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Uptake of acetylcholine was studied in a synaptic vesicle fraction isolated from rat brain. Hyposmotically treated P3 vesicles took up acetylcholine (ACh) in the presence of MgATP, and the uptake was inhibited by low temperature, ammonium ions, the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) and bafilomycin A1, a specific inhibitor of the vacuolar H(+)-ATPase. Uptake was also inhibited by drugs that bind allosterically to the ACh transporter, namely vesamicol (IC50 value of 170 nM) and 4-aminobenzovesamicol (IC50 value of 25 nM). KM and Vmax values for ACh active transport were estimated to be 5 mM and 4 nmol min-1 mg-1 of cholinergic vesciles, respectively. Active transport of ACh by synaptic vesicles partially purified from brain is mediated by a vesamicol-sensitive transporter and is dependent on a proton gradient generated by the vesicular H(+)-ATPase.

Pyridostigmine bromide (PB) was approved by the U.S. Food and Drug Administration (FDA) in 2003 as a pretreatment in humans against the lethal effects of the irreversible nerve agent soman (GD). Organophosphate (OP) chemical warfare agents such as GD exert their toxic effects by inhibiting acetylcholinesterase (AChE) from terminating the action of acetylcholine at postsynaptic sites in cholinergic nerve terminals (including crucial peripheral muscle such as diaphragm). As part of the post-marketing approval of PB, the FDA required (under 21CFR314, the "two animal rule") the study of a non-human primate model (the common marmoset Callithrix jacchus jacchus) to demonstrate increased survival against lethal GD poisoning, and protection of physiological hemi-diaphragm function after PB pretreatment and subsequent GD exposure. Marmosets (male and female) were placed in the following experimental groups: (i) control (saline pretreatment only), (ii) low dose PB (12.5 microg/kg), or (iii) high dose (39.5 microg/kg) PB. Thirty minutes after the PB dose, animals were challenged with either saline (control) or soman (GD, 45 microg/kg), followed 1 min later by atropine (2mg/kg) and 2-PAM (25mg/kg). After a further 16 min, animals were euthanized and the complete diaphragm removed; the right hemi-diaphragm was frozen immediately at -80 degrees C, and the left hemi-diaphragm was placed in a tissue bath for 4h (to allow for decarbamylation to occur), then frozen. AChE activities were determined using the automated WRAIR cholinesterase assay. Blood samples were collected for AChE activities prior to PB, before GD challenge, and after sacrifice. RBC-AChE was inhibited by approximately 18% and 50% at the low and high doses of PB, respectively, compared to control (baseline) activity. In the absence of PB pretreatment, the inhibition of RBC-AChE by GD was 98%. The recovery of hemi-diaphragm AChE activity after the 4h wash period (decarbamylation) was approximately 8% and 17%, at the low and high PB doses, respectively, compared with the baseline (control) AChE activity prior to PB pretreatment or soman exposure. The results suggest that PB pretreatment protects a critical fraction of AChE activity in the marmoset diaphragm, which is sufficient to allow the animal to breathe despite exposure to a dose of soman that is lethal in unprotected animals.

In chromaffin granule ghosts, the Na+/Ca2+ exchanger in the granule membrane can provide a high affinity (Km 1-3 microM) and high capacity (Vmax 50-100 nmol mg min-2) mechanism for Ca2+ accumulation. The activity of the Na+/H+ antiporter can be used to couple Ca2+ uptake via Na+/Ca2+ exchange to ATP-dependent proton translocation via the granule membrane H(+)-ATPase. Therefore, Ca2+ uptake can be indirectly linked to the proton pump. However, under conditions designed to mimic the environment of a granule in the cytosol of a chromaffin cell, measured rates of Ca2+ uptake are low, a free Ca2+ concentration of about 5 microM in the ghost matrix being attained. Under such circumstances, the granules seem unlikely to play a major role in calcium homeostasis in the intact cell.