Journal zyxwvutsrqponmlk oJNeurochemistry zyxwvutsrqponm Raven Press, Ltd., New zyxwvutsrqpo York zyxwvutsrqpo 0 199 1 International Society for Neurochemistry Nerve Growth Factor Potentiates Bradykinin-Induced Calcium Influx and Release in PC12 Cells *?Andrew B. Bush, *Laurence A. Borden, *Lloyd A. Greene, and *Frederick R. Maxfield *Department of Pathology and Center for Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, and ?Department of Pharmacology, New York Univer.rity Medical Center, New York, New York, U.S.A. Abstract: To investigate how the response to agonists changes during neuronal differentiation, we examined the effect of nerve growth factor (NGF) on bradykinin-induced calcium increases in PCI 2 cells. Short-term (I h) treatment with NGF increased the potency of bradykinin to raise intracellular cal- cium by about 10-fold,whereas long-term (1 week) treatment, which was associated with the expression of the differentiated phenotype, increased the potency about 100-fold. Neither treatment affected the maximal response to bradykinin. NGF alone had no acute effect on calcium levels. Short-term po- tentiation appeared to be mainly a result of greater release of calcium from intracellular stores, whereas the effect of long-term treatment apparently was due to increases in both release from intracellular stores and calcium influx. [3H]Bradykinin binding to intact PC 12 cells was unaltered by short-term NGF treatment, whereas differentiated cells displayed a 50% increase in receptor number and about a twofold increase in affinity as compared with cells not treated with NGF. The production of inositol phosphates in response to bradykinin correlated poorly with the calcium transients, in that large calcium responses were associated with small increases in inositol phosphates. Neither NGF treatment had a significant effect on the appearance of inositol phosphates in response to bradykinin. Experiments with permeabilized cells revealed that differentiated cells did not display a heightened response to exogenously added inositol I ,4,5-tns- phosphate. Our results demonstrate that NGF modulates the bradykinin signaling pathway without acutely activating this pathway itself. Key Words: Nerve growth factor-Bradyki- nin-Calcium-PC I2 cells-Inositol phosphates. Bush A. B. et al. Nerve growth factor potentiates bradykinin-in- duced calcium influx and release in PC zyx 12 cells. J. Neurochem. zy 57, zyxwvu 562-574 (1991). Neuronal differentiation is characterized by a wide range of morphological and biochemical changes. For example, increased expression of ion channels and neurotransmitter receptors is frequently observed as neurons mature in cell culture (Rohrer et al., 1985; Simmoneau et al., 1985). These receptors transduce the signals from extracellular agonists into intracellular responses. Although increases in the number of cell- surface receptors have been shown, little is known about how the internal cellular response to agonists is affected during development. The PC 12 pheochromocytoma cell line has been widely used as a model system for studying neuronal differentiation induced by nerve growth factor (NGF) (Greene and Tischler, 1976;Levi et al., 1988). Although the precise biochemical mechanisms for NGF-induced differentiation remain unclear, a large number of re- sponses to NGF treatment have now been described. Some responses, such as membrane ruffling and acti- vation of certain kinases, occur within minutes (Con- nolly et al., 1979; Halegoua and Patrick, 1980). Other responses, such as neurite outgrowth, require hours to days to become apparent. In this study, we have in- vestigated the effect of NGF on the response of PC12 cells to bradykinin. Because PC 12 cells respond to bra- dykinin with or without NGF treatment, this is an ex- cellent system for examining how responses to extra- cellular signals change during acute and prolonged ex- posure to NGF and during transition to a neuronal phenotype. Bradykinin is a nonapeptide formed from the cleav- age of kininogens and is involved in several physiolog- ical processes including inflammation, blood pressure regulation, and pain transmission (Regoli and BarabC, Received September 11, 1990; revised manuscript received January 4, 1991; accepted January 15, 1991. Address correspondence and reprint requests to Dr. F. Maxfield at Department of Pathology, Columbia University, 630 W. 168th Street, New York, NY 10032, U.S.A. Abbreviations used: ACE, angiotensin-converting enzyme; BSA, bovine serum albumin; [Ca"], free intracellular calcium; EGF, epi- dermal growth factor; fura-2/AM, fura-2 acetoxymethyl ester; IGF- 11, insulin-like growth factor 11; IP3, inositol I ,4,5-trisphosphate; NGF, nerve growth factor: PI, phosphoinositide. 562