Studies are proposed to unravel the intracellular mechanisms involved in the short and long term regulation by Ca2+ of cAMP accumulation in C6-2B rat glioma cells. During the previous grant period the applicant demonstrated that Ca2+ exerts a dramatic inhibitory action on cAMP accumulation and upon adenylyl cyclase in C6-213 cells. These cells are a useful model to study Ca2+ inhibition of adenylyl cyclase, since they almost exclusively contain type VI adenylyl cyclase, a recently cloned form which is inhibited by submicromolar concentrations of Ca2+. The applicant has proposed that Ca2+ plays an important role in the short and long term regulation of the concentration of cAMP in C6-2B cells. The first focus will be upon the mechanism by which Ca2+ regulates cAMP metabolism. Later in the project the applicant will extend these studies to ascertain the involvement of Ca2+ in hormone and/or cAMP induced refractoriness. Methodology based upon the applicant's previous work and that of others, is proposed for the high resolution simultaneous high speed ratio imaging of Ca2+ and cAMP in living cells at multiple planes of focus using convolution-deconvolution techniques to improve spatial image resolution based upon knowledge of the microscopic point spread function. Thus, detailed three dimensional intracellular time- concentration information about the interdependent kinetics of both second messengers will help the applicant to examine more localized second messenger changes which might not be detected by standard imaging techniques. The concentrations of both cAMP and Ca2+ will be experimentally modified to examine the short and long term interdependency between the two in terms of their steady state concentration and oscillatory behavior. Studies will involve pharmacological manipulation of Ca2+ and cAMP, direct microinjection of the second messengers or their modifiers or analogs, or modification of cAMP synthesis with antisense RNA complementary to type VI adenylyl cyclase, the major form of adenylyl cyclase in C6-2B cells and abundant in cardiac tissue. Further studies are aimed at defining this mechanism, at the level of adenylyl cyclase or on G protein-cyclase interactions, by examining the mechanism of Ca2+ inhibition of adenylyl cyclase in reconstituted preparations of G proteins and cyclase in phospholipid vesicles.
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