Cyclic Adenosine Monophosphate and Diacylglycerol Mutually Inhibitory Second Messengers in Cultured Rat Inner Medullary Collecting Duct Cells Isaac Teitelbaum With the technical assistance of Alexis Strasheim Department ofMedicine, University of Colorado School ofMedicine, Denver, Colorado 80262 Abstract ble for mediating the enhanced hydraulic conductivity of the apical membrane. Recent studies from this laboratory have Studies were performed to examine interactions between the demonstrated that in addition to AC activity, cultured inner adenylyl cyclase (AC) and phospholipase C (PLC) signaling medullary collecting duct cells also exhibit hormonal stimula- systems in cultured rat inner medullary collecting duct cells. tion of phospholipase C (PLC) activity upon exposure to epi- Stimulation of AC by either arginine vasopressin (AVP) or dermal growth factor (EGF) (1). forskolin or addition of exogenous cAMP inhibits epidermal It is now well established that AC and PLC are not auton- growth factor (EGF)-stimulated PLC. This inhibition is me- omous in their regulation of cellular events; rather, these two diated by activation of cAMP-dependent kinase as it is pre- signaling systems may interact in either positive or negative vented by pretreatment with the A-kinase inhibitor, fashion. For example, cAMP has been shown to inhibit phos- N-12-(methylamino)ethyll-5-isoquinoline-sulfonamide (H8) phoinositide hydrolysis in platelets (2) and neutrophils (3), but but not by the C-kinase inhibitor, 145-isoquinolinylsulfonyl)- it has no effect in monocytes (4). Conversely, activation of 2-methylpiperazine (H7). Exposure to EGF eliminates AVP- protein kinase C, one of the major consequences of phos- stimulated cAMP generation. This is not mediated by a cy- phoinositide hydrolysis, has been reported to inhibit hor- clooxygenase product as inhibition by EGF is observed even in mone-stimulated AC activity in some tissues (5-8), while it the presence of the cyclooxygenase inhibitor, flurbiprofen. In- potentiates AC activity in other tissues (9-12). hibition by EGF is not due to an increase in inositol trisphos- Potential interactions between the AC and PLC signaling phate (IP3) as exposure of saponin-permeabilized cells to exog- systems in renal tissues have not been extensively examined. enous IP3 is without effect. Inhibition by EGF is prevented by cAMP has been shown to modestly attenuate a,-adrenergic- pretreatment with the C-kinase inhibitor, H7, but not by the stimulated PLC activity in renal cortical slices (13), however, A-kinase inhibitor, H8. Exposure to the synthetic diacylglyc- the cell responsible was not identified. Furthermore, the other erol (DAG), dioctanoylglycerol, also inhibits AVP-stimulated signaling limb, i.e., the effect of stimulation of PLC on cAMP AC activity; therefore, inhibition by EGF is due to activation of generation, was not examined. Similarly, while activation of protein kinase C. Thus, in cultured rat inner medullary collect- protein kinase C has been shown to inhibit AVP-stimulated ing duct cells, cAMP and DAG function as mutually inhibitory water flow in the toad bladder (14) and in the rabbit cortical second messengers with each impairing formation of the other. collecting duct (15), in neither tissue has the effect of cAMP on (J. Clin. Invest. 1990. 86:46-51.) Key words: adenyl cyclase - hormonally stimulated PLC activity been examined. Accord- phospholipase C * protein kinase c * AVP * EOF * inner medul- ingly, the present study was undertaken to examine bidirec- lary collecting duct tional interactions between the AC and PLC signaling systems in a single cell type, the cultured rat inner medullary collecting Introduction tubule (RIMCT) epithelial cell. One of the major functions of the collecting duct is the stimu- Methods lation of adenylyl cyclase (AC)' activity in response to arginine vasopressin (AVP). The ensuing increase in cAMP is responsi- Cell culture. Cultures of RIMCT cells were prepared as previously described (1, 16). For studies on cAMP production, tissue from two Correspondence and reprint requests to Dr. I. Teitelbaum, C28 1, Uni- rats was used to plate one 24-well dish (Costar Data Packaging Corp., versity of Colorado School of Medicine, 4200 E. 9th Avenue, Denver, Cambridge, MA); for studies on inositol trisphosphate (1P3) production CO 80262. wells were plated at three times this density to obtain uniform con- Receivedfor publication 26 June 1989 and in revisedform 26 Jan- fluent cultures. uary 1990. Generation ofsamples for measurement ofIP3. At 96 h the Hams F1 2/Liebovitz L 15 medium in which the cells were initially grown was 1. Abbreviations used in this paper: AC, adenylyl cyclase; DAG, diac- aspirated and the cultures washed twice with sterile PBS. The cells were ylglycerol; DOG, dioctanoylglycerol; EGF, epidermal growth factor; then fed with inositol-free DME supplemented with 3H-myo-2-inositol H7, 1-(5-isoquinolinylsulfonyl).2-methylpiperazine; H8, N-[2-meth- (5 ACi/well, Amersham Corp., Arlington Heights, IL). Studies were ylamino)ethyl]-5-isoquinoline-sulfonamide; IPI, inositol monophos- performed after 24 h of labeling, as this time has been demonstrated to phate; IP2, inositol bisphosphate; IP3, inositol trisphosphate; PLA2, be sufficient for incorporation into the phospholipid pool. Media was phospholipase A2; PLC, phospholipase C; RIMCT, rat inner medul- aspirated from the cells and the cells were washed twice with 500 y1 of lary collecting tubule. PBS. The cells were then incubated for 15 min in PBS without or with desired test substances, e.g., AVP, forskolin, or ClPheScAMP. In stud- J. Clin. Invest. ies employing the protein kinase inhibitors (17) 145-isoquinolinylsul- © The American Society for Clinical Investigation, Inc. fonyl)-2-methylpiperazine (H7) or N-[2-(methylamino)ethyl]-5-iso- 0021-9738/90/07/0046/06 $2.00 quinoline-sulfonamide (H8) (Calbiochem-Behring Corp., La Jolla, Volume 86, July 1990, 46-51 CA), there was a 5-min preincubation with these compounds alone, 46 I. Teitelbaum E O00 followed by a 15-min incubation with these compounds plus any other 2500 1 desired agents. Upon completion of all preincubations, the buffer was aspirated 2000 and replaced with 500 gl of PBS without or with 100 nM EGF (Sigma 'P3 Chemical Co., St. Louis, MO). Previous studies have demonstrated 1500 that EGF-stimulated IP3 production in RIMCT cells peaks at 10 s (1). (CPM/well) Therefore, the rcaction was terminated after 10 s by the addition of 500 1000 1 dul of ice-cold 20% TCA. The cells were scraped off of the dish and s s z centrifuged at 1,000 g for 10 min. The supernate containing inositol 500 . g phosphates was washed four times with an equal volume of ether and l l R l stored at -20'C until analysis 0 r Is Determination ofinositol phosphates. Inositol phosphates were sep- EGFl l RblEGF EGF + + + arated by anion exchange chromatography as described by Berridge et AVPrForskolin | aPhscMP al. (18). In brief, the samples were thawed and brought to a pH of 6.0 or (loOnM) (1OpM) (O.1mM) greater with 50 mM Tris base. They were then applied to Dowex lx-8 columns (formate form) (Dow Corning Corp., Midland, MI). Serial Figure 1. Effect of cAMP on epidermal growth factor (EGF)-stimu- elutions were performed with water, 5 mM sodium tetraborate/60 mM lated IP3 production (n = 4). Cells are exposed to AVP, forskolin, or sodium formate, 0.1 M formic acid/0.2 M ammonium formate, 0.1 M ClPheScAMP for 15 min before a 10-s incubation with 100 nM formic acid/0.4 M ammonium formate, and 0.1 M formic acid/ 1.0 M EGF. Exposure to cAMP prevents stimulation by EGF. ammonium formate which elute, respectively, inositol, glycerylphos- phorylinositol, inositol phosphate, inositol bisphosphate (IP2), and IP3. Results are expressed as counts per minute of IP3 per well. 10 gM forskolin (868±57), or 0.1 mM ClPheScAMP Determination ofDAG levels. At 96 h after plating cells were fed (774±108). Pretreatment with ClPheScAMP results in a de- with Ham's/Leibovitz medium containing ['4C]arachidonic acid (6 crease in inositol monophosphate (IP1) (1,658±169 vs. MCi/ml) and 5% fetal calf serum. After 16-18 h the medium was 893±149 cpm/well; n = 4, P < 0.02) and IP2 (906±142 vs. aspirated and cells were washed twice with PBS. Cells were exposed to 513±49 cpm/well; n = 4, P < 0.05) as well. Similarly, as seen PBS containing the desired test substances for 15 min; PBS was aspi- in Fig. 2, pretreatment with ClPheScAMP decreases EGF- rated and the cells scraped into 1 ml of ice-cold methanol under N2. stimulated DAG production from 4.20±0.48 to 2.85±0.04% After 60 min at 4°C 2 ml of ice-cold chloroform was added, the cells of total = P < Thus whether were gassed with N2, and extracted overnight at 4°C. 750 ul phospholipid (n 3; 0.05). cAMP, sonicated, or stimulated inhibits sub- of H20 was added, samples were vortexed for 2-3 min, then centri- added exogenously endogenously, fuged at 1,200 rpm for 6 min. The lower phase that contained lipids sequent stimulation of PLC. was aspirated, dried under N2, and resuspended in 50 Mul of chloro- Mechanism of inhibition by cAMP. To determine whether form/methanol, 2: 1. the effect of cAMP is mediated by stimulation of protein ki- Samples were spotted on to standard silica plates and subjected to nase A, the effect of H8, a relatively specific inhibitor of cyclic thin layer chromatography using a petroleum ether/diethyl ether/gla- nucleotide-dependent kinases (17), was examined. As seen in cial acetic acid (70:30:1) solvent system. The plates were subjected to Fig. 3, 50 MM H8 itself has no effect on subsequent EGF-stim- autoradiography and the diacylglycerol (DAG) containing bands were ulated IP3 production. However, inhibition of PLC activity by scraped and quantitated by liquid scintillation counting (19).