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

K Ross, G Parker, M Whitaker, N J Reynolds

Index: Br. J. Dermatol. 158(1) , 31-7, (2008)

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Abstract

In many cells, depletion of intracellular calcium (Ca2+) reservoirs triggers Ca2+ entry through store-operated Ca2+ channels in the plasma membrane. However, the mechanisms of agonist-induced calcium entry (ACE) in keratinocytes are not fully understood.This study was designed to determine if pharmacological inhibition of calcium-independent phospholipase A (iPLA(2)) impairs ACE in normal human epidermal keratinocytes.Confocal laser scanning microscopy was used to monitor the dynamics of Ca2+ 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.Both ATP and UTP induced Ca2+ release in primary human keratinocytes. This was not followed by robust Ca2+ influx when the experiments were performed in low Ca2+ (70 micromol L(-1)) medium. Upon elevation of extracellular Ca2+ to 1.2 mmol L(-1), however, a biphasic response consisting of an initial Ca2+ 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 Ca2+ release in keratinocytes but failed to induce sustained Ca2+ entry even when extracellular Ca2+ was elevated to 1.2 mmol L(-1).Our results demonstrate for the first time an important role for iPLA(2) in regulating ACE in primary human keratinocytes.