Angiotensin. II, and Vasopressin in Rat. Cortical Collecting. Duct 1. Eberhard .... system seems to be active under basal conditions, it appar- ently does not.
Regulation of Na/H Exchange by Diadenosine Polyphosphates, Angiotensin II, and Vasopressin Cortical Collecting Duct 1 Eberhard
Schlatter,2
Sabine
Haxelmans,
leva
Ankorina,
E. Schlatter, S. Haxelmans, I. Ankorina, R. Kleta, Westf#{228}lischeWilhelms-UniversitOt MUnster, Medizinische Poliklinik, Experimentelle Nephrologie, M#{252}nster,Germany (J, Am. Soc. Nephrol.
Robert
exchange
for
collecting ducts (CCD) by basolateral Na/H
exchange. The influence of various agonists on pH1 and cellular Ca2 activity ((Ca2)1) in freshly isolated CCD cells was examined with BCECF and fura-2 fluorescence ratios. The recovery of pH1 per minute (ApH/ mm) after an acid load was 0.26 ± 0.03 (N = 53) in control conditions and was increased by the diadenosine polyphosphates Ap4A, Ap5A, Ap6A, the phorbol ester phorbol 12-myristat 13-acetate (PMA) (each 5 mol/L) and angiotensmn 11(100 nmol/L) by 0.05 ± 0.02(N= 10),O.1 1 ± 0.05(N= 13), 0.09 ± 0.02(N= 24), 8), respecipH/min 5 j.tmol/L) in pH/min of an inhib,.tmol/l, N =
8). Fura-2 fluorescence ratio was not significanhly changed with angiotensin II, Ap3A, or Ap4A but increased with vasopressmn, AIR, Ap5A, and Ap6A by 0.08 ± 0.02(N= 13),0.04 ± 0.02(N= 13),0.03 ± 0.01 (N= 14), and 0.03 ± 0.01 (N = 10), respectively. These data indicate that Na/H exchange in rat CCD is activated by the stimulation of a Ca2-independent protein kinase C and inhibited by protein kinase A. kinase
A, protein
kinase
C. BCECF,
(pH1)
of principal
fura-2,
HOE-694
T by
he
intracellular
pH
cortical collecting duct the Na /H exchanger,
(CCD) is regulated which extrudes
cells
of the mostly H
Received
2
Correspondence
Nephrologie,
February
15, 1995. to
Dr.
E.
Domagkstrasse
Accepted
May
2, 1995.
Schiotter.
Medizinische
3A, 48149,
MUnster,
Poiikilnik,
Germany.
1046.6673/0604-1223103.00/0
Journal of the American Society of Nephroiogy Copyright © 1995 by the American Society of Nephrology
Journal
of the American
Society
of Nephrology
Experimentelle
In
principal
from
rabbit
kidneys,
and
no such
are
used ment
as regulators of cell proliferation (6-8). This may be especially
epithelium rate,
during
cells
of this
is located (2-4). All of CCD seg-
data
from
rat
/H
in
has
exchange
in many
develop-
and important
a fairly high cellular as do the renal tubular epithelia, tubular damage or Inflammation.
Na
that
epithella
for
an
turnover especially
is regulated
by
a large number of hormones involving different intracellular transduction pathways (9). Angiotensin II (Al!) modulates Na / H #{247} exchange in opossum kidney cells (10,11) and in isolated proximal tubules from rabbit kidneys (12). The observed stimulation of exchange activity by All is apparently independent of cytosolic cAMP generation (10,11). In proximal LLC-PK1 and OK cells, agonists increasing cellular cAMP down-regulate Na/H activity (13,14). In the thick ascending limb again, agonists stimulating cytosolic cAMP production decrease
be opposite exchangers
Na
/H
activity (15). although
there
seem
to
effects present
of vasopressin (AVP) on the Na/H in the basolateral (activation) and luminal membrane (inhibItion) (16). In Madin-Darby canine kidney cells (17) and in intercalated cells of rabbit CCD (18), mlneralocorticoids stimulate Na/H exchange. In contrast to the proximal tubule, In A6 cells, the Na/H activity is increased byAVP (19). In general, Na /H exchange is regulated In renal as well as in other epithelial and nonepithelial cells different
protein
kinases
including
protein
kinase A, protein kinase C, and tyrosine klnase (9.2023). This diversity of regulation is complicated even further by the presence of various Isoforms of the Na/H exchanger in different cells, but also within one cell type (8,9,24-27). There is increasing evidence that Na* /H exchange activity is involved
1
(1,2).
CCD
through
Key Words: Protein
Na
available. Although this transport system seems to be active under basal conditions, it apparently does not contribute significantly to net Na transport in the rabbit CCD (1) and probably also not in the rat CCD (5). It is, however, generally accepted that pH1 and therefore the activity of the Na /H are
ABSTRACT
0.10 ± 0.03 (N = 7), and 0.09 ± 0.03 (N = tively. Vasopressin (10 nmol/L) decreased by 0.11 ± 0.03 (N = 9); AlP and Ap3A (each had no significant effect. The increase with Ap6A was abolished in the presence itor of protein kinase C, caiphosfin C (0.1
Kleta
nephron segment, the Na /H exchanger exclusively in the basolateral membrane these studies were performed in Isolated
ments
1995; 6:1223-1229)
In principal cells of rat cortical cellular pH (pH1) is regulated
and
in Rat
Cell also and
proliferation, regulated nucleotides
polyphosphates of Na /H
by these
in essential
for example, by vasoactive such as
hypertension
of mesangial
was
cells
is
agonists like All or AVP ATP and diadenosine
(31-34). In this study, exchange in the principal
agonist
(28-30).
the regulation cells of rat CCD
examined.
1223
Na’7H
Regulation
by DiadenosineS,
All, and
AVP
in Rat COD
METHODS Cells
and
CCD Wistar
mm while the 37#{176}C. Excitation
Solutions
cells
were
rats
and
freshly
(body
wt.
Sulzfeld. Germany) our laboratory and
by
isolated 100 to 200 the
from kidneys g; Charles
enzymatic
method
of female River Wiga, customary in (35). Isolated
described in detail before CCD clusters of 10 to 15 cells were fixed with a glass pipette In the heated perfusion chamber mounted on an inverted microscope (Axiovert 10; Zeiss, Jena, Germany) equipped with a 100X oil-Immersion lens. The standard bath solution contained (in mifiimoles per liter): NaC1. 145; K2HPO4. 1.6; KH2PO4. 0.4; calcium gluconate, 1.3; MgCI2. 1; and Dglucose. 5; pH was adjusted to 7.4. All experiments were performed in a running bath (0.5-mL volume) with an exchange rate of 20 times per minute at 37#{176}C.All standard chemicals used were obtained in the highest available purity from Sigma (Deisenhofen, Germany) and Merck (Darmstadt. Germany). 2’ .7’ -Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluo-
rescein-acetoxymethyl
ester
(BCECF-AM)
was obtained
Molecular Probes (Eugene. OR). fura-2-AM from Sigma. and pluronic F- 127 was purchased chem (Bad Soden, Germany). Diadenosine (Ap3A),
diadenosine
taphosphate
tetraphosphate
(Ap5A).
and
(Ap4A),
diadenosine
Measurements
diadenosine
pen-
hexaphosphate
were purchased from Sigma. HOE-694 by Hoechst AG (Frankfurt, Germany).
from
was obtained from Calbiotriphosphate
was
(Ap6A)
kindly
provided
of pH1
CCD cells were loaded with BCECF with the membranepermeable ester (BCECF-AM. 1 mol/L, dissolved in 0.1 gIL pluronic F- 127 in standard solution) at room temperature for 45 mm in the dark. Loaded COD cells were excited at 488 and 436 nm with a filter wheel (Physiologlsches Institut, Universlt#{228}tFrelburg, Germany) rotating at 10 Hz and a xenonquartz lamp (XBO 75 W; Zeiss) as the light source. Fluorescence
at
520
to
560
nm
was
detected
with
a single-photon
counting tube (H3460-04; Hamamatsu, Herrsching, Germany). and 10 consecutive datum points were averaged, yielding a time resolution of 1 Hz. The autofluorescence and the system noise were measured before the experiment and subtracted from the BCECF fluorescence signals. An iris diaphragm allowed the reduction of the area of measurement to a single cell. Measurements of pH1 were exclusively taken from those cells demonstrating a low fluorescence typical for principal cells (36). The fluorescence ratio was recorded after a
15-mm
equilibration
period
in
the
continuously
perfused
bath. Experiments were controlled and data were analyzed with an AT-486 computer system and specific software (U. Frobe. Universit#{228}t Freiburg. Germany). The calibration of the BCECF fluorescence signal was done in 12 separate experiments with the protonophore carbonyl cyanide
m-chlorophenyl-hydrazone
external before ratio
pH
pH values (36). In this on
=
the
0.86X
1 Mmol/L)
pH +
was
linear
and
followed
the
fura-2
equation
used
The
Ca2
J) activity of COD cells was measured with the Ca2-sensitlve dye fura-2 as described previously (37). COD cells were incubated with fura-2/acetoxyrnethyl ester (2 to 5 tmol/L) dissolved with 0.1 gIL pluronic F- 127 for 45 mm at room temperature In the dark. The incubation was followed by an equilibration period of 15
1224
intracellular
Ca2
(LCa2
was
control solution 360. and 380
recorded
at
500
to
at nm,
530
nm.
to judge
the leakage
or
bleaching
of fura-2,
the
loss
of
cells, or air bubbles in the area of measurement during the experiments (37). Calibration of (Ca2 ) was attempted at the end of each experiment by the incubation of the cells with the Ca2 ionophore ionomycin (1 gimol/L; Sigma. Deisenhofen, Germany)
in
the
presence
(1.3
mmol/L)
and
nominal
ab-
sence of Ca2 (with 5 mmol/L ethylene glycol-bis(13-arninoethyl ether) N.N,N’,N’-tetraacetic acid present) according to standard methods (38). For those experiments where a paired calibration at the end of the experiment was not successful, mean values of all calibrations were used to estimate
[Ca2J1.
Statistical
Analysis
Data are presented as fluorescence ratios (488/436 mit and 340/380 nm for pH1 and [Ca2l1, respectively) in the figures presenting original recordings or as mean values of calculated pH1 values or fura-2 fluorescence ratios or calculated [Ca2 ] values ± SE (N), where N refers to the number of experiments. A paired or nonpaired t test (two-sided) was used where appropriate to test for statistically significant differences. P 0.05 was set as the significance level.
RESU LTS Basal
Activity
of Na/H
A
total of 45
Exchange
rats were
used
Isolated CCD cell clusters fluorescence ratio as an 0.04 in resting conditions
were
experiments. tained in
By
the
of pH1 was
at
the
of the
value
beginning
calibration
experiments
ratio
(see
COD. (N
=
of this
corresponded
HOE-694 9) and
exchanger
acidified by
concentrations
0.09
±
cells
0.02
(N
of 1 and
10 jimol/L,
an
Load
ob-
in the
basolat-
to estimate
to the
COD
curve
Methods), this to an estimated cells of rat CCD. HOE-694 (39).
pH1 of 7.23 ± 0.04 for these principal The inhibitor of the Na /H exchanger specific for the isoform N}{E- 1 present eral membranes of the CCD, was used contribution
and 65 BCECF 2.78 ± of the
for this study, examined. The
indicator
use
separate
fluorescence
Recovery The
From
addition
solution
5.74.
of Intracellular
emission
with 340,
Measurements were taken from 5 to 10 cells with the aid of an Iris diaphragm. The ratio of the emissions after excitations at 340 and 380 nm at 10 Hz was calculated, and 10 consecutive data points were averaged, yielding a time resolution of 1 Hz. Signal noise and autofluorescence were measured and subtracted for each experiment. Fluorescence at 360 tim was
(pH
pH1 by 0.83
Measurements
were superfused wavelengths were
basal
the
pH1
of the
by
0.02 ± 0.01 pH units at respectively. 7)
=
and
between 6.5 and 8.0 as described in detail range, the dependence of the fluorescence
external ratio
(CCCP;
the
cells
of 20 mmol/L =
±
694. Figure experiment. these cells estimated
NH4
7.4) resulted
0.05
/NH3 transiently pH units (FIgure 1). cation was impaired NH4
Acid
(N
=
/NH3
to the
in a transient
64) pH
decreased The recovery
pH1 by 0.62
from
in
the removal
such
of
0.03
±
an acidifi-
HOE1 depicts the original recording of such an The activity of the Na/H exchanger in after NH4 /NH3-induced acidification was as the initial slope (first 120 s) of the increase in
the
presence
units;
control
Increase
Volume
of 1 pmol/L
6-
Number
4
‘
1995
Schiatter
et a)
4.
E
-
3-
,
0 0
2-
1-
2mm[J 2 mm
HOE 694 0#{149}
Figure 1. Original recording of the BCECF fluorescence ratio of a rat CCD cell. Effects of the addition and removal of NH4 (20 mmol/L) on fluorescence ratio and inhibition of the recovery of the fluorescence ratio from the acidification by HOE-694
(1 ,mol/L).
in pH1 after it had reached a minimal value. This ery rate (pH1/mIn) was 0.26 ± 0.03 (N 53) control conditions. In the presence of 1 ji.mol/L 694, ipH1/mIn was reduced by 0.13 ± 0.03 (N
1
recovunder
=
HOE-
,
Absence The
of Effects
of various by exposing CCD
examined in
of Agonists
influence
the
constantly
on
agonists
Basal
pH1
basal
pH1
on
2 mm
2. OrigInal recordings of the BCECF fluorescence ratio of a rat CCD cell. Effects of AVP (10 nmol/L) and All (100 nmol/L) on the recovery of the fluorescence ratio from the NH4 (20 mmol/l)-lnduced acidification.
for 3 to 5 mm None of the nmol/L;N = 5), AVP (10 6), Ap3A (N 5), Ap4A (N (N = 6), each 5 p.mol/L-
tested agonists-AlI (100 nmol/L; N = 7), ATP (N = = = 11), Ap5A (N = 6), and Ap6A significantly changed pH, under resting conditions. The stimulation of protein kinase C with the phorbol ester PMA (5 J.Lmol/L) also did not significantly alter pH1 (N = 7), whereas the inhibitor of protein kinase C, calphostin 0 (0.1 jLmol/L), reduced pH1 significantly by 0.08 ± 0.02 pH units (N = 7).
0.15-
0.10-
.c
All
0.05-
(n=8)
E
Influence of Agonists Acid Load The
influence
of pH, examined
the
of the
as
activity by
by the
depicted All (100
after
transient
modulations amounted
above
From
agonists
an
on the
/NH3-mnduced
indication Na
cytosolic
original
ratio and
All
an
of the
Recovery
acidification
for
their
ability
/H exchanger acidification. As
recordings
of the
to change
after it demonstrated
BCECF
average
to an
inhibition
on Ap5A,
recovery
pH, and
recovery, Ap6A
of pH1. When
C, calphostin C (0.1 laration of pH1 recovery ited, but pH, recovery
Journal
of the American
whereas
significantly
o
was
the inhibitor j.mol/L), was
32
±
by
was
Ap6A
even
Society of Nephrology
AVP (n9)
-0.10-
-0.15-
11%
9)
=
the
dinucleotides
accelerated of protein present, was not reduced
-0.05-
fluorescence
by
the
0.00-
‘I, 01 C
was
in Figure 2, AVP (10 nmol/L) decreased nmol/L) enhanced the recovery of pH, acidification by NH4/NH3. These of Na / H exchange activity by AVP and on
effect
0. C
recovery
(AVP; N = 8) and an activation by 37 ± 7% (All; N of the respective control values (Figure 3). ATP and diadenosine polyphosphate Ap3A had no significant Ap4A,
I
