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


OBJECTIVE
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 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.


RESULTS
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).


CONCLUSIONS
Our results demonstrate for the first time an important role for iPLA(2) in regulating ACE in primary human keratinocytes.
Ross
K
K
Dermatological Sciences, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle upon Tyne, U.K. kehinde.ross@newcastle.ac.uk
Parker
G
G
Whitaker
M
M
Reynolds
N J
NJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D008246:Lysophospholipids / Q000494:pharmacology; D018613:Microscopy, Confocal; D009281:Naphthalenes / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D054512:Phospholipases A2, Calcium-Independent / Q000037:antagonists & inhibitors / Q000502:physiology*; D011753:Pyrones / Q000494:pharmacology; D014544:Uridine Triphosphate / Q000494:pharmacology
D008246:Lysophospholipids; D009281:Naphthalenes; D010726:Phosphodiesterase Inhibitors; D011753:Pyrones; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D000255:Adenosine Triphosphate; D054512:Phospholipases A2, Calcium-Independent; C032881:lysophosphatidic acid; D002118:Calcium; D014544:Uridine Triphosphate