Using14C-labeled arginine to14C-labeled citrulline conversion assays in brain homogenates from 14- to 18-day-old and adult spontaneously hypertensive rats, we tested the hypotheses that maturation increases neuronal nitric oxide synthase (nNOS) activity and that this increase involves changes in cofactor availability and/or nNOS kinetics. nNOS activity (in pmol ⋅ mg−1 ⋅ min−1) was 46% higher in adults (19 ± 2) than in pups (13 ± 1). The addition of 264 μM calmodulin (CaM), 3 μM FAD, 3 μM flavin adenine mononucleotide (FMN), and 10 μM tetrahydrobiopterin (BH4) increased NOS activity by 3, 46, 45, and 88% in pups and by 19, 40, 36, and 102% in adults, respectively. All cofactor effects were significant except for CaM in the pup homogenates. Cofactor effects were not significantly different between pup and adult homogenates, except for BH4, which increased absolute NOS activity more in adults than in pups. Values of maximal enzyme velocity ( V max) for nNOS in the absence of added cofactors were greater in adults than in pups (104 ± 5 vs. 53 ± 3, P < 0.05). Addition of 3 μM FAD or 3 μM FMN increased pup V max values to 68 ± 2 and 99 ± 5, respectively, but had no effect in adults. BH4 did not affect V max in either group. Control values of the Michaelis-Menten constant ( K m) forl-arginine were greater ( P < 0.05) in pups (5.7 ± 0.4 μM) than in adults (4.3 ± 0.2 μM) and were significantly reduced by 10 μM BH4 to 3.8 ± 0.2 and 2.9 ± 0.1 μM, respectively. Neither FAD nor FMN affected K mvalues in either group. The results indicate that endogenous nNOS cofactor levels are not saturating in either pups or adults, changes in cofactor levels differentially affect NOS kinetics in pups and adults, and age-related differences in NOS activity result from fundamental differences in NOS kinetics. These findings support the general hypothesis that the increased vulnerability to ischemic stroke associated with maturation is due in part to corresponding increases in the capacity for nitric oxide synthesis.
Vascular responsiveness to nitric oxide synthesis inhibition in hypertensive rats.
