SUMMARY1. Excitatory inputs to amacrine cells in the salamander retinal slice preparation were examined using whole-cell patch pipette voltage-clamp techniques. In strychnine (500 nm) and bicuculline (100 /1M), two types of amacrine cell were easily distinguished by their light-evoked excitatory responses: transient and sustained.2. In transient amacrine cells the current-voltage (I-V) relation for the peak lightevoked current was non-linear with a negative slope region between -50 and -70 mV. Responses reversed near + 10 mV and were prolonged at more positive holding potentials.3. In DL-2-amino-phosphonoheptanoate (AP7, 30 ftM), a selective N-methyl-Daspartate (NMDA) receptor antagonist, both the negatively sloped region of the light I-V relation and the prolongation of the response at positive potentials were eliminated. In 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 2 /tM), a selective non-NMDA receptor antagonist, light-evoked currents at the most hyperpolarized holding potentials were eliminated. At potentials positive to -85 mV the lightevoked currents lacked a fast onset. The light I-V relation in CNQX had a negative slope region between -35 and -80 mV.

K+ channel activation has been associated with growth or differentiation in many cells. We have previously identified a 70-pS K+ channel that was found only in differentiated involucrin-positive cells. In this study we examined the role of K+ channels in Ca2+-induced keratinocyte differentiation. Consistent with our previous report, we found that a K+ conductance developed only in cells cultured in high extracellular Ca2+. Addition of charybdotoxin or verapamil blocked these K+ channels and inhibited Ca2+-induced differentiation, as assessed by cornified envelope formation or transglutaminase activity. These results suggest that K+ channel activation is necessary for Ca2+-induced differentiation. Finally, we used (125)I-labeled charybdotoxin to demonstrate the presence of K+ channels in intact human and mouse epidermis, hair follicles, and eccrine glands, indicating that these channels are found in keratinocytes both in vitro and in vivo. Thus K+ channels may moderate Ca2+ influx in more differentiated keratinocytes and may play a central role in keratinocyte differentiation.

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A B S T R AC T A sustained high voltage-activated (HVA), nifedipine-and cadmiumsensitive calcium current and a sustained calcium action potential (AP) were recorded from horizontal cells isolated from catfish retina, pH indicator dyes showed that superfusion with NH4CI alkalinized these cells and that washout of NH4C1 or superfusion with Na-acetate acidified them. HVA current was slightly enhanced during superfusion of NH4C1 but was suppressed upon NH4C1 washout or application of Na-acetate. When 25 mM HEPES was added to the patch pipette to increase intracellular pH buffering, the effects of NH4CI and Na-acetate on HVA current were reduced. These results indicated that intracellular acidification reduces HVA calcium current and alkalinization increases it. Sustained APs, recorded with high resistance, small diameter microelectrodes, were blocked by cobalt and cadmium and their magnitude varied with extraceUular calcium concentration. These results provide confirmatory evidence that the HVA current is a major component of the AP and indicate that the AP can be used as a measure of how the HVA current can be modified in intact, undialyzed cells. The duration of APs was increased by superfusion with NH4C1 and reduced by washout of NH4C1 or superfusion with Na-acetate. The Na-acetate and NH4CI washout-dependent shortening of the APs was observed in the presence of intracellular BAPTA, a calcium chelator, IBMX, a phosphodiesterase inhibitor, and in Na-free or TEA-enriched saline. These findings provide supportive evidence that intracellular acidification may directly suppress the HVA calcium current in intact cells. Intracellular pH changes would thereby be expected to modulate not only the resting membrane potential of these cells in darkness, but calcium-dependent release of neurotransmitter from these cells as well. Furthermore, this acidification-dependent suppression of calcium current could serve a protective role by reducing calcium entry during retinal ischemia, which is usually thought to be accompanied by intracellular acidosis.

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.