ACETYLCHOLINE DETECTION BY A MODIFIED HPLC-ED METHOD IMPROVES THE ASSESSMENT OF CHOLINERGIC FUNCTION IN THE MYENTERIC PLEXUS OF THE GUINEA-PIG COLON

Because of the low basal output, measurement of acetylcholine (ACh) re lease from enteric neurons usually requires cholinesterase inhibition, a condition which is known to interfere with feed-back mechanisms reg ulating ACh release. In this study, we resorted to a highly sensitive HPLC-ED method to determine the minimum requirement of physostigmine t o achieve reliable quantitation of spontaneous endogenous ACh overflow from the guinea-pig isolated colon. Furthermore, in order to assess t he degree of interference by physostigmine with cholinergic function, we assessed the effect of scopolamine and oxotremorine (in the presenc e of physostigmine) on spontaneous ACh overflow (to detect the presenc e of autoreceptors) and also measured the efficiency of the peristalti c reflex with different physostigmine concentrations. Spontaneous endo genous ACh overflow was detectable only with physostigmine concentrati ons greater than or equal to 10 nM. ACh overflow increased with increa sing physostigmine concentrations (10 nM-10 mu M range). Scopolamine s ignificantly enhanced the facilitatory effect of physostigmine concent rations greater than or equal to 10 nM; conversely, oxotremorine inhib ited ACh overflow. Peristaltic efficiency was not significantly affect ed by physostigmine concentrations less than or equal to 300 nM. In co nclusion, this modified HPLC-ED method allows ACh detection with minim al physostigmine concentrations (10-30 nM), which do not interfere wit h peristaltic activity, do not saturate autoreceptor feed-back mechani sms ;md therefore improve the assessment of cholinergic function in co lonic enteric neurons. (C) 1997 Elsevier Science Ireland Ltd.