Identification and Characterization of G Protein-Regulated Phospholipase C in Human Myocardium

The generation of the second messengers inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) by phosphoinositide-specific phospholipases C (PLCs) is a key mechanism by which many cellular functions such as intracellular calcium handling or growth and differentiation are modulated. In the myocardium, PLC plays a role in the mediation of positive inotropic effects and is possibly involved in the pathogenesis of myocardial hypertrophy. Among the variety of PLC isozymes known, the PLCβfamily is regulated by heterotrimeric G proteins. The aim of the present study was to identify and to characterize the PLCβisoform present in human myocardium. PLC activity in human myocardial membranes was dependent on the presence of Ca2+. Interestingly, PLC was markedly stimulated by GTPγS, used as an activator of G proteins. This stimulation was completely abolished by GDP. However, purifiedα-subunits from retinal transducin (αt), used as scavengers of freeβγ-subunits, did not abolish this effect indicating GTPγS stimulation being mediated by G proteinα-subunits. PLC activity was also stimulated by G proteinβγ-subunits purified from bovine retina (βγt). This stimulation was completely blocked by addition of purifiedαt. Reverse transcriptions and polymerase chain reactions (RT-PCR) provided evidence for PLCβ1mRNA being expressed in human myocardium, whereas PCR products corresponding to PLCβ2and PLCβ3mRNAs were not detected. It is concluded that PLCβ1mRNA is expressed in human myocardium. The functional properties of human myocardial PLC activity correspond well to the properties established for PLCβ1, i.e. sensitivity to G proteinα- as well asβγ-subunits. The presence of other as yet unidentified PLC isozymes is nevertheless possible. The identification of the PLCβisozyme present in human myocardium and the understanding of its regulation by G protein subunits sets the stage for the investigation of possible involvement of this system in the pathophysiology of myocardial hypertrophy.