Diminution of contractile response by kappa-opioid receptor agonists in isolated rat ventricular cardiomyocytes is mediated via a pertussis toxin-sensitive G protein.

Opioids directly decrease the contractile response of isolated ventricular cardiomyocytes to electrical stimulation. To investigate whether these effects are mediated via GTP-binding G(i/o) proteins we examined the influence of pertussis toxin on the effects of the kappa-opioid receptor agonist trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benz eneacetamide (U-50,488) methanesulphonate and on the as yet undescribed effects of the opioid peptide dynorphin A (1-8) on contraction. In isolated, electrically driven, rat ventricular cardiomyocytes both agents concentration dependently reduced cell shortening within 15 min, decreasing the contractile response by 79+/-4% (n=5) and 62+/-2% (n=6) of control values at maximal effective concentrations of 10 microM (U-50,488) and 1 microM [dynorphin A (1-8)], respectively. Pertussis toxin pre-treatment (200 ng/ml; 4.5-5 h) completely abolished the effects of U-50,488 and dynorphin A (1-8) on the contractile response, indicating that these effects are mediated via G(i/o) proteins. In addition, the non-selective opioid receptor antagonist (-)-naloxone and the kappa-opioid receptor antagonist nor-binaltorphimine antagonized the effects of U-50,488 and dynorphin A (1-8) on the contractile response. Furthermore, the mu- and delta-opioid receptor agonist (D-Ala2, D-Leu5)-enkephalin (DADLE) had no effects on contraction. These results indicate that the decrease in cell shortening is due to stimulation of kappa-opioid receptors. The direct effect of kappa-opioid receptor agonists on the contractile response thus represents an additional mechanism for decreasing cardiac contractility, besides the M-cholinoceptor- or adenosine receptor-mediated pathway. It is conceivable that increased release of endogenous dynorphins from the heart during hypoxia may protect the heart in a similar manner to adenosine.

Characterization of biochemical effects of CGS 21680C, an A2-adenosine receptor agonist, in the mammalian ventricle.

Effects of a putative A2-adenosine receptor agonist 2-[(p-2-carboxyethyl)-phenethylamino]-5'-N-ethyl-carboxamide-adeno sine (CGS 21680C) on force of contraction, protein phosphorylation, cyclic adenosine monophosphate (cAMP) content, and the activity of phosphodiesterase (PDE) isoenzymes in guinea pig ventricular (GPV) preparations were studied. CGS 21680C (1-100 microM) did not affect force of contraction in isolated electrically driven papillary muscles and was ineffective in increasing phosphorylation of phospholamban (PLB) and the inhibitory subunit of troponin (TnI) in [32P]-labeled GPV cardiomyocytes. However, under the same conditions, CGS 21680C (10 microM) increased cAMP content from 4.3 +/- 0.2 to 13.0 +/- 0.6 pmol/mg protein, and this effect was completely abolished by A2-adenosine receptor antagonist 9-chloro-2-(2-furanyl)-5,6-dihydro-1,2,4-triazolo-(1,5-c)quinazolin++ +-5-imine (CGS 15943A). CGS 21680C (10 microM) inhibited PDE isoenzymes I, II, III, IV by 7.0, 8.3, 4.7, and 23.2%, respectively. Similarly, rolipram (100 microM), a selective PDE IV inhibitor, increased cAMP content from 4.4 +/- 0.3 to 7.2 +/- 0.3 pmol/mg protein without affecting the phosphorylation state of PLB and TnI. We conclude that CGS 21680C increases cAMP content in GPV cardiomyocytes by activation of adenylyl cyclase or in part by inhibition of PDE IV activity. The increase in cAMP content by CGS 21680C or rolipram is ineffective in increasing phosphorylation of PLB and TnI. These results support the concept of compartments for cAMP or protein kinase A or both in cardiomyocytes that are not coupled to phosphorylation and contractility.

Positive inotropic effects of the calcium sensitizer CGP 48506 in guinea pig myocardium.

In isolated papillary muscles from reserpinized guinea pigs, CGP 48506 increased force of contraction in a concentration-dependent and reversible manner, starting at 10 mumol/l and reaching 364.14 +/- 46.10% of predrug values at 100 mumol/l. The positive inotropic effect of CGP 48506 was not sensitive to 10 mumol/l carbachol. The positive inotropic effect of CGP 48506 was accompanied by increases in time to peak tension and in time of relaxation amounting to 223.37 +/- 6.87% and 247.10 +/- 9.34% of control, respectively, at 100 mumol/l (n = 10). CGP 48506 sensitized trabeculae from guinea pig hearts to calcium with an EC50 value of 22 mumol/l. However, CGP 48506 (up to 300 mumol/l) did not affect the activity of cardiac PDE isoenzymes I to IV. Likewise, CGP 48506 (up to 100 mumol/l) did not increase phosphorylation of select cardiac regulatory proteins or cyclic AMP content in guinea pig ventricular cardiomyocytes and did not affect cardiac phosphorylase phosphatase activity. CGP 48506 is the first pharmacological agent with noteworthy calcium-sensitizing properties that has been found to be devoid of inhibitory activity on cardiac PDE.

Positive inotropic effects of the calcium sensitizer CGP 48506 in failing human myocardium.

In trabeculae carneae from failing human myocardium, CGP 48506 increased the force of contraction, which reached 310 +/- 41% of predrug values at 100 mumol/l. Its stereoisomer CGP 48508 did not affect the force of contraction (100 mumol/l). The positive inotropic effect of CGP 48506 was not sensitive to 10 mumol/l carbachol. The positive inotropic effect of CGP 48506 was accompanied by increases in time to peak tension and time of relaxation amounting to 175 +/- 4% and 205 +/- 15% of control, respectively, at 100 mumol/l. CGP 48506 but not CGP 48508 sensitized skinned trabeculae from failing human myocardium to calcium with an EC50 value of 10 mumol/l. However, CGP 48506 and CGP 48508 (up to 300 mumol/l) did not affect the activity of PDE isoenzymes I to IV from failing human myocardium. CGP 48506 is the first inotropic agent with calcium-sensitizing properties in the human heart that has been found to be devoid of inhibitory activity on human cardiac PDE isoenzymes.

Characterization of the phosphodiesterase inhibition by 2-(3-methoxy-5-methylsulfinyl-2-thienyl)-1H-imidazo-(4,5-c)-pyridine HCl and its sulfide- and sulfone derivatives in myocardial preparations from failing human hearts.

The effects of HN-10200 (2-(3-methoxy-5-methylsulfinyl-2-thienyl)-1H-imidazo(4,5-c)-pyridine HCl) and its derivatives HN-10201-sulfide and HN-10202-sulfone on the activities of the phosphodiesterase (PDE) isoenzyme activities isolated from ventricular myocardium of failing human hearts (end-stage myocardial failure, NYHA IV) were investigated. Four PDE isoenzymes (PDE I-IV) were separated by DEAE-sepharose chromatography. Milrinone, 3-isobutyl-1-methylxanthine (IBMX), and a derivative of pimobendan (2-(4-hydroxyphenyl)-5-(5-methyl-3-oxo-4,5-dihydro-2H-6-pyridazinyl)- benzimidazole HCl, PiD) were studied for comparison. Furthermore, the influence of HN-10200 on force of contraction and cAMP content of ventricular trabeculae of these hearts were determined. HN-10200 inhibited the activities of PDE I-IV concentration-dependently. The IC50 values were (mumol/l): 218.7, 283.1, 119.6, and 85.8 for PDE I-IV, respectively. The IC50 values of its derivatives were in the same range, i.e. the parent compound or its derivatives inhibited the PDE isoenzymes nonselectively. IBMX also inhibited PDE I-IV nonselectively, but was about ten times more potent based on IC50 values. In contrast, PiD was the most selective and potent PDE III inhibitor tested. Milrinone inhibited both, PDE III and IV, up to two orders of magnitude more potently than PDE I and II, HN-10200 (30 mumol/l) only marginally and insignificantly increased force of contraction and cAMP content of the ventricular trabeculae. Thus, HN-10200 and it's derivatives HN-10201-sulfide and HN-10202-sulfone are nonselective inhibitors of myocardial PDE I-IV. HN-10200 revealed only neglectable positive inotropic effects in preparations from failing human heart.

Effects of saterinone and its enantiomers R(+)-saterinone and S(-)-saterinone on the phosphodiesterase isoenzymes from ventricular tissue of failing human hearts and porcine hearts.

Stereoisomers often exhibit differences in their pharmacological activities. Therefore, the phosphodiesterase inhibitory effects of the cardiotonic agent saterinone in form of the racemate were investigated in comparison with the inhibitory properties of its enantiomers R(+)- and S(-)-saterinone. For this purpose the phosphodiesterase isoenzymes from ventricular tissue of failing human hearts and porcine hearts were separated by DEAE-sepharose anion exchange chromatography. Four different phosphodiesterase isoenzymes were isolated from failing human myocardium and designated phosphodiesterase I-IV. Three phosphodiesterase isoenzymes could be separated from ventricular tissue of porcine hearts. A Ca2+/calmodulin stimulated phosphodiesterase I was not detectable in porcine myocardium. In failing human hearts racemic saterinone was a potent inhibitor of phosphodiesterase III (IC50 0.02 mumol/l) and IV (IC50 0.03 mumol/l) as compared to the inhibition of phosphodiesterase I (IC50 37.3 mumol/l) and II (IC50 51.4 mumol/l). In comparison with the racemate, R(+)- and S(-)-saterinone showed only slight differences in their phosphodiesterase inhibitory effects. R(+)-saterinone inhibited phosphodiesterase III slightly but significantly more potently and selectively than did S(-)-saterinone. Compared to the inhibition of phosphodiesterase I and II both enantiomers were similarly potent and selective inhibitors of phosphodiesterase III and IV. Similar results were obtained in porcine hearts. It is concluded that the racemate saterinone and its enantiomers (R(+)- and S(-)-saterinone are virtually equipotent concerning the inhibition of phosphodiesterase isoenzymes isolated from failing human hearts or porcine ventricular tissue. The enantiomers of saterinone did not exhibit distinct stereoselectivity in their phosphodiesterase inhibitory effects.

High selectivity for inhibition of phosphodiesterase III and positive inotropic effects of MCI-154 in guinea pig myocardium.

MCI-154 (0.3-100 microM) exerted a concentration-dependent positive inotropic effect in isolated guinea pig papillary muscles (EC50 0.8 microM). The efficacy of MCI-154 (253% of predrug value) was 1.7-fold higher than that of saterinone but comparable to that of milrinone. Carbachol markedly reduced the increase in force of contraction (FOC) of MCI-154. In intact contracting papillary muscles, the positive inotropic effect was accompanied by an increase in cyclic AMP content to 0.78 +/- 0.09 pmol/mg wet weight (n = 10), corresponding to 150% of the basal value (0.51 +/- 0.05 pmol/mg wet weight, n = 21) in the presence of submaximal cyclic AMP phosphodiesterase (PDE) isoenzyme III inhibiting concentrations of MCI-154 (30 microM). MCI-154 (1-1,000 microM) concentration-dependently inhibited the activity of PDE III from homogenates of guinea pig myocardium. The IC50 was 3.8 microM. PDE I, II, and IV were not significantly affected up to 100 microM (PDE I and IV) and up to 1,000 microM (PDE II). In comparison, milrinone and saterinone were PDE III/IV-selective PDE inhibitors. Rolipram inhibited PDE IV only. IBMX and theophylline were nonselective PDE inhibitors. MCI-154 had only a marginal positive chronotropic effect. The frequency of spontaneously beating right auricles from guinea pig heart was increased by 8.7% at most (n = 5). MCI-154 increased Ca2+ sensitivity in chemically skinned porcine ventricular muscle fibers.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphodiesterase inhibition in ventricular cardiomyocytes from guinea-pig hearts.

1. The present study compared the cyclic nucleotide phosphodiesterase (PDE) activities in cardiomyocytes and ventricular cardiac tissue from guinea-pigs. The aim of the study was to determine whether PDE activities in ventricular tissue accurately reflect the isoenzymes present in cardiomyocytes. 2. In homogenates of cardiomyocytes and multicellular ventricular tissue, four distinct soluble PDE activities could be separated by DEAE-sepharose chromatography. 3. In multicellular cardiac tissue as well as in cardiomyocyte preparations, adenosine 3':5'-cyclic monophosphate (cyclic AMP) PDE isoenzymes I-IV were comparable in terms of substrate affinities, and inhibition or stimulation by guanosine 3':5'-cyclic monophosphate (cyclic GMP). However, in cardiomyocytes the Vmax values of PDE I-IV were lower by a factor of about 2 to 7 and the basal activities were lower by a factor of about 3 to 5 as compared to multicellular cardiac tissue. 4. To investigate whether the PDE I-IV activities were similarly inhibited by PDE inhibitors in both preparations, we studied the effects of 3-isobutyl-1-methylxanthine (IBMX), UD-CG 212 Cl (2-(4-hydroxy-phenyl)-5-(5-methyl-3-oxo-4, 5-dihydro-2H-6-pyridazinyl)benzimidazole HCl) and rolipram. UD-CG 212 Cl was a selective PDE III inhibitor in cardiomyocytes (IC50 0.3 mumol l-1) and in ventricular tissue (IC50 value 0.1 mumol l-1). Rolipram selectively inhibited PDE IV in cardiomyocytes (IC50 1.4 mumol ml-1) and in ventricular tissue (IC50 1.1 mumol l-1) whereas IBMX was a nonselective PDE inhibitor in both preparations.5. It is concluded that the PDE isoenzymes I-IV from multicellular ventricular tissue can be used as a representative system for investigating PDE inhibiting properties of PDE inhibitors in the myocardium since comparable PDE isoenzymes I-IV exist in guinea-pig ventricular cardiomyocytes and multicellular ventricular tissue.

Mechanism underlying the reduced positive inotropic effects of the phosphodiesterase III inhibitors pimobendan, adibendan and saterinone in failing as compared to nonfailing human cardiac muscle preparations.

The present study was performed to compare the effects of the new positive inotropic phosphodiesterase III inhibitors pimobendan, adibendan, and saterinone on the isometric force of contraction in electrically driven ventricular trabeculae carneae isolated from explanted failing (end-stage myocardial failure) with those from nonfailing (prospective organ donors) human hearts. In preparations from nonfailing hearts the phosphodiesterase inhibitors, as well as the beta-adrenoceptor agonist isoprenaline, the cardiac glycoside dihydro-ouabain, and calcium, which were studied for comparison, revealed pronounced positive inotropic effects. The maximal effects of pimobendan, adibendan, and saterinone amounted to 56%, 36% and 45%, respectively, of the maximal effect of calcium. In contrast, in preparations from failing hearts the phosphodiesterase III inhibitors failed to significantly increase the force of contraction and the effect of isoprenaline was markedly reduced. The effects of dihydroouabain and calcium were almost unaltered. The diminished effects of isoprenaline were restored by the concomitant application of phosphodiesterase inhibitors. To elucidate the underlying mechanism of the lack of effect of the phosphodiesterase III inhibitors in the failing heart we also investigated the inhibitory effects of these compounds on the activities of the phosphodiesterase isoenzymes I-III separated by DEAE-cellulose chromatography from both kinds of myocardial tissue. Furthermore, the effects of pimobendan and isoprenaline on the content of cyclic adenosine monophosphate (determined by radioimmunoassays) of intact contracting trabeculae were studied. The lack of effect of the phosphodiesterase inhibitors in failing human hearts could not be explained by an altered phosphodiesterase inhibition, since the properties of the phosphodiesterase isoenzymes I-III and also the inhibitory effects of the phosphodiesterase inhibitors on these isoenzymes did not differ between failing and nonfailing human myocardial tissue. Instead, it may be due to a diminished formation of cyclic adenosine monophosphate in failing hearts, presumably caused mainly by a defect in receptor-adenylate cyclase coupling at least in idiopathic dilated cardiomyopathy. Both the basal and the pimobendan-stimulated or isoprenaline-stimulated contents of cyclic adenosine monophosphate of intact contracting trabeculae from failing hearts were decreased compared with the levels in nonfailing hearts. However, under the combined action of isoprenaline and pimobendan the cyclic adenosine monophosphate level reached values as high as with each compound alone in nonfailing preparations, and in addition the positive inotropic effect of isoprenaline was restored. These findings may have important clinical implications. Along with the elevated levels of circulating catecholamines the positive inotropic effects of the phosphodiesterase inhibitors may be maintained in patients with heart failure.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of the triazolopyrimidine trapidil on force of contraction, beating frequency and phosphodiesterase I--IV activity in guinea-pig hearts.

The effects of the triazolopyrimidine trapidil (5-methyl-7-diethylamino-s-triazolo [1,5-alpha]pyrimidine, CAS 15421-84-8) on force of contraction, beating frequency and phosphodiesterase (PDE) activity were investigated in isolated preparations from guinea-pig hearts. The effects of 3-isobutyl-1-methylxanthine (IBMX), theophylline and milrinone were studied for comparison. Trapidil exerted a concentration-dependent (1000-3000 mumol(s)/l) positive inotropic effect (EC50 562.4 mumol(s)/l) in guinea-pig papillary muscles. The positive inotropic effect was accompanied by a shortening of the duration of contraction as described for IBMX, or isoprenaline. The efficacy of trapidil was lower than that of IBMX or milrinone. Both agents maximally enhanced force of contraction to a 3fold (milrinone) or even 6fold greater amount (IBMX). The potency of trapidil was almost in the same order of magnitude as that of milrinone. The positive inotropic effect of trapidil is at least partially due to a cyclic adenosine monophosphate (cAMP)-dependent mechanism because carbachol antagonized the increase in force of contraction. Trapidil concentration-dependently but nonselectively inhibited the activities of cAMP PDE isoenzymes I-IV as did theophylline or IBMX. Based on IC50 values (275 mumol(s)/l on the average) trapidil had a potency similar to that of theophylline while IBMX was about one order of magnitude more potent. Regarding the inhibition of PDE III, IBMX was 49fold and milrinone 114fold more potent than trapidil. Trapidil revealed only a marginal positive chronotropic effect. The frequency of spontaneously beating right auricles was increased by 13% at most. Trapidil did not produce any tachyarrhythmias or contractures. It is concluded that the positive inotropic effect of trapidil is mainly due to PDE inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)