PflfigersArch (1992) 421:82- 89
Joumal of Physiology 9 Springer-Verlag1992
Regulation of the cAMP signal transduction pathway by protein kinase C in rat submandibular cells Norman Fleming, Lynne Mellow, and Devinder Bhullar Department of Oral Biology,Universityof Manitoba, 780 BannatyneAvenue, Winnipeg, Manitoba, Canada R3E OW3 Received October 4, 1991/Receivedafter revision January 8, 1992/AcceptedJanuary 20, 1992
Abstract. Treatment of rat submandibular acinar cell extracts with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) caused the dose-dependent activation of protein kinase C (PKC), assessed by the phosphorylation of a novel and highly specific substrate. This effect was duplicated by a diacylglycerol, but not by the 4c~phorbol ester 4c~-phorbol 12,13-didecanoate. The TPA elevation of PKC was blocked by the PKC inhibitors H-7 and sangivamycin. In intact cells, TPA caused the translocation of PKC from cytosol to membrane, consistent with its known mode of activation. The/~-adrenergic agonist, isoproterenol, stimulated cAMP levels which were significantly reduced by preactivation of PKC. This inhibitory PKC effect was reversed by H-7. When cAMP was stimulated at the post-receptor level, however, by forskolin, NaF or GTP[TS], PKC did not inhibit, but rather enhanced the cyclic nucleotide response. Since PKC phosphorylated an endogenous protein of 55 kDa, the size of the/~ receptor, these findings indicate that, as in other cell types, PKC can desensitize adenylate cyclase by direct phosphorylation of the /~ receptor, but potentiate the cAMP response by a post-receptor mechanism. In mucin secretion studies in the model, TPA alone caused the cAMP-independent release of up to 44% total mucin, which was much less than additive with the isoproterenol response. When the cAMP-mucosecretory response was stimulated at the adenylate cyclase level by forskolin, however, the TPA + forskolin effects were additive. These findings on the modulation of cAMP by PKC indicate cross-talk regulation in the phosphoinositide-cAMP signal transduction pathways in submandibular acinar cells. Key words: Submandibular glands - Phorbol esters Protein kinase C - cAMP - Phosphoinositide - Signal transduction - /?-Adrenoceptors - Mucin secretion
Offprint requests to: N. Fleming
Introduction Rat submandibular glands are rich in mucus-secreting acinar cells. Maximal mucosecretory responses in the cells are elicited by/~-adrenergic stimulation [3, 9, 24], which activates the adenylate cyclase/cAMP signal transduction pathway. It was first shown by Bogart  that muscariniccholinergic agonists also elicited a significant mucosecretory response in the submandibular model. This was later confirmed in our laboratory, when it was found that carbachol (muscarinic) and agonists of two other classes, methoxamine (el-adrenergic) and substance P (tachykinin) could evoke the release of acinar cell mucin . It was further demonstrated that all three agonists shared a common mechanism of action by causing the receptorlinked activation of phospholipase C (PLC) to stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsPz) [91. Activation of the phosphoinositide signal transduction pathway leads to the generation of two separate second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol . Diacylglycerol, in turn, activates protein kinase C (PKC), a ubiquitous enzyme that regulates many physiological processes . We have found that probes for PKC activation, phorbol esters and exogenous diacylglycerols, are capable of stimulating the secretion of up to 30% total mucin from rat submandibular cells . These earlier studies confirmed that both the cAMP and phosphoinositide transduction pathways are involved in controlling mucin secretion in the submandibular gland. Much attention has recently been focused on crosstalk regulation between the pathways, particularly on the modulating effects of PKC on the cAMP response (see review by Houslay ). Depending on the cell type investigated and on the specific nature of the cAMP-elevating stimulus, PKC has been shown both to augment and to inhibit nucleotide levels . The present study was therefore undertaken to investigate whether PKC, activated by a phorbol ester, might regulate the cAMP response in submandibular acinar cells.
Some doubt has been expressed as to whether all the effects of phorbol esters are mediated by PKC . More specifically, Quissell et al.  were unable to find any evidence that phorbol esters either regulated mucin secretion or activated PKC in the rat submandibular cell model. The initial part of the present study was therefore designed to determine whether phorbol esters do activate PKC in submandibular cells. Our results confirm that a phorbol ester can activate PKC, as assessed by biochemical assay, cause the translocation of PKC from cytosol to membrane and phosphorylate an endogenous substrate that may be the/~adrenergic receptor. Furthermore, activation of PKC results in a modulation of the cAMP-mucosecretory response. Materials and methods Materials. Purified collagenase, CLSPA grade, was obtained from Worthington, Freehold, N.J., USA. Hanks' balanced salt solution, minimal essential medium amino acids and HEPES buffer were supplied by Gibco Canada Inc., Burlington, Ont., Canada. Acrylamide, bisacrylamide, TRIS, sodium dodecyl sulphate (SDS), glycine, ammonium persulphate, tetramethylethylenediamine, bromophenoI blue, 2-mercaptoethanol, dithiothreitol, molecular mass standards and Coomassie blue were from Bio-Rad Labs (Canada) Ltd., Mississauga, Ont., Canada. Forskolin and H-7 were obtained from Calbiochem, San Diego, Calif., USA. Sangivamycin was a gift from the N.C.I.C.A. Repository, Rockville, Md., USA. Cyclic AMP radioimmunoassay kits were a product of Dupont, Medical Products Dept., Wilmington, Del., USA. The protein kinase C assay kit, adenosine 5'-[7-32p]triphosphate and [32p]orthophosphate were supplied by the Amersham Corp., Arlington Heights, Ill., USA. All other chemicals were obtained from Sigma, St. Louis, Mo., USA.
Preparation of cells. Submandibular acinar cells from the pooled glands of two to four male Sprague Dawley rats were prepared by dissociation with collagenase in modified Hank's balanced salt solution as described previously . The cells were buffered to pH 7.3 by 15 m M HEPES and maintained at 37~ in a water bath shaker in culture medium saturated with 90% 02/]0% C02. Dispersed cells were washed in fresh culture medium and normally resuspended at a concentration of 10 mg wet weight/l-ml aliquot for experimental treatment. In preliminary experiments with the PKC assay, fresh, homogenized submandibular tissue was used. Protein Icinase C assay. PKC activity was measured by using a PKC assay kit (Amersham) with a high-affinity peptide substrate (eight amino acids, Mr = 1098). The PKC activator was 3 p-M 12-O-tetradecanoylphorbol 13-acetate (TPA) and assay buffer also contained 8mol/100mol I~-c~-phosphatidyl-I~-serine and 1 2 m M Ca 2+ in 50 mM TRIS/HC1. In the assay 25-p-1 samples of diluted cell extract (see below) were mixed with 25 p-1 activator buffer and 25 gl [3ZP]ATP (0.25 p-Ci 32p) at 25~ After 15 rain the reaction was halted with 100 p-1 acidic stop reagent. [3zp]Phosphorylated substrate in 125-gl aliquots was localized on small numbered squares of binding paper, which were then washed twice for i0 min in an excess of 5% acetic acid. The papers were placed in vials with 10 ml scintillation fluid and 32p activity was measured in a beta scintillation counter. Non-specific background phosphorylation was assessed in cell extracts exposed to the PKC substrate but not to the activator. In one series of experiments fresh submandibular glands were homogenized (1 : 10, w:v) in ice-cold sample buffer of 50 mM TRIS/ HC1, 5 mM EDTA, 10 mM EGTA, 0.3% w/v 2-mercaptoethanol, 2 gg/ml leupeptin and 50 gg/ml phenylmethylsulphonyl fluoride
(PMSF). The homogenate was centrifuged at 15 000 g and the clear supernatant fluid diluted to a concentration of 32 gg protein/25 lal. Doubling dilutions were then prepared from 3 2 - 1 gg protein/25 p-1 and these were assayed for PKC activity as outlined above. The same dilution range of samples was also exposed to the 4e-phorbol ester, 4c~-phorbol 12,13-didecanoate (PDD, 3 gM) and assayed for PKC. In a second series of experiments, gland tissue supernatant (8 lag protein) was incubated with a range of concentrations (10 n M 0.1 mM) of the PKC inhibitors H-7 [1-(5-isoquinolinesulphonyl)2-methylpiperazine dihydrochloride] or sangivamycin, for i0 rain before being assayed for PKC activity. In further studies, the effect of a range of concentrations of TPA on PKC activation in soluble submandubular gland extract was examined. The TPA in the assay kit (3 p.M) was replaced by 10 nM 10 p-M phorbol ester and assays were carried out as before. In addition, the capacity of the exogenous diacylglycerol, 1,2oleoylacetylglycerol (OAG), to activate PKC was examined.
PKC translocation experiments. The correlation between PKC activation and its translocation from the cytosol to membrane in intact cells was investigated. Collagenase-dispersed cells were treated with TPA (1 n M - 0 . 1 mM) for 15 rain. The cells were then centrifuged at 200 g for 30 s and the culture medium supernatant discarded. The packed cells were sonicated in 1.5 ml cold buffer for 7 s at 10% power (Microson Ultrasonic cell disruptor, XL2005, 0.3cm microprobe; Heat Systems Incorporated, Farmingdale, New York) then centrifuged at 15000 g for 10 min at 4 ~C. The clear supernatant was assayed for PKC as the cytosolic fraction. The remaining pellets were sonicated in 1.5 ml cold buffer for 20 s at 20% power and the resulting suspension assayed for PKC. The sonication conditions in these studies were crucial in obtaining maximal estimations of cytosolic and membrane-bound PKC without frothing, and were derived experimentally. PKC endogenous substrate phosphorylation studies. Collagenase-dispersed cells from four submandibular glands (approximately 120 mg protein) were incubated for I h at 37~ in 6 ml culture medium containing 180 p-Ci/ml [3aP]orthophosphate. The cells were washed in culture medium then resuspended in fresh unlabelled medium at a concentration of 10 mg protein/l-ml aliquot. The preparations were treated with 1 laM TPA or left untreated as controls, and aliquots were collected at times 0, 15, 30 and 45 rain. The cells were centrifuged at 200 g for 30 s, the supernatant discarded and the pellet washed in 4 ml phosphate-buffered saline. The samples were again centrifuged and the cells suspended in 1 ml stop buffer of 50 mM TRIS/HC1, I % w/v SDS, 30 mg/ml dithiothreitol and 10% v/v glycerol, pH 6.8. Samples were heated to 100~ for 20 rain then centrifuged at 15 000 g for 10 min. Supernatant aliquots containing 150 p-g protein were applied to each lane of a 1 0 % - 2 0 % lineargradient polyacrylamide gel with 5% stacking gel and electrophoresis was carried out at 2.5 W constant power overnight . The following standards were also run as molecular mass markers: phosphorylase b, 97.4kDa; bovine serum albumin, 66.2kDa; ovalbumin, 45.0kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa. The running buffer was standard TRIS/glycine/SDS pH 8.3 . The gels were fixed in 45% methanol/10% acetic acid, stained in 0.1% Coomassie blue in the same solvent, destained in a Bio-Rad model 556 destainer with charcoal filter and dried by fan between sheets of cellophane in a gel drying frame (Research Diagnostics, Flanders, N.J., USA). Autoradiography was carried out on the dried gels on Kodak X-OMAT A R film for 4 - 6 days at room temperature. Bands representing azP-labelled proteins on the developed autoradiographs were subjected to scanning densitometry on a Bio-Rad model 620 video densitometer.
Cyclic" AMP studies. Studies were carried out to determine whether PKC might regulate cAMP levels in submandibular cells. In the first series of experiments, the effect of TPA on the/~-adrenergic-induced
84 elevation of cAMP was examined. Aliquots (1 ml) of dispersed cells were subjected to the following treatments: 3 pM TPA for 20 min. 5 pM isoproterenol (IPR) for 10 min, TPA for 10 min followed by IPR for 10 min, and 0.1 mM H-7 for 5 min followed by TPA for 10 rain followed by IPR for 10 rain. Control cells received no treatment. Reactions were terminated by the addition of 250 ~t130% cold trichloroacetic acid and samples centrifuged at J 5 000 g to remove the precipitate. The supernatant was extracted three times with water-saturated ether; the aqueous phase was evaporated to dryness and then resolubilized in 5 ml 0.05 M sodium acetate buffer pH 6.2. Samples were further diluted x 10 in the same buffer and cAMP was measured by radioimmunoassay (Du Pont RIANEN cAMP 125I radioimmunoassay kit) [19, 20]. In a second series of experiments the potential effect of TPA in regulating cAMP levels that were stimulated at the post-receptor level were examined. As before, cells were preincubated for 10 min with 3 gM TPA or left as controls for 10 rain, then the cAMP pathway was stimulated at the level of the G~ regulatory protein (1 mM NaF or 0.1 mM GTP[yS]) or at the adenylate cyclase level (50 gM forskolin). Cells treated with NaF or GTP[TS] were first permeabilized by electroporation in the Bio-Rad Gene Pulser as described previously . Mucin secretion studies. The effect of TPA on the #-adrenergicstimulated secretion of mucin was examined. Aliquots of I ml cell suspension were exposed to 3 pM TPA for 50 rain, to 5 gM IPR for 40 min or to TPA for 10 rain followed by IPR for 40 rain. Controls were left untreated and sampled at times 0 and 50 min to establish background mucin levels. Samples were centrifuged at 200 g for 30 s and the pellet washed with I ml fresh culture medium, which was added to the initial supernatant. The supernatant and washings were diluted x 6 and assayed for mucin by the Alcian blue method described by Hall et al. . Standards were a series of dilutions of purified bovine submandibular gland mucin (Sigma), which was found in preliminary experiments to produce dilution response curves exactly parallel to those of rat submandibular gland extract. To allow ample headroom for an additive TPA + IPR response the experiments were repeated with the suboptimal doses of 0.1 IxM TPA and 10 nM IPR. In other experiments the effect of TPA on mucin secretion stimulated at the adenylate cyclase level by forskolin was investigated. Cells were exposed to 0.1 ~tM TPA for 50 min, 0.3 pM forskolin for 40 rain, or TPA for 10 min followed by forskolin for 40 min. Experimental results were examined statistically by two-way analysis of variance and means were compared by Duncan's multiple range test or by the Student t-test. Values of P < 0.05 were considered significant.
Log 2 [cell
Fig. 1. Activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol 13-acetate (TPA) in rat submandibular gland extracts. The abscissa represents a doubling dilution series of gland supematant containing 1 - 32 ~tg cell mucin/25-~l sample. PKC was assayed by its capacity to phosphorylate a synthetic peptide substrate (see Materials and methods). TPA results are means i SEM, n = 3. The 4~-phorbol ester 4~-phorbol 12,13-didecanoate (PDD) did not activate PKC (average values of two experiments). Nonspecific background phosphorylation levels ( - TPA) have been subtracted. 0 , 3 gM TPA; 9 3 gM PDD
r.) M -0.5
P K C assay
Fig. 2. Inhibitory effect of a range of concentrations of H-7 ( 9 and sangivamycin ( 9 on TPA-induced activation of PKC in submandibular gland supernatants. Preparations containing 8 pg cell protein were preincubated with the inhibitors for 10 min before PKC activation with 3 gM TPA. The negative values at 10 ~tM and 100 gM syngivamycin reflect an inhibition to below control PKC levels. Values are the means of two experiments. Non-specific background phosphorylation has been subtracted. In the absence of the inhibitors, the average TPA-induced level of phosphorylation was 2220 dpm 3zp
W h e n a r a n g e o f c o n c e n t r a t i o n s o f soluble s u b m a n d i b u lar g l a n d e x t r a c t ( 1 - 3 2 ~tg p r o t e i n / 2 5 - g l sample) was a s s a y e d for P K C activity after T P A a c t i v a t i o n , a concent r a t i o n / r e s p o n s e curve was o b t a i n e d (Fig. 1). M a x i m a l activity was d e t e c t e d in cell s u p e r n a t a n t s c o n t a i n i n g 16 gg p r o t e i n / s a m p l e , in w h i c h the 32p r e c o r d e d ( d p m ) repr e s e n t e d an a v e r a g e o f 357 p m o l labelled p h o s p h a t e t r a n s f e r r e d m i n - 1 m g p r o t e i n - 1, c o m p a r e d with 22 p m o l in n o n - T P A - s t i m u l a t e d c o n t r o l s , a m o r e t h a n 16-fold increase (n = 3). T h e 4 e - p h o r b o l ester, P D D , however, at the s a m e c o n c e n t r a t i o n as T P A in the a s s a y system (3 ~tM), d i d n o t a c t i v a t e P K C in a n y o f the e x t r a c t c o n c e n t r a t i o n s tested. P D D at h i g h e r a n d l o w e r doses was also ineffective in e n z y m e a c t i v a t i o n (see below).
B o t h P K C i n h i b i t o r s , H-7 a n d s a n g i v a m y c i n , r e d u c e d T P A - s t i m u l a t e d s u b s t r a t e p h o s p h o r y l a t i o n in a c o n c e n t r a t i o n - d e p e n d e n t w a y (Fig. 2). S a n g i v a m y c i n was the m o r e p o t e n t o f the two w i t h a n ICso o f a p p r o x i m a t e l y 70 n M c o m p a r e d with a figure o f a b o u t 2 ~tM for H-7. T h e 32p r a d i o a c t i v i t y s h o w n in Fig. 2 h a s the c o n t r o l ( n o n - T P A - s t i m u l a t e d ) values a l r e a d y s u b t r a c t e d . T h e negative results p r o v i d e d b y 10 p M a n d 100 g M s a n g i v a mycin therefore probably represent an inhibition of backg r o u n d p r o t e i n p h o s p h o r y l a t i o n levels to b e l o w c o n t r o l values. I n a n o t h e r series o f e x p e r i m e n t s , the T P A in the P K C a s s a y k i t was s u b s t i t u t e d with a r a n g e (1 n M -- 10 p M ) o f
ITPA] (M) -7 -6
Fig.& Effect of a range of concentrations of TPA (O) and the diacylglycerol oleoylacetylglycerol (9 (OAG) on the activation of PKC in rat submandibular acinar cell extracts containing 32 Ilg cell protein. Means __ SEM, n = 3
r,.) 0 . 0 -g
Fig. 5. Phosphorylation of an endogenous protein substrate of 55 kDa by TPA in rat submandibular acinar cells. Cells were prelabelled with 32p for 1 h, then treated with 1 pM TPA for up to 45 rain. Proteins were extracted and subjected to polyacrylamide gel electrophoresis followed by autoradiography. Protein bands were quantified by densitometry. The photograph shows the phosphorylation of a 55-kDa protein in TPA-treated cells at 45 min and the densitometer tracings on the left indicate that the protein is phosphorylated to 171% control level. By reference to published work and to the TPA inhibition of fl-adrenoceptor-induced cAMP observed in the present study, we suggest that the 55-kDa protein may be the fll-adrenergic receptor
Fig. 4. Effect of TPA concentration in causing the translocation of PKC from cytosol to membranes in rat submandibular acinar cells. Intact cells were exposed to TPA for 15 rain then sonicated and centrifuged. PKC was assayed in diluted 25-pl samples of both the cytosolic and membrane fractions by the addition of assay kit reagents, including the TPA activator at 3 gM. Values are the means of two experiments. 0, Cytosol; 9 membranes; m, total
experiments). Again, the maximal effect was reached around 1 gM concentration. The total P K C measured in cytosol plus m e m b r a n e did not change with T P A treatment (Fig. 4), indicating the phorbol ester effects were not related to enzyme synthesis or degradation.
T P A concentrations so that the dose/response of phorbol ester activation of enzyme could be investigated. All concentrations of T P A tested activated P K C , the maximal response being observed at a dose between i gM and 10 gM. This was consistent with the concentration of 3 IxM T P A used in the A m e r s h a m assay kit. Over the same concentration range, the 4 e-phorbol ester, P D D , did not activate P K C (results not shown). In the same series of experiments, kit T P A was substituted with a concentration range of the diacylglycerol, OAG. The diglyceride also stimulated P K C activity in a dose-dependent way, but was only a b o u t half as effective as T P A in promoting substrate phosphorylation at the optimal O A G dose of 500 gg/ml (n = 3, Fig. 3).
PKC endogenous substrate phosphorylation
PKC translocation studies T P A caused the dose-dependent translocation of P K C from the cytosol to the m e m b r a n e fraction of cultured submandibular acinar cells (Fig. 4, mean values of two
T P A caused the phosphorylation of a protein of approximately 55 k D a molecular mass. Increased phosphorylation of the protein was already evident by 15 rain and reached a m a x i m u m at 45 rain of the time course (Fig. 5). Scanning densitometry indicated that the 55-kDa peak was 171% of the control value at 45 rain. Other minor variations between the densitometer scans were not considered significant, since they were much smaller than this value and since bands were not obviously different on visual inspection. The 55-kDa band was sometimes difficult to detect, as high levels of control cell protein phosphorylation tended to obscure the background on autoradiographs. In some experiments, cells were preincubated for 10 min with 0.1 g M okadaic acid ( M o a n a Bioproducts, Honolulu, Hawaii, USA), a potent inhibitor of types I and 2A phosphatase, in an attempt to amplify phosphorylation of the 55-kDa band. The effects of okadaic acid were, however, non-specific, and resulted in an overall enhancement of background phosphorylation.
86 120 IPR 5 x 10-SM
r~ 1 0 0
TPA 3 x l O - 6 M
700 600 o
5 x i 0 5M
1 x lO-SM
1 x lO-4M
3 X 10-SM
forskolin produced an enhanced response of 644 +_ 38% control level (P < 0.01). Again, TPA alone had no effect. Cyclic A M P was also increased in electropermeabilized cells by stimulation at the level of the Gs regulatory protein with 1 m M N a F (164_+ 10% control, n = 3, P < 0.01), or0.1 m M GTP[TS ] (133 _ 8% control, n = 3, P < 0.05, Fig. 6b). These reduced responses, compared with those with IPR and forskolin, probably reflect some membrane damage caused by the electroporation process. The N a F response was significantly amplified to 268 • 13% by TPA pretreatment ( P < 0.01, Fig. 6b), while TPA produced a marginal increase in the GTP[TS] response from 133% to 150 • 2% control level, which was not statistically significant.
Mucin secretion studies
Fig. 6a, b. Modulation of the cAMP response by TPA in submandibular acinar cells, a A 10-rain exposure of cells to isoproterenol (IPR) produced a 4.5-fold increase in cAMP over control level. Preincubation with TPA reduced this response to 37% of its normal value. The reduction was significantly (P < 0.05) reversed to 60% normal value by the inhibitor H-7. H-7 alone had no effect on the IPR response (not shown), means + SEM, n = 4. b A 10-min exposure of intact cells to forskolin, or of electroporated cells to NaF or GTP[TS] stimulated significant increases in cAMP. For comparative purposes, all responses are calculated relative to the control level of 100%. TPA caused an enhancement of the forskolin and NaF responses (P < 0.01). The small increase observed with GTP[?S] + TPA was not statistically significant. Means _+ SEM, n = 3. TPA alone had no effect on cAMP levels
c A MP studies cAMP was assayed in control cells at a level of 70 _+ 6 (mean _+ SEM, n = 4) pmol cAMP/mg protein. The fladrenergic agonist, IPR (5 jaM), stimulated the nucleotide to approximately 4.5-fold this level, 321 + 31 pmol, in 10 min. For comparative purposes, the modulating effects of TPA on the cAMP response in different experiments were normalized to the IPR response of 100%. A 10-min preincubation with 3 jam TPA inhibited the subsequent IPR-stimulated cAMP response to 37 4- 6% of its normal value (Fig. 6a). This effect was significantly reversed to 60 _+ 8% (P < 0.05) by 5 min pretreatment with H-7 (Fig. 6a) indicating that TPA inhibition of the IPR-stimulated c A M P response was mediated by PKC. TPA alone had no effect on the resting cAMP levels of control cells. Pretreatment of cells with H-7 alone had no effect on the IPR-induced elevation of cAMP (results not shown). Forskolin (50 jaM), an agent that directly stimulates adenylate cyclase, elevated cAMP levels to 386 _+ 31% of control values (n = 3). In contrast to the IPR results, however, treatment of cells with TPA followed by
Non-stimulated control cells secreted an average of 16 gg mucin/mg cell protein over the incubation period. A concentration of 5 jaM IPR stimulated the release of 92 _ 10 gg mucin/mg cell protein, or 575 + 62% control value (n = 3, Fig. 7a). TPA (3 jaM) alone caused a mucosecretory response of 475_+ 41% of the control level. When cells were treated with both TPA and IPR, however, a response of only 650__ 50% c o n t r o l was evoked, far short of the calculated theoretical additive response of 1050 %, which would be expected if the TPA and IPR effects on secretion were independent of each other (Fig. 7a). To allow for the possibility that the maximal mucin response had already been reached with these combined TPA + IPR doses, the experiments were repeated with sub-optimal combinations of the agonists, so that ample headroom would exist for additive or supraadditive responses (Fig. 7b). The same trend was observed, however, with the following responses recorded: 0.1 jaM TPA, 180_+12% control; 1 0 n M IPR, 190_+20%; TPA + IPR, 214 __ 10% (n = 3). Here the theoretical additive response for independent stimuli would be 370%. In other experiments, a sub-optimal concentration of 0.3 jaM forskolin stimulated the cAMP pathway at the post-receptor level of adenylate cyclase to produce a release of 155 + 18% control level mucin (Fig. 7c). When cells were sequentially treated with TPA and forskolin, however, the level of mucin secretion observed was exactly an additive response (Fig. 7c).
Discussion Stimulation of rat submandibular acinar cells by agonists of several different classes causes the release of mucin [3, 8, 9, 24]. Two receptor-linked signal transduction pathways regulate the mucosecretory response./%Adrenergic stimulation activates the adenylate cyclase/cAMP pathway [10, 25] and causes the secretion of over 60% cell mucin . Muscarinic-, ~-adrenergic- and tachykinin agonists stimulate the phosphoinositide transduction pathway and provoke the release of 3 0 % - 4 5 % mucin [9, 19, 20]. The diacylglycerol product of PtdInsPz hy-
TPA 3 x 10-SM I P R 5 x 10 tim
700 500 300 100
TPA i x lO-VM IPR I x lO-SY
TPA I x IO-VM
Forskolin 3 x IO-VM
IO0 Fig. 7 a - e . Effect of TPA on the mucin secretion response in submandibular acinar cells, a Cells treated with an optimal concentration of TPA for 40 min secreted mucin to 475% control levels. The comparable figure for IPR-treated cells was 575%. The 650% response produced by TPA + IPR, however, was not significantly different from that of IPR alone, and far short of a theoretical additive response of 1050%. Means _ SEM, n = 3. b The experiments discussed in Fig. 6a were repeated with sub-optimal doses of TPA and IPR to ensure that the sub-additive secretion measured with TPA + IPR did not simply reflect the maximal possible mucosecretory response, with no headroom for further enhancement. The observed trend with lower doses, however, was exactly the same; i.e. TPA and IPR alone both stimulated reduced secretory responses. Their combined effect was not significantly different from that of IPR alone, and short of a theoretical additive response. Means i SEM, n = 3. e Cells stimulated with a sub-optimal concentration of forskolin at the adenylate cyclase level produced a mucosecretory response. When cells were sequentially treated with TPA then forskolin, an additive secretion of mucin was provoked, in contrast to the non-additive responses observed with TPA + IPR in Fig. 6a and b. Means • SEM, n = 3
drolysis activates protein kinase C, an enzyme that can regulate many cell processes [2, 22]. Phorbol esters are universally used to bypass the signal transduction sequence and activate PKC directly . Thus, we showed in an earlier study that the phorbol ester TPA, as well as exogenous diacylglycerols, stimulated mucin secretion in the submandibular model, and proposed that this re-
flected the activity of a PtdInsP2 - diacylglycerol PKC regulatory pathway . However, in a subsequent study on the same experimental model, Quissell et al.  found no evidence for a direct role for PKC in salivary mucin secretion, were unable to detect TPA-induced phosphorylation of endogenous substrate and speculated that the observed effects of TPA and diacylglycerols in our study  may have been non-specific and not mediated by PKC. The findings of these authors prompted us to re-examine the question of phorbol ester activation of PKC in submandibular acinar cells and, further, to investigate in the model, the potential PKC cross-talk regulation of the cAMP pathway that has been described in several other cell types . Several lines of evidence in the present study confirm PKC in submandibular cells can be activated by phorbol esters. Using a sensitive and highly specific biochemical assay for PKC, we have shown that TPA stimulates enzyme activity in a range of concentrations of cell extract. The 4e-phorbol ester PDD, which is known not to activate PKC, had no effect, indicating the specificity of the 4/~-TPA response. The TPA stimulation of PKC was dose-dependent on a range of 1 r i M - 1 gM phorbol ester, and was inhibited by the two PKC inhibitors H-7 and sangivamycin. Furthermore, TPA caused the translocation of PKC from cytosol to membranes in submandibular cells, consistent with its known mode of activation . Finally, the diacylglycerol, OAG, was also capable of activating PKC in a concentration-dependent response. The OAG effect was less potent than that of TPA. This may reflect a greater stability of TPA-enzyme complexes, compared with OAG-enzyme complexes , or TPA may have a greater selectivity for PKC isoforms found in salivary gland cells. In endogenous substrate studies, TPA caused the phosphorylation of a 55-kDa protein. The band was difficult to detect on autoradiographs and not evident in all preparations. This may reflect the high susceptibility of PKC to proteolysis, a problem also encountered in the PKC biochemical assay studies discussed above, where inclusion of the inhibitors PMSF and leupeptin in buffers was necessary to maintain enzyme activity. In secretion experiments, TPA at a concentration of 3 gM stimulated the release of mucin to 4.75-fold the control response, representing about 45% total cell mucin. TPA alone had no effect on cAMP levels. Taken together, these results present convincing evidence that TPA stimulates PKC in the submandibular model and that the enzyme plays a direct role, which is not cAMP-mediated, in the secretion of mucin. Our findings therefore support and extend our earlier work on the effects of TPA  and are not in agreement with the later observations of Quissell et al. . The reasons for these discrepant results are not apparent but may relate to the method of quantifying released mucin. In studies where significant secretory responses to muscarinic agents or phorbol esters have been measured, mucin release was assessed by mass measurement of the total secreted product, i.e. sialic acid assay , radioimmunoassay [8, 9] or Alcian blue assay (present study). In contrast, the [14C]glucosamine technique used by
88 Quissell et al.  labels only a small fraction of the total mucin pool, which is confined to newly synthesized glycoprotein in granules lying deep within the cell, close to the Golgi apparatus. It may be, then, that sub-optimal secretory stimuli, such as carbachol or TPA, cause the preferential release ofpresynthesized, non-labelled mucin located in more apical regions of the acinar cells, which cannot be quantified by scintillation counting. In studies on the potential regulation of the cAMP pathway by PKC, we found that TPA inhibited the IPRinduced elevation of nucleotide to less than 40% of its normal value. This is consistent with the PKC effect on cAMP in prostaglandin-stimulated B lymphocytes , and in fl-adrenergic-, prostaglandin- or histamine-stimulated pig epidermal cells . Also consistent with these reports and others were our findings that the TPA inhibitory response was evident only when the cAMP pathway was stimulated at the receptor level, and that a TPAinduced enhancement of cAMP occurred when cAMP was activated at the post-receptor level by forskolin, N a F or GTP[TS] [4, 14, 29]. Several studies have reported that the fi-adrenoceptor can be phosphorylated by PKC [4, 7, 13, 15, 21]. The phosphorylation has been correlated with an adenylate cyclase desensitization in duck erythrocytes  and in Chinese hamster fibroblasts transfected with human adrenoceptors , caused by impaired receptorGs coupling . Johnson et al.  have shown specifically that the P K C phosphorylation site on the hamster fl receptor is serine-261 on the third intracellular loop. It has been reported by Bylund et al.  that the fl receptor on rat submandibular cells is of the fil subtype. The mass of the fil receptor is 55 k D a [6, 28]. In the present study we demonstrated that TPA causes phosphorylation of a 55-kDa protein. When this is considered, together with our findings on TPA modulation of cAMP, in the light of current knowledge of receptor phosphorylation, we believe it highly probable that the 55-kDa protein substrate is the fll receptor on salivary acinar cells and that its PKC-induced phosphorylation uncouples the receptor from its signal transduction pathway to desensitize adenylate cyclase and lower cAMP. The opposite effect, i.e. a T P A enhancement of cAMP, was found if the pathway was activated at the Gs protein or adenylate cyclase level. This may reflect a second locus of interaction of P K C with the cAMP pathway. Many reports have shown that P K C can phosphorylate and inactivate Gi regulatory protein and so remove its inhibitory tonic effect on adenylate cyclase, leading to an elevation of cAMP levels [11, 17]. While we have no p r o o f that this mechanism operates in the submandibular model, it is the most likely explanation for our results. This dual effect of P K C in inhibiting receptor-elevated cAMP, but enhancing post-receptor-stimulated cAMP in the same cell, has also been reported in murine thymocytes  and in astrocytoma cells . The mucin-release studies with secretagogue combinations are rather more difficult to interpret, since T P A alone stimulated a significant secretory response. The TPA + IPR response was marginally above the level of secreted mucin provoked by IPR alone, but not significantly different. The combined T P A + IPR response was
far short of a theoretical additive secretion level. Based on the TPA inhibitory effect in the comparable cAMP studies, our interpretation is that in cells treated with TPA then IPR, a normal PKC-induced secretory response, but reduced IPR/cAMP-stimulated mucin release, occured. In cells treated with TPA then forskolin, however, an additive secretory response was demonstrated. This is again consistent with TPA inhibition of the IPR/cAMPstimulated mucin response occurring only when the pathway was stimulated at the receptor level. A supra-additive mucin response with TPA + forskolin, which might be predicted on the basis of the enhanced cyclic A M P levels stimulated by the two agents, was not observed. This may indicate that the TPA elevation of forskolin-stimulated cAMP measured at 20 min was not maintained throughout the mucin secretion period of 50 min. The results of this investigation are summarized in the following proposal. Phorbol esters activate protein kinase C in rat submandibular cells. P K C stimulates mucin secretion directly by a cAMP-independent action and also cross-reacts with the cAMP transduction pathway to regulate adenylate cyclase by a dual mechanism. Phosphorylation of the fi-adrenoreceptor causes adenylate cyclase desensitization while P K C action at a post-receptor locus, which may be the Gi protein, results in adenylate cyclase sensitization.
Acknowledgement. Supported by the Canadian Cystic Fibrosis Foundation.
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