Platelet-Derived Growth Factor (PDGF) through its biological activities of mitogenesis, chemotaxis, and vasoconstriction, has been implicated in the disease processes of cancer, atherosclerosis, and hypertension. PDGF elicits these activities through the existence of distinct PDGF isoforms (AB heterodimer and AA and BB homodimers) and receptors. However, the mechanism(s) by which PDGF binding to its receptors is transduced into biological activity is not clear. We have obtained preliminary data supporting a role for alterations in cytosolic free calcium ([Ca++]i) in PDGF-stimulated mitogenesis. The overall goal of this proposal is to test the hypothesis that early alterations in [Ca++]i which occur within seconds to minutes after PDGF binding, are necessary for PDGF stimulated mitogenesis, and that the amplitude and kinetics of the [Ca++]i response are modulated by other PDGF-induced alterations in cellular functions. Therefore our specific aims are: 1) Determination of the role of early PDGF-isoform-induced alterations in [Ca++]i in PDGF-stimulated mitogenic activity; and 2) Determination of the cellular mechanisms of modulation of PDGF-isoform-induced alterations in [Ca++]i. Using Multiparameter Digitized Video Microscopy (MDVM) which allows the real time observation of a number of cellular functions at the level of a single intact living cell, we will monitor [Ca++]i and DNA synthesis in the same vascular smooth muscle cell (VSMC) following treatment with PDGF isoforms. These induced alterations in [Ca++]i by manipulation of the extracellular ionic environment or pharmacologic antagonists, through the use of Ca++ ionophores, caged Ca++ or caged Ca++ buffer, and by microinjection of BAPTA-conjugated dextran. We will also monitor, in the same VSMC, [Ca++]i and plasma membrane potential (VM), to determine whether PDGF-isoform- induced alterations in [Ca++]i are modulated by VM, and [Ca++]i can also be modulated by pHi. The role of inositol trisphosphate (IP3), Ca++ itself and activated Protein Kinase C (PKC) in PDGF-isoform-induced alterations in [Ca++]i will be examined using caged IP3, caged Ca++ and caged diacylglycerol, in conjunction with the Ca++-sensitive fluorophore fluo-3. The studies proposed here will provide substantial new information regarding the role of calcium in cell growth, the importance of early changes in [Ca++]i in PDGF-stimulated mitogenesis, and whether alterations in other cellular functions caused by PDGF (e.g. VM, pHi, IP3 and PKC activation), via modulation of PDGF-induced alterations in [Ca++]i can influence PDGF mitogenic activity. Such information will be of importance as it may aid in the development of therapeutic modalities to treat PDGF- dependent disease conditions.
