In order to further elucidate the mechanisms that control vertebrate cell division, regulation of phosphodiesterase (PDE) activity by hormones and guanine nucleotides will be examined. Amphibian oocytes are naturally arrested in prophase I of their meiotic division, and provide an excellent model system for these studies, since they are exceptionally large cells, 1.3 mm in diameter, that are suitable for microinjection. Significant fluxes in intracellular levels of cAMP have been measured previously and correlated with the stages of the cell cycle and the process of cell division. This change in cAMP levels is one of the early events that follows hormone induction of the maturation process, and has been correlated with steroid-induced inhibition of adenylate cyclase activity. A complementary regulation of PDE activity may also be hypothesized. The in vivo role of PDE activity in decreasing cAMP will be assessed by micro-injecting 3H-cAMP into intact oocytes and following the kinetics of PDE activity both before and after induction of oocyte maturation by progesterone and insulin. Any involvement of calmodulin-dependent PDE will be examined by injection of purified bovine brain PDE and the calmodulin inhibitor, calmidazolium. The possible role of the ras gene product, p21, in regulating cell division via modulation of enzyme systems that regulate cAMP levels will also be investigated. Ras gene will be microinjected into oocytes, and expression of p21 will be assessed using Western immunoblots of oocyte homogenates and membrane preparations with monoclonal antibodies directed against p21. Gene expression will then be correlated with any effects on the time course of oocyte maturation, any concomitant changes in adenylate cyclase and PDE activities, and any changes in RIA-measured oocyte cAMP levels. The effects of the non-hydrolyzable guanine nucleotides, GTP S and Gpp(NH)p, upon in vitro oocyte PDE activity will also be examined. Finally, the role of guanine nucleotide binding proteins in regulating PDE activity will also be assessed using the bacterial toxins, pertussis toxin and cholera toxin, which have previously been used to examine the actions of guanine nucleotide binding proteins in regulating adenylate cyclase activity. These experiments will broaden our understanding of the roles of PDE and oncogene products in altering cAMP levels and thereby regulating cell division.
