Characterization of calcium signaling pathways in human preadipocytes

Abstract

Intracellular free Ca 2+ (Ca i 2+) is an important regulator of many cellular activities; however, Ca 2+ signaling is not well studied in human preadipocytes. The purpose of the present study was to characterize Ca 2+ signal pathways using a confocal scanning technique and RT-PCR. It was found that spontaneous Ca i 2+ oscillations were observed in 12.1% preadipocytes, and number of cells withCa 2+ oscillations was increased to 47.9% by 1% fetal bovine serum. Ca i 2+ oscillations were dependent on Ca 2+ entry mainly via stored-operated Ca 2+ (SOC) entry. They were suppressed by the SOC entry channel blocker La 3+, the phospholipase C (PLC) inhibitor U73122, the inositol trisphosphate receptor (IP3R) blocker 2-amino-ethoxydiphenyl borate, or the sarcoplasmic/endoplasmic reticulum Ca 2+ pump (SERCA) inhibitors thapsigargin and cyclopiazonic acid, but not by ryanodine. The IP3R activator thimerosal increased Ca i 2+ oscillations. In addition, the plasma membrane Ca 2+ pump (PMCA) inhibitor carboxyeosin and Na +-Ca 2+ exchanger (NCX) inhibitor Ni 2+ both suppressed Ca 2+ oscillations. RT-PCR revealed that the mRNAs for IP3R1-3, SERCA1,2, NCX3 and PMCA1,3,4, CaV1.2, and TRPC1,4,6, STIM1 and Orai1 (for SOC entry channels) were significant in human preadipocytes. The present study demonstrates that multiple Ca 2+ signal pathways are present in human preadipocytes, and provides a basis for investigating how Ca 2+ signals regulate biological and physiological activities of human preadipocytes. © 2009 Wiley-Liss, Inc.