependent protein kinase in right atrium from non-failing hearts. In right ventricle from failing hearts (-)-CGP 12177 increased contractile force (–logEC50[M] 7.4±0.1, Emax 34±3%, n=13 patients) and hastened the time to peak force (–logEC50[M] 7.6±0.1) and time to reach 50% relaxation (–logEC50[M] 7.4±0.1) in the presence of (-)-propranolol and 3-isobutyl-1-methylxanthine. Our results show that (-)-CGP 12177 increases contractility and hastens relaxation through a cyclic AMP pathway in human myocardium, consistent with mediation through a (-)-propranolol-resistant state of the β1-adrenoceptor. The reduction in heart failure of atrial inotropic potency of (-)-CGP 12177, as well as of the high-affinity and low-affinity binding sites for (-)-[3H]CGP 12177, is consistent with the β1-adrenoceptor nature of these sites.

Conservation of the cardiostimulant effects of (−)-norepinephrine across Ser49Gly and Gly389Arg beta1-adrenergic receptor polymorphisms in human right atrium in vitro ☆

OBJECTIVEnThe goal of this study was to determine whether the cardiostimulant effects of the endogenous beta(1)-adrenergic receptor (AR) agonist, (-)-norepinephrine are modified by polymorphic (Serine49Glycine [Ser49Gly], Glycine389Arginine [Gly389Arg]) variants of beta(1)-ARs in the nonfailing adult human heart.nnnBACKGROUNDnHuman heart beta(1)-ARs perform a crucial role in mediating the cardiostimulant effects of (-)-norepinephrine. An understanding of the significance of Ser49Gly and Gly389Arg polymorphisms in the human heart is beginning to emerge, but not as yet in adult patients who have coronary artery disease (CAD).nnnMETHODSnThe potency and maximal effects of (-)-norepinephrine at beta(1)-ARs (in the presence of beta(2)-AR blockade with 50 nM ICI 118,551 [erythro-DL-1(7-methylindan-4-yloxy)-3-isopropylamino-butan-2-ol]) for changes in contractile force and shortening of contractile cycle duration were determined in human right atrium in vitro from 87 patients undergoing coronary artery bypass grafting who were taking beta-blockers before surgery. A smaller sample of patients (n = 20) not taking beta-blockers was also investigated. Genotyping for two beta(1)-AR polymorphisms (Ser49Gly and Gly389Arg) was determined from a sample of blood taken at the time of surgery.nnnRESULTSn(-)-Norepinephrine caused concentration-dependent increases in contractile force and reductions in time to reach peak force and time to reach 50% relaxation. There were no differences in the potency or maximal effects of (-)-norepinephrine in the right atrium from patients with different Ser49Gly and Gly389Arg polymorphisms.nnnCONCLUSIONSnThe cardiostimulant effects of (-)-norepinephrine at beta(1)-ARs were conserved across Ser49Gly and Gly389Arg polymorphisms in the right atrium of nonfailing hearts from patients with CAD managed with or without beta-blockers.

Characterization and localization of atypical β-adrenoceptors in rat ileum

1 . Homogenate binding studies and receptor autoradiography have been used to examine the binding characteristics and localization of propranolol‐resistant (—)‐[125I]‐cyanopindolol (CYP) binding sites in rat ileum. 2 . Saturation studies with (—)‐[125I]‐CYP and homogenates of rat ileum identified a site with pKD 8.89 ± 0.08 and Bmax=50.3±4.1 fmol mg−1 protein (n = 6). Both β1 and β‐adrenoceptors (AR) were not detected in these preparations. 3 . (—)‐Isoprenaline infusion (400 μg kg−1 h−1) for 14 days caused no significant change in the density of (−)−[125I]‐CYP binding which was 48.9±12.8 and 40.6±12.3 fmol mg−1 protein in control and isoprenaline‐treated animals respectively (n= 6) (P=0.97). 4 . Competition for (—)‐[125I]‐CYP binding in the presence of 0.1 μm (—)‐propranolol gave affinity values for CYP, tertatolol, alprenolol, ICI 118551 and CGP 20712A that correspond to known affinities at atypical β‐ARs. Stereoselectivity ratios for tertatolol and alprenolol were low. 5 . Autoradiographic localization of propranolol resistant (—)‐[125I]‐CYP binding showed sites associated with the mucosa and to a lesser extent to the muscularis. A small population of β2‐ARs were detected located predominantly in the longitudinal and circular smooth muscle layers. 6 . This study identifies an (—)‐[125I]‐CYP binding site in rat ileum that is resistant to blockade by propranolol (0.1 μm), is located predominantly in the mucosa, shows resistance to downregulation by isoprenaline and has binding characteristics of the atypical β‐AR.

Densitometric analysis of β1-and β2- adrenoceptors in guinea-pig atrioventricular conducting system

Quantitative autoradiography was used to determine the densities of β1- and β2-adrenoceptors in the atrioventricular conducting system in guinea-pig. (−)[125I]Cyanopindolol (CYP) was used to label β1- and β2-adrenoceptors in the absence or presence of the β1-adrenoceptor selective antagonist CGP 20712A (100 nm) or the β2-adrenoceptor selective antagonist ICI 118,551 (70 nm) or the non-selective β-adrenoceptor antagonist (−)-propranolol (1 μm). Protein in discrete anatomical regions was determined using a densitometric method based on the dye Coomassie brilliant blue G. In the atrioventricular conducting system the proportion of β2-adrenoceptors determined by inhibition of total (−)[125I]-CYP binding by CGP 20712A (100 nm) ranged from 32.5% (atrioventricular node) to 48.7% (left bundle branch). In the atrioventricular node (16.8 fmol/mg protein), bundle of His (12.1 fmol/mg protein), right (17.4 fmol/mg protein) and left (21.1 fmol/mg protein) bundle branches and Purkinje cells there was a higher density of β2-adrenoceptors than in the interventricular septum (8.4 fmol/mg protein) and right atria (8.3 fmol/mg protein). The medial smooth muscle of the aorta, aortic valve, adventitia of the aorta, nerve tissue, tricuspid and mitral valves contained only β2-adrenoceptors. It is speculated that the use of β-adrenoceptor antagonists to control cardiac arrhythmias involving a defect in conduction in the atrioventricular node should take into consideration both β1- and β2-adrenoceptors.

Activation of calcineurin in human failing heart ventricle by endothelin-1, angiotensin II and urotensin II

1 The calcineurin (CaN) enzyme–transcriptional pathway is critically involved in hypertrophy of heart muscle in some animal models. Currently there is no information concerning the regulation of CaN activation by endogenous agonists in human heart. 2 Human right ventricular trabeculae from explanted human (14 male/2 female) failing hearts were set up in a tissue bath and electrically paced at 1u2003Hz and incubated with or without 100u2003nM endothelin‐1 (ET‐1), 10u2003μM, angiotensin‐II (Ang II) or 20u2003nM human urotensin‐II (hUII) for 30u2003min. Tissues from four patients were incubated with 200u2003nM tacrolimus (FK506) for 30u2003min and then incubated in the presence or absence of ET‐1 for a further 30u2003min. 3 ET‐1 increased contractile force in all 13 patients (P<0.001). Ang II and hUII increased contractile force in three out of eight and four out of 10 patients but overall nonsignificantly (P>0.1). FK506 had no effect on contractile force (P=0.12). 4 ET‐1, Ang II and hUII increased calcineurin activity by 32, 71 and 15%, respectively, while FK506 reduced activity by 34%. ET‐1 in the presence of FK506 did not restore calcineurin activity (P=0.1). 5 There was no relationship between basal CaN activity and expression levels in the right ventricle. Increased levels of free phosphate were detected in ventricular homogenates that were incubated with PKCɛ compared to samples incubated without PKCɛ. 6 Endogenous cardiostimulants which activate Gαq‐coupled receptors increase the activity of calcineurin in human heart following acute (30u2003min) exposure. PKC may contribute to this effect by increasing levels of phosphorylated calcineurin substrate.

Regulation of ß-adrenoceptors in the guinea-pig sinoatrial node

This study examined the changes of ß-adrenoceptors in the guinea-pig sinoatrial nodal region following 7 day (−)-isoprenaline (400 μg/kg/h s.c.) infusion and the relationship between ß-adrenoceptor desensitization and receptor down-regulation. Changes in ß1-and ß2-adrenoceptor density were measured using quantitative autoradiography and function in organ bath studies.(−)-Isoprenaline treatment produced a marked decrease in total (from 57.5 to 33.9 fmol/mg protein), ß1-(from 49.4 to 32.8 fmol/mg protein), and ß2-adrenoceptor density (from 8.1 to 1.05 fmol/mg protein) in the sinoatrial node. In adjacent right atrium, treatment produced no change in total (39.5 and 36.7 fmol/mg protein) or ß1-adrenoceptors (35.9 and 36.4 fmol/mg protein) but did decrease ß2-adrenoceptors (from 3.7 to 0.3 fmol/mg protein). Chronotropic effects were measured in spontaneously beating right atrium. Procaterol, a selective ß2-adrenoceptor agonist, caused a biphasic chronotropic response in control right atria, the first part of which was abolished in the tissue from treated animals. The maximum increase in right atrial rate to R0363, a ß1-adrenoceptor selective partial agonist, was reduced from 114 bpm in control to 43 bpm in treated animals. In electrically driven right atrium with the sinoatrial node removed procaterol failed to produce a positive inotropic response via ß2-adrenoceptors, but the maximum response to RO 363 was reduced from 0.75 g in the control tissue to 0.12 g in the treated tissue.This study showed that changes in ß2-adrenoceptor density following 7 day (−)-isoprenaline infusion are compatible with reduced functional responsiveness in the SA node. The reduction of al-adrenoceptor number in the SA node was also compatible with the reduced chronotropic response in this tissue. However the lack of effect on ß1-adrenoceptor density in the right atrium was not consistent with the decrease in Qt-adrenoceptor mediated inotropic response in this tissue. This suggests that ß-adrenoceptor desensitization is not always associated with receptor down-regulation but depends also on the changes in the cell signalling system beyond the level of the receptor which differ according to the cardiac location.

FUNCTION, CHARACTERIZATION AND AUTORADIOGRAPHIC LOCALIZATION AND QUANTITATION OF β-ADRENOCEPTORS IN CARDIAC TISSUES

1. This paper demonstrates the use of organ bath, radioligand binding and autoradiography to detect β1‐ and β2‐adrenoceptors in human and guinea‐pig cardiac tissues.

Persistent β-adrenoceptor blockade with alkylating pindolol (BIM) in guinea-pig left atria and trachea

The actions of alkylating pindolol (N8-bromoacetyl-N1-3-(4-indolyloxy)-2-hydroxypropyl[z]-1,8- diamino-p-menthane; BIM) have been examined on beta-adrenoceptors in guinea-pig left atria and trachea. In organ bath experiments, addition of BIM (greater than or equal to 0.1 microM), followed by washout, produced concentration-dependent rightward shifts of the dose-response curve to cumulative additions of (-)-isoprenaline and oxymethylene-isoprenaline and reductions in the maximal response. The apparent pA2 value for BIM was 9.23 +/- 0.20 (slope = 0.98 +/- 0.20). Changes in the maximal density of beta-adrenoceptor binding sites were determined in guinea-pig left atrial membranes using [125I]-cyanopindolol. BIM (0.1, 1.0 and 10 microM) produced 14, 23 and 41% reductions in Bmax with no change in KD. The binding of [125I]-BIM to guinea-pig left atrial membranes had a high non-specific binding component and a pseudo-Hill coefficient less than unity. The apparent KD value of [125I]-BIM at beta-adrenoceptor binding sites was 85.7 +/- 57.9 pM.