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Abstract
The layers of keratinocytes form an acid mantle on the surface of the skin. Herein, we investigated the effects of acidic pH on the membrane current and [Ca2+]c of human primary keratinocytes from foreskins and human keratinocyte cell line (HaCaT). Acidic extracellular pH (pHe≤ 5.5) activated outwardly rectifying Cl current (ICl,pH) with slow kinetics of voltage-dependent activation. ICl,pH was potently inhibited by an anion channel blocker 4,4‘-diisothiocyanostilbene-2,2’-disulphonic acid (DIDS, 73.5% inhibition at 1 μM). ICl,pH became more sensitive to pHe by raising temperature from 24°C to 37°C. HaCaT cells also expressed Ca2+-activated Cl− current (ICl,Ca), and the amplitude of ICl,Ca was increased by relatively weak acidic pHe (7.0 and 6.8). Interestingly, the acidic pHe (5.0) also induced a sharp increase in the intracellular [Ca2+] (Δ [Ca2+]acid) of HaCaT cells. The Δ [Ca2+]acid was independent of extracellular Ca2+, and was abolished by the pretreatment with PLC inhibitor, U73122. In primary human keratinocytes, 5 out of 28 tested cells showed Δ[Ca2+]acid. In summary, we found ICl,pH and Δ[Ca2+]acid in human keratinocytes, and these ionic signals might have implication in pathophysiological responses and differentiation of epidermal keratinocytes.
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Fig. 1.
Activation of outwardly rectifying Cl current by acidic pHe. Representative current traces obtained from primary keratinocytes (A) and HaCaT cells (B) by steplike pulses. The membrane voltage was held at −40 mV, and incremental step-like pulses from −100 to 100 mV (20 mV intervals, 400 ms duration, see inset). The currents (B-a) were obtained with Nagluconate solution in the bath (pH 4.2). (B-b) Representative current traces obtained by step-like pulses under different pHe. The current to voltage relations (I-V curves), measured at the end of each pulse, are shown in right panels. Step like pulses were applied in different conditions of extracellular anions (pH 4.2 Br−, and I−). Current traces presented in (C) were obtained from the same cell, respectively. I-V curves of ICl,pH obtained in NaCl, NaBr, and NaI solutions are shown in the right panel of (C) Each symbol represents mean±SEM of current amplitudes normalized to cell capacitance (pA/pF).
Fig. 2.
Effects of anion channel blockers on ICl,pH. (A, B) Representative current traces obtained by step-like pulses, same as in Fig. 1. ICl,pH was activated by pHe 5.0, and DIDS (1μM) or niflumic acid (NFA, 100μM) was added (right panels). (C, D) I-V curves of ICl,pH activated by pHe 5.0 and effects of DIDS (0.3μM and 1μM) or niflumic acid (30μM and 100μM). Each symbol represents mean±SEM (n=5, respectively) of current amplitudes normalized to the cell capacitance (pA/pF).
Fig. 3.
Effects of bath temperature on ICl,pH activation in HaCaT cells. (A) Representative current traces obtained by step like pulses, same as in Fig. 1. Current traces at pH 7.4, 5.5, and 5.0 were recorded under room temperature (24°C, left panels) and 37°C (right panels). The raise of temperature alone had insignificant effect on membrane conductance, whereas substantial increase was shown at pH 5.5. At pH 5.0, high temperature increased the activation kinetics with similar peak amplitude. All current trances shown were obtained from the same cell. (B) Mean amplitudes of normalized ICl,pH (pA/pF) measured at 100 mV under different pHe and temperature were compared (n=8) ∗indicate p-value<0.05.
Fig. 4.
Ca2+-activated Cl− current (ICl,Ca) of HaCaT cells and effects of acidic pHe. (A) Outward currents with slowly activating kinetics were induced by dialyzing cells against Ca2+-clamped CsCl solution (see inset). Lowering pHe to 7.0 and 6.8 increased the amplitude of outward current. After testing pHe effects, the inhibition of outward current by 500 μM DIDS was confirmed at pHe 6.8. (B) Summary of the effects of pHe on ICl,Ca of HaCaT cells. In each cell, the current amplitudes measured at the end of step-pulse were normalized to the control amplitude (pHe 7.4), and mean±SEM values were plotted (n=8). ∗indicate p-value<0.05.
Fig. 5.
Acidic pHe-induced increase of [Ca2+]c (Δ[Ca2+]acid). (A, B) Representative traces of the fluorescence ratio (F340/380) and the responses of HaCaT (A) and primary keratinyocyte (B) to pHe 5.0 and ATP (100 μM). The Δ[Ca2+]acid was persistent in the absence of extracellular Ca2+ (A). (C, D) Inhibition of Δ[Ca2+]acid by PLC inhibitor (U73122, 2 μM)), whereas no effect of the negative analogue (U73343, 2 μM).
Fig. 6.
Slow decrease of intracellular pH (pHi) in response to extracellular acidification. (A) A representative trace of BCECF emission ratio (F490/440) measured in HaCaT cell. pHe was changed from 7.4 to 5.5. or to 5.0 as indicated. (B) Summary of calibrated pHi (mean± SEM, n=8) of control (pHe 7.4) and 10 min after pHe changes (5.5 and 5.0).
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