Objective: Several recent studies have implicated a role of endogenous
nitric oxide (NO) in the pathophysiology of myocardial ischemic/reper
fusion injury. However, the mechanism by which NO exerts its beneficia
l/detrimental effects remains unknown. This study examined the intrace
llular signaling of NO by studying the role of the NO-cGMP signaling p
athway on the phospho-diesteratic breakdown and turnover of phosphoino
sitides during myocardial ischemia and reperfusion. Methods: Isolated
working rat hearts were made ischemic for 30 min followed by 30 min of
reperfusion. A separate group of hearts were pre-perfused with 3 mM L
-arginine for 10 min prior to ischemia. The release of NO was monitore
d using an on-line amperometric sensor. The aortic flow and developed
pressure were examined to determine the effects of L-arginine on ische
mic/reperfusion injury. For signal transduction experiments, sarcolemm
al membranes were radiolabeled by perfusing the isolated hearts with [
H-3] myoinositol and [C-14]arachidonic acid. Hearts were then perfused
for 10 min in the presence or absence of L-arginine via the Langendor
ff mode. Ischemia was induced for 30 min followed by 30 min of reperfu
sion. Experiments were terminated before L-arginine and after L-argini
ne treatment, after ischemia, and during reperfusion. Biopsies were pr
ocessed to determine the isotopic incorporation into various phosphoin
ositols as well as phosphatidic acid and diacylglycerol. cGMP was assa
yed by radioimmunoassay and SOD content was determined by enzymatic an
alysis. Results: The release of NO was diminished following ischemia a
nd reperfusion and was augmented by L-arginine. L-Arginine reduced isc
hemic/reperfusion injury as evidenced by the enhanced myocardial funct
ional recovery. cGMP, which remained unaffected by ischemia and reperf
usion, was stimulated significantly after L-arginine treatment. The cG
MP level persisted up to 10 min of reperfusion and then dropped slight
ly. Reperfusion of ischemic myocardium resulted in significant accumul
ation of radiolabeled inositol phosphate, inositol bisphosphate, and i
nositol triphosphate. Isotopic incorporation of [H-3]inositol into pho
sphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidyli
nositol-4,5-bisphosphate was increased significantly during reperfusio
n. Reperfusion of the ischemic heart prelabeled with [C-14]-arachidoni
c acid resulted in modest increases in [C-14]diacylglycerol and [C-14]
phosphatidic acid. Pretreatment of the heart with L-arginine significa
ntly reversed this enhanced phosphodiesteratic breakdown during ischem
ia and early reperfusion. However, at the end of the reperfusion the i
nhibitory effect of L-arginine on the phosphodiesterases seems to be r
educed. In L-arginine-treated hearts, SOD activity was progressively d
ecreased with the duration of reperfusion time. Conclusions: The resul
ts suggest for the first time that NO plays a significant role in tran
smembrane signaling in the ischemic myocardium. The signaling seems to
be transmitted via cGMP and opposes the effects of phosphodiesterases
by inhibiting the ischemia/reperfusion-induced phosphodiesteratic bre
akdown. This signaling effect appears to be reduced as reperfusion pro
gresses. These results, when viewed in the light of free radical chemi
stry of NO, suggest that such on- and off-signaling of NO may be linke
d to its interaction with the superoxide radical generated during the
reperfusion of ischemic myocardium.