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Cellular distribution of isoforms of protein kinase C (PKC) in pancreatic acini
ELSEVIER
B i o c h i ~mic~a e t B i o p h y s i c a A~ta Biochimica et Biophysica Acta 1269 (1995) 307-315
Cellular distribution of isoforms of protein kinase C (PKC) in pancreatic acini Bahar Bastani a, Liying Yang a, Joseph J. Baldassare a, Dale A. Polio b, Jerry D. Gardner b., a Di,::isionof Nephrology, Saint Louis UniversityHealth Sciences Center, St. Louis, MO 63104, USA b Departmentof Internal Medicine, Saint Louis University Health Sciences Center, 1402 South Grand Blvd., St. Louis, MO 63104, USA Received 12 June 1995; accepted 4 July 1995
Abstract As in a previous study (Biochim. Biophys. Acta 1224 (1994) 127-138), we used quantitative immunoblot analysis and found that rat pancreatic acini possess four different isoforms of PKC-alpha, delta, epsilon and zeta. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) caused translocation of each isoform from the cytosol to the membrane fraction. CCK-8 increased diacylglycerol (DAG) and caused translocation of PKC-8 and PKC-e but not that of PKC-a or PKC-sr. L-364,718, a CCK receptor antagonist, prevented as well as reversed the effects of CCK-8 on DAG and on translocation of PKC-8 and PKC-e. To explore the possibility that different isoforms of PKC might have. different distributions in rat pancreas, we used immunocytochemistry to determine the cellular distribution of different isoforms of PKC in intact pancreas as well as pancreatic acini. In intact pancreas, PKC-a and PKC-8 were detected in islet cells but not in duct or acinar cells. PKC-e was detected in the apical region of acinar cells and PKC-~" was detected over the luminal surfaces of acinar cells and the ductules that extend from the acinus. Neither PKC-e nor PKC-~" was detected in islets. In pancreatic acini PKC-a and PKC-8 were detected in islets or fragments of islets that contaminated the preparation but were not detected in acinar cells. PKC-e was detected in the apical region of acinar cells and adding 1 /,M TPA or 1 /,M CCK-8 accentuated the immunostaining but did not alter its cellular distribution. L-364,718 reversed the changes in immunostaining caused by CCK-8. PKC-sr was detected over the luminal surface of the acinar cells. TPA, but not CCK-8 or CCK-8 followed by L-364,718, increased the number of acini that showed staining of the luminal surfaces of acin~u"cells. Thus, the present results demonstrate that different isoforms of PKC are distributed differently in rat pancreas and that the different patterns of distribution can explain, at least in part, the different responses to CCK-8.
Keywords: Diacylglycerol; Translocation PKC; Cholecystokinin; Protein kinase C; Tetradecanoyl phorbol acetate; L-364,718
1. Introduction PKC was initially identified as a protein kinase that could be activated by calcium and phospholipids [1]. Subsequently it was found that PKC could be activated by diacylglycerol [1]. Activation of PKC has been proposed to be involved in a variety of cell responses including cell proliferation, secretion, smooth muscle contraction, regulation of gene expression ~md membrane transport [1]. During the last several years it has become apparent that PKC is actually a family o f related enzymes that are differentially expressed in various cell types [2]. Biochemical, immunologic and molecular cloning studies have demonstrated the existence of isoforms of PKC that can be divided into two classes based on whether or not they are
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activated by calcium. For example, PKC isoforms ~ , 131, /32 and 3~ are activated by calcium, and phospholipid; isoforms iS, E, ~" and 7 / a r e activated by phospholipid but not by calcium [2]. Isoform-specific properties such as rates of down-regulation, subcellular location, activation requirements and substrate specificities suggest that different isoforms may regulate unique cellular functions [2-5]. On the other hand, in cells that possess multiple isoforms, the particular function of an individual isoform has not yet been clearly established. Moreover, to date no endogenous isoform-specific substrate has been identified for any isoform of PKC. In a previous study [6] of rat pancreatic acini using quantitative immunoblot analysis we detected four different isoforms of PKC-c~, ~, E and ft. W e did not detect the /3, y or 7/ isoforms. W e also found that although CCK-8 caused a three-fold increase in cellular D A G , CCK-8 caused redistribution of PKC-t5 and PKC-e but not P K C - a
B. Bastani et al. /Biochimica et Biophysica Acta 1269 (1995) 307-315
308
or PKC-~" from the cytosol to the membrane fraction of homogenized acini. In our previous study [6] we considered the possibility that the isoforms whose distribution is not altered by CCK-8 might be in different cells from those in which the increase in DAG occurs. In the present study we examined this possibility by using immunocytochemistry to determine the cellular distribution of different isoforms of PKC in intact pancreas as well as in pancreatic acini. Our results indicate that PKC-c~, PKC-6, PKC-E and PKC-~" are each distributed differently in the pancreas and that the pattern of cellular distribution can explain, at least in part, whether or not a particular isoform undergoes translocation in response to CCK-8.
2. Materials
Male Sprague-Dawley rats (75-150 g) were obtained from Harlan-Sprague-Dawley, Indianapolis, IN; purified collagenase (Type CLSPA, 643 units/mg) from Worthington Biochemicals, Freehold, NJ; Hepes, TPA and soybean trypsin inhibitor from Sigma Chemical, St. Louis, MO; glutamine from Research Plus Laboratories, Danville, N J; COOH-terminal octapeptide of cholecystokinin (CCK-8), from Peninsula Laboratory, Belmont, CA; basal media (Eagle) amino acids (100-times concentrated) from GIBCO, Grand Island, NY; essential vitamin mixture (100-times concentrated) from Microbiological Associates, Bethesda, MD; bovine plasma albumin (fraction V) from Miles Laboratories, Elkhart, IN; rabbit polyclonal anti-PKC c~, /3, y, 6, e and ~" and the corresponding peptide antigens from GIBCO BRL, Grand Island, NY; rabbit polyclonal anti-PKC r/ from Sphinx Pharmaceutical Corp., Durham, NC; Hyperfilm MP and enhanced chemiluminescence (ECL) from Amersham, Arlington Heights, IL; poly(vinylidene difluoride) membrane sheets and goat anti-rabbit IgG from Bio-Rad Laboratories, Richmond, CA; sn-l,2-diacylglycerol kinase from Calbiochem, San Diego, CA; spectral grade methanol, chloroform and isopropyl alcohol from EM Science, Gibbstown, NJ; and reagent-grade glacial acetic acid from Fisher Scientific, Fair Lawn, NJ. Unless stated otherwise, the standard incubation solution contained 24.5 mM Hepes (pH 7.4), 98 mM NaC1, 6
mM KC1, 2.5 mM NaH2PO 4, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 0.5 mM CaC12, 1% ( w / v ) albumin, 0.1% ( w / v ) trypsin inhibitor, 1% ( v / v ) essential amino acid mixture, 0.1% ( w / v ) bacitracin and 1% ( v / v ) vitamin mixture. All incubations were performed with 100% 02 as the gas phase.
3. Methods
Tissue preparation. Dispersed acini from rat pancreas were prepared using the modifications [7] of the procedure published previously [8]. PKC isoforms. The various isoforms of PKC were measured by quantitative immunoblot analysis as described previously [6]. Acini from six pancreata were suspended in 5 ml of standard incubation solution. Samples (1 ml) were incubated for the times and with the agents indicated at 37°C. Values for the amount of a particular isoform associated with the membrane fraction were expressed as a percentage of the total fraction (i.e., cytosol plus membrane fraction). With each isoform that was detected the appropriate band could be abolished by preincubating the antiserum with the corresponding peptide antigen. The activity of each antiserum was confirmed by documenting its ability to detect the appropriate PKC isoform in supernatant prepared from rat brain homogenate as described previously [6]. Diacylglycerol (DAG). Acini from one pancreas were suspended in 13 ml of standard incubation solution and 1 ml samples were incubated at 37°C for the appropriate time with the agents indicated. Cellular DAG was determined using the mass assay described previously [6]. DAG was expressed as nmol/100 nmol phospholipid. Phospholipid. Phospholipid was measured using the phosphate assay of Ames [9] as described previously [6]. Immunocwtochemistry. Acini from two animals were centrifuged at 200 g for 10 min and after removing the supernatant were fixed in B5 (220 mM HgCI 2, 90 mM sodium acetate, 3.7% ( v / v ) formaldehyde) overnight and paraffin blocked. Slices from intact rat pancreas (2 mm thick) were fixed in the same manner. Tissue sections, 4 /zm thick, were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol (100%, 95%, 70% ( v / v ) ) and serially incubated in Lugol's iodine solution,
Table 1 Effect o f C C K - 8 and T P A on diacylglycerol and the distribution o f different isoforms of P K C in pancreatic acini Addition
None C C K - 8 (1 /zM) T P A ( 1 /xM)
Diacylglycerol
P K C Isoform (% m e m b r a n e )
( n m o l / 1 0 0 nmol phospholipid)
Alpha
Delta
Epsilon
Zeta
0.21 _ 0.03 0.61 _+ 0.08 * -
35 _ 4 37_+ 5 71 + 3 "
10 _+ 2 42 _+ 4 * 68+5 *
16 + 3 77 + 8 * 9+7 *
18 _+ 3 17 _+ 2 74+6 *
Pancreatic acini were incubated with the agents indicated for 10 min at 37°C. Values given are means + S.D. f r o m 4 separate experiments. * Significantly different ( P < 0.01) f r o m c o r r e s p o n d i n g value for no addition by S t u d e n t ' s paired t-test.
B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
5% ( w / v ) sodium thios.ulfate followed by phosphatebuffered saline (PBS; 20 mM sodium phosphate (pH 7.4), 150 mM NaC1). To block non-specific binding of the primary antibodies, sections were incubated in blocking solution (10% ( v / v ) calf serum, 10% ( v / v ) goat serum, 1% ( w / v ) poly(ethylene glycol) M r 20000, in PBS) for 30 min. Sections were then incubated overnight with rabbit PKC isoform-specific antisera, diluted 1:200 ( ( v / v ) in blocking solution). Samples were subsequently rinsed in PBS, reblocked for 15 rain and incubated in fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (diluted at 1:50 in blocking solution) for 20 rain. Sections were rinsed in PBS and mourned in a fresh mixture of paraphenylenediamine (PPD (2 mg/ml))in 50% glycerol ( v / v , in PBS). The slides were viewed using Nikon Optiphot-2 mercury epifluorescent microscope (Nikon Incorporation, Instrument Group, NY, USA) and a confocal microscope (Bio-Rad MRC 600).
.....
309
4. Results
CCK-8 caused a 3-fold increase in DAG in pancreatic acini and under identical conditions caused translocation of PKC-6 and PKC-e but not PKC-cr or PKC-sr (Table 1). The phorbol ester TPA caused translocation of each of the four different isoforms of PKC in pancreatic acini (Table 1). To examine the reversibility of the actions of CCK-8 on DAG and PKC-e acini were first incubated with CCK-8 for 10 min and then with the CCK receptor antagonist L-364,718 for another 10 min. As illustrated in Table 2, the effects of CCK-8 on DAG and PKC-e were reversed completely within 10 min after adding L-364,718. Results similar to those illustrated in Table 2 for PKC-e were also obtained with PKC-~ (data not shown). We were initially puzzled by the results in Table 1 showing that CCK-8 increased diacylglycerol but caused
~ • ....
Fig. 1. Sections f r o m rat pancreas i m m u n o s t a i n e d with rabbit polyclonal antiserum against P K C - a and P K C - 6 isoenzymes. In panel A - top, P K C - a staining is confined to some o f the peripheral islet cells, with no staining in the cells located in the center o f the islet or in the acini. Panel A - bottom s h o w s that there is n o staining fi)r PKC-ot over the pancreatic ducts. In panel B - top, P K C - 6 has a distribution similar to that for PKC-ot in that staining is in some o f the peripheral islet cells. Panel B - bottom s h o w s that there is no staining for P K C - 6 over the pancreatic ducts. In both panels magnification is x 400. A n t i s e r u m p r e a b s o r b e d with the synthetic i m m u n i z i n g peptide showed no staining (data not shown).
3 l0
B. Bastani et al./Biochimica et Biophysica Acta 1269 (1995) 307-315
Table 2 Reversibility of the effect of CCK-8 on diacylglycerol and PKC-e in pancreatic acini Addition
Diacylglycerol (nmol/100nmol pbospbolipid)
PKC-e (percent membrane)
None CCK-8 (1 /zM) L-364,718 (1 /zM) CCK-8 then L-364,718
0.22 __0.03 0.64_+0.09 * 0.21 _+0.04 0.26 _+0.05
19 _+4 68_+9 * 20_+3 18 _+4
Pancreatic acini were incubated at 37°C with no additions, CCK-8 or L-364,718 for 10 min or with CCK-8 for 10 min followed by L-364,718 for an additional 10 min. * Significantly different (P <0.01) from corresponding value for no addition by Student's paired t-test.
translocation o f only P K C - 6 and P K C - e and not P K C - a or PKC-~. W e considered the possibility that different isoforms o f P K C might be in different cell types, thereby accounting for the different responses to C C K - 8 . T o e x a m ine this possibility, we used i m m u n o c y t o c h e m i s t r y to determine the cellular distribution o f different isoforms o f P K C in the pancreas. As illustrated in Fig. l, we detected P K C - a and P K C - 6
in s o m e of the peripheral islet cells in sections from whole rat pancreas. Neither P K C isoform was detected in acinar cells or duct cells. Incubating 1 ~ g o f antibody with 4 0 - 1 0 0 p,g of the corresponding synthetic peptide antigen overnight at 4°C abolished the staining o f the peripheral islet cells (data not shown). W e also detected P K C - a and P K C - 6 in peripheral cells in islets or fragments o f islets that were present in our preparation o f pancreatic acini, but there was no detectable P K C - a or P K C - 6 in acinar cells or duct cells (data not shown). W e detected no PKC-/3 in any preparation by i m m u n o c y t o c h e m i s t r y (data not shown). P K C - e was detected in acinar cells but not in islet cells or duct cells in sections f r o m rat pancreas (Fig. 2). P K C - e was localized to the apical region of the acinar cell (Fig. 2B) where, on bright field microscopy, z y m o g e n granules could be identified. C o n f o c a l m i c r o s c o p y also identified P K C - e in the apical region o f the acinar cells (Fig. 2C). Incubating 1 /~g o f antibody with 10 /zg o f the P K C - e synthetic peptide antigen o v e r n i g h t at 4°C abolished subsequent i m m u n o s t a i n i n g for PKC-E (Fig. 2A). P K C - sr was detected in acinar cells and duct cells but not in islet cells in sections from rat pancreas (Fig. 3). PKC-~" was detected in the region o f the luminal surface o f the acinar cells as well as the luminal surface o f the
A
Fig. 2. Sections from rat pancreas immunostained with rabbit polyclonal antiserum against PKC-e. In panel A - top, antiserum preabsorbed with the immunizing PKC-e synthetic peptide shows no specific staining (magnification is × 400). Panel A - bottom is a confocal photomicrograph showing no specific staining for PKC-e when the antiserum was preabsorbed with the immunizing PKC-e synthetic peptide. In panel B - top, there is prominent vesicular staining confined to the apical region of the acinar cells (magnification is X400). Panel B - bottom shows that there is no staining for PKC-~ over the pancreatic ducts. In panel C, confocal microscopy illustrates that the staining has a vesicular pattern and is confined to the apical pole of the acinar cells where zymogen granules are located. In panels A - bottom and C, confocal magnification is indicated by the bar.
B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
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B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
ductules that extend from the acinus (Fig. 3B). Confocal microscopy confirmed that PKC-¢ was present at the luminal surface of acinar cells and associated ductules (Fig. 3C). Incubating 1 /zg of antibody with 10 /zg of the PKC-~" synthetic peptide antigen overnight at 4°C abolished subsequent immunostaining for PKC-~" (Fig. 3A). In pancreatic acini (Fig. 4), as in sections of whole pancreas (Fig. 2), PKC-e was localized to the apical region of the acinar cells (Fig. 4A). Incubating the acini
with 1 /.tM TPA (Fig. 4B) and with 1 /zM CCK-8 (Fig. 4C) accentuated the immunostaining in the apical region of the acinar cells but did not change the cellular distribution of the immunostaining. In acini that had been incubated with 1 /zM CCK-8 followed by 1 /~M L-364,718, the intensity of the staining in the apical region of the acinar cell was nearly the same as that in control acini, but there was a more diffuse cytoplasmic distribution of the immunostaining (Fig. 4D).
Fig. 4. Effects of CCK-8 and TPA on the immunostainingof PKC-e in rat pancreatic acini. Pancreatic acini were incubatedat 37°C for 10 min with no additions (A), 1 /xM TPA (B), 1 /xM CCK-8 (C) or l /.tM CCK-8 for l0 rain and then l /xM L-364,718 for an additional 10 min (D). In panel A, in control acini there is vesicularstainingpredominantlylocalizedto the apical region of the acinar cells. In panel B with TPA, and in panel C with CCK-8, there is accentuationof the vesicularstaining in the apical region of the acinar cells. In panel D with CCK-8 followed by L-364,718, the intensityof the apical stainingis comparableto that in panel A, but the staininghas a more diffuse cellular distributionthan in panels A-C. In each panel magnificationis X400.
B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
In pancreatic acini, PKC-,~ was localized to the luminal surface of the acinar cells (Fig. 5A). No ductule structures were detected in our preparation of acini, probably because the ductules are removed during processing whole pancreas to produce dispersed acini [7,8]. Incubating acini with 1 /zM TPA increased the number of acini that showed staining at the luminal surface of the acinar cells (Fig. 5B). Incubating acini with 1 /zM CCK-8 (Fig. 5C) or 1 /zM CCK-8 followed by 1 /xM L-364,718 (Fig. 5D) did not detectably alter the immunostaining of the acini.
313
5. Discussion
The present results demonstrate the different patterns of distribution of the various isoforms of PKC that can be detected by quantitative immunoblot analysis of pancreatic acini. That is, PKC-e is present in the apical region of acinar cells and PKC-~" is present on the luminal surfaces of acinar cells and of the ductular structures that extended from the acinar cells. Neither PKC-E nor PKC-~" is detectable in pancreatic islets. PKC-a and PKC-6 are de-
D
~•~i~ ~ ~i! ¸
Fig. 5. Effects of CCK-8 and TPA on the immunostaining of PKC-~" in rat pancreatic acini. Pancreatic acini were incubated at 37°C for 10 min with no additions (A), 1 /zM TPA (B), 1 ;xM CCK-8 (C) or l/xM CCK-8 for l0 min and then 1 /xM L-364,718 for an additional 10 minutes (D). In panel A in control acini, there is staining of the luminal surface of occasional acinar cells. In panel B with TPA there is an increase in the number of acinar cells that show staining of the luminal surface. In panels C and D with CCK-8 alone or after adding L-364,718 the luminal staining of acinar cells is similar to that in panel A. In each panel magnification is ::<400.
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B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
tectable in cells in the periphery of the islets but not in duct or acinar cells. The presence of PKC-a and PKC-S in our preparation of pancreatic acini is due to the islet cells that contaminate our preparation. As will be discussed later, the islet cells that contain PKC-ce probably differ from those that contain PKC-S. CCK-8 and TPA cause translocation of and enhance the immunostaining pattern of PKC-e and these effects of CCK-8 as well as the increase in DAG caused by CCK-8 can be reversed completely by adding L-364,718. TPA, but not CCK-8, also causes translocation of PKC-~ and increases the number of acinar cells that showed immunostaining of PKC-~" over the luminal surface. Our finding PKC-t~ and PKC-3 in peripherally located cells in the islets agrees with previous reports by others [10-15]. One previous study [14] detected PKC-~" and PKC-e immunoreactivity in islet cells in rat pancreas; however, in the present study we found PKC-~" immunoreactivity localized to the luminal surface of acinar cells and adjacent ductules and PKC-e immunoreactivity localized to acinar cells. This same previous study [14] also detected PKC-/3 immunoreactivity in islet cells. We detected no PKC-/3 immunoreactivity in pancreatic acini using quantitative immunoblot analysis [6] or in sections of whole pancreas or pancreatic acini using immunocytochemistry. The differences between the two studies in terms of PKC-/3 and the distribution of PKC-E and PKC-~" may reflect the use of different antisera because antisera that are satisfactory for immunoblot analysis may not be equally effective for immunocytochemistry. The present study was performed to attempt to explain the findings that although CCK-8 increases DAG in pancreatic acini and causes translocation of PKC-S and PKC-e as would be expected, CCK-8 does not cause translocation of PKC-a or PKC-~. This lack of translocation of PKC-cz and PKC-~" does not result from an inability of these isoforms to undergo translocation because TPA can cause translocation of both isoforms. We were not surprised by the inability of CCK-8 to cause translocation of PKC-~" because this isoform lacks a phorbol ester binding site and is not activated by DAG [3,16-18]. PKC-~" can, however, be activated by phosphatidylinositol 3,4,5-trisphosphate [19]. Since PKC-~" lacks a phorbol ester binding site and is not activated by DAG, we were initially surprised to find TPA-induced translocation of PKC-ff. Phorbol esters, however, have been found to cause translocation of PKC-~" in other systems [20,21]. It may be that, since activation of PKC is accompanied by autophosphorylation [22,23], TPA activation of some other isoform of PKC can phosphorylate PKC-ff; and thereby, induce its translocation. We considered several possible explanations for the inability of CCK-8 to cause translocation of PKC-a. One possibility is that CCK-8-induced translocation of PKC-a is transient and is reversed completely by the end of the incubation. Evidence for transient translocation of PKC-a
is that in IIC9 fibroblasts incubated with thrombin, translocation of PKC-a is maximal by 15-30 s and reversed completely by 60 s [20]. In other experiments we made measurements as early as 15 s and failed to detect CCK-8induced translocation of PKC-ct [6]. It is also possible that, although DAG can cause translocation of PKC-a, in our system the increase in cellular DAG is not in the proper functional compartment to cause translocation of PKC-a. In other systems [5,24-29] the increase in DAG derived from PIP 2 has been shown to cause translocation of PKC whereas the increase in DAG from phosphatidylcholine does not cause translocation of PKC. Others [20] have speculated that DAG derived from PIP 2 might have a different fatty acid composition from that derived from phosphatidylcholine and that these differences might account for the different effects of DAG on an isoform of PKC. Finally, the present findings that PKC-a is found only in certain cells in the islets but not in acinar cells or duct cells support the hypothesis that CCK-8 does not cause translocation of PKC-c~ because the increase in DAG occurs in different cells (e.g., acinar cells) from the islet cells that contain PKC-c~. Presumably, this occurs because the islet cells that contain PKC-a do not possess receptors for CCK. PKC-& like PKC-a, is also present in islet cells, but not in acinar cells or duct cells. Unlike PKC-a, however, PKC-~ is translocated by CCK-8. Presumably this occurs because the islet cells that possess PKC-8 also possess CCK receptors that when occupied by CCK-8 cause increased DAG and translocation of PKC-tS. These islet cells, however, must be different from those that possess PKC-a. Support for this possibility is the finding that PKC-a is found only in /3 cells within the islets in rat pancreas, whereas other PKC isoforms occur in other cell types within the islets [13]. Finally, knowing that PKC-a and PKC-~ are in islet cells and not in acinar cells has practical consequences. That is, this information could lead investigators to explore other isoforms, such as PKC-e, instead of the a and 3 isoforms as potential mediators of the actions of various agents on acinar cell function.
References [1] Nishizuka, Y. (1984) Nature 308, 693-698. [2] Kikkawa, U., Kishimoto, A. and Nishizuka, Y. (1989) Annu. Rev. Biochem. 58, 31-44. [3] Nishizuka, Y. (1988) Nature 334, 661-665. [4] Parker, P.J., Kour, G., Marais, R.M., Mitchell, F., Pears, C., Schaap, D., Stabel, S. and Webster, C. (1989) Mol. Cell. Endocrinol. 65, 1-11. [5] Kiley, S.C., Parker, P.J., Fabbro, D. and Jaken, S. (1991) J. Biol. Chem. 266, 23761-23768. [6] Pollo, D.A., Baldassare, J.J., Honda, T., Henderson, P.A., Talkad, V.D. and Gardner, J.D. (1994) Biochim. Biophys. Acta 1224, 127138.
B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
[7] Jensen, R.T., Letup, G.F. and Gardner, J.D. (1982) J. Biol. Chem. 257, 5554-5559. [8] Peikin, S.R., Rottman, A.J., Batzri, S. and Gardner, J.D. (1978) Am. J. Physiol. 235, 743-750. [9] Ames, B.N. (1966)Methods Enzymol. 8, 115-118. [10] Atsuko, I., Saito, N., Tanigu~chi, H., Chiba, T., Kikkawa, U., Saitoh Y. and Tanaka, C. (1989) Diabetes 38, 1005-1011. [11] Ganesan, S., Calle, R., Zawalich, K., Smallwood, J.I., Zawalich, W.S. and Rasmussen, H. (1990) Proc. Natl. Acad. Sci. USA 87, 9893-9897. [12] Koji, O., Hagiwara, M., Hachiya, T., Usauda, N., Nagata, T. and Hidaka, H. (1990) Endocrinology 126, 1235-1240. [13] Fletcher, D.J. and Ways, D.K. (1991) Diabetes 40, 1496-1503. [14] Wetsel, W.C., Khan, W.A., Merchenthaler, I., Rivera, H., Halpern, A.E., Phung, H.M., Negro-Vilar, A. and Hannun, Y.A. (1992) J. Cell Biol. 117, 121-133. [15] Arkhammer, P., Juntti-Berggren, L., Larsson, O., Welsh, M., Nanberg, E., Sjoholm, A., Kohler, M. and Berggren, P.O. (1994) J. Biol. Chem. 269, 2743-2749. [16] Nakanishi, H. and Exton, J.H. (1992) J. Biol. Chem. 267, 1634716354. [17] Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K. and Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. USA 86, 3099-3103.
315
[18] Ways, D.K., Cook, P.P., Webster, C. and Parker, P.J. (1992) J. Biol. Chem. 267, 4799-4805. [19] Nakanishi, H., Brewer, K.A. and Exton, J.H. (1993) J. Biol. Chem. 268, 13-16. [20] Crabos, M., Imber, R., Woodtli, T., Fabbro, D. and Erne, P. (1991) Biochim. Biophys. Res. Commun. 178, 878-883. [21] Baldassare, J.J., Henderson, P.A., Burns, D., Loomis, C. and Fisher, G.J. (1992) J. Biol. Chem. 267, 15585-15590. [22] Borner, C., Filipuzzi, I., Wartmann, M., Eppenberger, U. and Fabbro, D. (1989) J. Biol. Chem. 264, 13902-13909. [23] Mitchell, F.E., Marais, R.M. and Parker, P.J. (1989) Biochem. J. 261, 131-136. [24] Ha, K.-S. and Exton, J.H. (1993) J. Biol. Chem. 268, 10534-10539. [25] Matin, T.F., Hsieh, K.-P. and Porter, B.W. (1990) J. Biol. Chem. 265, 7623-7631. [26] Wright, T.M., Rangan, LA., Shin, H.S. and Raben, D.M. (1988) J. Biol. Chem. 263, 9374-9380. [27] Pessin, M.S. and Raben, D.M. (1989) J. Biol. Chem. 264, 87298738. [28] Raben, D.M., Pessin, M.S., Rangan. L.A. and Wright, T.M. (1991) J. Biol. Chem. 266, 3315-3321. [29] Pessin, M.S., Baldassare, J.J. and Raben, D.M. (1990) J. Biol. Chem. 265, 7959-7966.
B i o c h i ~mic~a e t B i o p h y s i c a A~ta Biochimica et Biophysica Acta 1269 (1995) 307-315
Cellular distribution of isoforms of protein kinase C (PKC) in pancreatic acini Bahar Bastani a, Liying Yang a, Joseph J. Baldassare a, Dale A. Polio b, Jerry D. Gardner b., a Di,::isionof Nephrology, Saint Louis UniversityHealth Sciences Center, St. Louis, MO 63104, USA b Departmentof Internal Medicine, Saint Louis University Health Sciences Center, 1402 South Grand Blvd., St. Louis, MO 63104, USA Received 12 June 1995; accepted 4 July 1995
Abstract As in a previous study (Biochim. Biophys. Acta 1224 (1994) 127-138), we used quantitative immunoblot analysis and found that rat pancreatic acini possess four different isoforms of PKC-alpha, delta, epsilon and zeta. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) caused translocation of each isoform from the cytosol to the membrane fraction. CCK-8 increased diacylglycerol (DAG) and caused translocation of PKC-8 and PKC-e but not that of PKC-a or PKC-sr. L-364,718, a CCK receptor antagonist, prevented as well as reversed the effects of CCK-8 on DAG and on translocation of PKC-8 and PKC-e. To explore the possibility that different isoforms of PKC might have. different distributions in rat pancreas, we used immunocytochemistry to determine the cellular distribution of different isoforms of PKC in intact pancreas as well as pancreatic acini. In intact pancreas, PKC-a and PKC-8 were detected in islet cells but not in duct or acinar cells. PKC-e was detected in the apical region of acinar cells and PKC-~" was detected over the luminal surfaces of acinar cells and the ductules that extend from the acinus. Neither PKC-e nor PKC-~" was detected in islets. In pancreatic acini PKC-a and PKC-8 were detected in islets or fragments of islets that contaminated the preparation but were not detected in acinar cells. PKC-e was detected in the apical region of acinar cells and adding 1 /,M TPA or 1 /,M CCK-8 accentuated the immunostaining but did not alter its cellular distribution. L-364,718 reversed the changes in immunostaining caused by CCK-8. PKC-sr was detected over the luminal surface of the acinar cells. TPA, but not CCK-8 or CCK-8 followed by L-364,718, increased the number of acini that showed staining of the luminal surfaces of acin~u"cells. Thus, the present results demonstrate that different isoforms of PKC are distributed differently in rat pancreas and that the different patterns of distribution can explain, at least in part, the different responses to CCK-8.
Keywords: Diacylglycerol; Translocation PKC; Cholecystokinin; Protein kinase C; Tetradecanoyl phorbol acetate; L-364,718
1. Introduction PKC was initially identified as a protein kinase that could be activated by calcium and phospholipids [1]. Subsequently it was found that PKC could be activated by diacylglycerol [1]. Activation of PKC has been proposed to be involved in a variety of cell responses including cell proliferation, secretion, smooth muscle contraction, regulation of gene expression ~md membrane transport [1]. During the last several years it has become apparent that PKC is actually a family o f related enzymes that are differentially expressed in various cell types [2]. Biochemical, immunologic and molecular cloning studies have demonstrated the existence of isoforms of PKC that can be divided into two classes based on whether or not they are
* Corresponding author. Fax: + 1 314 2685108. 0167-4889/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSD1 0167-4889(95)00120-4
activated by calcium. For example, PKC isoforms ~ , 131, /32 and 3~ are activated by calcium, and phospholipid; isoforms iS, E, ~" and 7 / a r e activated by phospholipid but not by calcium [2]. Isoform-specific properties such as rates of down-regulation, subcellular location, activation requirements and substrate specificities suggest that different isoforms may regulate unique cellular functions [2-5]. On the other hand, in cells that possess multiple isoforms, the particular function of an individual isoform has not yet been clearly established. Moreover, to date no endogenous isoform-specific substrate has been identified for any isoform of PKC. In a previous study [6] of rat pancreatic acini using quantitative immunoblot analysis we detected four different isoforms of PKC-c~, ~, E and ft. W e did not detect the /3, y or 7/ isoforms. W e also found that although CCK-8 caused a three-fold increase in cellular D A G , CCK-8 caused redistribution of PKC-t5 and PKC-e but not P K C - a
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or PKC-~" from the cytosol to the membrane fraction of homogenized acini. In our previous study [6] we considered the possibility that the isoforms whose distribution is not altered by CCK-8 might be in different cells from those in which the increase in DAG occurs. In the present study we examined this possibility by using immunocytochemistry to determine the cellular distribution of different isoforms of PKC in intact pancreas as well as in pancreatic acini. Our results indicate that PKC-c~, PKC-6, PKC-E and PKC-~" are each distributed differently in the pancreas and that the pattern of cellular distribution can explain, at least in part, whether or not a particular isoform undergoes translocation in response to CCK-8.
2. Materials
Male Sprague-Dawley rats (75-150 g) were obtained from Harlan-Sprague-Dawley, Indianapolis, IN; purified collagenase (Type CLSPA, 643 units/mg) from Worthington Biochemicals, Freehold, NJ; Hepes, TPA and soybean trypsin inhibitor from Sigma Chemical, St. Louis, MO; glutamine from Research Plus Laboratories, Danville, N J; COOH-terminal octapeptide of cholecystokinin (CCK-8), from Peninsula Laboratory, Belmont, CA; basal media (Eagle) amino acids (100-times concentrated) from GIBCO, Grand Island, NY; essential vitamin mixture (100-times concentrated) from Microbiological Associates, Bethesda, MD; bovine plasma albumin (fraction V) from Miles Laboratories, Elkhart, IN; rabbit polyclonal anti-PKC c~, /3, y, 6, e and ~" and the corresponding peptide antigens from GIBCO BRL, Grand Island, NY; rabbit polyclonal anti-PKC r/ from Sphinx Pharmaceutical Corp., Durham, NC; Hyperfilm MP and enhanced chemiluminescence (ECL) from Amersham, Arlington Heights, IL; poly(vinylidene difluoride) membrane sheets and goat anti-rabbit IgG from Bio-Rad Laboratories, Richmond, CA; sn-l,2-diacylglycerol kinase from Calbiochem, San Diego, CA; spectral grade methanol, chloroform and isopropyl alcohol from EM Science, Gibbstown, NJ; and reagent-grade glacial acetic acid from Fisher Scientific, Fair Lawn, NJ. Unless stated otherwise, the standard incubation solution contained 24.5 mM Hepes (pH 7.4), 98 mM NaC1, 6
mM KC1, 2.5 mM NaH2PO 4, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 0.5 mM CaC12, 1% ( w / v ) albumin, 0.1% ( w / v ) trypsin inhibitor, 1% ( v / v ) essential amino acid mixture, 0.1% ( w / v ) bacitracin and 1% ( v / v ) vitamin mixture. All incubations were performed with 100% 02 as the gas phase.
3. Methods
Tissue preparation. Dispersed acini from rat pancreas were prepared using the modifications [7] of the procedure published previously [8]. PKC isoforms. The various isoforms of PKC were measured by quantitative immunoblot analysis as described previously [6]. Acini from six pancreata were suspended in 5 ml of standard incubation solution. Samples (1 ml) were incubated for the times and with the agents indicated at 37°C. Values for the amount of a particular isoform associated with the membrane fraction were expressed as a percentage of the total fraction (i.e., cytosol plus membrane fraction). With each isoform that was detected the appropriate band could be abolished by preincubating the antiserum with the corresponding peptide antigen. The activity of each antiserum was confirmed by documenting its ability to detect the appropriate PKC isoform in supernatant prepared from rat brain homogenate as described previously [6]. Diacylglycerol (DAG). Acini from one pancreas were suspended in 13 ml of standard incubation solution and 1 ml samples were incubated at 37°C for the appropriate time with the agents indicated. Cellular DAG was determined using the mass assay described previously [6]. DAG was expressed as nmol/100 nmol phospholipid. Phospholipid. Phospholipid was measured using the phosphate assay of Ames [9] as described previously [6]. Immunocwtochemistry. Acini from two animals were centrifuged at 200 g for 10 min and after removing the supernatant were fixed in B5 (220 mM HgCI 2, 90 mM sodium acetate, 3.7% ( v / v ) formaldehyde) overnight and paraffin blocked. Slices from intact rat pancreas (2 mm thick) were fixed in the same manner. Tissue sections, 4 /zm thick, were deparaffinized in xylene, rehydrated in decreasing concentrations of ethanol (100%, 95%, 70% ( v / v ) ) and serially incubated in Lugol's iodine solution,
Table 1 Effect o f C C K - 8 and T P A on diacylglycerol and the distribution o f different isoforms of P K C in pancreatic acini Addition
None C C K - 8 (1 /zM) T P A ( 1 /xM)
Diacylglycerol
P K C Isoform (% m e m b r a n e )
( n m o l / 1 0 0 nmol phospholipid)
Alpha
Delta
Epsilon
Zeta
0.21 _ 0.03 0.61 _+ 0.08 * -
35 _ 4 37_+ 5 71 + 3 "
10 _+ 2 42 _+ 4 * 68+5 *
16 + 3 77 + 8 * 9+7 *
18 _+ 3 17 _+ 2 74+6 *
Pancreatic acini were incubated with the agents indicated for 10 min at 37°C. Values given are means + S.D. f r o m 4 separate experiments. * Significantly different ( P < 0.01) f r o m c o r r e s p o n d i n g value for no addition by S t u d e n t ' s paired t-test.
B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
5% ( w / v ) sodium thios.ulfate followed by phosphatebuffered saline (PBS; 20 mM sodium phosphate (pH 7.4), 150 mM NaC1). To block non-specific binding of the primary antibodies, sections were incubated in blocking solution (10% ( v / v ) calf serum, 10% ( v / v ) goat serum, 1% ( w / v ) poly(ethylene glycol) M r 20000, in PBS) for 30 min. Sections were then incubated overnight with rabbit PKC isoform-specific antisera, diluted 1:200 ( ( v / v ) in blocking solution). Samples were subsequently rinsed in PBS, reblocked for 15 rain and incubated in fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG (diluted at 1:50 in blocking solution) for 20 rain. Sections were rinsed in PBS and mourned in a fresh mixture of paraphenylenediamine (PPD (2 mg/ml))in 50% glycerol ( v / v , in PBS). The slides were viewed using Nikon Optiphot-2 mercury epifluorescent microscope (Nikon Incorporation, Instrument Group, NY, USA) and a confocal microscope (Bio-Rad MRC 600).
.....
309
4. Results
CCK-8 caused a 3-fold increase in DAG in pancreatic acini and under identical conditions caused translocation of PKC-6 and PKC-e but not PKC-cr or PKC-sr (Table 1). The phorbol ester TPA caused translocation of each of the four different isoforms of PKC in pancreatic acini (Table 1). To examine the reversibility of the actions of CCK-8 on DAG and PKC-e acini were first incubated with CCK-8 for 10 min and then with the CCK receptor antagonist L-364,718 for another 10 min. As illustrated in Table 2, the effects of CCK-8 on DAG and PKC-e were reversed completely within 10 min after adding L-364,718. Results similar to those illustrated in Table 2 for PKC-e were also obtained with PKC-~ (data not shown). We were initially puzzled by the results in Table 1 showing that CCK-8 increased diacylglycerol but caused
~ • ....
Fig. 1. Sections f r o m rat pancreas i m m u n o s t a i n e d with rabbit polyclonal antiserum against P K C - a and P K C - 6 isoenzymes. In panel A - top, P K C - a staining is confined to some o f the peripheral islet cells, with no staining in the cells located in the center o f the islet or in the acini. Panel A - bottom s h o w s that there is n o staining fi)r PKC-ot over the pancreatic ducts. In panel B - top, P K C - 6 has a distribution similar to that for PKC-ot in that staining is in some o f the peripheral islet cells. Panel B - bottom s h o w s that there is no staining for P K C - 6 over the pancreatic ducts. In both panels magnification is x 400. A n t i s e r u m p r e a b s o r b e d with the synthetic i m m u n i z i n g peptide showed no staining (data not shown).
3 l0
B. Bastani et al./Biochimica et Biophysica Acta 1269 (1995) 307-315
Table 2 Reversibility of the effect of CCK-8 on diacylglycerol and PKC-e in pancreatic acini Addition
Diacylglycerol (nmol/100nmol pbospbolipid)
PKC-e (percent membrane)
None CCK-8 (1 /zM) L-364,718 (1 /zM) CCK-8 then L-364,718
0.22 __0.03 0.64_+0.09 * 0.21 _+0.04 0.26 _+0.05
19 _+4 68_+9 * 20_+3 18 _+4
Pancreatic acini were incubated at 37°C with no additions, CCK-8 or L-364,718 for 10 min or with CCK-8 for 10 min followed by L-364,718 for an additional 10 min. * Significantly different (P <0.01) from corresponding value for no addition by Student's paired t-test.
translocation o f only P K C - 6 and P K C - e and not P K C - a or PKC-~. W e considered the possibility that different isoforms o f P K C might be in different cell types, thereby accounting for the different responses to C C K - 8 . T o e x a m ine this possibility, we used i m m u n o c y t o c h e m i s t r y to determine the cellular distribution o f different isoforms o f P K C in the pancreas. As illustrated in Fig. l, we detected P K C - a and P K C - 6
in s o m e of the peripheral islet cells in sections from whole rat pancreas. Neither P K C isoform was detected in acinar cells or duct cells. Incubating 1 ~ g o f antibody with 4 0 - 1 0 0 p,g of the corresponding synthetic peptide antigen overnight at 4°C abolished the staining o f the peripheral islet cells (data not shown). W e also detected P K C - a and P K C - 6 in peripheral cells in islets or fragments o f islets that were present in our preparation o f pancreatic acini, but there was no detectable P K C - a or P K C - 6 in acinar cells or duct cells (data not shown). W e detected no PKC-/3 in any preparation by i m m u n o c y t o c h e m i s t r y (data not shown). P K C - e was detected in acinar cells but not in islet cells or duct cells in sections f r o m rat pancreas (Fig. 2). P K C - e was localized to the apical region of the acinar cell (Fig. 2B) where, on bright field microscopy, z y m o g e n granules could be identified. C o n f o c a l m i c r o s c o p y also identified P K C - e in the apical region o f the acinar cells (Fig. 2C). Incubating 1 /~g o f antibody with 10 /zg o f the P K C - e synthetic peptide antigen o v e r n i g h t at 4°C abolished subsequent i m m u n o s t a i n i n g for PKC-E (Fig. 2A). P K C - sr was detected in acinar cells and duct cells but not in islet cells in sections from rat pancreas (Fig. 3). PKC-~" was detected in the region o f the luminal surface o f the acinar cells as well as the luminal surface o f the
A
Fig. 2. Sections from rat pancreas immunostained with rabbit polyclonal antiserum against PKC-e. In panel A - top, antiserum preabsorbed with the immunizing PKC-e synthetic peptide shows no specific staining (magnification is × 400). Panel A - bottom is a confocal photomicrograph showing no specific staining for PKC-e when the antiserum was preabsorbed with the immunizing PKC-e synthetic peptide. In panel B - top, there is prominent vesicular staining confined to the apical region of the acinar cells (magnification is X400). Panel B - bottom shows that there is no staining for PKC-~ over the pancreatic ducts. In panel C, confocal microscopy illustrates that the staining has a vesicular pattern and is confined to the apical pole of the acinar cells where zymogen granules are located. In panels A - bottom and C, confocal magnification is indicated by the bar.
B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
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B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
ductules that extend from the acinus (Fig. 3B). Confocal microscopy confirmed that PKC-¢ was present at the luminal surface of acinar cells and associated ductules (Fig. 3C). Incubating 1 /zg of antibody with 10 /zg of the PKC-~" synthetic peptide antigen overnight at 4°C abolished subsequent immunostaining for PKC-~" (Fig. 3A). In pancreatic acini (Fig. 4), as in sections of whole pancreas (Fig. 2), PKC-e was localized to the apical region of the acinar cells (Fig. 4A). Incubating the acini
with 1 /.tM TPA (Fig. 4B) and with 1 /zM CCK-8 (Fig. 4C) accentuated the immunostaining in the apical region of the acinar cells but did not change the cellular distribution of the immunostaining. In acini that had been incubated with 1 /zM CCK-8 followed by 1 /~M L-364,718, the intensity of the staining in the apical region of the acinar cell was nearly the same as that in control acini, but there was a more diffuse cytoplasmic distribution of the immunostaining (Fig. 4D).
Fig. 4. Effects of CCK-8 and TPA on the immunostainingof PKC-e in rat pancreatic acini. Pancreatic acini were incubatedat 37°C for 10 min with no additions (A), 1 /xM TPA (B), 1 /xM CCK-8 (C) or l /.tM CCK-8 for l0 rain and then l /xM L-364,718 for an additional 10 min (D). In panel A, in control acini there is vesicularstainingpredominantlylocalizedto the apical region of the acinar cells. In panel B with TPA, and in panel C with CCK-8, there is accentuationof the vesicularstaining in the apical region of the acinar cells. In panel D with CCK-8 followed by L-364,718, the intensityof the apical stainingis comparableto that in panel A, but the staininghas a more diffuse cellular distributionthan in panels A-C. In each panel magnificationis X400.
B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
In pancreatic acini, PKC-,~ was localized to the luminal surface of the acinar cells (Fig. 5A). No ductule structures were detected in our preparation of acini, probably because the ductules are removed during processing whole pancreas to produce dispersed acini [7,8]. Incubating acini with 1 /zM TPA increased the number of acini that showed staining at the luminal surface of the acinar cells (Fig. 5B). Incubating acini with 1 /zM CCK-8 (Fig. 5C) or 1 /zM CCK-8 followed by 1 /xM L-364,718 (Fig. 5D) did not detectably alter the immunostaining of the acini.
313
5. Discussion
The present results demonstrate the different patterns of distribution of the various isoforms of PKC that can be detected by quantitative immunoblot analysis of pancreatic acini. That is, PKC-e is present in the apical region of acinar cells and PKC-~" is present on the luminal surfaces of acinar cells and of the ductular structures that extended from the acinar cells. Neither PKC-E nor PKC-~" is detectable in pancreatic islets. PKC-a and PKC-6 are de-
D
~•~i~ ~ ~i! ¸
Fig. 5. Effects of CCK-8 and TPA on the immunostaining of PKC-~" in rat pancreatic acini. Pancreatic acini were incubated at 37°C for 10 min with no additions (A), 1 /zM TPA (B), 1 ;xM CCK-8 (C) or l/xM CCK-8 for l0 min and then 1 /xM L-364,718 for an additional 10 minutes (D). In panel A in control acini, there is staining of the luminal surface of occasional acinar cells. In panel B with TPA there is an increase in the number of acinar cells that show staining of the luminal surface. In panels C and D with CCK-8 alone or after adding L-364,718 the luminal staining of acinar cells is similar to that in panel A. In each panel magnification is ::<400.
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tectable in cells in the periphery of the islets but not in duct or acinar cells. The presence of PKC-a and PKC-S in our preparation of pancreatic acini is due to the islet cells that contaminate our preparation. As will be discussed later, the islet cells that contain PKC-ce probably differ from those that contain PKC-S. CCK-8 and TPA cause translocation of and enhance the immunostaining pattern of PKC-e and these effects of CCK-8 as well as the increase in DAG caused by CCK-8 can be reversed completely by adding L-364,718. TPA, but not CCK-8, also causes translocation of PKC-~ and increases the number of acinar cells that showed immunostaining of PKC-~" over the luminal surface. Our finding PKC-t~ and PKC-3 in peripherally located cells in the islets agrees with previous reports by others [10-15]. One previous study [14] detected PKC-~" and PKC-e immunoreactivity in islet cells in rat pancreas; however, in the present study we found PKC-~" immunoreactivity localized to the luminal surface of acinar cells and adjacent ductules and PKC-e immunoreactivity localized to acinar cells. This same previous study [14] also detected PKC-/3 immunoreactivity in islet cells. We detected no PKC-/3 immunoreactivity in pancreatic acini using quantitative immunoblot analysis [6] or in sections of whole pancreas or pancreatic acini using immunocytochemistry. The differences between the two studies in terms of PKC-/3 and the distribution of PKC-E and PKC-~" may reflect the use of different antisera because antisera that are satisfactory for immunoblot analysis may not be equally effective for immunocytochemistry. The present study was performed to attempt to explain the findings that although CCK-8 increases DAG in pancreatic acini and causes translocation of PKC-S and PKC-e as would be expected, CCK-8 does not cause translocation of PKC-a or PKC-~. This lack of translocation of PKC-cz and PKC-~" does not result from an inability of these isoforms to undergo translocation because TPA can cause translocation of both isoforms. We were not surprised by the inability of CCK-8 to cause translocation of PKC-~" because this isoform lacks a phorbol ester binding site and is not activated by DAG [3,16-18]. PKC-~" can, however, be activated by phosphatidylinositol 3,4,5-trisphosphate [19]. Since PKC-~" lacks a phorbol ester binding site and is not activated by DAG, we were initially surprised to find TPA-induced translocation of PKC-ff. Phorbol esters, however, have been found to cause translocation of PKC-~" in other systems [20,21]. It may be that, since activation of PKC is accompanied by autophosphorylation [22,23], TPA activation of some other isoform of PKC can phosphorylate PKC-ff; and thereby, induce its translocation. We considered several possible explanations for the inability of CCK-8 to cause translocation of PKC-a. One possibility is that CCK-8-induced translocation of PKC-a is transient and is reversed completely by the end of the incubation. Evidence for transient translocation of PKC-a
is that in IIC9 fibroblasts incubated with thrombin, translocation of PKC-a is maximal by 15-30 s and reversed completely by 60 s [20]. In other experiments we made measurements as early as 15 s and failed to detect CCK-8induced translocation of PKC-ct [6]. It is also possible that, although DAG can cause translocation of PKC-a, in our system the increase in cellular DAG is not in the proper functional compartment to cause translocation of PKC-a. In other systems [5,24-29] the increase in DAG derived from PIP 2 has been shown to cause translocation of PKC whereas the increase in DAG from phosphatidylcholine does not cause translocation of PKC. Others [20] have speculated that DAG derived from PIP 2 might have a different fatty acid composition from that derived from phosphatidylcholine and that these differences might account for the different effects of DAG on an isoform of PKC. Finally, the present findings that PKC-a is found only in certain cells in the islets but not in acinar cells or duct cells support the hypothesis that CCK-8 does not cause translocation of PKC-c~ because the increase in DAG occurs in different cells (e.g., acinar cells) from the islet cells that contain PKC-c~. Presumably, this occurs because the islet cells that contain PKC-a do not possess receptors for CCK. PKC-& like PKC-a, is also present in islet cells, but not in acinar cells or duct cells. Unlike PKC-a, however, PKC-~ is translocated by CCK-8. Presumably this occurs because the islet cells that possess PKC-8 also possess CCK receptors that when occupied by CCK-8 cause increased DAG and translocation of PKC-tS. These islet cells, however, must be different from those that possess PKC-a. Support for this possibility is the finding that PKC-a is found only in /3 cells within the islets in rat pancreas, whereas other PKC isoforms occur in other cell types within the islets [13]. Finally, knowing that PKC-a and PKC-~ are in islet cells and not in acinar cells has practical consequences. That is, this information could lead investigators to explore other isoforms, such as PKC-e, instead of the a and 3 isoforms as potential mediators of the actions of various agents on acinar cell function.
References [1] Nishizuka, Y. (1984) Nature 308, 693-698. [2] Kikkawa, U., Kishimoto, A. and Nishizuka, Y. (1989) Annu. Rev. Biochem. 58, 31-44. [3] Nishizuka, Y. (1988) Nature 334, 661-665. [4] Parker, P.J., Kour, G., Marais, R.M., Mitchell, F., Pears, C., Schaap, D., Stabel, S. and Webster, C. (1989) Mol. Cell. Endocrinol. 65, 1-11. [5] Kiley, S.C., Parker, P.J., Fabbro, D. and Jaken, S. (1991) J. Biol. Chem. 266, 23761-23768. [6] Pollo, D.A., Baldassare, J.J., Honda, T., Henderson, P.A., Talkad, V.D. and Gardner, J.D. (1994) Biochim. Biophys. Acta 1224, 127138.
B. Bastani et al. / Biochimica et Biophysica Acta 1269 (1995) 307-315
[7] Jensen, R.T., Letup, G.F. and Gardner, J.D. (1982) J. Biol. Chem. 257, 5554-5559. [8] Peikin, S.R., Rottman, A.J., Batzri, S. and Gardner, J.D. (1978) Am. J. Physiol. 235, 743-750. [9] Ames, B.N. (1966)Methods Enzymol. 8, 115-118. [10] Atsuko, I., Saito, N., Tanigu~chi, H., Chiba, T., Kikkawa, U., Saitoh Y. and Tanaka, C. (1989) Diabetes 38, 1005-1011. [11] Ganesan, S., Calle, R., Zawalich, K., Smallwood, J.I., Zawalich, W.S. and Rasmussen, H. (1990) Proc. Natl. Acad. Sci. USA 87, 9893-9897. [12] Koji, O., Hagiwara, M., Hachiya, T., Usauda, N., Nagata, T. and Hidaka, H. (1990) Endocrinology 126, 1235-1240. [13] Fletcher, D.J. and Ways, D.K. (1991) Diabetes 40, 1496-1503. [14] Wetsel, W.C., Khan, W.A., Merchenthaler, I., Rivera, H., Halpern, A.E., Phung, H.M., Negro-Vilar, A. and Hannun, Y.A. (1992) J. Cell Biol. 117, 121-133. [15] Arkhammer, P., Juntti-Berggren, L., Larsson, O., Welsh, M., Nanberg, E., Sjoholm, A., Kohler, M. and Berggren, P.O. (1994) J. Biol. Chem. 269, 2743-2749. [16] Nakanishi, H. and Exton, J.H. (1992) J. Biol. Chem. 267, 1634716354. [17] Ono, Y., Fujii, T., Ogita, K., Kikkawa, U., Igarashi, K. and Nishizuka, Y. (1989) Proc. Natl. Acad. Sci. USA 86, 3099-3103.
315
[18] Ways, D.K., Cook, P.P., Webster, C. and Parker, P.J. (1992) J. Biol. Chem. 267, 4799-4805. [19] Nakanishi, H., Brewer, K.A. and Exton, J.H. (1993) J. Biol. Chem. 268, 13-16. [20] Crabos, M., Imber, R., Woodtli, T., Fabbro, D. and Erne, P. (1991) Biochim. Biophys. Res. Commun. 178, 878-883. [21] Baldassare, J.J., Henderson, P.A., Burns, D., Loomis, C. and Fisher, G.J. (1992) J. Biol. Chem. 267, 15585-15590. [22] Borner, C., Filipuzzi, I., Wartmann, M., Eppenberger, U. and Fabbro, D. (1989) J. Biol. Chem. 264, 13902-13909. [23] Mitchell, F.E., Marais, R.M. and Parker, P.J. (1989) Biochem. J. 261, 131-136. [24] Ha, K.-S. and Exton, J.H. (1993) J. Biol. Chem. 268, 10534-10539. [25] Matin, T.F., Hsieh, K.-P. and Porter, B.W. (1990) J. Biol. Chem. 265, 7623-7631. [26] Wright, T.M., Rangan, LA., Shin, H.S. and Raben, D.M. (1988) J. Biol. Chem. 263, 9374-9380. [27] Pessin, M.S. and Raben, D.M. (1989) J. Biol. Chem. 264, 87298738. [28] Raben, D.M., Pessin, M.S., Rangan. L.A. and Wright, T.M. (1991) J. Biol. Chem. 266, 3315-3321. [29] Pessin, M.S., Baldassare, J.J. and Raben, D.M. (1990) J. Biol. Chem. 265, 7959-7966.