Effect of angiotensin II on calcium reabsorption by the
luminal membranes of the nephron
A.
Charbonneau,
M.
Leclerc, and
M. G.
Brunette
Maisonneuve-Rosemont Hospital; Guy-Bernier Research Center; and
University of Montreal, Montreal, Quebec, Canada H1T 2M4
 |
ABSTRACT |
In the rat and the rabbit, a number of
studies have reported the effects of angiotensin II (ANG II) on
Na+ reabsorption by the proximal (PT) and distal (DT)
convoluted tubules of the kidney. The aim of the present study was to
examine the effect of ANG II on Ca2+ uptake by the luminal
membranes of the PT and DT of the rabbit. Incubation of PT and DT with
10
12 M ANG II enhanced the initial Ca2+
uptake in the two segments. Dose-response experiments revealed, for
Ca2+ as well as for Na+ transport, a biphasic
action with a maximal effect at 10
12 M. Ca2+
transport by the DT luminal membrane presents a dual kinetic. ANG II
action influenced the high-affinity Ca2+ channel,
increasing maximal velocity from 0.72 ± 0.03 to 0.90 ± 0.05 pmol · µg
1 · 10 s
1
(P < 0.05, n = 3) and leaving the
Michaelis-Menten constant unchanged. The effect of ANG II was abolished
by losartan, suggesting that the hormone is acting through
AT1 receptors. In the PT, calphostin C inhibited the effect
of the hormone. It is therefore probable that protein kinase C is
involved as a messenger. In the DT, however, neither Rp cAMP,
calphostin C, nor econazole (a phospholipase A inhibitor) influenced
the hormone action. Therefore, the mechanisms involved in the hormone
action remain undetermined. Finally, we questioned whether ANG II acts
in the same DT segment as does parathyroid hormone on Ca2+
transport. The two hormones increased Ca2+ transport, but
their actions were not additive, suggesting that they both influence
the same channels in the same segment of the distal nephron, i.e., the
segment responsible for the high-affinity calcium channel.
renal calcium transport
 |
INTRODUCTION |
DURING THE LAST
DECADES, a considerable amount of data has been accumulated
concerning the activity of the renin-angiotensin system in the proximal
tubule (PT) of the nephron. By use of several techniques
[immunocytochemistry (20, 45), dosage of angiotensinogen and renin in the supernatant of PT cell cultures (39, 60), or detection of angiotensinogen mRNA by in situ hybridization (17, 28)], a number of studies clearly showed that this
nephron segment contains a complete renin-angiotensin system able to
synthesize renin and angiotensin-converting enzyme (ACE) (10,
49) and then secrete high concentrations of angiotensin II (ANG
II) into the tubular fluid (5, 43, 52). This angiotensin
synthesis is regulated by dietary Na+, as reported by
Ingelfinger et al. (27), Fox et al. (16), Jo
et al. (29), and Tank and colleagues (54,
55). Indeed, a low-sodium diet strongly increases both
angiotensinogen and renin mRNA in the rat renal cortex.
Conversely, endogenous ANG II has been shown to control Na+
and water reabsorption in the PT through the presence of in situ receptors (29). This effect, however, depends on the
hormone concentration; micropuncture and microperfusion experiments
have demonstrated that physiological concentrations (from
10
12 to 10
10 M) stimulate
Na+ reabsorption, whereas higher doses
(10
7 M) cause inhibition, a phenomenon which produces a
bell-shaped dose-response curve (21, 22, 46, 50, 51, 53,
58).
Distal tubules (DT) are also the site of ANG II receptors, as shown in
microdissection (40) and cell culture studies
(11). In this segment, Stop-flow (57),
microperfusion (25, 33, 34, 58), and micropuncture
experiments (37) detected either a decrease (37,
57), an increase (33, 34, 58), or no change
(25, 26) in Na+ transport, depending on the
hormone concentration utilized.
In contrast to this abundant literature concerning the action of ANG II
on Na+ reabsorption, very few studies hve questioned its
role in Ca2+ transport. Romero et al. (46)
suggested that the negative effect of high hormone concentrations on
Na+ transport might be due to Ca2+ entry into
the cell, but such a hypothesis has never been clearly confirmed. A
study using voltage-clamp techniques in cardiac myocytes (13) showed that intracellular administration of ANG II
increased Ca2+ cell content, but probably through a protein
kinase C (PKC) activation rather than a change in membrane
Ca2+ permeability. In adrenal glomerulosa cells, however,
Maturana et al. (38) recently reported that high doses of
ANG II (100 nM) induced a marked inhibition of L-type Ca2+
channel current.
The purpose of the present study was to examine the effect of ANG II on
Ca2+ transport by the PT and DT luminal membranes of the
rabbit kidney. Results indicate that ANG II influences Ca2+
uptake by both PT and DT membranes according to a bell-shaped curve.
This effect occurs after a few minutes of incubation and involves the
maximal velocity (Vmax) kinetic parameter,
supporting the hypothesis of an exocytosis mechanism at the origin of
this action. Both parathyroid hormone (PTH) and ANG II act on the
high-affinity component of Ca2+ uptake by the DT luminal
membrane. Because their actions are not additive, it is probable that
the two hormones act in the same segment of the DT.
 |
MATERIALS AND METHODS |
Preparation of tubule suspensions.
PT and DT suspensions were prepared by means of collagenase digestion
and Percoll-density gradient centrifugation techniques with fresh
rabbit kidneys obtained directly from the slaughterhouse. Slices of
cortex were incubated for 20 min at 37°C in a cell culture medium
(DMEM) containing 1 mg/ml collagenase type V and equilibrated with 95%
O2-5% CO2. The suspensions were filtered
through a tea strainer, and the filtrate was centrifuged at 200 g for a few seconds. The pellets were washed twice in
Krebs-Henseleit buffer (KHB) containing 0.5% BSA suspended in 40%
Percoll in the cell culture medium and centrifuged at 28,000 g for 30 min at 4°C. The DT and PT bands were separately
collected, washed twice in KHB with BSA, and incubated at 37°C for 10 min in the cell culture medium containing 2% fetal bovine serum, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and ANG II at the indicated
concentration. In the ANG II receptor or messenger inhibition
experiments, these various inhibitors were added to the tubule
incubation medium. Incubation was stopped by rapid dilution in ice-cold
KHB solution, and the tubules were washed three times with the same
solution and frozen at
80°C until the day of the experiment.
Luminal membrane purification.
On the day of the experiment, the frozen tubules were thawed and
homogenized with 10 strokes of a Potter homogenizer. The luminal
membranes from PT and DT were purified using the MgCl2 precipitation technique. After addition of 12 mM MgCl2
(final concentration), the suspension was stirred on ice for 20 min
(proximal tubules) or 10 min (distal tubule suspension) and centrifuged at 3,000 g for 20 min at 4°C. The supernatant was
collected and centrifuged at 28,000 g for 30 min at 4°C.
The sedimented membranes were washed twice in 280 mM mannitol with 20 mM Tris-HEPES, pH 7.4, suspended in this medium, and finally allowed to
vesiculate at 4°C for 1 h. Enzyme enrichments of each type of
preparation are presented in Table 1.
Ca2+ uptake by the luminal membrane
vesicles.
45Ca2+ uptake was measured by the rapid
Millipore (Bedford, MA) filtration technique. Uptake was initiated by
adding 25 µl of incubation medium at 35°C to 5 µl of membrane
suspension (~25 µg protein). The incubation medium contained either
120 mM NaCl and 20 mM choline chloride or 140 mM choline chloride, with
20 mM Tris-HEPES, pH 7.4, and, unless otherwise specified, 0.5 mM 45CaCl2. Uptake was stopped by the addition of
1 ml of ice-cold solution containing 150 mM KCl, 20 mM Tris-HEPES, pH
7.4, and 2 mM EGTA. The suspensions were then filtered through
Millipore filters (0.45 µM), the filters were rinsed with an
additional 5 ml of stop solution, and the retained radioactivity was
counted. Nonspecific binding was measured at time 0 under
the same conditions.
Enzyme marker measurements.
The purity of the various preparations was monitored by measurement of
the specific enzyme activities. Alkaline phosphatase activity was
determined according to the technique of Kelly and Hamilton
(31) and Na+-K+-ATPase to the
technique of Post and Sen (44).
Materials.
45CaCl2 was purchased from Mandel (NEN Life
Science Products, Boston, MA). Collagenase type V, ANG II,
DBcAMP, econazole, and all the other chemicals were from Sigma
(St. Louis, MO). Calphostin C was purchased from Calbiochem. Losartan
and PD-123319 were generously given by Merck, Sharp and Dohme and by
our colleague, Dr. John S. D. Chan, respectively.
 |
RESULTS |
Effect of ANG II on the time course of
Ca2+ uptake by PT luminal membranes.
The initial experiments were designed to evaluate the effect of PT and
DT incubation with ANG II on 0.5 mM Ca2+ uptake by the
corresponding luminal membranes. As shown in Fig. 1, 10
12 M ANG II
significantly increased the initial Ca2+ transport by the
PT membrane vesicles from 0.49 ± 0.03 to 0.66 ± 0.04 pmol · µg
1 · 10 s
1
(P < 0.005, n = 4). This initial
effect progressively decreased, to completely disappear after 180 s.

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Fig. 1.
Effect of 10 12 M angiotensin (ANG) II on
0.5 mM Ca2+ uptake by proximal tubule luminal membrane
vesicles. *P < 0.05, **P < 0.02, ***P < 0.01 vs. control (CTL) values (unpaired
Student's t-test); n = 4.
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Effect of ANG II on Ca2+ uptake by PT
luminal membranes: dose-response curve.
When PT were incubated with increasing concentrations of ANG II, the
rate of 0.5 mM Ca2+ uptake by the luminal membrane vesicles
was progressively enhanced at hormone concentrations from 0 to
10
12 M and then returned to the control values with ANG
II at 10
10 M and above, showing a bell-shaped curve (Fig.
2).

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Fig. 2.
Dose-response curve of effect of ANG II on 0.5 mM
Ca2+ uptake by proximal tubule luminal membranes.
*P < 0.05, ***P < 0.01 vs. control
values (unpaired Student's t-test); n = 4.
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Effect of ANG II on the time course of
Ca2+ uptake by the DT luminal membranes
in the presence or absence of Na+.
We have previously shown (9) that the presence of
Na+ in the incubation medium strongly decreases
Ca2+ uptake by the luminal membrane of DT, whereas it has
no effect on PT membranes. Then we were interested in investigating
whether the hormone affects Ca2+ transport by the distal
membrane, and if so, whether this action is influenced by the presence
of Na+. Ca2+ uptake was measured in two
different incubation media containing either 140 mM choline chloride
and no Na+ or 120 mM NaCl and 20 mM choline chloride. In
the absence of Na+, 10
12 M ANG II increased
Ca2+ transport from 0.52 ± 0.02 to 0.96 ± 0.04 pmol · µg
1 · 10 s
1
(P < 0.001, n = 4). The presence of
Na+ curtailed Ca2+ uptake, but the hormone
still enhanced this transport from 0.38 ± 0.02 to 0.63 ± 0.04 pmol · µg
1 · 10 s
1
(P < 0.005, n = 4; Fig.
3).

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Fig. 3.
Effect of 10 12 M ANG II on 0.5 mM Ca2+
uptake by distal tubule luminal membranes in the absence or presence of
100 mM NaCl. *P < 0.05, **P < 0.02, ***P < 0.005 vs. CTL values (unpaired Student's
t-test); n = 4.
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Dose-response curve in the DT luminal membranes.
In another series of experiments following the same protocol, we
examined the possibility of a biphasic action of ANG II on Ca2+ uptake in DT as in PT. Indeed, a similar dose-response
curve was observed in DT as in PT membrane vesicles (Fig.
4), again with a maximal response at ANG
II concentration of 10
12 M.

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Fig. 4.
Dose-response curve of effect of ANG II on 0.5 Ca2+ uptake by distal tubule luminal membranes in the
absence of Na+. *P < 0.05, ***P < 0.005 vs. control values (unpaired Student's
t-test); n = 4-6.
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Effect of incubation time with ANG II on
Ca2+ uptake by PT and DT luminal
membranes.
The effect of tubule incubation with ANG II on Ca2+ uptake
by their luminal membranes was relatively rapid. As shown in Fig. 5, a mere 10-min incubation sufficed to
provoke the observed modification of Ca2+ transport.

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Fig. 5.
Effect of 10 12 M ANG II on 0.5 mM Ca2+
uptake by proximal (PT) and distal tubule (DT) luminal membranes in the
absence of Na+. Variation with time of incubation.
*P < 0.05, **P < 0.02, ***P < 0.01 vs. control values (unpaired Student's
t-test); n = 3.
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Effect of ANG II on kinetic parameters of
Ca2+ uptake by the PT and DT membranes.
To further understand the mechanism involved in the effect of ANG II on
Ca2+ uptake by the luminal membranes of the two nephron
segments, we measured Ca2+ uptake using Ca2+
concentrations from 0.025 to 4.00 mM. Figure
6 shows the corresponding Eadie-Hofstee
plot obtained in the PT and DT membranes. In PT, ANG II affected the
Vmax value, leaving the value of the
Michaelis-Menten constant (Km) intact. As we
previously reported (9), Ca2+ uptake by DT
luminal membranes presents a dual kinetic with a low-affinity component
sensitive to calcitonin (61) and a high-affinity component
sensitive to PTH (32) and calbindin 28K (4).
Such a dual kinetic was again observed, with the ANG II targeting
exclusively the high-affinity component, increasing, as in PT, the
Vmax value (Table
2).

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Fig. 6.
Effect of 10 12 M ANG II on the Eadie-Hofstee plot of
Ca2+ uptake by DT luminal membranes in the absence of
Na+. V, velocity of uptake; S, substrate.
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Does ANG II also act directly on the luminal membrane
Ca2+ channels?
Unlike most hormone receptors that are exclusively expressed in
basolateral membranes, ANG II receptors have been shown to be present
on both the apical and basolateral membranes (6, 7).
Because the ANG II concentration is relatively high in the tubular
fluid, we questioned whether the hormone might have a direct effect on
luminal Ca2+ channels independent of the intracellular
machinery. In these experiments, ANG II was added to the membrane
suspensions either after the vesiculation step or in the incubation medium.
As shown in Fig. 7, Ca2+
uptake by the two site membranes was not influenced by the presence of
10
12 M ANG II in the incubation medium. Addition of the
hormone to the vesicle suspension after vesiculization also had no
effect (data not shown).

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Fig. 7.
Direct effect of 10 12 M ANG II in the incubation
medium of 0.5 Ca2+ uptake by the PT (left) and
DT (right) luminal membranes.
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Which receptors are involved in the effect of ANG II on the PT and
DT luminal membranes?
To characterize the type of receptors through which ANG II exerts its
enhancement of Ca2+ transport by the DT membranes, a new
series of experiments was performed using losartan and PD-123319 as
AT1- and AT2-specific inhibitors. The results
are presented in Fig. 8. As constantly observed, incubation of PT and DT with 10
12 M ANG II
significantly increased Ca2+ uptake from 0.39 ± 0.020 to 0.55 ± 0.015 in PT and from 0.46 ± 0.027 to 0.75 ± 0.041 pmol · µg
1 · 10 s
1
in DT membranes. In the presence of 10
6 M losartan,
however, the effect of the hormone was completely abolished (0.39 ± 0.023 and 0.50 ± 0.037 pmol · µg
1 · 10 s
1 in PT
and DT, respectively; not significant vs. control values). In contrast,
the AT2 inhibitor PD-123319 did not significantly interfere
with the influence of ANG II on Ca2+ uptake.

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Fig. 8.
Effect of 10 6 M losartan and 10 6 M
PD-123319 (AT1 and AT2 receptor inhibitors,
respectively) on the ANG II-dependent (10 12 M) 0.5 mM
Ca2+ transport by the PT and DT luminal membranes. **
P < 0.02, *** P < 0.005 vs. control
values (unpaired t-test); n = 3.
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Is ANG II action influenced by nitrendipine?
In previous unpublished experiments, we observed that the low-affinity
Ca2+ channel was inhibited predominantly by nitrendipine:
0.1 mM Ca2+ uptake was 1.59 ± 0.08 and 1.51 ± 0.09 mmol · 10 s
1 · µg
protein
1 in control and treated vesicles (NS) whereas 1.0 mM Ca2+ uptake was 2.59 ± 0.25 vs. 1.96 ± 0.35 mmol · 10 s
1 · µg
protein
1 in the two conditions, respectively
(P < 0.05, n = 3; unpaired t-test). In the present experiments, addition of 10 µM
nitrendipine to the vesicule suspensions again slightly decreased the
total Ca2+ uptake. However, this treatment did not prevent
the response to 10
12 M ANG II; 0.1 mM Ca2+
uptakes were 0.33 ± 0.04 vs. 0.42 ± 0.05 pmol · µg
1 · 10 s
1 in
membranes from control and treated tubules, respectively
(P < 0.02, n = 3; unpaired
t-test; Fig. 9).

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Fig. 9.
Effect of 10 µM nitrendipine on the ANG II-dependent
0.1 mM Ca2+ uptake by the DT luminal membranes. **
P < 0.02 (n = 3) vs. data obtained
with nitrendipine alone.
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What messengers are involved in ANG II actions in PT and DT?
Current evidence demonstrated that concentrations of ANG II within the
picomolar-to-nanomolar ranges inhibit the production of cAMP in PT
cells (35, 59), whereas at higher concentrations (nanomolar and above), the hormone rather activates phospholipase A
(14, 15). The decrease in cAMP is thought to be at the
origin of the effect on Na+ transport, because cAMP has
been shown to curtail Na+ uptake by the brush border
membranes (12). Much less is known about the mechanisms of
ANG II actions in DT cells.
In a new series of experiments, PT and DT suspensions were incubated
with ANG II and various messenger inhibitors, i.e., the cAMP antagonist
adenosine 3,5'-cyclic monophosphothioate (Rp-cAMP), calphostin C, and
econazole, and 0.5 mM Ca2+ uptakes were subsequently
measured by their respective luminal membrane vesicles. Results are
presented in Fig. 10, A and
B, and Table 3.

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Fig. 10.
Effect of 10 4 M Rp cAMPs,
10 7 M calphostin C (Cal.C), and 10 5 M
econazole (Econaz.) on the ANG II (10 12 M)-dependent 0.5 mM Ca2+ transport by luminal membranes from PT
(left) and DT (right). *P < 0.05, **P < 0.02, ***P < 0.01 vs. CTL values (unpaired Student's t-test);
n = 3.
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In the PT, inhibition of PKC by 10
7 M calphostin C
completely abolished the action of ANG II on 0.5 mM Ca2+
transport. In contrast, inhibition of neither cAMP by 10
4
M Rp cAMP nor phospholipase A by 10
5 M econazole
prevented the hormone effect, thus suggesting that PKC was the major,
if not the only, messenger responsible for the increase in
Ca2+ transport.
In the DT, however, different results were obtained. Inhibition of
cAMP, PKC, or phospholipase A did not modify the response to ANG II.
Because we had previously observed (23), as did others using different cell preparations (18), that
Ca2+ transport in the DT was extremely sensitive to the
association of cAMP and PKC, we questioned whether a combination of the
two inhibitors Rp cAMP and calphostin C would interfere with ANG II action. Results of an additional experiment indicated that the combination of 10
4 M Rp-cAMP and 10
7 M
calphostin C did not influence either 0.5 mM Ca2+ uptake
(Ca2+ uptakes: 0.44, 0.64 and 0.66 pmol · µg
1 · 10 s
1 in
membranes from control tubules and tubules treated with ANG II or ANG
II plus calphostin C and Rp cAMP, respectively).
Combined effect of ANG II and PTH.
In the kidney, the main Ca2+-regulating hormones are PTH,
calcitonin (CT), and calbindin 28K. All of them increase
Ca2+ uptake by the distal luminal membrane: CT opens the
low-affinity (61), whereas PTH (32) and
calbindin 28K (4) stimulate the high-affinity, channels.
We have shown that 10
12 M ANG II influences the
high-affinity component, as does PTH. Then we hypothesized that a lack
of additivity of ANG II and PTH should confirm an identical site and
mechanism of action of the two hormones. DT were incubated for 10 min
with either the carrier ANG II alone, PTH alone, or ANG II in
combination with PTH. Results are presented in Fig.
11. Both ANG II and PTH increased
Ca2+ uptake by the DT luminal membranes. However, the
combined action of the two hormones was not different from the effect
of each of them: Ca2+ uptakes were 0.49 ± 0.018, 0.61 ± 0.01, 0.71 ± 0.034, and 0.71 ± 0.032 pmol · µg
1 · 10 s
1 in the
four experimental conditions, respectively. It is therefore probable
that ANG II acts in the same segment as PTH, i.e., the late part of the
distal cortical tubule (40).

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Fig. 11.
Separate and combined effects of DT incubation with
10 12 M ANG II and 10 7 M parathyroid hormone
(PTH) on 0.5 mM Ca2+ uptake by their luminal membranes.
***P < 0.01 vs. CTL values (unpaired Student's
t-test); n = 3.
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Effect of ANG II on Na+ transport by
the PT and DT luminal membranes: interrelationship of the two cations.
Because Na+ and Ca2+ transports by the DT
luminal membrane are tightly related (9), we performed a
few additional experiments to investigate the effect of ANG II on
Na+ uptake by PT and DT membranes. As already reported, low
concentration of ANG II (10
12 M) significantly increased
1 mM Na+ transport by the PT brush border membrane (from
0.89 ± 0.02 to 1.07 ± 0.02 pmol · µg
1 · 10 s
1,
P < 0.01, n = 3). Unexpectedly,
however, at the same concentration, the hormone had the opposite effect
in the DT membranes: Na+ uptake fell from 0.60 ± 0.03 to 0.44 ± 0.05 pmol · µg
1 · 10 s
1 (P < 0.05, n = 3).
 |
DISCUSSION |
ANG II influences Ca2+ transport by
the PT and DT luminal membranes, but through which messengers and which
mechanisms?
The aim of the present study was to detect and characterize the effect
of ANG II on Ca2+ transport through the luminal membranes
of PT and DT. Results clearly showed that the hormone influences
Ca2+ uptake by the two membranes according to a bell-shaped
curve: physiological concentrations increase the cation transport with a maximal effect at 10
12 M, whereas at higher
concentrations, such a stimulation progressively declines.
The mechanisms involved in these actions remain unclear. ANG II has
been repeatedly reported to depress adenylate cyclase activity in
several types of PT cell preparations (56, 59). The fact
that ANG II decreases cAMP synthesis and increases Ca2+
transport by the luminal membrane suggests an inhibitory action of the
messenger, a hypothesis which is in contradiction to data previously
reported by our group (23). Indeed, we have been unable to
clearly demonstrate any action of DB-cAMP on Ca2+ uptake by
PT brush border membranes (23). Confirming the
noninvolvement of adenylate cyclase in the effect of ANG II on
Ca2+ transport by the PT, inhibition of the messenger
delivery by Rp cAMP did not prevent the hormone effect. Phospholipase A
is probably also not involved in the regulation of Ca2+
uptake by 10
12 angiotensin, since incubation with
econazole did not inhibit the hormone action.
Our results rather support the role of PKC in the effect of ANG II on
Ca2+ uptake by the PT. The hormone has been shown
repeatedly to activate PKC to produce its effect in PT (30, 36,
47, 48) and the ascending limb of the loop of Henle
(1). In the present study, addition of calphostin C to the
incubation medium with ANG II abolished the influence of the hormone on
Ca2+ transport, thus suggesting that PKC is the main, if
not the only, messenger active at that site.
The mechanisms involved in DT membranes are no easier to understand.
cAMP and PKC have been shown to interact with each other to stimulate
Ca2+ transport by the luminal membranes (19,
23). Here again, therefore, a decrease in cAMP delivery should
curtail Ca2+ uptake rather than the opposite, as observed
in our study, confirming the nonimplication of cAMP in the distal
effect, i.e., the lack of inhibitory effect of Rp cAMP on the ANG
II-dependent Ca2+ uptake. However, at variance with what
was observed in PT, calphostin C did not interfere with the hormone
action and association of Rp cAMP, and calphostin C also failed to
prevent the hormone action. Further experiments should explore other
messenger systems such as mitogen-activated protein kinases, the
various isoforms of phospholipases, the tyrosine kinases, or even
cytochrome P-450.
Another aspect of the mechanism involved in the action of ANG II is the
relatively short incubation time necessary to be efficient, i.e., 10 min in PT and 5 min in DT. The hypothesis of a direct action on the
luminal membranes has been excluded by the lack of effect of ANG II
added to the vesicle suspensions. It is possible, however, that the
hormone influences the density of transport molecules through an
indirect endo-/exocytosis mechanism independently of the synthesis of
any new molecule. Indeed, a similar mechanism has been shown by
Schelling and Linas (47) for Na+
transport by the PT luminal membranes. The fact that, in our kinetic
experiments, the hormones increase the Vmax
values without influencing the affinity (Km) is
compatible with this hypothesis.
ANG II opens the high-affinity Ca2+
channels in the DT.
Electrophysiology (43) and molecular biology experiments
(2) showed the presence of several types of
Ca2+ channels in the DT luminal membranes. Our laboratory
clearly detected a dual kinetics of Ca2+ transport by the
DT luminal membrane, a low-affinity channel sensitive to calcitonin
(61), and a high-affinity channel sensitive to PTH
(3) and the vitamin D-dependent calbindin 28K
(4). The high-affinity channel has been further
characterized by Hoenderop et al. (24), who identified the
channel ECaC mRNA which was co-localized with the calbindin-D 28K and,
therefore, probably the PTH receptors. In the present study, ANG II
opened the high-affinity Ca2+ channel, i.e., the channel
that is sensitive to PTH and calbindin 28K. The lack of additivity of
PTH and ANG II actions on Ca2+ transport as shown in Fig. 9
strengthens this hypothesis.
Nitrendipine does not curtail the action of ANG II.
As aforementioned, nitrendipine is an L-type Ca2+ channel
blocker that inhibits the low-affinity channel in the DT luminal
membrane. The inhibitor did not prevent the effect of ANG II on
Ca2+ transport. This observation is quite logical, because
ANG II rather opens the high-affinity component of Ca2+
uptake. It further confirms that ANG II and nitrendipine act in two
different segments of the distal nephron.
ANG II activates the
Na+/H+
exchanger in PT and curtails the exchanger activity in the DT.
In PT, the observed stimulation of the exchanger by ANG II first
confirms other data previously reported. In DT, however, the
hormone has an opposite effect, a finding which was unexpected. This is
another observation of a certain antagonist behavior of Ca2+ and Na+ transports through the distal
luminal membranes. Na+ decreases Ca2+ uptake
(23); conversely, Ca2+ partially blocks
Na+/H+ exchange (12). The
Ca2+ regulating hormones PTH (4), calcitonin
(61), and calbindin 28K (8), all of which
open the distal Ca2+ channels, also curtail the
Na+ entry by this membrane. Hypothetically, it is therefore
possible that these Ca2+ channels are, in fact, cation
channels through which Ca2+ competes with Na+
to be transported.
In summary, the present studies investigated the action of ANG II on
Ca2+ uptake by the PT and DT luminal membranes. At both
sites, ANG II provoked a biphasic response, with a stimulation of
Ca2+ transport of hormone concentrations below
10
12 M and a return to control values at higher
concentrations. In the DT, the presence of 100 mM NaCl in the
incubation medium decreased Ca2+ uptake but did not prevent
the effect of ANG II. The hormone increased the
Vmax without changing the
Km values. Addition of 10
6 M
losartan to the incubation medium prevented the effect of the hormone.
Inhibition of PKC stimulation curtailed the effect in the PT but not in
the DT. Finally, PTH, like ANG II, enhances Ca2+ transport;
however, the effects of the two hormones were not additive, suggesting
that they act on the same Ca2+ transport molecule, i.e.,
the high-affinity Ca2+ channel previously described in the
DT luminal membrane.
 |
ACKNOWLEDGEMENTS |
The authors are grateful to Jean-Marie Bélanger for providing
the rabbit kidneys and Claudette Plante for secretarial assistance.
 |
FOOTNOTES |
This study was supported by Grant MT-11-203 from the Medical
Research Council.
Address for reprint requests and other correspondence: M. G. Brunette, Maisonneuve-Rosemont Hospital, Research Centre, 5415, l'Assomption Blvd. Montreal QC, Canada H1T 2M4
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 November 2000; accepted in final form 5 February 2001.
 |
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