In our previous studies we showed phosphodiesterase inhibiors, which usually raise intracellular cyclic AMP levels, inhibit lactose systhesis up to 100% in mammary gland slices from lactating guinea pigs. Exogenous cAMP and DBcAMP inhibition is limited to about 30% as is treatment with catecholamines whichstimulate adenylate cyclase activity. Further studies showed that cAMP inhibition is specific (ATP, cGMP, and 5'-AMP have no effect) and inhibition by a potent analogue, 8-lCl-S-cAMP, is also limited to about 30% but at 1/100 the dosage. Additivity between DBcAMP and 1-methyl-3-isobutylxanthine (MIX) only occurs when the two concentrations are adjusted to yield about 20% inhibition each (10 to the minus 3rd power M DBcAMP and 10 to the minus 5th power M MIX). Cholera toxin has no effect on lactose synthesis in the range 0.001 to 10 g/ml. In the proposed project we will continue to investigate the inhibitory role of cAMP in lactation and whether such inhibition represents a physiological regulatory system analogous to that of progesterone withdrawal and the triggering of lactogenesis. First, we will ovariectomize rats in late pregnancy to induce lactogenesis and then follow the mammary gland cyclic nucleotide system (cAMP, and cGMP levels, activities of adenylate and guanylate cyclases, the phosphodiesterases and the cyclic nucleotide-dependent protein kinases) using lactose production as a measure of lactogenesis. Progesterone replacement in operated animals will be used to determine if blockage of induced lactogenesis prevents cyclic nucleotide changes. Second, the problem of a mixed cell population in mammary gland will be aproach by following the same parameters in cultured alveolar cells on floating collagen gels during hormonal induction of lactogenesis. Finally, the site(s) of cAMP action will be investigated, first, by studying its effects on glucose uptake by isolated acini; and second, by studying its effects on lactose synthetic system, including alpha-lactalbumin and galactosyl transferase, in floating collgen gel cultures of alveolar cells and cell-free preparations.