Departments of Medicine and Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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Mitogen-activated
protein (MAP) kinases mediate a variety of critical cellular events,
but their role in the regulation of epithelial transport is largely
undefined. Recently, we demonstrated that nerve growth factor (NGF)
inhibits HCO60%) the effects of NGF to inhibit basolateral Na+/H+ exchange activity and transepithelial
HCO
mitogen-activated protein kinase; epithelial transport; kidney; extracellular signal-regulated kinase; medullary thick ascending limb
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INTRODUCTION |
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PLASMA MEMBRANE NA+/h+ exchangers are involved in mediating a variety of cellular processes, including intracellular (pHi) and cell volume regulation, epithelial Na+ absorption, and cell growth (19, 38, 46). At least six NHE isoforms (NHE1-6) have been identified in mammalian cells (12). These differ in their tissue distribution, inhibitor sensitivity, and responses to regulatory stimuli (12, 38, 46). A prominent feature of Na+/H+ exchange is its stimulation by growth factors. This stimulation is rapid, occurs in a wide variety of cell types, and is observed with virtually all mitogens (12, 19, 38, 46). In addition, Na+/H+ exchangers activated by growth factors include NHE1, the ubiquitously expressed exchanger isoform present in nonpolar cells and in the basolateral membrane of epithelia, and NHE2 and NHE3, isoforms that are localized selectively in the apical membrane of certain epithelial cells in the kidney and gastrointestinal tract (38, 46). The mechanisms involved in growth factor stimulation of Na+/H+ exchange have been a subject of active investigation because of the close association of increased exchange activity with cell proliferation, transformation, hypertrophy, and adhesion (19, 21, 24, 30, 37, 38).
The medullary thick ascending limb (MTAL) of the mammalian kidney
participates in acid-base regulation by reabsorbing a sizable fraction
of the HCO
Mitogen-activated protein (MAP) kinases are essential intermediates in signaling pathways activated by multiple stimuli, including growth factors, hormones, neurotransmitters, cytokines, and environmental stress (32, 43). MAP kinase subgroups in mammalian cells include extracellular signal-regulated kinases (ERKs), c-Jun NH2-terminal kinases (JNKs), and p38 MAP kinases. These kinases are regulated through distinct signaling cascades, leading to the activation of a MAP kinase kinase (MEK or MKK) that activates MAP kinase through direct phosphorylation on threonine and tyrosine (43). Recent work supports a role for MAP kinase pathways in the regulation of Na+/H+ exchange activity (6, 20, 24, 27, 36). In particular, ERK has been demonstrated to play a key role in mediating stimulation of NHE1 by growth factors in a number of nonepithelial systems (6, 12, 20, 24, 27, 36). At present, however, it is unknown whether MAP kinase pathways are relevant for the regulation of Na+/H+ exchange activity and its related functions in renal tubules or other epithelia.
The purpose of the present study was to investigate the role of MAP
kinase pathways in the regulation of Na+/H+
exchange by NGF in the MTAL. We found that the novel inhibitory actions
of NGF on basolateral Na+/H+ exchange activity
and HCO
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METHODS |
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Tubule perfusion and measurement of net
HCO, 25 HCO
1 · mm
1. One to
three 10-min tubule fluid samples were then collected for each period
(initial, experimental, and recovery). The tubules were allowed to
reequilibrate for 5-10 min after a change in the composition of
the bath solution. The absolute rate of HCO
1 · mm
1) was
calculated from the luminal flow rate and the difference between total
CO2 concentrations measured in perfused and collected fluids (16, 17). An average HCO
Measurement of intracellular pH and
Na+/H+
exchange activity.
pHi was measured in isolated and perfused MTALs with the
pH-sensitive dye
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein as
described previously (18, 40, 41). For pHi
experiments, tubules were perfused and bathed in Na+-free
HEPES-buffered solution that contained (in mM) 145 N-methyl-D-glucammonium, 4 K+, 147 Cl, 2.0 Ca2+, 1.5 Mg2+, 1.0 phosphate, 1.0 SO
90% by bath
ethylisopropyl amiloride (EIPA; 50 µM) under all experimental conditions.
Tissue preparations and protein kinase assays.
MAP kinase activities were studied by using two previously described
preparations: 1) strips of tissue dissected from the inner
stripe of the outer medulla (the region of the kidney highly enriched
in MTALs), and 2) microdissected MTALs (4, 39). Use of the two preparations in combination permits comprehensive study
of MAP kinase activities under multiple experimental conditions plus
direct confirmation of key observations in dissected MTALs (4,
39). The tissue strips or tubules were incubated in vitro at
37°C in the control solution used for HCO-32P]ATP and myelin basic protein (ERK1/2),
glutathione S-transferase-c-Jun (JNK), or glutathione
S-transferase-activating transcription factor 2 (p38) as
substrates (29, 39, 44). Phosphorylated substrates were
isolated by SDS-PAGE, visualized by autoradiography, and quantified by
densitometry. Equal amounts of protein kinases in immunoprecipitates
were verified within experiments by immunoblotting with the same
antibodies used for immunoprecipitation, as previously described
(39). Anti-ERK1/2 antibody (anti-rat MAPK R2) was purchased from Upstate Biotechnology. Antibodies against JNK and p38
MAP kinase were kindly provided by R. J. Davis and have been described previously (29, 33, 39, 44).
Statistical analysis. Results are presented as means ± SE. Differences between means were evaluated by using Student's t-test for paired or unpaired data or ANOVA with the Newman-Keuls multiple range test, as appropriate. P < 0.05 was considered statistically significant.
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RESULTS |
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NGF increases ERK activity.
Strips of tissue from the inner stripe of the outer medulla were
incubated in vitro for 15 min in the absence (control) and presence of
0.7 nM NGF, and then MAP kinase activities were measured as described
(39). NGF increased ERK activity 1.9-fold but had no effect on JNK or p38 MAP kinase activity (Fig.
1).
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ERK activation is blocked by inhibitors of MEK.
To investigate the role of the ERK pathway in mediating NGF-induced
inhibition of HCO
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Inhibitors of ERK activation reduce inhibition of
HCO1 · mm
1
(n = 5, P < 0.001; Fig.
5A). In MTAL bathed with U0126
or PD-98059, NGF decreased HCO
1 · mm
1
(n = 9, P < 0.001; Fig.
5B). The net decrease in HCO
1 · mm
1
without inhibitors (Fig. 5A) vs. 1.8 ± 0.2 pmol · min
1 ·mm
1 with
inhibitors (Fig. 5B); P < 0.001]. The
basal rate of HCO
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Inhibitors of ERK activation reduce inhibition of basolateral
Na+/H+
exchange activity by NGF.
NGF inhibits HCO
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Inhibitors of ERK activation do not reduce inhibition of
HCO1 · mm
1
(n = 4, P < 0.025). In tubules bathed
with 15 µM U0126, addition of EIPA to the bath decreased
HCO
1 · mm
1
(n = 4, P < 0.005). These results
suggest that the interaction between basolateral and apical
Na+/H+ exchangers does not involve ERK
activation. Taken together, our findings support the conclusion that
the MEK1/2 inhibitors decrease NGF-induced inhibition of
HCO
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DISCUSSION |
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MAP kinase pathways mediate a variety of critical cellular events,
including growth, differentiation, survival and repair, inflammation,
and responses to toxins and physical stress (14, 32, 43).
In contrast, the role of MAP kinases in the regulation of epithelial
transport is largely undefined. Recently, we demonstrated that NGF
inhibits transepithelial HCO
NGF regulates the ERK, JNK, and p38 MAP kinase pathways in nerve cells,
and the integrated action of these signaling pathways plays a crucial
role in mediating NGF-induced control of neuronal cell proliferation,
differentiation, and survival (26, 31, 44, 45). In the
MTAL, we found that exposure to NGF caused selective activation of ERK,
with no effect on JNK or p38 MAP kinase. The activation of ERK is rapid
and sustained, consistent with a direct and continuous role in
mediating NGF-induced regulation of HCO
Stimulation of Na+/H+ exchange by growth
factors has been a virtually universal finding (12, 19, 38,
46). Increases in Na+/H+ exchange (NHE1)
activity early in the mitogenic response may increase pHi
and induce cell signals that are obligatory or permissive for
proliferation, differentiation, or hypertrophy (19, 24, 37,
38). In contrast, we found that NGF uniquely inhibits basolateral Na+/H+ exchange activity in the
MTAL, which results in inhibition of transepithelial
HCO5 min) and the
duration (at least 60 min) of ERK activation correlate temporally with NGF-induced inhibition of HCO
In contrast to its role in mediating inhibition of basolateral Na+/H+ exchange in the MTAL, ERK has been demonstrated to play an important role in activation of Na+/H+ exchange (NHE1) by growth factors in other cells (6, 20, 24, 27, 36). Several mechanisms have been identified that may contribute to ERK-dependent stimulation of NHE1 in these systems: 1) direct phosphorylation of NHE1 by ERK (24, 27); 2) direct phosphorylation of NHE1 by a downstream target of ERK [recent studies have shown that phosphorylation of NHE1 on serine-703 by p90RSK, a direct substrate of ERK, is important for growth factor-induced exchanger activation (36)]; and 3) ERK-dependent phosphorylation of accessory proteins that interact with and regulate NHE1 (6). The relative roles of these mechanisms in mediating ERK-dependent activation of NHE1 have not been defined; however, they provide an important framework for future analysis of how ERK uniquely inhibits Na+/H+ exchange activity in the MTAL. For example, it is possible that ERK activation leads to inhibition of Na+/H+ exchange in the MTAL due to activation of downstream targets or interaction with accessory proteins that differ from those in other cell types. Immunolocalization of protein expression (5, 7), localization of mRNA expression (9, 23), and functional studies of inhibitor kinetics (18) indicate that basolateral Na+/H+ exchange activity in the MTAL is mediated by NHE1. However, our studies do not rule out the possibility that NGF may act via ERK to inhibit an as yet unidentified exchanger that functions in parallel with and has an amiloride sensitivity similar to NHE1 in the basolateral membrane.
Although ERK plays a predominant role in mediating the inhibition of
basolateral Na+/H+ exchange by NGF, significant
inhibition of Na+/H+ exchange activity and
HCO
NGF inhibits transepithelial HCO
Although NGF and its receptors are expressed in the kidney, their role
in the regulation of kidney function remains to be determined
(17, 40). The magnitude of the change in
HCO
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ACKNOWLEDGEMENTS |
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We thank R. J. Davis for generously providing reagents and helpful advice on MAP kinase assays and L. Reuss for critical evaluation of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-38217.
2
In separate, paired experiments, bath addition of
U0126 or PD-98059 caused a small (~10%) decrease in
HCO
1 In some experiments, ERK activity with inhibitor+NGF was less than activity measured with inhibitor alone (illustrated by the U0126 experiment in Fig. 4A). This may reflect NGF activation of a MAP kinase phosphatase (11), which results in a decrease in MAP kinase activity when the normally opposing stimulatory action of MEK is eliminated by pretreatment with the MEK inhibitors. The reduced ERK activity with inhibitor+NGF was not a consistent finding in other experiments and overall was not statistically significant (Fig. 4B).
Address for reprint requests and other correspondence: D. W. Good, 4.200 John Sealy Annex 0562, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555-0562 (E-mail dgood{at}UTMB.edu).
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. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00133.2001
Received 27 April 2001; accepted in final form 19 December 2001.
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