Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells.

T L Lambert,R S Kent,A R Whorton

doi:10.1016/s0021-9258(18)66865-6

Abstract

Bradykinin stimulation of inositol polyphosphate production was followed using [3H]inositol-labeled porcine aortic endothelial cells grown in culture. Bradykinin stimulated a significant increase in inositol trisphosphate (IP3) production within 15 s. This increase reached a maximum value of 5-fold above control at 30 s and returned toward baseline by 90 s. Production of inositol bisphosphate increased with time reaching 4-fold by 60 s. Bradykinin stimulated the production of IP3 and inositol biphosphate in a dose-dependent manner with an EC50 of 9 X 10(-9) M. Labeled pools of phosphatidylinositol-4,5-bisphosphate (PIPP) decreased by 50% within 30 s, corresponding to the rise in IP3, while labeled lysophosphatidylinositol pools increased 3-fold by 60 s. Pertussis toxin, a protein which ribosylates GTP-binding proteins, did not inhibit bradykinin-stimulated inositol polyphosphate production. Incubation of labeled cells in the absence of extracellular Ca2+ also did not affect bradykinin-stimulated inositol polyphosphate production. Further, A23187, a Ca2+ ionophore, failed to stimulate PIPP metabolism. Finally, Ca2+ influx into cell monolayers occurred with a time course which paralleled rather than preceded the increase in IP3 levels. These data suggest that bradykinin stimulates phospholipase C metabolism of PIPP to IP3 by a mechanism which does not contain a pertussis toxin sensitive GTP-binding protein. Also, this receptor-linked phospholipase C activity does not appear to be activated by extracellular Ca2+ influx. The results support the proposal that IP3 production initiates Ca2+ mobilization and suggest that the calcium-dependent step in arachidonate release is distal to IP3 production.

Highlights

Ribosylution Assay-To determine whether pertussis toxin ADP production, porcine aortic endothelial cells were labeled for ribosylates endothelial cell membrane proteins and to demonstrate 72 h with [3H)inositol, treated with bradykinin, and extracted
The medium was removed and cells washed twice with Hanks' balanced salt solution (HBSS) (HL-60 cells washed by centrifugation; endothelial cell monolayers washed)
Maximal inhibition of these processes can beachieved by washing andincubatingendothelial cellsincalcium-free HBSS without resorting to EGTA chelatio(3n).T o examine whether inhibition of inositol polyphosphate production occurs in absence of extracellular calcium, cells were washed withcalcium-freebuffer, incubated incalcium-free orcalcium-supplemented bufferfor 5 min,andthenstimulated with bradykinin

Summary

RESULTS

60 promyelocytes were grown and passaged as described by Brandt et al (14). Ribosylution Assay-To determine whether pertussis toxin ADP production, porcine aortic endothelial cells were labeled for ribosylates endothelial cell membrane proteins and to demonstrate 72 h with [3H)inositol, treated with bradykinin, and extracted. The pellet was washed twice with the above buffer, resuspended in 100 mM potassium phosphate, pH 7.5, containing 5 mM MgClz and 50 units/ml of aprotinin, sonicated for 20 s at 0 "C and incubated (Fig. 2 A ) or as the ratioof stimulated divided by control (Fig. 2 B ) , a significant increase in IP3 production was seen by 15 s. This effectreached a maximum value a t 30 s and returned toward baseline by 90 s.

FRACTION NUMBER

BradySktiinminulation of Inositol Polyphosphute Production

BradySkitniminulation of InPosoiltyoplhosPphroadteuction

DISCUSSION