The influence of selective (milrinone: 10, 50, 100 microM) and nonselective phosphodiesterase (isobutylmethylxanthine: 0.1, 10, 100 microM) inhibitors and beta-adrenergic stimulation (isoproterenol: 0.01, 0.1 microM) on phospholamban and myofibrillar protein phosphorylation was studied in guinea pig hearts perfused with [32P]orthophosphate. Changes in protein phosphorylation were compared to alterations in tissue cyclic AMP (cAMP) levels and positive inotropic effects induced by these agents. Isoproterenol (0.01 microM), milrinone (50 microM), and isobutylmethylxanthine (100 microM) all produced similar, twofold increases in dP/dt and -dP/dt but only stimulation with isobutylmethylxanthine and isoproterenol was associated with significant increases in phospholamban phosphorylation. At these equipotent doses, the effects of isobutylmethylxanthine were associated with higher increases (3.1-fold) in cAMP than those observed with isoproterenol (twofold). Milrinone (50 microM) produced a 2.5-fold increase in cAMP levels but failed to change phospholamban phosphorylation. Higher doses of milrinone (100 microM) resulted in relatively high (4.1-fold) cAMP levels, and this was associated with increased (1.5-fold) phosphorylation of phospholamban. Phosphorylation of troponin I was significantly increased at 0.01 microM and 0.1 microM isoproterenol, while phosphorylation of C protein was observed only at 0.1 microM isoproterenol. Isobutylmethylxanthine and milrinone did not significantly increase phosphorylation of either troponin I or C protein at any of the doses studied. These findings indicate that cardiotonic agents acting via the cAMP pathway may produce similar inotropic responses at different levels of cAMP and phosphorylation of sarcoplasmic reticulum and myofibrillar proteins.

No abstract

Protein phosphorylation acts a pivotal mechanism in regulating the contractile state of the heart by modulating particular levels of autonomic control on cardiac force/length relationships. Early studies of changes in cardiac protein phosphorylation focused on key components of the excitation-coupling process, namely phospholamban of the sarcoplasmic reticulum and myofibrillar troponin I. In more recent years the emphasis has shifted towards the identification of other phosphoproteins, and more importantly, the delineation of the mechanistic and signaling pathways regulating the various known phosphoproteins. In addition to cAMP- and Ca(2+)-calmodulin-dependent kinase processes, these have included regulation by protein kinase C and the ever-emerging family of growth factor-related kinases such as the tyrosine-, mitogen- and stress-activated protein kinases. Similarly, the role of protein dephosphorylation by protein phosphatases has been recognized as integral in modulating normal cardiac cellular function. Recent studies involving a variety of cardiovascular pathologies have demonstrated that changes in the phosphorylation states of key cardiac regulatory proteins may underlie cardiac dysfunction in disease states. The emphasis of this comprehensive review will be on discussing the role of cardiac phosphoproteins in regulating myocardial function and pathophysiology based not only on in vitro data, but more importantly, from ex vivo experiments with corroborative physiological and biochemical evidence.

Factor Xa (FXa) has materialized as a key enzyme for the intervention of the blood coagulation cascade and for the development of new antithrombotic agents. FXa is the lone enzyme responsible for the production of thrombin and therefore is an attractive target for the control of thrombus formation. We have designed and synthesized a unique series of quinoxalinone FXa inhibitors. This series resulted in 3-[4-[5-((2S,6R)-2,6-dimethylpiperidin-1-yl)pentyl]-3-oxo-3,4-dihydroquinoxolin-2-yl]benzamidine (35) with 0.83 nM activity against FXa and excellent selectivity over similar serine proteases. An X-ray crystal structure of compound 35 bound to trypsin along with molecular modeling has led to a predicted binding conformation of compound 35 in FXa. Compound 35 has also been proven to be efficacious in vivo in both the rabbit veno-venous shunt and dog electrolytic injury models. In addition, it was shown that compound 35 did not significantly increase bleeding times in a rabbit model except at the highest doses and plasma concentrations were elevated in a dose dependent manner following a bolus dose and continuous intravenous infusion.

Communications to the Editor Design of a Functional Hexapeptide Antagonist of Endothelin Endothelin-1 (ET-1, Figure 1), a bicyclic 21-amino acid peptide, is a potent constrictor of vascular smooth muscle.1 2"3 Since the isolation of ET-1 from the supernatant of cultured porcine endothelial aortic cells, human genomic analysis has identified two structurally and functionally related isopeptides (ET-2 and ET-3).4 56Previous structure-activity analyses have shown the importance of the C-terminal L-tryptophan indole ring, its carboxylate, and the two cystine bridges (1-15 and 3-11