From the
In the rat medullary thick ascending limb (MTAL),
hyperosmolality inhibits transepithelial
HCO
In many cell types, Na
In the rat kidney, the medullary
thick ascending limb (MTAL)
The present study was designed to examine intracellular
signaling mechanisms involved in hyperosmotic regulation of
HCO
The tubule perfusion
protocol was as described previously
(16, 17) . In
brief, after mounting on pipettes, the tubules were equilibrated for
20-40 min at 37 °C in the initial perfusion and bath
solutions. Two to four 10-min tubule fluid samples were then collected
for determination of the HCO
Activation of Na
Evidence
supporting the conclusions of this study was obtained primarily from
experiments examining the effects of PTK inhibitors. Several findings
support the view that these agents influenced MTAL
HCO
Apical membrane Na
At least four unique mammalian isoforms of
Na
Although our results suggest an important role for
tyrosine phosphorylation in the inhibition of
HCO
The role of protein phosphorylation in activation of
Na
Genistein inhibited hyperosmotic regulation
of HCO
In summary, the effect of
hyperosmolality to inhibit MTAL HCO
Values are means ± S.E.
Mannitol (300 mM) or NaCl (75 mM) was added to
perfusate and bath in the absence or presence of bath genistein (7
µM). Numbers in parentheses are numbers of tubules.
V, fluid flow rate; [TCO
Values are
means ± S.E. In series A, genistein (7 µM) was
added to control bath solution; in series B, ethylisopropyl amiloride
(EIPA, 50 µM) was added to luminal perfusate in the
presence of bath genistein. Numbers in parentheses, V,
[TCO
We thank L. Reuss and M. Jennings for critical reading
of the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
absorption
(JHCO
) by inhibiting apical membrane
Na
/H
exchange. To examine signaling
mechanisms involved in this regulatory response, MTALs were isolated
and perfused in vitro with 25 mM
HCO
solutions (290 mosmol/kg
H
O). Osmolality was increased in lumen and bath solutions
by addition of 300 mM mannitol or 75 mM NaCl.
Addition of mannitol reduced JHCO
by 60%
and addition of NaCl reduced JHCO
by 50%.
With the protein tyrosine kinase (PTK) inhibitor genistein (7
µM) or herbimycin A (1 µM) in the bath,
addition of mannitol reduced JHCO
only by
11% and addition of NaCl reduced JHCO
only by 15%. Staurosporine (10
M) or
forskolin (10
M) in the bath had no effect
on inhibition of JHCO
by hypertonic NaCl.
Genistein had no effect on inhibition of
JHCO
by vasopressin (a cyclic
AMP-dependent process) or stimulation of
JHCO
by prostaglandin E
(a
protein kinase C-dependent process). Under isosmotic conditions,
addition of genistein or herbimycin A to the bath increased
JHCO
by 30% through stimulation of apical
membrane Na
/H
exchange. Addition of
the tyrosine phosphatase inhibitor molybdate (50 µM) to
the bath reproduced the inhibition of
JHCO
observed with hyperosmolality. These
data indicate that 1) the effect of hyperosmolality to inhibit MTAL
HCO
absorption through inhibition of
apical membrane Na
/H
exchange is
mediated via a PTK-dependent pathway that functions independent of
regulation by cyclic AMP and protein kinase C, and 2) a constitutive
PTK activity inhibits apical membrane Na
/H
exchange and HCO
absorption under
isosmotic conditions. Our results suggest that tyrosine phosphorylation
is a critical step in inhibition of the apical
Na
/H
exchanger isoform NHE-3 by
hyperosmolality.
/H
exchange is a primary transport pathway responsible for cell
volume regulation in hyperosmotic conditions
(1, 2, 3, 4) . In response to osmotic
shrinkage, parallel activation of Na
/H
and Cl
/HCO
exchangers results in net uptake of NaCl and water, thus returning cell
volume toward its original value
(5, 6, 7, 8, 9, 10, 11) .
The stimulation of Na
/H
exchange by
hyperosmolality is the result of an increase in the sensitivity of the
exchanger to internal H
(1, 5, 8) but does not appear to require direct phosphorylation of the
exchanger
(11) . The intracellular signaling mechanisms involved
in osmotic regulation of Na
/H
exchange are largely unknown.
(
)
of the loop of
Henle reabsorbs a sizable fraction of the
HCO
filtered at the glomerulus
(12, 13) . The H
secretion required for
this HCO
absorption is mediated virtually
completely by apical membrane Na
/H
exchange
(13, 14, 15) . Thus, the rate of
transepithelial HCO
absorption serves as
a measure of apical Na
/H
exchange
activity under steady-state transporting conditions. In recent studies,
we demonstrated that apical membrane Na
/H
exchange in the rat MTAL exhibits a unique functional response to
hyperosmolality. In contrast to the activation of
Na
/H
exchange observed in other cell
types, hyperosmolality markedly inhibited both apical membrane
Na
/H
exchange and net
HCO
absorption in MTAL segments
(15, 16) . The inhibition of apical
Na
/H
exchange could not be explained
by an increase in intracellular Na
activity or
intracellular pH (pH
) and was the result of a
decrease in the sensitivity of the exchanger to internal
H
, reflected by an acid shift in the pH
dependence curve
(15) . The inhibition by hyperosmolality
also was associated with a marked increase in the pH
sensitivity of the exchanger over the physiologic
pH
range, suggesting that this regulatory response
may be a specialized adaptation that enables the MTAL to regulate
pH
or luminal acidification in the hyperosmotic
environment of the renal medulla
(15) . However, the signal
transduction mechanisms by which hyperosmolality inhibits apical
membrane Na
/H
exchange have not been
identified.
absorption and apical membrane
Na
/H
exchange in the isolated,
perfused MTAL of the rat. The results indicate that the inhibition of
HCO
absorption by hyperosmolality is
mediated via a protein tyrosine kinase (PTK)-dependent pathway and that
regulation via this pathway occurs independent of regulation by cyclic
AMP and protein kinase C. In addition, a constitutive PTK activity
inhibits apical membrane Na
/H
exchange and HCO
absorption under
isosmotic conditions.
Materials
Stock solutions of genistein (20
mM), herbimycin A (1.8 mM), staurosporine (0.2
mM), and 5-( N-ethyl- N-isopropyl)-amiloride
(EIPA, 50 mM) were prepared in MeSO. Stock
solutions (1 mg/ml) of prostaglandin E
(PGE
)
and forskolin were prepared in ethanol. Arginine vasopressin (AVP) was
prepared as a 4
10
M stock solution
in water. The agents were diluted into bath and perfusion solutions to
final concentrations given under ``Results.'' Equivalent
concentrations of ethanol or Me
SO were added to control
solutions. All agents were purchased from Sigma, except genistein and
EIPA (Research Biochemicals International, Natick, MA) and herbimycin A
(Life Technologies, Inc.).
Tubule Perfusion
Medullary thick ascending limbs
from male Sprague-Dawley rats (50-80 g, Taconic, Germantown, NY)
were isolated and perfused in vitro as described previously
(12, 16, 17) . Single tubules were dissected
from the inner stripe of the outer medulla at 10 °C in control bath
solution (see below), transferred to a bath chamber on the stage of an
inverted stereomicroscope, and mounted on concentric glass pipettes for
microperfusion
(12, 16) . Tubule fluid emerging from the
distal end of the tubules was collected for timed intervals into
calibrated constriction pipettes for calculation of tubule fluid flow
rates and for analysis of total COconcentrations. The
luminal perfusion rate (normalized per unit of tubule length) averaged
1.5-1.7 nl/min/mm. In all experiments, the luminal perfusion
solution contained (in mM): 146 Na
, 4
K
, 122 Cl
, 25
HCO
, 2.0 Ca
, 1.5
Mg
, 2.0 phosphate, 1.2
SO
, 1.0 citrate, 2.0 lactate, and 5.5
glucose. The bath solution was identical except for addition of 0.2%
fatty acid free bovine albumin (Sigma). Osmolality of the solutions was
290 mosmol/kg H
O. Hyperosmotic solutions were prepared by
addition of 300 mM mannitol (final osmolality = 590
mosmol/kg H
O) or 75 mM NaCl (final osmolality
= 425 mosmol/kg H
O). Other experimental agents were
added to perfusion or bath solutions as described under
``Results.'' All solutions were equilibrated with 95%
O
, 5% CO
and were pH 7.45-7.47 at 37
°C. The length of the perfused tubule segments ranged from 0.49 to
0.70 mm and averaged 0.60 ± 0.01 mm.
transport
rate (initial period). The perfusion and/or bath solutions were then
changed to one of the experimental solutions (increase in osmolality,
addition of inhibitor, etc.), and the tubule was allowed to
re-equilibrate for 5-20 min. Two to four additional tubule fluid
samples were then collected (experimental period). Finally, the initial
solutions were returned to the perfusate and bath and the control
measurements repeated (recovery period). In separate experiments,
measurements of inner and outer tubule diameters were obtained for
calculation of epithelial cell volume
(15, 18, 19) . The protocol and conditions for
the cell volume experiments were virtually identical to those used for
HCO
transport experiments.
Analysis
Total carbon dioxide concentrations in
perfusion and bath solutions and in collected tubule fluid were
measured by microcalorimetry as described previously
(12) .
Transepithelial voltage was measured between calomel cells using 140
mM NaCl-agar bridges
(12, 16) . Because net
fluid transport is absent in MTALs studied in symmetric isosmotic or
hyperosmotic solutions
(12, 16) , absolute rates of
HCO absorption
(JHCO
, picomoles/min/mm)
(
)
were calculated as JHCO
= V ([TCO
]
[TCO
]
])/ L, where V is fluid collection rate (nanoliters/min),
[TCO
] is total carbon dioxide concentration
(mM) in perfused (o) and collected (c) fluid, and L is perfused tubule length (mm). A mean
HCO
absorption rate was calculated for
each period studied in a given tubule. When control measurements were
made at the beginning and end of an experiment, the values were
averaged. Single tubule values are presented in the figures. Results in
tables and text are means ± S.E. Differences between means were
evaluated using Student's t test for paired data, with
p < 0.05 considered statistically significant.
Effects of Genistein and Herbimycin A on Inhibition by
Mannitol
The effects on HCO
absorption of increasing osmolality with mannitol are shown in
and Fig. 1. Adding 300 mM mannitol to the
perfusion and bath solutions decreased
HCO
absorption by 60%, from 9.9 to 4.2
pmol/min/mm ( (A); Fig. 1 A). The inhibition
by mannitol was reversible and was similar to that observed previously
in the presence of vasopressin
(16) . In contrast, in tubules
bathed with the PTK inhibitor genistein (7 µM) or
herbimycin A (1 µM), adding 300 mM mannitol to
the perfusate and bath decreased HCO
absorption only by 11%, from 11.7 to 10.4 pmol/min/mm (
(B); Fig. 1 B). Addition of mannitol
decreased the transepithelial voltage in the absence or presence of
inhibitor ().
Figure 1:
Effects of adding mannitol
( Mann, 300 mM) to perfusate and bath on
HCO absorption in the absence
( A) or presence ( B) of protein tyrosine kinase
inhibitors. In B, genistein (7 µM) or herbimycin
A (1 µM) was present in the bath throughout the
experiments. Control and genistein solutions were 290 mosmol/kg
H
O; solutions containing mannitol were 590 mosmol/kg
H
O. Filled and open circles are mean
values for single tubules. Lines connect paired measurements
made in the same tubule. P values are for paired t test. Mean
values are in Table I (A and B).
Effects of Genistein on Inhibition by NaCl
To
determine whether the effect of genistein was specific for mannitol, we
examined the effects of increasing osmolality with NaCl, the solute
primarily responsible for the hyperosmolality of the renal outer
medulla in vivo. Adding 75 mM NaCl to the perfusion
and bath solutions decreased HCO
absorption by 50%, from 14.0 to 7.1 pmol/min/mm ( (C);
Fig. 2A). A similar inhibition was observed previously
with addition of 150 mM NaCl
(16) . In contrast, in
tubules bathed with 7 µM genistein, adding 75 mM
NaCl to the perfusate and bath decreased
HCO
absorption only by 15%, from 17.8 to
15.5 pmol/min/mm ( (D); Fig. 2 B). Thus, the
effect of genistein to block hyperosmotic inhibition was independent of
the solute used to produce hyperosmolality.
Figure 2:
Effects of adding NaCl (75 mM) to
perfusate and bath on HCO absorption in
the absence ( A) or presence ( B) of genistein. In
B, genistein ( Gen, 7 µM) was present in
the bath throughout the experiments. Control and genistein solutions
were 290 mosmol/kg H
O; solutions containing added NaCl were
425 mosmol/kg H
O. Filled circles, lines, and P values as in
Fig. 1. Mean values are in Table I (C and
D).
To determine whether the
influence of genistein on hyperosmotic inhibition of
HCO absorption was related to an effect
on cell volume, steady-state cell volume was determined in MTALs
studied under the same experimental conditions used in the preceding
HCO
transport experiments. In the absence
of genistein, the initial control volume was 0.30 ± 0.03 nl/mm
( n = 3). Addition of 75 mM NaCl to the
perfusate and bath decreased cell volume to 0.22 ± 0.02 nl/mm
( p < 0.001). The cells returned to their original volume
(0.29 ± 0.03 nl/mm) following 75 mM NaCl
removal.
(
)
In tubules bathed with 7
µM genistein, the initial cell volume was 0.29 ±
0.02 nl/mm, addition of 75 mM NaCl decreased cell volume to
0.22 ± 0.02 nl/mm ( p < 0.001), and cell volume
recovered to 0.29 ± 0.02 nl/mm following 75 mM NaCl
removal ( n = 3). Thus, genistein had no effect on cell
volume under isosmotic or hyperosmotic conditions. In both the absence
and presence of genistein, cell volume was stable in hyperosmotic NaCl
for up to 30 min and the decrease in cell volume was the result of an
increase in the tubule inner (luminal) diameter.
Effects of Staurosporine and Forskolin on Inhibition by
NaCl
HCO absorption in the rat
MTAL is inhibited by cyclic AMP and, under certain conditions,
stimulated by activation of protein kinase C
(13, 17, 20) . To determine whether these
signaling pathways are involved in inhibition of
HCO
absorption by hyperosmolality, the
effects of increasing NaCl concentration were examined in the presence
of staurosporine, a protein kinase C inhibitor, or forskolin, a direct
activator of adenylyl cyclase. At the concentrations used, forskolin
mediates maximal cyclic AMP-dependent inhibition of
HCO
absorption
(17) , and
staurosporine eliminates completely protein kinase C-dependent
regulation of HCO
transport
(20) .
The results are shown in Fig. 3. In tubules bathed with
10
M staurosporine, addition of 75
mM NaCl to the perfusate and bath decreased
HCO
absorption by 53%, from 12.7 ±
1.8 to 6.0 ± 1.6 pmol/min/mm ( n = 3;
Fig. 3A). In tubules bathed with 10
M forskolin, addition of 75 mM NaCl decreased
HCO
absorption by 75%, from 10.1 ±
0.4 to 2.5 ± 0.9 pmol/min/mm ( n = 3;
Fig. 3B). In both cases, the inhibition by NaCl was
reversible. Thus, the effect of hyperosmotic NaCl to inhibit
HCO
absorption was unimpaired by
pretreatment with staurosporine or forskolin.
Figure 3:
Effects
of adding NaCl (75 mM) to perfusate and bath on
HCO absorption in the presence of
staurosporine ( A) or forskolin ( B). Either
staurosporine ( Stauro, 10
M) or
forskolin ( Forsk, 10
M) was
present in the bath throughout the experiments. Staurosporine and
forskolin solutions were 290 mosmol/kg H
O; solutions
containing added NaCl were 425 mosmol/kg H
O. Filled
circles, lines, and p values as in Fig. 1. Mean values
are in the text.
Effects of Vasopressin and Prostaglandin E
Previously, we demonstrated that AVP
inhibits HCOin
the Presence of Genistein
absorption by 40-50%
via cyclic AMP
(17) and that this inhibition is reversed by
PGE
through activation of protein kinase C
(20, 21) . To determine whether regulation of
HCO
absorption by AVP or PGE
also may involve PTK, the effects of AVP and PGE
were
studied in the presence of genistein (Fig. 4). With 7
µM genistein in the bath, adding 10
M AVP to the bath decreased
HCO
absorption by 45%, from 14.9 ±
1.6 to 8.2 ± 1.1 pmol/min/mm ( p < 0.01;
Fig. 4A). With both AVP and genistein in the bath,
adding 10
M PGE
to the bath
increased HCO
absorption from 8.3
± 0.2 to 12.4 ± 0.4 pmol/min/mm ( p < 0.001;
Fig. 4B). The effects of AVP and PGE
were
reversible and are similar to results obtained previously in MTALs
studied in the absence of genistein
(17, 21) . Thus, at
the same concentration that nearly abolished inhibition by
hyperosmolality, genistein did not prevent AVP inhibition or PGE
stimulation of HCO
absorption. Effects of Genistein on Basal HCO
Absorption-The effects of PTK inhibitors on
HCO
absorption under control (isosmotic)
conditions are shown in and Fig. 5. Addition of 7
µM genistein or 1 µM herbimycin A to the bath
increased HCO
absorption by 30%, from
13.3 to 17.2 pmol/min/mm ( (A)). The stimulation of
HCO
absorption was reversible
(Fig. 5 A). Thus, a constitutive PTK activity appears to
play a role in determining the base-line
HCO
absorption rate.
Figure 4:
Effects of arginine vasopressin
( AVP, 10
M in the bath) and
prostaglandin E
( PGE
,
10
M in the bath) on
HCO
absorption in the presence of
genistein. Genistein ( Gen, 7 µM) was present in
the bath throughout the experiments. In PGE
experiments
( B), the bath also contained 10
M
AVP. All solutions were 290 mosmol/kg H
O. Filled
circles, lines, and p values as in Fig. 1. Mean values
are in the text.
Figure 5:
Effects of genistein on basal
HCO absorption. In A, either
genistein (7 µM) or herbimycin A (1 µM) was
added to control bath solution. In B, ethylisopropyl amiloride
( EIPA, 50 µM) was added to the luminal perfusate
in tubules bathed with 7 µM genistein. All solutions were
290 mosmol/kg H
O. Filled circles, lines, and p values as in Fig. 1. Mean values are in Table II (A and
B).
Further
experiments were performed to assess whether the increase in
HCO absorption with genistein was
mediated by an increase in apical membrane
Na
/H
exchange. The contribution of
apical Na
/H
exchange to
HCO
absorption was assessed by luminal
addition of EIPA, a potent Na
/H
exchange inhibitor. With 7 µM genistein in the bath
solution, addition of 50 µM EIPA to the luminal perfusate
decreased HCO
absorption by 80%, from
16.3 to 3.6 pmol/min/mm ( (B); Fig. 5 B). In
the absence of genistein, luminal EIPA decreased
HCO
absorption from 11.8 ± 0.4 to
2.8 ± 0.2 pmol/min/mm ( n = 3; p <
0.001).
(
)
Thus, the increase in
HCO
absorption observed with genistein
was inhibitable by luminal EIPA.
Effects of Molybdate
To assess further the role of
tyrosine phosphorylation in the regulation of
HCO absorption, we examined the effect of
molybdate, a potent protein tyrosine phosphatase inhibitor
(22, 23) . In tubules perfused and bathed in isosmotic
solution, addition of 50 µM sodium molybdate to the bath
decreased HCO
absorption by 48%, from
13.6 ± 1.1 to 7.1 ± 1.6 pmol/min/mm ( n =
4; p < 0.025). The inhibition by molybdate was fully
reversible and occurred in the absence of a change in transepithelial
voltage.
(
)
Thus, the effect of hyperosmolality to
inhibit HCO
absorption could be
reproduced with an agent that inhibits tyrosine phosphatases. In three
additional experiments, addition of molybdate to the bath had no effect
on HCO
absorption in tubules perfused and
bathed with hypertonic NaCl (5.4 ± 0.4 pmol/min/mm, NaCl
versus 5.3 ± 0.1 pmol/min/mm, NaCl + molybdate;
p = NS). Thus, the inhibitory effects of
hyperosmolality and molybdate were not additive, suggesting that the
two factors may act through a similar mechanism of action. These
results provide further support for a role for tyrosine phosphorylation
in the inhibition of HCO
absorption by
hyperosmolality.
/H
exchange
by hyperosmolality is an important mechanism for maintenance of cell
volume in many cell types
(1, 2, 3, 4) .
In contrast, we have shown recently that hyperosmolality inhibits
apical membrane Na
/H
exchange in the
MTAL of the rat
(15) . Inhibition of the apical
Na
/H
exchanger accounts functionally
for the effect of hyperosmolality to inhibit transepithelial
HCO
absorption
(15, 16) .
The results of the present study show that these inhibitory effects of
hyperosmolality are mediated via a PTK-dependent signaling pathway.
Furthermore, regulation of HCO
absorption
by hyperosmolality via the PTK pathway occurs independent of regulation
by cyclic AMP and protein kinase C. Tyrosine phosphorylation has been
suggested previously to be an important step in swelling-induced
activation of ion channels in a human intestinal cell line
(26) . Our data are the first to suggest that tyrosine
phosphorylation also may play an essential role in osmotic regulation
of Na
/H
exchange.
absorption via their actions on PTK
activity. First, virtually identical results were obtained with
genistein and herbimycin A, two chemically unrelated PTK inhibitors
with different mechanisms of action
(27, 28) . Second,
at the concentrations studied, both genistein and herbimycin A are
selective PTK inhibitors, with no significant activity against a
variety of other protein kinases, phosphatases, or
phosphodiesterase.
(
)(27, 28, 29, 30, 31, 32) .
The apparent specificity of these agents was supported in the present
study by the observation that genistein, at the same concentration that
nearly eliminated inhibition by hyperosmolality, had no effect on
regulation of HCO
absorption by AVP (a
cyclic AMP-dependent process) or PGE
(a protein kinase
C-dependent process). Third, both genistein and herbimycin A reversibly
stimulated HCO
absorption under isosmotic
conditions. This finding, along with the lack of effect of genistein on
regulation by AVP and PGE
, suggests that the effect of
these agents to inhibit hyperosmotic regulation was not the result of a
toxic or nonspecific metabolic effect on the MTAL cells. Taken
together, these results support the notion that genistein and
herbimycin A prevent the inhibition of
HCO
absorption by hyperosmolality via
their targeted actions to inhibit PTK activity. Role of PTK in Inhibition of HCO
Absorption by Hyperosmolality-In the rat, the osmolality
of the renal medulla can vary from 290 mosmol/kg H
O to more
than 1500 mosmol/kg H
O in response to changes in systemic
H
O balance. Thus, the osmolalities achieved in the present
study with NaCl (425 mosmol/kg H
O) and mannitol (590
mosmol/kg H
O) represent reasonable estimates of the values
expected to surround the MTAL in vivo. Previously, we
demonstrated that hyperosmolality produced with a variety of solutes
markedly inhibited MTAL HCO
absorption,
an effect that may be important physiologically for limiting delivery
of HCO
to the medullary interstitial
fluid during antidiuresis
(13, 16) . Results of the
present study indicate that hyperosmotic inhibition of
HCO
absorption was eliminated nearly
completely by inhibitors of PTK. These agents prevented inhibition of
HCO
absorption by both physiologic (NaCl)
and nonphysiologic (mannitol) osmotic agents, indicating that the
increase in osmolality rather than addition of a particular solute was
the signal that initiates activation of the PTK regulatory pathway.
Protein tyrosine phosphorylation thus appears to be a critical element
in the inhibition of HCO
absorption by
hyperosmolality. Further support for this conclusion was obtained from
the observation that the inhibitory effect of hyperosmolality could be
reproduced with molybdate, a potent tyrosine phosphatase inhibitor
(22, 23) . In addition, the lack of additivity of the
effects of hyperosmolality and molybdate suggests that these factors
inhibit HCO
absorption through a common
signaling mechanism, presumably an increase in tyrosine
phosphorylation.
/H
exchange mediates virtually all of MTAL
HCO
absorption
(13, 14, 15) . Furthermore, hyperosmolality
inhibits HCO
absorption through
inhibition of apical membrane Na
/H
exchange
(15) . The effect of the PTK inhibitors to block
hyperosmotic inhibition of HCO
absorption
is thus most likely the result of their preventing inhibition of apical
Na
/H
exchange. An alternative
possibility is that exposure to PTK inhibitors may unmask a second
H
secretory pathway that is stimulated by
hyperosmolality, thereby offsetting hyperosmotic inhibition of the
apical Na
/H
exchanger to maintain
HCO
absorption. This possibility seems
unlikely, however, since stimulation of
HCO
absorption by genistein was inhibited
by luminal EIPA. This indicates that PTK regulation of
HCO
absorption is mediated through
regulation of apical membrane Na
/H
exchange and that the apical exchanger mediates
HCO
absorption both in the absence and
presence of PTK inhibitors. Our results are thus most consistent with
the conclusion that hyperosmolality acts via a PTK-dependent signaling
pathway to inhibit apical membrane Na
/H
exchange, thereby inhibiting transepithelial
HCO
absorption. Hyperosmolality inhibits
the apical Na
/H
exchanger by
decreasing its sensitivity to internal H
, manifested
as an acid shift in the pH
dependence curve
(15) . The present results suggest that this osmotic-induced
acid shift requires PTK. Further work is needed to test this directly
and to identify the molecular mechanisms involved in PTK-dependent
regulation of the Na
/H
exchanger.
/H
exchange (NHE-1 through NHE-4)
have been identified
(33) . Recent preliminary studies suggest
that NHE-3 may be the Na
/H
exchanger
isoform in the apical membrane of the MTAL
(34, 35) , as
reported previously for the renal proximal tubule
(36) and
intestine
(37) . Based on our demonstration that hyperosmolality
directly inhibits apical Na
/H
exchange in the rat MTAL
(15) , we infer that
hyperosmolality inhibits NHE-3 in this nephron segment. This view is
supported by recent studies in transfected cell lines
(38) and
cultured renal epithelial cells
(39) which reported that
hyperosmolality inhibits NHE-3 but stimulates NHE-1 and NHE-2. The
present study provides the first evidence that the effect of
hyperosmolality to inhibit NHE-3 may depend critically on tyrosine
phosphorylation.
absorption, they do not establish the
nature of the link between PTK activity and hyperosmolality. An
increase in tyrosine phosphorylation in response to hyperosmolality
could result from stimulation of tyrosine kinase activity, inhibition
of protein tyrosine phosphatase activity, or both. Alternatively, the
role of PTK may be permissive, such that tyrosine phosphorylation is
essential for intact functioning of the regulatory mechanism but is not
a process regulated directly by hyperosmolality. In previous studies in
cultured intestinal cells, hypotonicity induced a rapid increase in
tyrosine phosphorylation that was potentiated by pretreatment with a
tyrosine phosphatase inhibitor, suggesting a primary role for
activation of tyrosine kinase
(26) . Analyses of signal
transduction mechanisms involved in osmotic activation of KCl
cotransport in red blood cells and Na
/H
exchange in lymphocytes also suggest a primary role for
regulation via a protein kinase
(4, 40, 41) .
Recent studies in yeast and in mammalian cell lines have demonstrated
that several members of the mitogen-activated protein (MAP) kinase, and
MAP kinase kinase families are activated by osmotic stress
(42, 43, 44, 45) . Furthermore, the
activation of MAP kinases involves tyrosine phosphorylation
(42, 44, 45) . These findings support the notion
that hyperosmotic regulation occurs via activation of protein kinases
and suggest that MAP kinases may be a component of a PTK-dependent
signaling pathway that mediates hyperosmotic inhibition of apical
membrane Na
/H
exchange in the rat
MTAL.
/H
exchange during cell volume
regulation is poorly understood. Stimulation of
Na
/H
exchange by hyperosmolality does
not appear to require direct phosphorylation of the exchanger (at least
for the NHE-1 isoform)
(11) ; however, phosphotransferase
reactions involving other proteins appear to be involved in the
activation process
(4, 6, 41, 46) .
These reactions have not been identified, although protein kinases A
and C do not appear to be involved
(4, 10) . In view of
the present results, it will be of interest to determine whether
activation of Na
/H
exchange may be a
PTK-dependent process.
absorption but had no effect on
basal (isosmotic) cell volume or on the extent of cell shrinkage in
response to hypertonic NaCl. Thus, the PTK inhibitors did not influence
HCO
absorption through effects on cell
volume, and cell shrinkage, by itself, was not sufficient to elicit the
inhibition of HCO
absorption. This latter
result supports our previous conclusion
(15) that an increase
in cell Na
activity secondary to cell shrinkage
appears to play little, if any, role in the hyperosmotic inhibition of
apical membrane Na
/H
exchange. Our
experiments do not address the question of whether a decrease in cell
volume may be necessary for activation of the PTK signaling pathway.
Regulation via the PTK-dependent Pathway Occurs Independent of
Regulation by Cyclic AMP and Protein Kinase C
AVP inhibits MTAL
HCO absorption by 40-50% by
increasing intracellular cyclic AMP
(17) . Cyclic AMP does not
appear to be involved in inhibition of
HCO
absorption by hyperosmolality, since
1) inhibition by hyperosmolality is additive to the maximal inhibition
that can be achieved with AVP or exogenous addition of 8-bromo-cyclic
AMP
(16, 17) , and 2) stimulation of cyclic AMP
production with forskolin has no effect on hyperosmotic inhibition of
HCO
absorption (Fig. 3 B).
We have also shown that the inhibition of
HCO
absorption by AVP is reversed by
PGE
through activation of protein kinase C
(20, 21) . Evidence against the involvement of protein
kinase C in hyperosmotic regulation includes 1) hyperosmolality has no
effect on stimulation of HCO
absorption
by PGE
(21) , and 2) staurosporine, at a
concentration that eliminates completely protein kinase C-dependent
regulation by PGE
(20) , has no effect on inhibition
by hyperosmolality (Fig. 3 A). A further dissociation of
the signaling pathways was obtained from experiments demonstrating that
genistein had no effect on either AVP inhibition or PGE
stimulation of HCO
absorption
(Fig. 4). Thus, a genistein-sensitive PTK does not appear to be
involved in regulation of HCO
absorption
by AVP and PGE
. Taken together, these results indicate that
hyperosmolality inhibits apical membrane
Na
/H
exchange and
HCO
absorption via a PTK-dependent
pathway that does not involve cyclic AMP or protein kinase C and that
operates independent of regulation by AVP and PGE
. These
findings are in contrast to results suggesting that AVP or cyclic AMP
is necessary for hyperosmotic activation of basolateral
Na
/H
exchange in the mouse MTAL
(19) but are consistent with evidence in other cell types that
protein kinases A and C are not involved in hyperosmotic stimulation of
Na
/H
exchange
(4, 10) . Role of PTK in Basal HCO
Absorption-Genistein or herbimycin A stimulated
HCO
absorption under isosmotic conditions
in the absence of a change in cell volume. As discussed above, this
stimulation is inhibited by luminal EIPA, indicating that genistein
increased HCO
absorption through
stimulation of apical membrane Na
/H
exchange. These data indicate that apical membrane
Na
/H
exchange is under basal control
by PTK, such that a constitutive PTK activity inhibits apical
Na
/H
exchange and
HCO
absorption under isosmotic conditions
in the absence of added agonist. Although it is reasonable to assume
that this is the same PTK pathway that is involved in hyperosmotic
inhibition of HCO
absorption, this
remains to be proven. Nonetheless, our results suggest that tyrosine
phosphorylation is an important determinant of apical membrane
Na
/H
exchange activity and
HCO
absorption in the MTAL under both
isosmotic and hyperosmotic conditions.
absorption through inhibition of apical membrane
Na
/H
exchange is mediated via a
PTK-dependent pathway. Regulation via this pathway occurs independent
of regulation by cyclic AMP and protein kinase C. A constitutive PTK
activity also controls basal HCO
absorption and apical Na
/H
exchange
activity under isosmotic conditions. These data support an important
role for protein tyrosine kinase in osmotic regulation of
Na
/H
exchange and suggest that
tyrosine phosphorylation is an essential step in inhibition of NHE-3 by
hyperosmolality in the MTAL.
Table:
Effects
of genistein on inhibition of HCOabsorption by hyperosmolality
]
,
total carbon dioxide concentration in collected tubule fluid;
JTCO
, absolute rate of total CO
absorption;
V
, transepithelial voltage, oriented lumen
positive with respect to bath. [TCO
] was 25.4
± 0.1 mM in perfusion fluid and 25.4 ± 0.1
mM in bath.
Table:
Effects of genistein
on basal HCOabsorption
]
, JTCO
, and
V
as in Table I. [TCO
] was
25.5 ± 0.1 mM in perfusion fluid and 25.1 ± 0.1
mM in bath.
, prostaglandin E
; AVP, arginine
vasopressin; MAP, mitogen-activated protein.
transport experiments, the fall in
total CO
concentration along the tubule lumen is due to a
decrease in the concentration of HCO
because, at physiologic pH, HCO
accounts
for 95% of the total CO
of the perfusate (the remaining 5%
is comprised primarily of dissolved CO
and carbonic acid).
Thus, total CO
absorption rates (JTCO
) are
virtually equal to HCO
absorption rates
(JHCO
), and the terms are used
interchangeably throughout the paper.
absorption in the absence or presence of vasopressin (Ref. 16 and
present study), suggesting, by analogy with the mouse data, that
hyperosmolality inhibits HCO
absorption
in the absence or presence of cell volume regulation.
absorption observed in the presence of EIPA is abolished by removal of
luminal Na
and is the result of incomplete inhibition
of the apical membrane Na
/H
exchanger
by EIPA at physiological Na
concentrations
(13-15).
absorption when added to the
bath at low concentrations (0.02-0.05 µM). However,
in contrast to results obtained with molybdate, the inhibition by
phenylarsine oxide was virtually complete, irreversible, and associated
with a sharp decrease in the transepithelial voltage, presumably
reflecting nonspecific metabolic effects as observed with this agent in
other cell types (25).
for
inhibition of tyrosine kinase activity ranges from 1 to 100
µM for genistein and from 0.1 to 10 µM for
herbimycin A, depending on the particular PTK or physiological process
being studied, the tissue preparation, and the conditions of the assay
( e.g. in vitro versus in vivo)
(27-32). In the present study, a low concentration of genistein
(7 µM) or herbimycin A (1 µM) was sufficient
to reduce hyperosmotic inhibition of HCO
absorption by 80%. Whether higher concentrations of the inhibitors
would have abolished the inhibition was not tested.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.