Comparison of the positive inotropic effects of serotonin,histamine, angiotensin II,endothelin and isoprenaline in the isolated human right atrium

Summary The receptor systems through which serotonin (5-HT), histamine, angiotensin II and endothelin increase the force of contraction were studied in isolated right atria from patients without apparent heart failure.All agonists increased the atrial force of contraction in a concentration-dependent manner; maximal effects, however, were significantly less than those evoked by isoprenaline or Ca²⁺. 5-HT and histamine, but not angiotensin II and endothelin, activated adenylate cyclase, whereas endothelin and angiotensin II stimulated inositol phosphate generation. Experiments with subtype-selective antagonists revealed that histamine effects were mediated by H₂-receptors (sensitive to ranitidine), 5-HT-effects by 5-HT₄-receptors (sensitive to SDZ 205-557) and angiotensin II effects by AT₁-receptors (sensitive to losartan).We conclude that in human right atria the force of contraction can be increased by cyclic AMP-dependent (histamine, 5-HT) and -independent (angiotensin II, endothelin) pathways. Compared to -adrenoceptors, however, all other receptor systems increase the force of contraction only submaximally indicating that the -adrenoceptor pathway is the most important physiological mechanism to regulate force of contraction and/or heart rate in the human heart.Correspondence to O. E. Brodde at the above address     

Trophic effect of angiotensin II in neonatal rat cardiomyocytes: role of endothelin-1 and non-myocyte cells

1. Angiotensin II (AII) and the endothelins (ET) are known to be potent trophic stimuli in various cells including cardiomyocytes. In order to characterize further these effects we studied, in neonatal rat ventricular cardiomyocytes, the effects of several endothelin-receptor antagonists and the AT₁-receptor antagonist losartan on AII- and endothelin-induced inositol phosphate (IP)-formation (assessed as accumulation of total [³H]-IPs in myo-[³H]-inositol prelabelled cells) and increase in rate of protein synthesis (assessed as [³H]-phenylalanine incorporation).
2. Endothelin (10 pM–1 μM) concentration-dependently increased IP-formation (max. increase at 100 nM ET-1: 130±14% above basal, n=25) and [³H]-phenylalanine incorporation (max. increase at 1 μM: 52±4% above basal, n=16) with an order of potency: ET-1>>ET-3. Both effects were antagonized by the ET_A/ET_B-receptor antagonist bosentan and the ET_A-receptor antagonist BQ-123, but not affected by the ET_B-receptor antagonist IRL 1038 and the AT₁-receptor antagonist losartan.
3. Pretreatment of the cells with 500 ng ml⁻¹ pertussis toxin (PTX) overnight that completely inactivated PTX-sensitive G-proteins did not attenuate but rather enhance ET-1-induced IP-formation. On the other hand, in PTX-pretreated cardiomyocytes ET-1-induced [³H]-phenylalanine incorporation was decreased by 39±5% (n=5).
4. AII (1 nM–1 μM) concentration-dependently increased IP-formation (max. increase at 1 μM: 42±7% above basal, n=16) and [³H]-phenylalanine incorporation (max. increase at 1 μM: 29±2%, n=9). These effects were antagonized by losartan, but they were also antagonized by bosentan and BQ-123.
5. In well-defined cultures of cardiomyocytes (not contaminated with non-myocyte cells) AII failed to increase [³H]-phenylalanine incorporation; addition of non-myocyte cells to the cardiomyocytes restored AII-induced increase in [³H]-phenylalanine incorporation.
6. We conclude that, in rat neonatal ventricular cardiomyocytes, (a) the ET-1-induced increase in rate of protein synthesis (through ET_A-receptor stimulation) involves at least two signalling pathways: one via a PTX-insensitive G-protein coupled to IP-formation, and the other one via a PTX-sensitive G-protein, and (b) the trophic effects of AII are brought about via local ET-1 secretion upon AT₁-receptor stimulation in neonatal rat ventricular non-myocyte cells.

     

Cardiac effects of beta-adrenoceptor antagonists with intrinsic sympathomimetic activity in humans: beta1- and/or beta2-adrenoceptor mediated?

The aim of this study was to find out whether cardiac responses to the beta-adrenoceptor antagonists with intrinsic sympathomimetic activity (ISA) xamoterol and celiprolol are mediated by cardiac beta1- or beta2-adrenoceptors or both. For this purpose we assessed, in six healthy male volunteers, the effects of xamoterol (100 and 200 mg, p.o.) and celiprolol (200, 600, and 1,200 mg, p.o.) on blood pressure, heart rate, and heart rate-corrected duration of the electromechanical systole (QS2c, as a measure of inotropism). Xamoterol, in both doses, increased systolic blood pressure and heart rate, transiently decreased diastolic blood pressure, and shortened QS2c; all these effects were attenuated after pretreatment of the volunteers with the beta1-adrenoceptor antagonist bisoprolol. Celiprolol, in all three doses, increased heart rate, decreased diastolic blood pressure, and shortened QS2c but only marginally increased systolic blood pressure. Bisoprolol did not attenuate these celiprolol effects but rather enhanced celiprolol effects on systolic blood pressure and heart rate. In a further set of experiments, we studied cardiovascular effects of celiprolol in six healthy volunteers whose beta2-adrenoceptors had been desensitized by a 2-week treatment with 3x5 mg/day terbutaline. Under these conditions, celiprolol failed to increase heart rate or to shorten QS2c. We conclude that, under resting conditions, in healthy volunteers, beta-adrenoceptor antagonists with ISA can exert increases in heart rate and contractility that are mediated by either cardiac beta1-adrenoceptor (xamoterol) or cardiac beta2-adrenoceptor (celiprolol) stimulation. Thus in the human heart, the ISA of beta-adrenoceptor antagonists can be a beta1- or beta2-adrenoceptor agonistic component.     

Endothelin-induced inositol phosphate formation in rat kidney. Studies on receptor subtypes, G-proteins and regulation during ontogenesis

The aim of this study was to characterize the properties of endothelin (ET)-receptor subtypes mediating inositol phosphate (IP)-formation in rat kidney and their regulation during ontogenesis. In renal cortical slices of adult rats (12–16 weeks old) ET's concentration-dependently increased IP-formation with an order of potency ET -1 ET 3. While the non-selective ET receptor antagonist bosentan (10 M) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 M) inhibited it only by 70%, the ET_B-receptor antagonist IRL 1038 (1 M) by 25%; combined application of BQ-123 + IRL 1038 caused complete inhibition of ET-1-induced IP-formation. Pretreatment of isolated renal cells with pertussis toxin (PTX, 500 ng/ml) overnight did not attenuate but significantly increased ET-1-induced IP-formation. Ontogenetic studies in renal slices from neonatal, 1, 2, 3, 6, 12 and 24 weeks old rats revealed that ET-1-induced IP-formation maturation-dependently declined being highest in neonatal rats (increase: 169% over basal) and lowest in 24 weeks old rats (increase: 47% over basal). This decline in ET-induced IP-formation was accompanied by a decrease in renal ET receptor number and the amount of immunodetectable G_q/11 (assessed by Western-blotting using the QL-antiserum). Moreover, ET receptor subtypes changed during the maturation process: from neonates to 12 weeks old rats number and functional responsiveness of ET_A-receptors declined, while that of ET_B-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ET_A- and ET_B-receptors and does not involve a PTX-sensitive G-protein. ET-induced IP-formation declines during the maturation process; this is associated with a decrease in ET-receptor number and the immunodetectable amount of G_q/11.     

Homogeneous class of beta-1 adrenergic receptors in rat kidney. Identification by (+/-)-125 iodocyanopindolol binding

The highly specific beta-adrenergic radio-ligand (+/-)-125 iodocyanopindolol (ICYP) was used to characterize the beta-adrenergic receptor subtype present in rat kidney. Binding of ICYP to membranes from rat kidney was of high affinity (KD = 68.9 pM) and saturable with 1.06 pmoles ICYP bound/g tissue wet wt at maximal occupancy of the sites. Analysis of inhibition of ICYP binding by beta 1- and beta 2-selective adrenergic drugs via pseudo-Scatchard ("Hoifstee') plots resulted in linear plots indicating the existence of a homogeneous population of beta-adrenergic receptors. From the resulting KD-values for practolol (2.2 microM), metoprolol (0.21 microM), zinterol (0.4 microM) and IPS 339 (0.046 microM) it is concluded that the beta-adrenergic receptor in rat kidney is of the beta 1-subtype. This subclassification is further supported by the fact that (-)-noradrenaline and (-)-adrenaline were equipotent in inhibiting ICYP binding. The beta-adrenergic agonists (-)-isoprenaline and zinterol bind to two distinct states of this beta 1-receptor, a high and low affinity state. GTP (10(-4) M) converts this heterogeneous binding into a homogeneous low affinity binding.     

In vitro and in vivo down-regulation of human plateletα 2-adrenoceptors by clonidine

Summary The effect of clonidine on the number of ₂-adrenoceptors in human platelet membranes, determined by³H-yohimbine binding, was investigatedin vitro andin vivo. Incubation of platelet membranes with clonidine (1–100 µM) for 16 h at 25 °C led to a concentration-dependent decrease in the number of³H-yohimbine binding sites of 10–25%; the affinity of³H-yohimbine to the sites was not changed (K_D approximately 3–4 nM). In such desensitized membranes, inhibition of³H-yohimbine binding by clonidine resulted in steep, monophasic displacement curves, which in comparison to the curves from control membranes (IC₅₀ for clonidine 90 nM), were shifted to the right (IC₅₀: 321 nM) and were not affected by 10^–4M guanosine-5-triphosphate (GTP).Treatment of 3 hypertensive patients with clonidine (3×150 µg/d for 7 days) reduced blood pressure and heart rate. Simultaneously, both³H-yohimbine binding sites on platelet membranes and plasma catecholamine levels decreased within three days and remained at a reduced level during treatment. After abrupt cessation of clonidine treatment, blood pressure, heart rate and plasma catecholamines rapidly increased, reaching values after two days similar to or higher than those before treatment.³H-yohimbine binding sites, however, initially decreased further before returning to control values. In platelet membranes derived from hypertensive patients treated with clonidine for at least three weeks, GTP (10^–4M) had no influence on inhibition of³H-yohimbine binding by (—)-adrenaline and clonidine. It is concluded that clonidine desensitizes ₂-adrenoceptors in human platelet membranesin vitro andin vivo. An important step in the desensitization process is the uncoupling of receptor occupancy by agonists and adenylate cyclase activity, as indicated by loss of the regulatory activity of GTP on desensitized membranes. The clonidine withdrawal syndrome may be caused by enhanced release of endogenous catecholamines not adequately regulated by presynaptic ₂-adrenoceptors, which have become subsensitive after chronic clonidine treatment.     

G-protein-coupled receptor kinase activity in human heart failure: effects of beta-adrenoceptor blockade

OBJECTIVES: In human end-stage heart failure as well as in experimental animal models of heart failure, G-protein-coupled receptor kinase activity (GRK) is increased while beta-adrenoceptor responsiveness is diminished. In animal studies, beta-adrenoceptor blockers reverse the GRK-mediated desensitization and down-regulation of myocardial beta-adrenoceptors. The aim of this study was to investigate whether alterations in GRK activity are an early or late accompaniment of human heart failure and whether also in humans beta-adrenoceptor blocker treatment is able to influence myocardial GRK activity. METHODS: We assessed in right atria, obtained from patients at different stages of heart failure, treated with or not treated with beta-adrenoceptor blockers, and in the four chambers of explanted hearts, obtained from patients with end-stage heart failure, beta-adrenoceptor density (by (-)-[(125)I]-iodocyanopindolol binding) and GRK activity (by an in vitro rhodopsin phosphorylation assay). RESULTS: With increasing severity of heart failure, plasma noradrenaline levels increased while myocardial beta-adrenoceptor density decreased with a maximum in GRK activity in end-stage heart failure. However, in relation to the progression of heart failure, we found that GRK activity transiently increased at an early stage of heart failure (NYHA I and II) but decreased back to control values in patients at NYHA III and IV. beta-Adrenoceptor blockers were able to reduce the early increase in GRK activity at NYHA I and II to control levels, whereas in those patients who did not have increased GRK activity (NYHA III and IV), they had only a marginal effect. CONCLUSION: According to our results, an increase in GRK activity is an early and transient event in the course of heart failure that can be prevented by beta-adrenoceptor blocker treatment.     

Down-regulated β-adrenoceptors in severely failing human ventricles: Uniform regional distribution,but no increased internalization

Summary In chronic heart failure cardiac -adrenoceptors are decreased. In this study we investigated whether a) in severely failing human ventricles -adrenoceptors are uniformly decreased or regional variations exist, and b) the -adrenoceptor decrease is caused by increased internalization or is a real loss in -adrenoceptors. For this purpose we assessed -adrenoceptor number and subtype distribution in a particulate fraction (mainly sarcolemmal plasma membranes) and a light vesicle fraction of right and left ventricular segments (obtained by cutting transversal, rings of 2 cm from the midventricular regions) of explanted hearts from 2 patients with end-stage congestive dilated cardiomyopathy (DCM) and one patient with end-stage ischemic cardiomyopathy (ICM). In all three hearts ventricular -adrenoceptor number was very low (7.5–10 and 21–26 fmol/mg protein in DCM, 15–22 fmol/mg protein in ICM compared to 68–74 fmol/mg protein in non-failing ventricles). -Adrenoceptors were uniformly decreased over the whole ventricular region and no considerable regional variations existed. The same held true for ₁- and ₂-adrenoceptors. In ICM decrease in -adrenoceptors was due to a concomitant reduction in ₁- and ₂-adrenoceptors, in DCM it was mainly caused by ₁-adrenoceptor down-regulation. In all ventricular segments investigated light vesicle -adrenoceptors amounted to about 5–7% of total ventricular -adrenoceptors and this was not significantly different from non-failing left ventricles. We conclude that a) in severely failing human ventricles -adrenoceptors are evenly down-regulated and no regional variations exist and b) the decrease in -adrenoceptors is not due to enhanced internalization but is a real loss of -adrenoceptors.Abbreviations DCM dillted cardiomyopathy - ICM ischemic cardiomyopathy - ICI 118,551 erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol hydrochloride - CGP 12177 (±)-4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride - ICYP (–) [¹²⁵I]-Iodocyanopindolol     

β-Adrenoceptor polymorphisms

There can be no doubt that ₁-, ₂- and ₃-adrenoceptor genes have genetic polymorphisms. Two single nucleotide polymorphisms have been described for the ₁- (Ser49Gly; Gly389Arg), three for the ₂- (Arg16Gly; Gln27Glu; Thr164Ile) and one for the ₃-adrenoceptor subtype (Trp64Arg) that might be of functional importance. The possibility that changes in expression or properties of the -adrenoceptors due to single nucleotide polymorphisms might have phenotypic consequences influencing their cardiovascular or metabolic function or may contribute to the pathophysiology of several disorders like hypertension, congestive heart failure, asthma or obesity is an idea that has attracted much interest during the last 10 years. At present, it appears that these -adrenoceptor polymorphisms are very likely not disease-causing genes, but might be risk factors, might modify disease and/or might influence progression of disease. The aim of this review is to provide an overview of the functional consequences of such -adrenoceptor polymorphisms in vitro, ex vivo and in vivo.     

Role of beta 1- and beta 2-adrenoceptors in hypertrophic and apoptotic effects of noradrenaline and adrenaline in adult rat ventricular cardiomyocytes

In adult rat ventricular cardiomyocytes alpha1-adrenoceptor (AR) stimulation causes increases in protein synthesis. On the other hand beta1-AR stimulation inhibits protein synthesis, and evokes apoptotic cell death. We studied, in adult rat ventricular cardiomyocytes, effects of noradrenaline (NA), adrenaline (ADR) and phenylephrine (PE) on protein synthesis (assessed by [3H]-phenylalanine incorporation into the cardiomyocytes) in relation to effects on early apoptosis (measured by Annexin V/propidium iodide staining). PE (10(-9)-10(-5) M) induced protein synthesis was not affected by the beta1-AR blocker CGP 20712A (CGP, 300 nM) or beta2-AR blocker ICI 118,551 (ICI, 55 nM). ADR (10(-9)-10(-5) M) induced protein synthesis was enhanced by CGP and decreased by ICI. Pretreatment of the cardiomyocytes with pertussis toxin (PTX) decreased NA- and ADR- induced protein synthesis, but did not affect PE-effects. NA (10(-5) M) and ADR (10(-5) M) caused a significant increase in the number of apoptotic cells; these effects were enhanced by PTX-treatment, abolished by CGP, but not significantly affected by ICI. Furthermore, there was a significant negative correlation between catecholamine-evoked apoptosis and catecholamine-induced hypertrophic effects. We conclude that, in ventricular cardiomyocytes of adult rats, growth-promoting effects of NA and ADR are composed of alpha1A-AR mediated increase in protein synthesis and beta1-AR mediated apoptosis that counteracts increases in protein synthesis. The role of beta2-adrenoceptor appears to be a balance of antiapoptotic effects via a PTX-sensitive pathway and proapoptotic effects via a GS-adenylyl cyclase pathway.