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.
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 beta-antagonist, inhibited this response, while prazosin and yohimbine (alpha 1- and alpha 2-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.
Osawa
Y
Y
Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan.
Koizumi
H
H
Fukaya
T
T
Yasui
C
C
Ohkawara
A
A
Ueda
T
T
D000241:Adenosine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000494:pharmacology*; D000420:Albuterol / Q000494:pharmacology; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D004837:Epinephrine / Q000494:pharmacology; D016257:Fura-2 / Q000031:analogs & derivatives; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000737:chemistry / Q000166:cytology / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D011433:Propranolol / Q000494:pharmacology
C049925:fura-2-am; D005576:Colforsin; D006632:Histamine; D011433:Propranolol; D000262:Adenylyl Cyclases; D000241:Adenosine; D000420:Albuterol; D002118:Calcium; D016257:Fura-2; D004837:Epinephrine