Citations

We recently reported that expression of recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts restores NO formation in canine cerebral arteries without endothelium in response to bradykinin ex vivo and in vivo. The present study was designed to further characterize the stimuli that can activate recombinant eNOS enzyme expressed in the adventitia of cerebral arteries. To stimulate recombinant eNOS, we used serum (0.1–10%), substance P (10−11–3 × 10−9M), and ANG II (10−7–10−5M) because they increase intracellular calcium concentrations in fibroblasts. Endothelium-denuded segments of canine basilar arteries were incubated with an adenoviral vector encoding β-galactosidase gene or eNOS gene for 30 min at 37°C. After 24 h, vasomotor activity and cGMP formation in eNOS or β-galactosidase arteries were examined by isometric force recording and by radioimmunoassay, respectively. In control arteries and β-galactosidase gene-transduced arteries, serum caused concentration-dependent contractions, whereas in recombinant eNOS gene-transduced arteries, serum produced concentration-dependent relaxations. Substance P and ANG II had no effect on vascular tone in control and β-galactosidase arteries but caused concentration-dependent relaxations as well as a significant increase in cGMP levels in eNOS arteries. These relaxations were blocked by the NOS inhibitor NG-nitro-l-arginine methyl ester. Chemical treatment or mechanical inactivation of adventitial function significantly attenuated substance P-induced relaxations and ANG II-induced relaxations. These findings demonstrate that serum, substance P, and ANG II cause adventitia-dependent relaxations in cerebral arteries expressing the recombinant eNOS gene. This mechanism of vasodilatation may have beneficial effects in the prevention and treatment of vascular disorders characterized by the diminished bioavailability of NO, such as cerebral vasospasm.

Resistance arteries are an important target for vascular gene therapy because they play a key role in the regulation of tissue blood flow. The present study was designed to determine the effects of recombinant endothelial (e) nitric oxide synthase (NOS) gene expression on vasomotor reactivity of small brain stem arteries (internal diameter, 253 ± 2.5 μm). Arterial rings were exposed ex vivo to an adenoviral vector (109and 1010plaque-forming units/ml) encoding eNOS gene or β-galactosidase gene. Twenty-four hours after transduction, vascular function was examined by isometric force studies. Transgene expression was evident mainly in adventitia. In arteries with endothelium transduced with eNOS gene but not with control β-galactosidase gene, relaxations to bradykinin and substance P were significantly augmented. Removal of endothelium abolished relaxations to bradykinin and substance P in control and β-galactosidase arteries. However, in endothelium-denuded arteries transduced with recombinant eNOS, bradykinin and substance P caused relaxations that were abolished in the presence of the NOS inhibitor NG-nitro-l-arginine methyl ester. In control arteries, endothelium removal augmented relaxations to the nitric oxide donors sodium nitroprusside and diethylamine NONOate. This augmentation was absent in eNOS gene-transduced arteries without endothelium. Our results suggest that, in small brain stem arteries, expression of recombinant eNOS increases biosynthesis of nitric oxide. Adventitia of small arteries is a good target for expression of recombinant eNOS. Genetically engineered adventitial cells may serve as a substitute source of nitric oxide in cerebral arteries with dysfunctional endothelium.

Our previous ex vivo and in vivo studies reported that expression of the recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts recovers NO production in arteries without endothelium in response to bradykinin. The present study was designed to characterize subtypes of bradykinin receptors on adventitial fibroblasts coupled to the activation of recombinant eNOS. Endothelium-denuded segments of canine basilar arteries were transduced with β-galactosidase (β-Gal) gene or eNOS gene ex vivo, using a replication-defective adenoviral vector (1010plaque-forming units/ml) for 30 min at 37°C. Twenty-four hours later, isometric force recording or cGMP measurement was carried out. B1bradykinin receptor agonist (des-Arg9-bradykinin, 10−10-10−8mol/l) did not significantly affect vascular tone in control or β-Gal gene-transduced canine basilar arteries without endothelium. In contrast, this agonist caused concentration-dependent relaxations in recombinant eNOS gene-transduced arteries without endothelium. Relaxations to B1receptor agonist in the eNOS arteries were abolished by B1receptor antagonist (des-Arg9-[Leu8]bradykinin, 6 × 10−9mol/l) but not by B2receptor antagonist (Hoe-140, 5 × 10−8mol/l). Bradykinin did not significantly alter vascular tone in control or β-gal arteries without endothelium, whereas this peptide (10−11-10−8mol/l) induced concentration-dependent relaxations, as well as an increase in cGMP formation in endothelium-denuded eNOS-transduced arteries. Stimulatory effects of bradykinin were prevented in the presence of a B2receptor antagonist but not in the presence of a B1receptor antagonist. B1and B2receptor antagonists had no effect on relaxations to substance P, confirming the selectivity of the compounds. Our results suggest that B1and B2bradykinin receptors are coupled to activation of recombinant eNOS expressed in adventitial fibroblasts.

Background and PurposeThe mechanisms underlying smooth muscle relaxations of cerebral arteries in response to nitric oxide (NO) and cyclic GMP (cGMP) are still not completely understood. The present study was designed to determine the role of potassium channels in the relaxations to NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), as well as 8-bromo-3′,5′-cGMP (a synthetic analogue of cGMP) and zaprinast (a selective cGMP phosphodiesterase inhibitor).MethodsRings of canine middle cerebral arteries without endothelium were suspended in Krebs-Ringer bicarbonate solution for isometric tension recording. The levels of cGMP were measured by radioimmunoassay. Relaxations to NO donors 8-bromo-cGMP and zaprinast were studied in the presence and in the absence of K+channel blockers charybdotoxin (large-conductance Ca2+-activated K+channels), glyburide (ATP-sensitive K+channels), 4-aminopyridine (delayed rectifier K+channels), and BaCl2(multiple types of K+channels).ResultsConcentration-dependent relaxations caused by NO donors (SIN-1 and SNP) were significantly reduced in arteries treated with BaCl2(3×10−4mol/L) or charybdotoxin (3×10−8mol/L). Relaxations to 8-bromo-cGMP were not affected by the same concentrations of BaCl2and charybdotoxin; however, they were reduced by higher concentrations of BaCl2(3×10−3mol/L) and charybdotoxin (10−7mol/L). Zaprinast-induced relaxations were significantly reduced by BaCl2(3×10−4mol/L) or charybdotoxin (3×10−8mol/L). Glyburide (10−5mol/L) and 4-aminopyridine (10−3mol/L) did not alter the relaxations to SIN-1 or SNP. The production of cGMP stimulated by SIN-1 in the vascular smooth muscle was not affected by BaCl2(3×10−3mol/L) or charybdotoxin (10−7mol/L).ConclusionsThese results indicate that in canine middle cerebral arteries, a significant portion of relaxations to NO liberated from nitrovasodilators is mediated by large-conductance Ca2+-activated K+channels. Other K+channels, sensitive to BaCl2, may also be involved in the mechanism of relaxations induced by NO.

This study was designed to determine the effect of subarachnoid hemorrhage (SAH) on potassium (K + ) channels involved in relaxations of cerebral arteries to nitrovasodilators. The effects of K + channel inhibitors on relaxations to 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP) were studied in rings of basilar arteries obtained from untreated dogs and dogs exposed to SAH. The levels of cyclic GMP were measured by radioimmunoassay. In rings without endothelium, concentration-dependent relaxations to SIN-1 (10 −9 − 10 −4 mol/L) and SNP (10 −9 − 10 −4 mol/L) were not affected by SAH, whereas increase in cyclic GMP production stimulated by SIN-1 (10 −6 mol/L) was significantly suppressed after SAH. The relaxations to SIN-1 and SNP were reduced by charybdotoxin (CTX; 10 −7 mol/L), a selective Ca 2+ -activated K + channel inhibitor, in both normal and SAH arteries; however, the reduction of relaxations by CTX was significantly greater in SAH arteries. By contrast, the relaxations to these nitrovasodilators were not affected by glyburide (10 −5 mol/L), an ATP-sensitive K + channel inhibitor, in both normal and SAH arteries. These findings suggest that in cerebral arteries exposed to SAH, Ca 2+ -activated K + channels may play a compensatory role in mediation of relaxations to nitric oxide. This may help to explain mechanisms of relaxations to nitrovasodilators in arteries with impaired production of cyclic GMP.