Joseph W. Vanable

Effects of amiloride analogues on adult Notophthalmus viridescens limb stump currents

We have previously investigated the relevance to limb regeneration of epidermally driven, Na+-dependent limb stump currents by blocking epidermal Na+ channels with amiloride, 3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide. In order to reduce Notophthalmus viridescens stump currents more effectively than with amiloride, we have examined six amiloride analogues. Of these, only benzamil, 3,5-diamino-6-chloro-N-[(benzylamino)aminomethylene]pyrazinecarboxamide, was more effective than amiloride. The concentration of benzamil that reduced stump currents to half their initial value was 0.034 microM, while this concentration for amiloride was 0.42 microM. We also found a contribution of calcium ions to these currents. When immersed in water whose Ca2+ concentration decreased stepwise from 1 to 0 mM, stump currents decreased significantly, although to a variable extent, depending on the batch of newts. With 30 microM benzamil and 0.5 mM calcium (in water that also contained 1.5 mM NaCl and 0.06 mM KCl) stump currents could be reduced to very low levels and, in many cases, changed to incurrents.

Electric field strength and epithelization in the newt (Notophthalmus viridescens).

There is convincing evidence that endogenous electric fields are necessary for normal wound epithelization, but it is unclear whether normal epithelization rates can be accelerated increasing normal field strengths. Although we confirmed that normal lateral fields are required for normal Notophthalmus viridescens epithelization rates, significant increases in epithelization were not achieved by increasing normal field strengths with direct current. With increases of less than 50%, epithelization rates were slightly increased. When the field strengths were augmented by 50%, epithelization was significantly retarded. This pattern was also observed when we applied direct current to wounds whose fields were nullified with benzamil. Epithelization was more rapid in wounds with field strengths raised to 20, 40, and 60 mV/mm than in benzamil-treated wounds without field augmentation. Epithelization was most rapid at 40 mV/mm, the normal value. When fields were augmented to 80 and 100 mV/mm, the epithelization rate diminished significantly. We also augmented field strengths by increasing the Na+ concentration in the medium surrounding the digits. Wounds healed more slowly in 10 mM Na+ than in 1.5 mM Na+, the normal pond water concentration. When field strengths in 10 mM Na+ were diminished to levels found in contralateral digits at 1.5 mM Na+ by applying direct current, the epithelization rate was restored to normal. We conclude that newt wound epithelization rates are nearly maximal at normal wound field strengths. Field strengths significantly higher than normal (50% more) result in significantly diminished rates of epithelization.