The contribution of reactive oxygen species and p38 mitogen-activated protein kinase to myofilament oxidation and progression of heart failure in rabbits.

BACKGROUND AND PURPOSE: The formation of reactive oxygen species (ROS) is increased in heart failure (HF). However, the causal and mechanistic relationship of ROS formation with contractile dysfunction is not clear in detail. Therefore, ROS formation, myofibrillar protein oxidation and p38 MAP kinase activation were related to contractile function in failing rabbit hearts. EXPERIMENTAL APPROACH AND KEY RESULTS: Three weeks of rapid left ventricular (LV) pacing reduced LV shortening fraction (SF, echocardiography) from 32 +/- 1% to 13 +/- 1%. ROS formation, as assessed by dihydroethidine staining, increased by 36 +/- 8% and was associated with increased tropomyosin oxidation, as reflected by dimer formation (dimer to monomer ratio increased 2.28 +/- 0.66-fold in HF vs. sham, P < 0.05). Apoptosis (TdT-mediated dUTP nick end labelling staining) increased more than 12-fold after 3 weeks of pacing when a significant increase in the phosphorylation of p38 MAP kinase and HSP27 was detected (Western blotting). Vitamins C and E abolished the increases in ROS formation and tropomyosin oxidation along with an improvement of LVSF (19 +/- 1%, P < 0.05 vs. untreated HF) and prevention of apoptosis, but without modifying p38 MAP kinase activation. Inhibition of p38 MAP kinase by SB281832 counteracted ROS formation, tropomyosin oxidation and contractile failure, without affecting apoptosis. CONCLUSIONS AND IMPLICATIONS: Thus, p38 MAP kinase activation appears to be upstream rather than downstream of ROS, which impacts on LV function through myofibrillar oxidation. p38 MAP kinase inhibition is a potential target to prevent or treat HF.

Presynaptic alpha-2C adrenoceptor-mediated control of noradrenaline release in humans: genotype- or age-dependent?

In vitro alpha-2CDel322-325 adrenoceptor (AR) polymorphism exhibits reduced functional responsiveness. We studied whether this is true also in vivo in humans. We assessed in nine young wild-type (WT) alpha-2C AR subjects (aged 23 years), 10 elder WT alpha-2C AR subjects (aged 63 years), and nine alpha-2CDel AR subjects (aged 28 years) clonidine (1 microg/kg intravenous (i.v.) bolus)-evoked plasma noradrenaline (pNA), heart rate (HR), and blood pressure (BP) changes. Clonidine-evoked pNA decreases were comparable in young WT alpha-2C and in alpha-2CDel AR subjects, but significantly lower (P=0.033) in elder subjects. Similarly, clonidine-evoked HR decreases were significantly larger in young WT alpha-2C and in alpha-2CDel AR subjects than in elder subjects, whereas clonidine-evoked BP decreases were larger in elder subjects. In conclusion, alpha-2CDel AR appears to play only a minor role in presynaptic regulation of NA release and/or to be not hypofunctional in vivo in humans, but functional responsiveness of presynaptic alpha-2 AR declines with ageing.

[Role of the L-arginine/nitric oxide system in the action of nebivolol]. / Bedeutung des L-Arginin/NO-Systems für die Wirkung von Nebivolol.

Beta-blockers exert their effects through blockade of beta-adrenoceptors. First-generation beta-blockers non-selectively block beta1- and beta2-adrenoceptors, while second-generation beta-blockers predominantly block beta1-adrenoceptors. Third-generation beta-blockers possess additional vasodilator properties. Nebivolol is a highly selective b1-blocker with additional vasodilator effects which are mediated by the endothelial L-arginine/nitric oxide system. The hemodynamic action of nebivolol differs from those of first and second-generation beta-blockers: it acutely decreases peripheral resistance (while first and second-generation beta-blockers cause an initial transient increase in peripheral resistance) and decreases in heart rate that are less pronounced than those evoked by first and second-generation beta-blockers. Beta-blockers are frequently used in the treatment of hypertension, coronary artery disease and chronic heart failure. Nebivolol should thus be a suitable drug for treating these diseases, especially since nitric oxide has vasodilator and anti-atherogenic effects. One long-term trial (the SENIORS study) has shown that nebivolol, compared with a placebo, reduced death and hospital admission in elderly patients with chronic heart failure. Additional studies are needed to investigate the clinical relevance of nitric oxide-mediated vasodilatation and to show if nebivolol is as efficacious as the other beta-blockers--metoprolol, bisoprolol and carvedilol--in the treatment of chronic heart failure.

Beta-adrenoceptor polymorphisms.

There can be no doubt that beta(1)-, beta(2)- and beta(3)-adrenoceptor genes have genetic polymorphisms. Two single nucleotide polymorphisms have been described for the beta(1)- (Ser49Gly; Gly389Arg), three for the beta(2)- (Arg16Gly; Gln27Glu; Thr164Ile) and one for the beta(3)-adrenoceptor subtype (Trp64Arg) that might be of functional importance. The possibility that changes in expression or properties of the beta-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 beta-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 beta-adrenoceptor polymorphisms in vitro, ex vivo and in vivo.

Regulation of equine lymphocyte beta-adrenoceptors under the influence of clenbuterol and dexamethasone.

In 12 healthy horses, the effects of the beta2-agonist clenbuterol and the glucocorticoid dexamethasone on the lymphocyte beta2-adrenoceptor density and affinity (determined by (-)-[125I]-iodocyanopindolol binding) as well as its responsiveness (assessed by lymphocyte cyclic AMP [cAMP] responses to 10 micromol/l (-)-isoprenaline) were studied. Clenbuterol treatment, 2 x 0.8 microg/kg/day i.v. for 12 days, decreased significantly ICYP binding sites by approximately 30-40%; concomitantly, lymphocyte cAMP response to (-)-isoprenaline was reduced. After withdrawal of clenbuterol, beta2-adrenoceptor density and responsiveness gradually increased, reaching predrug levels after 4 days. The effects of dexamethasone on clenbuterol-induced desensitisation were further investigated. Administration of dexamethasone (1 x 0.1 mg/kg/day, i.v. for 5 days) immediately after clenbuterol withdrawal accelerated beta2-adrenoceptor recovery: only 24 h after administration dexamethasone restored the number of binding sites and cAMP response to (-)-isoprenaline to levels statistically indistinguishable from values before clenbuterol treatment. Three days after dexamethasone administration, lymphocyte beta2-adrenoceptors were further increased about 2-fold the pretreatment values, and this increase declined gradually after dexamethasone withdrawal, reaching baseline values after 4 days. Furthermore, in groups exposed simultaneously to both drugs, dexamethasone completely prevented clenbuterol-induced decrease in lymphocyte beta2-adrenergic receptor density and responsiveness. No significant change was observed in the dissociation constant for ICYP in any of the situations. We conclude that dexamethasone (glucocorticoids) can reverse and prevent Clenbuterol-induced desensitisation (down-regulation) of the lymphocyte beta2-adrenoceptors and therefore, a combined therapy with clenbuterol and dexamethasone may be potentially beneficial in horses suffering from chronic obstructive pulmonary disease (COPD).

Noradrenaline-induced increase in protein synthesis in adult rat cardiomyocytes: involvement of only alpha1A-adrenoceptors.

Adult rat ventricular cardiomyocytes contain alpha1A- and alpha1B-adrenoceptors (ARs, 20%:80%, assessed by [3H]prazosin binding). We studied which alpha1-AR subtype mediates noradrenaline (NA)-induced increase in rate of protein synthesis, and which signalling pathway is involved. NA (10-9-10-4 M) concentration-dependently increased inositol phosphate (IP) formation (pEC50-value=6.1+/-0.1, n=5) and protein synthesis (assessed as [3H]phenylalanine incorporation; pEC50-value=6.6+/-0.1, n=6). NA-induced IP-formation was partly inhibited by the alpha1B-AR antagonist chloroethylclonidine (CEC, 30 microM; 33+/-9% inhibition, n=5); following CEC-treatment the alpha1A-AR-selective 5-methyl-urapidil (5-MU) inhibited NA-induced IP-formation with a pKi-value of 9.2+/-0.2 (n=6); the alpha1D-AR-selective BMY 7378 was only a weak antagonist (pKi-value <7). NA-induced increase in protein synthesis was insensitive to CEC whereas 5-MU inhibited it with a pKi-value of 9.1+/-0.2 (n=6). NA (1 microM)-induced increase in protein synthesis was inhibited by the protein kinase C (PKC) inhibitor bisindolylmaleimide (IC50-value: 206 nM), the PI 3-kinase inhibitors wortmannin (IC50=3.4 nM) and LY 294002 (IC50=10 microM), and p70s6-kinase inhibitor rapamycin (IC50=123 pM) but not by the p38 MAP-kinase inhibitor SB 203580 (10 microM) or the MEK-inhibitor PD 98059 (25 microM). Moreover, 5-MU (30 nM) but not CEC inhibited NA-induced activation of p70s6-kinase. We conclude that, in adult rat cardiomyocytes, alpha1A- and alpha1B-AR mediate NA-induced IP-formation but only alpha1A-ARs mediate increase in protein synthesis. Alpha1A-AR-mediated increase in protein synthesis involves activation of a PKC, PI 3-kinase and p70s6-kinase but not of ERK- or p38 MAP-kinase.

Enhanced protein phosphorylation in hypertensive hypertrophy.

OBJECTIVE: Chronic pressure overload in spontaneously hypertensive rats (SHR) is accompanied by heart hypertrophy and signs of heart failure. Since there is growing evidence for a possible pathophysiological role of altered protein phosphorylation in heart hypertrophy and failure, we studied here cardiac regulatory phosphoproteins and the kinases and phosphatases which regulate their phosphorylation state. METHODS: The experiments were performed in ventricles of SHR (12-13 weeks old) and age-matched normotensive Wistar-Kyoto rats (WKY). RESULTS: Basal as well as isoproterenol (Iso)-stimulated force of contraction (FOC) was markedly decreased in isolated electrically driven papillary muscles of SHR. Iso (3 micromol/l, 10 min) increased FOC by 0.91+/-0.20 mN in SHR and by 3.88+/-0.52 mN in WKY, respectively. Ca(2+)-uptake by sarcoplasmic reticulum (SR) at low ionized Ca(2+)-concentration was increased in homogenates from SHR. This was not due to altered expression of phospholamban (PLB), SR-Ca(2+)-ATPase and calsequestrin. However, PLB-phosphorylation at threonine-17 (PLB-PT-17) and the activity of Ca(2+)/calmodulin dependent protein kinase (Ca(2+)/Cam-PK) was increased in SHR. In addition, we found an enhanced protein kinase A (PKA)-dependent phosphorylation of the inhibitory subunit of troponin (TnI). In contrast, there was no difference in the activity or expression (protein- and mRNA-level) of protein phosphatases type 1 or type 2A between SHR and WKY. CONCLUSIONS: It is suggested that increased Ca(2+)/Cam-PK-activity with resulting increase of PLB-PT-17 enhanced SR-Ca(2+)-uptake in SHR and might contribute to the pathophysiological changes in cardiac hypertrophy of SHR.

Muscarinic receptors in the mammalian heart.

In the mammalian heart, cardiac function is under the control of the sympathetic and parasympathetic nervous system. All regions of the mammalian heart are innervated by parasympathetic (vagal) nerves, although the supraventricular tissues are more densely innervated than the ventricles. Vagal activation causes stimulation of cardiac muscarinic acetylcholine receptors (M-ChR) that modulate pacemaker activity via I(f) and I(K.ACh), atrioventricular conduction, and directly (in atrium) or indirectly (in ventricles) force of contraction. However, the functional response elicited by M-ChR-activation depends on species, age, anatomic structure investigated, and M-ChR-agonist concentration used. Among the five M-ChR-subtypes M(2)-ChR is the predominant isoform present in the mammalian heart, while in the coronary circulation M(3)-ChR have been identified. In addition, evidence for a possible existence of an additional, not M(2)-ChR in the heart has been presented. M-ChR are subject to regulation by G-protein-coupled-receptor kinase. Alterations of cardiac M(2)-ChR in age and various kinds of disease are discussed.

Impaired beta-adrenergic control of immune function in patients with chronic heart failure: reversal by beta1-blocker treatment.

BACKGROUND: In patients with chronic heart failure (CHF) and heightened sympathetic activity alterations in immune function have been described. OBJECTIVES: To find out whether, in CHF patients, beta-blocker treatment might beneficially affect immune function. METHODS: We studied activation of circulating lymphocytes (assessed as concanavalin A (CON A)-induced inositol phosphate (IP) formation and proliferation ([3H]-thymidine incorporation) from 8 CHF patients on standard medication (Group A, mean age 54 +/- 6 yrs, NYHA class II - IV, mean 3.1 +/- 0.3) and in 9 CHF patients on standard medication and additional treatment with the beta1-blocker metoprolol (Group B, mean age: 56 +/- 3 yrs, NYHA class II - IV, mean 2.9 +/- 0.2); 8 age-matched healthy volunteers (mean age 49 +/- 3 yrs) served as controls. RESULTS: Compared to controls, in group A isoprenaline-induced lymphocyte cyclic AMP-increase was reduced, CON A-evoked IP formation significantly enhanced and isoprenaline-induced inhibition of CON A-evoked IP formation and proliferation almost abolished. In group B, however, all these parameters were not significantly different from controls. CONCLUSION: In CHF patients lymphocyte cyclic AMP response to beta-adrenoceptor stimulation is blunted and the inhibitory effect of cyclic AMP on lymphocyte activation is almost abolished; this could result in a non-regulated increased production and release of proinflammatory cytokines that might contribute to the progression of the disease. Chronic treatment of CHF patients with the beta1-blocker metoprolol (at least partly) restores lymphocyte cyclic AMP responses to beta-adrenoceptor stimulation and the inhibitory effects of cyclic AMP on lymphocyte activation; the resulting "normalization" of the immune function might contribute to the beneficial effects of beta1-blockers in treatment of CHF.

In-vivo studies do not support a major functional role for the Gly389Arg beta 1-adrenoceptor polymorphism in humans.

beta 1-adrenoceptors play a pivotal role in regulating contractility and heart rate in the human heart. Recently, a polymorphism of the beta 1-adrenoceptor has been detected: at amino acid position 389 either Gly or Arg has been found with the Gly389 exhibiting reduced responsiveness upon agonist-induced stimulation in vitro. In order to find out whether the Gly389 polymorphism exhibits blunted responsiveness also in vivo we studied, in healthy volunteers, the effects of exercise on heart rate and heart rate-corrected duration of electromechanical systole (QS2c as a measure of inotropism) which, in humans, is mediated by beta 1-adrenoceptors stimulation. Twenty-four healthy volunteers (12 female, 12 male) homozygous for the Gly389 or Arg389 exercised on a bicycle in supine position (25, 50, 75 and 100 W for 5 min each), and heart rate and QS2c were assessed; in addition, plasma renin activity (PRA) was determined which is also regulated by beta 1-adrenoceptors in humans. Exercise caused work-load dependent increases in heart rate and PRA, and shortening of QS2c; however, these changes were not significantly different between the Gly389 and Arg389 polymorphism. Thus, these three beta 1-adrenoceptor responses did not differ between volunteers with the Arg389 versus the Gly389 polymorphism. Intragroup analysis, however, revealed that exercise induced increase in heart rate and shortening of QS2c were higher in female than in male volunteers. In conclusion, our data do not support the idea that the reduced responsiveness of Gly389 against agonist-induced stimulation observed in vitro is of major functional importance in vivo.

ET-receptor function in relation to blood pressure in spontaneously hypertensive and aortic-banded rats.

Endothelin-1 (ET-1), a potent endogenous vasoconstrictor, has been proposed to play a pathophysiologic role in hypertension. The aim of this study was to find out whether changes in ET-receptor function are cause or consequence of blood pressure elevation in hypertension. For this purpose, we assessed ET-receptor function [as ET-1-induced [3H]inositol phosphate (IP) accumulation] in slices of left ventricle and renal cortex and in rings of thoracic and abdominal aorta from spontaneously hypertensive rats (SHR) at the age of 8 weeks (i.e. developing hypertension), 12 and 24 weeks (established hypertension) vs. normotensive age-matched Wistar-Kyoto (WKY) rats, and from supra-renal aortic-banded (AOB) rats at the age of 8, 12 and 24 weeks (i.e. 4, 8 and 20 weeks after AOB) vs. sham-operated (SOP) age-matched WKY rats. In the SHR with established hypertension ET-1-induced IP formation was altered in all tissues investigated: it was significantly increased vs. WKY rats in left ventricle, and significantly decreased in renal and aortic tissues. Similarly, in AOB rats at all ages ET-1-induced IP formation was changed in those tissues that were under pressure load [heart (increase) and thoracic aorta (decrease)] vs. SOP rats, whereas in those tissues not under pressure load (kidney and abdominal aorta) ET-1-induced IP formation was not different between AOB and SOP rats. Moreover, in 8-week-old SHR (where hypertension is not yet established) ET-1-induced IP formation was not significantly different compared to WKY rats (with the exception of thoracic aorta). We conclude that, at least in SHR and AOB rats, changes in ET-1 signalling are secondary to the elevation in blood pressure.

Inositolphosphate formation in thoracic and abdominal rat aorta following Gq/11-coupled receptor stimulation.

Although thoracic and abdominal rat aorta are often used as a classical pharmacological preparation for the assessment of vascular drug effects, little is known on regional differences among these two parts of the aorta with regard to their reaction to Gq/11-coupled receptor activation. Thus, we determined, in rings from thoracic and abdominal aorta from 12-week-old male Wistar rats, the effects of noradrenaline (NA; 10(-8)-10(-4) M), endothelin-1 (ET-1; 10(-10)-10(-6) M) and the thromboxane A2 mimetic U 46619 (10(-8)-10(-5) M) on inositolphoshate (IP) formation (assessed as accumulation of total [3H]IPs in [3H]myoinositol prelabelled rings). NA, ET-1 and U 46619 concentration-dependently increased IP formation; maximum increases were, however, significantly more pronounced in thoracic than in abdominal aorta. Similarly, NA, ET-1 and U 46619 evoked significantly larger maximum contractions in thoracic than in abdominal aorta. NA-induced [3H]IP formation could be inhibited with BMY 7378 (10(-9)-10(-4) M) and with 5-methyl-urapidil (5-MU; 10(-9)-10(-5) M) both exhibiting biphasic concentration-inhibition curves. The pKi-values for BMY 7378 at the high affinity site were in thoracic aorta 8.93+/-0.28 (n=5), and in abdominal aorta 8.76+/-0.35 (n=4) and at the low affinity site 6.45+/-0.2 (thoracic aorta) and 6.55+/-0.27 (abdominal aorta). pKi-Values for 5-MU in thoracic aorta at the high affinity site were 8.25+/-0.34 (n=4), and at the low affinity site 6.61+/-0.39 . In abdominal aorta reliable pKi-values could not be calculated for 5-MU due to a low signal-to-noise ratio. On the other hand, in both preparations the ETA-receptor antagonist BQ-123 (10(-9)-10(-5) M) and the TP-receptor antagonist SQ 29548 (10(-9)-10(-5) M) inhibited ET-1- and U 46619-induced IP formation, respectively, with monophasic concentration-inhibition curves: pKi-values for BQ-123 were: 8.16+/-0.24 (thoracic aorta) and 8.10+/-0.35 (abdominal aorta) and for SQ 29548: 8.2+/-0.3 (thoracic aorta) and 8.5+/-0.3 (abdominal aorta). The amount of immunodetectable Gq/11-protein was similar in both tissues. We conclude that responses to NA, ET-1 and U 46619 (IP formation and contractile force) are larger in thoracic than in abdominal aorta. ET-1 effects on IP formation are mediated by ETA-receptors and U 46619 effects by TP-receptors. NA effects are mediated by alpha1D- and alpha1A-adrenoceptors; alpha1B-adrenoceptors seem to play a minor role.

Blunted cardiac responses to receptor activation in subjects with Thr164Ile beta(2)-adrenoceptors.

BACKGROUND: Recent evidence indicates that certain genotypes of beta(2)-adrenoceptors (AR) may indicate an increased risk of cardiovascular disease or an increased rate of disease progression. Of particular importance, the Thr164Ile polymorphism, which is found in approximately 4% of humans, shows decreased receptor signaling, blunted cardiac response when expressed in transgenic mice, and is associated with a decreased survival rate in patients with congestive heart failure. METHODS AND RESULTS: In this study, we compared functional activity, ie, chronotropic (heart rate increases) and inotropic (duration of the electromechanical systole) responses to intravenously administered terbutaline, in 6 subjects (4 women and 2 men) who were heterozygous for Thr164Ile with the responses in 12 volunteers (6 women and 6 men) who were homozygous for wild-type (WT) beta(2)-AR (ie, Arg16, Gln27, and Thr164). The beta(2)AR polymorphism significantly affected the dose-response curves for terbutaline-induced inotropic and chronotropic responses: compared with WT individuals, subjects with the Thr164Ile receptor had substantial blunting in maximal increases in heart rate (WT, 29.7+/-3.9 beats/min; Ile164, 20.7+/-1.9 beats/min; P:=0.016) and a shortening of the duration of electromechanical systole (WT, 51.9+/-4.5 ms; Ile164, 37.9+/-4.6 ms; P:=0.02). CONCLUSIONS: These data show that humans with the Ile164 genotype show blunted cardiac beta(2)-AR responsiveness, which may help explain the decreased survival of patients with this genotype in the setting of congestive heart failure.

Beta-adrenoceptor stimulation attenuates the hypertrophic effect of alpha-adrenoceptor stimulation in adult rat ventricular cardiomyocytes.

OBJECTIVES: The study investigated whether beta-adrenoceptor antagonists augment the hypertrophic response of cardiomyocytes evoked by norepinephrine. BACKGROUND: In adult ventricular cardiomyocytes, stimulation of alpha- but not beta-adrenoceptors induces myocardial hypertrophy. Natural catecholamines, like norepinephrine, stimulate simultaneously alpha- and beta-adrenoceptors. We investigated whether beta-adrenoceptor stimulation interferes with the hypertrophic response caused by alpha-adrenoceptor stimulation. METHODS: Adult ventricular cardiomyocytes isolated from rats were used as an experimental model. Hypertrophic parameters under investigation were stimulation of phenylalanine incorporation and protein mass, stimulation of 14C-uridine incorporation and RNA mass, and increases in cell shape. RESULTS: Norepinephrine (0.01 to 10 micromol/liter) increased concentration-dependent phenylalanine incorporation; pEC50 value was 5.9 +/- 0.1 (n = 8). The alpha1-adrenoceptor antagonist prazosin (0.1 micromol/liter) suppressed norepinephrine-induced increase in rate of protein synthesis. Conversely, propranolol (1 micromol/liter) and the beta1-adrenoceptor selective antagonists CPG 20712A (300 nmol/liter) or atenolol (1 micromol/liter) augmented increases in phenylalanine incorporation caused by norepinephrine. Addition of the beta2-adrenoceptor antagonist ICI 118,551 (55 nmol/liter) did not influence the hypertrophic effect of norepinephrine. Atenolol augmented the norepinephrine-induced increases of all hypertrophic parameters investigated (i.e., protein mass, uridine incorporation, RNA mass, cell volume, and cross-sectional area). In the presence of norepinephrine, inhibition of beta1-adrenoceptors increased the amount of protein kinase C-alpha and -delta isoforms translocated into the particulate fraction. The effect of pharmacological inhibition of beta1-adrenoceptors could be mimicked by Rp-cAMPS (adenosine-3', 5'-cyclic phosphorothiolate-Rp). The inhibitory effect of beta1-adrenoceptor stimulation on the alpha-adrenoceptor-mediated effect persisted in cardiomyocytes isolated from hypertrophic hearts of rats submitted to aortic banding. CONCLUSIONS: In isolated ventricular cardiomyocytes from rats, beta1-adrenoceptor stimulation attenuates the hypertrophic response evoked by alpha1-adrenoceptor stimulation.

Presence, distribution and physiological function of adrenergic and muscarinic receptor subtypes in the human heart.

The sympathetic and parasympathetic nervous system play a powerful role in controlling cardiac function by activating adrenergic and muscarinic receptors. In the human heart there exist alpha1-, beta1- and beta2-adrenoceptors and M2-muscarinic receptors and possibly also (prejunctional) alpha2-adrenoceptors. Beta1- and beta2-adrenoceptors are quite evenly distributed in the human heart while M2-receptors are heterogeneously distributed (more receptors in atria than in ventricles). Stimulation of beta1- and beta2-adrenoceptors causes increases in heart rate and force of contraction while stimulation of M2-receptors decreases heart rate (directly in atria) and force of contraction (indirectly in ventricles). Pathological situations (such as heart failure) or pharmacological interventions (for example, beta-blocker treatment) can alter the distribution of beta1- and beta2-adrenoceptors in the human heart, while M2-receptors are only marginally affected. On the other hand, relatively little is known on distribution and functional role of alpha1- and alpha2-adrenoceptor subtypes in the human heart.

The cardiac beta-adrenoceptor-G-protein(s)-adenylyl cyclase system in monocrotaline-treated rats.

In rats, injection of the alkaloid monocrotaline (MCT) causes right ventricular hypertrophy and cardiac failure. In order to study whether, in MCT-treated rats, changes in the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system might be comparable to those found in human primary pulmonary hypertension, we assessed in right and left ventricles from MCT-treated rats the components of the beta -adrenoceptor system: the receptor number and subtype distribution (by (-)-[(125)I]iodocyanopindolol binding), the G-proteins (by quantitative Western blotting), and the activity of adenylyl cyclase. A single injection of 60 mg/kg i.p. MCT caused in rats right ventricular hypertrophy (RVH); part of the rats developed cardiac failure (RVF). In these rats the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system was markedly changed beta -adrenoceptors were desensitized due to a decrease in receptor number, an uncoupling of the receptor from the G(s)-adenylyl cyclase system, a decrease in G(s)and a decrease in the activity of the catalytic unit of adenylyl cyclase. In general, these changes were more pronounced in right ventricles v left ventricles, and in rats with RVF v rats with RVH. On the other hand, cardiac muscarinic receptors and G(i)appeared not to be altered. We conclude that in MCT-treated rats changes in the cardiac beta -adrenoceptor-G-protein(s)-adenylyl cyclase system occur that resemble those observed in human primary pulmonary hypertension. Thus, MCT-treated rat appears to be a suitable animal model to study in more detail the pathophysiology of the development of right heart failure, and to identify new therapeutic possibilities.

Differential pattern of endothelin-1-induced inotropic effects in right atria and left ventricles of the human heart.

In human right atrium, endothelin A (ET(A)) receptors couple to both inositol phosphate formation and inhibition of adenylylcyclase, whereas in human left ventricle, ET(A) receptors couple only to inositol phosphate formation. To find out whether this might be of functional relevance, we studied, in right atria obtained from 32 patients undergoing coronary bypass grafting without apparent heart failure, and in right atria and left ventricles from eight patients with end-stage heart failure (NYHA IV) undergoing heart transplantation, the effects of endothelin-1 (ET-1) on basal force of contraction or on force of contraction increased by 1 microM forskolin. ET-1 (0.1 microM) exerted a positive inotropic effect in atrial and ventricular tissue; this could be antagonized by the ET(A)-receptor antagonist BQ 123, but not by the ET(B)-receptor antagonist BQ 788. In atrial, but not in ventricular tissue, this positive inotropic effect was preceded by a transient negative inotropic effect. This negative inotropic effect was inhibited by BQ 123, but not by BQ 788. It was significantly prolonged in forskolin-prestimulated atria, and was significantly larger in atria from failing hearts. We conclude that, because ET-1 inhibits adenylylcyclase and causes negative inotropic effects in atria but not in ventricles, adenylylcyclase inhibition might be responsible for the transient negative inotropic effect of ET-1.

Chamber-specific alterations of noradrenaline uptake (uptake(1)) in right ventricles of monocrotaline-treated rats.

1. In rats a single injection of the alkaloid monocrotaline (60 mg MCT kg(-1) body weight, i.p.) caused right ventricular hypertrophy and heart failure. The aim of this study was to find out whether, in these MCT-treated rats, the cardiac neuronal noradrenaline uptake (uptake(1)) might undergo chamber-specific alterations. 2. For this purpose we assessed in right and left ventricular slices, uptake(1) activity (by [(3)H]-noradrenaline accumulation), and in right and left ventricular membranes, uptake(1) carrier protein density (by [(3)H]-nisoxetine binding). 3. Uptake(1)-inhibitors blocked [(3)H]-noradrenaline accumulation in ventricular slices and [(3)H]-nisoxetine binding in ventricular membranes with the order of potency: desipramine > nisoxetine >> cocaine > or = GBR 12909, indicating that with both approaches noradrenaline uptake(1) was determined. 4. In right ventricular slices of MCT-treated rats uptake(1) activity was significantly lower than in control rats (84.7+/-8.2 vs 145.1+/-6.2 pmol noradrenaline mg(-1) tissue 15 min(-1); P<0.05). This was accompanied by a significant decrease in the density of [(3)H]-nisoxetine binding sites (73.7+/-14.4 vs 125.9+/-9.1 fmol mg(-1) protein; P:<0.05). 5. In left ventricular slices of MCT-treated rats uptake(1) activity was not significantly altered (131.2+/-10.5 vs 116.1+/-5.2 pmol noradrenaline mg(-1) tissue 15 min(-1)); similarly, also the density of [(3)H]-nisoxetine binding sites was unchanged (108+/-9.7 vs 123+/-7.7 fmol mg(-1) protein). 6. We conclude that in MCT-treated rats with right ventricular hypertrophy and heart failure uptake(1) activity is chamber-specifically reduced possibly due to a decrease in carrier protein density.

Cardiac beta-adrenoceptors in chronic uremia: studies in humans and rats.

OBJECTIVES: The purpose of this study was to elucidate whether cardiac beta-adrenergic effects may be blunted in patients on maintenance hemodialysis (HD) and may help to explain autonomic dysfunction. BACKGROUND: Patients on HD often suffer from autonomic dysfunction. METHODS: We investigated the cardiovascular response of five HD patients (age: 46.1+/-7.9 years) and six healthy volunteers (age: 48.2+/-7.5 years) to isoprenaline, pirenzepine and phenylephrine. For analysis of underlying mechanisms of beta-adrenoceptor hyporesponsiveness, six-week-old male Wistar rats were rendered uremic by 5/6-nephrectomy (n = 9; SNX) and were killed for removal of the heart after six to seven weeks. Sham-operated rats (n = 15) served as controls. RESULTS: In the patient study, isoprenaline (3.5, 7, 17, 35 ng/kg/min, i.v.) led to an increase in heart rate, and shortening of the heart rate corrected duration of the electromechanical systole (QS2c), both of which were significantly reduced in HD patients. Baroreflex sensitivity was significantly reduced in HD patients. The response to low parasympathomimetic doses of pirenzepine was unchanged. In the rat study, left ventricular strips were placed in an organ bath, electrically driven and exposed to isoprenaline (10(-11) to 10(-6) mol/liter). While pD2 values were unchanged, maximum effect at the highest concentration was significantly reduced in SNX rats. The response to carbachol was not altered, nor was the M2-cholinoceptor density. There was no difference in beta-adrenoceptor density, or in immunodetectable amount of Gs and Gi protein. Activation of adenylyl cyclase evoked by isoprenaline was significantly reduced in left ventricular membranes of SNX rats, whereas effects of 10 micromol/liter GTP, 10 mmol/liter NaF, 10 micromol/liter forskolin and 10 mmol/liter Mn2+ were not altered. CONCLUSIONS: Cardiac beta-adrenergic responses are blunted in chronic uremia due to reduced isoprenaline-dependent activation of adenylyl cyclase. This might be caused by an "uncoupling" of the receptor or by an inhibition of the receptor by uremic toxins.

Human cardiac beta1- or beta2-adrenergic receptor stimulation and the negative chronotropic effect of low-dose pirenzepine.

OBJECTIVES: The M1-muscarinic receptor antagonist pirenzepine in low doses (<1 mg intravenously) decreases heart rate. We investigated whether these effects of pirenzepine differ in volunteers with activated cardiac beta1-adrenergic receptors versus activated cardiac beta2-adrenergic receptors. METHODS: In 17 male volunteers (25 +/- 1 years) we studied effects of pirenzepine infusion (0.5 mg intravenous bolus followed by continuous infusion of 0.15 microg/kg/min) on heart rate and heart rate-corrected duration of electromechanical systole (QS2c, as a measure of inotropism) that had been stimulated by activation of cardiac beta1-adrenergic receptors (bicycle exercise in the supine position for 60 minutes at 25 W) or cardiac beta2-adrenergic receptors (continuous intravenous infusion of 100 ng/kg/min terbutaline). RESULTS: Bicycle exercise and terbutaline infusion significantly increased heart rate and shortened QS2c. When pirenzepine was infused 20 minutes after the beginning of the exercise or terbutaline infusion, heart rate decreased in both settings by approximately the same extent (approximately -10 to -14 beats/min), although exercise and terbutaline infusion continued; however, QS2c was not affected. Pirenzepine (0.05 to 1 mg intravenous bolus)-induced decrease in heart rate was abolished after 6 days of transdermal scopolamine treatment of volunteers. CONCLUSIONS: Low-dose pirenzepine decreased heart rate by muscarinic receptor stimulation, because this was blocked by scopolamine. Moreover, low-dose pirenzepine did not differentiate between cardiac beta1- or beta2-adrenergic receptor stimulation; however, low-dose pirenzepine did not affect cardiac contractility as measured by QS2c. Low-dose pirenzepine therefore exerted a unique pattern of action in the human heart: it decreased heart rate (basal and beta1- and/or beta2-adrenergic receptor-stimulated) without affecting contractility.