Proceedings of The Physiological Society

University of York (2002) J Physiol 539P, S021


Nitric oxide increases perineurial permeability to K+ via an NO-sGC-cGMP mechanism

P.I. Ashmore, M. Lalji, S.A. Khan, R. Hevawitharane and N.J. Abbott

BBB Group, Centre for Neuroscience Research, King's College London, London SE1 1UL, UK

The perineurium forms one component of the blood-nerve barrier, and contributes to regulation of the endoneurial microenvironment (Rechthand & Rapoport, 1987). Khan et al. (2001) showed that nitric oxide (NO) caused a small reversible increase in perineurial permeability to K+ (PK) in the frog sciatic nerve; the present study examines the underlying mechanisms.

Frogs (Rana pipiens or R. temporaria) were killed by an approved humane procedure (in accordance with UK legislation). The sciatic nerve was mounted in the groove across a 5-compartment Perspex chamber, with Vaseline seals between compartments (Abbott et al. 1997). The nerve was stimulated via compartments #1 and #2. Compound action potential (CAP) amplitude and direct current potential (DCP) were measured between compartments #3 (Ringer or test solution) and #5 via Ringer-agar bridges to Ag/AgCl electrodes. Compartment #4 contained Vaseline. Control Ringer (pH 7.4) contained (mM): 116 NaCl, 2.5 KCl, 1.8 CaCl2 and 10 Hepes. Changes in CAP and DCP were measured in response to elevated K+ Ringer pulses (110 mM KCl replacing NaCl) before and after the application of test solutions of NO, to establish the permeability of the perineurium to K+; a negative liquid junction potential indicated a tight perineurium, while a leaky perineurium generated a positive diffusion potential ‘step'.

The control perineurium showed low PK. A 30 min application of the NO donor DETANONOate (supplying 4 and 8 µM NO) caused an increase in PK (increase in step DCP from 0.02 ± 0.02 mV, mean ± S.E.M., n = 12 to 0.12 ± 0.03 mV, n = 21; P < 0.01, Student's unpaired t test). At 16 µM NO, step DCP increased to 0.24 ± 0.05 mV (n = 18, P < 0.001). Pre-incubation for 10 min with the soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]-Oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 2 µM) before NO application, inhibited the increase in step DCP (P < 0.05, n = 15) at 4 and 8 µM NO, but not at 16 µM NO (n = 5). 30 min application of 200 µM YC-1, a sGC stimulator, mimicked the NO-induced increase in PK, step DCP increasing significantly by 0.15 ± 0.02 mV (n = 3, P < 0.001). The effect of 200 µM YC-1 was also significantly inhibited by 2 µM ODQ (n = 3, P < 0.01).

This is the first evidence that the NO-induced increase in PK of the frog sciatic nerve occurs via an intracellular signalling mechanism involving sGC at the lower concentrations of NO (4 and 8 µM). At 16 µM NO free radicals may be involved. This study has physiological and pathological implications.

P.A. was supported by a Physiological Society Vacation Studentship.

Where applicable, experiments conform with Society ethical requirements