Search

Enzymes can be used in high-performance liquid chromatography post-column reactors to improve sensitivity and specificity of detection for some compounds by converting the compounds to easily detectable products. The enzymes can be covalently bound to a post-column reactor, but a simpler approach is to bind them by adsorption to an ion exchanger or a hydrophobic interaction support. This technique has been applied to electrochemical detection of acetylcholine by using adsorption of choline oxidase and cholinesterase to a 3-cm long commercially available weak anion-exchange cartridge. Conversion of acetylcholine to peroxide is quantitative during the 10-sec residence time in the cartridge. Enzyme elution from the cartridge is negligible when low ionic strength mobile phases are used. Fresh enzyme needs to be added to the cartridge at only 1-2 week intervals.

Acetylcholine (ACh) and vasoactive intestinal peptide (VIP) probably coexist in cholinergic neurons of rodent salivary glands. In this tissue, cholinergic drugs regulate release of both ACh and VIP from postganglionic cholinergic neurons. In the present study we investigated whether VIP could modulate the metabolism of ACh in mouse submandibular gland cholinergic neurons using ACh turnover rate (TRACh) as a parameter. The TRACh was estimated via measurement of the formation of [3H]ACh during constant rate infusion of [3H]choline. Choline and ACh were separated by reverse phase high-performance liquid chromatography and were detected electrochemically after enzymatic postcolumn reaction. We calculated that the TRACh was about 3 nmol/mg of protein per hr. Pilocarpine, a muscarinic agonist decreased the TRACh about 5-fold whereas atropine methyl Br, a muscarinic antagonist, caused a large increase in turnover. Turnover, therefore, appears to be regulated by a feedback mechanism triggered by occupancy of postsynaptic receptors. VIP infused i.v. (40 micrograms/kg/min) decreased the TRACh by about 50%. Atropine completely prevented the inhibition of the TRACh induced by VIP. These results suggest that, by changing postsynaptic or presynaptic muscarinic receptor function, VIP may participate in the control of ACh metabolism. Parasympathetic decentralization of salivary glands did not prevent the effect of either atropine or VIP on TRACh. This finding suggests that the central afferent input to the ganglionic cells is not required for the regulation of ACh metabolism and, therefore, the feedback loop probably acts via a postganglionic mechanism which is not elucidated by present experiments.

Choline oxidase and cholinesterase were found to retain their activity for 1-2 weeks at room temperature while adsorbed to a commercially available anion-exchange cartridge. These enzymes convert acetylcholine to H2O2. Acetylcholine can be measured in tissue extracts by separation at pH 7 on a polymeric reverse-phase high-performance liquid chromatography column, conversion of acetylcholine to H2O2 on a postcolumn enzyme-loaded anion-exchange cartridge, and electrochemical detection of the H2O2 formed.

In GH3 pituitary cell homogenates, acetylcholine (ACh) (IC50 200 nM) inhibits adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity in a concentration- and GTP-dependent manner. Maximal inhibition was obtained with 10 microM ACh and corresponded to approximately a 50% decrease in basal enzyme activity. ACh inhibition is antagonized by atropine and is mimicked by muscarinic receptor agonists, but not by nicotine. ACh reduces the adenylate cyclase stimulation by vasoactive intestinal peptide (VIP), without changing its EC50. In intact GH3 cells, ACh decreases the cyclic AMP content and the rate of prolactin release in a concentration-dependent manner. When the cells are simultaneously exposed to VIP and ACh, the VIP-induced increases in cyclic AMP accumulation and prolactin release are reduced by 80% and 40%, respectively. The potency of VIP is not significantly changed by the presence of ACh, and vice versa.

Bilateral (3 nmol/side) i.c.v. infusion of ethylcholine aziridinium ion (AF64A) causes a 70% decrease of hippocampal acetylcholine content lasting for longer than 30 days without changing the density of hippocampal recognition sites for muscarinic ligands. In hippocampal slices prepared from rats receiving i.c.v. AF64A, the activation of phosphoinositide turnover or the inhibition of cyclic AMP accumulation elicited by muscarinic receptor agonists is facilitated. This AF64A treatment also causes a long-lasting decrease of hippocampal norepinephrine and serotonin (5-HT) content. Even a smaller dose of AF64A (1.5 nmol/side) reduces the hippocampal 5-HT content. The number of alpha-1 adrenoceptor recognition sites is slightly increased by 3 nmol/side of AF64A and the stimulation of phosphoinositide turnover by norepinephrine is facilitated. In contrast the decrease of hippocampal 5-HT concentration elicited by AF64A fails to change the 5-HT receptor indices that were measured. These results indicate that in rat hippocampus muscarinic receptors and alpha-1 adrenoceptors are denervated by AF64A and that this denervation promotes a receptor supersensitivity. These results also suggest that we could not find appropriate conditions to express a complete specificity of AF64A in destroying cholinergic axons and therefore this drug cannot be used readily to induce a selective deficiency of central cholinergic transmission.