Pharmacological modulation of phosphorylation mechanisms in heart and vasculature

Abstract

AbstractChanges in the extent of phosphorylation of key regulatory proteins/enzymes that are intimately involved in either intermediary metabolism or contraction/relaxation in both cardiac and vascular smooth muscle (VSM) are regulated by cyclic nucleotides (cAMP/cGMP) or Ca2+. Ca2+ regulates phosphorylation by stimulation of kinase activity through two regulatory cofactors: the calcium‐binding protein calmodulin (which activates myosin light chain kinase—MLCK) or phosphatidylserine plus diacylglycerol (which activates protein kinase C—PKC). MLCK is different in VSM vs. cardiac muscle; it is larger and present in higher concentrations in VSM. Myosin light chain phosphorylation is requisite for rapid force generation in VSM and may also be related to stress maintenance. The primary regulatory mechanism in cardiac muscle is thin‐filament linked; myosin light chain phosphorylation is relatively slow and is not related to rapid changes in inotropy. PKC activation in VSM may be related to non‐rapid stress development via phosphorylation of a unique 25‐kDa substrate. Two isozymic forms of cAMP‐dependent protein kinase exist in both cardiac and VSM. Activation of cAMP protein kinase in response to cAMP‐related cardiotonic or vasodilator agents has been reported. However, differences in subsequent phosphorylation of selected substrates exists in cardiac muscle for agents such as PGE and isoproterenol. Activation of cGMP protein kinase has also been demonstrated in cardiac and vascular smooth muscle. A positive relationship appears to exist between kinase activation and relaxation of the latter tissue by ANF. Several substrates for cAMP protein kinase that may be linked to positive inotropy have been identified in cardiac muscle. Phosphorylation of phospholamban increases Ca2+ uptake by the sarcoplasmic reticulum. Phosphorylation of a 15‐kDa protein in the sarcolemma may be related to an increase in opening of voltage‐dependent Ca2+ channels. On the contractile proteins, phosphorylation of the troponin I subunit as well as phosphorylation of C‐protein may contribute to the increased rate of relaxation observed with β‐agonists. In VSM, phosphorylation of MLCK by cAMP protein kinase decreases affinity of MLCK for Ca2+ ‐calmodulin and inhibits light chain phosphorylation and subsequent actin‐myosin interactions. However, while MLCK phosphorylation has been demonstrated in intact smooth muscle, functional loss of Ca2+ ‐calmodulin binding and the relationship to relaxation by β‐agonists have not been apparent. Other substrates for cAMP and cGMP protein kinase have been identified in intact VSM and membranous preparations. Recently, naphthalene and isoquinoline sulfonamide protein kinase inhibitors have been disclosed, which demonstrate different relative potencies for these protein kinases. Moreover, some of these agents are vascular relaxants or platelet aggregation inhibitors. These agents may aid in the search for specific modulators of protein phosphorylation systems and also delineate the role of these systems in cardiovascular function.