cGMP is considered the only mediator synthesized by
soluble guanylyl cyclase (sGC) in response to
nitric oxide (NO). However, purified sGC can synthesize several other
cyclic nucleotides, including
inosine 3',5'-cyclic monophosphate (cIMP). The present study was designed to determine the role of cIMP in hypoxic contractions of isolated porcine coronary arteries. Vascular responses were examined by measuring isometric tension.
Cyclic nucleotides were assayed by HPLC tandem mass spectroscopy.
Rho kinase (ROCK) activity was determined by measuring the phosphorylation of
myosin phosphatase target subunit 1 using Western blot analysis and an ELISA kit. The level of cIMP, but not that of cGMP, was elevated by
hypoxia in arteries with, but not in those without, endothelium [except if treated with
diethylenetriamine (
DETA) NONOate]; the increases in cIMP were inhibited by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ).
Hypoxia (Po2: 25-30 mmHg) augmented contractions of arteries with and without endothelium if treated with
DETA NONOate; these hypoxic contractions were blocked by ODQ. In arteries without endothelium, hypoxic augmentation of contraction was also obtained with exogenous cIMP. In arteries with endothelium, hypoxic augmentation of contraction was further enhanced by
inosine 5'-triphosphate, the precursor for cIMP. The augmentation of contraction caused by
hypoxia or cIMP was accompanied by increased phosphorylation of
myosin phosphatase target subunit 1 at Thr(853), which was prevented by the ROCK inhibitor
Y-27632. ROCK activity in the supernatant of isolated arteries was stimulated by cIMP in a concentration-dependent fashion. These results demonstrate that cIMP synthesized by sGC is the likely mediator of hypoxic augmentation of coronary vasoconstriction, in part by activating ROCK.