In vivo production of nitric oxide correlates with NMDA-induced cerebral hyperemia in newborn sheep

Stimulation of N-methyl-D-aspartate (NMDA) receptors in brain increases nitric oxide production in vitro. We tested the hypothesis that nitric oxide participates in the increase in local cerebral blood flow (CBF) caused by infusion of NMDA in anesthetized newborn sheep. We used the combined hydrogen clearance and microdialysis technique for simultaneous measurement of local CBF, infusion of drugs, and measurement of interstitial levels of L- [¹⁴C]citrulline in the parietal cortex. Release of L-[¹⁴C]citrulline into the dialysate during continuous infusion of L-[¹⁴C]arginine was used as a marker of nitric oxide production in vivo. Citrulline recovery and CBF were measured hourly during a 4-h infusion of cerebrospinal fluid containing either 1) no additional drugs, 2) 1 mM NMDA, 3) 1 mM N(G)-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), 4) 1 mM NMDA + 1 mM L-NAME, 5) 0.1 mM 2-chloroadenosine (adenosine receptor agonist), or 6) 0.1 mM 2-chloroadenosine + 1 mM L-NAME. At 240 min of perfusion, CBF (ml · min^{- 1} · 100 g^-1; means ± SE) was as follows: control 52 ± 3, NMDA 116 ± 11, L-NAME 32 ± 5, NMDA + L-NAME 40 ± 4, 2-chloroadenosine 201 ± 63, and 2-chloroadenosine + L-NAME 129 ± 18. Citrulline recovery (fmol/min) at 240 min of perfusion was as follows: control 38 ± 12, NMDA 149 ± 21, L-NAME 9 ± 1, NMDA + L-NAME 39 ± 5, 2-chloroadenosine 13 ± 5, and 2- chloroadenosine + L-NAME 17 ± 1. Infusion of NMDA increased CBF and L- [¹⁴C]citrulline release, and these increases were inhibited by addition of L-NAME to the dialysate. In contrast, the cerebral vasodilator, 2- chloroadenosine, with or without L-NAME, increased CBF without an increase in L-[¹⁴C]citrulline release, thereby demonstrating that the effect of L-NAME to block NMDA-mediated increases in CBF was specific and not due to generalized vasoparalysis. These results demonstrate that the local increase in CBF caused by stimulation of cortical NMDA receptors is dependent on production of nitric oxide. This study also demonstrates that microdialysate- measured conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline is potentially useful as a marker of nitric oxide production in vivo.