Amiloride Blocks a Keratinocyte Nonspecific Cation Channel and Inhibits Ca++ -Induced Keratinocyte Differentiation

Abstract

Proliferation and differentiation in many cells are linked to specific changes in transmembrane ion fluxes. Previously, we have identified a nonspecific cation channel in keratinocytes, which is permeable to and activated by Ca++. To test whether this cation channel might serve as a pathway for Ca++ entry, we examined the effect of blocking this channel on membrane currents, markers of differentiation, and intracellular Ca++. In patch clamp studies, 10(-8) to 10(-6) M amiloride decreased the single-channel open probability. The same concentrations of amiloride inhibited the calcium-induced formation of cornified envelopes and activity of transglutaminase in a dose-dependent fashion. Amiloride inhibited the long-term rise of intracellular Ca++ induced by raised extracellular Ca++, without blocking the initial increase of intracellular Ca++. Amiloride at concentrations of 10(-7) to 10(-3) M did not change the resting intracellular pH of keratinocytes, although concentrations of 10(-6) M or greater inhibited the recovery from NH4(+)-induced acidification. To test whether the effect of amiloride was toxic, we measured DNA synthesis in the presence or absence of amiloride. DNA synthesis was unchanged, suggesting that amiloride's actions were not due to toxic effects. Although the exact mechanisms of amiloride's action remains to be determined, these experiments suggest that this compound may inhibit keratinocyte differentiation by blocking the nonspecific cation channel.

Cyclic AMP mediates keratinocyte directional migration in an electric field

“…The isoforms of adenylyl cyclase expressed by human keratinocytes are presently unknown but fetal rat keratinocytes do express isoforms VI and VIII (Takahashi et al, 1998), meriting further investigation of the adenylyl-cyclase isoforms expressed in human keratinocytes. The mechanism used by keratinocytes to sense and respond to an applied EF does have a very specific requirement for Ca 2+ influx that is not blocked by either amiloride or verapamil (Trollinger et al, 2002) and is, therefore, independent of the non-specific cation channel (Mauro et al, 1995) previously identified.…”

NHE1 Regulates the Stratum Corneum Permeability Barrier Homeostasis

“…Although intracellular pH has been linked to diverse cellular functions in various tissues, including cell proliferation, trans- formation, and differentiation (1,33,34), extracellular acidification also occurs in a regulated manner. The NHE family of Na ϩ /H ϩ exchangers is ubiquitous, and their role in acid-base balance is best understood in kidney (33), where concentrationdriven influx of Na ϩ provides the driving force for the NHEmediated extrusion of H ϩ into the extracellular domain.…”

“…Cultured keratinocytes express the sodium-proton exchanger (NHE) 1 class of non-energy-dependent transporters, which controls intracellular pH (1). Recently, the NHE1 isoform has been shown to be the only isoform in keratinocytes and epidermis (2).…”

Epoxyeicosatrienoic Acids Activate Transglutaminases in Situ and Induce Cornification of Epidermal Keratinocytes

“…Transglutaminase activities require Ca 2ϩ as cofactor (36,37,40). Amiloride blocks cell Ca 2ϩ influx, and by this mechanism it has been reported to inhibit Ca 2ϩ -induced keratinocyte differentiation and CE formation (41). Cystamine is a water-soluble primary amine that inhibits transglutaminase activities by competing with ⑀-lysines in endogenous protein substrates (42,43).…”