Adenylate cyclase induces intracellular calcium increase in single human epidermal keratinocytes measured by fluorescence microscopy using Fura 2-AM.

Abstract

Intracellular calcium ([Ca2+]i) is an important second messenger of extracellular signals to induce various cellular responses. Extracellular and intracellular Ca2+ are considered to be important for cellular differentiation and proliferation of epidermal keratinocytes. Several mechanisms which increase [Ca2+]i have been demonstrated in various tissues, but in epidermal keratinocytes these mechanisms are poorly understood. In epidermal keratinocytes the adenylate cyclase-cyclic AMP response is thought to regulate cell proliferation and differentiation. However, the series of reactions which follow the cyclic AMP response remain unknown. Beta-adrenergic agonists increase [Ca2+]i in cultured epidermal keratinocytes, and we have therefore studied whether stimulation of keratinocyte adenylate cyclase could induce [Ca2+]i increase, by using fluorescence microscopy with Fura 2-AM. Adenosine and histamine, which are known to be keratinocyte adenylate cyclase receptor agonists, induced transient [Ca2+]i increase, as did epinephrine. In addition, forskolin, a direct adenylate cyclase activator, and dibutyryl-cyclic AMP also induced an increase in [Ca2+]i. In a calcium-free medium epinephrine, adenosine, histamine and dibutyryl-cyclic AMP induced an increase in [Ca2+]i. These results suggest that cyclic AMP in human epidermal keratinocytes regulates [Ca2+]i, which is released from intracellular stores.