Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sodium butyrate and its derivatives are useful therapeutic
agents for the treatment of genetic diseases including urea cycle disorders, sickle cell disease, thalassemias, and possibly cystic fibrosis (CF). Butyrate partially restores cAMP-activated
Cl secretion in CF
epithelial cells by stimulating
F508 cystic fibrosis transmembrane
conductance regulator (
F508-CFTR) gene expression and increasing the
amount of
F508-CFTR in the plasma membrane. Because the effect of
butyrate on Cl
secretion by
renal epithelial cells has not been reported, we examined the effects
of chronic butyrate treatment (15-18 h) on the function,
expression, and localization of CFTR fused to the green fluorescent
protein (GFP-CFTR) in stably transfected MDCK cells. We report that
sodium butyrate reduced Cl
secretion across MDCK cells, yet increased apical membrane GFP-CFTR expression 25-fold and increased apical membrane
Cl
currents 30-fold.
Although butyrate also increased Na-K-ATPase protein expression
twofold, the drug reduced the activity of the Na-K-ATPase by 55%. Our
findings suggest that butyrate inhibits cAMP-stimulated
Cl
secretion across MDCK
cells in part by reducing the activity of the Na-K-ATPase.
green fluorescent protein; cystic fibrosis; gene expression; cystic fibrosis transmembrane conductance regulator; adenosine 3',5'-cyclic monophosphate
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE CYSTIC FIBROSIS transmembrane conductance regulator
(CFTR) is a cAMP-activated
Cl channel that is targeted
to the apical plasma membrane in many epithelial cells, including those
in the kidney (5, 8, 31). In the kidney, CFTR is expressed in all
nephron segments (31) and is important for transepithelial
Cl
transport (12, 33) and
enlargement of renal cysts in polycystic kidney disease (11). Mutations
in CFTR are responsible for the genetic disease cystic fibrosis (CF)
(22). Nearly 70% of individuals with CF express
F508-CFTR, a
mutation that prevents the trafficking of CFTR from the endoplasmic
reticulum to the plasma membrane (4, 35). Accordingly, cAMP does not
stimulate Cl
secretion in
CF epithelial cells. Although CF does not cause major alterations in
renal function, individuals with CF do exhibit renal dysfunction
including decreased ability to excrete a salt load, decreased ability
to maximally dilute and concentrate urine, increased proximal tubule
sodium reabsorption, and altered drug excretion (reviewed in Ref. 31).
Recently, it was suggested that butyrate and the butyrate analog
4-phenylbutyrate may be useful therapeutic agents for treatment of CF
in individuals expressing
F508-CFTR (3, 26, 27). These agents
partially restored cAMP-activated
Cl
secretion in nasal,
bronchial, and pancreatic epithelial cells expressing
F508-CFTR by
stimulating
F508-CFTR gene expression and increasing the amount of
F508-CFTR protein in the plasma membrane (3, 26, 27). The effect of
butyrate on Cl
secretion by
renal epithelial cells has not been reported.
The short-chain fatty acid sodium butyrate has numerous and diverse
effects on cellular physiology including modulation of protein kinase
and phosphatase activities (6, 23, 24, 28), stimulation of microtubule
and microfilament formation (1), and transcriptional activation of
numerous gene products (15) including heat shock proteins (9) and
alkaline phosphatase (2). Because little is known about the effects of
butyrate on CFTR-mediated
Cl secretion in renal
epithelial cells, we examined the effects of sodium butyrate on the
function and expression of wild-type CFTR in MDCK cells, a model for
the renal distal tubule and collecting duct that secrete
Cl
. Since expression of
endogenous CFTR in renal epithelia is either low and difficult to
detect or below the limit of detection of currently available
techniques (5, 31), we fused CFTR to the green fluorescent protein
(GFP-CFTR) and stably transfected MDCK cells with the GFP-CFTR cDNA
(20). GFP, a 27-kDa protein from the jellyfish
Aequorea victoria, generates a
striking green fluorescence, is resistant to photobleaching, and does
not require any exogenous cofactors or substrates to fluoresce (32).
Previously, we demonstrated that fusion of GFP to CFTR does not alter
CFTR function or localization (20). We report that sodium butyrate reduced Cl
secretion across
MDCK cells, yet increased apical membrane GFP-CFTR expression 25-fold
and increased apical membrane
Cl
currents 30-fold.
Although butyrate also increased Na-K-ATPase protein expression
twofold, butyrate reduced the activity of the Na-K-ATPase by 55%. Our
findings suggest that butyrate inhibits cAMP-stimulated
Cl
secretion across MDCK
cells in part by reducing the activity of the Na-K-ATPase.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. MDCK type I cells stably transfected with GFP-CFTR and parental untransfected MDCK I cells, which express low levels of endogenous CFTR (19), were grown in culture on Transwell filter-bottom cups as described previously (20). Sodium n-butyrate (5 mM; Sigma, St. Louis, MO) was added to apical and basolateral cell culture chambers in complete cell culture media for 15-18 h. Fresh solutions of butyrate were prepared immediately before use for each experiment. Similar results, which are not pooled with presented data, were found in MDCK C7 cells, a clonal line of MDCK type I cells (generous gift of Dr. Hans Oberleithner) (10), stably transfected with GFP-CFTR.
Measurement of short-circuit current.
Short-circuit current
(Isc) was
measured across monolayers of MDCK cells grown on Transwell filter-bottom cups in the presence of amiloride
(105 M) in the apical
solution to inhibit electrogenic sodium transport as described (14,
20). Under these conditions, cAMP-stimulated Isc across
monolayers of MDCK cells is referable to CFTR-mediated Cl
secretion (20, 30).
Butyrate was not present in bathing solutions during measurements of
Isc. A
cAMP-stimulating cocktail [100 µM 8-chlorophenylthio-cAMP
(CPT-cAMP), 100 µM IBMX, and 20 µM forskolin] was applied to both apical and basolateral solutions to stimulate Isc. CPT-cAMP was
obtained from Boehringer-Mannheim (Indianapolis, IN). IBMX and
forskolin were purchased from Sigma (St. Louis, MO).
To examine the effect of butyrate on the activity of the Na-K-ATPase,
the apical membrane was permeabilized with nystatin (200 µg/ml).
Isc measured
under these conditions represents the activity of the Na-K-ATPase, as
described previously (25). To examine the effect of butyrate on apical
membrane Cl currents, the
basolateral membrane was permeabilized with nystatin (200 µg/ml).
When the basolateral membrane is permeabilized with nystatin,
Isc measured in
the presence of a transepithelial
Cl
ion gradient directed
from the apical to the basolateral solution (140 mM vs. 14 mM:
Cl
replaced on a equimolar
basis with gluconate: Ca2+ was
increased from 0.5 to 2 mM in the gluconate-containing solution to
maintain Ca2+ activity similar in
both solutions) represents the
Cl
current across the
apical membrane, as described previously (7, 13).
Confocal microscopy. Fluorescent images were acquired using a Zeiss Axioskop microscope (Thornwood, NY) equipped with a laser-scanning confocal unit (model MRC-1024; Bio-Rad, Hercules, CA), a 15-mW krypton-argon laser, and a ×63 PlanApochromat/1.4 NA oil-immersion objective. GFP fluorescence was excited using the 488-nm laser line and collected using a standard FITC filter set (530 ± 30 nm). Propidium iodide fluorescence was excited using the 568-nm laser line and collected using a standard Texas Red filter set (605 ± 32 nm). Images from vehicle- and butyrate-treated monolayers were collected using the same values for laser power, photomultiplier gain, iris, and black level. Acquired images were imported into Adobe Photoshop v3.0 for image processing and printing.
Cell surface biotinylation. Biotinylation of apical cell surface glycoproteins was performed as described (20). Following biotinylation, monolayers were solubilized in lysis buffer (50 mM Tris · HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40 and containing the Complete Protease Inhibitor cocktail; Boehringer Mannheim), scraped from filters, and spun at 14,000 g for 4 min to pellet insoluble material. Aliquots of cell lysates were removed for SDS-PAGE analysis, and the remainder of the supernatants were brought to a volume of 900 µl with lysis buffer and precipitated with 100 µl of a 50% slurry of streptavidin-agarose beads (Pierce, Rockford, IL) overnight at 4°C with end-over-end rotation. Beads were pelleted by brief centrifugation for 30 s at 14,000 g and washed three times with lysis buffer. Biotinylated proteins were eluted by boiling for 5 min in 50 µl of Laemmli sample buffer (0.24 M Tris · HCl, pH 8.9, 16% glycerol, 0.008% bromophenol blue, 5.6% SDS, and 80 mM dithiothreitol).
SDS-PAGE and Western blotting. Cell
lysates and biotinylated proteins were separated on 4-15%
Tris · HCl gradient gels (Bio-Rad) and transferred to
PVDF Immobilon membranes (Millipore, Bedford, MA). Membranes were
blocked overnight at 4°C in 5% nonfat dry milk in TBS/0.02%
Tween-20 and incubated with CFTR COOH-terminal (1:1,000) monoclonal
antibody (Genzyme, Cambridge, MA), Na-K-ATPase 1-subunit (1:1,000)
monoclonal antibody (Upstate Biotechnology, Lake Placid, NY), or
Na-K-2Cl cotransporter (1:10,000) monoclonal antibody T4 (17) (generous
gift of Dr. Bliss Forbush III; Yale University, New Haven, CT) followed
by anti-mouse horseradish peroxidase-conjugated secondary antibodies
(1:5,000-1:10,000; Amersham, Arlington Heights, IL). Blots were
developed by enhanced chemiluminescence (Amersham) using Hyperfilm ECL
(Amersham). Densitometric analysis of band intensities was performed
with public domain NIH Image v1.57 software.
Statistical analyses. Differences between means were compared by either paired or unpaired two-tailed Student's t-test as appropriate using Instat v2.01 statistical software (GraphPad, San Diego, CA). Data are expressed as the mean ± SE. P < 0.05 is considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Butyrate inhibits electrogenic
Cl secretion in MDCK
cells.
We examined the effect of butyrate on basal and cAMP-stimulated
Isc in parental,
untransfected MDCK cells and in MDCK cells stably expressing GFP-CFTR.
In parental untransfected cells, a cAMP-stimulating cocktail rapidly
increased Isc,
which reached a peak at 2 min and declined to an elevated state above
basal Isc (Fig.
1A;
Table 1). Treatment of monolayers with
butyrate (5 mM for 15-18 h) reduced basal and cAMP-stimulated
Isc (Fig. 1A; Table 1). Thus butyrate
inhibited cAMP-stimulated
Cl
secretion mediated by
endogenous CFTR. In cells stably transfected with GFP-CFTR, butyrate (5 mM for 15-18 h) also reduced basal and cAMP-stimulated
Isc (Fig.
1B; Table 1). In contrast, acute butyrate treatment (5 mM for 15 min) had no effect on cAMP-stimulated Isc in GFP-CFTR
stable transfectants (cAMP peak, 16.6 ± 3.3 µA/cm2 for vehicle-treated vs.
12.2 ± 2.1 µA/cm2 for
butyrate-treated monolayers, P > 0.05; cAMP steady state, 6.3 ± 0.9 µA/cm2 for vehicle-treated vs.
5.9 ± 0.6 µA/cm2 for
butyrate-treated monolayers, P > 0.05; n = 7-8 monolayers per
group).1
Thus chronic, but not acute, butyrate treatment inhibited
cAMP-stimulated Cl
secretion mediated by CFTR.
|
|
|
|
|
Effect of butyrate on Na-K-ATPase activity and
Cl currents.
In the next series of experiments, we tested the hypothesis that sodium
butyrate reduced Cl
secretion across MDCK cells by inhibiting the activity of the Na-K-ATPase and/or apical CFTR
Cl
channels. To examine the
effect of butyrate on the function of the Na-K-ATPase, we permeabilized
the apical membrane with nystatin and measured
Isc. Butyrate
reduced the Isc
attributed to the Na-K-ATPase from 52.2 ± 4.3 µA/cm2
(n = 4) in vehicle-treated cells to
23.6 ± 1.9 µA/cm2 in
butyrate-treated cells (n = 4, P < 0.001). To examine the effect of
butyrate on apical membrane
Cl
channels, we
permeabilized the basolateral membrane with nystatin (200 µg/ml) and
measured Isc in
the presence of a transepithelial Cl
ion gradient directed
from the apical to the basolateral solution. Butyrate dramatically
increased Cl
currents
across the apical membrane from 9.2 ± 5.2 µA/cm2
(n = 3) in vehicle-treated cells to
274.6 ± 31.4 µA/cm2 in
butyrate-treated cells (n = 3, P < 0.005). Taken
together, these results suggest that butyrate inhibits CFTR-mediated
Cl
secretion in part
by reducing the activity of the Na-K-ATPase.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major new finding of this report is that chronic butyrate treatment
reduced cAMP-stimulated Cl
secretion across polarized MDCK cells expressing endogenous CFTR and
MDCK cells stably expressing GFP-CFTR
Cl
channels. Paradoxically,
sodium butyrate increased apical membrane GFP-CFTR protein expression
25-fold and increased apical membrane Cl
currents 30-fold.
Although butyrate also increased Na-K-ATPase protein expression
twofold, it reduced the activity of the Na-K-ATPase by 55%. Our
findings suggest that butyrate inhibits cAMP-stimulated Cl
secretion across MDCK
cells in part by reducing the activity of the Na-K-ATPase. We have made
similar observations in human airway serous epithelial cells
(16).
Several investigators have examined the effect of butyrate on wild-type
CFTR-mediated transepithelial
Cl secretion
in nonrenal epithelial cells. In colonic T84 cells expressing endogenous wild-type CFTR, acute butyrate
treatment (20-30 min) reduced cAMP-stimulated
Cl
secretion by inhibiting
an apical membrane CFTR-like
Cl
conductance (7).
Prolonged butyrate treatment (24 h) also inhibited
Cl
secretion in T84 cells;
however, the effect of butyrate was mediated by downregulation of
Na-K-2Cl cotransporter expression and activity without affecting CFTR
expression and function (18). The apparent discrepancy between these
studies on T84 cells may be attributed to differences in the dose or
duration of butyrate treatment. In contrast, in the present study in
MDCK cells, acute butyrate treatment had no effect on cAMP-stimulated
Cl
secretion, whereas
prolonged butyrate treatment reduced cAMP-stimulated Cl
secretion in part by
inhibiting the Na-K-ATPase. Thus prolonged butyrate treatment inhibits
CFTR-mediated Cl
secretion
in MDCK and T84 cells.
Our data suggest that butyrate inhibits
Cl secretion across MDCK
cells in part by reducing the activity of the Na-K-ATPase. In MDCK
cells, the Na-K-ATPase maintains a low intracellular concentration of
Na+, which is important for
providing the driving force for
Cl
entry into the cell
across the basolateral membrane via the Na-K-2Cl cotransporter.
Cl
then exits the cell
across the apical membrane through CFTR
Cl
channels (30). By
reducing the activity of the Na-K-ATPase, we speculate that butyrate
increases intracellular Na+
concentration and thereby reduces
Cl
entry into the cell
across the basolateral membrane via the Na-K-2Cl cotransporter. The
decrease in Cl
entry would
reduce intracellular Cl
concentration and thereby inhibit
Cl
secretion across the
apical membrane, despite an increase in the amount of CFTR in the
apical membrane. The cellular mechanism(s) whereby butyrate inhibits
Na-K-ATPase activity is unknown. Butyrate has numerous and diverse
effects on cell physiology including modulation of protein kinase and
phosphatase activities (6, 23, 24, 28), stimulation of microtubule and
microfilament formation (1), and transcriptional activation of numerous
gene products (15), including heat shock proteins (9) and alkaline phosphatase (2). Additional experiments, beyond the scope of this
report, are required to elucidate the cellular mechanism(s) whereby
butyrate inhibits Na-K-ATPase activity in MDCK
cells.2
The observations in this report raise the question: why does butyrate
allow cAMP to stimulate Cl secretion in CF cells? In CF epithelial
cells cAMP has no effect on
Cl secretion, because of an
absence of CFTR in the apical plasma membrane. However, butyrate allows
cAMP to activate Cl
secretion in CF nasal, bronchial, and pancreatic epithelial cells expressing
F508-CFTR by increasing the amount of
F508-CFTR
protein in the plasma membrane (3, 26, 27). The data in the present study appear to be at odds with the butyrate studies on CF cells. If
butyrate also reduced Na-K-ATPase activity in CF cells, then one would
predict that cAMP would not stimulate
Cl
secretion in CF cells,
even though butyrate enhanced the amount of
F508-CFTR in the apical
membrane. We propose that the effects of butyrate may be cell-type
dependent such that butyrate may not change the activity of
Na-K-ATPase in nasal, bronchial, and pancreatic epithelial
cells. Thus the butyrate-induced increase in
F508-CFTR expression in
the apical membrane would be sufficient to allow cAMP to stimulate
Cl
secretion. It is also
possible that butyrate may reduce Na-K-ATPase activity in CF cells;
however, the increase in apical
Cl
conductance, subsequent
to the addition of
F508-CFTR to the membrane, is sufficient to
increase Cl
secretion even
though Na-K-ATPase activity may fall with butyrate treatment.
Additional studies are required to examine these possibilities.
The observation that butyrate has a negative effect on cAMP-stimulated
Cl secretion by MDCK cells
has potential implications for the use of butyrate to examine CFTR
function in vitro and for the clinical use of butyrate in vivo.
Butyrate-induced upregulation of CFTR expression from viral-based
promoters in transfected cells may, as this study suggests, actually
reduce Cl
secretion.
Clinical use of butyrate, although useful for treatment of patients
with CF, urea cycle disorders, sickle cell disease, thalassemia, and
cancer (21), may actually compromise wild-type CFTR function in kidney epithelia.
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Alice Givan and Ken Orndorff for assistance with confocal microscopy. We thank Dr. Bliss Forbush III for providing Na-K-2Cl cotransporter monoclonal antibody and Kerry O'Brien, Melissa Levak, and Jerod Denton for valuable technical assistance.
![]() |
FOOTNOTES |
---|
These studies were supported by the National Institutes of Health Grants DK/HL-45881 and by the Cystic Fibrosis Foundation. B. D. Moyer was supported by a predoctoral fellowship from the Dolores Zohrab Liebmann Foundation. J. Loffing was supported by a fellowship from the Swiss National Science Foundation. Confocal microscopy was performed at Dartmouth Medical School, in the Herbert C. Englert Cell Analysis Laboratory, which was established by a grant from the Fannie E. Rippel Foundation and is supported in part by Norris Cotton Cancer Center Core Grant CA-23108.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1 Currents were smaller in experiments examining the effect of acute butyrate on cAMP-stimulated Isc compared with experiments examining the effect of chronic butyrate on cAMP-stimulated Isc. Although current variations were observed between passage numbers, similar numbers of vehicle- and butyrate-treated monolayers, derived from the same passage number, were examined on the same day in all Isc experiments.
2
Although sodium butyrate had no effect on
Na-K-2Cl protein expression in MDCK cells, it is possible that the drug
reduced the activity of the cotransporter. Such an effect would be
expected to contribute to the fall in cAMP-stimulated
Cl secretion observed in
butyrate-treated cells. Additional studies are required to determine
whether butyrate inhibits the activity of the Na-K-2Cl cotransporter.
Address for reprint requests and other correspondence: B. A. Stanton, Dept. of Physiology, Dartmouth Medical School, Hanover, NH 03755 (E-mail: Bruce.A.Stanton{at}Dartmouth.edu).
Received 21 October 1998; accepted in final form 12 March 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Altenburg, B. C.,
D. P. Via,
and
S. H. Steiner.
Modification of the phenotype of murine sarcoma virus-transformed cells by sodium butyrate.
Exp. Cell Res.
102:
223-231,
1976[Medline].
2.
Barka, T.
Effect of sodium butyrate on the expression of genes transduced by retroviral vectors.
J. Cell. Biochem.
69:
201-210,
1998[Medline].
3.
Cheng, S. H.,
S. L. Fang,
J. Zabner,
J. Marshall,
S. Piraino,
S. C. Schiavi,
D. M. Jefferson,
M. J. Welsh,
and
A. E. Smith.
Functional activation of the cystic fibrosis trafficking mutant [/delta]F508-CFTR by overexpression.
Am. J. Physiol.
268 (Lung Cell. Mol. Physiol. 12):
L615-L624,
1995
4.
Cheng, S. H.,
R. J. Gregory,
J. Marshall,
S. Paul,
D. W. Souza,
G. A. White,
C. R. O'Riordan,
and
A. E. Smith.
Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis.
Cell
63:
827-834,
1990[Medline].
5.
Crawford, I.,
P. C. Maloney,
P. L. Zeitlin,
W. B. Guggino,
S. C. Hyde,
H. Turley,
K. C. Gatter,
A. Harris,
and
C. F. Higgins.
Immunocytochemical localization of the cystic fibrosis gene product CFTR.
Proc. Natl. Acad. Sci. USA
88:
9262-9266,
1991[Abstract].
6.
Cuisset, L.,
L. Tichonicky,
P. Jaffray,
and
M. Delpech.
The effects of sodium butyrate on transcription are mediated through activation of a protein phosphatase.
J. Biol. Chem.
272:
24148-24153,
1997
7.
Dagher, P. C.,
R. W. Egnor,
A. Taglietta-Kohlbrecher,
and
A. N. Charney.
Short-chain fatty acids inhibit cAMP-mediated chloride secretion in rat colon.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1853-C1860,
1996
8.
Denning, G. M.,
L. S. Ostedgaard,
S. H. Cheng,
A. E. Smith,
and
M. J. Welsh.
Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.
J. Clin. Invest.
89:
339-349,
1992[Medline].
9.
Garcia-Bermejo, L.,
N. E. Vilaboa,
C. Perez,
A. Galan,
E. De Blas,
and
P. Aller.
Modulation of heat-shock protein 70 (HSP70) gene expression by sodium butyrate in U-937 promonocytic cells: relationships with differentiation and apoptosis.
Exp. Cell Res.
236:
268-274,
1997[Medline].
10.
Gekle, M.,
S. Wunsch,
H. Oberleithner,
and
S. Silbernag.
Characterization of two MDCK-cell subtypes as a model system to study principal cell and intercalated cell properties.
Pflügers Arch.
428:
157-162,
1994[Medline].
11.
Hanaoka, K.,
O. Devuyst,
E. M. Schwiebert,
P. D. Wilson,
and
W. B. Guggino.
A role for CFTR in human autosomal dominant polycystic kidney disease.
Am. J. Physiol.
270 (Cell Physiol. 39):
C389-C399,
1996
12.
Husted, R. F.,
K. A. Volk,
R. D. Sigmund,
and
J. B. Stokes.
Anion secretion by the inner medullary collecting duct. Evidence for involvement of the cystic fibrosis transmembrane conductance regulator.
J. Clin. Invest.
95:
644-650,
1995[Medline].
13.
Hwang, T.-C.,
E. M. Schwiebert,
and
W. B. Guggino.
Apical and basolateral ATP stimulates tracheal epithelial chloride secretion via multiple purinergic receptors.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1611-C1623,
1996
14.
Kizer, N. L.,
B. Lewis,
and
B. A. Stanton.
Electrogenic sodium absorption and chloride secretion by an inner medullary collecting duct cell line (mIMCD-K2).
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F347-F355,
1995
15.
Kruh, J.
Effects of sodium butyrate, a new pharmacological agent, on cells in culture.
Mol. Cell. Biochem.
42:
65-82,
1982[Medline].
16.
Loffing, J.,
B. Moyer,
and
B. A. Stanton.
Phenylbutyrate increases CFTR expression but inhibits chloride secretion in human airway serous (Calu-3) epithelial cells (Abstract).
Ped. Pulmonol. Suppl.
17:
275,
1998.
17.
Lytle, C.,
J.-C. Xu,
D. Biemesderfer,
and
B. Forbush III.
Distribution and diversity of Na-K-Cl transport proteins: a study with monoclonal antibodies.
Am. J. Physiol.
269 (Cell Physiol. 38):
C1496-C1505,
1995
18.
Matthews, J. B.,
I. Hassan,
S. Meng,
S. Y. Archer,
B. J. Hrnjez,
and
R. A. Hodin.
Na-K-2Cl cotransporter gene expression and function during enterocyte differentiation: modulation of Cl secretory capacity by butyrate.
J. Clin. Invest.
101:
2072-2079,
1998
19.
Mohamed, A.,
D. Ferguson,
F. S. Seibert,
H. Cai,
N. Kartner,
S. Grinstein,
J. R. Riordan,
and
G. L. Lukacs.
Functional expression and apical localization of the cystic fibrosis transmembrane conductance regulator in MDCK I cells.
Biochem. J.
322:
259-265,
1997[Medline].
20.
Moyer, B. D.,
J. Loffing,
E. M. Schwiebert,
D. Loffing-Cueni,
P. A. Halpin,
K. H. Karlson,
I. I. Ismailov,
W. B. Guggino,
G. M. Langford,
and
B. A. Stanton.
Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator, tagged with green fluorescent protein in Madin-Darby canine kidney cells.
J. Biol. Chem.
273:
21759-21768,
1998
21.
Newmark, H. L.,
and
C. W. Young.
Butyrate and phenylacetate as differentiating agents: practical problems and opportunities.
J. Cell. Biochem. Suppl.
22:
247-253,
1995[Medline].
22.
Riordan, J. R.,
J. M. Rommens,
B. Kerem,
N. Alon,
R. Rozmahel,
Z. Grzelczak,
J. Zielenski,
S. Lok,
N. Plavsic,
J.-L. Chou,
M. L. Drumm,
M. C. Iannuzzi,
F. S. Collins,
and
L.-C. Tsui.
Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA.
Science
245:
1066-1073,
1989[Medline].
23.
Rivero, J. A.,
and
S. E. Adunyah.
Sodium butyrate induces tyrosine phosphorylation and activation of MAP kinase (ERK-1) in human K562 cells.
Biochem. Biophys. Res. Commun.
224:
796-801,
1996[Medline].
24.
Rivero, J. A.,
S. E. Adunyah,
and
K. J. Ceesay.
Phenylbutyrate, sodium butyrate and phenylacetate affect the levels of p34cdc2 kinase and PKC in K562 erythroleukemic cells.
Proc. Am. Assoc. Cancer Res.
37:
509,
1996.
25.
Rokaw, M. D.,
E. Sarac,
E. Lechman,
M. West,
J. Angeski,
J. P. Johnson,
and
M. L. Zeidel.
Chronic regulation of transepithelial Na+ transport by the rate of apical Na+ entry.
Am. J. Physiol.
270 (Cell Physiol. 39):
C600-C607,
1996
26.
Rubenstein, R. C.,
M. E. Egan,
and
P. L. Zeitlin.
In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing F508-CFTR.
J. Clin. Invest.
100:
2457-2465,
1997
27.
Rubenstein, R. C.,
and
P. L. Zeitlin.
A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in F508-homozygous cystic fibrosis patients.
Am. J. Respir. Crit. Care Med.
157:
484-490,
1998
28.
Russo, G. L.,
V. D. Pietra,
C. Mercurio,
F. D. Ragione,
D. R. Marshak,
A. Oliva,
and
V. Zappia.
Down-regulation of protein kinase CKII activity by sodium butyrate.
Biochem. Biophys. Res. Commun.
233:
673-677,
1997[Medline].
29.
Shima, D. T.,
K. Haldar,
R. Pepperkok,
R. Watson,
and
G. Warren.
Partitioning of the Golgi apparatus during mitosis in living HeLa cells.
J. Cell Biol.
137:
1211-1228,
1997
30.
Simmons, N. L.
Renal epithelial Cl secretion.
Exp. Physiol.
78:
117-137,
1993[Medline].
31.
Stanton, B. A.
Cystic fibrosis transmembrane conductance regulator (CFTR) and renal function.
Wien Klin. Wochenschr.
109:
457-564,
1997[Medline].
32.
Tsien, R. Y.
The green fluorescent protein.
Annu. Rev. Biochem.
67:
509-544,
1998[Medline].
33.
Vandorpe, D.,
N. Kizer,
F. Ciampolillo,
B. Moyer,
K. Karlson,
W. B. Guggino,
and
B. A. Stanton.
CFTR mediates electrogenic chloride secretion in mouse inner medullary collecting duct (mIMCD-K2) cells.
Am. J. Physiol.
269 (Cell Physiol. 38):
C683-C689,
1995[Abstract].
34.
Wacker, I.,
C. Kaether,
A. Kromer,
A. Migala,
W. Almers,
and
H.-H. Gerdes.
Microtubule-dependent transport of secretory vesicles in real time with a GFP-tagged secretory protein.
J. Cell Sci.
110:
1453-1463,
1997
35.
Welsh, M. J.,
and
A. E. Smith.
Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis.
Cell
73:
1251-1254,
1993[Medline].