CYCLIC-GMP ACCUMULATION IN PULMONARY MICROVASCULAR ENDOTHELIAL-CELLS MEASURED BY INTACT CELL PRELABELING

Cyclic GMP accumulation in cultured rat pulmonary microvascular endoth elial cells (RPMVEC) was studied with a new prelabeling method develop ed using intact platelets and smooth muscle cells (1). [H-3]-hypoxanth ine was used to radiolabel the cellular guanine nucleotide pool. Neutr al alumina and Dowex-50 double column chromatography was used to purif y and quantitate the levels of [H-3]-cyclic GMP. Changes in cyclic GMP metabolism in short and long term RPMVEC cultures were studied using rat atrial naturetic factor 8-33 (ANF) and sodium nitroprusside (SNP) in the presence and absence of cyclic nucleotide (CN) phosphodiesteras e (PDE) inhibitors. In RPMVEC exogenous hypoxanthine was incorporated into both low (65% uptake) and high (34% uptake) passage cells in a ti me-dependent manner reaching maximum incorporation near 8 hours. Basal cyclic GMP values in both groups were 0.003% of the total cellular tr itium (9 x 10(6) and 4 x 10(6) cpm/10(6) cells, respectively). ANF tre atment of prelabeled RPMVEC resulted in a 10- to 12-fold increase in [ H-3]-cyclic GMP in the absence of CN PDE inhibitors (EC(50)=5.4 nM). H owever, incubation with SNP showed no changes in cellular cyclic GMP a ccumulation. Several relatively selective CN PDE inhibitors had no eff ect on ANF or SNP induced cyclic GMP accumulation in RPMVEC. The ANF i nduced cGMP accumulation was verified by radioimmunoassay. These studi es confirm the utility of the hypoxanthine prelabeling technique to mo nitor intact microvascular EC cyclic GMP accumulation. Cultured RPMVEC show little or no functional soluble guanylate cyclase or cyclic GMP PDE activity.