I LSEVIER J. Lipid Mediators Cell Signalling 11 (1995) 25-39

MEDIATORS ANDCELLSICNALLING

Inhibition of prostaglandin E,-responsive adenylyl cyclase in embryonal human kidney 293 cells by phorbol esters

JesGs Sinchez-Yagiie * , Marina C. Rodriguez, Angel Hernhndez-Hernhndez, Marcia1 Llanillo

Departamento de Bioquimica y Biologia Molecular, Facultad de Biologia, Uniuersidad de Salamanca, Campus U. Miguel de Unamuno, Camp0 Chart-o s/n, 37007 Salamanca, Spain

Received 14 January 1994; revision received 27 May 1994; accepted 30 May 1994

Abstract

A clonal primary embryonal human kidney cell line, 293, increased CAMP production in response to prostaglandin E, (PGE,) (0.02-2 PM). The purpose of this study was to show the effects of tumor-promoting phorbol esters (e.g., 4P-phorbol l%-myristate 13-acetate, PMA) on PGE,-stimulated CAMP production. Pretreatment with PMA (0.2-200 nM) for 30 min markedly reduced PGE,-stimulated CAMP production in the presence of 0.5 mM isobutylmethylxanthine. The reduction by PMA was dose- and time-dependent. PMA seems to attenuate the increase in CAMP accumulation elicited by PGE, primarily, if not entirely, by inhibiting adenylyl cyclase activity, since we were unable to demonstrate an effect of PMA on the degradation half-life of CAMP in intact 293 cells. The action of PMA had some specificity for the agonist used; thus, PMA inhibited PGE,-activated adenylyl cyclase but had no effect on the forskolin-activated enzyme. Co-pretreatment with PMA and H-7, an inhibitor of protein kinase C (PKC), partially prevented the PMA-induced attenuation of the PGE,-stimulated CAMP accumulation, and 1-oleoyl-2-acetylglycerol, a synthetic diacyl- glycerol analog, partially mimicked the PMA action. Thus, PMA appeared to decrease CAMP production by a PKC-mediated mechanism, inhibiting adenylyl cyclase activity at a point other than the catalytic subunit of the enzyme in the kidney 293 cell line.

Keywords: Prostaglandin E,; Phorbol esters; Receptor desensitization; 293 cells

* Corresponding author. Tel. (23)-294400, ext. 1937; Fax (23)-294579.

0929-7855/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0929-7855(94)00025-8

26 J. Scinchez-Yagiie et al. /J. Lipid Mediators Cell Signalling I1 (I 995) 25-39

1. Introduction

Hormone stimulation of many cellular processes is mediated by activation of adenylyl cyclase activity and increased production of adenosine 3’S’-cyclic monophosphate (CAMP). CAMP production is prevented or switched off by several mechanisms, e.g., degradation of the message by phosphodiesterases, inhibition via attenuating hormones or by mechanisms termed refractoriness or desensitization. Desensitization includes a homologous, hormone-specific and a heterologous non- specific variant. In most cells, both events occur simultaneously. Under physiologi- cal conditions cells are often exposed simultaneously to a number of different agonists, which may result in a cooperative interaction between different signalling pathways and may be important in eliciting an integrated cellular response. Recent evidence suggests that interactions between adenylyl cyclase and other transmem- brane signalling systems may further modulate cellular responses to hormones. For example, increased membrane phosphoinositide turnover leading to the activation of protein kinase C (PKC) has been shown to influence the activity of the adenylyl cyclase-CAMP effector system in a number of different tissues (Nishizuka, 1986). The use of a tumor promotor - phorbol ester, a putative direct activator of PKC, has facilitated this kind of study. Phorbol ester can exert both inhibitory (Heyworth et al., 1984; Rebois and Patel, 1985) and stimulatory (Cronin and Canonico, 1985; Sugden et al., 1985) effects on agonist-induced CAMP production in different cell types. The intercommunication between different intracellular signal transduction pathways is thought to occur, at least partly, through PKC-induced phosphoryla- tion of components of the adenylyl cyclase system, including the stimulatory hormone receptor (Kelleher et al., 1984; Sibley et al., 1984), the inhibitory guanine nucleotide-binding-regulatory protein (Gil (Katada et al., 1985), and the catalytic subunit (Yoshimasa et al., 1987).

Prostaglandin E, (PGE,) can exert both stimulatory (Wandji et al., 1992) and inhibitory (Watanabe et al., 1991) effects on CAMP metabolism, probably mediated through distinct stimulatory and inhibitory prostaglandin receptors (Smith and Garcia Perez, 1985) and in addition, to the stimulatory and inhibitory guanine nucleotide regulatory proteins, G, and Gi, respectively (Haga et al., 1977; Mu- rayama and Ui, 1983; Anderson et al., 1984). The fact that PGE, has been shown to increase the CAMP level in many tissues and cells suggests that the EP, receptor subtype is ubiquitously distributed and mediates various PGE, actions in many tissues and cells. In the kidney, there is good evidence that PGE, plays a critical physiologic and pathophysiologic role in inhibiting CAMP-mediated vasopressin action in the collecting duct, where PGE, can be stimulated by vasopressin itself (Hebert et al., 1991). The effects of PGE, on water permeability in the collecting duct are quite consistent with a triple effector model, with PGE, receptors coupled to three signalling pathways via specific G proteins G,, Gi and G, (Hebert et al., 1991). It remains to be shown that separate PGE, receptors mediate these different effects although there is suggestive evidence that this may be the case (Sonnenburg and Smith, 1988). We have found no data on the actions of phorbol esters in human kidney cells in which prostaglandins mediate increases in intracel-

J. SLinchez-Yagiie et al. /J. Lipid Mediators Cell Signalling II (1995) 25-39 27

lular CAMP. Therefore, we used tumor embryonal human kidney 293 cells as a study model under cell culture conditions. It should also be noted that 293 cells have been used as a useful system by us (Sanchez Yagiie et al., 1993; Hipkin et al., 1993) and others (Luttrell et al., 1993) as a system for transfection and expression of cDNAs encoding different G-protein-coupled receptors, which allows the study of intercommunications between receptors after phorbol ester treatment.

2. Materials and methods

2.1. Supplies

Fibronectin, H-7 (1-(5-isoquinoline sulfonylj-2-methylpiperazine dihydrochlo- ride), isobutylmethylxanthine (IBMX), 4P-phorbol 12-myristate 13-acetate (PMA) and prostaglandin E, (PGE,) were obtained from Sigma (St. Louis, MO). Forskolin was purchased from Calbiochem. l-Oleoyl-2-acetylglycerol (OAG), which was from Serdary Research Lab. (Ontario, Canada), was dried once under an N, stream and dissolved again in dimethyl sulfoxide (DMSO). Tissue culture plastic ware and supplies were obtained from Corning and Gibco, respectively. All other reagents were obtained from commonly used suppliers and were of the best grade available.

2.2. Cells

Human embryonal kidney 293 cells (ATCC CRL 1573) were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Dulbecco’s modified Eagle’s medium with 10 mM Hepes, 50 Fg/ml gentamycin, and 10% heat-inactivated newborn calf serum, pH 7.4. Cells were plated on day 0 in 6 X 35-mm wells and maintained at 37°C in a humidified atmosphere containing 5% CO,. All experiments were performed on day 3. At this time the cell density was 1.0-1.2 X lo6 cells/well.

2.3. Standard experimental protocols

All experiments were initiated by washing the wells twice with 2 ml portions of warm assay medium (Waymouth’s MB752/1 modified to contain 20 mM Hepes, 1 mg/ml albumin, pH 7.4). All experiments were performed in a total volume of 1 ml of assay medium containing 0.5 mM IBMX. PMA and PGE, (stock solutions of 2 and 5 mM in DMSO, respectively) were dissolved in 0.15 M NaCl, 20 mM Hepes, 1 mg/ml albumin, pH 7.4 (solution A), immediately prior to the experiments. The final concentration of DMSO in cultures was usually 0.05%. Forskolin was dis- solved in ethanol and added to the wells as a 200-fold concentrated solution. H-7 (stock solution of 50 mM in DMSO) and OAG (stock solution of 1 M in DMSO) were dissolved in solution A immediately before starting the experiment. IBMX was dissolved directly in the assay medium.

28 .I. Sa’nchez-Yagiie et al. /J. Lipid Mediators Cell Signalling 11 (1995) 25-39

2.4. Rate of CAMP accumulation

After washing, the wells were preincubated (37°C) in a humidified incubator for 15 min without CO,. In some experiments, H-7 was added to this first preincuba- tion. PMA, OAG or vehicle were then added when neccesary and the wells were floated on a 37°C water bath exposed to room air for the indicated periods of time (standard incubation time was 30 min). The indicated agonists (PGE, or forskolin) were then added and the incubation was continued at 37°C. The reaction was stopped at different times after addition of the agonist as follows: the wells were placed on ice and 1 ml of ice-cold 1 N HClO, containing 1 mM theophylline was added to each well. Cells were scraped off and transferred to tubes. The wells were then washed once with 0.5 ml of 0.5 N HClO, containing 1 mM theophylline. The tubes with the cells were freeze-thawed once, and centrifuged. 1 ml of the supernatants were transfered to new tubes, neutralized with 0.5 ml of 0.72 M KOH/0.6 M KHCO, and centrifuged again. Supernatants were then chro- matographed on a combination of alumina and Dowex colums as described by Lorenz and Wells, 1983. The fractions containing the CAMP were diluted, acety- lated and assayed by radioimmunoassay with a highly sensitive radioimmunoassay kit (Biomed. Technols., Inc, Stonghton, MA).

The rates of CAMP accumulation (Fig. 2 and 6) in the presence of PGE, were calculated by linear regression of the linear portions of the data shown. The regression coefficients of all lines shown were at least 0.97.

2.5. Rate of CAMP degradation

Cells were plated in 6 X 35-mm wells precoated with fibronectin. After washing, the wells were preincubated (37°C) with or without 200 ng/ml PMA for 15 min. The wells then received 2 PM PGE,, and incubation was continued for an additional 15 min. After this time the wells were placed on ice and PGE, and PMA were removed by washing twice with 2-ml portions of cold Hanks’ balanced salt solution for 2 min, and once with 2 ml of cold assay medium. Finally, the wells received 1 ml of warm assay medium with or without 200 ng/ml PMA and were transferred to a 37°C water bath. At the times indicated the reactions were terminated by placing the wells on ice and adding perchloric acid as described above. The medium was not aspirated before stopping reactions. Consequently, we were sure of measuring CAMP degradation because we did not take into considera- tion potential loses of intracellular CAMP due to release of CAMP to the medium, which would artifactually decrease intracellular CAMP levels.

The rates of CAMP degradation (Fig. 4) were calculated by performing a weighted nonlinear regression of the raw data as described by Duggleby (1981).

2.6. Membrane isolation

Whole 293 cell membranes were prepared from cells that had been preincu- bated with or without PMA. After incubation of cell monolayers with or without

J. Sbnchez-Yagiie et al. /J. Lipid Mediators Cell Signalling I1 (I 995) 25-39 29

PMA for 30 min at 37°C the dishes were placed on an ice tray, and cell monolayers were washed three times with 5-ml portions of 0.25 M sucrose, 25 mM Tris-HCl and 1 mM EDTA, pH 7.4. Then, membranes were prepared and adenylyl cyclase activity determined as described before (Sanchez-Yagiie et al., 1992, 19931, except that CAMP was assayed as indicated above. Protein concentrations were measured using the method of Bradford (19761, using bovine serum albumin as standard.

2.7. Data presentation and analysis

All data presented are the means (+ S.E.) of two to three experiments. In each experiment triplicate wells were used for each point. When smaller than the actual symbols, error bars are not shown.

3. Results

3.1. Effects of PA44 on PGE,-elicited CAMP accumulation in 293 cells.

The first set of experiments performed corresponded to dose-response curves for PGE,-stimulated CAMP accumulation to demonstrate that 293 cells respond to PGE, with increased adenylyl cyclase activity (data not shown). In the presence of IBMX, basal activity was 15 pmol/106 cells and the maximal activity attained was 890 pmol/106 cells. The EC,, for PGE, was 0.04 ng/ml.

For the cells incubated with IBMX, CAMP accumulation proceeds in a linear fashion for at least lo-15 min (Fig. 1). Thus, the rate of CAMP accumulation at early time points following the addition of PGE, can be used to assess the activity of adenylyl cyclase in intact 293 cells. The data presented in Fig. 1 were obtained using this experimental approach and show that a short preincubation of 293 cells

"0 5 10 15

Incubation time (min)

Fig. 1. Effects of PMA on the PGE,-activated rate of CAMP accumulation in 293 cells. 293 cells were washed and preincubated in assay medium with 0.5 mM IBMX for 45 min at 37°C. PMA (200 nM) was added to half of the cultures during the last 30 min of this preincubation. At this time (shown as time = 01, all cells received 2 PM PGE,, and the incubation was continued at 37°C. Total CAMP was determined at the times indicated as described under section 2.

30 .I. Sa’nchez-Yagiie et al. /J. Lipid Mediators Cell Signalling I I (1995) 25-39

a 0

0 1.5 30 45 60 Minutes with PMA

Fig. 2. Effects of duration of pretreatment with PMA on PGEs-stimulated CAMP production in 293 cells. 293 cells were washed and preincubated in assay medium with 0.5 mM IBMX for 15 min at 37°C. After this preincubation PMA (200 nM) was added at different times, such that the length of the preincubation with PMA varied as indicated. At the end of the preincubation all cells received 2 yM PGE,. Total CAMP was determined 10 min after the addition of PGE,. (0) Total CAMP content of cells incubated without PMA or PGE,.

with PMA results in an inhibition of the rate of PGE,-enhanced CAMP accumula- tion. This rate was 46.2 pmol/min per lo6 cells for the cells incubated with PGE, only, and 25.6 pmol/min per lo6 cells for those incubated with PGE, plus PMA. Thus, the degree of inhibition of CAMP accumulation was 44%.

We next preincubated 293 cells in the presence of IBMX for increasing periods of time with a fixed concentration of PMA and the cells were finally incubated with PGE, for 10 min prior to measuring CAMP accumulation. The results of these experiments are shown in Fig. 2 and reveal that the average degree of inhibition was 42%, and also that the inhibiting effect of PMA can be easily detected within 5 min of addition of PMA and is maximally expressed in about 15-30 min.

The results obtained so far suggested that the ability of PMA to attenuate the rate of CAMP accumulation enhanced by PGE, involved inhibition of CAMP synthesis, since all experiments were carried out in the presence of IBMX. Nevertheless, in an attempt to measure directly the rate of degradation of CAMP in intact 293 cells, we developed a method similar to another described for G protein-coupled receptors (Pereira et al., 1988). Due to the particular experimental protocol followed to study this problem (see section 2) and because 293 cells tend to come off the plates after several washings, it was neccessary to plate cells with fibronectin as a support for the cells to stay bound to the wells during the time of the experiments. We first studied the time necessary to attain maximal levels of intracellular CAMP after stimulation of 293 cells with PGE, in the absence of IBMX. This time was proved to be 15 min (data not shown). To measure the effects of PMA on the rate of CAMP degradation, we first preincubated 293 cells with PGE, (at 37°C) in the presence or absence of PMA. After washing at 4°C the cells were incubated at 37°C in the presence or absence of PMA, and the amount of CAMP remaining was then determined at different times. As can be seen in Fig. 3, we were unable to demonstrate an effect of PMA on the degradation half-life of

J. S&chez-Yagiie et al. /J. Lipid Mediators Cell Signalling II (1995) 25-39 31

080 . . Time (min)

Fig. 3. Effects of PMA on the rate of degradation of CAMP in 293 cells. 293 cells were preincubated (30 min at 37°C) in assay medium with or without PMA (200 ng/ml) as indicated. 2 PM PGE, was added to all wells during the last 15 min. At the end of this preincubation PMA and PGE, were removed, the cells were then extensively washed and finally preincubated (37°C) in the presence or absence of PMA (200 ng/ml) as indicated. CAMP accumulation was determined as a function of time after prostaglandin removal. The lines shown were calculated using a nonlinear regression procedure as described under section 2.

CAMP in intact 293 cells (1.17 min in control cells vs 1.08 min in PMA-treated cells). This conclusion was also supported by the finding that PMA had no effect on the ability of forskolin to increase CAMP accumulation in intact 293 cells (cf. Fig. 5). Although these findings do not allow us to formally exclude the possibility that PMA might increase CAMP degradation, our data do indicate that the ability of PMA to attenuate the rate of CAMP accumulation enhanced by PGE, involves an inhibition of CAMP synthesis.

In additional experiments we examined the effects of a single concentration of PMA on the accumulation of CAMP enhanced by increasing concentrations of

i

- -PMA - +PMA

800

- Basal - +PMA 600

400

200

0 E 0 0.2 2 20 200

[PM.41 (nW

Fig. 4. Effects of increasing concentrations of PGE, and PMA on the accumulation of CAMP in 293 cells. 293 cells were washed and preincubated with 0.5 mM IBMX for 15 min at 37°C. Then: (left panel) PMA (200 nM) was added to half of the cultures, and the incubation was kept at 37°C for another 30 min. The indicated concentrations of PGE, were then added and total CAMP was measured 10 min after the addition of PGE,. (Right panel) The indicated concentrations of PMA were added and the cultures were kept at 37°C for 30 min prior to adding 2 FM PGE,. Total CAMP was measured 10 min after the addition of PGE,. The dashed line shows the total CAMP content of cells incubated with no additions.

32 _I. Sa’nchez-Yagiie et al. /J. Lipid Mediators Cell Signalling 11 (1995) 25-39

- -PMA - +PMA

0 5 10 15

Incubation time (min)

Fig. 5. Effects of PMA on the forskolin-activated rate of CAMP accumulation in 293 cells. 293 cells were washed and preincubated in assay medium with 0.5 mM IBMX for 45 min at 37°C. PMA (200 nM) was added to half of the cultures during the last 30 min of this preincubation. At this time (shown as time = 0) all cells received forskolin (200 PM) and the incubation was continued at 37°C. Total CAMP was measured at the indicated times.

PGE,. These results are illustrated in Fig. 4 (left panel) and show that an inhibitory effect was reliably detected at concentrations of PGE, higher than 0.02 PM. The degree of inhibition appeared to decline as the concentration of PGE, was increased (from 68-72% inhibition at 0.02-0.05 PM PGE, to 41% inhibition at 2 I_LM PGE,). The results shown in the right panel of Fig. 4 reveal the effects of increasing concentrations of PMA on the accumulation of CAMP elicited by a single concentration of PGE,. An inhibitory effect was reliably detected at 0.2 ng/ml PMA.

3.2. Some studies on the mechanism by which Pitt4 decreases the levels of CAMP in intact 293 cells

The results presented in Fig. 5 show that PMA had no effect on the ability of forskolin to elicit an increase in CAMP accumulation. They also demonstrate that the time course of accumulation of CAMP was faster when the cells were incubated with forskolin than with PGE,. In the presence of forskolin higher levels of CAMP were found at all times studied. Finally, it should be stressed that, as noted above, these results argue against an effect of PMA on CAMP degradation. Thus, if the ability of PMA to attenuate the PGE,-provoked increase in CAMP accumulation were partially due to an increase in CAMP degradation (see above), one would expect to observe the same phenomenon regardless of the compound used to enhance CAMP accumulation.

In general, the action of PMA is thought to be mediated by PKC. To test this possibility, the effects of H-7, a nonspecific inhibitor of PKC, on the effects of PMA on PGE,-stimulated CAMP accumulation were examined. After pretreat- ment with different concentrations of H-7 (10-100 PM) for 45 min and with PMA (200 ng/ml) for the last 30 min, cells were stimulated with 2 PM PGE, for 10 min. As can be seen in Fig. 6, H-7 prevented the inhibitory effect of PMA on PGE,-stimulated CAMP accumulation in 293 cells. To provide further evidence

J. S&chez-Yagiie et al. /J. Lipid Mediators Cell Signalling II (1995) 25-39 33

A B C D E

Fig. 6. H-7 reversal of the inhibition by PMA of PGE,-stimulated cAMP production in 293 cells. 293 cells were washed and preincubated in assay medium with 0.5 mM IBMX for 15 min at 37°C in the absence (vehicle only, A and B) or in the presence of different concentrations of H-7 (C, 10 PM; D, 50 PM; E, 100 PM). Vehicle (A) or PMA (200 nM) was added (B-E) and the incubation was kept at 37°C for another 30 min. At the end of the preincubation, all cells received 2 PM PGE,. Total CAMP was determined 10 min after the addition of PGE,.

that PGE,-stimulated CAMP responses are inhibited after the activation of protein kinase C we also examined the effects of the synthetic diacylglycerol analog OAG. Pretreatment of 293 cells with OAG for 30 min significantly inhibited the subse- quent CAMP response at 2 PM PGE, over the lo-100 PM range (Fig. 7). Although we do not know why we were unable to reproduce the inhibition entirely, it is possible that this could have been due to the lack of solubility of the analog.

Another approach that can be used to study the effects of PMA on the adenylyl cyclase system is one in which cells are preincubated with PMA followed by extensive washing. The effect of PGE, on adenylyl cyclase system is then deter- mined by measuring adenylyl cyclase activity in cell membranes rather than by measuring CAMP accumulation in intact cells. The results presented in Fig. 8 show that preincubation of intact cells with PMA does not decrease the ability of PGE, to stimulate adenylyl cyclase activity in isolated membranes. In fact, the basal as well as NaF or PGE,-stimulated adenylyl cyclase activity of membranes prepared

A B C D E

Fig. 7. OAG mimics the inhibition by PMA of PGE,-stimulated CAMP production in 293 cells. 293 cells were washed and preincubated in assay medium with 0.5 mM IBMX for 45 min at 37°C. Vehicle (A), PMA (200 nM, B) or OAG (1, 10 and 100 PM, corresponding to C. D and E) were added during the last 30 min of this preincubation. At this time all cells received 2 PM PGE,. Total CAMP was measured 10 min after the addition of PGE,.

34 _I. Sa’nchez-Yagiie et al. /.I. Lipid Mediators Cell Signalling 11 (1995) 25-39

h 600

.Z

.s e,

500

; g 400

ax v e 300 a$

z% 200

z E czz 100

P 0 GTP EE2 NaF

GTP AICI 3

Additions to assay

CM preincubation:

P %%

Fig. 8. Effect of PMA on adenylyl cyclase activity of 293 cells. Cells were preincubated for 30 min at 37°C with the indicated additions. The concentration of PMA using during the preincubation was 200 nM. At the end of this incubation membranes were prepared and assayed for adenylyl cyclase acivity (see section 2) in the presence of 100 PM GTP, 100 NM GTP and 5 PM PGE,, or 10 mM NaF and 10 PM AICl, as indicated. Each point shows the average + S.E. of nine determinations (three experiments with triplicate wells in each).

from PMA-treated 293 cells is the same as or higher than that of the control cells. Therefore, this experimental approach does not allow for the measurement of the PMA-induced uncoupling, and only the one described in Fig. 1 could be used for such purposes. Some of us have recently published similar results for other G protein-coupled receptors stably expressed in 293 cells (Hipkin et al., 1993; Quintana et al., 1994).

4. Discussion

The characteristics of prostaglandin receptors have not been fully investigated, although they have been identified and partly characterized in various tissues and cells (Robertson, 1986). PGE, receptors are pharmacologically subdivided into three subtypes, EP,, EP,, and EP,, and these subtypes are suggested to be different in their signal transduction; they are presumed coupled to stimulation of phospholipase C, and stimulation and inhibition of adenylyl cyclase, respectively. Recently, the mouse EP,, EP, and EP, receptors have been cloned and it was demonstrated that those receptors are G protein-coupled rhodopsin-type receptors (Sugimoto et al., 1992; Honda et al., 1993; Watabe et al., 1993). In the kidney, PGE, exerts both stimulator-y and inhibitory effects on CAMP metabolism (Ed- wards et al., 1981; Garcia Perez and Smith, 1981; Torikai and Kurokawa, 1981, 1983; Anderson et al., 1984; Smith and Garcia Perez, 1985). PGE, activates adenylyl cyclase of microdissected cortical and medullary collecting tubules and thin limbs (Torikai and Korokawa, 19811, but inhibits arginine vasopressin-induced CAMP production in the thick ascending limb (Edwards et al., 1981), the cortical collecting tubule (Smith and Garcia-Perez, 1985; Torikai and Korokawa, 1983; Garcia-Perez and Smith, 1981) and, possibly, the medullary collecting tubule (Edwards et al., 1981). These and other results suggest that there are both stimulatory (probably EP,) and inhibitory (probably EP,) PGE, receptors in

.I. Sa’nchez-Yagiie et al. /J. Lipid Mediators Cell Signal&g II (1995) 25-39 35

kidney, and the possibility that stimulatory PGE, receptors mediate CAMP-de- pendent heterologous desensitization of arginine vasopressin receptors. In this regard, the molecular cloning and intrarenal localization of rat EP, subtype receptor has been recently described (Takeuchi et al., 1993).

In the studies presented here, we used 293 embryonal kidney cells as a study model under cell culture conditions, and describe the first set of experiments designed to study the mechanism(s) by which PMA attenuates the increase in CAMP accumulation elicited by PGE, in this cell line.

It is well known that PMA and other phorbol esters have multiple effects on intracellular signal transduction pathways, although this multiplicity is related to the experimental designs (e.g., cell types, duration of PMA treatment, intact cells, or a cell-free system). Phorbol esters have been used as probes of PKC function in many systems. PKC is present in inactive form in the cytosol and associates with the plasma membrane when intracellular free Ca2+ concentrations rise (Hug and Sarre, 1993). Once associated with the membrane, it is fully activated by diacylglyc- erol, a product of agonist-induced inositol phospholipid breakdown (Bell and Burns, 1991). Tumor-promoting phorbol esters substitute for diacylglycerol and may therefore directly activate the enzyme. Several reports have demonstrated that PGE, can increase inositol trisphosphate production (presumably activating phos- phatidylinositol hydrolysis) and intracellular free calcium concentrations in differ- ent cells (Kawase et al., 1991; Ito et al., 1991) may be due to the presence of EP,-type receptors in these cells. Since diacylglycerols are formed together with inositol phosphates (Cockcroft and Thomas, 1992) PKC would also be expected to become activated when these cells are exposed to PGE,. This kinase activation could in turn modulate CAMP formation through specific agonists of the adenylyl cyclase system. In this regard, it is interesting that in the kidney increased intracellular calcium levels and PKC activation regulate CAMP generation in the collecting duct, and also modulate vasopressin-stimulated osmotic water flow. Although we have not investigated the possible increases in cytosolic Ca*+ in 293 cells after PGE, treatment, preliminary experiments did not reveal rises in inositol trisphosphate production after this treatment (data not shown), which may indicate a lack of EP,-type receptors in this cell line. Nevertheless, the obtaining of stable transfected 293 cell lines with the lutropin/choriogonadotropin (LH/CG) or the follitropin (FSH) receptors that responds to human choriogonadotropin (hCG) or FSH, respectively, with increased levels of inositol phosphates (Hipkin et al., 1993; Quintana et al., 1994; see below) should allow us to study attenuation of adenylyl cyclase activity by a PKC-linked hormonal pathway in this cellular model.

The finding that PMA inhibits the rate of CAMP accumulation elicited by PGE, in 293 cells incubated with phosphodiesterase inhibitors shows that this phe- nomenon is due (at least in part) to an inhibition of PGE,-activated adenylyl cyclase. The findings that PMA (i) does not stimulate CAMP degradation in intact 293 cells (Fig. 3) and (ii) does not inhibit forskolin-activated CAMP accumulation in intact 293 cells (Fig. 5) indicate that PMA does not attenuate the PGE,-elicited CAMP accumulation by stimulating CAMP degradation and suggest that the catalytic subunit of adenylyl cyclase is not involved in this phenomenon.

36 J. S&chez-Yagiie et al. /J. Lipid Mediators Cell Signalling 11 (1995) 25-39

The time course and dose responsiveness of the effects of PMA on 293 cells demonstrated here are similar to those reported in other systems (Warhurst et al., 1988; Kawase et al., 1991a,b). This, taken together with the observation that the effects of PMA can be antagonised by H-7 and partially mimicked by OAG, provides strong evidence that attenuation of the PGE, response is mediated by PKC activation. Similar conclusions have been reported for the decrease in PGE, response after PKC stimulation in the colonic cell line T84 (Warhurst et al., 1988). In this cell line, the mediation of the effects of phorbol ester (PDB) through the adenylyl cyclase system was also investigated using membranes isolated from control and PDB-treated cells, similar to the approach described here. However, while in T84 cells it was possible to demonstrate a decrease in the PGE,-stimu- lated adenylyl cyclase from cells pretreated with PDB, it was impossible in 293 cells (see section 3) as has been published before for other G protein-coupled receptors expressed in this cell line (Hipkin et al., 1993; Quintana et al., 1994). Nevertheless, in 293 and T84 cells, as well as in other cell lines (e.g., UMR 106 cell line, Freyaldenhoven et al., 1992), GTP- and fluoride-stimulated adenylyl cyclase activity has been described to be ‘increased in membranes derived from cells pretreated with phorbol esters up to values as high as 20-30%. Therefore, the adenylyl cyclase assay in membranes derived from cells pretreated with PMA cannot be used to study the PMA-induced uncoupling of the PGE,-stimulated activity in the 293 cell system. In other cell lines, such as the clonal osteoblast-like cell line MOB 3-4 or the H-T-29 colonic cell line, phorbol ester attenuation of receptor-stimulated CAMP production (PGE, and vasoactive intestinal peptide receptors, respectively) appears to be due to a direct action on the receptors to decrease their apparent affinity (Kawase et al., 1991; Bozou et al., 1987). In these cases, phorbol ester reduction of the PGE,-stimulated CAMP production could not be prevented by coincubation with H-7 and the decrease in the apparent affinity of the receptors could not be mimicked by OAG treatment. A role for Gi in mediating the effects of PMA on receptor responsive adenylyl cyclase has been suggested in some cases (Choi and Toscano, 1988; Freyaldenhoven et al., 19921, but not in others (Freyaldenhoven et al., 1992). Finally, another mechanism that should be considered is the PMA-dependent phosphorylation of the PGE, recep- tor. Phorbol-ester-induced phosphorylation of the P-adrenergic receptor, in associ- ation with desensitization of isoproterenol-stimulated adenylyl cyclase activity, has been demonstrated in avian (Kelleher et al., 1984; Sibley et al., 1984) and mammalian (Bouvier et al., 1987) erythrocytes. In several cell lines with stimulatory receptors linked to adenylyl cyclase, the altered function after PMA treatment has been attributed to receptor phosphorylation by PKC (Bozou et al., 1987; Ya- mashita et al., 1988; Findlay et al., 1989; Raymond, 1991). Nevertheless, it is important to stress that such a PKC-mediated covalent modification, although possible, has never been demonstrated for the prostaglandin receptor(s). Addition- ally, this PKC-mediated phosphorylation could be associated, or not, to a decrease in the number of PGE, receptors in 293 cells. Finally, it is important to note that although so far there is no evidence for adenylyl cyclase inhibition by a PKC-linked hormonal/neurotransmitter naturally expressed in this cell line, stably transfected

J. Scinchez-Yagiie et al. /J. Lipid Mediators Cell Signalling II (1995) 25-39 37

293 cell lines with the LH/CG or the FSH receptors have recently been obtained, showing that these cell lines respond to hCG or FSH with increased levels of both CAMP and inositol phosphates (Hipkin et al., 1993; Quintana et al.‘, 1994). Therefore, activation of these gonadotropin receptors would presumably result in the activation of PKC, because they generate at least one of the two second messengers (inositol phosphates) associated with this signalling system. In these cells, directly derived from 293 cells, it was also demonstrated that PMA is not only able to decrease the ability of gonadotropins to stimulate CAMP synthesis (uncoupling), but also that phosphorylation of both receptors does indeed corre- late with a functional uncoupling. This fact should now allow us to study the effect(s) of PKC on PGE,-responsive adenylyl cyclase in this cell line, with more physiological significance.

In conclusion, our results indicate that PMA inhibits the CAMP accumulation produced by PGE, stimulation of adenylyl cyclase in human kidney 293 cells by an activation of PKC that somehow could inhibit CAMP synthesis but not CAMP degradation. The specific point of lesion in the receptor-G protein-adenylyl cyclase system is now under study, although the catalytic subunit of the enzyme can be ruled out because the inhibition in CAMP synthesis cannot be reproduced in PMA-treated 293 cells with the cyclase activator forskolin.

Acknowledgements

The authors wish to thank N. Skinner for his assistance in the preparation of the manuscript. Financial support to The University of Salamanca is acknowl- edged.

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