Isoleucine-atrial natriuretic factor

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.

Mastoparan activated membrane-bound guanylate cyclase and potentiated the effect of atrial natriuretic factor (ANF) and ATP on guanylate cyclase activity in rat lung membranes. Mastoparan is a cationic, amphiphilic tetradecapeptide with an amidated carboxyl terminus. It takes the alpha-helical conformation upon interacting with the membrane. Several analogs were synthesized to study the role of the positive charges, the carboxyl amino group and the alpha-helical conformation of mastoparan in the activation of guanylate cyclase. The results showed that substitution of the C-terminal amide group of mastoparan with a carboxyl group significantly reduced its potency on the activation of guanylate cyclase. Replacement of three lysine residues of mastoparan with aspartic acid or serine residues completely abolished the stimulatory effect of mastoparan. When the alanine at position 10 of mastoparan was substituted by a proline, the resulting analog had no effect on guanylate cyclase activity. These results demonstrate that the positive charges and the helical structure of mastoparan are critical determinants for the activation of guanylate cyclase.