ANG II controls
Na+-K+(NH+4)-2Cl
cotransport via 20-HETE and PKC in medullary thick ascending
limb
Hassane
Amlal1,2,
Christian
LeGoff1,
Catherine
Vernimmen1,
Manoocher
Soleimani2,
Michel
Paillard1, and
Maurice
Bichara1
1 Physiologie et Endocrinologie
Cellulaire Rénale, Institut National de la Santé et de la
Recherche Médicale Unité 356, Université Pierre et
Marie Curie and Hôpital Broussais, 75270 Paris Cedex 06, France; and 2 Department of
Internal Medicine and Molecular Genetics, University of Cincinnati
School of Medicine, Cincinnati, Ohio 45267
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ABSTRACT |
Cell pH was monitored in medullary thick ascending limbs to
determine effects of ANG II on
Na+-K+(NH+4)-2Cl
cotransport. ANG II at 10
16
to 10
12 M inhibited
30-50% (P < 0.005),
but higher ANG II concentrations were stimulatory compared with the
10
12 M ANG II level
cotransport activity; eventually,
10
6 M ANG II stimulated
34% cotransport activity (P < 0.003). Inhibition by 10
12
M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase,
or cytochrome P-450-dependent
monooxygenase blockade; 10
12 M ANG II had no effect
additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE).
Stimulation by 10
6 M ANG II
was abolished by PLC and protein kinase C (PKC) blockade and was
partially suppressed when the rise in cytosolic
Ca2+ was prevented. All ANG II
effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus
10
12 M ANG II inhibits
via 20-HETE, whereas
5 × 10
11 M ANG II stimulates
via PKC
Na+-K+(NH+4)-2Cl
cotransport; all ANG II effects involve
AT1 receptors and PLC activation.
intracellular pH; intracellular calcium; cytochrome
P-450-dependent monooxygenase; phospholipase C; diacylglycerol lipase; protein kinase C; angiotensin
II receptors; DUP-753; PD-123319
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INTRODUCTION |
ANG II HAS A KEY ROLE IN the renal control of
Na+, acid-base, and water balance
not only by controlling aldosterone secretion and affecting renal
hemodynamics but also by acting directly on tubular transports. In the
proximal tubule, in which ANG II has basolateral and apical receptors,
low ANG II concentrations
(
10
10 M) stimulate but
high ANG II concentrations
(
10
7 M) inhibit NaCl,
NaHCO3, and water absorption
(reviewed in Ref. 7). The latter effects occur through several
transduction pathways acting on various carriers. For example, the
proximal apical
Na+/H+
antiport system, which is critical to
NaHCO3, NaCl, and water absorption, is stimulated by low ANG II concentrations through both
decrease in cAMP production and activation of protein kinase C (PKC),
and it is inhibited by high ANG II concentrations through activation of
arachidonic acid metabolism via the cytochrome
P-450/monooxygenase pathway (reviewed
in Ref. 7). These ANG II experimental effects are physiologically or
pathophysiologically relevant, since ANG II concentrations up to 2 × 10
8 M have been
measured in the proximal tubular fluid, concentrations that are much
higher than the 10
11 to 5 × 10
10 M plasma ANG
II concentrations because of intrarenal generation of ANG II (6, 24,
25).
ANG II may also act at tubular sites distal to the proximal tubule
because specific ANG II binding sites and mRNA for ANG II receptors are
present in the distal segments of the nephron (22, 23, 27), but
possible distal tubular effects of ANG II have been directly addressed
in only a few studies. Recent work has established that ANG II
stimulates Na+,
HCO
3, and water absorption in the rat
distal tubule accessible to micropuncture (18, 19, 29). In the rabbit
cortical collecting duct microperfused in vitro,
10
7 M ANG II stimulates
luminal alkalinization (HCO
3 secretion
by B-type intercalated cells) (30). No direct evidence is available, to
our knowledge, regarding possible ANG II effects on thick ascending
limb (TAL) transepithelial transports. However, specific ANG II binding
sites and mRNA for ANG II receptor subtype 1 (AT1) are present in the TAL,
particularly in the medullary TAL (MTAL) (22, 23, 27). In that segment,
the
Na+-K+-2Cl
cotransporter is a major apical carrier responsible for luminal uptake
and thus transcellular absorption of NaCl and for much of the luminal
step of transcellular MTAL NH+4 absorption
(10, 11) because it can function in a
Na+-NH+4-2Cl
mode (2, 3, 14); the
Na+-K+(NH+4)-2Cl
cotransport activity thus contributes to the degree of medullary hyperosmolality and thus water excretion as well as to the process of
NH3-NH+4 accumulation in the renal
medulla, which is critical to NH+4 and thus
net acid urinary excretion.
These considerations prompted us to directly assess possible effects of
ANG II on
Na+-K+(NH+4)-2Cl
cotransport activity of freshly harvested MTAL cells. To this purpose,
we have monitored intracellular pH
(pHi) in suspensions of rat
MTALs by use of the pH-sensitive fluorescent probe
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF);
rates of pHi changes due
specifically to NH+4 transport were
determined to quantify the bumetanide-sensitive cotransport activity,
as previously reported (1). The results show for the first time that
ANG II inhibits the MTAL
Na+-K+(NH+4)-2Cl
cotransport activity compared with untreated controls at the very low
concentration of
10
12 M
but that higher ANG II concentrations (
5 × 10
11 M) progressively
stimulate cotransport activity compared with the level seen with
10
12 M ANG II; the
inhibitory basal effect is caused by cytochrome P-450/monooxygenase-derived products
of arachidonic acid [20-hydroxyeicosatetranoic acid
(20-HETE)], whereas the stimulatory effect is mediated by PKC.
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METHODS |
Isolation of rat MTAL tubules.
The method used has been previously described in detail (17). In brief,
eight kidneys of four anesthetized male Sprague-Dawley rats
(200-300 g body wt) were bathed in situ for 1-2 min with ice-cold dissecting solution before rapidly removing them to avoid anoxic damage to medullary tissues and to improve cell viability. The
kidneys were then cut into thin slices along the corticopapillary axis
into ice-cold Hanks' solution supplemented with 24 mM
HCO
3 and bubbled with 95%
O2-5%
CO2, pH 7.38. Small tissue pieces
of inner stripes of outer medulla were then subjected at
37°C to successive 6-min periods of collagenase digestion (0.40 g/l); the MTAL tubules were harvested by sieving the supernatants
through a nylon mesh (75-µm opening) to separate MTAL fragments from
isolated cells and small fragments of other medullary tissues and were resuspended in an appropriate volume of the desired medium. The final
suspension contained almost exclusively MTAL tubules 75-200 µm
in length (>95%), occasional thin descending limb fragments, very
few medullary collecting tubules, and virtually no isolated cells.
Because the present study deals with possible tubular effects of ANG
II, we have checked by electron microscopy that there were no proximal
tubule fragments in this MTAL suspension obtained from carefully
dissected inner stripes of outer medulla. (Electron microscopy was
performed by Gerard Feldmann and Alain Moreau, Institut National de la
Santé et de la Recherche Médicale Unité 327, Paris,
France.) MTAL fragments were suspended at 37°C in a
CO2-free medium composed of (in
mM) 125 NaCl, 15 choline chloride, 3 KCl, 0.8 K2HPO4,
0.2 KH2PO4,
1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose, and 5 L-leucine; this solution was
adjusted to pH 7.4 with Tris, contained 0.1 g/l BSA, and was bubbled
with 100% O2; in some solutions, 10 mM BaCl2 isosmotically replaced
choline chloride.
Because the purpose of the present study was to assess possible acute
effects of ANG II, this hormone (or its vehicle for control studies)
was applied to the cells at 37°C 5 min before the measurements. To
assess the mechanism of the observed effect, inhibitors (or their
vehicles) were in some cases applied 3 min before ANG II.
Measurements of pHi.
pHi was estimated with use of
BCECF as previously described in detail (17). In brief, aliquots of
BCECF-loaded tubules were diluted into glass cuvettes containing 2 ml
of the experimental medium, and BCECF fluorescence was monitored with a
Jobin Yvon JY3D spectrofluorometer equipped with a water-jacketed,
temperature-controlled cuvette holder and magnetic stirrer.
Fluorescence intensity was recorded at one emission wavelength, 525 nm,
whereas the excitation wavelength was alternated manually at regular
intervals between two wavelengths, 504 and 450 nm. The fluorescence
ratio values (F504/F450)
were converted into pHi values
with calibration curves that were established daily by the
high-K+ medium-nigericin
and/or Triton X-100 methods, as described previously (17).
The
Na+-K+(NH+4)-2Cl
cotransport activity was assessed by estimating the
bumetanide-sensitive rate of intracellular acidification caused by
entry into the cells of NH+4 after abrupt
application of 4 mM NH4Cl to the
cells (1-3). After NH4Cl
addition, a very rapid cellular alkalinization first occurred (see Fig.
2) due to immediate predominant
NH3 entry, which ended when
intracellular and extracellular
NH3 concentrations were equal (NH3 equilibrium); this was
followed by a relatively rapid and profound fall in
pHi, the initial rate of which is
determined almost exclusively by the rate of
NH+4 entry that causes
H+ accumulation within the cells
because the intracellular NH3
amount that tends to rise above the
NH3 equilibrium value leaves the cell as fast as NH+4 enters (the
extracellular NH3 concentration,
which determines the NH3
equilibrium value, is constant in our experiments). Indeed, it is
generally accepted that NH3
readily permeates plasma membranes by lipid phase diffusion (at least
the basolateral membrane in the MTAL), which thus may not be rate
limiting during the secondary acidification phase. In the presence of 1 µM amiloride plus 10 mM Ba2+ to
block MTAL NH+4 carriers other than
Na+-K+ (NH+4)-2Cl
cotransport [NH+4 conductance,
K+/NH+4(H+)
antiport, and
NH+4(K+)-Cl
cotransport; Refs. 1-3], a noticeable cell acidification
followed the initial cell alkalinization after
NH4Cl addition (see Fig. 2); cell
acidification was due to
Na+-K+(NH+4)-2Cl
activity because, in the presence of 1 µM amiloride plus 10 mM Ba2+ + 0.1 mM furosemide (or
bumetanide), 4 mM NH+4-induced cell
acidification was abolished (Fig. 1D
in Ref. 2). We have previously demonstrated that the
NH+4-induced initial rate of acidification
(dpHi/dt)
is not affected by changes in the activity of the other MTAL
pHi regulatory mechanisms such as
Na+/H+
antiport (1, 2). Therefore, the
Na+-K+(NH+4)-2Cl
cotransport activity was defined as the bumetanide-sensitive 4 mM
NH+4-induced
dpHi/dt
observed in the presence of 1 µM amiloride plus 10 mM
Ba2+. In the experiments to be
described, the NH+4-induced dpHi/dt
was taken to directly reflect changes in the transmembrane H+-equivalent flux (=
dpHi/dt ×
i × cell volume,
where
i is the intrinsic cell
buffering power) because the
i × cell volume product is constant in these acute experiments that
lasted only a few minutes, as previously demonstrated (1). The results
concerning the NH+4-induced
dpHi/dt
are presented both as absolute values in the text and the legends of
Figs. 1-3, 6-8, and 10-12 and as percent of the mean
control value of each experiment in Figs. 3-8 and 10-12 to
facilitate comparisons.
Measurements of intracellular
Ca2+
concentration.
Intracellular Ca2+ concentration
([Ca2+]i)
was estimated with use of the
Ca2+-sensitive fluorescent probe
fura 2 exactly as previously described (5). In brief, fura 2-loaded
tubules were diluted into glass cuvettes placed in the
spectrofluorometer described above, and fluorescence intensity was
recorded at a 495-nm wavelength (10-nm bandwidth), whereas the
excitation wavelength was manually changed at 3-s intervals between
340- and 380-nm wavelengths (4-nm bandwidth). At the end of each run
(<90 s), 5 mM EGTA was added first, to estimate and correct for the
fluorescence of fura 2 that leaked out of the cells, as previously
described (5). Then 15 mM Tris and 6.5 µM digitonin were added to
permeabilize the cell plasma membrane and determine the minimum fura 2 fluorescence ratio, which was followed by addition of 5.5 mM
CaCl2 to determine the maximum
fura 2 fluorescence ratio. The signals generated by fura 2-loaded cells
were corrected for autofluorescence determined at the two excitation
wavelengths on a fura 2-free MTAL suspension aliquot. Values of
[Ca2+]i
were calculated with the usual equation (5).
Materials.
Collagenase CH grade II was obtained from Boehringer Mannheim (Maylan,
France). BCECF-AM,
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM, and fura 2-AM were obtained from Molecular
Probes (Eugene, OR). 20-HETE, 17-octadecynoic acid
(17-ODYA), oleyloxyethyl phosphorylcholine, RHC-80267, U-73122,
U-73343, W-7, and W-5 were from Biomol Research Laboratories (Plymouth
Meeting, PA).
N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide
(H-89) and SKF-525A were from Calbiochem (La Jolla, CA). ANG II,
8-bromo-cAMP, staurosporine, calphostin C, 4-bromophenacyl bromide,
amiloride, furosemide, ouabain, bumetanide, nigericin, and all other
chemicals were obtained from Sigma-Chimie (La Verpillière,
France). Nonpeptide ANG II receptor antagonists losartan (DUP-753) and
PD-123319 were provided by Dr. R. D. Smith from DuPont and Dr. J. A. Keiser from Parke-Davis, respectively.
Statistics.
Results are expressed as means ± SE. Statistical significance
between experimental groups was assessed by Student's
t-test or by one-way ANOVA completed
by a t-test using the within-groups residual variance of one-way ANOVA, as appropriate.
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RESULTS |
Concentration-dependent effects of ANG II.
In the absence of NH+4 and in the presence of
0.1 mM furosemide to block
Na+-K+-2Cl
cotransport, 10
12 and
10
6 M ANG II had no effect
on resting pHi or on the
pHi recovery following
intracellular acidification caused by abrupt addition of 40 mM
potassium acetate to MTAL suspensions (Fig.
1), a condition in which
pHi recovery occurs through
Na+/H+
antiport and H+-ATPase activities;
ANG II also had no effect on pHi
recovery in the presence of 200 nM bafilomycin
A1 added to block the MTAL H+-ATPase (not shown). As shown in
Figs. 2-4, ANG II did
not change the magnitude of the initial
NH3-induced cell alkalinization
following abrupt exposure to NH4Cl
but affected the
Na+-K+(NH+4)-2Cl
cotransport activity. In the presence of 1 µM amiloride plus 10 mM
Ba2+,
10
16 to
10
12 M ANG II reduced the
NH+4-induced
dpHi/dt
by 30-50% compared with untreated controls
(P < 0.005; Figs.
2A and 3).1
However, higher ANG II concentrations (
5 × 10
11 M) progressively
stimulated the NH+4-induced dpHi/dt
compared with the level observed with
10
12 M ANG II (Fig. 3);
eventually, 10
6 M ANG II
significantly stimulated the NH+4-induced dpHi/dt
above the level of untreated controls (Fig. 3). Thus it appeared that
ANG II activated an inhibitory transduction pathway at very low
concentrations (
10
12 M)
and that another stimulatory pathway was activated by
5 × 10
11 M ANG II;
10
12 and
10
6 M ANG II are used
hereafter as representative of the inhibitory and stimulatory ANG II
concentrations, respectively. In contrast, ANG II at
10
12 and
10
6 M (Figs.
2B and 4) had no effect on
NH+4-induced cell acidification in the
presence of 0.1 mM bumetanide, a condition in which
NH+4 enters the cell through transport pathways other than
Na+-K+(NH+4)-2Cl
cotransport. Taken together, these results demonstrate that the ANG II
effects described above resulted from alterations of the Na+-K+(NH+4)-2Cl
cotransport activity specifically but not from effects on other NH+4 or
H+ carriers or on
NH3 permeability. To assess
whether ANG II acts on
Na+-K+(NH+4)-2Cl
cotransport directly or indirectly through effects on other
Na+,
K+, or
Cl
MTAL carriers and
attendant changes in intracellular ion concentrations, ANG II was
applied after MTAL cells were exposed to 1 mM ouabain for 3 min to
block the
Na+-K+-ATPase.
Under this experimental condition,
10
12 M ANG II still
inhibited ~26% (P < 0.007) and
10
6 M ANG II still
stimulated ~28% (P < 0.008) the
NH+4-induced dpHi/dt
in the presence of 1 µM amiloride plus 10 mM
Ba2+ compared with untreated
controls (Fig. 4). Note that control NH+4-induced
dpHi/dt
values were lower in the presence of ouabain than in its absence
(
1.01 ± 0.06 vs.
1.78 ± 0.05 pH units/min;
P < 0.05) as a result of
Na+-K+-ATPase
blockade and subsequent changes in intracellular
Na+,
K+, and
Cl
concentrations. Thus
effects observed in the presence of 1 µM amiloride plus 10 mM
Ba2+ are hereafter referred to as
effects on the
Na+-K+(NH+4)2Cl
cotransport activity.

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Fig. 1.
Cell intracellular pH (pHi)
recovery from an acid load. Medullary thick ascending limb (MTAL) cells
were incubated in a CO2-free
medium, and pHi was monitored in
presence of 0.1 mM furosemide; at time
0, cells were acutely acidified by
entry of acetic acid after addition of potassium acetate. There was no
difference in basal pHi or in
initial rate of pHi recovery (in
pH units/min) between control cells (1.03 ± 0.10, n = 7) and cells exposed for 5 min to
1 pM (1.08 ± 0.07, n = 8) or 1 µM ANG II (1.08 ± 0.08, n = 7).
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Fig. 2.
Cell acidification caused by NH+4 entry. MTAL
cells were incubated in a CO2-free
medium in presence or absence of 1 pM ANG II for 5 min; addition at
time
0 of
NH4Cl caused an immediate cell
alkalinization (NH3 entry)
followed by cell acidification due to NH+4
entry (see METHODS for detailed
explanations). A: in presence of
amiloride + Ba2+, initial rate of
acidification
(dpHi/dt,
in pH units/min) due to
Na+-NH+4-2Cl
cotransport activity was decreased by ANG II ( 1.29 ± 0.17, n = 14) vs. control ( 1.93 ± 0.23, n = 12)
(P < 0.001).
B: in presence of bumetanide to block
Na+-NH+4-2Cl
cotransport,
dpHi/dt
was not affected by ANG II [ 2.08 ± 0.23 vs.
2.20 ± 0.30; not significant (NS)].
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Fig. 3.
Concentration-dependent effects of ANG II on
Na+-NH+4-2Cl
cotransport. Initial rate of cell acidification
(dpHi/dt)
caused by
Na+-NH+4-2Cl
cotransport activity in presence of 1 µM amiloride + 10 mM
Ba2+ was maximally reduced by
10 14 M ANG II; from this
low level, higher ANG II concentrations ( 5 × 10 11 M) were stimulatory.
Absolute value of control
dpHi/dt
(100%) was 1.78 ± 0.05 pH units/min
(n = 39); each point in presence of
ANG II represents mean ± SE of 5-12 values expressed
as % of mean control value of each of 14 MTAL suspensions.
* P < 0.003, compared with
untreated control; P < 0.10 and
§ P < 0.0001, compared with
10 12 M ANG II.
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Fig. 4.
Direct effects of ANG II on
Na+-NH+4-2Cl
cotransport. Inhibition by 1 pM and stimulation by 1 µM M ANG II of
Na+-NH+4-2Cl
cotransport activity [NH+4-induced
initial rate of cell acidification
(dpHi/dt)
in presence of amiloride + Ba2+] were still observed in
cells treated with ouabain 3 min before ANG II. No effect of ANG II was
observed on
dpHi/dt
in presence of bumetanide to block
Na+-K+(NH+4)-2Cl
cotransport. Each bar in presence of ANG II represents mean ± SE of
6-12 values expressed as % of mean control value (0).
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To establish by which ANG II receptor these effects occurred, we used
the nonpeptide ANG II receptor antagonists losartan and PD-123319 to
block AT1 and
AT2 receptors, respectively. As shown in Fig.
5,
10 µM losartan abolished both the inhibitory and stimulatory ANG II
effects, whereas 10 µM PD-123319 did not prevent them from occurring.
These results are consistent with all ANG II effects being mediated
through AT1 receptor occupancy.

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Fig. 5.
Effects of ANG II receptor antagonists. The
AT1 antagonist losartan (Los.),
but not the AT2 antagonist
PD-123319 (PD.), abolished inhibitory and stimulatory effects of 1 pM
and 1 µM ANG II, respectively, on
Na+-NH+4-2Cl
cotransport activity [NH+4-induced
initial rate of cell acidification
(dpHi/dt)
in presence of 1 µM amiloride + 10 mM
Ba2+]. C, control; each bar
represents mean ± SE of 8-12 values expressed as % of mean
control value.
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The ANG II inhibitory effect is mediated through the arachidonic
acid pathway.
We first established that the cAMP/cAMP-dependent protein kinase
(cAMP/PKA) pathway does not contribute to the ANG II effects under our
experimental conditions. To this purpose, MTAL cells were exposed to
0.5 mM 8-bromo-cAMP or 15 µM H-89 3 min before ANG II; we have
previously shown that 8-bromo-cAMP stimulates the cotransport activity
through a PKA-dependent mechanism and that 15 µM H-89 completely
blocks PKA in MTAL cells (1). As shown in Fig.
6,
10
12 M ANG II still
decreased and 10
6 M ANG II
still increased the cotransport activity despite the presence of either
8-bromo-cAMP or H-89.

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Fig. 6.
cAMP/protein kinase A (cAMP/PKA) pathway has no role in ANG II effects.
Pretreatment of MTAL cells with 0.5 mM 8-bromo-cAMP or 15 µM H-89, an
inhibitor of PKA, did not prevent 1 pM and 1 µM ANG II from
inhibiting and stimulating, respectively,
Na+-NH+4-2Cl
cotransport activity [NH+4-induced
initial rate of cell acidification
(dpHi/dt)
in presence of 1 µM amiloride + 10 mM
Ba2+]. Each bar represents
mean ± SE of 9-11 values expressed as % of mean control
value. Absolute control values were 1.97 ± 0.12 and
0.81 ± 0.07 pH units/min (P < 0.001) in presence of 8-bromo-cAMP and H-89, respectively.
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Because we (1) and others (8) had previously demonstrated that
arachidonic acid and its metabolites derived from the cytochrome
P-450-dependent monooxygenase
pathway inhibit
Na+-K+(NH+4)-2Cl
cotransport activity, we tested for a possible involvement
of that pathway in the inhibitory effect of low concentrations of ANG
II in the following ways. First, as shown in Fig.
7, 3 µM SKF-525A, a compound that blocks
the cytochrome P-450-dependent monooxygenase and abolishes the arachidonic acid effect in MTAL cells
like econazole (1), suppressed the inhibitory effect of
10
12 M ANG II; we have
previously shown that SKF-525A had no effect per se (1). Similarly,
17-ODYA, another inhibitor of the cytochrome P-450-dependent monooxygenase, had no
effect per se but abolished the inhibitory effect of
10
12 M ANG II on the
Na+-K+
(NH+4)-2Cl
cotransport activity [the NH+4-induced
dpHi/dt (in pH units/min) was
1.94 ± 0.06 in control
(n = 9),
1.94 ± 0.06 in the
presence of 5 µM 17-ODYA (n = 5),
and
1.97 ± 0.06 in the presence of
10
12 M ANG II + 5 µM
17-ODYA (n = 6)]. Second,
10
12 M ANG II caused no
further inhibition in the presence of 1 µM 20-HETE, one of the main
cytochrome P-450/monooxygenase-derived products of arachidonic acid in the MTAL (8), which itself inhibited
(P < 0.004) the cotransport activity
(Fig. 7). Thus the effects of 20-HETE and
10
12 M ANG II were not
additive. Third, to determine whether ANG II causes the release of
arachidonic acid from membrane lipids through activation of
phospholipase A2
(PLA2) or diacylglycerol (DAG) lipase, we used known inhibitors of these enzymes. As shown in Fig.
8, 3.3 µM 4-bromophenacyl bromide and 5 µM oleyloxyethyl phosphorylcholine, which both inhibit
PLA2 in MTAL cells (1), did not
prevent 10
12 M ANG II from
decreasing the
Na+-K+(NH+4)-2Cl
cotransport activity; these two compounds had no effect per se. In
contrast, 50 µM RHC-80267, an inhibitor of DAG lipase (26) that had
no effect per se on
Na+-K+(NH+4)-2Cl
cotransport activity, abolished the inhibitory effect of
10
12 M ANG II (Fig. 8).
Finally, that 10
12 M ANG II
activates phospholipase C (PLC) to produce DAG is shown below (see Fig.
12).

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Fig. 7.
Role of arachidonic acid pathway in inhibitory ANG II effect. Blockade
of cytochrome P-450-dependent
monooxygenase by 3 µM SKF-525A (SKF) abolished inhibitory effect of 1 pM ANG II on
Na+-NH+4-2Cl
cotransport activity [NH+4-induced
initial rate of cell acidification
(dpHi/dt)
in presence of 1 µM amiloride + 10 mM
Ba2+]; 1 pM ANG II had no
additional inhibitory effect in presence of 20-hydroxyeicosatetranoic
acid (20-HETE), one of the main monooxygenase-derived products of
arachidonic acid in MTAL, which itself inhibited
Na+-NH+4-2Cl
cotransport activity. Each bar represents mean ± SE of 6-8
values expressed as % of mean control value. Absolute control values
were 0.87 ± 0.09 (left)
and 1.02 ± 0.12 (right)
pH units/min.
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Fig. 8.
Diacylglycerol (DAG) lipase, but not phospholipase
A2
(PLA2), is involved in
inhibitory ANG II effect. Inhibitory effect of 1 pM ANG II on
Na+-NH+4-2Cl
cotransport activity [NH+4-induced
initial rate of cell acidification
(dpHi/dt)
in presence of 1 µM amiloride + 10 mM
Ba2+] was abolished by the
DAG lipase blocker RHC-80267 (RHC; 50 µM) but not by the
PLA2 blockers 4-bromophenacyl
bromide (BPB; 3.3 µM) and oleyloxyethyl phosphorylcholine (OPCH; 5 µM); RHC, BPB, and OPCH had no effect per se. Each experimental bar
represents mean ± SE of 6-11 values expressed as % of mean
control value for each experimental series. Absolute control values
(n = 23) were 0.87 ± 0.05 (n = 6), 1.05 ± 0.08 (n = 6), and 1.34 ± 0.11 pH
units/min (n = 11) for BPB, OPCH, and
RHC experiments, respectively.
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|
These results thus strongly suggest that
10
12 M ANG II enhances the
production of DAG, which is in turn deacylated by DAG lipase to yield
arachidonic acid that inhibits
Na+-K+(NH+4)-2Cl
cotransport activity through 20-HETE.
PKC is responsible for the stimulatory effect of ANG II.
We have previously shown that pharmacological activation of PKC by
phorbol 12,13-dibutyrate stimulates the MTAL
Na+-K+(NH+4)-2Cl
cotransport activity (1). We thus raised the hypothesis that PKC could
be responsible for the stimulation caused by ANG II. Indeed, results
obtained in our laboratory have established that ANG II increases
[Ca2+]i
in rat MTAL cells through AT1
receptor occupancy (abolished by losartan but not by PD-123319; G. Lazar, unpublished results); for example, Fig.
9 shows that
10
6 M ANG II caused
[Ca2+]i
to rapidly increase, which was followed by a return toward the basal
value. The ANG II-induced increase in
[Ca2+]i
was abolished by 30 µM W-7, an inhibitor of
Ca2+/calmodulin-dependent protein
kinase (Fig. 9). This suggests that the ANG II-induced increase in
[Ca2+]i
was due to liberation of Ca2+ from
intracellular organelles because W-7 has been shown to deplete intracellular
D-myo-inositol
1,4,5-trisphosphate-sensitive Ca2+
stores (28). In contrast, the ANG II-induced
Ca2+ spike persisted when
extracellular free Ca2+ was
removed by 5 mM EGTA; the ANG II-induced
Ca2+ spike was nevertheless
reduced by ~34% under the latter experimental condition
(P < 0.05; not shown).

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Fig. 9.
Effect of ANG II on intracellular
Ca2+ concentration
([Ca2+]i).
At time
0, 1 µM ANG II was added to MTAL
cells pretreated or not for 6 min with 30 µM W-7, a
Ca2+/calmodulin-dependent protein
kinase blocker; ANG II-induced rise in
[Ca2+]i
was abolished by W-7; n = 8 in absence
and 4 in presence of W-7.
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Figure 10 shows that 10 nM staurosporine,
which blocks PKC but does not by itself affect PKA or
Na+K+(NH+4)-2Cl
cotransport in MTAL cells at this concentration (1), not only abolished
the stimulation by 10
6 M
ANG II but also unmasked the underlying inhibitory influence of low ANG
II concentrations, compared with untreated controls; in contrast,
staurosporine did not affect the inhibitory effect of
10
12 M ANG II (Fig. 10).
Similarly, calphostin C, another inhibitor of PKC, had no effect per se
but turned the stimulatory effect of
10
6 M ANG II on the
Na+K+(NH+4)-2Cl
cotransport activity into an inhibitory effect [the
NH+4-induced dpHi/dt
(in pH units/min) was
1.82 ± 0.04 in control
(n = 9),
1.75 ± 0.09 in the
presence of 2 µM calphostin C (n = 5; not significantly different from control value), and
1.38 ± 0.09 in the presence of
10
6 M ANG II + 2 µM
calphostin C (n = 6;
P < 0.005 compared with control or
calphostin C)]. Conversely, blockade of cytochrome P-450-dependent
monooxygenase by 3 µM SKF-525A slightly but significantly enhanced
the stimulatory effect of
10
6 M ANG II [the
NH+4-induced
dpHi/dt
increased from
1.84 ± 0.04 (10
6 M ANG II;
n = 5) to
1.97 ± 0.03 pH
units/min (10
6 M ANG II + 3 µM SKF-525A; n = 5);
P < 0.03; not
shown], as expected from the suppression of an inhibitory
influence. Note that, in the presence of staurosporine,
10
12 and
10
6 M ANG II inhibited
cotransport activity to the same extent (Fig. 10); these results
confirm that the ANG II inhibitory effect via the arachidonic pathway
described above was maximal at
10
12 M ANG II.

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Fig. 10.
Role of PKC in ANG II effects. Stimulatory effect of 1 µM ANG II on
Na+-NH+4-2Cl
cotransport activity [NH+4-induced
initial rate of cell acidification
(dpHi/dt)
in presence of 1 µM amiloride + 10 mM
Ba2+] was turned into an
inhibitory effect by the PKC blocker staurosporine (10 nM); inhibitory
effect of 1 pM ANG II was not affected by staurosporine. Each bar in
presence of staurosporine represents mean ± SE of at least 8-9
values expressed as % of mean control value. Data in absence of
staurosporine are those of Fig. 4. Absolute control value in presence
of staurosporine was 1.28 ± 0.08 pH units/min.
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|
Usually, PKC is acutely activated by a hormonal agent after PLC
activation and attendant simultaneous DAG accumulation within the
plasma membrane and increase in
[Ca2+]i
triggered by
D-myo-inositol
1,4,5-trisphosphate; both DAG accumulation and
Ca2+ increase are able to activate
PKC. To determine the contribution of
Ca2+ to the activation of PKC by
ANG II, we used BAPTA to clamp
[Ca2+]i
and W-7 to suppress the ANG II-induced increase in
[Ca2+]i,
as described above; cells were loaded with BAPTA by 15-20 min of
preincubation in the presence of 50 µM BAPTA-AM and then washed
before ANG II addition. As shown in Fig.
11, in cells pretreated with BAPTA as
well as with 30 µM W-7, the stimulatory effect of 10
6 M ANG II was
reduced significantly but incompletely, since the underlying
inhibitory ANG II influence was not seen; W-5, an inactive structural
analog of W-7, did not prevent significant stimulation by
10
6 M ANG II. Furthermore,
W-7 had no additional effect in the presence of 10 nM staurosporine
that totally suppressed the stimulatory effect of high ANG II
concentrations (Fig. 11). It is thus clear that, under our experimental
conditions, the stimulatory effect of
10
6 M ANG II is entirely
due to PKC activation and that the PKC activation is due to the
combined actions of DAG and
Ca2+.

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Fig. 11.
Role of cytosolic Ca2+.
Stimulatory effect of 1 µM ANG II on
Na+-NH+4-2Cl
cotransport activity [NH+4-induced
initial rate of cell acidification
(dpHi/dt)
in presence of 1 µM amiloride + 10 mM
Ba2+] was prevented
partially in BAPTA- and W-7-treated MTAL cells compared with complete
abolition (significant inhibition being unmasked) caused by 10 nM
staurosporine (PKC blockade); W-7 had no further effect in presence of
staurosporine. W-5, an inactive structural analog of W-7, did not
prevent stimulation of
Na+-NH+4-2Cl
cotransport activity by 1 µM ANG II. Each bar represents mean ± SE of 6-9 values expressed as % of mean control value. Absolute
control values were 0.95 ± 0.24 (left), 1.22 ± 0.08 (middle), and 1.01 ± 0.03 pH units/min (right).
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|
It thus appeared that both the inhibitory and stimulatory
effects of the various concentrations of ANG II were possibly secondary to PLC activation. To confirm this point, we used the PLC inhibitor U-73122 (21). As shown in Fig. 12, 1 µM
U-73122, but not its inactive structural analog U-73343, abolished both
the inhibitory and stimulatory effects of
10
12 and
10
6 M ANG II, respectively.

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Fig. 12.
Role of PLC in ANG II effects. Inhibition of PLC by 1 µM U-73122
abolished both inhibitory and stimulatory effects of 1 pM and 1 µM
ANG II, respectively, on
Na+-NH+4-2Cl
cotransport activity [NH+4-induced
initial rate of cell acidification
(dpHi/dt)
in presence of 1 µM amiloride + 10 mM
Ba2+]; U-73343, an inactive
structural analog of U-73122, did not prevent ANG II effects. Each bar
represents mean ± SE of 10-13 values expressed as % of mean
control value. Absolute control values were 0.78 ± 0.04 (left) and 0.68 ± 0.05 pH
units/min (right).
|
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 |
DISCUSSION |
This study is the first, to our knowledge, in which possible
acute (5 min) effects of ANG II on rat MTAL
Na+-K+(NH+4)-2Cl
cotransport were tested for.
Na+-K+(NH+4)-2Cl
cotransport activity was assessed in rat MTAL
CO2-free suspensions as the
bumetanide-sensitive NH+4-induced initial rate of cell acidification caused by abrupt exposure to
NH4Cl. We have previously
documented the validity of this approach in which the acute
NH+4-induced initial
pHi changes are not affected by
the activity of other pHi
regulatory mechanisms including H+
carriers (1, 2). In addition, ANG II specifically altered the
Na+-K+(NH+4)-2Cl
cotransport activity, since no effect of ANG II was observed in the
presence of bumetanide (or furosemide) on basal
pHi,
NH+4-induced cell acidification, or
pHi recovery from an acid load.
That ANG II did not affect basal
pHi and the global response to an
acid load, which we wanted to ascertain, does not exclude the
possibility that ANG II might affect one
H+ carrier or another
specifically; for example, it is known that Na+/H+
exchange (NHE) activity of MTAL cells in suspension is mediated by both
NHE-1 and NHE-3 (4), carriers that may be affected by ANG II in
opposite directions to maintain cell pH.
The
Na+-K+(NH+4)-2Cl
cotransport activity was inhibited by very low ANG II concentrations,
i.e., maximal inhibition (~50%) compared with untreated controls was
observed with 10
14 M ANG
II; higher ANG II concentrations (
5 × 10
11 M) progressively
stimulated cotransport activity from the low level seen with
10
14 to
10
12 M ANG II. Because
plasma or intrarenal ANG II concentrations are
10
11 M, an in vivo
increase in the medullary ANG II concentration, even moderate, should
stimulate MTAL
Na+-K+(NH+4)-2Cl
cotransport, a hypothesis that is consistent with the previous demonstration of a primary antidiuretic action of ANG II (16). The ANG
II effects occurred through AT1
receptor occupancy inasmuch as they were abolished by losartan but
insensitive to PD-123319; this result is consistent with the previously
established presence in the rat MTAL of specific ANG II binding sites
(23) and mRNA for AT1 receptors
(22, 27). However, a biphasic effect of ANG II occurred over a very
wide range of ANG II concentrations, which could suggest that different
types of AT1 receptor are present in the MTAL. Under this hypothesis, high- and low-affinity receptors would be activated by
10
12 M and
5 × 10
11 M ANG II,
respectively, with the low-affinity receptor corresponding to classical
AT1 receptors. Further studies are
needed to address this issue. ANG II appeared to act on the cotransport
activity directly and not indirectly through changes in intracellular
ion concentrations, since the ANG II effects were observed virtually unchanged in the presence of ouabain added 3 min before ANG II. Furthermore, the cAMP/PKA pathway was not involved in the ANG II
effects, since these effects were still observed in the presence of
exogenous cAMP or H-89 that blocks PKA. We have previously demonstrated
that the rat MTAL
Na+-K+(NH+4)-2Cl
cotransport activity is controlled not only by cAMP/PKA but also by
arachidonic acid products and PKC (1).
Activation of the MTAL arachidonic acid pathway was responsible for the
Na+-K+(NH+4)-2Cl
cotransport inhibition caused by low ANG II concentrations. We (1) and
others (8) have shown that arachidonic acid inhibits Na+-K+(NH+4)-2Cl
cotransport in the MTAL via cytochrome
P-450/monooxygenase-derived 20-HETE;
in the MTAL, cyclooxygenase activity is low, lipoxygenase activity is
absent, and there is no epoxyeicosatrienoic acid production (8, 20). In
the present study, the inhibitory ANG II effect was quantitatively
comparable to those of arachidonic acid and 20-HETE (1), was abolished
by the monooxygenase inhibitors SKF-525A and 17-ODYA but not by PKA or
PKC blockers, and was not additive to that of 20-HETE; furthermore, the
stimulatory effect of high ANG II concentration was enhanced by
cytochrome P-450/monooxygenase blockade. Because the ANG II inhibitory effect was abolished by PLC or
DAG lipase but not PLA2 blockade,
it appears that very low ANG II concentrations stimulate PLC after
AT1 receptor occupancy sufficiently to produce DAG, which is in turn deacylated by DAG lipase
to yield arachidonic acid; then the main cytochrome
P-450/monooxygenase-derived products,
20-HETE and/or 20-carboxy-arachidonic acid in the
MTAL, inhibit the
Na+-K+(NH+4)-2Cl
cotransporter. Note that when PKC was blocked by staurosporine (Fig.
10) or calphostin C, 10
6 M
ANG II inhibited the cotransport activity to the same extent as did
10
12 M ANG II; this
confirms that the cotransport is maximally inhibited through the
arachidonic acid pathway by an ANG II concentration as low as
10
14 to
10
12 M. Indeed, in a recent
study (20), ANG II increased 20-HETE production in the rat MTAL to
similar extents at 5 × 10
11 and 5 × 10
8 M (to ~260 and
~210% of the control value, respectively). Thus arachidonic acid
products would exert a basal fixed inhibitory tonus on
Na+-K+(NH+4)-2Cl
cotransport, the activity of which would then be regulated by stimulatory influences such as those of the PKA and PKC pathways. The
cellular mechanisms by which 20-HETE and/or
20-carboxy-arachidonic acid inhibits MTAL cotransport are unknown, but
they do not, however, require
Na+-K+-ATPase,
PKA, or PKC activities, since they are not affected by the presence of
ouabain, H-89, or staurosporine.
Activation of PKC was responsible for the stimulation of
Na+-K+(NH+4)-2Cl
cotransport by ANG II. PKC activation by a phorbol ester stimulates the
MTAL
Na+-K+(NH+4)-2Cl
cotransport activity (1). In this study, the stimulatory ANG II effect
was insensitive to the PKA blocker H-89 but was abolished by the PKC
inhibitors staurosporine and calphostin C as well as by PLC
blockade. Furthermore, stimulation of
Na+K+(NH+4)-2Cl
cotransport by high ANG II concentrations was suppressed, although incompletely, in cells loaded with BAPTA or treated with W-7 in which
the ANG II-induced rise in
[Ca2+]i
was abolished; W-7 had no inhibitory effect additive to that of
staurosporine. These results thus strongly suggest that, after AT1 receptor occupancy and PLC
activation, concentrations of ANG II
5 × 10
11 M stimulate MTAL
Na+K+(NH+4)-2Cl
cotransport through DAG- and
Ca2+-activated PKCs. Both
Ca2+-responsive and
Ca2+-unresponsive PKC isoforms
appeared to be involved in the ANG II stimulatory effect, since the ANG
II-induced rise in
[Ca2+]i
is necessary for complete but not for partial stimulation. Results
obtained on MTAL suspensions in our laboratory, with use of immunoblots
on particulate and cytosolic fractions with antibodies specific to
various PKC isoforms (13), have established that the
Ca2+-responsive PKC
and the
Ca2+-unresponsive PKC
, PKC
,
and PKC
are present in rat MTAL cells (Z. Karim and J. Poggioli,
unpublished results); in the rat proximal tubule in which the same
isoforms of PKC are present, ANG II induces the translocation of PKC
and PKC
(13). The hypothesis of direct stimulation of the MTAL
Na+-K+(NH+4)-2Cl
cotransporter by several PKC isoforms is consistent with the presence
in renal apical cotransporter sequences of several consensus PKC
phosphorylation sites within both the
NH2- and COOH-terminal domains
(9).
In a recent study (20), ANG II inhibited at a low concentration (5 × 10
11 M) and
stimulated at a high concentration (5 × 10
8 M) the rat MTAL apical
K+ channel; the inhibitory effect
was mediated via the 20-HETE pathway, whereas the stimulatory effect
appeared to involve nitric oxide but not PKC (20). In that work, ANG II
was also shown, through undefined cellular pathways, to lower the
intracellular Na+ concentration at
10
10 M but to enhance
intracellular Na+ concentration at
10
9 to
10
7 M. The latter effects
could not be secondary to the ANG II actions on the apical
K+ channel through modulation of
luminal K+ recycling and
subsequent secondary effects on
Na+-K+-2Cl
cotransport, since the tubules were not perfused, but were consistent with a direct regulation of
Na+-K+-2Cl
cotransport by ANG II as described in the present study. However, possible effects of ANG II on other
Na+ carriers are not excluded.
In conclusion, the present results lead us to propose a working model
for the AT1- and PLC-mediated
effects of ANG II on the MTAL
Na+-K+(NH+4)-2Cl
cotransporter (Fig. 13). Very low ANG II
concentrations through high-affinity receptor occupancy impose a basal
inhibitory tonus via DAG lipase- and cytochrome
P-450/monooxygenase-derived
arachidonic acid products (20-HETE and/or
20-carboxy-arachidonic acid). This basal inhibitory tonus, which is
maximal at 10
14 to
10
12 M ANG II, is
counterbalanced by the stimulatory influence of PKC, which is
progressively activated through the combined actions of augmented DAG
accumulation and increase in
[Ca2+]i
caused by low-affinity receptor occupancy by higher ANG II concentrations (>10
12 M).
These ANG II effects may be physiologically relevant, since the
circulating and proximal tubular fluid ANG II concentrations range from
10
11 to 2 × 10
8 M (6, 24, 25). ANG II
could thus have important effects on MTAL transports because of the
major role of the
Na+-K+ (NH+4)-2Cl
cotransporter on the latter. As stated above, a rise in the local ANG
II concentration should stimulate the
Na+-K+(NH+4)-2Cl
cotransporter and enhance NaCl and NH+4
absorption by the MTAL and consequently medullary osmolarity and
medullary NH+4 accumulation; this could
explain the primary antidiuretic action of ANG II additive to that of
antidiuretic hormone (16). By these effects on MTAL transports, ANG II
could thus contribute to the renal control of water and acid-base
balance, particularly in states of extracellular fluid volume
contraction in which the renin-ANG system is activated. It must be
emphasized that effects of ANG II on NaCl absorption by the MTAL could
not contribute to the renal control of
Na+ balance if they were not
accompanied by similar effects of ANG II in the cortical segment of the
TAL (CTAL), which is unknown at present. Indeed, changes in the NaCl
load delivered to the CTAL may induce changes in CTAL transports that
may balance those in the MTAL so that the total NaCl amount reabsorbed
may remain constant (15); this is observed, for example, with
vasopressin, which affects MTAL but not CTAL transports (15). Notably,
it is unknown at present whether ANG II receptors are present in the
basolateral or apical membrane of the MTAL (or both); further studies
are needed to address this issue as well as that of the possible
presence of different subtypes of
AT1 receptors in the MTAL.

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Fig. 13.
Working model for ANG II effects in rat MTAL. ANG II effects on
Na+-NH+4-2Cl
cotransport are mediated by AT1
receptor occupancy and PLC activation that produces DAG and increases
[Ca2+]i.
At 10 12 M (high-affinity
receptor), ANG II maximally inhibits cotransport through arachidonic
acid (A.A.; via cytochrome
P-450/monooxygenase-derived products)
produced from DAG by DAG lipase. At higher ANG II concentrations ( 5 × 10 11 M;
low-affinity receptor), DAG accumulation and increase in
[Ca2+]i
progressively activate PKC, which counterbalances arachidonic acid
inhibitory ( ) pathway to stimulate (+) cotransport.
|
|
 |
ACKNOWLEDGEMENTS |
We thank Dr. R. D. Smith (DuPont) and Dr. J. A. Keiser
(Parke-Davis) for having kindly provided losartan and PD-123319,
respectively.
 |
FOOTNOTES |
This study was supported by grants from Institut National de la
Santé et de la Recherche Médicale, Université Paris
6, Fondation pour la Recherche Médicale Française, and
Fondation de France.
Parts of this work were presented at the 26th Annual Meeting of the
American Society of Nephrology (Boston, MA, November 1993) and
published in abstract form (H. Amlal, C. Vernimmen, C. LeGoff, M. Paillard, and M. Bichara. J. Am. Soc. Nephrol. 4:
862, 1993).
1
The time course of
NH+4-induced cell acidification was
curvilinear but could be well fitted from the 3rd to the 12th or 15th
second to the linear equation form
pHi = C
k · ln(t),
in which C is pHi at time
(t) = 1, k is a constant, and the rate of
change in pHi at any time
ti is
dpHi/dt =
k/ti; r values from these linear fits were
0.90. Initial rate of pHi acidification at the third second was thus defined from the latter equation as
dpHi/dt =
k/3 in pH units per second
and expressed as pH units per minute, which thus represents the slope
of a line tangent to the curve at the third second. Fitting the first
12 or 15 s of the pHi time course
to a linear function relating pHi to ln(t) requires no assumption
regarding the mechanisms of the NH+4-induced
pHi response but merely provides a
straightforward means of quantitatively comparing experimental groups.
Address for reprint requests: M. Bichara, INSERM U.356, Centre de
Recherches Biomédicales des Cordeliers, 15 Rue de l'Ecole de
Médecine, 75270 Paris Cédex 06, France.
Received 21 March 1997; accepted in final form 5 January 1998.
 |
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