ATPase in cortical distal tubule via Vi receptor. .... theta glass tubing (R & D Optical Systems, Spencerville, MD,. USA) .... Copenhagen ABL 5 blood gas system.
Kidney International, Vol. 52 (1997), pp. 1035—1041
Luminal arginine vasopressin stimulates NatH exchange and HtATPase in cortical distal tubule via Vi receptor M.L.M. BARRETO-CHAVES and MARGARIDA DE MELLO-AIRES Department of Physiology and Biophysics, Instituto de Ciências Biomédicas, University of São Paulo, São Paulo, Brazil
Luminal arginine vasopressin stimulates Na-H exchange and H+ ATPase in cortical distal tubule via Vi receptor. Bicarbonate reabsorption was evaluated by stationary microperfusion of in vivo early distal (ED) and late distal (LD) segments of rat kidney. Intratubular pH was recorded by double-barreled H ion-exchange resin/reference (1 M KC1) microelec-
trodes for the determination of HCO3 reabsorption. In the presence of luminal arginine vasopressin (AVP, 10 M), a significant increase in 0.061 to HCO3 reabsorption was observed both in ED (from 0.931 2.12 1.67
Thus, it is possible that the difference in AVP response of Na-H exchangers may vary with the cell type being studied. Since the concentration of AVP in the luminal fluid may exceed 1,000-fold that of plasma [10, 11], and considering the possible existence of functional luminal vasopressin receptors [12, 13], the present study was designed to determine whether luminal AVP (10— M) regulates acid-base transport in the rat cortical distal
s] 0.111 nmol cm2 s. The addition of the Vi-receptor tubule. For this purpose, we evaluated the kinetics of HC03
0.171 nmol cm2 .
and LD segments [from 0.542
0.086 to
antagonist [(d (CH2)5, Tyr (Et)2) arginine vasopressin] (10 M) to luminal
reabsorption by the stationary microperfusion technique in in vivo early distal (ED) and late distal (LD) segments. In order to detect
segments. 5-(N, N-hexamethylene) amiloride (10—a M) added to luminal perfusion inhibited luminal AVP-mediated stimulation in ED (by 63.7%) and LD (by 34.1%) segments. The addition of Bafilomycin A1 (2 X iO M) to the luminal perfusion did not affect luminal AVP-mediated stimulation in ED segments, but reduced it (by 31.7%) in LD segments. Our
which specific process AVP stimulates distal bicarbonate reabsorption, we also examined the effects of the Vi and V2 receptor antagonists [(d (CH2)5, Tyr (Et)2) arginine vasopressin and (Adamantaneacetyl1,O-Et-D-Tyr2,Val4, Aminobutyryl6, Arg8'9) vasopressin, respectively], of the specific blocker of Na*H± exchanger [5-(N, N-hexamethylene) amiloride, HMA] and of the specific inhibitor of vacuolar H-ATPase [bafilomycin A1] on luminal AVP stimulation in ED and LD segments.
perfusion blocked luminal AVP mediated stimulation in ED and LD
results indicate that luminal AVP acts to stimulate the Na-H exchange in ED and LD segments via activation of Vi receptors, as well as the vacuolar H-ATPase in LD segments.
The major action of arginine vasopressin (AVP) in the kidney is to increase water permeability in the cortical (CCD) and inner medullaty collecting ducts (IMCD) [1]. However, AVP has also been shown to be involved in tubular fluid acidification. In the rat kidney, AVP inhibits bicarbonate reabsorption in the thick ascending limb [2, 3] and stimulates proton secretion (or bicarbonate reabsorption) in the distal tubule [2, 4] and CCD [2, 51. The nature of the mechanism underlying AVP action on bicarbonate reabsorption is, however, not yet clearly defined. In mesangial cells AVP stimulates the Na-H exchanger [6, 7] as well as the Natdependent or independent CIIHCO3 - exchangers [6]. In A6 cells (an amphibian distal nephron cell line), however, the addition of AVP either at low (subnanomolar) or at high (micro-
METHODS
ascending limbs showed that AVP stimulates the hasolateral cells while simultaneously inhibiting the apical Na1 -H1 antiporter [91.
perfusion with AVP and with AVP plus specific inhibitors. In each tubule, only one perfusion solution was used.
Male Wistar rats weighing 180 to 300 g were anesthetized with mactin (Byk-Gulden, Konstanz, Germany), 100 mg/kg ip. Wistar rats were obtained from Instituto de Ciências Biomédicas. They received a rat pellet diet and water ad libitum until the time of the experiment. The rats were prepared for in vivo micropuneture as described previously [14]. In brief, the left jugular vein and left
carotid artery were cannulated for infusions and blood with-
drawal, respectively. A tracheostomy was performed. The kidney was isolated by a lumbar approach and immobilized in situ by Ringer-Agar in a Lucite cup. During the experiment, the rats received venous saline containing 3% mannitol at 0.05 mI/mm. We studied the kinetics of HCOI reabsorption in ED (distal molar) concentrations inhibits the basolatcral Na-H-exchange convoluted tubule) and LD (connecting tubule and initial collectactivity [8]. Studies in isolated perfused mouse medullary thick ing duct) segments. The experiments were done during luminal
HC03 reabsorption was calculated from continuous measurement of luminal p1-I by means of microelectrodes in a fluid column Key words:
AVP, distal tubule, HCO3 reabsorption, Na-H exchange,
isolated by castor oil in the tubule lumen that followed the
Received for publication December 27, 1996 and in revised form May 15, 1997 Accepted for publication May 19, 1997
intratuhular p1-I changes toward the steady-state level [14, 15]. Briefly, the microperfusion procedure involved impalement of a proximal loop with a double-barreled micropipette made from theta glass tubing (R & D Optical Systems, Spencerville, MD, USA), One barrel was filled with Sudan black-colored castor oil,
© 1997 by the International Society of Nephrology
and the other was filled with the luminal perfusion solution
H-ATPase.
1035
1036
Chaves
and Mello-Aires: At/P in distal I1CO reabsorption
colored with 0.05% FD & C green. Initially, the luminal perfusion
was used to detect ED or LD loops. A double-barreled microeletrode was then inserted into the ED or LD loop. Afterward, luminal perfusion was performed at a rate sufficient to elevate luminal distal tubule loop pH to near that of the original perfusion
solution (pH 8). Then a column of oil was injected into the
Table 1. Transcpithelial potential difference in early and late distal tubule during luminal perfusion with AVP (10° M), Anti-Vi (10 M), Anti-V2 (106 M), HMA (10 M) and Bafilomycin Al (2 >< NI)
iO
Control
Early distal
Late distal
—17.5 0.75
—45.4 2.46 (17/6) 1.20
(20/6)
—15.0 0.65
proximal tubule lumen, blocking the flow of fluid. Intratubular pH AVP was measured as the voltage difference between the two barrels of
—46.7
(32/11)
(19/9)
the microeletrode made from Hilgenberg (Malsfeld, Germany) double-barreled asymmetric glass capillaries. The larger barrel contained a H f-sensitive ion-exchange resin (Fluka, Buchs, Swit-
Anti-Vi
zerland), and the smaller one contained 1 M KCI colored by FD &
AVP + Anti-VI
0.38 (20/5)
—41.3
1.19
—44.6
—14.5
—18.5
Anti-V2
2.62 (21/6)
(27/8)
—15.2 0.82
C green (reference barrel). Transepithelial electrical potential AVP + Anti-V2 difference (V1) was defined as the difference between the reference barrel and ground. This parameter was an additional crite- AVP + HMA rion for the recognition of ED or LD segments [16]. Intratubular pH was measured concomitantly in the same tubule with Vt. AVP + BAF Voltages were read by a World Precision Instruments (New Values are means Haven, CT, USA) model FD223 differential electrometer, the
—38.9
1.58
(17/6) —34.9 0.39
(19/5) —20.7
1.46
(24/6)
1.07
(27/6)
(19/5)
—16.3 0.94
—39.9
1.18
(17/6)
(22/7)
—12.9 0.99
—48.0
(18/5)
1.39
(22/7)
s; (N of measurements/N of tubuks). No
output of which was digitized in I second intervals by means of an analog-to-digital conversion board (Data Translation model DT 2801, Marlborough, MS, USA) mounted on a Dell 333 D microcomputer.
statistical differences were observed between the different experimental groups and the respective control.
stationary level. Along this curve, intratubular concentrations of HCO3 were calculated from intratubular pH values and from the systemic partial pressure of carbon dioxide (PCO2) by use of the Henderson-Hasselbalch equation at I second intervals, since we have previously demonstrated that renal cortical PCO2 is similar to that of arterial blood [17]. The rate of tubular acidification was expressed as the half-time of the reduction of the injected HC03
OsnabrUck, Germany), were dissolved in dimethyl sulfoxide [15]
The luminal pH fell from its initial value of 8 toward the (obtained from Prof. K. Altendorf, University of Osnabruck,
levels to their stationary level (t1/2). Net HC03 reabsorption (JHCO3) was calculated from the equation
JHCO1 = k([HC03]
—
and added to luminal perfusion solution (HMA = bafilomycin A1 = 2
1O s and
x iO M).
The pH and PCO2 in samples of blood collected from the
carotid artery were measured every 30 minutes with a Radiometer Copenhagen ABL 5 blood gas system. In the groups of animals
with luminal perfusion with the hormone, we also measured
urinary flow and Na excretion. Na
in urine collected
from
urinary bladder was measured by flame photometry.
]) r/2 where k is the rate constant of the reduction of luminal bicarbon-
Statistics Data are means SE. Differences between experimental groups
ate [k = ln2/(t112)], r is the tubule radius, and [HC03]1 and [HCO3] are the concentrations of the injected HC03 and
were evaluated by analysis of variance (one-way) with contrasts by
HC03 at the stationary level, respectively. The pH microeletrodes were calibrated before and after every impalement on the kidney's surface by superfusion with 20 mivi phosphate Ringer buffer solutions containing 130 mtvi NaCI at 37°C. Their pH values were adjusted to 6.5, 7.0, and 7.5 with 0.1
(mean of several measurements) when urine or blood collection
[HCO3
N NaOH or HCI. The mean slope of 49 microelectrodes under our experimental conditions was 60.1 2.28 mV per pH unit in the range of 6.5 to 7.0 and 57.7 2.39 mV in the range of 7.0 to 7.5. No significant interference of the presence of cations or anions was observed [15]. The luminal control perfusion solution contained (in mM): 100 NaCl, 25 NaHCO3, 5 KCI, 1 CaCl2 and 1.2 MgSO4. The osmolality was adjusted to 300 mOsm/kg H20 with raffinose. AVP (mol wt 1.084; Sigma Chemical Company, St. Louis, MO,
the Bonferroni technique, using N as the number of animals
was performed or the number of perfused tubules (mean of several perfusions). RESULTS
To detect whether the dose of AVP used during luminal microperfusion would cause an effect on renal function and to follow acid-base conditions, we measured urine flow (V), Na' excretion and systemic acid-base parameters during the microperfusion experiments. During luminal perfusion with the hormone, these data were similar to the control values [V, 0.149 0.014 ml
min kg'; urinary sodium flow, 8.81 0.978 j.tEq min' kg'; pH, 7.40 0.14; PCO2, 39.2 0.65 mm Hg and HCO3, 24.8
1.1 mM, N
=
12].
USA) was added to luminal solution (10—p M). VI-receptor
Table I gives mean data of V, in ED and LD segments during tubular perfusions in all experimental conditions. No statistical specific antagonist (Anti-VI, [d (CH2)5, Tyr (Et)2] arginine vasopressin) as well as V2-receptor specific antagonist (Anti-V2, differences were observed between the different experimental [AdamantaneacetyP, O-Et-D-Tyr2, Val4, Aminobutyryl6, Arg8'9] groups and the respective control. vasopressin) were obtained from Sigma Chemical Company and Mean intratubular steady-state pH levels are given in Table 2.
added to the luminal perfusion solution (10 NI and 10' M,
In both ED or LD segments, values found during luminal
respectively). 5-(N,N-hexamethylene) amiloride (HMA; Merck, microperfusions in all experimental groups were not significantly Sharp, & Dohme, Rahway, NJ, USA), as well as bafilomycin A1 different from the control value.
1037
Chaves and Mello-Aires: A VP in distal HCO3 reabsorption
Table 2. Intratuhular steady-state pH in early and late distal tubule during luminal perfusion with AVP (10 M), Anti-Vl (1O si), Anti-V2 (106 M), HMA (10 M) and Bafilomycin (2 X iO M) ________
Control
AVP + HMA
6.77 0.06 (20/6) 6.88 0.12 (19/9) 6.56 0.07 (20/5) 6.46 0.08 (27/8) 6.45 0.19 (19/5) 6.59 0.18 (27/6) 6.68 0.08
AVP + BAF
(22/7) 6.70 0.07
AVP
Anti-Vi Anti-V2
AVP + Anti-Vi AVP + Anti-V2
3.5
L
10
6.53 0.27 (17/6) 6.58 0.07 (32/11) 6.57 0.06 (24/6) 6.74 0.11 (21/6) 6.66 0.14 (17/6) 6.79 0.18 (19/5) 6.46 0.14 (17/6) 6.50 0.05
4.0
j:
12
Late distal
Early distal
________
A 14
3.0
2.5
8
2.0
4
-
2
0
C
B
I
(N=20/6)
14
-
Anti-Vi (N=20/5)
-
An i-V2 (N=27/8)
L
10
statistical differences were observed between the different experimental groups and the respective control.
8 6
3.5 3.0
10
2.5
8
2.0
6
1.5
4
1.0
4
I0 C.-
2
0
0
C,)
0 N.)
0.5
2
-
-
Anti-Vi
-
t
9
C,)
0
1.5 1.0
0.5 0.0 4.0 3.5
12
(18/5) (22/7) — ________ __________ Values are means SE; (N of measurements/N of tubules). No
—
12
t
I0 C-
I C-
3.0 0 9 2.5 C,) 2.0 0 1.5 1.0
C-i
N.)
In
0.5 0.0
Anti-V2 (N=17/6) (N=24/6) (N=21/6) Fig. 2. Effect of luminal Anti-Vi 10 M or Anti-V2 iO M on HC03 reabsorption in early and late distal tubule. Experiments were done in early A) and late (B) distal tubules. Values are means SE; N, number of microperfusions/number of tubules. Symbols are as in Figure 1. C
0.0
0 C
AVP
(F\!—_2016) (N=19/9)
AVP (N=1 7/6)(N=32/i 1) Late distal C
Early distal Fig. 1. Effect of luminal arginine vaSopressin io— M on HC03 reab-
sorption in early and late distal tubule. Values are means SE; N, number of microperfusions/number of tubules; (fl) t112, half-time of reduction of concentration of the injected HC03; () JHCO3, net HC01 reabsorption. *P < 0.01 compared with control. Abbreviations are: C, control; AVP, arginine vasopressin.
stimulates distal bicarbonate reabsorption at the apical membrane, in the following experiments luminal perfusion with VI and V2-receptor specific antagonists were used.
Effect of Anti-Vi 10 M and Anti-V2 106 M
Figure 2 shows the effects of luminal Vi or V2-reeeptor antagonists on tubular acidification in both ED (Fig. 2A) and LD (Fig. 2B) segments. In ED tubules, when Anti-Vi was used the t112 was 12.1 0.551 seconds (20/5) and JHCO3 was 0.82 0.037
nmol cm2 s , and when Anti-V2 was administered the t112 was
Effect of AVP 10 M Figure 1 demonstrates that AVP added to the luminal solution significantly stimulates HCO3 reabsorption in ED or LD segments. In ED segments, the half-time of acidification (t112) decreased from 10.2 0.531 seconds (N of perfusions/N of tubules = 20/6) during control luminal perfusion to 4.81 0.341 seconds (19/9) (P < 0.01) when AVP was added to lumen, and consequently JHCO3 increased from 0.931 0.061 to 2.12 0.171 nmol cm 2 s (P < 0.01). In LD segments the t112 decreased from 10.6 0.432 seconds (17/6) during control luminal perfusion to 4.33 0.201 seconds (32/11) (1' < 0.01) when AVP was used, and JI-1C03 increased from 0.542 0.086 to 1.67 0.111 nmol cm2 (P < 0.01). To determine and investigate which specific receptor AVP 1
9.23
0.291 seconds (27/8) and the JHCO3 was 0.94
0.112
nmol cm2 s. In LD tubules, with Anti-Vi luminal solution the t1,2 was 12.6
0.672 seconds (24/6) and JHCOI was 0.69 I
0,038 nmol cm2 s , and when Anti-V2 was used the t1,2 was 0.521 seconds (21/6) and the JHCO3 was 0.53 0.073 These data demonstrate that VI or V2-receptor antagonists have no intrinsic effects on luminal acidification in both ED and LD segments. Figure 3 shows that luminal AVP stimulates HC03 reabsorption both in ED (Fig. 3A) and LD (Fig. 3B) segments via activation of Vi receptors. In the group where AVP plus Anti-Vi was administered, the parameters of acidification were not signifi10.3
nmol cm 2,
s.
cantly different from the control group: in the ED segment the t1,2 was 11.5 0.772 seconds (19/5) and JHCO was 0.891 0.075 nmol' cm2 and in the LD segment the ti!22 was 10.5 0.611
1038
Chaves and Mello-Aires: A VP in distal HCO3 reabsorption
A 14
I
12
1
10
*
8
*//
6
4
i_Ei
2 0
C
AVP
A
4.0
#
ii
*.
AVP+
AVP+
3.5
C-
22 20
3.0
C)
18
2.5 2.0
I 9
C.,
:3
0
1.5 1.0
(I)
0.5
0.0
B 14 12 10
#
.
I
.
.1
8
.
*
*
6 4
0
C
AVP
2.5
I
I CD
0
I
AVP÷ AVP+
(N=17/6) (N=32/1 1) Anti-Vi
*
14
2.5
#
i2. 12
2.0
10 8 6 4 2 0
1.5 1.0
tF
10
•I
C—
*
*
2
3.0
3.0
16
4.0 3.5
3.5
I CC)
9
C,)
:3
0 C)
'F',
0, 0.5
AVP AVP+HMA AVP÷BA :00 B (N=20/6) (N=1 9/9) (N=22/7) (N=1 8/5) 12 4.0
(N=20/6) (N=19/9) Anti-Vi
Anti-V2 (N=1 9/5) (N=27/6)
4.0
Anti-V2
(N=17/6) (N=19/5) Fig. 3. Effect of luminal arginine vasopressin i0 M plus Anti-Vi i0 M or Anti-V2 10—6 M on HCO, reabsorption. Experiments were done in early 1A) and late (B) distal tubules. Values are means SE; N, number of microperfusions/number of tubules. Abbreviations and symbols are as in Figure 1. P < 0.01 compared with control. #P < 0.01 compared with
AVP.
8 6
4 2 0
I
.
#
. [111 .
3.5
C-
3.0
C)
2.5
2.0
I 9
C,)
:3
0
1.5
1.0
1/)
0.5 0.0
AVP AVP+HMA AVP+BAF (N=17/6) (N=32/11)(N=17/6) (N=22/7) Fig. 4. Effect of luminal arginine vasopressin iO M plus 5-(N, NC
hexamethylene) amiloride (HMA, iO M) or plus Bafilomycin A1 (2 x io— M) on HC03 reabsorption. Experiments were done in early (A) and late (B) distal tubules. Values are means SE; N, number of microperfusions/number of tubules. Abbreviations are: C, control; AVP, arginine vasopressin; HMA, 5-(N,N-hexamethylene) amiloride; BAF, Bafilomycin A1. Symbols are: (LII) t112, half time of reduction of concentration of the
injected HC03; () JHCO3, net HC03 reabsorption. *p < 0.01 compared with control. #P < 0.01 compared with AVP.
seconds (17/6) and JHCO3 was 0.804 0.065 nmol cm2 s. However, in the group where AVP plus Anti-V2 was adminis-
tered, the parameters of acidification were similar to the AVP group values: in the ED segment the t,2 was 4.89 0.25 1 second and in the (27/6) and JHCO3 was 1.66 0.101 nmol cm 2 LD segment the t112 was 4.40 0,231 second (19/5) and JHCO3 was 1.70 0.151 nmol cm2 s'.
It is known that in ED segments, HCO3 reabsorption is mediated mostly by Na-H exchange [15, 181, and in LD segments by Na -H exchange [151 and vacuolar H-ATPase [15, 18]. In the next series of experiments, we determined the specific mechanisms by which luminal AVP was stimulating HCO3 reabsorption. Effect of HMA 10" M Figure 4 summarizes the results obtained in ED (Fig. 4A) and LD (Fig. 4B) segments during luminal perfusion with AVP plus
compared to AVP), and JHCO3 decreased to 0.771 0.071 nmol cm2 s' (P < 0.01). In LD segments, HMA also inhibited the stimulation induced by AVP. The t112 increased to 6.55
0.511 s (17/6) (P < 0.01 as compared to AVP) and JHCO3 0.122 nmol em 2 . S (P < 0.01). These decreased to 1.10 data show that in ED and LD tubules AVP stimulates the Na-H exchanger responsible for HC03 reabsorption. Effect of bafilomycin A1 2 x iO M Data obtained during luminal perfusion with AVP plus bafilomycin A1, a highly specific inhibitor of vacuolar H -ATPase, are given in Figure 4 for the ED and LD segments. Bafilomycin A1 did
not inhibit the stimulation of ED HC01 reabsorption induced by AVP. The t1,2 was 5.59 0.331 s (18/5), and JHCO1 was 1.68 0.091 nmol cm2 s1. However, in LD segments, the t1,2 value was significantly increased to 6.94 0.422 s (22/7) (P < 0.01
HMA, a specific blocker of Na-H exchange. In ED segments, against AVP), resulting in a marked reduction in JHCO3 to HMA inhibited the stimulation of HCO3 reabsorption induced 1.14 0.082 nmol cm2• s (P < 0.01). These results indicate by AVP: the t112 increased to 11.3 0.832 s (22/7) (P < 0.01 as that AVP stimulates H1 -ATPase in LD segments.
Chaves and Mello-Aires: AVP in distal HC03 reabsorption
DISCUSSION
In the present study, we have investigated the effect of arginine vasopressin (AVP) on the kinetics of HCO reabsorption in both early distal (ED) and late distal (LD) segments. We examined the effect of luminal perfusion of the hormone in vivo by a stopped-
1039
identification of V 1-receptors for AVP in distal nephron, especially in the cortical portion of the collecting duct [25]. It has generally been accepted that the receptors for AVP are located in the basolateral membranes of renal tubular epithelia.
However, by an electrophysiological study Naruse et al [261
reported for the first time that AVP acts not only on the
flow microperfusion technique, which is not affected by the basolateral membrane, but also on the apical membrane of the glomerular filtration rate. This procedure also avoids systemic cortical collecting duct. In confirmation of this initial observation, alterations caused by hormone infusion, confirmed by absence of
changes in urine flow, Na excretion, and systemic acid-base values during experiments where luminal perfusion was per-
the authors obtained evidence that V1 receptors mediated luminal action of AVP in cortical ascending limb, distal convoluted tubule, connecting tubule and cortical collecting duct [27]. In addition,
formed. Furthermore, the perfusion solutions contain raffinose to luminal AVP has effects on intracellular calcium in medullary reach isotonicity, a non-reabsorbable molecule, in order to pre- ascending limb [28] and cortical collecting duct [29]. Taken vent the water flow induced by the hormone. Another advantage together, these data with the present results suggest that luminal of the luminal perfusion is to ascertain that the measurements of AVP might have a significant role in the regulation of HC01 acidification kinetics are performed at well-determined hormonal reabsorption of the ED and LD segments, via V1 receptors.
levels, as it is known that some peptides have a short half-life when injected parenterally. This is important since AVP is partially catabolized in distal nephron segments [19], and an intact structure of AVP is required for binding to the receptor [201. In all experimental groups no significant differences from the
Effect of AVP plus HMA
We chose to use HMA as a specific blocker of the Na exchanger, with an inhibition constant of > 400 /tM for Na control group were observed in the intratubular steady-state pH channels and 0.16 jtM for the Na-H exchanger [30]. In a values in both ED and LD segments. Changes in this parameter are related to several factors, including the rates of H secretion and HC03 reabsorption. These findings suggest that the main driving force for H secretion across the apical membrane is not altered during our experiments. Effect of AVP in early and late distal tubules
previous study Fernandez et a! [15], by in vivo microperfusion in
rats, found that net JHCO3 is decreased in both ED [from
0.68 0.13 (N = 8/8) to 0.33 0.07 (N = 16/8) nmol cm2 s] and LD segments [from 0.75 0.09 (N = 10/6) to 0.35 0.08 (N = 11/6) nmol cm2 s 'j during luminal perfusion with HMA (10 M). Thus, their data showed that mostly ED, but also partly LD segments, possess a mechanism of Na-H exchange respon-
Our results indicate that AVP has a direct effect stimulating sible for JHCO3. The present results obtained in ED segments HC03 reabsorption in ED and LD segments, expressed by a suggest that AVP stimulates the Na-H exchanger at the apical significant fall in acidification half-time and consequent increase in JHCO3 (Fig. 1). This effect was observed when the hormone was perfused luminally, which we show here for the first time. However, the present demonstration of AVP-induced enhance-
ment of HC01 reabsorption by distal tubules is not the first in vivo study of this kind. During hypotonic volume expansion in the
rat, Bichara et al [2] examined the acute effects of systemic
membrane, since luminal perfusion with AVP plus HMA (at the same dose used by Fernandez et a! [15], i0 M) reduced JHCO3 by a mean value of 63.7% compared with AVP alone (Fig. 4A). However, the inhibitory effect of HMA in LD segments (34.1%) was smaller than its effect in ED segments (Fig. 4B). This result is
in accordance with the data indicating that in ED segments HCO3 reabsorption is mediated predominantly by Na-H
exchange [15, 18]. The significant but smaller effect of HMA on LD action of AVP supports the existence of a minority participathe other hand, our results complement the in vitro rat CCD tion of Na/H exchange in this segment [15]. It is interesting that studies of Tomita et a! [5] and the findings of Levine et al on distal no significant effect of HMA on intratubular steady-state pH was tubules of normally fed rats [4], which showed AVP-stimulated found, but acidification t112 values were markedly increased. Work HCO3 reabsorption. However, since in these studies the nature from our laboratory has shown that the steady-state pH gradient of mechanisms underlying AVP stimulation was not defined, we depends on the driving force for H secretion (in this case, the decided to determine and then study which specific process AVP Na gradient across apical membrane), which is apparently unchanged by HMA [311. The observed increase in acidification stimulates distal UC03 reabsorption. DDAVP (1-desamino-8-D-arginine vasopressin), showing that net total CO2 secretion was reversed to net HC03 reabsorption. On
11/2 is compatible with reduction of the density of exchangers or of
Effect of AVP plus Anti-Vi or Anti-V2
their turnover at the apical membrane, so that it takes a longer period of time to reach the same maximal gradient. This modifi-
We chose the Vi-receptor specific antagonist (Anti-VI, [d (CH2)5, Tyr (Et)2] arginine vasopressin) to use as blocker of Vi-receptors [21, 221, and as blocker of V2-receptors the V2-
cation appears to be caused by the inhibitor, which competes with Na for the reaction site on the transporters, effectively reducing the number of active transporters.
receptor specific antagonist (Anti-V2, [Adamantaneacetyl1, 0-EtD-Tyr2, Va!4, Aminobutyryl6, Arg8'9 vasopressin) [23, 24]. We believe that the effect of AVP during luminal perfusion occurs via
in distal segments and that this action is mediated by V1-reccptors
Our results showing that AVP stimulates Na-H exchangers
agree with the signal-transduction pathways that have been deactivation of VI receptors at the luminal membrane, since the fined by a number of workers. It has been demonstrated that the stimulatory effect in response to luminal AVP in ED and LD effect mediated by stimulation of V7 receptors is sensitive at segments was prevented by simultaneous luminal perfusion with basolateral AVP concentrations of about 10 -12 to io' M [26, 27] VI-antagonist (Fig. 3). These data are in accordance with the and results in an increase in intracellular cAMP concentration. By
1040
Chaves and Mello-Aires: AVP in distal HCO reabsorption
contrast, a higher concentration of AVP (10— 10 to iO° M), such as the concentration used in the present work, is required to cause a measurable increase in cytoplasmic Ca24 concentration in the collecting ducts by stimulation of V1 receptors [32]. On the other hand, luminal AVP acts on intracellular Ca24 in CCD principal cells [29] and medullary thick ascending limb [28]. Based on these observations and ott our findings, we believe that the effects of luminal AVP (10-v M) observed in our study are mediated by V1
receptors in luminal membrane, rather than by V2 receptors, which are sensitive to much lower AVP levels and, by liberating
cAMP, are expected to inhibit Na-H4 exchange. On the other hand, it has been shown that stimulation of V1 receptors increases
cytoplasmic Ca24 concentration [32] and a rise in intracellular Ca24 stimulates Na4-H4 exchange in many cells and increases HC03 reabsorption in proximal convoluted tubule [33]. Furthermore, V2-receptors have not yet been identified in apical membrane [34], and recently Yoshitomi et al [35] demonstrated that
AVP stimulates luminal Na4 conductance mediated by Vireceptor in the rabbit CCD.
intercalated cells is associated with luminal alkalinization, it appears unlikely that the stimulatory effect of AVP on ATPase activity in the rat LD segments shown in the present work should occur in this subpopulation of intercalated cells, particu-
larly since it is known that, in rats, the majority of intercalated cells is of the A type [37].
The mechanism behind the effect of AVP on H4-ATPase activity in LD segments is unknown. The role of an increase in cell Ca2 has been mentioned above [32]. However, the precise nature of the signaling pathways involved in mediating the effect of AVP on H4-ATPase activity in LD segments remains to be established.
We have not investigated the possible effect of AVP on H4-K4-ATPase, since it was shown that this transporter is expressed only in potassium depletion in this segment [15, 18]. On the other hand, the possible physiological role of luminal AVP action should also be considered. The concentration of AVP
in the luminal fluid may exceed that of plasma, even under physiological conditions. Data from kinetic studies and from renal clearances suggested that the tubular secretion of vasopressin into
the distal ncphron is an important component of the renal Effect of AVP plus bafilomycin A1 clearance of this hormone in humans [38], dogs [39, 40] and rats Bafilomycin Al has been shown to be a highly specific inhibitor [41]. In humans, AVP clearance is in the range of 0.1 to 2 of the vacuolar type of H4-ATPase [36]. In ED segments of rat, by mi/kg/mm, and urine AVP levels range from 5 to 500 ps [29]. means of in vivo microperfusion experiments, no significant effect Ando, Tabei and Asano [12] reported that water permeability
of luminal bafilomycin A1 (2 x iO M) on net JHCO3 was
stimulated by basolateral AVP was inhibited by luminal AVP observed; in LD loops, however, JHCO1 values were signifi- within this concentration range. Luminal AVP at the concentracantly decreased [from 1.18 0.120 (N = 19/11) to 0.60 0.054 tion of 100 p increased intracellular Ca24 [29] and changed ionic (N = 35/9) nmol cm2 s 1j [15]. Thus, these results indicate conductances of the microperfused rabbit CCD [26, 27]. These that in LD tubule an H4-ATPase of the vacuolar type also data taken together with the present results suggest that luminal contributes to tubular acidification. Our results suggest that in ED AVP might have a significant role in the regulation of tubule segments, bafilomycin A1 (2 >< i0— M) did not impair the transport of water and ions, especially when AVP concentration is stimulation induced by AVP (Fig. 4A). However, our data indi- high. cate that AVP stimulates H-ATPase in LD segments, since In conclusion, we performed in vivo stationary microperfusion luminal perfusion with AVP plus bafilomycin reduced JHCO3 by studies in the cortical ED and LD tubules of the rat to evaluate a mean value of 31.7% compared with AVP alone (Fig. 4B). the effects of luminal AVP (10° NI) on HCO reabsorption. Our These findings are consistent with data showing that this ATPase results indicate for the first time that luminal AVP significantly occurs predominantly in LD segments and that bafilomyciri A1 at stimulates HC03 reabsorption in in vivo ED and LD segments the used concentration had no significant effect on ED acidifica- via activation of VI receptors. Luminal AVP acts stimulating tion [15, 18]. The inhibition of electrogenic H4 secretion is Na' -H4 exchange mainly in ED segments. Evidence has also expected to increase the lumen-negative V1; however, Table 1 been obtained for stimulation of the vacuolar H4-ATPase in LD shows no significant alteration of V during perfusions with segments. The present work, however, does not allow us to ascribe bafilomycin, suggesting that the transfer of H4 possibly is being the results with AVP to any specific cell type that might contribute electrically neutralized by the conductance of other co- or to acidification in cortical distal tubule. counter-ions. The absence of a significant effect of bafilomycin on
V has been shown before [15], suggesting that H -ATPase does
ACKNOWLEDGMENTS
not participate in V1 in a major way. Nevertheless, in contrast with our results, Ando, Tabei and Asano [12] have shown that luminal i0° M AVP hyperpolarized collecting duct transepithelial poten-
de São Paulo, Consciho Nacional de Desenvolvimento CientIflco e
tial difference, an effect reverted by acetazolamide. They suggested that AVP might impair electrogenic H' secretion in this segment. The reason for these differences is not clear, however, it should be remembered that the cited authors did not measured H secretion directly. The effect of AVP on electrogenic vacuolar H4-ATPase in LD epithelium is compatible with the presence of intercalated cells in both connecting segment and initial collecting duct. However, the
Reprint request.s to Margarida de Mello Aires, Department of Physiolo and Biophysics, Instituto de Ciências Biomédicas, University of São Paulo, SP 05508-900, Brazil.
present study does not establish the cellular site of the AVP effect.
Stimulation of H4-ATPase activity in type A intercalated cells would increase tubule fluid acidification, an effect compatible with
our present results. Because bicarbonate secretion by the type B
This work was supported by Fundaçbo de Amparo a Pesquisa do Estado
Tccnológico and Coordenacão de Aperfeiçoamento de Pessoal de Nivel Superior. We thank Dr. Gerhard Malnic, University of São Paulo, Brazil, for careful reading of the manuscript and for helpful suggestions.
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