SKF-96231

Although guanosine 3′,5′-cyclic monophosphate (cGMP) acts as a relaxant second messenger, the regulation of intracellular cGMP has not been comprehensively studied in human airway smooth muscle. We studied the production of cGMP by cultured human airway smooth muscle cells (HASMC) after stimulation with activators of soluble guanylyl cyclase [sodium nitroprusside (SNP) and S-nitroso- N-acetylpenicillamine (SNAP)] and particulate guanylyl cyclase [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and Escherichia coli heat stable enterotoxin (STa)]. cGMP was measured by enzyme-linked immunosorbent assay. Both SNP (10−6to 10−3M) and SNAP (10−6to 10−3M) caused concentration-dependent elevation of cGMP in the presence of the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (10−3M), with cGMP increasing 6- and 15-fold in response to SNP and SNAP, respectively, at the highest concentration tested (10−3M). The increases in cGMP in response to SNP (5 × 10−5M) and SNAP (10−5M) were inhibited by hemoglobin (Hb; 5 × 10−5M), a nitric oxide scavenger, and methylene blue (MB; 5 × 10−4M), an inhibitor of guanylyl cyclase. cGMP accumulation after SNAP was abolished by both Hb and MB. The response to SNP was inhibited by 79% with Hb and was abolished with MB. ANP, BNP, and CNP (10−9to 10−5M) + phosphoramidon (10−6M) caused a concentration-dependent elevation in cGMP with an order of potency ANP > BNP > CNP. cGMP formation in the presence of the highest concentration of the most potent natriuretic peptide (10−5M ANP) was two- to threefold greater than with the highest concentration of SNAP. The increase in cGMP seen with natriuretic peptides was similar in the presence or absence of phosphoramidon, a neutral endopeptidase (NEP) inhibitor, suggesting that NEP is not playing a role in modulating the effect of natriuretic peptides in HASMC. STa (400 IU/ml) had no effect on cGMP levels. SNAP- and ANP-induced cGMP accumulation was increased by the selective type V PDE inhibitors SKF-96231 and zaprinast, suggesting that type V PDE is responsible for cGMP breakdown in HASMC. These results suggest that cultured HASMC contain both soluble and particulate guanylyl cyclases. The order of potency of the natriuretic peptides ANP > BNP > CNP suggests that type A particulate membrane-bound guanylate cyclase predominates.

Guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue.We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C‐type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)).Both 10−710−3m and 10−710−3m SNAP generated a concentration‐dependent marked elevation in cyclic GMP production when incubated with 10−3m 3‐isobutyl‐1‐methylxanthine (IBMX) (both greater than 25×baseline values with highest drug concentration).The increase in production of cyclic GMP in response to 10−6m SNP and 10−5m SNAP was markedly inhibited by both 5×10−5m haemoglobin (102% and 92% inhibition) and 5×10−5m methylene blue (82% and 84% inhibition).The increase in cyclic GMP in response to 10−3m SNP was measured following co‐incubation with the phosphodiesterase inhibitors 10−710−3m IBMX, 10−710−4m milrinone and 10−710−4m SKF 96231. Only 10−410−3m IBMX significantly increased cyclic GMP levels.Cyclic GMP production was also significantly elevated from baseline by 10−5m ANP, 10−5m BNP, 10−5m CNP and 200 iu ml−1 of E. coli STa toxin in the presence of 10−3m IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (24 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed.These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.British Journal of Pharmacology (1997) 120, 1249–1254; doi:10.1038/sj.bjp.0701040