Jurrrnal uf Ntwruchemrsrry Raven Press, Ltd.. New York 0 1988 International Society for Neurochemistry

Activators of Protein Kinase C Act at a Postreceptor Site to Amplify Cyclic AMP Production in Rat Pinealocytes

David Sugden and David C . Klein

Section on Neuroendocrinolog.v, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development. National Institutes of Health, Bethesda, Maryland, U.S.A.

Abstract: Activation of a,-adrenoceptors appears to am- plify 8-adrenergic stimulation of cyclic AMP (CAMP) accu- mulation in rat pinealocytes severalfold by a mechanism involving activation of a Ca2+-, phospholipid-dependent protein kinase (protein kinase C). The mechanism of action of protein kinase C was investigated in this report using intact cells. Activation of protein kinase C with 48-phorbol 17-myristate 13-acetate (PMA; M) or the a,-adrener- gic agonist phenylephrine (PE; M ) did not inhibit cAMP efflux in 8-adrenergically stimulated cells. The am- plification of the @-adrenergic cAMP response by these agents also occurred in the presence of isobutylmethylxan- thine (lo-’ M ) and Ro 20-1724 ( M ) , an observation suggesting that inhibition of cAMP phosphodiesterase ac- tivity is not the mechanism of action. Furthermore, al- though PMA (lo-’ M ) caused a sixfold increase in the magnitude of the cAMP response to isoproterenol, it did not alter the ECS0 of the response (1.7 X M ) , a result indicating that protein kinase C activation does not alter

8-adrenoceptor sensitivity. The cAMP response following cholera toxin pretreatment (60-120 min) was rapidly and markedly enhanced by a,-adrenergic agonists (cirazoline > PE > methoxamine), by phorbol esters (PMA > 48- phorbol 12,13,-dibutyrate + 4a-phorbol 12,13didecano- ate), and by synthetic diacylglycerols ( I ,2-dioctanoylglyce- rol > I-oleoyl 2-acetylglycerol + diolein). The cAMP re- sponse to forskolin ( lo-’ M) was also increased by PE (3 X M) and PMA ( M). Together, these observa- tions indicate that protein kinase C activation amplifies @-adrenergic stimulation of pinealocyte cAMP production at a site beyond the receptor, perhaps on a regulatory gua- nine nucleotide binding protein or adenylyl cyclase itself. Key Words: Protein kinase C-Cyclic AMP-Rat pinealo- cytes-m,-Adrenoceptor activation-8-Adrenoceptor. Sugden D. and Klein D. C. Activators of protein lunase C act at a postreceptor site to amplify cyclic AMP production in rat pinealocytes. J. Neurocbem. 50, 149-155 (1988).

There is growing evidence that two enzymes of central importance in receptor-mediated transmem- brane signaling, adenylate cyclase and Ca2+-, phos- pholipid-dependent protein kinase (protein kinase C), may interact in both antagonistic and synergistic manners (Nishizuka, 1984). Direct activation of pro- tein kinase C with 4P-phorbol 12-myristate 13-ace- tate (PMA) reduces hormone-activated adenylate cy- clase activity in hepatocytes (Heyworth et al., 1984, 1985) and Leydig cells (Mukhopadhyay and Schu- macher, 1985; Rebois and Patel, 1985). However, in

other cell types, PMA enhances the cyclic AMP (CAMP) response (Bell et al., 1985; Cronin and Can- onico, 1985; Nabika et al., 1985). Of the latter type, one striking example is the rat pinealocyte (Sugden et at., 1985; Vanecek et al., 1985).

In this cell, intracellular cAMP is regulated by nor- epinephrine (NE), which is released into the perivas- cular space from the sympathetic nerves innervating the tissue. cAMP controls melatonin synthesis by reg- ulating the activity of arylalkylamine N-acetyltrans- ferase (EC 2.3. I .87). the rate-limiting enzyme in the

Received December 26. 1986: revised manuscript received July IS, 1987: accepted July 2 I , 1987.

Address correspondence and reprint requests to Dr. D. C. Klein at Section on Neuroendocnnology. Laboratory of Developmental Neurobiology, National Institute of Child Health and Human De- velopment, National Institutes of Health, Bethesda, MD 20892, U.S.A.

The present address of Dr. D. Sugden is Department of Physiol- ogy, Kings College London (KQC), University of London, Camp den Hill Road. London W8 7AH, U.K.

Ahhrrviotions used: CAMP, cyclic AMP [Ca”], , free cytosolic

Ca” concentration: CT. cholera toxin: diC8G, sn- I ,2dioctanoyl- glycerol: DMEM, Dulbecco’s modified Eagle’s medium: IBMX. isobutylmethylxanthine: ISO. isoproterenol: NE. norepinephrine: Ni and Ns, inhibitory and stimulatory regulatory guanine nucleo- tide binding protein, respectively: OAG. sn- I-oleoyl 2-acetylglyce- rol: PDBu. 48-phorbol I2,I3-dibutyrate: 4a-PDD, 4a-phorbol 12, I3didecanoate: PE. phenylephnne: PMA, 48-phorbol 12-myr- istate 13-acetate: protein kinase C. Ca’+-. phospholipiddependent protein kinase: Ro 20-1 724. (+)-4-(3-butoxy-4-methoxybenzyl)-2- imidazolidinone.

149

140 D. SUGDEN AND D. C. KLEIN

conversion of serotonin to melatonin (Klein et al., 198 I). The mechanism through which cAMP is con- trolled by NE involves both a,- and 8-adrenoceptors. 8-Adrenoceptor stimulation increases cAMP accu- mulation by activating adenylate cyclase. The in- crease in cAMP is markedly enhanced by a,-adreno- ceptor stimulation, which alone has no effect on cAMP (Vanecek et al., 1985). The mechanism by which a,-adrenoceptor stimulation enhances cAMP accumulation involves an increase in free cytosolic Ca2+ concentration ([Ca2+Ii), which apparently is due to an increase in net influx rather than release of intracellular stores (Sugden et al., 1986, 1987). In ad- dition, a,-adrenergic receptor activation also in- creases the activity of phospholipase C, which gener- ates diacylglycerol (Smith et al., 1979; Zatz, 1985). The increases in [Ca2+Ii and diacylglycerol are thought to translocate and activate protein kinase C (Nishizuka, 1984; Sugden et al., 1985). An involve- ment of protein kinase C in cAMP regulation is indi- cated by the finding that the potentiating effect of a,-adrenergic agonists can be duplicated by direct ac- tivators of the kinase such as PMA and sn-1-oleoyl 2-acetylglycerol (OAG).

Activation of protein kinase C presumably results in the phosphorylation and subsequent modification of the activity of a protein involved in regulating in- tracellular cAMP content. Possible mechanisms of action, including an increase in cAMP synthesis and an inhibition of cAMP degradation or export, were investigated in this report.

EXPERIMENTAL PROCEDURES

Materials Cholera toxin (CT) was purchased from Schwarz-Mann

(Cambridge, MA, U.S.A.). OAG and sn- 1,2-dioctanoyI- glycerol (dicaprylin; diCsG) were obtained from Avanti Polar Lipids, Inc. (Birmingham, AL, U.S.A.). Phorbol esters, isobutylmethylxanthine (IBMX), and diolein were from Sigma (St. Louis, MO, U.S.A.), and forskolin was from Calbiachem (San Diego, CA, U.S.A.). Ro 20-1724 was supplied by Hohann-La Roche (Nutley, NJ, U.S.A.). All other drugs and chemicals were obtained from sources previously identified (Sugden and Klein, 1984; Vanecek et al., 1985).

Cell culture procedures Female Sprague-Dawley rats (50-100 animals per batch

-7 weeks old; weighing 200-250 g) were purchased from Charles River Breeding Laboratories (Wilmington, MA, U.S.A.). Rats were housed under diurnal lighting condi- tions (light/dark 12:12; lights on at 6 a.m.) for 3-7 days before use. Pinealocytes were prepared by trypsinization as previously described (Buda and Klein, 1978; Vanecek et al., 1985) with minor modifications. Glands were incubated with trypsin ( I mg/ml; type 111; Sigma) for 15 min and then for a further 10 min afler tituration. In some batches of cells, ascorbate (0.1 mg/ml) and DL-prOpranOlOl ( 1 0-6 M) were included in the trypsinizing medium. Cells were washed twice following dispersion to remove propranolol from the medium.

Cells were incubated (2 X I O5 cells/ml; 37"C, 95% air/% C02) in suspension culture in Dulbecco's modified Eagle's medium (DMEM) containing fetal calf serum (10% vol/ vol) for 24 h before use in an experiment. Viability, judged by trypan blue exclusion, exceeded 95% in all experiments. Aliquots of cells (0.5 ml; lo5 cells) were treated with drugs as indicated in the tables and figure legends. CT was dis- solved in distilled water and stored (4°C) as a 1 mg/ml stock solution. Diacylglycerols (10 mg/ml in chloroform or chlo- roform/methanol) were dried under N2 and then suspended in dimethyl sulfoxide (1% vol/vol) by brief, vigorous soni- cation on ice immediately before addition to the cells. Phorbol esters were dissolved in dimethyl sulfoxide or eth- anol, and serial dilutions were made with water. The high- est concentration of dimethyl sulfoxide or ethanol in the medium (0.1% vol/vol) did not alter basal cAMP levels, and had no effect on the magnitude of the cAMP response to isoproterenol (ISO) ( M). Following treatment, cells were centrifuged ( 1 ,OOO g, 2 min), the medium was aspir- ated, and the cell pellets were immediately frozen on solid C02. Samples were prepared for assay by boiling in dilute acetic acid ( 5 mM) and then were frozen (-70°C). In some experiments, aliquots of medium were prepared for assay in an identical manner.

Assay of cAMP Samples were diluted when necessary with acetic acid (5

mM) and then were assayed in duplicate for cAMP by radioimmunoassay following acetylation as described pre- viously (Harper and Brooker, 1975; Vanecek et al., 1985) using an antibody generously provided by Dr. Kevin Catt (NICHD). The cross-reactivity of the antibody for cyclic GMP was 0.07%. Recovery of cAMP standards (1.9-500 fmol) added to cell homogenates averaged 102 -t 9%. Pro- tein content was measured by a dye binding method using bovine serum albumin as the standard (Bradford, 1976).

RESULTS

Release of cAMP to the extracellular medium The possibility that activation of protein kinase C

might potentiate the B-adrenergic cAMP response by antagonizing the efflux of cAMP into the medium was examined (Table 1). An inhibition of cAMP ef- flux would result in an enhanced intracellular accu- mulation of CAMP, which may be erroneously as- sumed to be secondary to an increase in cAMP syn- thesis. Treatment with IS0 ( M) elevated cAMP accumulation in cells and in the medium. Addition of phenylephrine (PE) ( M) or PMA (lo-' M) to ISO-treated cells potentiated cAMP accumulation both in the medium and in cells, an observation indi- cating that cAMP transport out of the cell is not in- hibited by PE or PMA.

Role of phosphodiesterase Incubation of pinealocytes with a high concentra-

tion oftwo phosphodiesterase inhibitors, IBMX ( M) and Ro 20- 1724 ( M), elevated both basal and agonist-induced cAMP levels (Table 2), but did not prevent the potentiation of j3-adrenergic stimula- tion produced by PE ( M) or PMA (lo-' M). In addition, phosphodiesterase activity measured in pi-

J. Neurmhem.. Vd. 50, No. 1. 1988

PROTEIN KINASE C AND PINEAL CAMP RESPONSE I51

TABLE 1. Release of CAMP into the medium

cAMP

Cellular Medium Treatment (pmol/105 cells) (pmol/tube)

Control 0.25 t 0.01 0.13 ? 0.01 IS0 1.71 t 0 . 0 7 1.44 t 0.10 PE 0.33 ? 0.03 0. I9 ? 0.06 IS0 + PE 16.5f 1.81" 4.65 t 0.03" NE 25.3 t 3.71 7.75 ? 0.67 PMA 0.27 f 0.01 0.35 t 0.02 IS0 + PMA 33.2 ? 0.18" 13.3 ? 1.0la

Pinealocytes (2 X IO'/ml) were centrifuged (I.OO0 g, 5 min. 22°C). and the medium was aspirated and replaced with fresh DMEM containing fetal calf serum (10% vol/vol). Triplicate ali- quots of cells ( lo5 cells, 0.5 ml/tube) were then equilibrated (37°C. 95% air/5% C02) for 15 min before addition of the drugs for a further I5 min. The concentration of adrenergic agonists (KO. NE. and PE) was M, and the concentration of PMA was lo-' M . Data are mean 2 SEM values from three cell aliquots. For further details. see Experimental Procedures.

" Significantly different from IS0 alone (p < 0.05).

nealocyte homogenates using cAMP (100 p M ) as substrate in the presence or absence of Ca2+ showed no alteration in activity following either NE ( 1 0-5 M) or PMA ( M) treatment (10 min) of cells (D. Sugden and V. Manganiello, unpublished data). With control, NE, and PMA treatment, cAMP phosphodi- esterase activity (pmol/min/105 cells) was 23.5, 20.3, and 20.4 in the absence of Ca2+; in the presence of Ca2+, the respective values were 45.1, 43.5, and 45.1 (mean of duplicate determinations). These re- sults lend no support to the hypothesis that activation of protein kinase C potentiates P-adrenergic stimula- tion of cAMP by inhibiting phosphodiesterase activity. Effect of PMA on the B-adrenergic response

PMA treatment produces a large, dose-related en- hancement of the cAMP response to a single concen-

TABLE 2. Effects ofphosphodiesterase inhihirnrs

cAMP (nmol/mg protein)

Treatment -PDEI +PDEI

Control 0.012 t 0.001 0.031 ? 0.009 IS0 0.382 +- 0.01 I 1.30 f 0.136 PE 0.01 8 t 0.003 0.089 f 0.024 IS0 t PE 0.95 t 0.052" 2.32 ? 0.095" NE I .44 ? 0.087 2.84 t 0. I5 PM A 0.0 I9 t 0.007 0.092 ? 0.0 I9 IS0 + PMA I .84 t 0.024" 4.42 ? 0.147'

Pinealorytes were incubated with the phosphodiesterase inhibi- tors (PDEI) IBMX M) and Ro 20-1724 M ) or with vehicle for I5 min; drugs were then added, and cells were collected 15 min later. The concentration of adrenergic agonists (ISO, NE, and PE) was M, and the concentration of PMA was lo-' M. Data are mean f SEM values from three samples.

* Significantly different from IS0 alone (p < 0.05).

tration of IS0 M) (Sugden et al., 1985). The present experiment was designed to determine if an optimal concentration of PMA (lo-' M) altered the EC50 of the cAMP response to ISO. IS0 increased pinealocyte cAMP content in a dose-related manner (Fig. 1). The maximal response was increased sixfold by PMA (Fig. 1, left), but the ECJo ( 1.7 X lo-* M) was unchanged (Fig. 1, right).

cAMP response to protein kinase C activators in CT-treated cells

Preliminary studies (Vanecek et al., 1986) indi- cated that PE enhanced cAMP accumulation in CT- treated pinealocytes. In each of the studies presented here (Figs. 2-7), CT ( 1 &ml) produced a small but significant increase in pinealocyte cAMP content fol- lowing a lag period. This lag is probably necessary for the toxin to bind to the cell and penetrate the cell membrane (Gill, 1977). The action of CT requires the intact molecule, as treatment of pinealocytes with pu- rified A or B subunit does not mimic the effects of the holotoxin (authors' unpublished data). The CT-stim- ulated increase was markedly amplified by treatment ( 1 5 min) with either PE ( M) or PMA (lo-' M) (Fig. 2). Similar effects of PE and PMA were also apparent at higher and lower concentrations of CT (Fig. 3).

Treatment of cells with the selective a,-adrenergic agonists cirazoline, methoxamine, and PE had no ef- fect on cAMP content, other than a small effect of the highest concentration of PE ( M). The latter is known to be due to nonselective activation of B- adrenoceptors and can be blocked by propranolol (Vanecek et al., 1985). When the effects of each al-

-log IlSOl

FIG. 1. Dose-response study of the effect of IS0 on cAMP levels in PMA-treated and control pinealocytes. Pinealocytes (lo5 cells/ tube) were incubated (37%. 95% air/5% C02) with IS0 or IS0 plus PMA (lo-' M ) for 15 min and then rapidly centrifuged (2 min. 1,000 9). Cell pellets were frozen immediately on solid C o n , stored (-7O"C), and assayed in duplicate for cAMP as described in Experimental Procedures. Data are mean f SEM (bars) values from triplicate samples. The absence of error bars i n d i t e s that the SEM fell within the symbol. For calculating the fractional re- sponse for each dose-response curve, the cAMP content at lo-' M IS0 was used.

152 D. SUGDEN A N D D. C. KLEIN

I ' 1 1 I

i 2t p1

TIME (rnmulnl

FIQ. 2. Tlme course of CT stimulation of plnealocyte cAMP

the percoda mdcated Celts were then treated wtth vehale (0 001% vd/vdbmemyl Wnoxlde). PMA (10 ' M). or PE (10 ' M ) for a twher 10 mh Data are mean ? SEM (bars) values from Iripllcclte sampks. See Flg. 1 for hvther detds

IeWJts CT (1 &ml) was dded to pneabcytes (1 d CdS/tUbe) for

adrenergic agonist were tested in CT-treated cells, it was found that cAMP content increased >100-fold (Fig. 4). The order of potency (cirazoline > PE > methoxamine) was identical to that reported for the potentiation of 0-adrenergk stimulation of CAMP by these agents (Vanecek et al., 1985). Cirazoline produced a smaller maximal effect than PE or meth- oxamine, an observation possibly reflecting some other action of cirazoline or indicating that this agent is not a full agonist in this tissue. The a,-selective antagonist pmosin ( M) completely prevented PE (lo-' M) amplification of the CT ( 1 pg/ml; 60 min of pretreatment) response. However, neither the arselective antagonist yohimbine ( M) nor the

I+-

4- 0 0.1 1 10 CHOLERA TOXIN (mglml)

- log IADRENERGIC AGONIST I

FIG. 4. Potentiation of CT stimulation of cAMP accumulation by selective a,-adrenergic agonists. cdls were pretreated with CT (1 pg/ml) or water for 90 min and then the concentratis of agonists ind i ted for 15 min. See Fig. 1 for further details.

&selective antagonist propranoIoI ( M) was ef- fective (CAMP values are presented as the mean ? SEM of triplicate samples in pmol/mg of protein): CT, 0.036 f 0.001; CT plus PE, 1.147 f 0.177; CT plus PE plus prazosin, 0.066 f 0.005: CT plus PE plus yohimbine, 0.965 ? 0.086; and CT plus PE plus propranolol, 0.863 _+ 0.029. These experiments con- firm the involvement of an aI-adrenoceptor.

Both PMA and 4&phorbol 12,13-dibutyrate (PDBu), which had no effect on cAMP levels alone,

rf' I 1 1 I

+PDBU f n " Lq/ ' 1 I I I

0 9 8 7 6 5

-1Og IPHORBDL ESTER1

FIG. 5. Potentiation of CT stimulation of CAMP accumulation by phorbor esters. Cells were pretreated with water or CT (1 pg/rnl) for 60 min. and then Me indicated concentration of each phorbol estw was added for a further 15 min. The veh i i s used (0.loh vd/vol ethanol or dimethyl sulfoxide) da not alter the cAMP re- sponse to CT. Data are mean f SEM (bars) values from triplicate cell samples.

1. N r v u - k n . . Vd. 50. No. 1. 1988

PROTEIN KINASE C AND PINEAL CAMP RESPONSE 153

-log IDIACYLGLYCEROLI

FIG. 6. Potentiation of CT stimulation of cAMP accumulation by synthetic diacylglycerols. Cells were pretreated with CT (1 pg/ml) or water for 90 min. and then the concentrations of the diacyl- glycerols indicated were added for a further 15 min. Diacylglyce- rols were suspended in dimethyl sulfoxide (1% vol/vol) by vigor- ous sonication on ice immediately before addition to the cells. Data are mean * SEM (bars) values from triplicate cell samples.

markedly elevated cAMP accumulation in CT- treated cells. The concentrations of PMA and PDBu in CT-treated cells that produced half-maximal po- tentiation (1.3 X lo-’ and 3.4 X M, respectively) are similar to the reported half-maximal concentra- tions necessary to potentiate 8-adrenergic cAMP ac- cumulation in pinealocytes (Sugden et al., 1985) and to activate protein kinase C in vitro (Kraft et al., 1982). 4a-Phorbol 12,13-didecanoate (4a-PDD) ( M) also had a small effect. 4a-PDD was pre- viously found not to potentiate 8-adrenergic stimula- tion of cAMP accumulation (Sugden et al., 1985). The small response in the present study may reflect

TIME (minutes1

FIG. 7. Time course of the potentiating effect of PE. PMA. and OAG on cAMP levels in CT-treated pinealocytes. Cells were pre- treated with CT (1 pg/ml. 60 min). and then PE (3 X 1 0-6 M). PMA

M), or OAG (lo-‘ M) was added. At various times after addition of PE. PMA. or OAG, triplicate aliquots of cells (lo5) were collected as described in Fig. 1, and cAMP wntent was assayed. Data are from a representative experiment repeated three times and are mean * SEM (bars) values from three samples of pineal- ocytes.

contamination with the 48 isomer of this compound. Two synthetic diacylglycerols, OAG and diC8G, also potentiated CT stimulation of cAMP levels (Fig. 6). The order of potency was diCsG > OAG 9 diolein.

We have previously described the time course of PE and PMA potentiation of 8-adrenergic stimula- tion of cAMP accumulation (Sugden et a]., 1985; Vanecek et al., 1985). However, the transient nature of the 8-adrenergic response itself confounds an accu- rate assessment of the time course of the potentiating effect. An alternative approach was to use CT treat- ment because the pineal cAMP content increases slowly during the first 2 h of treatment with CT ( I pg/ml) (Fig. 2). We found that 2 min after addition of PE ( M) to CT-treated cells an increase in cAMP level was apparent (Fig. 7). The response peaked at 5 min, declined substantially by 20 min, and was virtually absent by 60 min. In con- trast, PMA (lo-’ M) failed to elevate the cAMP re- sponse after 2 min of treatment, although a marked response was evident by 5 min (Fig. 7). The response to PMA, although more prolonged than that to PE and OAG, also declined markedly by 60 min.

Potentiation of the cAMP response to forskolin by PMA and PE

Forskolin ( 10-6-10-3 M) increased the cAMP con- tent in a dose-related manner, although the magni- tude of the effect (about fivefold at M) was small (Fig. 8). Both PE (3 X M) and PMA (lo-’ M) significantly amplified the cAMP response to forsko- lin (Fig. 8); the potentiation by PMA was fivefold greater than that by PE.

M) or OAG (

DISCUSSION The present study examined several possible mech-

anisms by which protein kinase C activation en-

I PMAllO 7Ml

0.5

. PE13X10 SMI

0 6 5 3 LOG IFORSKOLIN

FIG. 8. Stimulation of pinealocyte cAMP levels by forskolin in the presence and absence of PE and PMA Pinealocytes were incu- bated with forskolin. forskolin plus PMA (1 0 M), or forskolin plus PE (3 X 10 M) for 15 min Data are mean ? SEM (bars) values from three samples of cells (105/tube) Additon of PMA (10 M) or PE (3 X 10 M) alone d d not elevate cAMP levels in this expenment

154 D. SUGDEN AND D. C. KLEIN

hances @-adrenoceptor stimulation of intracellular cAMP content. The results clearly point to a postre- ceptor site of action. The possibility that activation of protein kinase C raises cAMP levels by inactivating a cAMP extrusion mechanism, such as the transport system found in avian erythrocytes and some cul- tured mammalian cells (Brunton and Buss, 1980), thus reducing cAMP efflux, can be eliminated. In addition, the results suggest that protein kinase C ac- tivation increases cAMP production rather than deg- radation. Furthermore, concentration-response stud- ies using IS0 indicate that an increase in @-adreno- ceptor sensitivity is not responsible for the potentiation of the cAMP response. This is consistent with findings from other cell types, which show that although PMA treatment can increase the phosphor- ylation of the @-adrenoceptor (Sibley et al., 1984), it reduced rather than increased the sensitivity of 8- adrenergic stimulation of adenylate cyclase in mouse epidermis (Garte and Belman, 1980) and rat glioma C6 cells (Mal low et al., 1980).

The suggestion that protein kinase C acts at a post- receptor site receives strong support from our studies using CT. CT activates adenylate cyclase by stabiliz- ing the active, GTP-bound form of the (stimulatory) regulatory guanine nucleotide binding protein, Ns (Selinger and Cassel, I98 I ) , thus obviating receptor stimulation. Activation of protein kinase C by phor- bol esters, synthetic diacylglycerols, or a,-adrenergic agonists markedly increased cAMP accumulation in pinealocytes treated with CT. Studies with forskolin also support the conclusion that protein kinase C acts at a postreceptor site. This is because forskolin acti- vates adenylate cyclase directly (Seamon and Daly, 1981) and also acts on Ns (Bouhela et al., 1985), and in our studies, we found that the cAMP response to forskolin was significantly increased by PE and PMA.

The data, taken together, support the hypothesis that protein kinase C activation increases cAMP syn- thesis by acting either on adenylate cyclase itself or on Ns, perhaps to facilitate the interaction of the regula- tory protein with adenylate cyclase. Evidence from other cell types in which protein kinase C activation alters CAMP content also points to a postreceptor site of action (Heyworth et al., 1984; Bell et al., 1985; Mukhopadhyay and Schumacher, 1985). Our at- tempts to demonstrate that treatments that activate protein kinase C enhance the stimulation of adenyl- ate cyclase activity in pinealocyte membranes have so far been unsuccessful (S. Beckner and D. Sugden, unpublished data). In contrast, it has been possible to demonstrate this in other systems using PMA to acti- vate protein kinase C (Bell et al., 1985; Naghshineh et al., 1986; Olianas and Onali, 1986; Yoshimasa et al., 1987).

Although pinealocytes d o not appear to have a re- ceptor-operated mechanism for inhibition of cAMP stirnulation, an - 39,000-dalton protein can be ADP-ribosylated in pineal membrane preparations in

the presence of pertussis toxin (D. Sugden, unpub- lished data). These observations are interesting in view of the recent finding that phosphorylation of the inhibitory regulatory guanine nucleotide binding protein (Ni) by protein kinase C can impair its ability transduce an inhibitory hormonal signal to adenylate cyclase (Jakobs et al., 1985; Katada et al., 1985). If such a mechanism were to operate in pinealocytes, it would require that 6-adrenergic stimulatjon activate not only Ns, but also Ni. Protein kinase C activation would remove the inhibitory influence of 8-adrener- gic stimulation (via Ni) and allow full expression of the stimulatory signal (via Ns). 8-Adrenoceptor-me- diated activation of Ni has been observed previously in a reconstitution system (Asano et al., 1984), and the coupling of the P-adrenoceptor to both Ni and Ns has been observed in adipocytes (Murayama and Ui, 1983). The question of whether this mechanism functions in the pinealocyte could be resolved using pertussis toxin.

An alternative possibility is that the P or y subunit of Ns might be the site of action of protein kinase C. However, the purified /3 subunit of Ni, unlike the free Ni a subunit, has been found not to be a substrate for the enzyme (Katada et al., 1985), and the 8 subunits of the different N proteins appear to be very similar (Bourne et al., 1987). Thus, the suggestion that the 0 subunit of pinealocyte Ns mediates the action of pro- tein kinase C on cAMP seems unlikely. As indicated above, it seems possible, based on recent studies using a smooth muscle cell line (Yoshimasa et al., 1987), that protein kinase C might act directly on adenylate cyclase.

An interesting aspect of the present study concerns the time course of the amplification of the cAMP response in CT-treated cells. Activation of protein kinase C rapidly potentiated the cAMP response, as was seen in earlier studies (Sugden et al., 1985; Vane- cek et al., 1985), but the potentiation declined by 20 min and was absent by 60 min. This transient poten- tiation by protein kinase C activators may indicate that the activated kinase is rapidly inactivated or that it produces a secondary effect resulting in a reduction of pinealocyte cAMP content, e.g., activation of cAMP phosphodiesterase or desensitization of /3- adrenergic receptors. Clearly these mechanisms may be of considerable importance in determining the time course of CAMP production by the physiological stimulus NE.

Acknowledgment: The authors would like to thank Dr. A. Louise Sugden for assistance with the time course ex- periment.

REFERENCES Asano T., Katada T., Gilman A. G., and Ross E. M. (1984) Acti-

vation of the inhibitory GTP-binding protein of adenylate cy- clase Gi by beta adrenergic receptors in reconstituted phospho- lipid vesicles. J. Biol. Chem. 259,935 1-9354.

Bell J. D., Buxton 1. L. O., and Brunton L. L. (1985) Enhancement ofadenylate cyclase activity in S49 lymphoma cells by phorbol

J. Neworhem.. Vd. SO. No. I . 1988

PROTEIN KINASE C AND PINEAL cAMP RESPONSE 155

esters: putative effect of C kinase on as GTP-catalytic subunit interaction. J. Biol. Chem 260,2625-2628.

Bouhela R.. Guillon G., Homburger V., and Bockaert J. (1985) Forskolin-induced change of the size of adenylate cyclase. J. Biol. Chrm. 260, 1090 I - 10904.

Bourne H . R., Masters S. B.. and Sullivan K. A. (1987) Mamma- lian G proteins: structure and function. Biochem. Soc. Trans. 1 5 3 5 - 3 8 .

Bradford M. M. (1976) A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the princi- ple of protein-dye binding. Anal. Biochem. 72, 248-254.

Brunton L. L. and Buss J. E. (1980) Export of cyclic AMP by mammalian reticulocytes. J. Cyclic Nrrcleoride Res. 6, 369-377.

Buda M. and Klein D. C. ( 1978) A suspension culture of pinealo- cytes: regulation of N-acetyltransferase activity. Endocrinol- o,yy 103, 1483-1493.

Cronin M. J. and Canonico P. L. ( 1985) Tumor promoters enhance basal and growth hormone releasing factor stimulated cyclic AMP levels in anterior pituitary cells. Biochem. Biophys. Res. Commun. 129, 404-4 10.

Carte S. and Belman S. J. (1980) Tumor promoter uncouples 8- adrenergic receptor from adenyl cyclase in mouse epidermis. Natirre284, 171-173.

Gill D. M . (1977) Mechanism of action of cholera toxin. Adv. Cyclic Nucleoride Res. 8, 85-1 18.

Harper J. F. and Brooker G. ( I 975) Femtomole sensitive radioim- munoassay for cyclic AMP and cyclic GMP after 2-0-acetyla- tion by acetic anhydride in aqueous solution. J . Cyclic Nu- cleolide Rex 1, 207-2 18.

Heyworth C. M.. Whetton A. D.. Kinsella A. R., and Houslay M. D. (1984) The phorbol ester 12-0-tetradecanoyl phorbol- 13-acetate inhibits glucagon-stimulated adenylate cyclase ac- tivity. FEBS Lett. 170, 38-42.

Heyworth C. M., Wilson S. P., Gawler D. J., and Houslay M. D. ( 1985) The phorbol ester TPA prevents the expression of both glucagon desensitization and the glucagon-mediated block of insulin stimulation of the peripheral plasma-membrane cy- clic-AMP phosphodiesterase in rat hepatocytes. FEBS Letf. 187, 196-199.

Jakobs K. H.. Bauer S., and Watanabe Y. (1985) Modulation of adenylate cyclase of human platelets by phorbol ester impair- ment of the hormone-sensitive inhibitory pathway. Eur. J. Biochem. 151,425-430.

Katada T., Gilman A. G., Watanabe Y., Bauer S.. and Jakobs K. H. (1985) Protein kinase C phosphorylates the inhibitory gua- nine-nucleotide-binding regulatory component and appar- ently suppresses its function in hormonal inhibition of ade- nylate cyclase. Eur. J. Biochem. 151, 43 1-437.

Klein D. C.. Auerbach D. A,, Namboodiri M. A. A.. and Wheler G. H. T. (1981) Indoleamine metabolism in the mammalian pineal gland, in The Pineal Gland: Anatomy and Biochemistry (Reiter R. J., ed), pp. 199-227. CRC Press. Boca Raton, Flor- ida.

Kraft A. S.. Anderson W. B.. Cooper H. L.. and Sando J. J. ( I 982) Decrease in cytosolic calciurn/phospholipiddependent pro- tein kinase activity following phorbol ester treatment of EL4 thymoma cells. J . Biol. Chem. 257, 13193-13 196.

Mallorga P., Tallman J. F., Henneberry R. C., Hirata F., Strittmat- ter W. T., and Axelrod J. (1980) Mepacrine blocks 8-adrener- gic agonist-induced desensitization in astrocytoma cells. Proc. Narl .-lrad. Sci. USA 77, I34 I - 1345.

Mukhopadhyay A. K. and Schumacher M. (1985) Inhibition of human chorionic gonadotropin-stimulated adenylate cyclase in purified mouse Leydig cells by the phorbol ester phorbol- 12-myristate- 13-acetate. FEES Left. 187, 56-60.

Murayama T. and Ui M. (1983) Loss of the inhibitory function of

the guanine nucleotide regulatory component of adenylate cy- clase due to its ADP ribosylation by islet activating protein pertussis toxin in adipocyte membranes. J. Bid . Chem. 258, 33 19-3326.

Nabika T., Nara Y., Yamori Y., Lovenberg W., and Endo J. (1985) Angiotensin I I and phorbol ester enhance isoproterenol- and vasoactive intestinal peptide-induced cyclic AMP accurnula- tion in vascular smooth muscle cells. Biochem Biophys. Res. Commim. 131, 30-36.

Naghshineh S., Noguchi M., Huang K.-P., and Londos C. (1986) Activation of adipocyte adenylate cyclase by protein kinase C. J. Biol. Chem. 261, 14534-14538.

Nishizuka Y. (1984) The role of protein kinase C in cell surface signal transduction and tumor promotion. Nature 308, 693-698.

Olianas M. C. and Onali P. (1986) Phorbol esters increase GTP- dependent adenylate cyclase activity in rat brain striatal membranes. J. Neurochem. 47, 890-897.

Rebois R. V. and Patel J. (1985) Phorbol ester causes desensitiza- tion of gonadotropin-responsive adenylate cyclase in a murine Leydig tumor cell line. J. Biol. Chem. 260, 8026-8031.

Seamon K. and Daly J. W. (1981) Activation of adenylatecyclase by the diterpene fonkolin does not require the guanine-nu- cleotide regulatory protein. J. B i d . Chem. 256, 9799-9801.

Selinger Z. and Cassel D. (1981) Role of guanine nucleotides in hormonal activation of adenylate cyclase. Adv. Cyclic Nuclee tide Res. 14, 15-22.

Sibley D., Nambi P., Peters J. R., and Lefkowitz R. J. (1984) Phorbol diesten promote beta-adrenergic receptor phosphor- ylation and adenylate cyclase desensitization in duck erythro- cytes. Biochem. Biophys. Res. Commun. 121.973-979.

Smith T. L., Eichberg J., and Hauser G. (1979) Postsynaptic local- ization of the alpha receptor-mediated stimulation of phos- phatidylinositol turnover in pineal gland. Life Sci. 24, 2 179-2 184.

Sugden A. L., Sugden D.. and Klein D. C. (1986) Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes. J. Biol. Chem. 261, 11608-1 1612.

Sugden A. L.. Sugden D., and Klein D. C. (1987) a,-Adrenoceptor activation elevates cytosolic calcium in rat pinealocytes by increasing net influx. J. Biol. Chem. 262, 741-745.

Sugden D. and Klein D. C. (1984) Rat pineal a,-adrenocepton: identification and characterization using [ '251]iod0-2[,!3-(4- hydroxyphenyl)-ethylaminomethyl]tetralone. Endocrinology 114,435440.

Sugden D., Vanecek J., Klein D. C., Thomas T. P., and Anderson W. B. (1985) Activation of protein kinase C potentiates i s prenaline-induced cyclic AMP accumulation in rat pinealo- cytes. Nafure 314, 359-360.

Vanecek J.. Sugden D.. Weller J. L., and Klein D. C. (1985) Atypi- cal synergistic nl- and 8-adrenergic regulation of adenosine Y.5'-monophosphate and guanosine Y.5'-monophosphate in rat pinealocytes. Endocrinology 116, 2 167-2 173.

Vanecek J.. Sugden D., Weller J. L., and Klein D. C. (1986) See- saw signal processing in pinealocytes involves reciprocal changes in the a,-adrenergic component of the cyclic GMP response and the 8-adrenergic component of the cyclic AMP response. J. Neirrochem. 47, 678-686.

Yoshimasa T.. Sibley D. R., Bouvier M.. Lefkowitz R. J.. and Caron M. G. (1987) Cross-talk between cellular signaling pathways suggested by phorbol-ester-induced adenylate- cyclase phosphorylation. Noritre 327, 67-70.

Zatr M. ( 198s) Denervation supersensitivity of the rat pineal l o norepinephrine-stimulated ['H]inositide turnover revealed by lithium and a convenient procedure. J . Neurochem 45, 95- 100.