Because of the low basal output, measurement of acetylcholine (ACh) release from enteric neurons usually requires cholinesterase inhibition, a condition which is known to interfere with feed-back mechanisms regulating ACh release. In this study, we resorted to a highly sensitive HPLC-ED method to determine the minimum requirement of physostigmine to achieve reliable quantitation of spontaneous endogenous ACh overflow from the guinea-pig isolated colon. Furthermore, in order to assess the degree of interference by physostigmine with cholinergic function, we assessed the effect of scopolamine and oxotremorine (in the presence of physostigmine) on spontaneous ACh overflow (to detect the presence of autoreceptors) and also measured the efficiency of the peristaltic reflex with different physostigmine concentrations. Spontaneous endogenous ACh overflow was detectable only with physostigmine concentrations greater than or equal to 10 nM. ACh overflow increased with increasing physostigmine concentrations (10 nM-10 mu M range). Scopolamine significantly enhanced the facilitatory effect of physostigmine concentrations greater than or equal to 10 nM; conversely, oxotremorine inhibited ACh overflow. Peristaltic efficiency was not significantly affected by physostigmine concentrations less than or equal to 300 nM. In conclusion, this modified HPLC-ED method allows ACh detection with minimal physostigmine concentrations (10-30 nM), which do not interfere with peristaltic activity, do not saturate autoreceptor feed-back mechanisms ;md therefore improve the assessment of cholinergic function in colonic enteric neurons.
Acetylcholine detection by a modified HPLC-ED method improves the assessment of cholinergic function in the myenteric plexus of the guinea-pig colon.
GIARONI, CRISTINA;MARINO, FRANCA;COSENTINO, MARCO;LECCHINI, SERGIO;FRIGO, GIANMARIO
1997-01-01
Abstract
Because of the low basal output, measurement of acetylcholine (ACh) release from enteric neurons usually requires cholinesterase inhibition, a condition which is known to interfere with feed-back mechanisms regulating ACh release. In this study, we resorted to a highly sensitive HPLC-ED method to determine the minimum requirement of physostigmine to achieve reliable quantitation of spontaneous endogenous ACh overflow from the guinea-pig isolated colon. Furthermore, in order to assess the degree of interference by physostigmine with cholinergic function, we assessed the effect of scopolamine and oxotremorine (in the presence of physostigmine) on spontaneous ACh overflow (to detect the presence of autoreceptors) and also measured the efficiency of the peristaltic reflex with different physostigmine concentrations. Spontaneous endogenous ACh overflow was detectable only with physostigmine concentrations greater than or equal to 10 nM. ACh overflow increased with increasing physostigmine concentrations (10 nM-10 mu M range). Scopolamine significantly enhanced the facilitatory effect of physostigmine concentrations greater than or equal to 10 nM; conversely, oxotremorine inhibited ACh overflow. Peristaltic efficiency was not significantly affected by physostigmine concentrations less than or equal to 300 nM. In conclusion, this modified HPLC-ED method allows ACh detection with minimal physostigmine concentrations (10-30 nM), which do not interfere with peristaltic activity, do not saturate autoreceptor feed-back mechanisms ;md therefore improve the assessment of cholinergic function in colonic enteric neurons.
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