Pleiotypic Control by Cyclic AMP: Interaction with Cyclic GMP and Possible Role of Microtubules

Abstract

Previous studies have shown that exogenous dibutyryl cyclic AMP inhibits the uptake of uridine, leucine, and 2-deoxyglucose by cultured mouse fibroblasts. 3':5'-cyclic GMP is shown here to counteract these inhibitory effects as well as the inhibition ofprecursor transport and leucine incorporation into proteins produced by prostaglandin E1. We conclude, therefore, that cyclic GMP antagonizes the "pleiotypic" effects of cyclic AMP in these cells.Colcemid and vinblastine, but not cytochalasin B., reverse the transport inhibition caused by cyclic AMP without affecting the intracellular concentrations of cyclic AMP. These results suggest the possibility that cyclic AMP regulates the membrane transport of certain substrates by influencing the organization of microtubules.Recent work from various laboratories has implicated cAMP in the regulation of cellular growth. In a preceding communication (1) we presented evidence that cAMP exerts this control by coordinately influencing several biochemical processes related to growth. This set of reactions was defined as the "pleiotypic" program, and its regulation as pleiotypic control (2). Earlier we had singled out this coordinated control as being of general significance since the same set of reactions is affected in various cell types by specific hormones and growthpromoting factors (2). The fact that cAMP controls the pleiotypic reactions provides a basis for relating differentiation, malignant transformation, and hormonal control of growth (1). We report here some observations pertaining to the mechanisms of cAMP control of the pleiotypic parameters.cGMP antagonizes the cAMP stimulated synthesis of flgalactosidase in cell-free bacterial extracts (3, 4). In certain animal cells as well, cGMP appears as if it might also directly or indirectly counteract the effects of cAMP (5-9). We have, therefore, investigated the interaction between these cyclic nucleotides on the pleiotypic reactions and conclude that here, too, cGMP overcomes the actions of cAMP. We also suggest, on the basis of studies with inhibitors of microtubule assembly, that the cyclic nucleotides may influence some membrane transport processes by their effects on colcemid-and vinblastine-sensitive structures, possibly the microtubular apparatus itself. MATERIALS AND METHODSThe Balb/c 3T3 mouse fibroblasts and their SV 40-transformed derivatives were grown in MEM plus 10% calf serum.The rates of precursor transport and incorporation into macromolecules were measured as described (2) In 3T3 cells, the rates of transport of uridine, leucine, and 2-deoxyglucose are depressed during serum deprivation and stimulated by the addition of serum again. We have reported(1) that (Bu)2cAMP + theophylline or PGE1 added to cells maintained in serum-containing medium mimic the response to serum starvation. These effectors also inhibit serumstimulated uptake of precursor by previously starved cells (1). We now show that cGMP antagonizes the effects of both (Bu)2cAMP + theophylline and PGE1 on these biochemical processes...

The role of cyclic nucleotides in the development and function of rat mammary tissue

“…In studies with rat mammary gland explants in culture [2] cyclic AMP inhibited the increase of enzyme activities normally associated with lactogenesis, in particular the activities of those enzymes involved in lipogenesis, and also markedly depressed the synthesis of DNA, RNA and fatty acids. This analogy with the pleiotypic response observed for other cells grown in culture [3], and the reversal by cyclic GMP of the pleiotypic inhibition of 3T3 cells treated with dibutyryl cyclic AMP [4] , led us to investigate the simultaneous fluctuations of both cyclic nucleotides throughout the lactation cycle. differentiation of the mammary gland during pregnancy, its response to hormones and the promotion of specific cell programmes for the synthesis of the characteristic components of milk may all be mediated by modification of the ratio between the two nucleotides.…”

“…Some of these activities may be highly relevant to the mammary gland a) the role of cyclic AMP as promoter of differentiation could be of special significance to the development of the gland during pregnancy, a stage at which the level of this nucleotide is high; b) the rise of cyclic GMP after the onset of lactation may explain the wave of mitosis which follows shortly after parturition in the rat [23], a process which is also significant (in addition to increasing the number of secretory cells present) in that it may, as suggested by Stockdale and Topper [24] , make these cells specially susceptible to environmental factors capable of modifying the metabolic pattern; c) the complex endocrine control of the mammary gland and the tissue response to hormones may be expressed through the dualistic system of opposing cyclic AMP and cyclic GMP levels which could, as has already been shown for other cells grown in culture [3,4] , also modulate the pleiotypic control of mammary cells [2] .…”

Identification of a calcium-binding protein as a calcium-dependent regulator of brain adenylate cyclase.

“…The possible relationship of CDR to Ca2+-dependent guanylate cyclase activity (23) requires systematic exploration. In spite of these limitations, the potential applicability of the present findings to the bidirectional cyclic nucleotide control of cell proliferation (22,24,25) should be noted. Anderson and Pastan (26) have evidence for the existence of an adenylate cyclase activating factor that is present in fibroblasts grown in culture.…”

Effects of prostaglandin on substrate uptake and cell division in human diploid fibroblasts

“…These experiments also indicate that this inhibition may be associated with the decrease in cell division caused by PGE1. A similar action by PGE1, that of inhibiting serum-stimulated uptake of uridine in 3T3 cells, was also noted by Kram & Tomkins (1973). This activity by PGE1 may indeed be a part of a synchronized effect on the metabolic events leading to incubation; o, -E1-24, corresponding cultures not treated with PGE1.…”

“…For example, Kram & Tomkins (1973) demonstrated that after serum stimulation, those cultures of Balb/c 3T3 cells that included PGE1 exhibited a much lower uptake ofleucine, uridine and adenosine than did the cultures to which PGE1 was not added. PGE1 is capable of reversibly decreasing cell division and the incorporation of [3H]thymidine into DNA in the WI38 human embryo lung fibroblasts (Kurtz et al, 1974).…”