The specific aim of this proposal is to elucidate the mechanism by which autacoids that influence cyclic nucleotides (cAMP and cGMP) production modulate the function of glomerular mesangial cells in culture. Mesangial cells possess properties similar to other inflammatory cells and are potentially capable of oxygen radical production and lysosomal enzyme release. These mediators may inflict auto-injury or injury to neighboring cells or extracellular structures that maintain the integrity of the glomerular basement membrane barrier. Experiments will be performed to quantitate the production of superoxide anion, hydrogen peroxide and hydroxyl radical in response to soluble surface stimuli e.g. chemotactic peptides or phorbol esters and particulate stimuli e.g. opsonized zymosan. In addition the kinetics of the release of two marker lysosomal enzymes Beta-glucoronidase and Beta-glucosidase will be studied. We will explore the role of the cyclic nucleotides (cAMP and cGMP) as mediators of the effect of the renal autacoids and neurotransmitters histamine, isoproterenol, adenosine, prostaglandin E2 and carbamylcholine on lysosomal enzyme release and oxygen radical production by rat mesangial cells in culture. We will determine if these hormones increase or decrease radical production or enzyme release, if these effects parallel their effects on cyclic nucleotides, and if cyclic nucleotide analogues mimic the effect of the hormones. Since calcium subserves a critical role in oxygen radical production and lysosomal enzyme release by inflammatory cells. We will explore the role of extracellular and intracellular calcium by studying the effect of calcium channel blockers and agents that antagonize the release of intracellular calcium, on radical production and enzyme release by mesangial cells. In addition, we will attempt to measure simultaneous changes in cytoplasmic calcium concentration using quin 2, a newly synthesized intracellular fluorescent calcium indicator. These studies will elucidate the kinetics of oxygen radical production and lysosomal enzyme release and determine the role of calcium in modulating these processes, in glomerular mesangial cells. Such information will enhance our understanding of the inter-relationship between inflammatory mediators and intrinsic glomerular cells.
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