Store operated calcium entry in NIH-3T3 cells.

roles in regulating cell functions such as chemotaxis, motility or secretion. Stimulation by neurotransmitters or hormones activates intracellular production of inositol 1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to the IP3 receptor on the Ca store (endoplasmic reticulum ; ER) membrane and releases the sequestered Ca to the intracellular space. Increased intracellular Ca functions as a second messenger for activation of many important downstream effectors. DAG activates protein kinase C (PKC), a key enzyme for diverse physiological functions. Capacitative or store operated calcium entry (SOCE) is a process by which depletion of intracellular Ca stores activates a Ca influx pathway (1). It is an essential pathway for refilling ER Ca stores after releasing store Ca and also serves as a direct activator of downstream effectors. SOCE has been investigated by use of inhibitors of sarcoplasmic-endoplasmic Ca-ATPase (SERCA), the most common of which is the irreversible inhibitor, thapsigargin. Application of thapsigargin to cells induces sustained SOCE, generally assessed by use of fluorescent Ca indicators. Also thapsigargin can be used to study the small, Ca selective Ca release-activated Ca current (ICRAC) by the patch clamp technique. Therefore, thapsigargininduced SOCE is a useful approach to investigate mechanisms of SOCE. Immortal NIH3T3 cells have been established from the embryonic NIH Swiss mice fibroblasts. The NIH-3T3 cell is a useful cell line for investigation of transformation, stress fiber formation, or feeder cells of keratinocytes. NIH-3T3 cells have epidermal growth factor (EGF) receptors (2), fibroblast growth factor (FGF) receptors (3), platelet derived growth factor (PDGF) receptors (3), bombesin receptors (3) bradykinin receptors (4) and ATP receptors (5), all of those receptors require calcium influx. Stimulation of those receptors mobilizes stored Ca through production of IP3. However, in a series of papers, it was reported that thapsigargin-induced SOCE in NIH-3T3 cells was not sensitive to several kinds of inhibitors (Table 1) (6-9), indicating that in NIH-3T3 cells, SOCE appears to be independent of PKC, calmodulin, microtubules or actin cytoskeleton.