Characterization of Bradykinin‐Induced Phosphoinositide Turnover in Neurohybrid NCB‐20 Cells

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Abstract
Phosphoinositide hydrolysis was studied in neurohybrid NCB‐20 cells prelabeled with myo‐[3H]inositol. Among nearly 20 neurotransmitters and neuromodulators examined, only bradykinin, carbachol, and histamine significantly increased the accumulation of [3H]inositol monophosphate (IPO in the presence of lithium. The EC50 of bradykinin was 20 nM and the saturating concentration was approximately 1 μM. The bradykinin response was robust (10‐fold) and was potently and selectively blocked by a bradykinin antagonist, B 4881 [D‐Arg‐(Hyp3 Thi5,8, D‐Phe7)‐bradykinin], with a Ki of 10 nM. This effect of bradykinin appeared to be additive to that mediated by activation of muscarinic cholinergic and histamine Hi receptors. The accumulation induced by bradykinin or carbachol was dependent on the presence of calcium in the incubation medium; less than twofold stimulation was observed in the absence of exogenous calcium. Bradykinin‐induced [3H]IPi accumulation required high concentration of lithium to elicit its maximal stimulation; the concentration of lithium required for half maximal effect was about 13 mM, similar to the value reported previously for carba‐chol‐induced accumulation in the same cell line. In contrast, using related neurohybrid NG108‐15 cells, brady‐kinin‐induced [3H)IPi accumulation was found to require much less lithium. In the presence of lithium, bradykinin also evoked a transient increase in the production of [3H]‐inositol bis‐ and trisphosphate. Basal and bradykinin‐in‐duced phosphoinositide breakdown was inhibited by 4β‐phorbol 12, 13‐dibutyrate, but was unaffected by the biologically inactive 4β‐phorbol. Pretreatment of cells with pertussis toxin induced only about 30% loss of the brady‐kinin‐induced [3H]IP1 accumulation, without affecting basal activity. These data might suggest that more than one type of GTP binding protein is involved in the accumulation of IP1. Preincubation of bradykinin (400 nM) with cells resulted in a time‐dependent loss of the ability of bradykinin to stimulate phosphoinositide hydrolysis; more than 50% of the activity was lost after 45 min exposure at 37°C. The bradykinin response was markedly attenuated by prestimulation with bradykinin; this desensitization was time‐dependent with a maximal effect observed after about 1 h prestimulation. Thus, the robust response of bradykinin on phosphoinositide turnover in NCB‐20 cells may serve as another interesting model in the study of its role in some bradykinin‐mediated physiological events.

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