Inhibition of calcium-independent phospholipase A impairs agonist-induced calcium entry in keratinocytes

BACKGROUND: In many cells, depletion of intracellular calcium (Ca(2+)) reservoirs triggers Ca(2+) entry through store-operated Ca(2+) channels in the plasma membrane. However, the mechanisms of agonist-induced calcium entry (ACE) in keratinocytes are not fully understood. OBJECTIVES: This study was designed to determine if pharmacological inhibition of calcium-independent phospholipase A (iPLA(2)) impairs ACE in normal human epidermal keratinocytes. METHODS: Confocal laser scanning microscopy was used to monitor the dynamics of Ca(2+) signalling in keratinocytes loaded with the calcium-sensitive dye Fluo-4. Cells were stimulated with extracellular nucleotides [adenosine triphosphate (ATP) or uridine triphosphate (UTP)] or with lysophosphatidic acid (LPA), a bioactive lipid that regulates keratinocyte proliferation and differentiation. RESULTS: Both ATP and UTP induced Ca(2+) release in primary human keratinocytes. This was not followed by robust Ca(2+) influx when the experiments were performed in low Ca(2+) (70 μmol L(−1)) medium. Upon elevation of extracellular Ca(2+) to 1·2 mmol L(−1), however, a biphasic response consisting of an initial Ca(2+) peak followed by an elevated plateau was observed. The plateau phase was inhibited when cells were treated with bromoenol lactone, a specific pharmacological inhibitor of iPLA(2). These findings indicate that iPLA(2) activity is required for ACE in keratinocytes. LPA also evoked Ca(2+) release in keratinocytes but failed to induce sustained Ca(2+) entry even when extracellular Ca(2+) was elevated to 1·2 mmol L(−1). CONCLUSION: Our results demonstrate for the first time an important role for iPLA(2) in regulating ACE in primary human keratinocytes.