Abstract
The effect of W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride] on Ca2+ signaling in Madin-Darby canine kidney cells was investigated. W-7 (0.1–1 mM) induced a [Ca2+]i increase, which comprised an initial increase and a plateau. Ca2+ removal inhibited the Ca2+ signals by 80%, suggesting that W-7 activated external Ca2+ influx and internal Ca2+ release. Pretreatment with the mitochondrial uncoupler carbonylcyanidem-chlorophenylhydrazone (2 μM) and the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (1 μM) abolished the internal Ca2+ release induced by 0.5 mM W-7; conversely, pretreatment with W-7 prevented thapsigargin and carbonylcyanide m-chlorophenylhydrazone from releasing internal Ca2+. W-7 (0.2 mM) induced Mn2+ quench of fura-2 fluorescence, which was inhibited by La3+ (0.1 mM) by 80%. La3+ (0.1 mM) partly inhibited 0.2 mM W-7-induced [Ca2+]i increase. Addition of 5 mM Ca2+ induced a significant [Ca2+]i increase after pretreating with 0.2 to 1 mM W-7 in Ca2+-free medium for 5 min, suggesting that W-7 induced capacitative Ca2+ entry. W-7 (0.5 mM) potentiated the capacitative Ca2+ entry induced by 1 μM thapsigargin by 15%. Pretreatment with aristolochic acid (40 μM) to inhibit phospholipase A2 reduced 0.5 mM W-7-induced internal Ca2+ release and external Ca2+ influx by 25 and 80%, respectively. Inhibition of phospholipase C with U73122 (2 μM) or inhibition of phospholipase D with propranolol (0.1 mM) had no effect on the internal Ca2+ release induced by 0.5 mM W-7. It remains unclear whether W-7 induced [Ca2+]i increases via inhibition of calmodulin. Three other calmodulin inhibitors (phenoxybenzamine, trifluoperazine, and fluphenazine-N-chloroethane) did not alter resting [Ca2+]i.
Footnotes
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Send reprint requests to: Dr. Chung-Ren Jan, Department of Medical Education and Research, Veterans General Hospital-Kaohsiung, 386 Ta Chung 1st Rd., Kaohsiung, Taiwan 813. E-mail:crjan{at}isca.vghks.gov.tw
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↵1 This work was supported by National Science Council Grant NSC89-2320-B-075B-009 and Veterans General Hospital-Kaohsiung Grant and VTY Joint Research Program, Tsou's Foundation Grant VGHKS89-13 (to C.-R.J.).
- Abbreviations:
- W-7
- N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride
- MDCK
- Madin-Darby canine kidney
- IP3
- inositol 1,4,5-trisphosphate
- ER
- endoplasmic reticulum
- fura-2/AM
- 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid pentaacetoxymethyl ester
- U73122
- 1-(6-((17b-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione
- CCCP
- carbonylcyanide m-chlorophenylhydrazone
- Received May 4, 1999.
- Accepted September 29, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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