Cyclic Adenosine Monophosphate and Diacylglycerol

Home , Phospholipase C

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). DAG ClPheScAMP is not observed in the presence of H8 was expressed as a percentage of total phospholipids. vs. In contrast, a Studies were in (2,124±158 2,183±162, NS). H7, relatively Determination of cAMP production. performed inhibitor of kinase C which itself has no at 300 mosM/kg in the presence of selective protein (17), Krebs-Ringer's buffer, pH 7.4, H20 to inhi- 0.5 mM isobutylmethylxanthine to inhibit phosphodiesterase. Studies effect on EGF-stimulated IP3 production, fails prevent using exogenous IP3 require cell permeabilization. These studies were bition by ClPheScAMP (2,206±115 vs. 842±109, P < 0.001). performed in a buffer designed to simulate the intracellular milieu This indicates that inhibition of EGF-stimulated PLC activity (composition in millimoles: 20 NaCl, 100 KCl, 5 Mg sulfate, 1 by cAMP is mediated by activation of A-kinase. NaH2PO4, 25 Na bicarbonate, and 1.0 EGTA, pH 7.2) containing saponin (50 ug/ml). Incubation with effector solutions and subsequent determinations of cAMP and protein were performed as reported pre- 5 (p 0.05 viously (16, 20). Results are expressed as femtomoles cAMP per mi- crogram protein. 4 I Statistical analysis. In studies on cAMP production, values obtained in triplicate wells were meaned for an n of one; in studies on IP3 or DAG production each well was considered an n of one. Compari- sons between two treatment groups were by the unpaired t test. Com- phospholipid) 2 parisons between three or more treatment groups were done by one- way analysis of variance (ANOVA) followed by appropriate contrasts of a priori assumptions (21). In all circumstances P < 0.05 was considered significant. Data are presented as mean±SEM. 0 Basal EGF EGF + Results CiPheScAMP Effect ofcAMP on EGF-stimulated PLC activity. The effect of (O.1mM) cAMP on EGF-stimulated IP3 production is portrayed in Fig. Figure 2. Effect of cAMP on EGF-stimulated DAG production (n 1. EGF significantly increases IP3 production from 975±102 to = 3). Cells are exposed to ClPheScAMP for 15 min before and dur- 2,371±63 cpm/well (P < 0.001). Stimulation by EGF is not ing incubation with EGF. ClPheScAMP inhibits EGF-stimulated observed in cells pretreated with 100 nM AVP (1,024±158), DAG production.

Inhibitory Messengers in the Renal Medulla 47 2500 rP ( 0.005 r ( .00 125 l1 'P3 2000 . cAMP (CPMtwell) 100. 1500 (fmol.g protein) 75

1000 50

500 25 0 IE - +I + + 0 arPhescMr - - (0.1mM) 0 .1.5 1 2 ! 10 Control H8 (50p.M) H7 (50p.M) [EGFJ nM

Figure 3. Effect of protein kinase inhibitors on cAMP inhibition of Figure 5. Dose-response curve for inhibition of AVP-stimulated PLC (n = 4). Cells are exposed to H7 or H8 for 5 min before and cAMP generation by EGF (n = 3). The indicated concentration of during exposure to ClPheScAMP. Pretreatment with H8, but not EGF is added simultaneously with 10-7 M AVP. with H7, prevents inhibition by cAMP.

Effect of stimulation ofPLC on A VP-stimulated AC activ- The role ofPGE2. To examine a potential role for EGF- ity. The effect of EGF on basal and AVP-stimulated cAMP stimulated PGE2 production in mediating inhibition of AC, accumulation is depicted in Fig. 4. EGF has no effect on basal the effect of EGF was examined in the presence of the cy- cAMP accumulation; however, simultaneous addition of EGF clooxygenase inhibitor, flurbiprofen (5 MM; Sigma Chemical eliminates the response to either a submaximal (10-9 M, Co.), which inhibits ionomycin-stimulated PGE2 production 56.2±3.30 vs. 28.92±3.14 fmol/gg protein, P < 0.001) or a > 95% (1). Inhibition of AC by EGF is not mediated by a maximal (lo0- M, 151.15±23.32 vs. 37.05+4.91, P < 0.001) cyclooxygenase product as even in the presence of flurbipro- concentration of AVP. fen, EGF eliminates the response to l0-' M AVP Though all studies were performed with 100 nM EGF, (156.27±27.05 vs. 38.9±10.20 fmol/ug protein; n = 3, P AVP-stimulated AC activity was, in fact, inhibited at much < 0.02). lower concentrations of EGF. As seen in Fig. 5, inhibition by The role ofIP3. RIMCT cells were permeabilized with sa- EGF was observed at a concentration as low as 0.1 nM with a ponin as described above and the ability of AVP to stimulate calculated IC50 of 0.93 nM. AVP-stimulated AC activity was cAMP accumulation was assessed in the absence or presence completely inhibited by 5 nM EGF. of 10 MM IP3. Saponization itself had no effect on AC, as AVP Mechanism of inhibition by EGF. In addition to stimulat- still increased cAMP accumulation from 58.19±4.94 to ing PLC, EGF also stimulates phospholipase A2 (PLA2) in 146.10±24.57 fmol/Mg protein (n = 5; P < 0.01). Exposure to RIMCT cells with resultant PGE2 production (22). PGE2 has IP3 had no effect on either basal (49.47±4.87 vs. 58.18±4.94; n been suggested to inhibit AC in the rabbit cortical collecting = 5, NS) or AVP-stimulated cAMP accumulation duct (23). Stimulation of PLC results in the formation of DAG (166.15±28.76 vs. 146.10±24.57; n = 5, NS). Therefore, inhi- and IP3. IP3 increases cytosolic Ca2+ by releasing Ca2" from bition of AVP-stimulated AC activity by EGF was not a con- nonmitochondrial pools (24). DAG activates protein kinase C sequence of an IP3-induced increase in cytosolic Ca2". (25). Previous studies have demonstrated inhibition of AVP- The role ofDAG. To examine a potential role for a DAG- stimulated cAMP accumulation (16, 26) or water flux (27) mediated increase in the activity of protein kinase C in inhibi- upon increasing cytosolic Ca2+. Activation of protein kinase C tion of AC by EGF, the effect of protein kinase C inhibition is known to inhibit stimulation of AC in a variety of tissues was examined. As seen in the upper panel of Fig. 6, pretreat- (5-8). Studies were undertaken, therefore, to determine which ment with the C-kinase inhibitor, H7, prevented inhibition of of these second messengers, PGE2, IP3, or DAG, mediates AVP-stimulated AC activity by EGF. In contrast, in a separate EGF inhibition of AVP-stimulated AC activity. set of experiments shown in the lower panel of Fig. 6, the A-kinase inhibitor, H8, conferred no protection against inhibi- 200 tion by EGF. r P( To confirm that activation of protein kinase C results in inhibition of cAMP 150 AVP-stimulated AC activity, the effect of the (finol/pg) synthetic highly permeable compound sn-1,2-dioctanoylglycerol (DOG) (28) was examined. As shown in Fig. 7, simulta- 100. Ir (uOAM neous addition of 100 nM DOG inhibited AVP-stimulated AC activity from 100.89±9.33 to 58.29±2.71 (n = 3, P < 0.02). 50 F'O~~~~~~1 Inhibition by DOG was not observed in cells pretreated with rmn H7, as AVP-stimulated cAMP generation remained at 100.08±7.03 fmol/ug even upon exposure to DOG. EGF + + (1OOnM) BASAL AVP (10-9M) AVP (10-7M) Discussion Figure 4. Effect of EGF on AVP-stimulated cAMP generation (n = 6). EGF (100 nM) is added simultaneously with AVP. Exposure to The role of phosphoinositide hydrolysis in the function of the EGF eliminates AVP-stimulated cAMP generation. collecting duct and its potential interactions with AC have not

48 I. Teitelbaum 1O50 PLC signaling systems in a single renal cell type with a product F(0.4I of each system inhibiting function of the other. cAMP Il Stimulation of AC by either AVP or forskolin or the provi- 1oo0 sion of exogenous cAMP all result in a decrease in EGF-stimu- (fmoI/~g) lated IP3 production (Fig. 1). It has been suggested that production ofDAG, the endogenous activator ofprotein kinase C, not 5o may result only from the hydrolysis of polyphosphoinosi- tides but of other phospholipids (e.g., phosphatidylcholine) as well (30). It is important to note, therefore, that pretreatment with ClPheScAMP inhibits EGF-stimulated DAG production 0 J- as as source EGF (Fig. 2) well IP3 formation. While the of the DAG (MO) is unknown, it is clear that inhibition of PLC by cAMP results in a decrease in the levels of both second and H7 (50 FIM) messengers, IP3 DAG. The exact locus of inhibition by cAMP is not known. so However, it is likely that cAMP decreases IP3 levels by impairing the formation of IP3 rather than by enhancing its degrada- tion, as the levels of IP, and IP2 are also decreased. The de- so cAMg crease in PLC activity upon exposure to cAMP is a conse- OM0144 quence of an A-kinase-mediated phosphorylation event as 40 inhibition by cAMP is not observed in the presence of the A-kinase inhibitor, H8 (Fig. 3). Although the Ki of H8 for cAMP-dependent protein kinase is only modestly lower than 20 that of H7 (1.2 vs. 3.0 AM) (17), the specificity of H8 for A-kinase is demonstrated by the inability of H7 to prevent 0o inhibition of PLC by cAMP. Conversely, inhibition of AC by EGF which is known to function activation of oon1M) BASAL AW (1O-7M) DOG, by protein kinase C, is prevented by H7 but not by H8 despite the only HS (50 p.M) modest difference in their respective Kis for protein kinase C Figure 6. Effect of protein kinase inhibitors on EGF-inhibition of (6.0 vs. 15.0 MM). AC. Cells are exposed to H7 (top, n = 6) or H8 (bottom, n = 5) for 5 Similar to its ability to inhibit PLC, cAMP has also been min before and during incubation without or with EGF. H7 prevents shown to inhibit PLA2 in cultured RIMCT cells (31) as well as inhibition by EGF; H8 does not. other tissues (32, 33). The concentration of ClPheScAMP that inhibits PLC (0.1 mM) is the same as that which inhibits PLA2 in this same tissue (31) and is comparable to that which stimu- been fully defined. Bradykinin has been shown to stimulate lates water flux in the toad bladder (34). PLC in cultured rabbit papillary duct cells (29) but potential Exposing RIMCT cells to AVP in the presence of EGF interactions with the AC signaling system were not examined. results in abolition of AVP-stimulated cAMP generation (Fig. Recent studies from this laboratory have demonstrated EGF- 4). As EGF eliminates the response to AVP even in the back- stimulated phosphoinositide hydrolysis in the cultured rat ground of cyclooxygenase inhibition, it is clear that the effect inner medullary collecting duct (1). The data reported herein of EGF is not a consequence of its stimulation of PGE2 pro- are the first to describe communication between the AC and duction (22). This is in accord with previous studies in RIMCT cells that do not support an inhibitory effect of PGE2 on AC in this tissue. Sato and Dunn have that at a 125 reported PGE2 high r- p 0.02C concentration enhances cAMP accumulation in RIMCT cells but observed no inhibition at concentration Simi- 100 any (35). cAMP larly, previous studies from this laboratory have shown that in 75 RIMCT cells exposed to the calcium ionophore A23187, (fmovIg) which very potently stimulates PGE2 production, AVP-stimu- 50 lated cAMP accumulation is not enhanced upon inhibition of cyclooxygenase with meclofenamate (16). 25 Studies were then performed to determine whether one of the second messengers produced upon stimulation of PLC in 0 J response to EGF, IP3 or DAG, is responsible for mediating inhibition of AC by EGF. Exposure of saponin-permeabilized DOG DOG cells to exogenous IP3 had no effect on AVP-stimulated cAMP + H7 accumulation. Release of Ca2+ from cellular stores has been reported to occur in response to the concentration of IP3 (10 7. Effect of DOG on AVP-stimulated cAMP Figure generation (n used in this The ofthe increase = 3). DOG (100 nM) was added simultaneously with IO-' M AVP; MM) study (36, 37). magnitude to in RIMCT cells was not H7 was present for 5 min before incubation with DOG. DOG inhib- in cytosolic Ca2+ in response IP3 ited AVP-stimulated cAMP generation; inhibition was not observed determined. Therefore, the present study does not unequivo- in the presence of H7. cally exclude a role for Ca2+ in mediating inhibition by EGF.

Inhibitory Messengers in the Renal Medulla 49 However, as inhibition of protein kinase C (see below) results in complete restoration of AVP-responsiveness in the presence of EGF, the role of any other mediator is likely to be minimal. ATP IP3 The lack of inhibition byIP3 differs from previous studies in C kiiase 4-DAG Figure 8. Schematic rep- the collecting duct in which increased cytosolicCa2+ inhibited resentation of the interac- the response to AVP (16, 27). This most likely reflects the fact tions between the AC and that the amount ofCa2+ released byIP3 and, therefore, the PLC signaling systems in subsequent increase in cytosolicCa2+ is much less than that the cultured rat inner obtained with a calcium ionophore (36, 37), as had been em- medullary collecting duct. ployed in earlier studies (16, 27). Rather than an effectofIP3, inhibition of AC by EGF appears to be due to activation of protein kinase C as EGF has inhibition of AVP-stimulated cAMP generation by activation no effect in the presence of the C-kinase inhibitor, H7 (Fig. 6). of protein kinase C suggests a possible role for protein kinase C We have previously reported that EGF-stimulatedIP3 produc- in "turn off" of the AVP signal. tion in RIMCT cells is first detected at10 nM EGF (1). Yet, as In summary, the data presented herein demonstrate for the seen in Fig. 5, inhibition of AVP-stimulated AC activity is first time the existence of this bidirectional signaling system in observed with as little as 0.1 nM EGF and is complete at 5 nM. cultured inner medullary collecting tubule cells (Fig. 8). Stim- As activation of PLC results in hydrolysis of not only phos- ulation of AC results in the formation of cAMP which, via phatidylinositol bisphosphate but of phosphatidylinositol activation of A-kinase, inhibits PLC; conversely, stimulation monophosphate and phosphatidylinositol as well (and perhaps of PLC results in the formation of DAG which, via activation other phospholipids, e.g., phosphatidylcholine) production of of C-kinase, inhibits AC. The exact loci of inhibition by these DAG, on a molar basis, is likely to exceed productionofIP3 respective kinases remain to be determined. (30). Therefore, inhibition of AC by concentrations of EGF below the threshold for detectionofIP3 production itself sug- Acknowledaments gests that the inhibition is a consequence of DAG production. Activation of protein kinase C has been shown to inhibit The author would like to thank Mr. Douglas Holmes and Dr. Stewart AVP-stimulated water flow in the toad bladder (14) and in the A. Metz for assistance with the determination of DAG levels, and Ms. isolated perfused rabbit cortical collecting tubule (15). Though Linda M. Benson for expert secretarial assistance. a pre-cAMP site appears operant in the toad bladder (14), the This work was supported by grant DK-38464 from the National predominant site of action of protein kinase C in both tissues is Institutes of Health. post-cAMP. Likewise, the ability of EGF to inhibit AVP- and cAMP-stimulated water flux in the rabbit cortical collecting References tubule to a similar extent (38) suggests a predominantly post- cAMP site of action. The studies reported herein demonstrate 1. Teitelbaum, I., A. Strasheim, and T. Berl. 1990. Epidermal that, apart from any post-cAMP effects, EGF, via activation of growth factor-stimulated phosphoinositide hydrolysis in rat inner protein kinase C, profoundly inhibits the generation of cAMP medullary collecting tubule cells. Regulation by G protein, calcium, response to AVP. This finding is consistent with other stud- and protein kinase C. J. Clin. Invest. 85:1044-1050. in 2. Takai, Y., K. Kaibuchi, K. Sano, and Y. Nishizuka. 1982. Coun- ies that have shown protein kinase C-mediated inhibition of teraction of calcium-activated, phospholipid-dependent protein kinase AVP-stimulated AC activity in Madin-Darby canine kidney activation by adenosine 3',5'-monophosphate and guanosine 3',5'- cells (39) or cultured rabbit cortical collecting ducts (40). How- monophosphate in platelets. J. Biochem. (Tokyo). 91:403-406. ever, in marked contrast to those studies in which inhibition of 3. Bianca, V. D., P. De Togni, M. Grezeskowiak, L. M. Vicentini, AC is observed only after more prolonged exposure (15 min to and F. Di Virgilio. 1986. Cyclic AMP inhibition of phosphoinositide 6 h) to activators of protein kinase C, in the present study turnover in human neutrophils. Biochim. Biophys. Acta. 886:441-447. inhibition by EGF or DOG requires no preincubation but is 4. Godfrey, R. W., R. M. Manzi, D. E. Gennaro, and S. T. Hoff- observed upon addition of these agents simultaneously with stein. 1987. Phospholipid and arachidonic acid metabolism in zymo- AVP. This indicates a direct inhibitory effect of protein kinase san-stimulated human monocytes: modulation by cAMP. J. Cell. rather than one mediated by a secondary phenomenon such Physiol. 131:384-392. C 5. Beckner, S. K., and W. L. Farrar. 1987. Inhibition of adenylate as stimulation of cell proliferation, as had been previously cyclase by IL 2 in human T lymphocytes is mediated by protein kinase suggested (40). Furthermore, unlike the other studies in which C. Biochem. Biophys. Res. Commun. 145:176-182. activation of protein kinase C attenuates but does not elimi- 6. Williams, K. A., W. Murphy, and R. J. Haslam. 1987. Effects of nate AVP-stimulated cAMP generation, exposure of RIMCT activation of protein kinase C on the agonist-induced stimulation and cells to EGF abolishes the response to AVP. DOG is less potent inhibition of cyclic AMP formation in intact human platelets. Bio- an itxhibitor of AC, perhaps reflecting the need for permeation chem. J. 243:667-678. of the cell membrane. Finally, in none of the previous studies 7. Hollingsworth, E. B., and J. W. Daly. 1987. Inhibition of recep- on the effect of protein kinase C was the other signaling limb, tor-mediated stimulation of cyclic AMP accumulation in neuroblas- i.e., the effect of cAMP on phosphoinositide hydrolysis, exam- toma-hybrid NCB-20 cells by a phorbol ester. Biochim. Biophys. Acta. ined. 930:272-278. 8. Yamashita, A., T. Kurokawa, Y. Une, and S. Ishibashi. 1988. The physiologic correlates of the biochemical observations Phorbol ester regulates stimulatory and inhibitory pathways of the described herein require further examination. As the role of hormone-sensitive adenylate cyclase system in rat reticulocytes. Eur. J. phosphoinositide hydrolysis in the function of the collecting Pharmacol. 151:167-175. duct is itself unclear, one can only speculate upon the signifi- 9. Magnaldo, I., J. Pouyssegur, and S. Paris. 1988. Thrombin exerts cance of its modulation by cAMP. In contrast, the profound a dual effect on stimulated adenylate cyclase in hamster fibroblasts, an

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