Tolerance to nitric oxide vasodilators in cerebral artery smooth muscle cells SMQ

We characterized tolerance to the NO producing vasodilators, rutroglycerin (NTG) and sodium nitroprusside (SNP) in cultured cerebral artery SMC by measuring intracellular cGMP following chronic exposure to these drugs. SMC were isolated from pig basilar. anterior and middle cerebral, and internal carotid arteries. Cells were identified as smooth muscle by hislochemical stain for smooth muscle α-actin. Intracellular cGMP was extracted wiih 0.1 N HC1 and measured by radioimmunoassay Acute concentration-response curves of cGMP to NTG (0.1 nM to 1 mM) and to SNP (0.1 jiM to 1 mM) were determined in cells pretreated for 1 hr. with 100 uM NTG and compared to response curves in untreated cells. Basal cGMP level was 2.1 ±0.4 pmol/mg/cell protein (n=16). Both agents increased cGMP but SNP was more effective. MaxJmum concentration of SNP (1 mM) increased cGMP to 163 ±5.9 pmol/mg vs 21 ± 2.4 pmol/mg for 1 mM NTG (P<0.01) in control cells. Cells pretreated with 100 uM NTG for 1 hr were unresponsive to acutely applied NTG up lo 1 mM but remained responsive to SNP. However, the response curve to SNP was significantly depressed by approximately 25% without a change in the EC₅₀. Following washout of NTG. the response of cGMP lo SNP returned to control within 12 hr. while the response to NTG required 48 hr. These results indicate that cerebral artery SMC in culture express soluble guanylyl cyclase and the enzymes necessary lo metabolize NTG lo NO. Prolonged exposure of the cells to NTG produced a depressed response of cGMP to rechallenge with NTG. This depressed response of intracellular cGMP could represent a mechanism of vascular tolerance to NO producing vasodilators.