Interactions between second messenger systems have been characterized in NIH 3T3 cells. Activation of protein kinase C with phorbol esters results in this cell line in a significant reduction in forskolin-induced formation of cyclic AMP. Such interaction is mediated by the gamma-isoform of protein kinase C. Two distinct receptors for prostaglandins are present in NIH 3T3 cells. One receptor is coupled to phospholipase C. This receptor has a profile of selectivity of PGF2alpha>>PGE2. Stimulation of phospholipase C by PGF2alpha is only partially inhibited by pertussis toxin pretreatment. Experiments in permeabilized cells demonstrate a requirement of a guanine nucleotide for PGF2alpha stimulation, indicating that activation of phospholipase C involves G protein. The second receptor that interacts with prostaglandins in NIH 3T3 cells causes a stimulation of adenylate cyclase. This receptor has a profile of selectivity of PGF2alpha=PGE2. At concentrations of 1-10 muM, activation of cyclic AMP formulation by PGF2alpha is significantly lower than that obtained with PGE2. The inhibitory action of high concentrations of PGF2alpha is attributed to activation of protein kinase C through phospholipase C activation and subsequent formation of diacylglycerol, since in the presence of a phorbol ester, PGE2-induced formation of cyclic AMP is comparable to that attained with high concentrations of PGF2alpha alone. Overnight treatment of NIH 3T3 cells with PGF2alpha results in a down regulation no only of prostaglandin-induced stimulation of phospholipase C, but also of guanine nucleotide-induced activation in permeabilized cells. This result suggests that effects on G protein(s) would therefore permit molecular characterization of the mechanism of down regulation. With the use of polymerase chain reaction (PCR), the expression of G proteins was examined in NIH 3T3 cells. Sequences corresponding to common regions in previously cloned G proteins were utilized as PCR primers. A sequence corresponding to Galpha11, which mediates phospholipase C activation in vitro, was identified in NIH 3T3 cells. Full length cDNAs for coding regions were obtained, introduced into mammalian expression vectors and transfected into NIH 3T3 cells. Transfected cells showed an enhanced response of phospholipase C to PGF2- when compared to vector-transfected controls. This result provides evidence for Galpha11 involvement in phospholipase C activation in the action of PGF2alpha in NIH 3T3 cells.
