C. Kennedy et al., BRADYKININ-INDUCED TRANSLOCATION OF CYTOPLASMIC PHOSPHOLIPASE A(2) INMDCK CELLS, Canadian journal of physiology and pharmacology, 75(6), 1997, pp. 563-567
The nonapeptide bradykinin (BK) plays an important role in the product
ion of eicosanoids within the collecting duct of the nephron. We have
shown previously that BK can initiate a complex signaling cascade that
causes the release of arachidonic acid (AA) from MDCK-D1 cells, a can
ine cell line of distal tubule and collecting duct origin. This releas
e is dependent upon early activation of specific upstream enzymes, inc
luding phosphatidylcholine-specific phospholipase C (PC-PLC) and phosp
holipase D (PLD). Ultimately, the release of this precursor of eicosan
oids is effected by recruitment of the cytoplasmic 85-kDa form of phos
pholipase A(2) (cPLA(2)). This enzyme is thought to translocate from t
he cytosol to cellular membranes following stimulation by agonists tha
t cause elevations of intracellular calcium ([Ca2+](i)). The present s
tudy was undertaken to examine the dependence of AA release upon Ca2influx in BK-stimulated MDCK cells. For this purpose, cells were incub
ated with 1 mu M BK for 1 min and lysed in Ca2+-free Tris buffer. The
high-speed 100 000 x g pellet was extracted with 10 mM octyl glucoside
and the cPLA(2), protein level was determined. Previous results from
our laboratory indicated that BK induced a 1.81-fold increase in cPLA(
2) activity associated with cellular membranes, while in the present s
tudy, Western blotting with a specific cPLA(2) antibody demonstrated a
similar elevation in protein detected with these same membranes. A se
lective inhibitor of receptor-mediated Ca2+ entry, SK&F 96365, was use
d to resolve the role of extracellular Ca2+ in BK's ability to evoke A
A release. Pretreatment of cells with SK&F 96365 resulted in an inhibi
tion of greater than 60% of the BK response. Taken together, these res
ults strongly suggest that BK-mediated AA release in MDCK-D1 cells is
at least partly contingent upon translocation of cPLA(2) to membranes
initiated by an influx of extracellular Ca2+.