title: [Changes in the intracellular free calcium of cultured human epidermal keratinocytes].
abstract: Changes in the intracellular free calcium ([Ca2+]i) of cultured normal human epidermal keratinocytes (NHEK) were investigated in order to determine whether the adenylate cyclase cAMP (AC) system and phospholipase C activating system are involved in increasing [Ca2+]i. NHEK were obtained from neonatal foreskin and grown in serum-free medium (K-GM) supplemented with 2% bovine pituitary extract. [Ca2+]i was measured by fluorescence ratio imaging microscopy using Fura-2 as the indicator. In the case of the AC system, transient increases in [Ca2+]i were observed in response to stimulation with epinephrine, norepinephrine, isoproterenol and salbutamol. Methoxamine, clonidine and dobutamine did not induce any [Ca2+]i increase. The [Ca2+]i increase evoked by epinephrine was inhibited by pretreatment with propranolol, but not by prazosin or yohimbine, indicating that epinephrine-induced [Ca2+]i elevation via beta 2-adrenergic stimulation. Similar changes were observed when NHEK were stimulated with histamine, adenosine, GTP gamma S, forskolin and dibutyryl cAMP respectively. The absence of extracellular Ca2+ had no effect on the epinephrine-induced [Ca2+]i increase. It appears that activated protein kinase A, based on cAMP accumulation via stimulatory GTP binding protein, elicited the release of Ca2+ from intracellular stores. On the other hand, when drugs known to activate phospholipase C in a wide variety of cell types were tested, a transient increase in [Ca2+]i was demonstrated in response to the addition of thrombin, bradykinin and substance P. This reaction was not affected by the presence of EGTA, suggesting that these drugs raise [Ca2+]i via phosphatidylinositol breakdown. Vasopressin, angiotensin II, serotonin and acetylcholine did not induce any increase in [Ca2+]i. On the basis of these studies, it was concluded that NHEK possess the mechanism which increase [Ca2+]i via AC system and phospholipase C activating system. It seems probable that this rise in [Ca2+]i initiates a calcium-dependent cellular response, such as activation of calcium/calmodulin dependent kinase, and subsequently regulates the proliferation and differentiation of human epidermal keratinocytes.
authors.affiliation: Department of Dermatology, Hokkaido University School of Medicine, Sapporo, Japan.
authors.forename: C
authors.initials: C
authors.lastname: Yasui
mesh_terms: D000241:Adenosine / Q000494:pharmacology; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D001920:Bradykinin / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D000078404:Epidermal Cells; D004837:Epinephrine / Q000494:pharmacology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism*; D013373:Substance P / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D000316:Adrenergic alpha-Agonists; D000318:Adrenergic beta-Agonists; D013373:Substance P; D000001:Calcimycin; D003994:Bucladesine; D006632:Histamine; D000242:Cyclic AMP; D010738:Type C Phospholipases; D013917:Thrombin; D000241:Adenosine; D001920:Bradykinin; D002118:Calcium; D004837:Epinephrine