Hemicholinium-3 mustard reveals two populations of cycling choline cotransporters in Limulus
Section snippets
Materials
Horseshoe crabs (Limulus polyphemus) were obtained from Marine Biological Laboratories (Woods Hole, MA) and were maintained in moist excelsior at an ambient temperature of 4–6°C. The pharmacological reagents used in this study, chelerythrine, staurosporine and HC-3, were purchased from Sigma Chemical Company (St. Louis, MO). Methyl-[3H]choline chloride (sp. act.=60–90 Ci/mmol) and methyl-[3H]HC-3 diacetate salt (sp. act.=120 Ci/mmol) were purchased from Dupont New England Nuclear (Wilmington,
Effect of 200 μM hemicholinium-3 mustard on 0.1 and 50 μM [3H]choline transport
Gylys and Jenden16 have shown that HCM selectively inhibits HACU in rat brain synaptosomes. To assess the specificity of HCM inhibition in the Limulus brain hemi-slice preparations, we determined the HCM inhibitory effect at two concentrations of [3H]choline, 0.1 and 50 μM. The high-affinity cotransport component of choline uptake at these two concentrations was determined by subtracting out that transport which occurs in the absence of Na+. At 0.1 μM choline, cotransport activity comprises
Discussion
In an effort to elucidate the mechanisms involved in the regulation of the ChCoT, mustard derivatives of choline and HC-3 have been developed and used as probes in numerous in vitro and in vivo studies.9., 35., 36., 37. One such compound, HCM, has been determined to be an irreversible, highly selective inhibitor of HACU.16., 17., 44. Gylys et al.17 reported that HCM is highly selective for the ChCoT, showing no measurable effect on Na+-independent (i.e. low-affinity) choline transport, nor the
Acknowledgements
This work was supported by NIH grants (MBRS) RR08037 and (RIMI) P20-RR11808.
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