494
KIDNEY
[29]
can be taken into account explicitly in the static head method by an appropriate control run (see Figs. 4 and 5). As in the steady state method, internal leaks will result in an underestimate of coupling and charge stoichiometries by the static head procedure. Concluding Remarks In conclusion, it is worth rcemphasizing that stoichiometric determinations must be tailored to the coupled transport system under investigation and to the experimental system in which it is found. When characterizing a new transporter in our laboratory we typically carry out an activation method experiment first. This experiment provides us not only with a stoichiometric determination but also with information concerning the activator concentration dependence of the transporter. We then attempt the direct method and the steady state method since these are often technically easier experiments than the static head method. Since most of the vesicle systems we work with are not capable of maintaining electrochemical gradients over time our steady state experiments cannot be interpreted quantitatively~; however, they can provide qualitative evidence for energetic coupling by demonstrating concentrative uptake of substrate due to activator or electrical gradients,s,21 Finally, we attempt the static head method. Although this method is often technically ditficult, it provides a direct thermodynamic demonstration of coupled transport and is free from many of the problems associated with other methods of measuring stoichiometry. 2~ y. Fukuhara and R. J. Turner, Am. J. Physiol. 245, F374 (1983).
[29] P h o s p h a t e T r a n s p o r t in E s t a b l i s h e d R e n a l Epithelial Cell Lines By J. BmER, K. MALMSTR6M, S. R~SHKIN, and H. MURER Introduction Phosphate (P.~ is reabsorbed from the lumenal fluid of the renal proximal tubule via a secondary active transport mechanism involving sodium/ phosphate cotransport across the microviUar brush border membrane. Numerous properties of this transport system, such as its kinetic characteristics and functional changes in response to hormones (e.g., parathyroid METHODS IN ENZYMOLOGY, VOL. 191
Copynsht © 1990 by A~lemi~ Press, Inc. All rishts ofreprodu~on in any form reserved.
[29]
PHOSPHATE TRANSPORT IN CULTURED CELLS
495
hormone), acid/base changes, and nutritional stimuli (e.g., phosphate diet), have been described. ~-3 For the elucidation of the regulatory mechanisms underlying the functional changes of the Na/Prcotransport system, isolated renal cortex brush border membrane vesicles (see also Chapter 26 of this volume for isolation procedures) have been used extensively. In particular, isolated renal cortex brush border membrane vesicles have been used to test for protein phosphorylation4,s and ADP ribosylation6-s as possible post-translational regulatory events. This kind of approach, however, has turned out to be unsuccessful, mainly because of the lack of (yet unknown) cellular factors in the isolated membranes and because the isolated membrane vesicles are closed and of a right-side-out orientation.9 This makes it difficult if not impossible to study posttranslational modifications of the Na/Pi cotransporter at its cytosolic domain. In the past few years, cultured renal cells have opened new possibilities to explore the regulatory events underlying the functional changes of the Na/Pi-cotransport system. This chapter will describe the basic methodology for measuring Pi transport in established renal epithelial cells and in apical membrane vesicles prepared thereof. It is by these methods that it has become possible to describe the basic mechanisms involved in the regulation of the Na/Prcotransport system by parathyroid hormone and by the concentration of extracellular phosphate. So far such studies have been performed with the following renal cell lines (see Table I): OK cells, derived from an adult American opossum~°; LLC-PK~ cells, derived from pig kidneyH; JTC-12 cells, derived from a monkey kidney~2; and MDCK cells, derived from dog kidney./3 Similarly, regulation of the Na/Pi-cotransport system can be studied in primary cultures of proximal tubular epithelial cells, as demonstrated by t j._p. Bonjour and J. Caverzasio, Rev. Physiol. Biochem. Pharmacol. 100, 162 (1984). 2 p. Gmaj and H. Muter, Physiol. Rev. 66, 36 (1986). a C. L. Mizgala and G. A. Quamm¢, Physiol. Rev. 65, 431 (1985). 4 j. Biber, K. Malmstr0m, V. Scalera, and H. Muter, PfluegersArch. 398, 221 (1983). 5 M. R. Hammerman and K. A. I-Iruska, J. Biol. Chem. 257, 992 (1982). 6 M. R. Hammerman, Am. J. Physiol. 251, F385 (1986). 7 S. A. Kempson and N. P. Curthoys, Am. J. Physiol. 245, C449 (1983). s p. Gmaj, J. Biber, S. Angielski, G. Stange, and H. Muter, Pfluegers Arch. 400, 60 (1984). 9 W. Haase, A. Schaefer, H. Muter, and R. Kline, Biochem. J. 172, 57 (1978). to H. Koyama, C. Goodpastute, M. M. Miller, R. L. Teplitz, and A. D. Riggs, In Vitro 14, 239 (1978). H R. N. Hull, W. R. Cherry, and G. W. Weaver, In Vitro 12, 670 (1976). t2 T. Takaoka, H. Katsuta, M. Endo, K. Sato, and H. Okumura, Jpn. J. Exp. Med. 32, 351 (1962). 13C. R. Gaush, W. C. Hard, and T. F. Smith, Proc. Soc. Exp. Biol. Med. 122, 931 (1966).
496
KIDNEY
[29]
TABLE I BASIC CHARACTERISTICS OF N A / P i COTRANSPORT IN ESTABLISHED RENAL EPITHELIAL CELL LINES a
Kinetic parametersc Cell lind OK OK (filter) Apical Basolateral LLC-PK1
JTC-12 MDCK
~Km for Pi(/zM)
V~
Na/Pi transport regulated by
86 _+ 8 94 _+ 7
10.3 + 1.8; 37 ° (1) 2.1 + 0.2; 37 o (3)
Parathyroid hormone (1, 2) and Pi deprivation (4)
340 _ 50 5000
1.1 + 0.04; 37* (13) 2.3 (13)
95 5- 15 125 196 5- 33
0.83 5- 0.14; 22* (5) 1.56; 37"C (9) 0.82 ± 0.07; 37* (10)
120
0.152; 37* (11)
Pi deprivation (12)
0.53 -_. 0.05; 37 ° (10)
Pi deprivation (10)
374 + 48
Pi deprivation
The data are taken from the references given in parantheses: (1) K. Malmstr6m and H. Murer, Am. J. Physio1251, C23 (1986); (2) J. A. Cole, S. L. Eber, R. E. Poelling, P. IC Thorne, and L. R. Forte, Am. J. Physiol. 253, E221 (1987); (3) J. Caverzasio, R. Rizzoli, and J.-P. Bonjour, J. Biol. Chem. 261, 3233 (1986); (4) J. Biber, J. Forgo, and H. Murer, Am. J. Physiol. 255, C155 (1988); (5) J. Biber, C. D. A. Brown, and H. Murer, Biochim. Biophys. Acta 735, 325 (1983); (6) J. Caverzasio, C. D. A. Brown, J. Biber, J.-P. Bonjour, and H. Murer, Am. J. Physiol. 248, F122 (1985); (7) J. Biber and H. Murer, Am. J. Physiol. 249, C430 (1985); (8) C. A. Rabito, Am. J. Physiol. 245, F22 (1983); (9) L. Noronha-Blob, C. Filburn, and B. Sacktor, Arch. Biochem. Biophys. 234, 265 (1984); (I0) B. Escoubet, K. Djabali, and C. Amiel, Am. J. Physiol. 256, C322 (1989); (11) Y. Takuwa and E. Ogata, Biochem. J. 230, 715 (1985); (12) Y. Takuwa, Y. Takeachi, and E. Ogata, Clin. Sci. 71, 307 (1986); (13) S. Reshkin, J. Forgo, and H. Murer, PfluegersArch., in press (1990). b If not indicated otherwise, cells have been grown to confluency in Petri dishes. c Vm~ is given in nanomoles Pi per milligram per minute at 22 or 37 ° as indicated. d All parameters were determined at an extracellular pH of 7.2 to 7.4.
various laboratories. 14-17 The methodological background for cultivating and using primary cell cultures of proximal tubular cells will, however, not be described here. For the successful use of an established renal epithelial cell line some premises must be considered. 18,~9 ~4M. A. Wagar, J. Seto, S. D. Chung, S. Hiller-Grohol, and M. Taub, £ Cell. Physiol. 124, 411 (1985). ,s L. Noronha-Blob and B. Sacktor, J. Biol. Chem. 261, 2164 (1986). ~e Y. Kinoshita, M. Fukase, A. Miyauchl, M. Takenaka, M. Nakada, and T. Fujita,Endocrinology (Baltimore) 119, 1954 (1986). ~7G. Friedlander and C. Amid, J. Biol. Chem. 264, 3935 (1989). ~sj. S. Handier, Kidneylnt. 30, 208 (1986). ~9L. M. Sakhrani and L. G. Fine, Miner. ElectrolyteMetab. 9, 276 (1983).
[29]
PHOSPHATE TRANSPORT IN CULTURED CELLS
497
1. Morphologically, cells must be polarized (apical pole being morphologically separated from the basolateral pole by tight junctions). As a consequence, an asymmetric distribution of various enzymatic and transport activities should be observed. 2. From (1) it follows that vectorial transport of water and other solutes should be observed. Very often vectorial solute transport into the space between the plastic surface and the basolateral membranes leads to the formation of the so-called domes. 3. Identification of metabolic and transport functions and hormonal responses typical for specific nephron segments2°m helps to classify the respective cell line as a model system for studying the cellular functions of a specific nephron segment. It should be mentioned here that no established renal cell line exists so far that exactly resembles a certain nephron segment
Transport Studies with intact Cell Monolayers Comments
1. Since any activity of cells in culture might vary depending on the "cell age" and/or on the state of confluency, it is important to establish first the exact culture conditions (e.g., days in culture) which will allow one to investigate a particular question. To illustrate that the rate ofNa/P i cotransport is not necessarily proportional to the state of confluency, initial Na/Pi cotransport in the cell lines LLC-PKI and OK is shown (Fig. 1) as a function of days in culture. 2. The time dependency of the uptake of a solute should be established as well. Because phosphate is metabolized rapidly within the cell, transport measurements should be performed within the linear range of uptake and in as short a time as possible. As illustrated in Fig. 2, the uptake of Pi into OK cells grown in Petri dishes is linear for at least 6 min. In order to completely exclude any possible contribution by metabolic activities, the basic observations, such as apparent Km and Vm~ values, as obtained with intact cell nonolayers, should be confirmed with isolated plasma membrane (apical or basolateral) vesicles. 3. Transport is best measured at 37 °. Yet results obtained at 25 ° are qualitatively similar. 4. When transport experiments are performed with cells grown as a monolayer on Petri dishes, uptake of the added solute occurs preferentially 20 F. Morel and A. Doucet, Physiol. Rev. 66, 377 (1986). 21 G. Wirthenson and W. G. Guder, Physiol. Rev. 66, 469 (1986).
498
KIDNEY
[29] i
A
2000
i
B
18
7.c
16
I
E
.E
E
14
1500
.E @
12 10
~
~
1000
8 o
E c
6 500
4
E
2i I
I
2
I
I
4
d a y s of culture
I
I
6
1
2
I
6
I
8
110
d a y s of culture
FIG. 1. Influence of days in culture on net sodium-dependent Pi uptake in OK (A) and LLC-PKI cells (B). At day 0 the cells were seeded as described in Refs. 25 and 26. In both cases confluency was reached a t ~ day 5. Media were changed every second day.
through the apical membrane. In order to measure uptake of a solute through both the apical and the basolateral membranes, cells are best grown on filters, such that the growth medium has access to both sides of the cell. = With the appropriate controls for tightness, it will be possible to measure transport through the apical or basolateral membrane separately. This approach has been used to study polarized Na/P i cotransport in LLC-PK123 and OK cells. 24
Experimental Procedures Cells Grown in Petri Dishes. Cells are grown to confluency in plastic culture dishes using standard protocols. 25-~s We routinely used dishes (35-mm diameter) containing at confluency approximately 0.5 nag of total cellular protein. 22 R. E. Steele, A. S. Preston, J. P. Johnson, and J. S. Handler, Am. J. Physiol. 251, C136 (1986). 23 C. A. Rabito, Am. J. Physiol. 245, F22 (1983). 24 S. Reshkin and H. Murer, Pfluegers Arch., in press (1990). 25 K. Malmstr6m and H. Muter, Am. J. Physiol. 251, C23 (1986). 26j. BiDer, C. D. A. Brown, and H. Muter, Biochim. Biophys. Acta 735, 325 (1983).
[29]
PHOSPHATE TRANSPORT IN CULTURED CELLS
499
I r"
50
~ 40 E
d, -
,/
30
t-
,/
e~
oe -
20
e~
-~ E e-
10
3
6
9
12
Time (min)
FIG. 2. Time course of Pi uptake into OK cells grown in Petri dishes. Pi transport was determined either in the presence of NaCI (@) or N-methyl-v-glucamine (MGA) (O). (From Malmstr6m and Muter. 25)
The growth medium is aspirated off and the cell monolayer is quickly rinsed twice with incubation buffer lacking the transport solute (in mM): 137 NaC1 (replaced by 137 N-methyl-D-glucamine-HC1 for sodium-independent transport), 5.4 KCI, 2.8 CaC12, 1.2 MgSO4, and 10 HEPES/Tris, pH 7.4. Transport is initiated by adding 1 ml of the above medium containing 0.1 mM KH232po4 (0.5 to 1.0 #Ci/ml). The Petri dish is agitated slowly (at the desired temperature) and at the given time point the radioactive medium is aspirated off and the monolayer is washed four times with 2 ml of an ice-cold stop solution (in raM): 137 NaCI, 14 Tris-HC1, pH 7.4. To measure total radioactivity taken up, the cells are solubilized with 1 ml of 0.5% Triton X-100. Homogeneous solub'flization is normally achieved within 30 min, such that 100 to 300 #1 of the homogenate can be used directly for scintillation counting. Total protein is determined by a modified Lowry procedure.27 The washing procedure is controlled (blank value) by adding 1 ml of the isotope-containing buffer, which afterward is aspirated off immediately. As an example, the background for 32p obtained after four washes is of the order of 200 to 500 clam (compared to - 15,000 cpm of total uptake) and cannot be reduced further by more extensive washing. Cells Grown on Permeant Filter Supports. For this purpose MillicellCM filter inserts (12-ram diameter, 0.45-gm pore size; Millipore, Bedford, 2: j. R. Dulley and P. A. Grieve, Anal. Biochem. 64, 176 (1975).
500
KIDNEY
[29]
MA) are used. If cells will not grow on bare filters, filters must be covered with a layer of collagen. To grow OK cells on Millicell-CM filters, rat tail collagen (R-type, 2 mg/ml; Serva, Heidelberg, FRG) is diluted four-fold in sterile ethanol:water (1 : 1) and 50/tl of this solution is added under sterile conditions, such that the total area is equally covered. Collagen-coated filter inserts are placed in a 24-well culture plate and permitted to dry overnight. The next day, 300 ~1 of a suspension of trypsinized cells ( - 5 × 105 cells/ml) is added to each filter insert. Place the inserts in the incubator for 2 hr. After this period, the medium is aspirated off and 500/~1 of fresh medium is added to each side of the insert. If necessary, the medium should be changed every second day. Prior to the experiment the monolayer is checked at the light microscopic level for intactness. Tests for monolayer permeability: There are a number of ways to measure the tightness of the monolayer during its development and after confluency. Transepithelial diffusion can be estimated by measuring the movement across the monolayer of large nontransported/nonmetabolized compounds, such as inulin. 2s On the other hand, transepithelial movement of 22Na can be measured with bare filters and filters containing an intact monolayer.29 Usually substantial movement of radioactivity occurs within the first 5 min through bare filters, which is equilibrated in about 30 min. In contrast, when an intact monolayer has been formed only about 5% of the radioactivity can be found in the transcompartment after 30 min. If the cell monolayer is incubated for 30 rain in a calcium-free medium (containing in addition 1 mM EGTA) prior to the addition of the radioactivity, evidence that the impermeability results from the formation of tight junctions can be obtained. If high enough, the determination of transepithelial electrical resistance would be another way to monitor monolayer integrity. Transport studies: With some modifications, to facilitate the use of the filter inserts, the procedure and solutions are essentially as described for the transport studies with cells grown in plastic Petri dishes (see above). Just prior to uptake, the filter inserts are removed from the growth medium and gently rinsed on both sides twice in sodium-free uptake solution. Then uptake solutions (but without the substrate) are added to the appropriate filter insert compartment (500/tl to each side). Uptake is initiated by mixing 50/tl of the same uptake solution containing the I 1-fold amount of the desired substrate to one or the other compartment. Uptake is stopped and unspecifically bound radioactivity is effectively removed from the relatively high plastic surface area by rapid aspiration of 28M. J. Caplan, H. C. Anderson, G. E. Palade, and J. D. Jamieson, Cell (Cambridge, Mass.) 46, 623 (1986). 29j. G. Haggerty, N. Agarwal, R. F. Reilly, E. A. Adelberg, and C. W. Slayman, Proc. Natl. Acad. Sci. U.S.A. 85, 6797 (1988).
[29]
PHOSPHATE TRANSPORT IN CULTURED CELLS
501
the uptake solution and careful rinsing of the filter and filter insert in an ice-cold isotonic solution containing a high concentration (up to 100 mM) of the substrate. Total radioactivity incorporated into the monolayer is assessed by counting the whole filter insert which has been mixed with scintillation cocktail. Non-specific binding (blanks) is assessed by measuring zero time uptake by starting uptake and immediately aspirating off the uptake solution. Total counts in the uptake solution are measured for each filter by removing 10 ~1 of the complete uptake solution.
Isolation of Apical Membrane Vesicles Comments
1. For successful isolation of apical membrane vesicles it is necessary to start with a large quantity of cells ( - 2 × 108). Usually, sufficient membrane vesicles (-0.5 nag of protein) can be obtained when starting with cells cultivated either in one or two roller bottles (each 840 cm 3) or in one or two large Petri dishes (each 625 cm2). Alternatively, cells might also be cultivated on microcarrier beads such as Cytodex (Pharr~acia; Piscataway, NJ) or Biosilon (Nunc). 2. Characterization of the isolated apical membranes is best performed by the determination of enzymatic activities for which the cellular localization is well established. For example, in the established cell lines LLC-PK~ and MDCK the enzyme alkaline pbosphatase (EC 3.1.3.1) and leucine aminopeptidase (EC 3.4.11.2) have been localized by immunohistochemistry in the apical membrane-a°,31whereas the Na+K+-ATPase (EC 3.6.1.3) is exclusively localized in the basolateral membrane. 32-34 For the isolation of transport-competent apical membrane vesicles from the cell lines OK and LLC-PKt the following two procedures have been used successfully. 2s,3s The enzymatic characterization of these preparations is illustrated in Table II. Other very similar procedures have been 3oC. A. Rabito, J. I. Kreisberg, and D. Wight, £ Biol. Chem. 259, 574 (1984). 31 D. Louvard, Proc. Natl. Acad. Sci. U.S.A. 77, 4132 (1980). 32j. W. Mills, A. D. C. MeKnight, J. H. l)ayer, and D. A. Ausiello, Am. J. Physiol. 236, C157 (1979). 33 j. F. Lanb, P. Odgen, and N. L. Simmons, Biochim. Biophys. Acta 644, 333 (1981). C. A. Rabito and R. Tehao, Am. £ Physiol. 2311,C43 (1980). 3~C. D. A. Brown, M. Bodmer, J. Biber, and H. Muter, Biochim. Biophys. Acta 769, 471 (1984).
502
KIDNEY
129]
-H
÷1 ÷l
+1
÷1 ÷l
+1
+l +1
÷l
~ ÷1
,...1 m
r..)
-'d o cl z .
KIDNEY
[29]
can be taken into account explicitly in the static head method by an appropriate control run (see Figs. 4 and 5). As in the steady state method, internal leaks will result in an underestimate of coupling and charge stoichiometries by the static head procedure. Concluding Remarks In conclusion, it is worth rcemphasizing that stoichiometric determinations must be tailored to the coupled transport system under investigation and to the experimental system in which it is found. When characterizing a new transporter in our laboratory we typically carry out an activation method experiment first. This experiment provides us not only with a stoichiometric determination but also with information concerning the activator concentration dependence of the transporter. We then attempt the direct method and the steady state method since these are often technically easier experiments than the static head method. Since most of the vesicle systems we work with are not capable of maintaining electrochemical gradients over time our steady state experiments cannot be interpreted quantitatively~; however, they can provide qualitative evidence for energetic coupling by demonstrating concentrative uptake of substrate due to activator or electrical gradients,s,21 Finally, we attempt the static head method. Although this method is often technically ditficult, it provides a direct thermodynamic demonstration of coupled transport and is free from many of the problems associated with other methods of measuring stoichiometry. 2~ y. Fukuhara and R. J. Turner, Am. J. Physiol. 245, F374 (1983).
[29] P h o s p h a t e T r a n s p o r t in E s t a b l i s h e d R e n a l Epithelial Cell Lines By J. BmER, K. MALMSTR6M, S. R~SHKIN, and H. MURER Introduction Phosphate (P.~ is reabsorbed from the lumenal fluid of the renal proximal tubule via a secondary active transport mechanism involving sodium/ phosphate cotransport across the microviUar brush border membrane. Numerous properties of this transport system, such as its kinetic characteristics and functional changes in response to hormones (e.g., parathyroid METHODS IN ENZYMOLOGY, VOL. 191
Copynsht © 1990 by A~lemi~ Press, Inc. All rishts ofreprodu~on in any form reserved.
[29]
PHOSPHATE TRANSPORT IN CULTURED CELLS
495
hormone), acid/base changes, and nutritional stimuli (e.g., phosphate diet), have been described. ~-3 For the elucidation of the regulatory mechanisms underlying the functional changes of the Na/Prcotransport system, isolated renal cortex brush border membrane vesicles (see also Chapter 26 of this volume for isolation procedures) have been used extensively. In particular, isolated renal cortex brush border membrane vesicles have been used to test for protein phosphorylation4,s and ADP ribosylation6-s as possible post-translational regulatory events. This kind of approach, however, has turned out to be unsuccessful, mainly because of the lack of (yet unknown) cellular factors in the isolated membranes and because the isolated membrane vesicles are closed and of a right-side-out orientation.9 This makes it difficult if not impossible to study posttranslational modifications of the Na/Pi cotransporter at its cytosolic domain. In the past few years, cultured renal cells have opened new possibilities to explore the regulatory events underlying the functional changes of the Na/Pi-cotransport system. This chapter will describe the basic methodology for measuring Pi transport in established renal epithelial cells and in apical membrane vesicles prepared thereof. It is by these methods that it has become possible to describe the basic mechanisms involved in the regulation of the Na/Prcotransport system by parathyroid hormone and by the concentration of extracellular phosphate. So far such studies have been performed with the following renal cell lines (see Table I): OK cells, derived from an adult American opossum~°; LLC-PK~ cells, derived from pig kidneyH; JTC-12 cells, derived from a monkey kidney~2; and MDCK cells, derived from dog kidney./3 Similarly, regulation of the Na/Pi-cotransport system can be studied in primary cultures of proximal tubular epithelial cells, as demonstrated by t j._p. Bonjour and J. Caverzasio, Rev. Physiol. Biochem. Pharmacol. 100, 162 (1984). 2 p. Gmaj and H. Muter, Physiol. Rev. 66, 36 (1986). a C. L. Mizgala and G. A. Quamm¢, Physiol. Rev. 65, 431 (1985). 4 j. Biber, K. Malmstr0m, V. Scalera, and H. Muter, PfluegersArch. 398, 221 (1983). 5 M. R. Hammerman and K. A. I-Iruska, J. Biol. Chem. 257, 992 (1982). 6 M. R. Hammerman, Am. J. Physiol. 251, F385 (1986). 7 S. A. Kempson and N. P. Curthoys, Am. J. Physiol. 245, C449 (1983). s p. Gmaj, J. Biber, S. Angielski, G. Stange, and H. Muter, Pfluegers Arch. 400, 60 (1984). 9 W. Haase, A. Schaefer, H. Muter, and R. Kline, Biochem. J. 172, 57 (1978). to H. Koyama, C. Goodpastute, M. M. Miller, R. L. Teplitz, and A. D. Riggs, In Vitro 14, 239 (1978). H R. N. Hull, W. R. Cherry, and G. W. Weaver, In Vitro 12, 670 (1976). t2 T. Takaoka, H. Katsuta, M. Endo, K. Sato, and H. Okumura, Jpn. J. Exp. Med. 32, 351 (1962). 13C. R. Gaush, W. C. Hard, and T. F. Smith, Proc. Soc. Exp. Biol. Med. 122, 931 (1966).
496
KIDNEY
[29]
TABLE I BASIC CHARACTERISTICS OF N A / P i COTRANSPORT IN ESTABLISHED RENAL EPITHELIAL CELL LINES a
Kinetic parametersc Cell lind OK OK (filter) Apical Basolateral LLC-PK1
JTC-12 MDCK
~Km for Pi(/zM)
V~
Na/Pi transport regulated by
86 _+ 8 94 _+ 7
10.3 + 1.8; 37 ° (1) 2.1 + 0.2; 37 o (3)
Parathyroid hormone (1, 2) and Pi deprivation (4)
340 _ 50 5000
1.1 + 0.04; 37* (13) 2.3 (13)
95 5- 15 125 196 5- 33
0.83 5- 0.14; 22* (5) 1.56; 37"C (9) 0.82 ± 0.07; 37* (10)
120
0.152; 37* (11)
Pi deprivation (12)
0.53 -_. 0.05; 37 ° (10)
Pi deprivation (10)
374 + 48
Pi deprivation
The data are taken from the references given in parantheses: (1) K. Malmstr6m and H. Murer, Am. J. Physio1251, C23 (1986); (2) J. A. Cole, S. L. Eber, R. E. Poelling, P. IC Thorne, and L. R. Forte, Am. J. Physiol. 253, E221 (1987); (3) J. Caverzasio, R. Rizzoli, and J.-P. Bonjour, J. Biol. Chem. 261, 3233 (1986); (4) J. Biber, J. Forgo, and H. Murer, Am. J. Physiol. 255, C155 (1988); (5) J. Biber, C. D. A. Brown, and H. Murer, Biochim. Biophys. Acta 735, 325 (1983); (6) J. Caverzasio, C. D. A. Brown, J. Biber, J.-P. Bonjour, and H. Murer, Am. J. Physiol. 248, F122 (1985); (7) J. Biber and H. Murer, Am. J. Physiol. 249, C430 (1985); (8) C. A. Rabito, Am. J. Physiol. 245, F22 (1983); (9) L. Noronha-Blob, C. Filburn, and B. Sacktor, Arch. Biochem. Biophys. 234, 265 (1984); (I0) B. Escoubet, K. Djabali, and C. Amiel, Am. J. Physiol. 256, C322 (1989); (11) Y. Takuwa and E. Ogata, Biochem. J. 230, 715 (1985); (12) Y. Takuwa, Y. Takeachi, and E. Ogata, Clin. Sci. 71, 307 (1986); (13) S. Reshkin, J. Forgo, and H. Murer, PfluegersArch., in press (1990). b If not indicated otherwise, cells have been grown to confluency in Petri dishes. c Vm~ is given in nanomoles Pi per milligram per minute at 22 or 37 ° as indicated. d All parameters were determined at an extracellular pH of 7.2 to 7.4.
various laboratories. 14-17 The methodological background for cultivating and using primary cell cultures of proximal tubular cells will, however, not be described here. For the successful use of an established renal epithelial cell line some premises must be considered. 18,~9 ~4M. A. Wagar, J. Seto, S. D. Chung, S. Hiller-Grohol, and M. Taub, £ Cell. Physiol. 124, 411 (1985). ,s L. Noronha-Blob and B. Sacktor, J. Biol. Chem. 261, 2164 (1986). ~e Y. Kinoshita, M. Fukase, A. Miyauchl, M. Takenaka, M. Nakada, and T. Fujita,Endocrinology (Baltimore) 119, 1954 (1986). ~7G. Friedlander and C. Amid, J. Biol. Chem. 264, 3935 (1989). ~sj. S. Handier, Kidneylnt. 30, 208 (1986). ~9L. M. Sakhrani and L. G. Fine, Miner. ElectrolyteMetab. 9, 276 (1983).
[29]
PHOSPHATE TRANSPORT IN CULTURED CELLS
497
1. Morphologically, cells must be polarized (apical pole being morphologically separated from the basolateral pole by tight junctions). As a consequence, an asymmetric distribution of various enzymatic and transport activities should be observed. 2. From (1) it follows that vectorial transport of water and other solutes should be observed. Very often vectorial solute transport into the space between the plastic surface and the basolateral membranes leads to the formation of the so-called domes. 3. Identification of metabolic and transport functions and hormonal responses typical for specific nephron segments2°m helps to classify the respective cell line as a model system for studying the cellular functions of a specific nephron segment. It should be mentioned here that no established renal cell line exists so far that exactly resembles a certain nephron segment
Transport Studies with intact Cell Monolayers Comments
1. Since any activity of cells in culture might vary depending on the "cell age" and/or on the state of confluency, it is important to establish first the exact culture conditions (e.g., days in culture) which will allow one to investigate a particular question. To illustrate that the rate ofNa/P i cotransport is not necessarily proportional to the state of confluency, initial Na/Pi cotransport in the cell lines LLC-PKI and OK is shown (Fig. 1) as a function of days in culture. 2. The time dependency of the uptake of a solute should be established as well. Because phosphate is metabolized rapidly within the cell, transport measurements should be performed within the linear range of uptake and in as short a time as possible. As illustrated in Fig. 2, the uptake of Pi into OK cells grown in Petri dishes is linear for at least 6 min. In order to completely exclude any possible contribution by metabolic activities, the basic observations, such as apparent Km and Vm~ values, as obtained with intact cell nonolayers, should be confirmed with isolated plasma membrane (apical or basolateral) vesicles. 3. Transport is best measured at 37 °. Yet results obtained at 25 ° are qualitatively similar. 4. When transport experiments are performed with cells grown as a monolayer on Petri dishes, uptake of the added solute occurs preferentially 20 F. Morel and A. Doucet, Physiol. Rev. 66, 377 (1986). 21 G. Wirthenson and W. G. Guder, Physiol. Rev. 66, 469 (1986).
498
KIDNEY
[29] i
A
2000
i
B
18
7.c
16
I
E
.E
E
14
1500
.E @
12 10
~
~
1000
8 o
E c
6 500
4
E
2i I
I
2
I
I
4
d a y s of culture
I
I
6
1
2
I
6
I
8
110
d a y s of culture
FIG. 1. Influence of days in culture on net sodium-dependent Pi uptake in OK (A) and LLC-PKI cells (B). At day 0 the cells were seeded as described in Refs. 25 and 26. In both cases confluency was reached a t ~ day 5. Media were changed every second day.
through the apical membrane. In order to measure uptake of a solute through both the apical and the basolateral membranes, cells are best grown on filters, such that the growth medium has access to both sides of the cell. = With the appropriate controls for tightness, it will be possible to measure transport through the apical or basolateral membrane separately. This approach has been used to study polarized Na/P i cotransport in LLC-PK123 and OK cells. 24
Experimental Procedures Cells Grown in Petri Dishes. Cells are grown to confluency in plastic culture dishes using standard protocols. 25-~s We routinely used dishes (35-mm diameter) containing at confluency approximately 0.5 nag of total cellular protein. 22 R. E. Steele, A. S. Preston, J. P. Johnson, and J. S. Handler, Am. J. Physiol. 251, C136 (1986). 23 C. A. Rabito, Am. J. Physiol. 245, F22 (1983). 24 S. Reshkin and H. Murer, Pfluegers Arch., in press (1990). 25 K. Malmstr6m and H. Muter, Am. J. Physiol. 251, C23 (1986). 26j. BiDer, C. D. A. Brown, and H. Muter, Biochim. Biophys. Acta 735, 325 (1983).
[29]
PHOSPHATE TRANSPORT IN CULTURED CELLS
499
I r"
50
~ 40 E
d, -
,/
30
t-
,/
e~
oe -
20
e~
-~ E e-
10
3
6
9
12
Time (min)
FIG. 2. Time course of Pi uptake into OK cells grown in Petri dishes. Pi transport was determined either in the presence of NaCI (@) or N-methyl-v-glucamine (MGA) (O). (From Malmstr6m and Muter. 25)
The growth medium is aspirated off and the cell monolayer is quickly rinsed twice with incubation buffer lacking the transport solute (in mM): 137 NaC1 (replaced by 137 N-methyl-D-glucamine-HC1 for sodium-independent transport), 5.4 KCI, 2.8 CaC12, 1.2 MgSO4, and 10 HEPES/Tris, pH 7.4. Transport is initiated by adding 1 ml of the above medium containing 0.1 mM KH232po4 (0.5 to 1.0 #Ci/ml). The Petri dish is agitated slowly (at the desired temperature) and at the given time point the radioactive medium is aspirated off and the monolayer is washed four times with 2 ml of an ice-cold stop solution (in raM): 137 NaCI, 14 Tris-HC1, pH 7.4. To measure total radioactivity taken up, the cells are solubilized with 1 ml of 0.5% Triton X-100. Homogeneous solub'flization is normally achieved within 30 min, such that 100 to 300 #1 of the homogenate can be used directly for scintillation counting. Total protein is determined by a modified Lowry procedure.27 The washing procedure is controlled (blank value) by adding 1 ml of the isotope-containing buffer, which afterward is aspirated off immediately. As an example, the background for 32p obtained after four washes is of the order of 200 to 500 clam (compared to - 15,000 cpm of total uptake) and cannot be reduced further by more extensive washing. Cells Grown on Permeant Filter Supports. For this purpose MillicellCM filter inserts (12-ram diameter, 0.45-gm pore size; Millipore, Bedford, 2: j. R. Dulley and P. A. Grieve, Anal. Biochem. 64, 176 (1975).
500
KIDNEY
[29]
MA) are used. If cells will not grow on bare filters, filters must be covered with a layer of collagen. To grow OK cells on Millicell-CM filters, rat tail collagen (R-type, 2 mg/ml; Serva, Heidelberg, FRG) is diluted four-fold in sterile ethanol:water (1 : 1) and 50/tl of this solution is added under sterile conditions, such that the total area is equally covered. Collagen-coated filter inserts are placed in a 24-well culture plate and permitted to dry overnight. The next day, 300 ~1 of a suspension of trypsinized cells ( - 5 × 105 cells/ml) is added to each filter insert. Place the inserts in the incubator for 2 hr. After this period, the medium is aspirated off and 500/~1 of fresh medium is added to each side of the insert. If necessary, the medium should be changed every second day. Prior to the experiment the monolayer is checked at the light microscopic level for intactness. Tests for monolayer permeability: There are a number of ways to measure the tightness of the monolayer during its development and after confluency. Transepithelial diffusion can be estimated by measuring the movement across the monolayer of large nontransported/nonmetabolized compounds, such as inulin. 2s On the other hand, transepithelial movement of 22Na can be measured with bare filters and filters containing an intact monolayer.29 Usually substantial movement of radioactivity occurs within the first 5 min through bare filters, which is equilibrated in about 30 min. In contrast, when an intact monolayer has been formed only about 5% of the radioactivity can be found in the transcompartment after 30 min. If the cell monolayer is incubated for 30 rain in a calcium-free medium (containing in addition 1 mM EGTA) prior to the addition of the radioactivity, evidence that the impermeability results from the formation of tight junctions can be obtained. If high enough, the determination of transepithelial electrical resistance would be another way to monitor monolayer integrity. Transport studies: With some modifications, to facilitate the use of the filter inserts, the procedure and solutions are essentially as described for the transport studies with cells grown in plastic Petri dishes (see above). Just prior to uptake, the filter inserts are removed from the growth medium and gently rinsed on both sides twice in sodium-free uptake solution. Then uptake solutions (but without the substrate) are added to the appropriate filter insert compartment (500/tl to each side). Uptake is initiated by mixing 50/tl of the same uptake solution containing the I 1-fold amount of the desired substrate to one or the other compartment. Uptake is stopped and unspecifically bound radioactivity is effectively removed from the relatively high plastic surface area by rapid aspiration of 28M. J. Caplan, H. C. Anderson, G. E. Palade, and J. D. Jamieson, Cell (Cambridge, Mass.) 46, 623 (1986). 29j. G. Haggerty, N. Agarwal, R. F. Reilly, E. A. Adelberg, and C. W. Slayman, Proc. Natl. Acad. Sci. U.S.A. 85, 6797 (1988).
[29]
PHOSPHATE TRANSPORT IN CULTURED CELLS
501
the uptake solution and careful rinsing of the filter and filter insert in an ice-cold isotonic solution containing a high concentration (up to 100 mM) of the substrate. Total radioactivity incorporated into the monolayer is assessed by counting the whole filter insert which has been mixed with scintillation cocktail. Non-specific binding (blanks) is assessed by measuring zero time uptake by starting uptake and immediately aspirating off the uptake solution. Total counts in the uptake solution are measured for each filter by removing 10 ~1 of the complete uptake solution.
Isolation of Apical Membrane Vesicles Comments
1. For successful isolation of apical membrane vesicles it is necessary to start with a large quantity of cells ( - 2 × 108). Usually, sufficient membrane vesicles (-0.5 nag of protein) can be obtained when starting with cells cultivated either in one or two roller bottles (each 840 cm 3) or in one or two large Petri dishes (each 625 cm2). Alternatively, cells might also be cultivated on microcarrier beads such as Cytodex (Pharr~acia; Piscataway, NJ) or Biosilon (Nunc). 2. Characterization of the isolated apical membranes is best performed by the determination of enzymatic activities for which the cellular localization is well established. For example, in the established cell lines LLC-PK~ and MDCK the enzyme alkaline pbosphatase (EC 3.1.3.1) and leucine aminopeptidase (EC 3.4.11.2) have been localized by immunohistochemistry in the apical membrane-a°,31whereas the Na+K+-ATPase (EC 3.6.1.3) is exclusively localized in the basolateral membrane. 32-34 For the isolation of transport-competent apical membrane vesicles from the cell lines OK and LLC-PKt the following two procedures have been used successfully. 2s,3s The enzymatic characterization of these preparations is illustrated in Table II. Other very similar procedures have been 3oC. A. Rabito, J. I. Kreisberg, and D. Wight, £ Biol. Chem. 259, 574 (1984). 31 D. Louvard, Proc. Natl. Acad. Sci. U.S.A. 77, 4132 (1980). 32j. W. Mills, A. D. C. MeKnight, J. H. l)ayer, and D. A. Ausiello, Am. J. Physiol. 236, C157 (1979). 33 j. F. Lanb, P. Odgen, and N. L. Simmons, Biochim. Biophys. Acta 644, 333 (1981). C. A. Rabito and R. Tehao, Am. £ Physiol. 2311,C43 (1980). 3~C. D. A. Brown, M. Bodmer, J. Biber, and H. Muter, Biochim. Biophys. Acta 769, 471 (1984).
502
KIDNEY
129]
-H
÷1 ÷l
+1
÷1 ÷l
+1
+l +1
÷l
~ ÷1
,...1 m
r..)
-'d o cl z .
