Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney

467

Biochimica et Biophysica Acta, 5 4 1 ( 1 9 7 8 ) 4 6 7 - - 4 8 2 © Elsevier/North-Holland Biomedical Press

BBA 28573

HORMONE-SPECIFIC RESPONSES AND BIOSYNTHESIS OF SULFOLIPIDS IN CELL LINES DERIVED FROM MAMMALIAN KIDNEY *

I N E O I S H I Z U K A a, K E I K O T A D A N O YOSHITAKA NAGAI b

a, N A O K A Z U

N A G A T A b, Y U K I O N I I M U R A a a n d

a Department of Biochemistry, Teikyo University School of Medicine, 2-11-1 Kaga, ltabashi-ku, Tokyo 173 and b Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashi-ku, Tokyo 173 (Japan) (Received January 16th, 1978)

Summary The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule. The level of 3':5'-cyclic AMP in JTC-12.P3 (monkey kidney)cells increased in 2 min as much as 2.5--5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1--34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin EL (14 pM) and isopropylnorepinephrine (10 pM) were also found to increase the concentration of cyclic AMP by more than 3 0 - a n d 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell. In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E1 was observed. The cell lines JTC-12, MDCK and MDBK, when incubated with H235SO4, * A part of this work was presented at the 49th Annual Meeting of Japanese Biochemical Society (Sept. 1976 in Sapporo), abstract: Seikagaku Vol. 48 (1976) p. 645 and at the 19th Japanese Congress on Biochemistry of Lipids (June 1977 in Kyoto), abstract: Proceedings for the Japanese Congress on Biochemistry of Lipids, VoL 19, pp. 159--162.

468 incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate. The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.

Introduction The cellular population in the kidney is very heterogeneous. As is well known, epithelial cells in the kidney have each specific cellular functions inherent to the portion of a nephron [1]. So it is often difficult, especially in biochemical studies, to get meaningful knowledge by analyzing whole kidney or even an anatomically defined portion such as renal cortex or medulla. One approach to solve this problem is to examine the homogeneous kidney cell lines retaining physiological and biochemical features of the cells originated from the specific part of nephron. In the Laboratory of Cancer Cell research in the Institute of Medical Science, the University of T o k y o (Director, Prof. H. Katsuta), many epithelial cell lines were isolated from mammalian kidney [2]. The morphological characteristics of these cell lines, however, cannot be the only criteria to ascribe these epithelial cell lines to a certain portion of nephron. In the present study, we measured the cellular levels of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and examined the responses to hormones in several cell lines isolated from mammalian kidney. Among various hormones, parathyroid hormone, arginine vasopressin, calcitonin and cathecholamines have been shown to stimulate renal adenylate cyclase. It was proposed that the adenylate cyclase sensitive to parathyroid hormone is limited to cells in cortex [3--5], but that of calcitonin is distributed also in the outer medulla [6]. Arginine vasopressin, on the other hand, stimulates adenylate cyclase of either outer and inner medulla [7]. As the second specific marker of kidney epithelial cells, the metabolism of sulfatides in these cell lines was studied. Sulfatides are the main glycolipid component characteristic for the kidney [8,9] and the concentration on dry weight basis was found to be higher in inner medulla and papilla portion of the bovine kidney as compared to cortex or outer medulla [8]. Fibroblasts are known not to synthesize sulfatides [10,11]. Although the exact distribution in nephron of such sulfolipids is n o t known, it may serve as a good marker of tubular cell, because sulfatides are known to be the c o m p o n e n t of cell membranes specific to the tissue, e.g. myelin [12] or spermatogenic cells of mammals [13]. Materials and Methods Bovine parathyroid hormone trichloroacetic acid powder (Wilson, 263 units/ mg) and synthetic bovine type (1--34) tetratriaconta peptide (3840 units/mg, Beckman), porcine calcitonin (60 MRCU/mg, Armour), DL-isopropylnorepinephrine (Sigma), synthetic fll-24 corticotropin (Cortrosyn, Daiichi Seiyaku, Tokyo), arginine vasopressin (20 units/mg, Park Davies), cyclic [3H]AMP (20 Ci/mmol, Radiochemical Center, Amhersham), 1-methyl-3-isobutylxanthine

469 (IBMX, Aldrich) and carrier-free H2~5SO4 in 0.05 M HC1 (Isotope Kyokai, T o k y o ) were obtained commercially. Glucagon, the cyclodextrin derivative of prostaglandin E1 and cyclic AMP radioimmunoassay kit were gifts from Novo Industry, Ono Yakuhin (Osaka) and Yamasa Shoyu (Choshi), respectively. Cells, media and culture conditions. The cell line JTC-12.P3 was isolated from cyanomologous m o n k e y kidney cells [2,14--16]. The inclusion of " P 3 " in the name of cell lines indicates those cultured in the synthetic media containing so-called non-essential amino acids in addition to essential amino acid. No evident alteration has been found in the morphology of JTC-12.P3 as compared to JTC-12 [16]. In this paper this cell line was cultured in the medium containing serum. The MDCK cell line, isolated from the whole normal adult dog kidney [17], was provided by Dr. J. Leighton (Cancer Bioassay Laboratory, Department of Pathology, The Medical College of Pennsylvania) and maintained in culture by serial passage in serum-free medium [18] or as frozen aliquots of cells in complete medium with 10% (v/v) dimethyl sulfoxide. MDBK cells were isolated by Madin and Darby [19] from bovine kidney. Fibroblasts were prepared from rat lung (RLG) or guinea pig kidney (GPK-1) [20]. The culture medium used throughout the experiments consisted of DM-160 [2] ( K y o k u t o Seiyaku, T o k y o ) supplemented with 10 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) buffer, pH 7.4, kanamycin sulfate (60 pg/ml, Meiji Seiyaku, T o k y o ) and 10% Donner Calf Serum (Flow Laboratory, Rockvill, Md.), except for JTC-12 cell line, which was cultured with 5% serum. Cells were grown at 37°C in stationary monolayer culture. Usually cells grown to confluency (10 s cells or 50 pg protein/cm 2) were used for experiments. The medium was renewed twice a week. TD-40 type of flasks (40 cm 2) with silicone rubber stoppers (Ikemoto Rika, T o k y o ) [21] were used. Cyclic A M P determination. Cells were detached from flasks by treatment with 200 units/ml of trypsin (Mochida Seiyaku, T o k y o ) by incubation for several minutes at room temperature. The suspension of 2 • 104/cm 2 cells was transferred to new TD-40 flasks that contained 10 ml of growth medium and airtight stoppered, usually 4 or 5 days before use for experiments. For most studies in which cyclic AMP was measured, the medium containing serum was removed and equilibrated with 4 ml of serum-free growth medium containing 10 mM theophylline for 10 min or 2 mM IBMX for 30 min [22]. Compounds to be tested were then added and the incubation was continued at 37°C. The duration of culture and special conditions employed for each of the experiment are described in the legend to the figures. To terminate the incubation the medium was quickly removed by aspiration and cultures and aliquots of medium (1.0 ml) were immediately treated with 1.5 ml of 7.5% and 0.1 ml of 75% cold trichloroacetic acid, respectively, to extract cyclic AMP. Tracer a m o u n t of cyclic [3H]AMP (5000 cpm, 0.25 pmol) was added for measuring loss of cyclic AMP during its extraction and fractionation. The cells were collected by suspending in trichloroacetic acid with a rubber policeman and the flask was washed with an additional 1 ml of trichloroacetic acid. After freezing and thawing twice at --70°C, extract of cyclic AMP was obtained by centrifuging at 1000 × g for 10 min at 4 °C. The precipitate was

470 dissolved in 0.1 M NaOH and heated at 80°C for 1 h for the measurement of protein [23] using bovine serum albumin as the standard. After trichloroacetic acid was removed by shaking three times with 7 ml portions of water-saturated diethyl ether, the deproteinized supernatant was applied on a Dowex 50-W X8 column (H ÷ form, 0.5 X 8 cm) and eluted with water. The first 4 ml, which did not contain any radioactivities, was discarded and the following 4 ml was used for the cyclic AMP assay. This fraction was lyophilized and the residue was dissolved in 0.5--2.0 ml of 50 mM Tris • HC1 buffer, pH 7.4, containing 10 mM MgC12, and aliquots were assayed for cyclic AMP by the m e t h o d of Gilman [ 24]. In several experiments, it was confirmed that prior incubation for 30 min at 37°C with 10 munits/ml of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) from beef heart (Boehringer, Mannheim) destroyed completely the cyclic AMP content in the sample within the limits of sensitivity of the assay (0.125 pmol). The recovery of cyclic AMP from the cells estimated by added tracer was 70- 80%. The medium was lyophilized after trichloroacetic acid was removed by extraction with 5 volumes of water-saturated diethyl ether three times. The residue was redissolved in 0.25 ml of water and 50-pl aliquots of the solution were used for the estimation of cyclic AMP by radioimmunoassay [25]. All experiments were repeated more than twice with a minimum of duplicate samples. Results are expressed as the mean -+S.E.M. Significance of the differences was examined by Student's t-test and P values less than 0.05 were taken as significant. Sulfolipid labelling pattern with H23sSO¢. Carrier-free H23sSO4 was diluted with 100 mM phosphate buffer, pH 7.2, as described previously [26]. Aliquots of the above solution, containing 40 pCi of radioisotope, were added to the medium (the final concentration, 5 pCi/ml) at a nearly confluent stage of cell culture, and incubated for 24 h. Cell sheets were washed with phosphatebuffered saline and harvested by scraping with a rubber policeman. Then the cell suspensions were centrifuged at 80 × g and washed three times with phosphate-buffered saline. The cell pellets, 2--3 mg of protein, were extracted with 90 pl of water and 1.9 ml of a mixture of chloroform and methanol (2 : 1, v/v), sonicated for a few min, then 1.0 ml of methanol was added and centrifuged at 1000 × g for 10 min. The cell residue was suspended in 0.1 M NaOH and stored for protein determination. The extract was evaporated to dryness, redissolved in 4 ml of chloroform/methanol (2 : 1, v/v) and partitioned with 1 ml of 0.88% KC1. The lower organic layer was partitioned twice more with the "theoretical upper layer" as described elsewhere [12]. The final organic layer was then evaporated to dryness in a rotary evaporator. The residue was redissolved in a small a m o u n t of chloroform/methanol (2 : 1, v/v) mixture and analyzed by thin-layer chromatography of silica gel G, developed with the solvent system: chloroform/methanol/acetone/acetic acid/water (10 : 2 : 4 : 2 : 1, v/v). The autoradiogram was taken as described [12]. The reference aSS-labelled sulfatides and seminolipids were prepared from rat brain [12] and testis [13]. Synthetic cholesterol [3SS]sulfate was the gift from Dr. Iwamori of this institute [27]. The aSS-labelled kidney sulfolipids containing sulfatides, cholesterol sulfate and the sulfate ester of lactosylceramide were prepared according to the m e t h o d described previously [12].

471 Results

Cultures o f epithelial kidney cell lines and fibroblasts The morphological appearance of cultured epithelial cell lines isolated from mammalian kidney, i.e. MDCK [17,18,28], MDBK [19], JTC-12.P3 [2,14--16] are in compliance with those already reported (Fig. la). MDCK is unique in forming domes by active transport of water from apical to basolateral surface of the cell layer. Both R L G and GPK-1 cell lines are morphologically fibroblasts isolated from rat lung and guinea pig kidney, respectively, and lost contact inhibition (Figs. l b and c). Effects o f various hormones on intracellular levels o f cyclic A M P in cell lines JTC-12 cells were incubated with a number of hormones for 5 min in the presence of 10 mM theophylline (Table I). The concentrations of each agonist applied were those which produced maximal cyclic AMP accumulation in the isolated rat or rabbit kidney tissue (parathyroid hormone, porcine calcitonin, arginine vasopressin, and prostaglandin El) [29--31] or fat cells (corticotropin and glucagon or isopropylnorepinephrine) [32]. Significant increases in cyclic AMP levels in JTC-12 cells were caused by parathyroid hormone, prostaglandin El, and isopropylnorepinephrine. The response to prostaglandin E1 (40-fold) was more dramatic than the response to parathyroid hormone (Wilson) 4--10fold) or L-isopropylnorepinephrine (9-fold). Experiments on another cell lines are also listed in Table I. MDBK has a high basal level of cyclic AMP (61 pmol/ mg protein) and none of the hormone tested caused any significant increase. MDCK had also relatively higher basal levels of cyclic AMP and responded to arginine vasopressin and prostaglandin El with increase of 12- and 22-fold, respectively. Slight but significant increase of cyclic AMP in MDCK was also induced by parathyroid hormone and isopropylnorepinephrine. Prostaglandin E1 enhanced cyclic AMP levels in both GPK-1 and RLG, but either parathyroid hormone, porcine calcitonin or arginine vasopressin did not show such a stimulatory effect on these fibroblastic cell lines. It is noted that porcine calcitonin, which is know to stimulate adenylate cyclase of cortex as well as of outer medulla had no effect on cyclic AMP in these cell lines. Effects o f parathyroid hormone as a function o f dose or time on cyclic A M P metabolism in kidney cell lines in the presence o f theophylline or I B M X The effect of parathyroid hormones on cyclic AMP metabolism in JTC-12 cells in the presence of theophylline was analyzed further. A significant increase of cyclic AMP by parathyroid hormone (Wilson) was evident at a concentration of as low as 0.05 unit/ml and the effect was maximal at 0.5--5 units/ml (Fig. 2). Synthetic parathyroid hormone (1-34) [33] showed a very similar dose vs. response relationship. Thus the sensitivity of the cells to parathyroid hormone seems to be similar or even more pronounced as compared to that of renal cortical adenylate cyclase of rat, which has been shown to be stimulated detectably with 0.14 unit/ml of parathyroid hormone with log-linear response over the range of 0.14--1.13 units/ml [34]. The time course of the effect of parathyroid hormone on intracellular levels of cyclic AMP was as shown in Fig. 3. Increase of cyclic AMP was clear already

i~! ¸

~Q

U

473 TABLE I THE EFFECTS FIBROBLASTS

OF

VARIOUS

HORMONES

ON EPITHELIAL

CELL LINES FROM

KIDNEY

AND

C u l t u r e s o f e a c h cell line w e r e g r o w n to c o n f l u e n c y . T h e c y c l i c A M P c o n t e n t s in t h e m o n o l a y e r o f cells w e r e m e a s u r e d a f t e r 5 m i n o f i n c u b a t i o n w i t h h o r m o n e s in t h e p r e s e n c e o f 10 m M t h e o p h y l l i n e . E a c h m e a n _+ S.E. is o f t r i p l i c a t e i n c u b a t i o n s . Incubation conditions

Control P a r a t h y r o i d h o r m o n e (Wilson), 1 u n i t / m l P o r c i n e c a l c i t o n i n , 50 m u n i t s / m l Arginine vasopressin, 5 munits/ml I s o p r o p y l n o r e p i n e p h r i n e , 10 pM P r o s t a g l a n d i n E l , 23 # M

Cellular cyclic A M P ( p m o l / m g p r o t e i n w i t h e a c h cell line) JTC-12

MDCK

MDBK

GPK-1

RLG

11.7 _+0.2 54.9 ±0.8 *,** 12.1 ±1.0 * * * 12.1 _+1.1 t 112.2 +6.9 * 465.7 _+13.8 * , i t

44.0 ±1.7 63.3 t6.3 * 43.7 +3.8 533.2 _+26.9 * 86.9 -+2.4 * 987.3 _+59.1 *

60.9 ±5.8 83.2 -+9.6 65.5 ±10.1 88.3 +11.8 n.d.

22.4 _+2.1 13.5 +2.1 21.0 -+1.3 13.9 -+1.9 n.d.

27.2 _+3.3 24.4 _+1.7 17.5 _+1.8 26.1 _+1.2 n.d.

80.9 +6.8

90.4 -+11.5 *

795.0 _+30.6 *

0 . 0 1 f r o m t h e c o n t r o l value. I n c u b a t i o n t i m e , 2 rain. Porcine calcitonin, 100 munits/ml. A r g i n i n e v a s o p r e s s i n , 10 m u n i t s / m l . P r o s t a g l a n d i n E l , 14 p M . n.d., n o t d e t e r m i n e d .

* t' ~

** *** t tt

at 1 min of test incubation and reached maximum at 2 min. If no phosphodiesterase inhibitor was added, the increase of cyclic AMP by parathyroid hormone was only 30% of basal concentration and the response was quite transient. The maximal increase was at 30 s to 1 min after the addition of parathyroid hormone and returned to basal level already at 2 min (Fig. 3a). In the presence of 10 mM theophylline, the increase of cyclic AMP by synthetic parathyroid hormone (1--34) was maximal at 1--2 min of incubation and started to level off faster than that by parathyroid hormone {Wilson) (Fig. 3b). The absen'ce of hormonal stimulation of any kind in MDBK cells and the small parathyroid hormone effect in MDCK cells (Table I) could be attributed to experimental technique in at least two ways. (A) Incomplete inhibition of cyclic nucleotide phosphodiesterase activity [35]. (B) Hormonal stimulation has been shown to result in rapid efflux of cyclic AMP. To solve these problems 2 mM IBMX was used as an inhibitor of phosphodiesterase activity instead of 10 mM theophyUine and cellular as well as media cyclic AMP contents were assayed. The time course of cyclic AMP accumulation in response to 1.9 units/ ml of parathyroid hormone (1-34) in JTC-12 cells is shown in Fig. 4. The increase of cellular cyclic AMP was clear at 1 min of incubation. The maximum level was reached in 2--5 min, forming a plateau in contrast to the results with F i g . I . M o r p h o l o g y o f J T C - 1 2 cells a n d f i b r o b l a s t s . (a) J T C - 1 2 , c o n f l u e n t . ( b ) G P K - 1 , 1 d a y b e f o r e c o n f l u e n c y , (c) R L C , 1 d a y b e f o r e e o n f l u e n e y . P h a s e c o n t r a s t , XlO0. F o r t h e m e d i a a n d c u l t u r e c o n d i t i o n s , see M a t e r i a l s a n d M e t h o d s .

474

60

.t-.!

/

,

40 PTH ( Wdson )

=8 20

0

~/ /-

0

!

//

/

1

/

!

./

00

j

01

,95

1

5

PTH. U / m l F i g . 2. T h e e f f e c t s o f v a r i o u s c o n c e n t r a t i o n s o f p a r a t h y r o i d h o r m o n e i n t h e i n c u b a t i o n m e d i a o n c y c l i c A M P a c c u m u l a t i o n i n J T C - 1 2 cells i n t h e p r e s e n c e o f t h e o p h y l l i n e . T h e c u l t u r e a n d a s s a y c o n d i t i o n s f o r cyclic AMP were similar to those of Table I except for the concentration of cyclic AMP was measured 2 rain after the addition of parathyroid hormone. • -e with parathyroid hormone (1o34) and o ...... o parathyroid hormone (Wilson), both in the presence of 10 mM theophylline.

10 mM theophylline (Fig. 3). The cyclic AMP secreted into media in 10 min was less than 10% of cellular cyclic AMP. Both the cellular cyclic AMP concentration and the a m o u n t of cyclic AMP effluxed into media were determined as listed in Table II. The use of 2 mM IBMX as the phosphodiesterase inhibitor instead of theophylline revealed that cyclic AMP levels of MDBK cells were elevated slightly but significantly by porcine calcitonin (1.7-fold), arginine vasopressin (2.3-fold), isopropylnorepi-

.

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,

0

,

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,

2 8 Incubation time, rain

,

10

.

0

.

.

.

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2 4 6 8 Incubation time, rain

10

F i g . 3. T i m e c o u r s e o f c h a n g e s o f c e l l u l a r c y c l i c A M P l e v e l s i n c u l t u r e o f J C T - 1 2 cells a f t e r i n c u b a t i o n w i t h a, p a r a t h y r o i d h o r m o n e ( W i l s o n ) a n d b , p a r a t h y r o i d h o r m o n e ( 1 - 3 4 ) i n t h e p r e s e n c e o f t h e o p h y l l i n e . C e l l s w e r e g ~ o w n f o r 5 d a y s t o c o n f l u e n c e . T h e a v e r a g e c e l l u l a r p r o t e i n w a s a p p r o x . 5 5 / ~ g / c m 2. T h e amount of hormone used was, parathyroid hormone (Wilson), 1 unit/ml and parathyroid hormone (1-34), 1.9 units/ml. , and ...... , the values with and without hormone; e, with 10 mM theophylline and 9, w i t h o u t a n y p h o s p h o d i e s t e r a s e i n h i b i t o r i n t h e m e d i a .

475

I0 S

1oo 0"" 0 @

.c_

E r80 u

I

lO ~

104

o

.c Q.

60

E E

cL 40 D-" <~ .u

/

'~ 100

{

Q.

b r l PTH [ ] ContrOt

~, 80

3"20

'~" 40

o_ uR

0

2 4 6 8 Incubation time, min

10

2o o

7 1 2 3 Days in culture

F i g . 4. E f f e c t of p a r a t h y r o i d h o r m o n e on t h e c y c l i c A M P levels of J T C - 1 2 cells a n d t h e i n c u b a t i o n m e d i u m a f t e r v a r i o u s p e r i o d s o f e x p o s u r e o f t h e cells t o p a r a t h y r o i d h o r m o n e ( 1 - 3 4 ) in t h e p r e s e n c e o f I B M X . C u l t u r e a n d a s s a y c o n d i t i o n s w e r e as d e s c r i b e d in t h e t e x t e x c e p t f o r t h e p h o s p h o d i e s t e r a s e i n h i b i t o r , w h i c h w a s c h a n g e d to 2 m M I B M X i n s t e a d o f 10 m M t h e o p h y l l i n e . I n c u b a t i o n m e d i a a n d cell s h e e t s w e r e f i x e d a n d a n a l y z e d s e p a r a t e l y f o r c y c l i c A M P . T h e v a l u e s s h o w n are t r i p l i c a t e a v e r a g e s w i t h s t a n d a r d deviations. • -', cell, p a r a t h y r o i d h o r m o n e t r e a t e d ; o o, m e d i u m , p a r a t h y r o i d h o r m o n e treated; • ...... $, cell, c o n t r o l ; o . . . . . . o, m e d i u m , c o n t r o l . Fig. 5. T h e e f f e c t s o f d u r a t i o n on a 7 d a y p e r i o d o f s u b c u l t u r e o n t h e m a g n i t u d e of t h e e f f e c t o f p a r a t h y r o i d h o r m o n e o n c y c l i c A M P c o n t e n t o f J T C - 1 2 cells s u b c u l t u r e d a t 2 - 104 c e l l s / c m 2. (a) F o r t h e cell n u m b e r , t h e cells f r o m d u p l i c a t e d i s h e s w e r e c o u n t e d . • e, cell n u m b e r / c m 2 ; ©. . . . . . o cell p r o t e i n , p g / c m 2. (b) A t i n d i c a t e d d a y s d u r i n g t h e s u b c u l t u r e p e r i o d , cells w e r e i n c u b a t e d w i t h or w i t h o u t 1.9 u n i t s / m l of p a r a t h y r o i d h o r m o n e ( 1 - 3 4 ) f o r 2 r a i n in t h e p r e s e n c e of 10 m M t h e o p h y l l i n e . T h e m e d i u m o f t h e c u l t u r e w a s c h a n g e d 24 h b e f o r e t h e a s s a y o f cellula~ c y c l i c A M P c o n t e n t . O p e n b a r , c o n trol. S h a d o w e d b a r , r e s p o n s e to p a r a t h y r o i d h o r m o n e . T h e v a l u e s a r e m e a n + S.E. a n d t r i p l i c a t e d e t e r minations.

nephrine (2.7-fold) and prostaglandin E, (3.1-fold). The cellular cyclic AMP levels of MDCK cell line were elevated slightly (1.5-fold) as in the presence of theophylline (Table I). With MDBK and MDCK cells, the efflux of cyclic AMP into media was less than 11 and 20%, respectively, which were comparable to results with JTC-12 cell line (Fig. 4). Effects of parathyroid hormone on JTC-12 cells in relation to duration o f subculture Cultures of JTC-12 cells were initiated with 2 • 104 cells/cm 2 and basal as well as parathyroid hormone-stimulated levels of cyclic AMP were measured on the 1st, 2nd, 3rd, 5th and 7th day of subculture (Fig. 5). Since the amount of

476 T A B L E II C Y C L I C A M P L E V E L S IN T H E C E L L S A N D IN T H E I N C U B A T I O N THE CELLS TO VARIOUS HORMONES

MEDIA AFTER

EXPOSURE

OF

The cyclic A M P c o n t e n t s o f t h e cells a n d m e d i a were m e a s u r e d after 10 rain of i n c u b a t i o n w i t h h o r m o n e s i n t h e P r e s e n c e o f 2 m M 3 - i s o b u t y l - l - m e t h y l x a n t h i n e . E a c h m e a n ~ S.E. is o f t r i p l i c a t e i n c u b a t i o n s . Cell line and incubation conditions

Cyclic AMP (pmol/mg cell protein) Cells

Media

Cells plus media

4.9 5.8 12.2 8.2 14.9 12.4

48.9 60.3 87,1 109,8 135,6 146.9

MDBK Control P a r a t h y r o i d h o r m o n e ( 1 - - 3 4 ) , 1.9 u n i t s l m l Porcine calcitonin, 50 munits/ml Arginine vasopressin, 5 munits/ml I s o p r o p y l n o r e p i n c p h r i n e , 10 ~M Prostaglandin El, 14 pM MDCK Control Parathyroid hormone

(1--34)1.9

units/ml

44.0 54.5 74.9 101.6 120.7 134.5

± 6.3 ± 11.7 ± 2.5 ± 8.0 + 1.2 ._~ 9.3

44.2 ± 5.2 64.6 ÷ 12.5

* * * *

~- 0 . 5 ± 0.5 ± 1.9 ~ 0.5 ± 3.0 _+ 2 . 8

4.7 ~ 0.6 1 6 . 0 * 5.6

* * * **

48.9 80.6

* P < 0.01. ** P < 0 . 0 5 .

protein recovered from each flask correlated well with the number of cells per flask until the 7th day of culture (Fig. 5a), the cyclic AMP values in pmol/mg protein reflect as well the amount of cyclic AMP per cell (Fig. 5b). The basal levels of cyclic AMP were slightly higher on the 5--7th day of subculture as compared to those of the 1--3rd day. The increment of cyclic AMP by parathyroid hormone was 55 pmol/mg protein on the first day, decrease to 28 pmol on the second day and then increase steadily until the 7th day when the increment was maximal.

Biosynthesis of sulfatides in cultured kidney cells The kidney epithelial cells (JTC-12 and MDCK) incorporated H;3sSO4 into the lipid fraction. As shown in the autoradiogram (Fig. 6), the radioactivities were incorporated mainly into sulfatides and an unknown lipid, which migrated closely to phosphatidylcholine in the solvent system used for the separation of lipids. The fatty acids of sulfatides from rat kidney are known to be composed mainly of h y d r o x y long chain acids. In contrast, in these cell lines non-hydroxy fatty acids may be the predominant components. The unidentified lipid may be sulfocholine lipid, reported to be synthesized in rat kidney [36]. In addition, a small amount of sulfolipid corresponding to the sulfate ester of lactosylceramide was also detected in JTC-12 cells, and cholesterol sulfate in MDCK cells. The migration behaviors of these sulfolipids were also compared in another solvent system: chloroform/methanol/2.5 M ammonia (70 : 25 : 4, v/v) or as peracetylated derivatives [37] in the solvent system: chloroform/methanol/ water (85 : 15 : 1.5, v/v) [38] to confirms the assignment given above. The radioactivities measured for each spot of lipids were listed in Table III. MDBK cell line was also proved to synthesize small amount of sulfatide by thin-layer autoradiography. Thus these cell lines were proved to retain the feature of

tl. 4

u_

O

O

v

tO

¢q

Fig. 6. T h e a u t o r a d i o g r a m of t h i n - l a y e r c h r o m a t o g r a p h y of lipid e x t r a c t s f r o m J T C - 1 2 a n d M D C K cells i n c u b a t e d w i t h H 235 SO4. T h e lipids w e r e e x t r a c t e d f r o m t h e cell as d e s c r i b e d in t h e t e x t a n d a n a l y z e d b y t h i n - l a y e r c h r o m a t o g r a p h y of silica gel G ( 6 0 F 2 S4, M e r c k ) , d e v e l o p e d w i t h t h e s o l v e n t s y s t e m : c h l o r o form/methanol/acetone/acetic a c i d / w a t e r ( 1 0 : 2 : 4 : 2 : 1, v / v ) . T h e origin (O) a n d t h e s o l v e n t f r o n t (F) are m a r k e d w i t h a r r o w s . (a) L a n e 1 a n d 6, r a t k i d n e y sulfolipids, t h r e e m a j o r b a n d s f r o m a b o v e to origin are c h o l e s t e r o l s u l f a t e , s u l f a t i d e s a n d t h e sulfate ester of c e r a m i d e d i h e x o s i d e ; lane 2, c h o l e s t e r o l sulfate, t w o f a i n t b a n d s of m o r e p o l a r c o m p o u n d s are a r t i f a c t of o r g a n i c synthesis~ lane 3, J T C - 1 2 ; l a n e s 4 a n d 5, M D C K . (b) L a n e 1, J T C - 1 2 i n c u b a t e d w i t h 0.5 m M b u t y r a t e ; lanes 2 a n d 3, J T C - 1 2 ; lane 4, R L G (fibroblast); lane 5, r a t k i d n e y sulfolipids.

478

TABLE III THE RADIOACTIVITY AND MDCK CELLS

I N C O R P O R A T I O N O F H~ 5 SO 4 I N T O I N D I V I D U A L S U L F O L I P I D S O F J T C - 1 2

T h e c o n d i t i o n s f o r t h i n - l a y e r c h r o m a t o g r a p h y w e r e s i m i l a r t o Fig. 6. T h e r a d i o a c t i v i t y o f t h e s p o t w a s d e t e r m i n e d b y s c i n t i l l a t i o n c o u n t i n g o f t h e s c r a p e d silica gel p o w d e r in A q u a s o l ( N e w E n g l a n d N u c l e a r ) . Cell line

JTC-12 MDCK t RLG * ** *** T

Incorporation (cpm/mg protein) Cholesterol-S *

Sulfatides

C D H - S **

O t h e r s ***

n.d. 147 n.d.

574 1049 n.d.

98 n.d. n.d.

473 348 n.d.

Cholesterol sulfate. Ceramide dihexoside sulfate. C o n t a i n an u n i d e n t i f i e d s u l f o l i p i d . In t h e case o f M D C K , cells w e r e c u l t u r e d in D M - 1 2 0 m e d i u m [ 2 ] . n.d., not detected.

mammalian kidney to synthesize sulfolipids. In contrast, the lipid fractions extracted from fibroblasts were n o t labelled by the isotope. When JTC-12 cells were incubated with 0.5 mM butyrate in addition to the ordinary culture medium, the pattern of incorporation of sulfate into sulfolipids changed remarkably (Fig. 6b, lane 1). Decrease of sulfatide and relative increase of ceramide dihexoside sulfate were observed. Discussion

Cultured cell lines are useful model for studying various aspects of cellular function. It seems, therefore, important to examine the characteristics of cells and to localize the origin of cells. This is indispensable in the study on kidney cells because the cellular function and metabolic activity are quite varied along a nephron [39]. The reactions of cultured cell lines against various agents such as hormones or bacterial toxins were reviewed [40], however, kidney cell lines reactive to parathyroid hormone have not been reported except for a brief description by Borle [41]. The present studies could show characteristic differences in cyclic AMP metabolism among several cell lines of mammalian kidney origin. Sensitivity to parathyroid hormone was shown most clearly in JTC-12 cells and only minimally in MDCK cells (Table I). In contrast, the increase of cyclic AMP by arginine vasopressin was observed in MDCK and MDBK cells and n o t in JTC-12 cells (Tables I and II). The fibroblastic cells even of kidney origin (GPK-1) did not respond to either hormones, though prostaglandin El, a supposedly ubiquitous stimulator of adenylate cyclase [42] especially for fibroblasts and glioma cells [40], induced a marked increase of cyclic AMP in these cells. It is generally accepted that the cells of proximal convoluted tubule are the main target of parathyroid hormone [4,43,44]. On the other hand, the effect of antidiuretic hormone, which activates adenylate cyclase predominantly in medulla [3,5,45], is explained by its action on tubular cells of collecting duct [43,46]. Thus, it seems probable that JTC-12 cells and MDCK

479 cells originated from tubuli of the renal cortex and medulla, respectively. Recent demonstration by Morel and his associates [39,47--49] on the precise localization of hormone-sensitive cells along the rabbit nephron seems interesting in interpreting our current results. The studies of the authors and micropuncture studies [44] suggest that the effect of parathyroid hormone is localized as a proximal or cortical collecting tubule. However, we do not know yet whether their results on rabbit can be applied on nephrons of m o n k e y or of other mammals. There are other parameters, which can serve as the marker of specific cell populations of kidney. They are, the activity of (Na ÷ + K÷)-ATPase [50], biosynthesis of prostaglandins from arachidonate [51], or the ability of the transport of amino acids [52]. Effects of antidiuretic hormone (ADH) on MDCK cell line were studied by Mita and Endo [53], and the presence of ADH-sensitive adenylate cyclase was demonstrated. On the other hand, Borle [41] studied the effect of parathyroid hormone or calcitonin on calcium transport in cultured m o n k e y kidney cells but referred to the effect on cyclic AMP only by a brief description that the nucleotide in the cell was increased by parathyroid hormone when theophylline was present in the incubation medium. In the present study, the response of cyclic AMP to parathyroid hormone in JTC-12 cells was observed also in the absence of phosphodiesterase inhibitors with synthetic parathyroid as Potts et al. [33] showed on rat renal cortical adenylate cyclase. Moreover, the dose relation of parathyroid h o r m o n e action on elevating cyclic AMP in JTC-12 cells was very close to that reported on freshly isolated rat renal adenylate cyclase [5]. Cyclic AMP was not increased by porcine calcitonin in any cell lines examined in the present study except for MDBK. A greater relative concentration of calcitonin receptors was shown at the corticomedullary junction [54], and calcitonin-sensitive renal adenylate cyclase is now considered to be distinct from the enzyme sensitive to parathyroid hormone, although several former observations indicated that the effects of calcitonin and parathyroid hormone were not additive in enhancing renal adenylate cyclase activity or in increasing cellular cyclic AMP [6,30]. The results obtained in the present study that the JTC-12 cells responded only to parathyroid hormone but n o t to porcine calcitonin and MDBK responded to porcine calcitonin and arginine vasopressin but not to parathyroid hormone may be evidence that the cells sensitive to parathyroid h o r m o n e and calcitonin are distinct. The effect of parathyroid hormone to induce an increase of cyclic AMP in JTC-12 cells was barely detected in the absence of theophylline (Fig. 3a) or IBMX (Fig. 4). Such a marked dependency of parathyroid hormone action on phosphodiesterase inhibitors is unusual as compared to the results obtained in renal cortical tissue of various species [29--31]. It is u n k n o w n whether (1) the stimulatory effect of parathyroid h o r m o n e on adenylate cyclase was diminished, (2) cellular efflux of cyclic AMP into media was increased or (3) phosphodiesterase activity was too d o m i n a n t in JTC-12 cells. Both the second and third possibilities were, however, considered to be less possible according to the results obtained with 2 mM IBMX and by measuring also the efflux of cyclic AMP into media (Fig. 4). In the presence of 10 mM theophylline the cyclic AMP levels of MDBK cells were not significantly stimulated by any of the hor-

480 mone tested. By the use of 2 mM IBMX, small but significant responses to arginine vasopressin, porcine calcitonin, isopropylnorepinephrine and prostaglandin E1 were observed. This phenomenon may suggest that, in the presence of theophylline cyclic AMP efflux under stimulation by hormones may be somewhat higher in MDBK cells than in the other cells. This may lead at least to the conclusion that MDBK cell, which usually described as "epitheloid" cell, originated from renal epithelia. Otten et al. [ 55] reported that cyclic AMP levels rose when non-transformed cells ceased growth at confluency. In our case, the response to parathyroid hormone of the cyclic AMP concentration/mg protein increased steadily until the 7th day. Confluency was attained at the 5th day. But protein synthesis takes place, even after confluency, about 20 h after the addition of fresh serum [56], thus may lead to production of receptor protein on the cell surface. In liver [ 57] and several other tissues, prostaglandin E i action appeared to be best explained by assuming an interaction with a specific receptor that does not share any property with polypeptide hormone or catecholamine receptors [42]. The results with these kidney cells (Tables I and II) might serve as an evidence that prostaglandin E~ receptor and parathyroid hormone, calcitonin or ADH receptors are at least in kidney in a single cell species. As the marker of specific tissue or cells, enzyme activity is often employed. But the activity depends usually on the assay procedures and n o t always reliable. For instance, in the case of myelin from mammalian brain, the marker enzyme most frequently used, is 2':3'-cyclic adenosine m onophosphate phosphodiesterase. But there is objection to the specificity of the enzyme to myelin [58]. Rather, glycolipids such as ganglioside G7 [59] or cerebroside to sulfatide ratio [60] serve as the good marker. Recently Dawson et al. [61] demonstrated high levels of incorporation of H23sSO4 into cultured cloned cell lines from mouse neuronal t u m o r and suggested that the original t u m o r was of oligodendrocyte or Schwann cell origin. For these reasons, sulfolipids were considered as the good marker for also kidney cells. In this study renal epithelial cell lines were proved to synthesize sulfolipids providing another evidence for the preservation of the specific lipid metabolism of kidney. It may be also worthwhile to note that, as is shown in Fig. 6, 0.5 mM butyrate changed the labelling pattern of sulfolipids. Ceramide dihexoside sulfate was significantly more strongly labelled. Similar situation with the ganglioside GM3 was reported by Fishman et al. [62,63]. Karlsson et al. [64] and Umeda et al. [9] suggested the close relationship between sulfatides and (Na*+ K*)-ATPase (EC 3.6.3.0) activity. The specific association of sulfatides with a particular function has been proposed by Karlsson et al. [64] that sulfatides may be implicated in some manner in the activity of (Na÷+ K÷)-ATPase of mammalian tissues. This hypothesis needs further exploration. After the completion of this manuscript, Sarli~ve et al. [50] reported on the analysis of sulfatide contents, the activities of phosphoadenosine phosphosulfate-galactosyl ceramide sulfotransferase and (Na ÷ + K÷)-ATPase of glomeruli and tubuli isolated from rabbit kidney. They concluded that both the concentration of sulfatides and sulfotransferase activity were highest in medulla and tubule. Thus it seems for us that these cell lines, JTC-12, MDCK and MDBK,

481

onginated from renal epithelia. They may serve as t h e good model to study various aspects of kidney, especially concerned with specific function of nephron.

Acknowledgements The authors are greatly indebted to Professor H. Katsuta and Dr. T. Takaoka (Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo) for kind suggestions for tissue culture and for the gift of various cell lines used for this experiment. We are also grateful for valuable suggestions by Dr. H. Matsuzawa, Faculty of Agruculture, University of Tokyo; Ms. T. Ohsawa and Dr. N. Kimura of Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology.

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