SECRETION OF CYCLIC-GMP BY CULTURED EPITHELIAL AND FIBROBLAST CELL-LINES IN RESPONSE TO NITRIC-OXIDE

LLC-PK1 epithelial cells and RFL-6 fibroblasts secreted both cyclic AM P (cAMP) and cyclic GMP (cGMP) when costimulated with forskolin and 3- morpholinosydnonimine (a chemical nitric oxide generator). Intracellul ar cAMP levels as high as 1100 and 12,000 pmol/10(6) cells were achiev ed for the two cell types, respectively. These levels were high enough to reach approximately 50% saturation of the cAMP transporter and inh ibited transport of cGMP to an equal extent, suggesting that the two c yclic nucleotides compete for a common transport system. The rates of secretion of cGMP and cAMP from LLC-PK1 cells increased in proportion to their rates of synthesis as concentrations of stimulant were varied , but increased only 25% relative to intracellular concentrations in r esponse to inhibition of phosphodiesterases by 3-isobutylmethylxanthin e. It is proposed that secretion of cyclic nucleotides is not simply p roportional to the total intracellular pool in these cells, but rather is coupled to synthesis. In support of this model, oxyhemoglobin was used to trap nitric oxide and block activity of guanylate cyclase in c ells treated with 3-morpholinosydnonimine. As a result, secretion of c GMP ceased within 1 min, whereas intracellular levels decreased slowly over 60 min. Probenecid [p-(dipropylsulfamoyl)benzoic acid] is a nons elective antagonist of anion transport that inhibited secretion of cAM P in both cell types but, unexpectedly, blocked synthesis of cGMP, and this was reflected in direct inhibition of soluble guanylate cyclase in cell lysates. Two heat-stable, high molecular weight factors that c onfer sensitivity to probenecid were identified, and these factors inc reased the sensitivity of guanylate cyclase to nitric acid by an order of magnitude.