Chloride Channel Inhibition Reduces Tail Regeneration in the Developing Vertebrate Ambystoma Mexicanum

Abstract

Regeneration is dependent on ion signaling which may be activated by endogenous bioelectric fields (EF) in response to the wounding. EF modulation by electrical stimulation (ES) or pharmacological ion channel manipulation alters regenerative capacity. Satellite cell proliferation during ES in the invertebrate, P. clarkii was dependent on K+ and Ca2+ flux (unpublished results). We hypothesize that regeneration in the vertebrate, Ambystoma mexicanum are mediated by intact cellular ion channel activity. Ambystoma hatchlings were raised in welled plates and Holtfreter's solution (2 ml). Tails were amputated 2mm proximal of the tip and imaged on days 0, 2, 4, and 9. Ion channel regulation of regeneration was assessed by inhibiting K+ channels with tetraethylammonium (TEA), Na+/K+ ATPase with ouabain (OB), and Cl− channels with DIDS. Uncut controls measured drug effects on normal growth. Tail tip growth determined regenerative capacity in cut hatchlings. OB and TEA had no effect on regeneration. Cl− channel blockade (DIDS) decreased regeneration from 0.276±0.003 to 0.193±0.002 mm/day (p<0.05) but did not alter uncut, control growth. Thus, ion channels may be critical to vertebrate regeneration by modulating proliferation via cell volume control. Specific ion channel gene expression will further assess the effects of bioelectric fields on vertebrate regeneration capacity.