The long range goal of the proposed studies is to elucidate subcellular mechanisms by which certain cardioactive hormones and agents regulate myocardial cell function. The data we have obtained previously in this on-going project, in conjunction with that reported by others, has permitted us to establish a model for the interaction of phosphoinositide metabolism with the handling of Ca by the sarcoplasmic reticulum. Alpha-adrenergic hormones stimulate the breakdown of phosphatidylinositolbisphosphate to diacylglycerol and inositol trisphosphate. The IP3 stimulates the release of Ca from the SR and DAG activates protein kinase C. This response is modulated by an inhibition of phosphatidylinositol (PI) formation in the SR by elevated Ca2+ and, in addition, a reduction of IP3 is brought about by activation of IP3 phosphatases. Experiments are proposed to test various aspects of this model. Moreover, evidence that the SR also forms PIP and PIP2 as well as PI suggests that these substances may affect the SR Ca pump and this will be tested. Ca transport into the isolated SR is known to be stimulated by phosphorylation of the cytoplasmic segment of the membrane-bound protein phospholamban. Mild treatment with trypsin, which cleaves the cytoplasmic segment, also stimulates Ca transport but trypsin has no effect if the phospholamban has been phosphorylated prior to treatment. These observations support the hypothesis that unphosphorylated cytoplasmic segment has an inhibitory effect on Ca transport through some specific interaction with the surface of the SR membrane. This model will be tested in a number of ways including the following. The cytoplasmic segment and portions thereof will be chemically synthesized and the effects of their phosphorylated and unphosphorylated forms on trypsin activated SR will be determined. A panel of polyclonal antibodies will be obtained which will be antigenic to defined regions on the cytoplasmic segment of phospholamban. These will be used to monitor the release of the peptides upon activation of the Ca pump by treatment of membranes with different proteases as well as to determine which sequences are important in the Ca pump activation by antibody alone. These studies should allow detection of changes in the conformation of phospholamban upon phosphorylation and in the aging heart. In a third project, we will specifically test whether the in vitro increase in 32P incorporation into phospholamban in microsomes prepared from rat hearts from aged animals, previously observed, is due to a change in phospholamban.
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