The Amine-Containing Cutaneous Irritant Heptylamine Inhibits the Volume-Regulated Anion Channel and Mobilizes Intracellular Calcium in Normal Human Epidermal Keratinocytes

Abstract

Many amines are skin irritants and cause contact dermatitis. However, little is known about their mechanisms of action in keratinocytes except that they induce the release of the inflammatory mediators cytokines and ATP. Here, we tested whether volume-regulated anion channels (VRACs) in primary cultures of normal human epidermal keratinocytes are modulated by the referenced amine-containing cutaneous irritant heptylamine. Under isotonic conditions, we isolated the VRAC current (I(VRAC)) from other conductances using a high Ca(2+)-buffering internal solution. I(VRAC) ran up after patch rupturing and reached a plateau within 15 min. It was reversibly and dose-dependently inhibited by heptylamine with an IC(50) value of 260 microM. Cell-swelling caused by the application of a hypotonic solution increased 2.7-fold I(VRAC) and reduced the inhibition of VRAC by heptylamine with a dose-response curve shifted approximately 10-fold to the right. In addition, we showed, using cell-attached patch recordings, that adding heptylamine to the bath inhibited VRAC activity. This suggests that heptylamine diffuses into the membrane to inhibit VRAC. Finally, we demonstrated that heptylamine induced Ca(2+)-store depletion and that VRAC inhibition was not caused by the increase in cytosolic Ca(2+). Taken together, these results identify heptylamine as a blocker of VRAC and suggest that Ca(2+)-store depletion may be involved in mechanisms of irritant contact dermatitis caused by heptylamine.