Adenylate cyclase induces intracellular Ca²⁺ increase in single human epidermal keratinocytes of the epidermal sheet as measured by digital imaging microscopy using Fura 2-AM

Summary

Intracellular Ca²⁺ ([Ca²⁺]_i) is thought to act as a second messenger of transmembrane signalling systems. However, no measurement of [Ca²⁺]_i has been made in intact epidermal keratinocytes. We have developed a method for measuring [Ca²⁺]_i in human keratinocytes from pure epidermal sheet by the application of digital imaging fluorescence microscopy with the use of Fura 2-AM. Normal human pure epidermal sheets were obtained by dispase treatment. Epinephrine and salbutamol induced transient [Ca²⁺]_i increases. Propranolol, a Β-antagonist, inhibited this response, while prazosin and yohimbine (alpha₁- and alpha₂-antagonists, respectively) did not affect the response. Histamine and adenosine, also receptor agonists of the epidermal adenylate cyclase system, induced a similar [Ca²⁺]_i increase, as did forskolin, a direct activator of adenylate cyclase. These data coincide with those previously presented for cultured human epidermal keratinocytes, and reveal that adenylate cyclase activation induces an increase of [Ca²⁺]_i in intact epidermal cells. This technique enables the kinetics of [Ca²⁺]_i in various skin disorders to be investigated.