title: Novel effects of clotrimazole on Ca2+ signaling in Madin Darby canine kidney cells.
abstract: The effect of clotrimazole on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ indicator. Clotrimazole (1-30 microM) induced a concentration-dependent [Ca2+]i increase. The [Ca2+]i increase comprised an initial rise and a slow decay. External Ca2+ removal partly inhibited the Ca2+ signals by reducing both the initial rise and the decay phase, indicating that clotrimazole triggered both Ca2+ influx and Ca2+ release. Pretreatment with 30 microM clotrimazole in Ca2+-free medium abolished the Ca2+ release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, and conversely, pretreatment with thapsigargin prevented clotrimazole from releasing more Ca2+. This suggests that the thapsigargin-sensitive Ca2+ store is the source of clotrimazole-induced Ca2+ release. Clotrimazole (10 microM) triggered Mn2+ quench of fura-2 fluorescence which was partly inhibited by 1 mM La3+. Addition of 3 mM Ca2+ induced a [Ca2+]i increase after preincubation with 10 microM clotrimazole in Ca2+-free medium, indicating that clotrimazole activated capacitative Ca2+ entry. However, 10 and 30 microM clotrimazole inhibited 1 microM thapsigargin-induced capacitative Ca2+ entry by 21% and 74%, respectively. Pretreatment with 40 microM aristolochic acid to inhibit phospholipase A2 reduced 30 microM clotrimazole-induced Ca2+ release by 51%, but inhibiting phospholipase C with 2 microM U73122 had little effect. This implies that clotrimazole induces Ca2+ release in an IP3-independent manner, which could be modulated by phospholipase A2-coupled events.
authors.affiliation: Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, Taiwan. crjan@isca.vghks.gov.tw
authors.forename: C R
authors.initials: CR
authors.lastname: Jan
authors.forename: C J
authors.initials: CJ
authors.lastname: Tseng
authors.forename: K J
authors.initials: KJ
authors.lastname: Chou
authors.forename: H T
authors.initials: HT
authors.lastname: Chiang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002460:Cell Line; D003022:Clotrimazole / Q000494:pharmacology*; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D007202:Indicators and Reagents; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors; D054467:Phospholipases A2; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
chemical_list: D000935:Antifungal Agents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D000252:Calcium-Transporting ATPases; D003022:Clotrimazole; D002118:Calcium; D016257:Fura-2