- Email: [email protected]
[31] Characterization of hormone-sensitive Madin—Darby canine kidney cells
360
HORMONALLY RESPONSIVE CELLS
[31]
8 hr. This amount of time is sufficient to observe induction by butyrate but short enough to prevent toxic effects of metabolic inhibitors. The induction of glucagon receptors by PGEj and Ro 20-1724, but not butyrate, occurs much better in defined media as compared to serum. In fact, if serum is added to defined media containing PGEI, induction of glucagon receptors is inhibited in a manner dependent on the concentration of serum. In contrast, the induction by butyrate is identical in both defined and serum-containing media. High levels of cyclic AMP also decrease the induction of glucagon responsiveness by butyrate. This observation may explain why concentrations of PGE1 and Ro 20-1724 greater than 1 ~M do not induce as extensively as do lower concentrations of these agents. Induction by any agent can be prevented by inhibitors of phosphodiesterase, cholera toxin, or cyclic AMP analogs, if any of these agents are added along with the inducer.
Applications of Inducible Hormone Receptors Obviously, a system where hormone receptors can be manipulated provides a convenient model system to study the biogenesis of receptors, from their transcription to their packaging and insertion into the plasma membrane. Also of interest is an understanding of the mechanism of the induction process by butyrate, which causes numerous changes in many cell types, 7 as well as more physiological regulators of differentiation, such as prostaglandins and cyclic AMP. Because glucagon receptors disappear as a result of viral transformation, the understanding of this phenomenon may provide insight into the transformation process.
[31] C h a r a c t e r i z a t i o n o f H o r m o n e - S e n s i t i v e M a d i n - D a r b y C a n i n e K i d n e y Cells
By
MICHAEL C. LIN, SUZANNE
K.
BECKNER,
and FREDERICK J. DARFLER
Cultured cells, especially the established lines, provide a continuous and homogeneous system for studying hormone action in intact cells. The cell line known as Madin-Darby canine kidney cells (MDCK cells), l derived from normal dog kidney more than 20 years ago, is ideal for this type t C. R. Gaush, W. L. Hard, and T. F. Smith, Proc. Soc. Exp. Biol. Med. 122, 931 (1966).
METHODS IN ENZYMOLOGY,VOL. 109
Copyright © 1985by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-182009-2
[31]
HORMONE-SENSITIVE MDCK CELLS
361
of research, since it retains differentiated functions in culture 2 and, in addition, responds to several hormones, including glucagon, vasopressin, fl-adrenergic agonists, and prostaglandins. 3,4 The maintenance and general properties of MDCK cells were previously described by Taub and Saier. 5 This chapter will deal mainly with the optimal culture conditions for maintaining hormone responsiveness, the measurement of intraceUular cyclic AMP, and the characteristics of several types of hormone sensitivity in MDCK cells. Maintenance of Cell Cultures. Stock cells are kept in 100-mm culture dishes (Costar #3100) at 37° under 5% CO2/95% air with 80% humidity. For subculture, medium is removed from stock cells, the monolayer culture rinsed with 10 ml phosphate-buffered saline (PBS) and 5 ml trypsin (0.05%)-EDTA (0.02%) is added. Trypsin digestion proceeds for 5 to 20 min at 37° (depending on the culture age of stock cells), until cells begin to detach from the dish when shaken. The cells are quantitatively detached by pipetting or scraping and suspended with Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (Gibco or Hyclone) at the desired cell density for plating. The addition of medium containing 5% serum terminates the trypsin digestion. The optimal cell density for plating is about 0.5 to 2 x 105 cells/35 mm dish. Cells can be maintained in 5% serum or in defined medium as described below. The serum-free medium for MDCK cells has been reported by Taub et al. 6 This defined medium consists of DMEM : Ham's F-12 (1 : 1), 5/~g/ml insulin, 5/~g/ml transferrin, 50 nM hydrocortisone, 5 pM triiodothyronine, 0.1/xM prostaglandin EI(PGE0, 10 nM selenium dioxide, and 10 mM HEPES buffer, pH 7.4. Since PGEI is not required for growth under our conditions, it is routinely omitted from our defined medium to maximize hormone sensitivity of cells. To maintain cells in serum-free conditions, MDCK cells are rinsed with PBS 4 hr after plating in 5% serum and defined medium is added. Measurement of Hormone Responsiveness. The surface of MDCK cells is polarized 5 ; it is believed that hormone receptors are on the serosal surface facing the culture dish. Therefore, to assure accessibility of hormone receptors, monolayer cultures which are 80% confluent are used for experiments. Normally 2 to 3 days after plating, MDCK cells in 35-mm dishes are rinsed with 3 ml PBS and incubated with 3 ml DMEM without 2 j. Leighton, Z. Brada, L. W. Estes, and G. Justh, Science 163, 472 (1969). 3 M. J. Rindlerl L. M. Chuman, L. Shaffer, and M. H. Saier, Jr., J. CellBiol. 81, 635 (1979). 4 M. C. Lin, S. M. Koh, D. D. Dykman, S. K. Beckner, and T. Y. Shih, Exp. Cell Res. 142, 181 (1982). 5 M. Taub and M. H. Saier, Jr., this series, Vol. 58, p. 552. 6 M. Taub, L. M. Chuman, M. H. Saier, Jr., and G. Sato, Proc. Natl. Acad. Sci. U.S.A. 76, 3338 (1979).
362
HORMONALLY RESPONSIVE CELLS
[31]
serum or hormone supplement at 37 ° for 2 hr. When cultured in 5% serum, MDCK cells produce substantial quantities of PGEz and PGF2~, which can reach a concentration of 0.1/zM in the medium. This level of prostaglandins not only causes high basal concentrations of cyclic AMP but also leads to desensitization of hormone sensitivity, effects not seen when cells are cultured in defined medium. To minimize these effects, cells are incubated for 2 hr with DMEM prior to measurement of hormone sensitivity. After this incubation, the DMEM is replaced with 2 ml fresh DMEM containing 20/zM Ro 20-1724 [4-(3-butoxy-4-methoxy-benzyl)-2-imidazolidinone, Hoffmann-La Roche], an inhibitor of cyclic AMP phosphodiesterase, and 20 mM HEPES buffer, pH 7.4. After 30 min at 25 ° (room temperature), a small aliquot of hormone solution is added to the desired concentration to initiate the reaction. After 3 min, the medium is quickly removed and I to 2 ml boiling water is added which serves to terminate the reaction and to extract cyclic AMP. Dishes are scraped and each sample is brought to an identical volume (usually 2 ml) and boiled for 5 min. After centrifugation, the concentration of cyclic AMP in the supernatant is measured by a radioimmunoassay. Since the intracellular concentration of cyclic AMP is low in MDCK cells, the addition of a phosphodiesterase inhibitor simplifies detection of cyclic AMP by eliminating a concentration step. Radioimmunoassay of Cyclic AMP. Cyclic AMP is measured by radioimmunoassay as described previously 7 with two modifications: (1) cyclic AMP is acetylated to increase the sensitivity of the assay and (2) antirabbit Ig antiserum is used to separate bound from free [125I]succinyl cyclic AMP. Briefly the procedure is as follows: samples or standards (5 to 2000 fmol/tube) in 25 to 100 ~1 are made up to 200 /zl with 50 mM acetate buffer, pH 6.2, and acetylated with 5/zl acetic anhydride : triethylamine (1:2). After 15 min at room temperature, [125I]succinyl cyclic AMP (Meloy Labs, VA), 10,000 cpm in 100/.d, and 100/xl cyclic AMP antiserum, sufficient to bind 30-60% of the radioactive ligand, are added. After 4 hr at room temperature, carrier rabbit serum and second antiserum (anti-rabbit Ig from sheep, goat or burro, Meloy Labs) are added. After standing overnight at 4°, 2 ml of cold 10 mM acetate buffer, pH 6.2, is added, the tubes are centrifuged, and the radioactivity in the pellets counted. We have used the antiserum against cyclic AMP obtained from Collaborative Research and New England Nuclear with good results. Unfortunately, antiserum from Collaborative Research is no longer available. Other sources are Meloy Labs (Springfield, VA), Sigma (St. Louis, MO), Miles (Elkhart, IN), and Research Products International Corp. 7 A. L. Steiner, this series, Vol. 38, p. 96.
[31]
HORMONE-SENSITIVE MDCK CELLS
363
(Elk Grove Village, IL). The antiserum from New England Nuclear is precoated with the second antibody and thus the assay requires only one incubation. Hormone Responsiveness of MDCK Cells. The production of cyclic AMP in the presence of hormone is linear for about 3 min as shown in Fig. 1. During that time period, more than 90% of the cyclic AMP produced remains inside the cells. Therefore, hormone responsiveness is measured in terms of the increase in intracellular cyclic AMP produced in 3 min in the presence of hormone over the basal level. The responsiveness of MDCK cells to several hormones is shown in the table. Cells are more responsive and have lower basal concentrations of cyclic AMP when they are grown in the defined medium than in 5% fetal bovine serum. Concentration-dependent activation of cyclic AMP production is shown in Fig. 2 for three hormones. The concentrations required for half-maximal activation by glucagon, isoproterenol, and PGE] are 20, 80, and 100 nM, respectively. The binding of []25I]glucagon to MDCK cells, as described in the legend to Fig. 3, is also half maximal at 20 nM (Fig. 3).
N
-6 E
25
< ~
20
Q.
u ~:
15
..J
~ < I--z
10 /
•
NO ADDITION
I
I
I
I
1
I
I
1
2
3
4
5
6
7
INCUBATION TIME, MIN FIG. 1. Time course for cyclic AMP production in MDCK cells. Intracellular cyclic AMP produced in the absence or presence of glucagon (2/~M) was measured by a radioimmunoassay as described.
364
HORMONALLY RESPONSIVE CELLS
[31]
GLUCAGON J
PGE! ~...._.
/
,,,',,-
/-
.,/o--
"
. . . .
-
z o
z o
o
.~M
I
I
l
I I 10-e
1
K
1 I I 10-7
1
I
I
I I 10-6
I
I
I
CONCENTRATION OF HORMONE, M
FIG. 2. Concentration-dependent activation of cyclic AMP production by glucagon (@), isoproterenol (©), and prostaglandin E~ (A). Intraceilular cyclic AMP was measured 3 min after the addition of hormones at concentrations indicated. Hormone response is expressed as described in footnote d to the table. HORMONE RESPONSIVENESS OF MDCK CELLS
Intracellular cyclic AMP" (fmol/dish) Culture medium
No addition
+Glucagon b
+Vasopressin
+ Isoproterenol
+PGE1
5% fetal bovine serum Serum-freec
74
454 (6.2) d
268 (3.7)
440 (6.0)
466 (6.4)
85 (9.4)
385 (43)
409 (45)
9
386 (43)
a The concentration of intracellular cyclic AMP was assayed as described in the text. Each value represents the average of three determinations which agreed within 5%. b Concentrations used in the assay: 2 p,M glucagon, 2/xM vasopressin, 2 /xM isoproterenol, and 5/zM prostaglandin Ej. ' Serum-free medium contains hormone supplements as described in the text. a Values in parentheses represent hormone response which is the ratio of cyclic AMP produced in the presence of hormone to that in its absence.
I
[31]
HORMONE-SENSITIVE MDCK CELLS
365
~a0 O
e d
z D o DD
20
Z
o <
3
10
O
I
1
I
I
10-8
I
I
I
I
I
10-7
CONCENTRATION OF GLUCAGON, M
FIG. 3. Concentration-dependent binding of [125I]glucagon to MDCK cells. Monolayer cultures (in 35 mm dishes) were incubated in DME medium containing 20 mM HEPES buffer, pH 7.4, 0.2% bovine serum albumin, and 1 m M bacitracin for 20 min at 37° with 1 n M [~25I]glucagon (New England Nuclear) and native glucagon at the concentrations indicated. After two washings with 2 ml of the same medium (4°), bound [t2~I]glucagon was extracted with 1 ml 0.5 N NaOH and the radioactivity was counted. Nonspecific binding was estimated by the inclusion of 2/.~M unlabeled glucagon.
Comments. The MDCK cell line is one of the few lines that retain sensitivity toward numerous hormones, particularly the peptide hormones, glucagon and vasopressin. In contrast, glucagon responsiveness of cell lines derived from liver is often diminished. MDCK cells have been used as a model system for studying hormone regulation of kidney functions. 6,8 The availability of a differentiated, hormone-responsive cell line facilitates studies of the cascade of biochemical events subsequent to cyclic AMP elevation and its correlation with specific kidney functions. In a companion report, we show that certain hormone responses of MDCK cells are lost following viral transformation. 9
8 j. S. Lever, Proc. Natl. Acad. Sci. U.S.A. 76, 1323 (1979). 9 S. K. Beckner, F. J. Darfler, and M. C. Lin, this series [30].
HORMONALLY RESPONSIVE CELLS
[31]
8 hr. This amount of time is sufficient to observe induction by butyrate but short enough to prevent toxic effects of metabolic inhibitors. The induction of glucagon receptors by PGEj and Ro 20-1724, but not butyrate, occurs much better in defined media as compared to serum. In fact, if serum is added to defined media containing PGEI, induction of glucagon receptors is inhibited in a manner dependent on the concentration of serum. In contrast, the induction by butyrate is identical in both defined and serum-containing media. High levels of cyclic AMP also decrease the induction of glucagon responsiveness by butyrate. This observation may explain why concentrations of PGE1 and Ro 20-1724 greater than 1 ~M do not induce as extensively as do lower concentrations of these agents. Induction by any agent can be prevented by inhibitors of phosphodiesterase, cholera toxin, or cyclic AMP analogs, if any of these agents are added along with the inducer.
Applications of Inducible Hormone Receptors Obviously, a system where hormone receptors can be manipulated provides a convenient model system to study the biogenesis of receptors, from their transcription to their packaging and insertion into the plasma membrane. Also of interest is an understanding of the mechanism of the induction process by butyrate, which causes numerous changes in many cell types, 7 as well as more physiological regulators of differentiation, such as prostaglandins and cyclic AMP. Because glucagon receptors disappear as a result of viral transformation, the understanding of this phenomenon may provide insight into the transformation process.
[31] C h a r a c t e r i z a t i o n o f H o r m o n e - S e n s i t i v e M a d i n - D a r b y C a n i n e K i d n e y Cells
By
MICHAEL C. LIN, SUZANNE
K.
BECKNER,
and FREDERICK J. DARFLER
Cultured cells, especially the established lines, provide a continuous and homogeneous system for studying hormone action in intact cells. The cell line known as Madin-Darby canine kidney cells (MDCK cells), l derived from normal dog kidney more than 20 years ago, is ideal for this type t C. R. Gaush, W. L. Hard, and T. F. Smith, Proc. Soc. Exp. Biol. Med. 122, 931 (1966).
METHODS IN ENZYMOLOGY,VOL. 109
Copyright © 1985by Academic Press, Inc. All rights of reproductionin any form reserved. ISBN 0-12-182009-2
[31]
HORMONE-SENSITIVE MDCK CELLS
361
of research, since it retains differentiated functions in culture 2 and, in addition, responds to several hormones, including glucagon, vasopressin, fl-adrenergic agonists, and prostaglandins. 3,4 The maintenance and general properties of MDCK cells were previously described by Taub and Saier. 5 This chapter will deal mainly with the optimal culture conditions for maintaining hormone responsiveness, the measurement of intraceUular cyclic AMP, and the characteristics of several types of hormone sensitivity in MDCK cells. Maintenance of Cell Cultures. Stock cells are kept in 100-mm culture dishes (Costar #3100) at 37° under 5% CO2/95% air with 80% humidity. For subculture, medium is removed from stock cells, the monolayer culture rinsed with 10 ml phosphate-buffered saline (PBS) and 5 ml trypsin (0.05%)-EDTA (0.02%) is added. Trypsin digestion proceeds for 5 to 20 min at 37° (depending on the culture age of stock cells), until cells begin to detach from the dish when shaken. The cells are quantitatively detached by pipetting or scraping and suspended with Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal bovine serum (Gibco or Hyclone) at the desired cell density for plating. The addition of medium containing 5% serum terminates the trypsin digestion. The optimal cell density for plating is about 0.5 to 2 x 105 cells/35 mm dish. Cells can be maintained in 5% serum or in defined medium as described below. The serum-free medium for MDCK cells has been reported by Taub et al. 6 This defined medium consists of DMEM : Ham's F-12 (1 : 1), 5/~g/ml insulin, 5/~g/ml transferrin, 50 nM hydrocortisone, 5 pM triiodothyronine, 0.1/xM prostaglandin EI(PGE0, 10 nM selenium dioxide, and 10 mM HEPES buffer, pH 7.4. Since PGEI is not required for growth under our conditions, it is routinely omitted from our defined medium to maximize hormone sensitivity of cells. To maintain cells in serum-free conditions, MDCK cells are rinsed with PBS 4 hr after plating in 5% serum and defined medium is added. Measurement of Hormone Responsiveness. The surface of MDCK cells is polarized 5 ; it is believed that hormone receptors are on the serosal surface facing the culture dish. Therefore, to assure accessibility of hormone receptors, monolayer cultures which are 80% confluent are used for experiments. Normally 2 to 3 days after plating, MDCK cells in 35-mm dishes are rinsed with 3 ml PBS and incubated with 3 ml DMEM without 2 j. Leighton, Z. Brada, L. W. Estes, and G. Justh, Science 163, 472 (1969). 3 M. J. Rindlerl L. M. Chuman, L. Shaffer, and M. H. Saier, Jr., J. CellBiol. 81, 635 (1979). 4 M. C. Lin, S. M. Koh, D. D. Dykman, S. K. Beckner, and T. Y. Shih, Exp. Cell Res. 142, 181 (1982). 5 M. Taub and M. H. Saier, Jr., this series, Vol. 58, p. 552. 6 M. Taub, L. M. Chuman, M. H. Saier, Jr., and G. Sato, Proc. Natl. Acad. Sci. U.S.A. 76, 3338 (1979).
362
HORMONALLY RESPONSIVE CELLS
[31]
serum or hormone supplement at 37 ° for 2 hr. When cultured in 5% serum, MDCK cells produce substantial quantities of PGEz and PGF2~, which can reach a concentration of 0.1/zM in the medium. This level of prostaglandins not only causes high basal concentrations of cyclic AMP but also leads to desensitization of hormone sensitivity, effects not seen when cells are cultured in defined medium. To minimize these effects, cells are incubated for 2 hr with DMEM prior to measurement of hormone sensitivity. After this incubation, the DMEM is replaced with 2 ml fresh DMEM containing 20/zM Ro 20-1724 [4-(3-butoxy-4-methoxy-benzyl)-2-imidazolidinone, Hoffmann-La Roche], an inhibitor of cyclic AMP phosphodiesterase, and 20 mM HEPES buffer, pH 7.4. After 30 min at 25 ° (room temperature), a small aliquot of hormone solution is added to the desired concentration to initiate the reaction. After 3 min, the medium is quickly removed and I to 2 ml boiling water is added which serves to terminate the reaction and to extract cyclic AMP. Dishes are scraped and each sample is brought to an identical volume (usually 2 ml) and boiled for 5 min. After centrifugation, the concentration of cyclic AMP in the supernatant is measured by a radioimmunoassay. Since the intracellular concentration of cyclic AMP is low in MDCK cells, the addition of a phosphodiesterase inhibitor simplifies detection of cyclic AMP by eliminating a concentration step. Radioimmunoassay of Cyclic AMP. Cyclic AMP is measured by radioimmunoassay as described previously 7 with two modifications: (1) cyclic AMP is acetylated to increase the sensitivity of the assay and (2) antirabbit Ig antiserum is used to separate bound from free [125I]succinyl cyclic AMP. Briefly the procedure is as follows: samples or standards (5 to 2000 fmol/tube) in 25 to 100 ~1 are made up to 200 /zl with 50 mM acetate buffer, pH 6.2, and acetylated with 5/zl acetic anhydride : triethylamine (1:2). After 15 min at room temperature, [125I]succinyl cyclic AMP (Meloy Labs, VA), 10,000 cpm in 100/.d, and 100/xl cyclic AMP antiserum, sufficient to bind 30-60% of the radioactive ligand, are added. After 4 hr at room temperature, carrier rabbit serum and second antiserum (anti-rabbit Ig from sheep, goat or burro, Meloy Labs) are added. After standing overnight at 4°, 2 ml of cold 10 mM acetate buffer, pH 6.2, is added, the tubes are centrifuged, and the radioactivity in the pellets counted. We have used the antiserum against cyclic AMP obtained from Collaborative Research and New England Nuclear with good results. Unfortunately, antiserum from Collaborative Research is no longer available. Other sources are Meloy Labs (Springfield, VA), Sigma (St. Louis, MO), Miles (Elkhart, IN), and Research Products International Corp. 7 A. L. Steiner, this series, Vol. 38, p. 96.
[31]
HORMONE-SENSITIVE MDCK CELLS
363
(Elk Grove Village, IL). The antiserum from New England Nuclear is precoated with the second antibody and thus the assay requires only one incubation. Hormone Responsiveness of MDCK Cells. The production of cyclic AMP in the presence of hormone is linear for about 3 min as shown in Fig. 1. During that time period, more than 90% of the cyclic AMP produced remains inside the cells. Therefore, hormone responsiveness is measured in terms of the increase in intracellular cyclic AMP produced in 3 min in the presence of hormone over the basal level. The responsiveness of MDCK cells to several hormones is shown in the table. Cells are more responsive and have lower basal concentrations of cyclic AMP when they are grown in the defined medium than in 5% fetal bovine serum. Concentration-dependent activation of cyclic AMP production is shown in Fig. 2 for three hormones. The concentrations required for half-maximal activation by glucagon, isoproterenol, and PGE] are 20, 80, and 100 nM, respectively. The binding of []25I]glucagon to MDCK cells, as described in the legend to Fig. 3, is also half maximal at 20 nM (Fig. 3).
N
-6 E
25
< ~
20
Q.
u ~:
15
..J
~ < I--z
10 /
•
NO ADDITION
I
I
I
I
1
I
I
1
2
3
4
5
6
7
INCUBATION TIME, MIN FIG. 1. Time course for cyclic AMP production in MDCK cells. Intracellular cyclic AMP produced in the absence or presence of glucagon (2/~M) was measured by a radioimmunoassay as described.
364
HORMONALLY RESPONSIVE CELLS
[31]
GLUCAGON J
PGE! ~...._.
/
,,,',,-
/-
.,/o--
"
. . . .
-
z o
z o
o
.~M
I
I
l
I I 10-e
1
K
1 I I 10-7
1
I
I
I I 10-6
I
I
I
CONCENTRATION OF HORMONE, M
FIG. 2. Concentration-dependent activation of cyclic AMP production by glucagon (@), isoproterenol (©), and prostaglandin E~ (A). Intraceilular cyclic AMP was measured 3 min after the addition of hormones at concentrations indicated. Hormone response is expressed as described in footnote d to the table. HORMONE RESPONSIVENESS OF MDCK CELLS
Intracellular cyclic AMP" (fmol/dish) Culture medium
No addition
+Glucagon b
+Vasopressin
+ Isoproterenol
+PGE1
5% fetal bovine serum Serum-freec
74
454 (6.2) d
268 (3.7)
440 (6.0)
466 (6.4)
85 (9.4)
385 (43)
409 (45)
9
386 (43)
a The concentration of intracellular cyclic AMP was assayed as described in the text. Each value represents the average of three determinations which agreed within 5%. b Concentrations used in the assay: 2 p,M glucagon, 2/xM vasopressin, 2 /xM isoproterenol, and 5/zM prostaglandin Ej. ' Serum-free medium contains hormone supplements as described in the text. a Values in parentheses represent hormone response which is the ratio of cyclic AMP produced in the presence of hormone to that in its absence.
I
[31]
HORMONE-SENSITIVE MDCK CELLS
365
~a0 O
e d
z D o DD
20
Z
o <
3
10
O
I
1
I
I
10-8
I
I
I
I
I
10-7
CONCENTRATION OF GLUCAGON, M
FIG. 3. Concentration-dependent binding of [125I]glucagon to MDCK cells. Monolayer cultures (in 35 mm dishes) were incubated in DME medium containing 20 mM HEPES buffer, pH 7.4, 0.2% bovine serum albumin, and 1 m M bacitracin for 20 min at 37° with 1 n M [~25I]glucagon (New England Nuclear) and native glucagon at the concentrations indicated. After two washings with 2 ml of the same medium (4°), bound [t2~I]glucagon was extracted with 1 ml 0.5 N NaOH and the radioactivity was counted. Nonspecific binding was estimated by the inclusion of 2/.~M unlabeled glucagon.
Comments. The MDCK cell line is one of the few lines that retain sensitivity toward numerous hormones, particularly the peptide hormones, glucagon and vasopressin. In contrast, glucagon responsiveness of cell lines derived from liver is often diminished. MDCK cells have been used as a model system for studying hormone regulation of kidney functions. 6,8 The availability of a differentiated, hormone-responsive cell line facilitates studies of the cascade of biochemical events subsequent to cyclic AMP elevation and its correlation with specific kidney functions. In a companion report, we show that certain hormone responses of MDCK cells are lost following viral transformation. 9
8 j. S. Lever, Proc. Natl. Acad. Sci. U.S.A. 76, 1323 (1979). 9 S. K. Beckner, F. J. Darfler, and M. C. Lin, this series [30].