Acta Physiol Scand 1992, 146, 135-140
Protein kinase C activity in rat renal proximal tubule cells C. F. M E N D E Z " ? , A. H A N S S O N ? , G. S K O G L U N D t , M. I N G E L M A N - S U N D B E R G t and A. A P E R I A " * Department of Pediatrics, St Goran's Children's Hospital, Karolinska Institute, and t Department of Physiological Chemistry, Karolinska Institute, Stockholm, Sweden MENDEZ, C. F., HANSSON, A., SKOGLUND, G., INGELMAN-SUNDBERG, M. & APERIA,A. 1992. Protein kinase C activity in rat renal proximal tubule cells. Acta Physiol Scand 146, 135-140. Received 19 December 1991 accepted 17 March 1992. ISSN 0001-6772. Departments of Pediatrics and Physiological Chemistry, Karolinska Institute, Sweden. The presence of protein kinase C (PKC) in proximal tubule cells of the rat kidney is established by means of immunodetection and by the demonstration of calcium- and phospholipid-dependent, staurosporine-inhibitable histone phosphorylation. The calcium-dependence of renal PKC is described. Maximal activation of the enzyme (178.2 and 258.8 pmol P, mg-' min-' for cytosol and membrane respectively) was achieved with 5 PM of Ca2+.Phorbol 12,13 dibutyrate (PDBu) translocated PKC from cytosol to membrane in a dose- and time-dependent fashion, while 4cc-phorbol 12,13-didecanoate produced no significant effect on translocation. Cytosolic PKC activity was compared in immature and mature tissues (10- and 40-day-old kidneys). Basal activity was found to be significantly higher (P< 0.05) in immature cells (272.8 vs. 157.5 pmol M for 15 min reduced immunoreactivity in the soluble Pi mg-' min-'). PDBu at fraction of both groups, which was accompanied by a significant decrease in kinase activity. We speculate that the high PKC activity in the infant kidney plays a role in cell growth. Key laords : ontogeny, protein kinase C, proximal tubule cells, rat kidney.
Protein kinase C (PKC) is a calcium- and phospholipid-dependent protein kinase expressed in all mammalian cells. The activation of this kinase is a common mechanism for transducing into the cell various extracellular signals, such as those from certain hormones, neurotransmitters, growth factors and other biologically active substances (Nishizuka 1986). Indirect evidence suggests that in kidney tissue P K C plays an important role in regulating specific membrane transporters. Phorbol esters have been shown to regulate phosphate and bicarbonate transport in proximal convoluted tubules (Boneh et al. 1989, Wang & Chan 1990, Liu & Cogan 1990, Baum & Hays 1988). Both Na+/H+ exchanger (Mellas & Hammerman 1986, Weinman et al. 1989, Livne
METHODS
Correspondence : Professor Anita Aperia, Department of Paediatrics, St. Goran's Children's Hospital, 112 81 Stockholm, Sweden.
Tissue preparation. Male Sprague-Dawley rats were used for the studies. The animals, aged 10 and 40 days, were anaesthetized with thiobutabarbital (Inactin; Byk-Gulden, Koblenz, FRG) 50 mg kg-l and the
et al. 1991) and Na+,K+-ATPase (Bertorello &
Aperia 1989) have been suggested to be effector proteins for P K C in the kidney. Although there is abundant indirect evidence that PKC regulates ion transporters in the renal proximal tubule, the characteristics of activation of proximal tubular P K C have not been well defined. The purpose of the present study has been to characterize protein kinase C activity in a defined population of proximal tubule cells from the rat kidney. We also compare the activity of this protein kinase in immature and mature renal tissues.
135
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C. F . Mendez et al.
kidneys quickly removed. The organs were decapsulated and a 200 pm thick slice was taken from the outer cortex, using a Stadie-Riggs microtome. The tissue slices were homogenized by sonication, 1.5 s set 4 using a Branson sonifier 2 5 0 (Branson Corp. , C T , USA-\),in a buffer containing in mat: 1 20, EGT.4 2, EDT.4 2 , PXISF 1,2-mercaptcjethanol 10. and aprotinin 10000 K I E 1 ', p H 7.4 room temperature. In some studies where brain tissue was used for comparison, the whole brain was quickly removed and homogenized in 10 nil homogenization buffer, using a motor-dri\-en Teflon pestle and glass tube homogenizer. Separntroti of soluble and partirulntr .fiac.trons. Separation of cytosol from the particulate fraction nas achie\ed by centrifugation a t 1OOOOOg a t 2 "C, using a TST-60 rotor in a Sorvall O'TD-Combi ultracentrifuge. 'The hornogenatc was first centrifuged for 1 h and the supernatant sa\-ed as the c!-tosolic compartment where Triton-S 100 was added to give a final concentration of 1O o . The remaining pellet \+as resuspended in the homogenization buffer 0.1 Triton-X 100 b>-brief sonication and centrifuged for 1 h. The resulting supernatant was considered to he the membrane fraction. In a pilot stud!- we investigated the optimal detergent concentration necessar!. to detect PKC acti\-it!-. Addition of 1no of Triton to the c\ tosolic fraction and of 0.1 n o to the membranes elicited the highest calcium- and phospholipitldependent activity (187 and 155 pmol P, min-' mg-', respectivelv), while the actkit!- in the remaining pellet became negligible. For PKC determination. the preparations M-ere diluted at least 10 times into the assay buffer, s o that the Triton concentration never exceeded 0.1 {I,) in the actix-ity assaj-. T h e fractions were stored on ice in the presence of gl!-cerol at a final concentration of LOO,. Under these conditions, P K C activitl- did not change significantly during a i-day period. Activities were 184 and 154 pmol Pamg protein-' min-' for da!-s 1 and 5 , respectivel!-. T h e protein content was measured b!Bradford's method (Bradford lYih), using the conimercial dye reagent Bio rad (Kichmond, CL-\,US.-\). Protein kinusr C assay. The standard reaction mixture (final volume 100 ,MI) contained: Tris HCI LO m u , X'igCI, 10 m u , EGT.l 1 msi, histone €31 -50 pg, proteins 1&20 pg. Phospholipids (PS/I>) concentration was phosphatidylserine 5 pg7 dioleo! 1glycerol 0.1 p g . Unless otherwise stated, free CaL* concentration was adjusted to 0.1 mhi, using the dissociation constant given by Bartfai (1979). Basal actit-it! was measured in the presence of 0.5 m>i EGT.4 instead of calcium and in the absence of lipids. Solutions were freshly prepared. The lipids were sonicated in Tris HCI 20 mhi for 1.5-20 s. T h e reaction a a s initiated by adding [y-"P].1TP (final concentration 10 p s i ; specific activity approainiatcl! 300 cpm pmol-') and by raising the tem-
+
perature of the tube from 0 to 30 "C. In pilot experiments, the transfer of '32Pto histone was found to be linear with respect to time for the first 2 min. The reaction was therefore carried out for 2 min and stopped b!- the addition of 20,d of EDTA 125 mM ATP 5 mx1-l and the return of the tube to 0 "C. A 60 /)I aliquot from each tube was pipetted onto a 2 cm2 piece of phosphocellulose paper. Unincorporated "P was removed by washing the papers three times in phosphoric acid 75 mhi and once in ether: ethanol 1 :4, after Tvhich they were dried under a stream of air. T h e papers were placed in scintillation vials along with 5 nil scintillation liquid and the radioactivity counted in a scintillation counter. Gel rler.trophoresis. After solubilization (95 "C for 3 min), the samples were analysed on one-dimensional SDS-polyacrylamide slab gel electrophoresis, using 7.5 O O (w/v) acrylaniide, according to Laemmli (1970). .4fter electrophoresis, the gels were stained with coomassie blue, destained, dried, and exposed to Hyperfilm-MP X-ray films for autoradiography in Kodak X-Omatic intensifying screens at - 80 "C. After SDS-PAGE, proteins were electrophoretically transferred to nitrocellulose sheets. Yon-specific binding was blocked by the incubation of membranes in PBS containing lo:, bovine serum albumin. P K C was detected by means of a polyclonal rabbit antiserum raised against a s!-nthetic peptide that corresponds to a conserved region present in the a, p and y types of P K C (residues 185-204 in human type PKC) (Skoglund r t ul. 1988). After washing of the filters, the antibody was added at a 1 : 100 dilution in PBS l"/b bovine serum albumin. Specific binding was detected by using alkaline phosphatase conjugated goat anti-rabbit IgG with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) as substrate. Statistical analysis. Results are expressed either as pmol P, mg-' min-', or as a percentage ofits respective control, S S E M . When more than one mean value was to be compared, the significance was determined b>- analysis of variance (ANOVA). When a single value was compared to its control, significance was established using the Student's independent test. Reagents. Phorbol 12,13-dibutyrate (PDBu), 4aphorbol 12,13-didecanoate, Tris[hydroxymethyl]aminomethane (Tris), ethylene glycol bis(/l-aminoeth$ ether) h',N,hr',N'-tetraacetic acid (EGTA), ethplenediaminetetraacetic acid (EDTA), phenylmethyl-sulphonyl fluoride (PMSF), Histone HI (type 111 S, Sigma), L-a-phosphatidyl-L-serine, 1,2dioleoyl-sn-glycerol (C18 : l), adenosine triphosphate grade I1 (ATP) and Staurosporine were supplied by Sigma. RlgCI,, CaCI,, glycerol, Triton-X 100 and phosphoric acid came from Merck, P 81 phosphocellulose paper from Whatman, and [y3'PP]ATP (specific activity 3000 Ci mmol-I), Hybond-C nitrocellulose membranes and Hyperfilm-MP from
PKC in kidney proximal tubule cells Amersham (Amersham, UK). Alkaline phosphatase conjugated goat anti-rabbit IgG, 5-bromo-4-chloro3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) were from Bio Rad. Dulbecco's modified Eagle medium (DME) was purchased from Gibco, 2mercapto-ethanol from Carl Roth KG, and Aprotinin (Trasylola) from Bayer.
137
with the particulate fraction. PDBu M changed that distribution to 22.8 k 2.4 and 77.2+2.4y0 for cytosol and membrane, respectively. These results were confirmed by
RESULTS Cytosol from the renal cortex phosphorylates histone in a calcium- and phospholipid-dependent manner (Fig. 1). Calcium and phospholipids alone failed to phosphorylate histone. Calcium/phospholipid-dependent phosphorylation was inhibited by 100 nM staurosporine. Phosphorylation of endogenous substrates was negligible as compared to histone.
Calcium afinity T h e calcium-dependence of renal cytosolic P K C activation is shown in Figure 2. Activity (expressed as pmol Pi mg protein-' min-' k SEM) was 33.6f 8.2 in the absence of calcium. Maximal activation occurred within a narrow range of free calcium concentrations, approximately 1-5 ,UM. Maximal PKC activity was 178.2 f8.2 for the cytosol and 258.8 f 17.2 for the membrane. Under calcium-saturating conditions, the activity in brain cytosol was 1230 f51 pmol Pi mg-' min-l.
Fig. 1. Protein kinase C-catalysed phosphorylation of histone. Histone HI (50yg) was incubated with cytosolic preparation in the presence and the absence of PKC cofactors and of 100 nM staurosporine, under the conditions specified in Methods. The samples were then analysed by SDS-PAGE and autoradiography. 300
1
Efect of phorbol 12,13-dibutyrate on the subcellular distribution of protein kinase C Phorbol 12,13-dibutyrate translocates PKC from the cytosol to the membrane. When the tissue was incubated in the presence of PDBu, at concentrations ranging from lo-* to M for 15 min, PKC activity decreased in the cytosol and increased accordingly in the particulate fraction in a dose-dependent manner. PDBu M significantly reduced the activity in the cytosol to 2 6 f 4 y 0 of control (157.5k23.5 and 40.9 f8.5 pmol Pi mg-' min-l for control and PDBu M, respectively). These differences were significant at the 1yo level for cytosol, and at the 5% level for membrane, using the ANOVA test (Fig. 3a). I n the control slices, 62.1 f5.9% of the total activity was recovered in the cytosolic fraction, while the remaining 37.9f 5.9% was associated
0
1
10
100
200
1000
[Free calcium] pM
Fig. 2. Calcium dependence of PKC activation. Protein kinase C activity was assayed in the soluble (-O-) and particulate (-0-) fractions under conditions described in Methods. Free calcium concentration was adjusted to the values indicated, using the dissociation constant given by Bartfai (1979). Results are shown as the meankSEM of three independent experiments in which duplicates were
obtained.
138
C. F . Mendez et a1 A
-*-**-
A
150
T
-**-
-7
-6 OJ
Log [PDBu] (M)
200
I
1
Conlrol
~
PDBu
Control PDBu
_
10 days
_
_
40 days
Fig. 4. (a) Tissue slices from 10- and 40-day-old kidnel-s nere incubated in the presence of M PDBu for 15 min or the vehicle alone C!-tosolic P K C activity was determined as described in Xlethods. Results are expressed as the mean S E M of four independent experiments performed in duplicates. ** Significantl!- different from control value ( P < 0.01); *significantly different from homologous control ( P < 0.0.5). (b) Immunoblot analysis of immature and mature proximal tubule cells. Tissue was homogenized as described in Methods. Samples (0.3 mg) were electrophoretically separated and transferred to nitrocellulose paper. Blot was probed with a polyclonal antibody that recognizes several isoforms of PKC.
(a),
0 1
2.5
5
15
Time (min)
Fig. 3. Etfect of phorbol 12,l.i-dibut!-rate on the suhcellular distribution of protein hinase C. (a) Tissuc slices were incubated with vehicle or different PDBu concentrations during 1.i min a t room temperature. fractions nere Soluble ( w ) and particulate (0) obtained and assa!ed for P K C activit!-. as described in .2lethods. .ictivit! is espressed as ( I , ) of control. (b) 'l'ime-course of the PDBu effect. Slices nere exposed to 10 ' \i PIIBu for the times indicated and P K C activit! was determined in both c! tosol (-O-) m d Control experiments were permembrane (-0 tormed for each time-point, and PKC activit! is espressed as 'lo of its respective control value. Each point represents the mean of three to five esperimmts performed in duplicates 5 SEhI. T h e level of signific:?ncc V J S 0.01 for cytosol and 0.05 for membrme, using the .i\Ol-:l test.
immunodetection (data not shon n). T h e translocation TI as time-dependent, as sho\-r n in Figure
3 (b) T h c inacti\e phorbol ester, 4z-phorbol 12,13didecmoate produced no significant effect o n
(m).
PKC translocation, when used a t a concentration of 1 0 - " ~during 15 m i n (143.3f27.6 pmol P, mg-' min-'). Protein kinuse C activity in the developing kidnq I n this protocol we compared cytosolic PKC actkit!- i n proximal tubule cells from immature kidneys ( 10-day-old rats) a n d mature kidneys (40-day-old rats). As shown i n Figure 4(a), PKC activity was significantly higher ( P < 0.05) in immature than i n mature cells, (272.8 2 24.6 vs. 157.52 & 23.5 pmol Pi mg-' min-' for 10- a n d
PKC in kidney proximal tubule cells
139
study we used slices from the outermost cortex, a preparation known to consist of 85-95?/, proximal tubule cells (Aperia et al. 1981). We show a dose-dependent stimulation of P K C by PDBu with a half-maximal stimulation occurring around M, and a maximal effect at M. These results are in line with the observation by Hammerman et al. showing that PDBu binds to basolateral membranes of proximal tubules with a K,i of 6.2 x lo-@M reaching maximal binding at a concentration of M (Hammerman et al. 1986). Our choice of PDBu for this study was based on the finding that DISCUSSION kidney membranes bind this phorbol ester with This study establishes the presence of protein an affinity equal to 12-O-tetradecanoylphorbol kinase C in proximal tubule cells by immuno- 13-acetate (TPA) and higher than for other detection with an antibody raised against a phorbol esters (Hammerman et al. 1986). The consensus sequence that is present in several ontogeny of the protein kinase C system seems to PKC isoforms, and by demonstrating that the be tissue-specific. Cytosolic P K C activity has Ca2+- and phospholipid-dependent activity is been found to increase during maturation in attenuated in the presence of 100 nM of stauro- brain tissue (Hashimoto et al. 1988, Noguchi sporine. Moreover, kinase activity was trans- et al. 1988), pineal gland (Sugden 1989) and cat locatable to the membrane following phorbol visual cortex (Sheu et al. 199O), whereas liver ester treatment, while the inactive phorbol ester, and heart show a decline in P K C activity after 4a-phorbol 12,13-didecanoate did not change birth (Noguchi et al. 1988). Here we report a decline in cytosolic PKC the cellular distribution. Previous studies in the rat kidney (Hise & activity during maturation, with activities nearly Mehta 1988; Hise & Mehta 1989) report Ca2+ twice as high in immature kidney cells. A similar and phospholipid-dependent activity which but less pronounced decrease was noted by Hise accounts for about one third of the activity found & Mehta (Hise & Mehta 1988). At 10 days of age in this study. This difference is probably due to the renal cortex has a rapid growth which is differences in tissue manipulation or in cell type. mainly due to cell multiplication (Celsi et al. PKC is known to be substrate for Ca,+- 1986). We speculate that the high PKC activity activated proteinases (Adachi et al. 1990). Renal in the infant kidney plays a role in cell growth. tubule cells are rich in proteinases (Carone et al. It has been previously demonstrated (Fukuda 1979) which are released during the homogen- et al. 1991), that the PDBu-dependent inhibition ization procedure. In the previous studies on rat of Na+,K+-ATPase is absent in immature kidney, proteinase inhibitors were not used and proximal tubule cells. In view of these findings, the concentration of chelators was 10-fold lower we find it less likely that P K C plays a role in the than in the present study. Appropriate chelation regulation of ion transport in the immature of calcium is important for obtaining precise and kidney cells. Huang et al. (1990), in studies performed on stable free calcium concentrations during the assay. Kidney tissue is composed mainly of cerebellar tissue, have demonstrated differential epithelial cells from the renal tubule. However, expression of P K C isoforms during development. the various segments of the tubule have different I n view of our observation of differences in the functions and the epithelial cells that comprise apparent molecular weight of the immunothose segments differ in their transporting reactive material present in mature and immature characteristics. It is therefore important to define cells, it is tempting to speculate that different carefully the tissue preparation, so that the isoforms of P K C have different roles concerning activities obtained will reflect the situation in a the regulation of growth and ion transport. The specific segment of the nephron. Whole kidney expression of PKC isoforms and the role of PKC or whole kidney cortex preparations were used in in kidney growth are important topics for future previous studies on renal P K C activity. I n this studies.
40-day-old rats, respectively). When the cells were exposed to PDBu, M for 15 min, activity in the soluble fraction decreased significantly in both groups ( P < 0.01) and to a similar extent. This decrease in P K C activity was due to the disappearance of the enzyme from the cytosolic fraction as shown in Figure 4(b). It was also repeatedly noted that the immunoreactive material present in the cytosolic fraction of 10and 40-day-old kidneys, showed slight differences in their apparent molecular weights.
1-10
C. F. Mendez et al.
of protein kinase C in rat. 3 Neurosci 8 ( S ) , 1678-1683. ADACHI. y., hfURACHI, T . , MAKI, ISHII, K. 8( HISE,kl. & MEHTA,P. 1988. Activity of calcium/ phospholipid dependent protein kinase during rat HATANAKA, $1.1990. Calpain inhibitors block TP.4kidney development. Lzfe S c i 43 (IS), 1479-1483. dependent down-regulation of protein kinase C. HISE,M. & MEHTA,P. 1989. Activity of calciumBiomedical Res 11 (j), 313-317. sensitive phospholipid-dependent protein kinase C APERIA, A , , LARSSON, L. & ZETTERSTROM. R. 1981. following nephron loss. Kidney Int 36, 216-221. Hormonal induction of Na-K-ATPase in devel- HUANG, F., YOUNG,W.I., YOSHIDA,Y. & HUANG, K. oping proximal tubular cells. .4m 3 Physiol 241, 1990. Developmental expression of protein kinase F3-iGF360. C isozymes in rat cerebellum. Deu Brain Res 52, BARTFAI,T. 1979. Preparation of metal-chelate 121-30. complexes and the design of stead>--state kinetic LAEMZLLI, U.K. 1970. Cleavage of structural proteins experiments involving metal nucleotide complexes. during the assembly of the head of bacteriophage .4dr C'yrlir .\:urleotide Res 10, 219-242. T4. ,\'ature 227, 68&685. BALW, M. & HAYES,S. 1988. Phorbol myristate LK:, F. & COGAN,M. 1990. Role of protein kinase C acetate and dioctanoylglycerol inhibit transport in in proximal bicarbonate absorption and angiotensin rabbit proximal convoluted tubule. A m 3 Physiol signaling. A m 3 Physiol 258, F925-F933. LIVNE,A , , SARDET, C. & POUYSSEGLJR, J. 1991. T h e 254, F9-Fl4. Na+/H+ exchanger is phosphorylated in human BERTORELLO, A. & APERIA,A. 1989. Na--K+-L.\TPase platelets in response to activating agents. F E B S is an effector protein for protein kinase C in renal Lett. 284 (2), 219-22. proximal tubule cells. .4m 3 Physiol 256, M. 1986. Phorbol esterMELLAS,S. & HAMMERMAN, F3'7GF373. induced alkalinization of canine renal proximal BONEH,.I.,MANDLA,S. & TENENHOUSE, H. 1989. tubule cells. Am 3 Physiol 250, F451-F459. Phorbol myristate acetate activates protein kinase C, stimulates the phosphorylation of endogenous NISHIZUKA,Y. 1986. Studies and perspectives of protein kinase C. Science 233, 305-312. proteins and inhibits phosphate transport in mouse NOGUCHI, A., DEGUIRE, J. & ZAN?\BONI, P. 1988. renal tubules. Biochim Bioph-)Is .4cta 1012, 308-316. Protein kinase C in the developing rat liver, heart BRADFORD, h1.M 1976. A rapid and simple method and brain. Dec Pharmacol Ther 11, 37-43. for the quantitation of quantities of protein utilizing SHEU,F., KASAMATSU, T. & ROUTTENBERG, A. 1990. the principle of protein-dj-e. .4nnIyt Biorhem 72, Protein kinase C activity and substrate (Fl/GAP2-18-34, 43) phosphorylation in developing cat visual cortex. F., PETERSON, D., OPARIL, S. & PULLMAN, T. CARONE, Brain Res 524, 144-8. 19i9. Renal tubular transport and catabolism of SKOGLUND, G., PATARROYO, M., FORSBECK, K., proteins and peptides. Kidney Int 16, 271-278. NILSSON,K. & INGELMAN-SUNDBERG, M. 1988. CEJ.Sl, G., J.4KOBsSO\i, B. & APERIA, '4. 1986. Influence Evidence for separate control by phorbol esters of of age on compensatory renal growth in rats. CD18-dependent adhesion and translocation of Pediatric Res 20, 347-330. protein kinase C in U-937 cells. Cancer Res 48, 3 168-3 172. FLKLDA,Y., BERTORELLO, A. & ~\PERIA, A. 1991. SUGDEN, D. 1989. Ontogeny of pineal protein kinase C Ontogeny of the regulation of Na', K'-ATPase activity. Biochem Biophys Res Cornmun 159 (2), activity in the renal proximal tubule cell. Pedintric 701-706. Res 30 (2), 131-134. HAMMERM4N, AT., ROGERS, s.,MORRISSEY, J. & GAVIN, WANG, T. & CHAN, Y. 1990. Time- and dosedependent effects of protein kinase C on proximal J. 111 1986. Phorbol ester-stimulated phosphorylbicarbonate transport. 3 Membrane Biol 117, ation of basolateral membranes from canine 131-139. kidney. Am 3 Ph,ysiol 250, F1073-F1081. 'IZIEINMAN, E., DUBINSKY, W. & SHENOLIKAR, S. 1989. HASHIMOTO, T., ASE, K., SAWAMUR.4, s.,KIKKAWA, Regulation of the renal Na+-H+ exchanger. Hosp S.4IT0, N., TANAKA, c. & NlSHlZUK.4, Y. 1988. Prnct Off24 (3), 157-161, 164, 167. Postnatal development of a brain-specific subspecies
REFERENCES
c.,
Protein kinase C activity in rat renal proximal tubule cells C. F. M E N D E Z " ? , A. H A N S S O N ? , G. S K O G L U N D t , M. I N G E L M A N - S U N D B E R G t and A. A P E R I A " * Department of Pediatrics, St Goran's Children's Hospital, Karolinska Institute, and t Department of Physiological Chemistry, Karolinska Institute, Stockholm, Sweden MENDEZ, C. F., HANSSON, A., SKOGLUND, G., INGELMAN-SUNDBERG, M. & APERIA,A. 1992. Protein kinase C activity in rat renal proximal tubule cells. Acta Physiol Scand 146, 135-140. Received 19 December 1991 accepted 17 March 1992. ISSN 0001-6772. Departments of Pediatrics and Physiological Chemistry, Karolinska Institute, Sweden. The presence of protein kinase C (PKC) in proximal tubule cells of the rat kidney is established by means of immunodetection and by the demonstration of calcium- and phospholipid-dependent, staurosporine-inhibitable histone phosphorylation. The calcium-dependence of renal PKC is described. Maximal activation of the enzyme (178.2 and 258.8 pmol P, mg-' min-' for cytosol and membrane respectively) was achieved with 5 PM of Ca2+.Phorbol 12,13 dibutyrate (PDBu) translocated PKC from cytosol to membrane in a dose- and time-dependent fashion, while 4cc-phorbol 12,13-didecanoate produced no significant effect on translocation. Cytosolic PKC activity was compared in immature and mature tissues (10- and 40-day-old kidneys). Basal activity was found to be significantly higher (P< 0.05) in immature cells (272.8 vs. 157.5 pmol M for 15 min reduced immunoreactivity in the soluble Pi mg-' min-'). PDBu at fraction of both groups, which was accompanied by a significant decrease in kinase activity. We speculate that the high PKC activity in the infant kidney plays a role in cell growth. Key laords : ontogeny, protein kinase C, proximal tubule cells, rat kidney.
Protein kinase C (PKC) is a calcium- and phospholipid-dependent protein kinase expressed in all mammalian cells. The activation of this kinase is a common mechanism for transducing into the cell various extracellular signals, such as those from certain hormones, neurotransmitters, growth factors and other biologically active substances (Nishizuka 1986). Indirect evidence suggests that in kidney tissue P K C plays an important role in regulating specific membrane transporters. Phorbol esters have been shown to regulate phosphate and bicarbonate transport in proximal convoluted tubules (Boneh et al. 1989, Wang & Chan 1990, Liu & Cogan 1990, Baum & Hays 1988). Both Na+/H+ exchanger (Mellas & Hammerman 1986, Weinman et al. 1989, Livne
METHODS
Correspondence : Professor Anita Aperia, Department of Paediatrics, St. Goran's Children's Hospital, 112 81 Stockholm, Sweden.
Tissue preparation. Male Sprague-Dawley rats were used for the studies. The animals, aged 10 and 40 days, were anaesthetized with thiobutabarbital (Inactin; Byk-Gulden, Koblenz, FRG) 50 mg kg-l and the
et al. 1991) and Na+,K+-ATPase (Bertorello &
Aperia 1989) have been suggested to be effector proteins for P K C in the kidney. Although there is abundant indirect evidence that PKC regulates ion transporters in the renal proximal tubule, the characteristics of activation of proximal tubular P K C have not been well defined. The purpose of the present study has been to characterize protein kinase C activity in a defined population of proximal tubule cells from the rat kidney. We also compare the activity of this protein kinase in immature and mature renal tissues.
135
136
C. F . Mendez et al.
kidneys quickly removed. The organs were decapsulated and a 200 pm thick slice was taken from the outer cortex, using a Stadie-Riggs microtome. The tissue slices were homogenized by sonication, 1.5 s set 4 using a Branson sonifier 2 5 0 (Branson Corp. , C T , USA-\),in a buffer containing in mat: 1 20, EGT.4 2, EDT.4 2 , PXISF 1,2-mercaptcjethanol 10. and aprotinin 10000 K I E 1 ', p H 7.4 room temperature. In some studies where brain tissue was used for comparison, the whole brain was quickly removed and homogenized in 10 nil homogenization buffer, using a motor-dri\-en Teflon pestle and glass tube homogenizer. Separntroti of soluble and partirulntr .fiac.trons. Separation of cytosol from the particulate fraction nas achie\ed by centrifugation a t 1OOOOOg a t 2 "C, using a TST-60 rotor in a Sorvall O'TD-Combi ultracentrifuge. 'The hornogenatc was first centrifuged for 1 h and the supernatant sa\-ed as the c!-tosolic compartment where Triton-S 100 was added to give a final concentration of 1O o . The remaining pellet \+as resuspended in the homogenization buffer 0.1 Triton-X 100 b>-brief sonication and centrifuged for 1 h. The resulting supernatant was considered to he the membrane fraction. In a pilot stud!- we investigated the optimal detergent concentration necessar!. to detect PKC acti\-it!-. Addition of 1no of Triton to the c\ tosolic fraction and of 0.1 n o to the membranes elicited the highest calcium- and phospholipitldependent activity (187 and 155 pmol P, min-' mg-', respectivelv), while the actkit!- in the remaining pellet became negligible. For PKC determination. the preparations M-ere diluted at least 10 times into the assay buffer, s o that the Triton concentration never exceeded 0.1 {I,) in the actix-ity assaj-. T h e fractions were stored on ice in the presence of gl!-cerol at a final concentration of LOO,. Under these conditions, P K C activitl- did not change significantly during a i-day period. Activities were 184 and 154 pmol Pamg protein-' min-' for da!-s 1 and 5 , respectivel!-. T h e protein content was measured b!Bradford's method (Bradford lYih), using the conimercial dye reagent Bio rad (Kichmond, CL-\,US.-\). Protein kinusr C assay. The standard reaction mixture (final volume 100 ,MI) contained: Tris HCI LO m u , X'igCI, 10 m u , EGT.l 1 msi, histone €31 -50 pg, proteins 1&20 pg. Phospholipids (PS/I>) concentration was phosphatidylserine 5 pg7 dioleo! 1glycerol 0.1 p g . Unless otherwise stated, free CaL* concentration was adjusted to 0.1 mhi, using the dissociation constant given by Bartfai (1979). Basal actit-it! was measured in the presence of 0.5 m>i EGT.4 instead of calcium and in the absence of lipids. Solutions were freshly prepared. The lipids were sonicated in Tris HCI 20 mhi for 1.5-20 s. T h e reaction a a s initiated by adding [y-"P].1TP (final concentration 10 p s i ; specific activity approainiatcl! 300 cpm pmol-') and by raising the tem-
+
perature of the tube from 0 to 30 "C. In pilot experiments, the transfer of '32Pto histone was found to be linear with respect to time for the first 2 min. The reaction was therefore carried out for 2 min and stopped b!- the addition of 20,d of EDTA 125 mM ATP 5 mx1-l and the return of the tube to 0 "C. A 60 /)I aliquot from each tube was pipetted onto a 2 cm2 piece of phosphocellulose paper. Unincorporated "P was removed by washing the papers three times in phosphoric acid 75 mhi and once in ether: ethanol 1 :4, after Tvhich they were dried under a stream of air. T h e papers were placed in scintillation vials along with 5 nil scintillation liquid and the radioactivity counted in a scintillation counter. Gel rler.trophoresis. After solubilization (95 "C for 3 min), the samples were analysed on one-dimensional SDS-polyacrylamide slab gel electrophoresis, using 7.5 O O (w/v) acrylaniide, according to Laemmli (1970). .4fter electrophoresis, the gels were stained with coomassie blue, destained, dried, and exposed to Hyperfilm-MP X-ray films for autoradiography in Kodak X-Omatic intensifying screens at - 80 "C. After SDS-PAGE, proteins were electrophoretically transferred to nitrocellulose sheets. Yon-specific binding was blocked by the incubation of membranes in PBS containing lo:, bovine serum albumin. P K C was detected by means of a polyclonal rabbit antiserum raised against a s!-nthetic peptide that corresponds to a conserved region present in the a, p and y types of P K C (residues 185-204 in human type PKC) (Skoglund r t ul. 1988). After washing of the filters, the antibody was added at a 1 : 100 dilution in PBS l"/b bovine serum albumin. Specific binding was detected by using alkaline phosphatase conjugated goat anti-rabbit IgG with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) as substrate. Statistical analysis. Results are expressed either as pmol P, mg-' min-', or as a percentage ofits respective control, S S E M . When more than one mean value was to be compared, the significance was determined b>- analysis of variance (ANOVA). When a single value was compared to its control, significance was established using the Student's independent test. Reagents. Phorbol 12,13-dibutyrate (PDBu), 4aphorbol 12,13-didecanoate, Tris[hydroxymethyl]aminomethane (Tris), ethylene glycol bis(/l-aminoeth$ ether) h',N,hr',N'-tetraacetic acid (EGTA), ethplenediaminetetraacetic acid (EDTA), phenylmethyl-sulphonyl fluoride (PMSF), Histone HI (type 111 S, Sigma), L-a-phosphatidyl-L-serine, 1,2dioleoyl-sn-glycerol (C18 : l), adenosine triphosphate grade I1 (ATP) and Staurosporine were supplied by Sigma. RlgCI,, CaCI,, glycerol, Triton-X 100 and phosphoric acid came from Merck, P 81 phosphocellulose paper from Whatman, and [y3'PP]ATP (specific activity 3000 Ci mmol-I), Hybond-C nitrocellulose membranes and Hyperfilm-MP from
PKC in kidney proximal tubule cells Amersham (Amersham, UK). Alkaline phosphatase conjugated goat anti-rabbit IgG, 5-bromo-4-chloro3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) were from Bio Rad. Dulbecco's modified Eagle medium (DME) was purchased from Gibco, 2mercapto-ethanol from Carl Roth KG, and Aprotinin (Trasylola) from Bayer.
137
with the particulate fraction. PDBu M changed that distribution to 22.8 k 2.4 and 77.2+2.4y0 for cytosol and membrane, respectively. These results were confirmed by
RESULTS Cytosol from the renal cortex phosphorylates histone in a calcium- and phospholipid-dependent manner (Fig. 1). Calcium and phospholipids alone failed to phosphorylate histone. Calcium/phospholipid-dependent phosphorylation was inhibited by 100 nM staurosporine. Phosphorylation of endogenous substrates was negligible as compared to histone.
Calcium afinity T h e calcium-dependence of renal cytosolic P K C activation is shown in Figure 2. Activity (expressed as pmol Pi mg protein-' min-' k SEM) was 33.6f 8.2 in the absence of calcium. Maximal activation occurred within a narrow range of free calcium concentrations, approximately 1-5 ,UM. Maximal PKC activity was 178.2 f8.2 for the cytosol and 258.8 f 17.2 for the membrane. Under calcium-saturating conditions, the activity in brain cytosol was 1230 f51 pmol Pi mg-' min-l.
Fig. 1. Protein kinase C-catalysed phosphorylation of histone. Histone HI (50yg) was incubated with cytosolic preparation in the presence and the absence of PKC cofactors and of 100 nM staurosporine, under the conditions specified in Methods. The samples were then analysed by SDS-PAGE and autoradiography. 300
1
Efect of phorbol 12,13-dibutyrate on the subcellular distribution of protein kinase C Phorbol 12,13-dibutyrate translocates PKC from the cytosol to the membrane. When the tissue was incubated in the presence of PDBu, at concentrations ranging from lo-* to M for 15 min, PKC activity decreased in the cytosol and increased accordingly in the particulate fraction in a dose-dependent manner. PDBu M significantly reduced the activity in the cytosol to 2 6 f 4 y 0 of control (157.5k23.5 and 40.9 f8.5 pmol Pi mg-' min-l for control and PDBu M, respectively). These differences were significant at the 1yo level for cytosol, and at the 5% level for membrane, using the ANOVA test (Fig. 3a). I n the control slices, 62.1 f5.9% of the total activity was recovered in the cytosolic fraction, while the remaining 37.9f 5.9% was associated
0
1
10
100
200
1000
[Free calcium] pM
Fig. 2. Calcium dependence of PKC activation. Protein kinase C activity was assayed in the soluble (-O-) and particulate (-0-) fractions under conditions described in Methods. Free calcium concentration was adjusted to the values indicated, using the dissociation constant given by Bartfai (1979). Results are shown as the meankSEM of three independent experiments in which duplicates were
obtained.
138
C. F . Mendez et a1 A
-*-**-
A
150
T
-**-
-7
-6 OJ
Log [PDBu] (M)
200
I
1
Conlrol
~
PDBu
Control PDBu
_
10 days
_
_
40 days
Fig. 4. (a) Tissue slices from 10- and 40-day-old kidnel-s nere incubated in the presence of M PDBu for 15 min or the vehicle alone C!-tosolic P K C activity was determined as described in Xlethods. Results are expressed as the mean S E M of four independent experiments performed in duplicates. ** Significantl!- different from control value ( P < 0.01); *significantly different from homologous control ( P < 0.0.5). (b) Immunoblot analysis of immature and mature proximal tubule cells. Tissue was homogenized as described in Methods. Samples (0.3 mg) were electrophoretically separated and transferred to nitrocellulose paper. Blot was probed with a polyclonal antibody that recognizes several isoforms of PKC.
(a),
0 1
2.5
5
15
Time (min)
Fig. 3. Etfect of phorbol 12,l.i-dibut!-rate on the suhcellular distribution of protein hinase C. (a) Tissuc slices were incubated with vehicle or different PDBu concentrations during 1.i min a t room temperature. fractions nere Soluble ( w ) and particulate (0) obtained and assa!ed for P K C activit!-. as described in .2lethods. .ictivit! is espressed as ( I , ) of control. (b) 'l'ime-course of the PDBu effect. Slices nere exposed to 10 ' \i PIIBu for the times indicated and P K C activit! was determined in both c! tosol (-O-) m d Control experiments were permembrane (-0 tormed for each time-point, and PKC activit! is espressed as 'lo of its respective control value. Each point represents the mean of three to five esperimmts performed in duplicates 5 SEhI. T h e level of signific:?ncc V J S 0.01 for cytosol and 0.05 for membrme, using the .i\Ol-:l test.
immunodetection (data not shon n). T h e translocation TI as time-dependent, as sho\-r n in Figure
3 (b) T h c inacti\e phorbol ester, 4z-phorbol 12,13didecmoate produced no significant effect o n
(m).
PKC translocation, when used a t a concentration of 1 0 - " ~during 15 m i n (143.3f27.6 pmol P, mg-' min-'). Protein kinuse C activity in the developing kidnq I n this protocol we compared cytosolic PKC actkit!- i n proximal tubule cells from immature kidneys ( 10-day-old rats) a n d mature kidneys (40-day-old rats). As shown i n Figure 4(a), PKC activity was significantly higher ( P < 0.05) in immature than i n mature cells, (272.8 2 24.6 vs. 157.52 & 23.5 pmol Pi mg-' min-' for 10- a n d
PKC in kidney proximal tubule cells
139
study we used slices from the outermost cortex, a preparation known to consist of 85-95?/, proximal tubule cells (Aperia et al. 1981). We show a dose-dependent stimulation of P K C by PDBu with a half-maximal stimulation occurring around M, and a maximal effect at M. These results are in line with the observation by Hammerman et al. showing that PDBu binds to basolateral membranes of proximal tubules with a K,i of 6.2 x lo-@M reaching maximal binding at a concentration of M (Hammerman et al. 1986). Our choice of PDBu for this study was based on the finding that DISCUSSION kidney membranes bind this phorbol ester with This study establishes the presence of protein an affinity equal to 12-O-tetradecanoylphorbol kinase C in proximal tubule cells by immuno- 13-acetate (TPA) and higher than for other detection with an antibody raised against a phorbol esters (Hammerman et al. 1986). The consensus sequence that is present in several ontogeny of the protein kinase C system seems to PKC isoforms, and by demonstrating that the be tissue-specific. Cytosolic P K C activity has Ca2+- and phospholipid-dependent activity is been found to increase during maturation in attenuated in the presence of 100 nM of stauro- brain tissue (Hashimoto et al. 1988, Noguchi sporine. Moreover, kinase activity was trans- et al. 1988), pineal gland (Sugden 1989) and cat locatable to the membrane following phorbol visual cortex (Sheu et al. 199O), whereas liver ester treatment, while the inactive phorbol ester, and heart show a decline in P K C activity after 4a-phorbol 12,13-didecanoate did not change birth (Noguchi et al. 1988). Here we report a decline in cytosolic PKC the cellular distribution. Previous studies in the rat kidney (Hise & activity during maturation, with activities nearly Mehta 1988; Hise & Mehta 1989) report Ca2+ twice as high in immature kidney cells. A similar and phospholipid-dependent activity which but less pronounced decrease was noted by Hise accounts for about one third of the activity found & Mehta (Hise & Mehta 1988). At 10 days of age in this study. This difference is probably due to the renal cortex has a rapid growth which is differences in tissue manipulation or in cell type. mainly due to cell multiplication (Celsi et al. PKC is known to be substrate for Ca,+- 1986). We speculate that the high PKC activity activated proteinases (Adachi et al. 1990). Renal in the infant kidney plays a role in cell growth. tubule cells are rich in proteinases (Carone et al. It has been previously demonstrated (Fukuda 1979) which are released during the homogen- et al. 1991), that the PDBu-dependent inhibition ization procedure. In the previous studies on rat of Na+,K+-ATPase is absent in immature kidney, proteinase inhibitors were not used and proximal tubule cells. In view of these findings, the concentration of chelators was 10-fold lower we find it less likely that P K C plays a role in the than in the present study. Appropriate chelation regulation of ion transport in the immature of calcium is important for obtaining precise and kidney cells. Huang et al. (1990), in studies performed on stable free calcium concentrations during the assay. Kidney tissue is composed mainly of cerebellar tissue, have demonstrated differential epithelial cells from the renal tubule. However, expression of P K C isoforms during development. the various segments of the tubule have different I n view of our observation of differences in the functions and the epithelial cells that comprise apparent molecular weight of the immunothose segments differ in their transporting reactive material present in mature and immature characteristics. It is therefore important to define cells, it is tempting to speculate that different carefully the tissue preparation, so that the isoforms of P K C have different roles concerning activities obtained will reflect the situation in a the regulation of growth and ion transport. The specific segment of the nephron. Whole kidney expression of PKC isoforms and the role of PKC or whole kidney cortex preparations were used in in kidney growth are important topics for future previous studies on renal P K C activity. I n this studies.
40-day-old rats, respectively). When the cells were exposed to PDBu, M for 15 min, activity in the soluble fraction decreased significantly in both groups ( P < 0.01) and to a similar extent. This decrease in P K C activity was due to the disappearance of the enzyme from the cytosolic fraction as shown in Figure 4(b). It was also repeatedly noted that the immunoreactive material present in the cytosolic fraction of 10and 40-day-old kidneys, showed slight differences in their apparent molecular weights.
1-10
C. F. Mendez et al.
of protein kinase C in rat. 3 Neurosci 8 ( S ) , 1678-1683. ADACHI. y., hfURACHI, T . , MAKI, ISHII, K. 8( HISE,kl. & MEHTA,P. 1988. Activity of calcium/ phospholipid dependent protein kinase during rat HATANAKA, $1.1990. Calpain inhibitors block TP.4kidney development. Lzfe S c i 43 (IS), 1479-1483. dependent down-regulation of protein kinase C. HISE,M. & MEHTA,P. 1989. Activity of calciumBiomedical Res 11 (j), 313-317. sensitive phospholipid-dependent protein kinase C APERIA, A , , LARSSON, L. & ZETTERSTROM. R. 1981. following nephron loss. Kidney Int 36, 216-221. Hormonal induction of Na-K-ATPase in devel- HUANG, F., YOUNG,W.I., YOSHIDA,Y. & HUANG, K. oping proximal tubular cells. .4m 3 Physiol 241, 1990. Developmental expression of protein kinase F3-iGF360. C isozymes in rat cerebellum. Deu Brain Res 52, BARTFAI,T. 1979. Preparation of metal-chelate 121-30. complexes and the design of stead>--state kinetic LAEMZLLI, U.K. 1970. Cleavage of structural proteins experiments involving metal nucleotide complexes. during the assembly of the head of bacteriophage .4dr C'yrlir .\:urleotide Res 10, 219-242. T4. ,\'ature 227, 68&685. BALW, M. & HAYES,S. 1988. Phorbol myristate LK:, F. & COGAN,M. 1990. Role of protein kinase C acetate and dioctanoylglycerol inhibit transport in in proximal bicarbonate absorption and angiotensin rabbit proximal convoluted tubule. A m 3 Physiol signaling. A m 3 Physiol 258, F925-F933. LIVNE,A , , SARDET, C. & POUYSSEGLJR, J. 1991. T h e 254, F9-Fl4. Na+/H+ exchanger is phosphorylated in human BERTORELLO, A. & APERIA,A. 1989. Na--K+-L.\TPase platelets in response to activating agents. F E B S is an effector protein for protein kinase C in renal Lett. 284 (2), 219-22. proximal tubule cells. .4m 3 Physiol 256, M. 1986. Phorbol esterMELLAS,S. & HAMMERMAN, F3'7GF373. induced alkalinization of canine renal proximal BONEH,.I.,MANDLA,S. & TENENHOUSE, H. 1989. tubule cells. Am 3 Physiol 250, F451-F459. Phorbol myristate acetate activates protein kinase C, stimulates the phosphorylation of endogenous NISHIZUKA,Y. 1986. Studies and perspectives of protein kinase C. Science 233, 305-312. proteins and inhibits phosphate transport in mouse NOGUCHI, A., DEGUIRE, J. & ZAN?\BONI, P. 1988. renal tubules. Biochim Bioph-)Is .4cta 1012, 308-316. Protein kinase C in the developing rat liver, heart BRADFORD, h1.M 1976. A rapid and simple method and brain. Dec Pharmacol Ther 11, 37-43. for the quantitation of quantities of protein utilizing SHEU,F., KASAMATSU, T. & ROUTTENBERG, A. 1990. the principle of protein-dj-e. .4nnIyt Biorhem 72, Protein kinase C activity and substrate (Fl/GAP2-18-34, 43) phosphorylation in developing cat visual cortex. F., PETERSON, D., OPARIL, S. & PULLMAN, T. CARONE, Brain Res 524, 144-8. 19i9. Renal tubular transport and catabolism of SKOGLUND, G., PATARROYO, M., FORSBECK, K., proteins and peptides. Kidney Int 16, 271-278. NILSSON,K. & INGELMAN-SUNDBERG, M. 1988. CEJ.Sl, G., J.4KOBsSO\i, B. & APERIA, '4. 1986. Influence Evidence for separate control by phorbol esters of of age on compensatory renal growth in rats. CD18-dependent adhesion and translocation of Pediatric Res 20, 347-330. protein kinase C in U-937 cells. Cancer Res 48, 3 168-3 172. FLKLDA,Y., BERTORELLO, A. & ~\PERIA, A. 1991. SUGDEN, D. 1989. Ontogeny of pineal protein kinase C Ontogeny of the regulation of Na', K'-ATPase activity. Biochem Biophys Res Cornmun 159 (2), activity in the renal proximal tubule cell. Pedintric 701-706. Res 30 (2), 131-134. HAMMERM4N, AT., ROGERS, s.,MORRISSEY, J. & GAVIN, WANG, T. & CHAN, Y. 1990. Time- and dosedependent effects of protein kinase C on proximal J. 111 1986. Phorbol ester-stimulated phosphorylbicarbonate transport. 3 Membrane Biol 117, ation of basolateral membranes from canine 131-139. kidney. Am 3 Ph,ysiol 250, F1073-F1081. 'IZIEINMAN, E., DUBINSKY, W. & SHENOLIKAR, S. 1989. HASHIMOTO, T., ASE, K., SAWAMUR.4, s.,KIKKAWA, Regulation of the renal Na+-H+ exchanger. Hosp S.4IT0, N., TANAKA, c. & NlSHlZUK.4, Y. 1988. Prnct Off24 (3), 157-161, 164, 167. Postnatal development of a brain-specific subspecies
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