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Our recent findings indicate that, in rat neonatal ventricular cardiomyocytes, endothelin-1 (ET-1) induces increases in the rate of protein synthesis in a partly pertussis toxin (PTX)-sensitive manner, and that angiotensin II-evoked increases in the rate of protein synthesis are brought about via local secretion of ET-1. The aim of this study was to find out: (1) whether noradrenaline (NA) and the thromboxane A2 (TXA2)-mimetic U 46619-induced increases in the rate of protein synthesis may be also partly PTX-sensitive and/or mediated by ET-1, and (2) whether the growth-promoting effects of NA and U 46619 as well as ET-1 might involve activation of the same set of protein kinase C (PKC) isozymes. For this purpose we first studied the effects of NA and U 46619 on inositol phosphate (IP)-formation (assessed as accumulation of total [3H]IPs in myo-[3H]inositol prelabelled cells) and on the rate of protein synthesis (assessed as [3H]phenylalanine incorporation) (1) in the presence and absence of the ETA-receptor antagonist BQ-123, and (2) in nontreated and PTX-pretreated cells. Second, we assessed the effects of the PKC-inhibitors bisindolylmaleimide I and Gö 6976 and of phorbol-12-myristate-13-acetate (PMA; 1 µM overnight)-pretreatment on U 46619-, NA- and ET-1-induced increases in the rate of protein synthesis. NA (0.01–10 µM) concentration-dependently increased IP-formation (maximum increase: 115±23% above basal, n=4) and [3H]phenylalanine incorporation (maximum increase: 40±3% above basal, n=20). Both responses were antagonized by the α1-adrenoceptor antagonist prazosin (1 µM), but were not significantly affected by BQ-123 (1 µM). U 46619 (0.01–100 µM) concentration-dependently increased IP-formation (maximum increase: 89±12% above basal, n=8) and [3H]phenylalanine incorporation (maximum increase: 33±4% above basal, n=16). Both responses were slightly but significantly antagonized by the TP-receptor antagonist SQ 29548 (1 µM), but were not affected by BQ-123 (1 µM). Pretreatment of the cardiomyocytes with 250 ng ml–1 PTX overnight did not significantly affect NA- and U 46619-evoked increases in IP-formation and [3H]phenylalanine incorporation. The PKC-inhibitor bisindolylmaleimide I (5 µM) as well as pretreatment of the cells with PMA (1 µM) significantly reduced the effects of NA, U 46619 and ET-1 on the rate of protein synthesis; in contrast, the PKC-inhibitor Gö 6976 (5 µM) was without any effects. We conclude that, in rat neonatal ventricular cardiomyocytes, stimulation of Gq/11-coupled receptors increases the rate of protein synthesis; this involves activation of the same PKC-isozymes (very likely PKC-δ and/or -ε). NA and U 46619 cause their growth-promoting effects in a PTX-insensitive manner; ET-1 is not involved in their effects. [ABSTRACT FROM AUTHOR]

In rat ventricular cardiomyocytes beta2-adrenoceptors (AR) couple to Gs- and Gi-protein, and evidence has accumulated that beta2-AR agonists can differentially activate either Gs- or Gs- and Gi-protein. In this study, in isolated adult rat ventricular cardiomyocytes, we assessed the effects of pertussis toxin (PTX) on beta2-AR agonist (terbutaline (TER), salbutamol (SAL) and fenoterol (FEN)) evoked inhibition of phenylephrine (PE)-induced increase in the rate of protein synthesis (assessed as [3H]phenylalanine incorporation) to find out which beta2-AR agonist activates selectively Gs- or Gs- and Gi-protein. PE (1 microM) increased the rate of protein synthesis from 100% to 130+/-2% (n = 34). FEN, TER and SAL (1 nM-10 microM) inhibited PE-induced increase in the rate of protein synthesis concentration-dependently. FEN inhibited PE effects almost completely (from 132+/-3 to 101+/-1%), whereas TER and SAL caused only partial inhibition (from 131+/-2 to 114+/-2 and 129+/-1 to 111+/-2%, respectively). Pretreatment of cardiomyocytes with PTX (250 ng ml(-1) for 16 h at 37 degrees C) did not affect FEN effects, but converted TER- and SAL-evoked partial inhibition into complete inhibition. Inhibitory effects of the three beta2-AR agonists were markedly attenuated by beta1-AR selective antagonist CGP 20712A (CGP) (300 nM); in contrast, beta2-AR selective antagonist ICI 118,551 (55 nM) inhibited the inhibitory effects of the three beta2-AR agonists only in PTX-pretreated cardiomyocytes,with beta1-AR blocked by CGP. We conclude that, in adult rat ventricular cardiomyocytes, FEN activates selectively the Gs protein-pathway, while TER and SAL activate the Gs- and Gi-protein pathways. Part of the effects of these three beta2-AR agonists appears to be mediated by beta1-AR.

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.

In monocrotaline (MCT)-treated rats the beta-adrenoceptor-G-protein-adenylyl cyclase system-determined in crude membrane preparations from whole ventricular tissue-was desensitized not only in right (RV) but also in left ventricles (LV). This study aimed to assess the specific contribution of cardiomyocytes to these beta-adrenoceptor changes. Six-week-old male Wistar rats were treated with 60 mg/kg body weight MCT intraperitoneally; within 4-6 weeks, rats developed marked RV hypertrophy. Cardiomyocytes were isolated from RVs and LVs. In RV cardiomyocytes of MCT-treated rats, beta-adrenoceptor density was significantly reduced whereas it was unaltered in LV cardiomyocytes. Reduction of RV cardiomyocyte beta-adrenoceptors was due to a selective beta(1)-adrenoceptor reduction. Isoprenaline (100 microM)-induced cAMP increase was significantly reduced in RV but not in LV cardiomyocytes of MCT-treated rats. G protein-coupled receptor kinase activity was increased in RV but not in LV cardiomyocytes. alpha(1)-Adrenoceptor density and noradrenaline-induced increase in inositol phosphate formation were significantly reduced only in RV but not in LV cardiomyocytes from MCT-treated rats. It is concluded that in cardiomyocytes of MCT-treated rats, cardiac beta-adrenoceptors and alpha -adrenoceptors are chamber-specifically desensitized only in the RV. Thus, changes in cardiac beta-adrenoceptors determined in membrane preparations from whole tissue homogenates do not correctly reflect changes occurring in cardiomyocytes.

1 Muscarinic receptors (M-receptors) in the mammalian heart are predominantly of the M(2)-subtype. The aim of this study was to find out whether there might exist an additional myocardial non-M(2)-receptor. 2 For this purpose, we assessed, in adult rat isolated ventricular cardiomyocytes, carbachol-induced [(3)H]-inositol phosphate (IP) formation, and its inhibition by M-receptor antagonists. 3 Carbachol (10(-7)-10(-3) mol l(-1)) increased IP-formation (maximal increase: 14+/-3% above basal, n=6). This increase was significantly enhanced by pretreatment with pertussis toxin (PTX, 250 ng ml(-1) for 20 h): maximal increase was 31+/-5%, pEC(50)-value was 5.08+/-0.33 (n=6). 4 In PTX-pretreated cardiomyocytes 100 micromol l(-1) carbachol-induced IP-formation was inhibited by atropine (pK(i)-value: 8.89+/-0.10) and by the M(3)-receptor antagonist darifenacin (pK(i)-value: 8.67+/-0.23) but was not significantly affected by the M(1)-receptor antagonist pirenzepine (1 micromol l(-1)) or the M(2)-receptor antagonists AF-DX 116 and himbacine (1 micromol l(-1)). 5 In conclusion, in adult rat cardiomyocytes there exists an additional, non-M(2)-receptor, that is coupled to activation of the phospholipase C/IP(3)-pathway; this receptor is very likely of the M(3)-subtype.

Treatment of rats with monocrotaline (MCT) leads to pulmonary hypertension, right ventricular (RV) hypertrophy, and finally to RV heart failure. This is associated with characteristic changes in right ventricular beta-adrenoceptors (beta-AR), neuronal noradrenaline transporter (NAT) density and activity (uptake1), and G protein-coupled receptor kinase (GRK) activity. This study aimed to find out factors that determine beta-AR, uptake1, and GRK changes. Thus, 6-week-old rats were treated with 50 mg/kg MCT subcutaneous or 0.9% saline. Within 13 to 19 days after MCT application (group A), RV weight (222+/-6 versus 147+/-5 mg) and RV/left ventricular (LV) weight ratio (0.42+/-0.01 versus 0.29+/-0.01) were significantly increased, whereas plasma noradrenaline, RV beta-AR density, RV NAT density and activity, and RV GRK activity were not significantly altered. Twenty-one to twenty-eight days after MCT (group B), however, not only RV weight (316+/-4 versus 148+/-2 mg) and RV/LV weight ratio (0.61+/-0.01 versus 0.3+/-0.01) were markedly increased but also plasma noradrenaline (645+/-63 versus 278+/-18 pg/mL); now, RV beta-AR density (13.4+/-1.3 versus 26.5+/-1.1 fmol/mg protein), RV NAT density (50.9+/-11.3 versus 79.6+/-2.9 fmol/mg protein), and RV NAT activity (65.4+/-7.4 versus 111.8+/-15.9 pmol [3H]-NA/mg tissue slices/15 min) were significantly decreased and RV-membrane GRK activity (100+/-15 versus 67+/-6 [32P]-rhodopsin in cpm) significantly increased. LV parameters of MCT-treated rats were only marginally different from control LV. We conclude that in MCT-treated rats ventricular hypertrophy per se is not sufficient to cause characteristic alterations in the myocardial beta-AR system often seen in heart failure; only if ventricular hypertrophy is associated with neurohumoral activation beta-ARs are downregulated and GRK activity is increased.

The involvement of elongation factor-2 (EEF-2), a key-protein of peptide-chain elongation, in the slowing down of protein synthesis during cardiac ageing was addressed. EEF-2 was measured in rat heart extracts and isolated rat cardiomyocytes (CM) from newborn and adult rats using sodium-dodecylsulphate polyacrylamide gel electrophoresis after specific labeling with [32P]ADP-ribosylation or immunoblot. The age-dependent proportional content of several eucaryotic elongation factor-2 (eEF-2) subtypes in rat CM and rat heart extracts was compared using one-dimensional isoelectric focusing. EEF-2 was considerably reduced in the hearts of adult compared to neonatal rats (P<0.01). EEF-2 was also significantly decreased in isolated CM from adult versus newborn rats and during prolonged cultivation of neonatal CM. Cellular ageing was combined with reduced protein synthesis. During adolescence the eEF-2 variants shifted to acidic subtypes. Young adult and old rats revealed similar amounts and subtype distribution of cardiac eEF-2. Only the more acidic eEF-2 variants appeared to contain phosphorylated eEF-2. We concluded that total cardiac eEF-2 and its subtype pattern might play an important role in developmental and age-related proteomic changes.

In adult rat ventricular cardiomyocytes, noradrenaline exerts dual effects on protein synthesis: increases via alpha(1)-adrenoceptors and decreases via beta(1)-adrenoceptors. Carvedilol and bucindolol are beta-blockers with additional alpha(1)-adrenoceptor blocking activities. We studied the effects of carvedilol and bucindolol on noradrenaline-induced protein synthesis (assessed by [(3)H]phenylalanine incorporation) in adult rat ventricular cardiomyocytes. Radioligand binding studies with [(125)I]iodocyanopindolol and [(3)H]prazosin revealed that carvedilol had a much higher affinity to alpha(1)-adrenoceptors than bucindolol (beta(1)-/alpha(1)-adrenoceptor ratio for carvedilol, 1:2.7; for bucindolol, 1:43). Noradrenaline-evoked increases in protein synthesis were enhanced by propranolol (1 microM) and beta(1)-adrenoceptor-selective antagonists bisoprolol (1 microM) and CGP 20712A [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)-ethyl-amino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propranol methanesulfonate] (300 nM). Carvedilol (100 pM-10 microM) inhibited 1 microM noradrenaline-induced increase in protein synthesis with monophasic concentration-inhibition curves independent of whether CGP 20712A was present or not; K(i) values for carvedilol were 5 to 6 nM. In contrast, bucindolol (100 pM-10 microM) inhibited l microM noradrenaline-induced increase in protein synthesis with a bell-shaped concentration-inhibition curve; it increased noradrenaline-induced protein synthesis at 10 nM, although at concentrations >100 nM it was inhibited. In the presence of 300 nM CGP 20712A or 1 microM propranolol, however, bucindolol inhibited 1 microM noradrenaline-induced increase in protein synthesis with monophasic concentration-inhibition curves; K(i) values were 40 to 75 nM. On the other hand, both carvedilol and bucindolol inhibited 1 microM phenylephrine-induced protein synthesis with monophasic concentration-inhibition curves; K(i) values were 4 (carvedilol) and 45 nM (bucindolol). These results indicate that, at low (beta-adrenoceptor blocking) concentrations, bucindolol can enhance noradrenaline-induced protein synthesis whereas it is inhibited by carvedilol.

In rats, injection of monocrotaline (MCT) causes pulmonary hypertension that leads to right ventricular failure. The aim of the present study was to characterize the responses of various vessels (the pulmonary artery, the thoracic aorta and small mesenteric arteries) to noradrenaline (NA; 10(-10)-10(-5) M) and carbachol (10 microM) in MCT-treated rats. For this purpose 6-week-old male Wistar rats ( n=13) were treated with 60 mg/kg MCT i.p. After 4-6 weeks the rats were killed and the heart, lungs and vessels removed and compared with those from age-matched saline-treated control rats ( n=47). First, the alpha(1)-adrenoceptor subtype(s) involved in the vascular NA-responses were characterized in normal rats using the alpha(1)-adrenoceptor subtype-selective antagonists 5-methylurapidil (5-MU; competitive alpha(1A)-adrenoceptor antagonist; 10(-8)-10(-6) M), BMY 7378 (competitive alpha(1D)-adrenoceptor antagonist; 10(-7)-10(-6) M) and chloroethylclonidine (CEC; irreversible alpha(1B)-adrenoceptor antagonist; 30 microM). In the pulmonary artery the pA(2) for BMY 7378 was 7.93, while that for 5-MU could not be calculated. CEC suppressed the NA-induced contraction significantly. In the thoracic aorta, the pA(2) for BMY 7378 was 8.06, while 5-MU was less effective (pA(2) 7.31). CEC again suppressed the NA-induced contraction significantly. In mesenteric arteries, CEC was ineffective whereas 5-MU induced a significant, rightwards shift of the concentration/response curve for NA (pA(2) 8.05). BMY 7378 had a lower pA(2) (6.6). MCT-treated rats developed an increased right ventricular pressure, obliteration of pulmonary vessels and inflammatory lung infiltration. In the pulmonary artery, but not in the thoracic aorta or mesenteric artery of MCT-treated rats NA-induced contraction was attenuated. In addition, carbachol-induced relaxation was reduced in the pulmonary and mesenteric arteries. In conclusion, NA-induced contraction is mediated predominantly by alpha(1A)-adrenoceptors in small mesenteric arteries, by alpha(1D)-adrenoceptors in the thoracic aorta (with a contribution from alpha(1A)- and alpha(1B)-adrenoceptors) and by alpha(1D)- and alpha(1B)-adrenoceptors in pulmonary arteries. MCT leads to reduced NA-responsiveness exclusively in the pulmonary artery that does not, however, account for the development of pulmonary hypertension, and to a more generalized endothelial dysfunction which may contribute to the pathogenesis of pulmonary hypertension in this model.

Patients with chronic renal failure develop an autonomic dysfunction with impaired baroreflex control and attenuated cardiovascular beta-adrenoceptor response to noradrenaline. In rats that underwent 5/6-nephrectomy (SNX), cardiac beta-adrenoceptor responsiveness was reduced as well. Therefore, the aim of this study was to further investigate the mechanism underlying cardiac beta-adrenoceptor desensitization in SNX rats. For this purpose, right and left ventricular beta-adrenoceptor density, activity of the G-protein-coupled receptor kinase, and activity and density of the neuronal noradrenaline transporter (uptake1) were assessed in SNX rats. Seven weeks after SNX, rats had developed left heart hypertrophy. Plasma creatinine, urea, and noradrenaline levels were significantly increased; left and right ventricular noradrenaline content was significantly decreased when compared with sham-operated control rats. In these SNX rats, left, but not right, ventricular beta-adrenoceptor density was significantly reduced, and membrane-associated G-protein-coupled receptor kinase activity was significantly increased compared with sham-operated rats. Although right and left ventricular activity of uptake1 was unchanged, the neuronal noradrenaline transporter density was significantly reduced in both ventricles of SNX versus sham-operated rats. An increase in left ventricular G-protein-coupled receptor kinase activity, possibly triggered by enhanced cardiac noradrenaline release, might be responsible for the decrease in left ventricular beta-adrenoceptor responsiveness in SNX rats.

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.

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.

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.

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.

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., (Copyright 2000 Academic Press.)

The mechanism underlying endothelin-1 (ET-1)-induced increases in intracellular Ca2+ concentrations in the human neuroblastoma cell-line SK-N-MC was investigated. ET-receptor agonists increased inositol phosphate (IP)-formation (assessed as accumulation of total [3H]-IPs in [3H]-myo-inositol prelabelled cells) and intracellular Ca2+ (assessed by the FURA-2 method) with an order of potency: ET-1 > sarafotoxin 6b (S6b)> ET-3 = S6c; the ETA-receptor antagonist BQ-123 inhibited both responses with apparent pKi-values of 8.3 and 8.6, respectively, while the ETB-receptor antagonist BQ-788 did not. Pretreatment of the cells with pertussis toxin (PTX, 500 ng ml(-1) overnight) reduced ET-1-induced Ca2+ increases by 46+/-5%, but rather enhanced ET-1-induced IP-formation. Chelation of extracellular Ca2+ by 5 mM EGTA did not affect ET-1-induced IP-formation. However, in the presence of 5 mM EGTA or SKF 96365, an inhibitor of receptor mediated Ca2+ influx (1.0-3.0 x 10(-5) M) ET-1-induced Ca2+ increases were inhibited in normal, but not in PTX-treated cells. [125I]-ET-1 binding studies as well as mRNA expression studies (by RT-PCR) detected only ETA-receptors whereas expression of ETB-receptor mRNA was marginal. ET-1 (10(-8) M) inhibited isoprenaline-evoked cyclic AMP increases; this was antagonized by BQ-123, not affected by BQ-788 and abolished by PTX-treatment. We conclude that SK-N-MC cells contain a homogeneous population of ETA-receptors that couple to IP-formation and inhibition of cyclic AMP formation. Stimulation of these ETA-receptors increases intracellular Ca2+ by at least two mechanisms: a PTX-insensitive IP-mediated Ca2+ mobilization from intracellular stores and a PTX-sensitive influx of extracellular Ca2+.

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 microM) completely suppressed ET-induced IP-formation, the ETA-receptor antagonist BQ-123 (10 microM) inhibited it only by 70%, the ETB-receptor antagonist IRL 1038 (1 microM) 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 sites 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 Gq/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 ETA-receptors declined, while that of ETB-receptors increased. We conclude that in adult rat renal cortex ET-induced IP-formation is mediated by activation of both ETA- and ETB-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 Gq/11.

The aim of this study was to find out whether, in neonatal rat cardiomyocytes, platelet-activating factor (PAF) can stimulate eicosanoid formation. For this purpose neonatal cardiomyocytes were incubated for 60 min at 37 degrees C in HANKS buffer with PAF (10-1000 nM), and the eicosanoids thromboxane A2 (TXA2) and prostacyclin (PGI2) were assessed in the supernatant as TXB2 and 6-keto-PGF1 alpha, respectively, by an enzyme immunoassay. PAF caused concentration-dependent release of PGI2; TXA2, however, was significantly released only at the highest concentration of PAF (1000 nM). Acetylsalicylic acid (556 microM) and the PAF antagonist WEB 2086 (10 microM) significantly attenuated PAF-induced eicosanoid formation. We conclude that in neonatal rat cardiomyocytes PAF can induce eicosanoid formation and this effect is brought about by activation of a specific PAF receptor.

Free radical-induced oxidative stress has been linked to ischemia-reperfusion injury of the myocardium. The .OH radical is considered the most damaging radical and can be increased in cells by treatment in vitro with H2O2. The purpose of the present study was to determine if aliphatic halocarbons enhance H2O2-induced oxidative injury in isolated cardiac myocytes from neonatal rats. Oxidative damage was assessed by measuring release of thiobarbituric acid-reactive substances (TBARS) from lipid peroxidation, loss of lactate dehydrogenase (LDH) through damaged sarcolemmal membranes, and alterations in intracellular calcium ([Ca2+]i) transients in electrically stimulated (1 Hz, 10 ms, 60 V) myocytes. H2O2 increased TBARS release and LDH leakage in a concentration-dependent (20-200 microM) manner. Continuous suffusion with H2O2 first altered the configuration of [Ca2+]i transients, then eliminated them, and finally caused [Ca2+]i overload (basal [Ca2+]i exceeded peak systolic [Ca2+]i of control). The time to [Ca2+]i overload was inversely associated with concentration, and the shortest time to overload was obtained with 100 microM H2O2. A 1-h preincubation of myocytes with the iron chelator deferoxamine inhibited all effects of H2O2. 1,1,1-Trichloroethane, carbon tetrachloride, or halothane at 1 mM significantly and reversibly reduced [Ca2+]i transients but did not influence TBARS release or LDH leakage. Simultaneous exposure of myocytes to H2O2 and halocarbons did not affect the myocyte response to H2O2 exposure. Results indicate that the three halocarbons tested do not enhance H2O2-induced oxidative injury in isolated cardiac myocytes.

The experiments were carried out in order to clarify the mechanisms of attenuation of hypertension development by means of diets enriched with polyunsaturated fatty acids (PUFA) in spontaneously hypertensive rats (SHR). Female SHR were fed a linoleic acid rich (LAr) diet (13.3 cal % LA, sunflower oil), a linolenic acid rich (LNAr) diet (18.8 cal % LNA, 3.9 cal % LA; linseed oil) and a PUFA deficient diet (0.5 cal % LA, hydrogenated palm kernel fat), respectively, during the last week of pregnancy and during the suckling period. Corresponding diets were given to the male offspring up to an age of 16 weeks. Our results demonstrate that the attenuation of hypertension development in LAr and LNAr fed male SHR was paralleled by an increased in-vitro uptake of 14C-norepinephrine into cardiac and aortic tissues as well as an increased degradation rate of 14C-norepinephrine in cardiac tissue. Ex vivo prostaglandin (PG) formation was reduced after LNAr diet in the aorta (PGF2 alpha, PGI2-like material) and in the kidney medulla (PGE, PGF2 alpha). It is concluded that an increased catecholamine inactivation may play a role in the attenuation of hypertension development in LAr and LNAr diet fed SHR.

The influence of trapidil (T) and two selected 5,7-disubstituted s-triazolo (1,5-a)pyrimidine derivatives (TD: AR 12456 and AR 12463) on arachidonic acid(AA)- and prostaglandin endoperoxide analogue U 46619-induced blood pressure changes in normotensive rats was investigated in comparison with the cyclooxygenase inhibitor acetylsalicylic acid (ASA) and the thromboxane A2 (TXA2) antagonist BM 13177. ASA and AR 12456 completely eliminated the second blood pressure depression after injection of AA and simultaneously diminished TXA2, TXB2 and 6-keto-PGF1a formation in murine blood, whereas BM 13177 prevented the return of the blood pressure to preinjection level after the initial brief fall in arterial pressure. BM 13177 and AR 12463 reduced the rise in U 46619-provoked blood pressure by 75% and 58%, respectively. Trapidil had no effect on blood pressure changes stimulated by AA and U 46619.

Trapidil and four selected 5,7-disubstituted s-triazolo (1,5-a)pyrimidine derivatives (AR 12456, AR 12463, AR 12464, AR 12465) which have already been shown to possess inhibitory effects on arachidonic acid(AA)-induced aggregation and thromboxane A2(TXA2) formation in human and rabbit platelets in vitro diminished the AA-stimulated TXA2 production in murine blood in vivo. The inhibitory effects of the derivatives were generally stronger than those of trapidil. The strongest inhibitor of TXA2 formation was the derivative AR 12456. These in vivo results correspond well with our previous in vitro findings.