Cyclic GMP accumulation in pulmonary microvascular endothelial cells measured by intact cell prelabeling

Cyclic GMP accumulation in cultured rat pulmonary microvascular endothelial cells (RPMVEC) was studied with a new prelabeling method developed using intact platelets and smooth muscle cells (1). [3H]-hypoxanthine was used to radiolabel the cellular guanine nucleotide pool. Neutral alumina and Dowex-50 double column chromatography was used to purify and quantitate the levels of [3H]-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) phosphodiesterase (PDE) inhibitors. In RPMVEC exogenous hypoxanthine was incorporated into both low (65% uptake) and high (34% uptake) passage cells in a time-dependent manner reaching maximum incorporation near 8 hours. Basal cyclic GMP values in both groups were 0.003% of the total cellular tritium (9 x 10(6) and 4 x 10(6) cpm/10(6) cells, respectively). ANF treatment of prelabeled RPMVEC resulted in a 10- to 12-fold increase in [3H]-cyclic GMP in the absence of CN PDE inhibitors (EC50 = 5.4 nM). However, incubation with SNP showed no changes in cellular cyclic GMP accumulation. Several relatively selective CN PDE inhibitors had no effect on ANF or SNP induced cyclic GMP accumulation in RPMVEC. The ANF induced cGMP accumulation was verified by radioimmunoassay. These studies confirm the utility of the hypoxanthine prelabeling technique to monitor intact microvascular EC cyclic GMP accumulation. Cultured RPMVEC show little or no functional soluble guanylate cyclase or cyclic GMP PDE activity.