Biochimica et BiophysicaActa 888 (1986) 171-175 171 Elsevier BBA 11666 Bradykinin-induced changes in myo-inositol 1,2-(cyclic)phosphate in rabbit papillary collecting tubule cells James A. Shayman, Richard J. Auchus and Aubrey R. Morrison * Departments of Internal Medicine and Pharmacology, Washington Universi(v Medical School, St. Louis, MO 63110 (U.S.A.) (Received April 7th, 1986) Key words: Bradykinin; Inositol 1,2-(cyclic)phosphate; (Rabbit) Rabbit renal pipillary collecting tubule cells were harvested and grown in primary cultures. When labeled with myo-12-3H]inositol and extracted under neutral conditions, a metabolite undetected under acidic extraction was observed on resolution by anion-exchange chromatography and which eluted under similar conditions with authentic DL-myo-inositoI 1,2-(cyclic)phosphate; the mass spectrum of its pentakis(trimethyl- silyl) derivative contains an identical ratio of selected ion fragments to the authentic standard. Bradykinin, demonstrated previously to increase labeling of free inositoi polyphosphates, increases labeling of inositol 1,2 cyclic phosphate but over a time course subsequent to the formation of inositol trisphosphate. These observations are consistent with the model that bradykinin induces hydrolysis of phosphatidylinositol 4,5-bisphosphate which precedes hydrolysis of phosphatidylinositol in renal papillary collecting tubule cells. Introduction myo-Inositol 1,2-(cyclic)phosphate has been characterized as a product of phospholipase C- mediated hydrolysis of phosphatidylinositol [1]. Although it has previously been considered as a possible second messenger [2], its presence and changes in intracellular levels of this compound have been difficult to document clearly [3]. Stan- dard procedures for the recovery of inositol phos- phates involve the use of acid conditions which cause the nonenzymatic hydrolysis of myo-inositol 1,2-(cyclic)phosphate to inositol 1-phosphate and inositol 2-phosphate [4]. Because these latter com- pounds are not characteristically resolved by Dowex anion-exchange chromatography, it is dif- ficult to infer how myo-inositol 1,2-(cyclic)phos- phate changes during hormonal stimulation. For these reasons we sought to identify myo-inositol * To whom correspondence should be addressed. 1,2-(cyclic)phosphate and document possible bradykinin-induced changes in intracellular levels of this metabolite in primary cultrues of rabbit renal papillary collecting tubule cells. The char- acterization of these cultures and documentation of polyphosphoinositide metabolism have previ- ously been reported [5]. Materials and Methods Renal papillary collecting tubule cells were harvested from female New Zealand white rabbits and grown in 24-well culture dishes (Costar, Cam- bridge, MA) as previously reported [5]. Cells were grown at a seeding density of 5 • 10 4 cells per dish and grown in inositol-free, serum-free medium. After 4-5 days, confluent cells were labeled with 8 ~tCi per well of myo-[2-3H]inositol (spec. act. 16.5 Ci/mmol, Amersham, Niles, IL). After 24 h (a period demonstrated to result in steady-state labeling [5]), cells were washed four times with Krebs buffer (pH 7.5, 37°C) containing 10 mM