Dale B. Evans

Multiple molecular forms of cyclic nucleotide phosphodiesterase in cardiac and smooth muscle and in platelets. Isolation, characterization, and effects of various reference phosphodiesterase inhibitors and cardiotonic agents.

Multiple molecular forms of cyclic nucleotide phosphodiesterase have been identified previously in several tissues and cell types using a variety of different isolation methods. In the present study, the different molecular forms of phosphodiesterase (PDE) were isolated from cardiac muscle (guinea pig left ventricle), vascular smooth muscle (bovine coronary arteries) and human platelets using the same isolation procedure in each instance. These enzymes were then characterized kinetically, and the effects of various reference PDE inhibitors and cardiotonic agents on each form were examined. A low Km, low Vmax form of phosphodiesterase (PDE I) was found in all three tissue/cell types. PDE I activity was stimulated by calmodulin in cardiac and smooth muscle, but not in platelets. In smooth muscle and platelets, PDE I preferentially hydrolyzed cyclic GMP, whereas cardiac muscle PDE I hydrolyzed cyclic AMP and cyclic GMP equally. A high Km, high Vmax form of phosphodiesterase (PDE II) was found in cardiac muscle and platelets, but not in smooth muscle. PDE II activity was not stimulated by calmodulin and there was no substrate specificity. A low Km, low Vmax cyclic AMP-specific form of phosphodiesterase (PDE III) was found in all three tissue/cell types. The activity of PDE III was not stimulated by calmodulin. The reference inhibitors theophylline and papaverine exerted non-specific inhibitory effects on all forms of phosphodiesterase. Other reference inhibitors (M & B 22,948 and dipyridamole) and several cardiotonic agents (AR-L 57, CI-914, CI-930, amrinone, and MDL 17,043) exerted selective inhibitory effects on only one molecular form of phosphodiesterase. The degree of selectivity was often dependent upon the tissue or cell from which the molecular form of phosphodiesterase was isolated. These studies demonstrate that there is heterogeneity regarding the number of phosphodiesterases present in various tissue/cell types, as well as their substrate specificity and their ability to be stimulated by calmodulin, and these different molecular forms of phosphodiesterase can be selectively inhibited by different pharmacological agents. The possibility exists that such selective inhibitors may produce discrete changes in cyclic AMP or cyclic GMP levels, and that these changes may be produced in specific tissues and/or cells.

Adenosine receptors mediating cardiac depression

Several adenosine analogs were evaluated for their effects on rate and contractility in guinea pig isolated atria. Among adenosine agonists, (-)-N-(1-methyl-2-phenylethyl) adenosine (l-phenylisopropyladenosine; l-PIA) and N-cyclohexyladenosine (CHA), decreased rate and force at nanomolar concentrations, whereas 2-chloroadenosine, N,N-dimethyladenosine (N6-dimethyladenosine) and (+)-N-(1-methyl-2-phenylethyl)adenosine (d-phenylisopropyladenosine; d-PIA) were less potent cardiac depressants. The degree and order of potency of these agonists suggest that the cardiac depressant effects of adenosine are mediated via A1-receptors. The cardiac depressant effects of CHA and l-PIA were antagonized by theophylline and 1,3-diethyl-8-phenylxanthine (DPX).

Phosphodiesterase isozyme inhibition, activation of the cAMP system, and positive inotropy mediated by milrinone in isolated guinea pig cardiac muscle

The purpose of the present study was to examine the interrelationships among phosphodiesterase (PDE) isozyme inhibition, cAMP formation, activation of cAMP-dependent protein kinase (cAPK), and positive inotropy in isolated guinea pig cardiac muscle mediated by the cardiotonic/vasodilator agent, milrinone. Milrinone was a potent and selective inhibitor of the low Km cAMP PDE isozyme (peak III) isolated by diethylaminoethyl ether cellulose chromatography, with IC50 values of 0.7 microM for peak III PDE and 100 microM for peak I PDE. In isolated papillary muscles frozen at peak inotropic responses, positive and significant correlations were evident between isometric force development as a function of cAMP content (r = 0.72, p less than 0.05) or cAPK activity ratio, an index of activation of cAPK (r = 0.79, p less than 0.001), for concentrations of milrinone from 0.1-1000 microM. Similar correlations were evident in muscles frozen at peak inotropic responses for the beta-adrenoreceptor agonist isoproterenol (r = 0.96, p less than 0.001; r = 0.98, p less than 0.001, respectively), but not for ouabain or Bay K-8644. The temporal sequence of these events was also quantitated for concentrations of milrinone (100 microM) and isoproterenol (3 nM) that produced approximately a 100% increase in isometric force. Whereas early time interval of force development (30 s, 1 min, isoproterenol; 30 s milrinone) were not accompanied by significant increases in either cAMP content or cAPK activity ratio, peak increases in force development for both isoproterenol (2 min) and milrinone (1 min) were related to peak increases in cAPK activity ratios. In summary, these results show that significant increases in cAMP content or cAPK activation are correlated with positive inotropy in isolated guinea pig papillary muscles with milrinone. These correlations occur at concentrations of milrinone that inhibit cardiac PDE isozymes and are similar to the known cAMP-dependent cardiostimulant isoproterenol. These data support the hypothesis that selective PDE isozyme inhibition is a mechanism by which milrinone effects positive inotropy.

Differential Vasorelaxant Effects of Milrinone and Amrinone on Contractile Responses of Canine Coronary, Cerebral, and Renal Arteries

The vasorelaxant effects of milrinone and amrinone in canine coronary, cerebral, and renal arterial rings or strips contracted by either K+-depolarization, U46619 (a thromboxane mimetic), or prostaglandin F2α (PGF2α) were quantitated. Milrinone was more potent as a vasorelaxant in coronary arteries relative to cerebral or renal arteries regardless of the mode of contraction; amrinone was coronary selective with K+ contraction only. When comparing potency in arteries contracted by different agonists, milrinone was significantly more potent as a vasorelaxant in all three arteries contracted by either U46619 or PGF2α than in arteries contracted by K+ depolarization, whereas amrinone was only selective for U46619-induced contractions in cerebral arteries. This profile of activity for milrinone was similar to that of sodium nitrite and isoproterenol and dissimilar from the calcium entry blocking agents nimodipine and nifedipine. In conclusion, this study shows that coronary vascular selectivity exists for milrinone and amrinone. Moreover, the relaxant profiles of milrinone and amrinone, with different sources of vascular smooth muscle, are unlike those of calcium entry blocking agents and more similar to the profiles of agents that modulate cyclic nucleotide levels.

Studies aimed at elucidating the mechanism of action of CI-914, a new cardiotonic agent

CI-914 is a novel positive inotropic agent whose cardiotonic activity is not due to inhibition of Na+, K+-ATPase or to stimulation of cardiac beta-receptors. CI-914 also has no direct effect on sarcoplasmic reticulum, mitochondria or adenylate cyclase activity. CI-914 does, however, exert a potent inhibitory effect on cardiac phosphodiesterase activity. In evaluating the effect of this agent on the different molecular forms of phosphodiesterase present in cardiac muscle, CI-914 was found to selectively inhibit PDE III, which is a low Km, cAMP-specific form of the enzyme (IC50 = 6.1 microM). This inhibitory effect was found to be competitive with respect to the substrate. Papaverine and theophylline on the other hand were found to inhibit all three forms of phosphodiesterase present in cardiac muscle. The role of phosphodiesterase inhibition in mediating the positive inotropic response to CI-914 is supported by the finding that this agent: (i) significantly elevates cyclic AMP levels in ventricular tissue; (ii) shifts the normal concentration-response to the beta-receptor stimulant isoproterenol to the left: and (iii) restores contractility to K+-depolarized papillary muscles.

Selective effects of adenosine receptor agonists upon coronary resistance and heart rate in isolated working rabbit hearts

Dose dependent changes in heart rate (HR) and coronary resistance (CR) were determined for adenosine, 5-N-ethylcarboxamide adenosine (NECA) and l-N6-phenylisopropyladenosine (l-PIA) over a dose range of 1 X 10(-9) to 1 x 10(-5) M. Changes in CR were determined under controlled metabolic demand conditions (constant mean aortic pressure, constant mean left atrial pressure, and constant HR). Decreases in HR were determined by allowing the paced hearts to beat spontaneously between doses for 15 seconds. Adenosine significantly decreased CR and HR at greater than or equal to 1 X 10(-5) M. NECA significantly decreased both CR and HR at greater than or equal to 3 X 10(-8) M. l-PIA significantly decreased HR at greater than or equal to 3 X 10(-8) M; however a dose at greater than or equal to 3 X 10(-6) M was required to significantly decrease CR. These results provide evidence that the coronary vasodilator action of adenosine may primarily be mediated by A2 receptors. Furthermore, the data are in support of previous observations that the bradycardic action of adenosine is principally mediated via A1 receptors.

The effect of the cardiotonic imazodan (CI-914) on myocardial and peripheral hemodynamics in the anesthetized dog

Summary: The purpose of this study was to evaluate the effect of the new cardiotonic, imazodan (CI-914), on myocardial hemodynamics and oxygen consumption, and peripheral blood flow distribution. Organ blood flow was measured by the radiolabeled-microsphere-reference-withdrawal technique and myocardial oxygen consumption calculated from arterial and coronary sinus O2 content and blood flow. At inotropic levels, CI-914 decreases mean arterial pressure with a minimal increase in heart rate. CI-914 decreases coronary vascular resistance and increases supply to demand ratio, indicating an active coronary vasodilation. CI-914 does not alter peripheral blood flow distribution, thus suggesting equivalent organ vasodilation. These data suggest that CI-914 may be useful in the treatment of congestive heart failure because of its positive inotropic, coronary vasodilator, and peripheral vasodilator properties.