STIMULATION-INDUCED H-3 L-CITRULLINE ACCUMULATION IN ISOLATED LONGITUDINAL MUSCLE MYENTERIC PLEXUS PREPARATIONS OF RAT SMALL-INTESTINE - A MEASURE TO CHARACTERIZE NITRERGIC NEURONS

Nitric oxide (NO) synthase activity in rat isolated longitudinal muscl e myenteric plexus preparations of the small intestine was determined by measuring the accumulation of H-3-L-citrulline during 30 min incuba tion with H-3-L-arginine. In untreated preparations a significant amou nt of H-3-L-citrulline accumulated in the tissue, about 2000 dpm/30 mg per 30 min, accounting for about 1.7% of the tissue radioactivity. In termittent electrical field stimulation (15 Hz, 10 s trains with 10 s intervals for total of 20 min) caused a threefold increase in H-3-L-ci trulline accumulation. The NO synthase inhibitor N-omega-nitro-L-argin ine methyl ester (L-NAME) reduced the spontaneous accumulation of H-3- L-citrulline by 65% and prevented the electrically evoked increase. Re moval of extracellular calcium or addition of tetrodotoxin blocked the electrically evoked increase in H-3-L-citrulline accumulation without affecting spontaneous accumulation, Application of the calcium ionoph ore A 23187 (10 mu mol/l) or 45 mmol/l potassium caused a twofold incr ease in the accumulation of H-3-L-citrulline. The muscarine receptor a gonist oxotremorine (1 mu mol/l) had no effect on spontaneous accumula tion of H-3-L-citrulline, but inhibited the electrically evoked increa se by about 50%, and this effect was blocked by scopolamine. A substan tial amount of H-3-L-citrulline (15 000 dpm) accumulated also in the i ncubation media, and this was increased 1.7-fold by the presence of A 23187 and 2.7-fold by electrical stimulation. However, electrically ev oked increase in H-3-L-citrulline was not prevented by tetrodotoxin, i n contrast to observation on tissue levels. In conclusion, during incu bation with H-3-L-arginine tissue levels of H-3-L-citrulline in rat is olated longitudinal muscle myenteric plexus preparations, but not accu mulation in incubation media may be used as a biochemical marker of th e activity of nitrergic intestinal neurons which appear to be inhibite d via muscarine receptors.