0013.7227/92/1313-1429$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine
Vol. 131, No. 3 in U.S.A.
Printed
Society
Increases in Cellular Sodium Concentration by Arginine Vasopressin and Endothelin in Cultured Rat Glomerular Mesangial Cells* SAN-E
ISHIKAWA,
Division Japan
of
Endocrinology
KOJI
OKADA,
AND TOSHIKAZU
and Metabolism,
Department
SAITO of
Medicine,
ABSTRACT The present study was undertaken to determine whether arginine vasopressin (AVP), angiotensin-II, and endothelin (ET) increase the cellular sodium concentration ([Na+]i) in cultured rat glomerular mesangial cells. [Na+]i was measured using the fluorescence indicator dye sodium-binding benzofuran isophthalate. These three vasoconstrictor hormones increased cellular free calcium ([Ca*‘]i) and [Na+]i in a dosedependent manner ([Na+]i: basal, 11.5; 10e7 M AVP, 20.5; 10T7 M angiotensin-II, 13.8; and lo-’ M ET, 21.2 mM). The mobilization of [Caz+]i was faster than that of [Na+]i. The AVP-induced increase in [Na+ ]i was completely blunted by the potent V1 antagonist d(CH.&Tyr(Me)AVP. Vasoconstrictor hormones produced a biphasic cellular pH (pHi) change, characterized by a transient acidification, followed by a sustained alkalinization. The Ca*+-free condition mark-
V
ASOCONSTRICTOR hormones, such as arginine vasopressin (AVP) and angiotensin-II, produce cell contraction of glomerular mesangial cells (l-3). Schor et al. (4) demonstrated in a micropuncture study that the glomerular capillary ultrafiltration coefficient (Kf) is modified by AVP and angiotensin-II when injected intraarterially into rats. Recently, it has becomewell known that AVP and angiotensin-11act on glomerular mesangial cells mediated via their cellular second messenger,cellular free calcium ([Ca”]i) (57). In the absenceof bicarbonate, these hormones produce a biphasic celIular pH (pHi) change, characterized by a transient acidification, followed by a prolonged alkalinization that is both Na+ dependent and amiloride sensitive (8). The change in pHi is dependent on the Na+/H+ exchange across the plasma membrane, and the hormonally induced Na+/H+ exchange per seis controlled by [Ca’+]i (9, 10). Similar results were obtained in homologous cells of vascular smooth muscle (11-14). It is of great value to evaluate the role of cellular sodium ([Na+]i) in the cellular action of vasoconstrictor hormones in glomerular mesangial cells, becausecellular alkalinization is suggestedto depend on Na+ entry in exchange for H+ excretion, which is closely related to cell proliferation and cell contraction (3, 8). The fluorescence indicator dye Received January 28, 1992. Address requests for reprints to: San-e Ishikawa, M.D., Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School, 3311-l Yakushiji Minamikawachi-machi, Tochigi 329-04, Japan. * This work was supported by a grant from the Ministry of Education, Science, and Culture of Japan. The study will be presented at 9th International Congress of Endocrinology in Nice, France, August 1992.
Jichi Medical
School, Tochigi
329-04,
edly reduced AVP- and ET-induced increases in [Ca’+]i and [Na+]i and biphasic changes in pHi. In the Na+-free state, the hormonally mobilized [Ca”]i was significantly enhanced. Basal [Na+]i decreased to below 3 mM, and there was little increase in [Na+]i after the addition of vasoconstrictor hormones, suggesting that the source of [Na+]i is extracellular space. Only early acidification was obtained in the absence of a sustained alkalinization. The [Na+]i mobilization was closely related to the biphasic change in pHi. These results indicate that AVP, ET, and angiotensin-II increase [Na+]i in glomerular mesangial cells, and that the early mobilization of [Na+]i depends on Na+/Ca’+ exchange, and the sustained phase depends on Na+/H+ exchange. The hormonally mobilized [Ca”‘]i is essential for the activation of Na+/H+ exchange, and an increase in [Na+]i is suggested to play an important role in cellular alkalinization. (Endocrinology 131: 1429-1435,1992)
sodium-binding benzofuran isophthalate (SBFI) enables us to determine [Na’Ji in intact cells (15). The present study was undertaken to determine whether the vasoconstrictor hormones AVP, angiotensin-II, and endothelin (ET) affect [Na+]i in cultured rat glomerular mesangial cells. Whether Ca*+ and Na+ modulate the vasoconstrictor hormone-induced increasein [Na+]i was also examined. Materials and Methods Cell culture The experimental procedure was modified from the method of Kreisberg and Kamovsky (16). Male Sprague-Dawley rats, weighing 150-l 75 g, were used. Kidneys were removed under sterile conditions, and cortical tissues were cut away from the medulla. They were minced with physiological saline solution (PSS; 140 mu NaCl, 4.6 mu KCl, 1 mu MgC&, 2 mu CaC12, 10 mu glucose, and 10 mM HEPES, pH 7.4) by a sharp razor blade, and then passed through a series of steel sieves with decreasing pore sizes (60 and 200 mesh) with the glomeruli appearing on top of the 200-mesh sieve. ‘The minced renal cortical tissues were incubated with 3 ml collagenase (1 mg/ml; Worthington Biochemicals, Freehold, NJ) for 60 min at 37 C. After centrifuging the tubes at 500 x g for 4 n&at room temperature, the pellets we& resuspended with Dulbecco’s Modified Eagle’s Medium (Flow Laboratories, McLean, VA) supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and lOb*pg/ml streptomycin. The dispersed glomeruli were harvested into 35 x IO-mm plastic dishes with the medium and kept in a humidified incubator at 37 C under 95% air and 5% COZ. After the culture cells were confluent; they were subcultured using CaZc- and Mg’+-free Hanks’ solution containing 0.025% trypsin and 0.01% EDTA. The dispersed cells were collected into culture tubes and centrifuged at 500 x g for 5 min at room temperature. The pellets were resuspended in Dulbecco’s Modified Eagle’s Medium containing 20%
1429
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AVP, ET, AND
1430 TABLE in cultured
1. AVP-
and ET-l-induced rat glomerular mesangial
increases cells
in [Ca”]i
[Ca*+]i (nM)
and [Na+]i
[Na+]i
(mM)
Control 1 X lo-’ M AVP lx10”~AVP 1 x 10-r M AVP
106.9 166.0 346.7 503.9
k + f f
6.1 (6) 6.6 (6)” 33.8 (6)” 30.8 (6)’
11.5 15.1 19.9 20.5
f f f f
0.4 0.4 1.0 1.0
(10) (6)” (6)” (10)’
Control 1 x 1O-g M ET-1 1 x lo-* M ET-1 1 x 10-r M ET-1
108.8 184.8 261.4 787.1
f f + +.
6.7 (6) 15.4 (6)” 35.1 (6)” 98.2 (6)”
9.9 16.3 21.2 18.7
+ f + f
0.8 2.3 2.0 1.1
(6) (6)b (6)” (6)”
Values are the mean parentheses. a P < 0.01 us. control. b P < 0.05 vs. control.
TABLE cultured
a SEM.
The
number
of observations
2. Effects of AVP analogs on [Ca”+]i rat glomerular mesa&al cells
[Na+li IN GLOMERULI
and [Na+]i
is in
in
Measurement
of
En&. Voll31.
1992 No 3
[Na+]i
The experimental procedure was similar to that described in our previous studies (14,20). The cells were rinsed twice with 1 ml PSS and loaded with 10 PM SBFI/AM (Molecular Probes, Inc., Eugene, OR) in a volume of 0.25 ml for 3 h at 37 C. SBFI/AM was dissolved in PSS containing 0.02% pluronic F-127, a nonionic detergent. After aspirating the SBFI/AM solution, the glass slides were rinsed and then placed in a 1 x l-cm quartz cuvette with the aid of a special holder in a fluorescence spectrophotometer (CAF-110). The dual wavelength excitation method for measurement of SBFI fluorescence was used. The fluorescence was monitored at 500 nm, with excitation wavelengths of 340 and 380 nm in the ratio mode, in a manner similar to the monitoring of the effect of calcium on fura-2. After a stable fluorescence signal was achieved, the effecters were added. Basal and hormone-stimulated peak values were determined. [Na+]i was calibrated by equilibrating [Na+]i with the extracellular Na+ concentration, using 1 X lo+’ M gramicidin. The reference standard solutions were made from appropriate mixtures of Na+ and KC solutions, based on the solution of PSS. The total concentrations of Na+ and K+ were adjusted to 135 mM. The [Na+]i was determined by the relation between the ratio and the authentic [Na’]i.
[Ca*+]i (nM) Basal Control 1 x 1o-B M d(CH&Tyr(Me)AVP 1 x lo-’ M dDAVP
1 X 1O-7M AVP 1 X lo-’
106.0 f 5.2
496.9 f 22.8”
107.6 + 7.9 104.6 f 14.7
106.1 f 6.4
M
dDAVP
104.5 + 14.7 [Na+]i (mM)
Basal Control
1 x lo-’
M
AVP 1 X lo-’ M dDAVP
11.7 f 0.5
21.5 f 0.8”
10.9 + 0.7 12.0 f 0.6
10.9 f 0.7
1Xlo”M
d(CH&Tyr(Me)AVP 1 X 10-r M dDAVP
12.8 + 1.2
Values are the mean f SEM (n = 6). a P < 0.01 us. the basal level. fetal bovine serum, penicillin, and streptomycin and cultured in a humidified incubator. The culture cells at 3-10 passages were subjected to the following studies on days 7-10 of the subculture. For measurements of [Ca”]i, [Na+]i and pHi, the cells were cultured on thin glass slides (13 mm in diameter; Matsunami Kogyo Co., Osaka,
Measurement
of
pHi
The experimental procedure was similar to that reported in our previous studies (20,21). The cells were rinsed twice with 1 ml PSS and loaded with 2 2’,7’-bis-(2-carboxymethyI)S(and W 6)carboxyfluorescein, acetoxymethyl ester (BCECF/AM; Molecular Probes), for 60 min at 37 C. BCECF/AM was dissolved in PSS. The complete intracellular hydrolysis of BCECF/AM to BCECF was judged by changes in the excitation and emission spectra. The fluorescence was monitored at 530 nm, with excitation wavelengths of 450 and 500 nm in the ratio mode. After measurement of the basal pHi level, the effecters were added. Since autofluorescence of unloaded cells was less than 1% of the total fluorescence of BCECF-loaded cells, autofluorescence did not affect the pHi calculations. The fluorescent signal was calibrated at several pH values (6.6, 7.0, and 7.4) in KC1 solution (140 nut KCl, 4.6 mu NaCl, 1 ~I-IM MgC&, 2 nut CaC&, 10 mu glucose, and 10 mu HEPES) containing the K+/H+ ionophore nigericin (10 pg/ml).
Statktics All values of [Ca’+]i, [Na+]i, and pHi were analyzed by an analysis of multiple variance, using Scheffe’s method and Student’s t test. P < 0.05 was considered significant.
Japan). Measurement
of
[Ca”+li
The experimental procedure was similar to that used in our previous studies (17, 18). The cells were rinsed twice with 1 ml PSS and loaded with 5 PM fura-Z/AM (Dojin Biochemicals, Kumamoto, Japan) in a volume of 250 ~1 for 60 min at 37 C. After aspiration of the fura-Z/AM solution, the glass slides were rinsed and then placed in a 1 X l-cm quartz cuvette with the aid of a special holder in a fluorescence spectrophotometer (CAF-110, Japan Spectroscopic Co., Tokyo, Japan). The dual wavelengh excitation method for measurement of furafluorescence was used. The fluorescence was monitored at 500 nm, with excitation wavelengths of 340 and 380 nm in the ratio mode. The effecters were added after a stable fluorescence signal (R) was achieved. AVP (grade VI, Sigma, St. Louis, MO), ET-1 (Peptides Institute, Osaka, Japan), angiotensin-II (Peptides Institute), and atial natriuretic peptide (Peptides Institute) were used. From the ratio of fluorescence at 340 and 380 nm, the [Ca’+]i was determined, as described by Grynkiewicz et al. (19), using the following expression: [Ca’+]i (nM) = & X [(R - R,,,&R,,,,, R)] x 8, where R is the ratio of fluorescence of the sample at 340 and 380 nm, and R max and R,, were determined by treating the cells with 5 X 10m5 M digitonin and I X 10e2 M MnC12, respectively. The term @ is the ratio of fluorescence of furaat 380 nm in zero and saturating Ca’+ concentrations. K,, is the dissociation constant of furafor Ca’+, assumed to be 224 nM at 37 C (19).
Results AVP caused an increase in [Ca’+]i in a dose-dependent manner in glomerular mesangial cells in culture (Table 1). AVP (1 X lo-’ M) increased [Ca’+]i to 503.9 + 30.8 from 106.9 f 6.1 nM (P < 0.01). The [Ca’+]i promptly rose and reached a peak level a few seconds after the addition of AVP. The sustained phase of [Ca’+]i gradually decreased, but remained high above the basal level during the 15min observation period. As shown in Table 1, AVP alsoincreased [Na’]i in glomerular mesangial cells. Such an increase was obtained with AVP in a dose-dependent manner (Table 1). An increase in [Na+]i reached a peak level approximately 1 min after exposure to AVP, followed by sustainedelevation of [Na+]i, which lasted at least 15 min. Quite similar results were obtained with ET-l (Table 1). It was clear that the AVPor ET-l-induced peak value of [Na+]i appeared later than that of [Ca’+]i. Table 2 shows the effects of AVP analogs on AVP-mobilized [Ca”+]i or [Na+]i to elucidate whether Vi or V2 receptors
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AVP, ET, AND [Na+]i IN GLOMERULI
Control
[Na+Jr
1431
6
.
p+]l
Control
Ml) 800
(mM) 20 -
.--I
-
---
400 200 looI
OmtvlCCa*~~~..
OmM [3=a*+lo .
,/ 50 -
16
L
lo-
10 i I
500 -
OmMCNa+lo v
I
:
\yy+
400 -
200 ----y-----!
--.-I I I
100 -
I
5min /
1 min The effects of Ca’+- and Na+-free conditions on 1 X lo-’ M AVP-induced increases in [Na+]i (A) and [Ca*+]i (B) in cultured rat glomerular mesa&al cells. Upper panel, Control; middle panel, Ca*+-free condition; lower panel, Na+-free condition. FIG.
1.
are involved in the cellular action of AVP in glomerular mesangial cells. The selective and potent AVP V, antagonist
[1-(/3-mercapto-/3,/3-cyclopentamethylenepropionic acid)2(0-methyl)tyrosine]AVP[ [l X 10m6M; d(CH&Tyr(Me)AVP] (22) completely blocked the AVP-induced increasesin [Ca’+] i and [Na+]i. In contrast, the Vz agonist l-deamino-8-o-AVP (1 x lo-’ M; dDAVP) (23) did not affect either [Ca’+]i or [Na+]i. Next, we examined the hormonally mobilized [Ca’+]i and [Na+]i under Ca*+-free conditions to clarify whether cellular free Ca*+mobilization is essentialfor the hormonally induced increase in [Na+]i. Cells were preincubated for lo-15 min with the Ca*+-free medium containing 1 mu EGTA. As
shown in Fig. 1, in the Ca*+-free state, the 1 X lo-’ M AVPmobilized [Ca’+]i was markedly diminished as [Ca’+]i increased from 29.1 f 4.1 to 76.7 + 11.4 nM (P < 0.01). The sustained elevation of [Ca’+]i disappeared. Similarly, the mobilization of [Na+]i by 1 X lo-’ M AVP was also remarkably blunted. [Na+]i was elevated to only 1.8 ITLM(mean value), which was significantly less than the value in the control group. Similar results were obtained with 1 X lo-’ M ET-l (Fig. 2). In addition, Ca*+ ionophore ionomycin (1 x 10m6M) increased [Na+]i from 10.2 f 1.0 to 24.1 + 2.3 mu (n = 6; P < 0.01). Such an increase by ionomycin was totally absent in cells pretreated with the Ca*+-free medium containing 1 mu EGTA.
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1432
AVP,
A
ET,
AND
[Na+]i
B
Control
~8?J126 (mM)20.
Endo. 1992 Voll31 l No 3
IN GLOMERULI
800
l
400 -
15 lo-
200 5loo20-
16lo-
200 -
5loo-
OmM ma+ Jo 50 -
lo-
! !
5.
10.
/ I
O-
5min 400 -
200 -
100.
FIG. 2. The effects of Ca*+- and Na+-free conditions on 1 X low7 M ET-l-induced increases in [Na+]i (A) and [Ca*+]i (B) in cultured rat glomerular mesangial cells. Upper panel, Control; middle panel, Ca*+-free condition; lower panel, Na+-free condition.
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AVP, ET, AND Fontrot
[Na+]i IN GLOMERULI
1433
PHI
co/
Control i ;
7.2 -
: ,..,..
,.. :
___.i. :. ._L
:.
:
I... .!.. 1::.
;. ..j.:.f. !
i . . .._ j-..
,I,_
;
i..;..
j;
j
;...
T.‘.
7.2. 7.2 -
7.1OmMCCa*+]o
7.2 -
I :
,.
OmM 1 -i--T-~-~-1
1
7.1.
HatJO
;.. :. .:
I [
:
:
:
lmin FIG. 4. Changes in pHi after the addition of 1 x lo-? M ET-l in cultured rat glomerular mesangial cells. Upper panel, Control; lower panel, Ca*+-free condition.
lmin
3. Changes in pHi after the addition of 1 x 10T7 M AVP in cultured rat glomerular mesangial cells. Upper panel, Control; middle panel, Ca*+-free state; lower panel, Na+-free state. FIG.
TABLE 3. The changes in pHi in response to AVP and ET-l in cultured rat glomerular mesangial cells pHi 1 X lo-’
Control Ca’+-free state Na+-free state
AVP
Initial acidification
(nadir)
beak)
7.16 f 0.01 7.17 3z 0.02
7.12 f 0.01” 7.15 f 0.01”
7.23 + 0.01” 7.18 f 0.02
7.19 f 0.02
7.12 + 0.02”
7.17 + 0.03
1 X lo-’ Initial acidification
Basal
(nadir) Control Ca*+-free state Na+-free state
M
Basal
7.16 f 0.02 7.17 f 0.01 7.19 + 0.02
7.13 f 0.01” 7.16 + 0.02 7.11 zk 0.03”
Sustained alkalinization
M
ET-1 Sustained alkalinization
beak) 7.25 + 0.03”
7.17 + 0.01 7.18 f 0.03
Values are the mean k SEM (n = 6). “P < 0.01 us. the basal level. A similar
under Na+-free conditions. AVP-mobilized [Ca”+]i was enhanced under
study was performed
The 1 X lo-’
M
Na+-free conditions. The peak value of [Ca’+]i was significantly greater in the Na+-free state than in the controls (Fig. 1). In the ET-1 study, the peak level of [Ca’+]i in the Na’free condition was almost equal to that in the controls, but the sustained phase of [Ca’+]i was higher in the Na+-free state than in the controls (Fig. 2). In contrast, exposure to the Na+-free solution decreasedbasal [Na+]i to 2.5 + 0.2 mM and enormously reduced the responseof [Na+]i to AVP and ET1 (Figs. 1 and 2). Another vasoactive hormone, 1 X 10e7M angiotensin-II, also increased [Ca’+]i to 198.7 + 16.1 from 91.7 f 7.4 nM (n = 6; P < O.Ol), significantly less than that produced by 1 x lo-’ M AVP or ET-l. A mild elevation of [Na+]i was found after the addition of 1 X lo-’ M angiotensin-II. The increases in [Na+]i and [Ca’+]i induced by angiotensin-II were 100 times less than those caused by AVP or ET-l. Atria1 natriuretic peptide had no effect on [Ca’+]i and [Na+]i in glomerular mesangial cells (data not shown). Figure 3 depicts the change in pHi after 1 X lo-’ M AVF was added. The typical alteration in pHi is shown in the upper panel. After exposure of cells to 1 X lo-’ M AVP, the initial acidification occurred during the 3-min observation period, followed by sustained alkalinization. The basal pHi was 7.16 + 0.01 (n = 6), and the minimal and maximal pHi values were 7.12 f 0.01 and 7.23 + 0.01, respectively (P < 0.01 us. the basal pHi). In the Ca2+-free condition, both the initial acidification and the sustained alkalinization were markedly blunted (Fig. 3 and Table 3). Lastly, the pHi study was carried out in cells treated with the Na+-free condition. AVP (1 X 10e7M) caused an initial acidification (7.19 -r- 0.02
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AVP, ET, AND
1434
[Na’li
to 7.12 + 0.02) without any elevation of pHi in the sustained phase. Similar results were obtained with 1 X 10m7M ET-l (Table 3 and Fig. 4). Discussion It is well known that [Ca’+]i is the cellular second messenger for AVP, ET-l, and angiotensin-II in glomerular mesangial cells, in which mobilization is dependent on the breakdown of phosphatidylinositol(5-7, 24, 25). As for AVP, the Vi receptors are involved in the cellular action of AVP (3,5). The biological activities of AVP and ET-1 to mobilize [Ca’+] i are 100 times greater than that of angiotensin-II. This finding confirms the study of contraction of isolated mesenteric artery by Altura and Altura (26). The study, using Ca’+free medium, suggestedthe sourceof Ca2+.A [Ca’+]i transient produced by AVP or ET-l was one seventh lessin the Ca2+free condition than in the control condition, and the sustained elevation of [Ca”]i disappeared in the Ca*‘-free state. The early mobilization of [Ca”]i is derived from both intraand extracellular Ca*+, and the sustained phase depends to a great extent on extracellular Ca*+. Such a hormonally mobilized [Ca”]i results in contraction of glomerular mesangial cells (6, 27). However, little is known about what mechanismsare involved in the cell contraction. The present study further evaluated the interaction of [Ca’+]i and [Na+]i to explore the role of [Na+]i in the cellular action of vasoconstrictor hormones. As shown in Figs. 3 and 4, AVP and ET-1 produced an initial cellular acidification, followed by a sustained cellular alkalinization in glomerular mesangial cells. Such a biphasic pHi change is based on ion exchanges across the plasma membrane. An initial cellular acidification depends on the activation of Ca*+-ATPase. Vasoconstrictor hormones cause a huge increase in [Ca2+]i, which produces efflux of 45Ca2+ (6). During this period, Na+ gets into the cells in exchange for Ca’+. The Na+/Ca’+ exchange promotes an initial increase in [Na+]i in glomerular mesangial cells (9, 10). The Na’/Ca2+ exchange was supported by studies in Ca2+-free and Na+free conditions. The hormonally induced mobilization of [Ca’+]i was remarkably attenuated in the Ca2+-free state, providing the reduction in Ca2+-ATPase activity. The vasoconstrictor hormone-produced cellular acidification and [Na+]i mobilization were markedly reduced. In the Na’-free state AVP- or ET-l-mobilized [Ca”]i was significantly augmented, which depends on the reversed exchange of Ca2+ and Na’ (9,10,14,28,29). The basallevel of [Na+]i decreased to below 3 mM, and there was little increment in [Na+]i after the addition of AVP or ET-1 in glomerular mesangial cells, As the changes in [Na+]i and [Ca”+]i are of millimolar and nanomolar magnitudes, respectively, the dependence on Na+/Ca’+ exchange cannot be biochemically interpreted. Further study will be necessary to explore the exact mechanism. For example, the possibility that there is any difference in the turnover of Ca2+ efflux and Na+ influx or that the vasoconstrictor hormones directly open the Na+ channel of plasma membrane could be evaluated. Also, it is possibile that the Ca2+-free condition decreases[Ca’+]i mobilization by AVP or ET-l, which reduces cellular signal transduction
IN GLOMERULI
Endo. Voll31.
1992 No 3
and, thus, attenuates the cellular action of hormones. Next, we focused on the relation between the sustained cellular alkalinization and the mobilization of [Ca’+]i in response to vasoconstrictor hormones in cultured glomerular mesangial cells. The sustained cellular alkalinization disappeared under the Na+-free condition. The Na+-free state abolished three different vasoconstrictor hormone-induced increases in [Na+]i, suggesting that the source of Na+ is extracellular fluid. As demonstrated previously (9, lo), the Na+/H+ exchange participates the hormonally induced cellular alkalinization. The depletion of exchangable Na+ blocks the Na+/H+ exchange and results in the absenceof cellular alkalinization. Therefore, the change in [Na+]i is cIosely related to that in pHi. Under the Ca2+-free condition, the reduced mobilization of [Ca’+]i by vasoconstrictor hormones blunted the hormonally induced increase in [Na+]i and cellular alkalinization. [Ca’+]i acts as a cellular secondmessenger and activates the Na+/H+ exchanger in glomerular mesangial cells. In addition, the Na+/H+ exchanger is independent of protein kinase-C, because the inhibitor of protein kinase-C, l-(5-isoquinolinesulfonyl)2-methyl-piperazine dihydrochloride (H-7), did not affect the AVP-induced cellular alkalinization (our unpublished observation). In summary, we demonstrated that three vasoconstrictor hormones, AVP, ET-l, and angiotensin-II, increase[Na+]i in a dose-dependent manner in cultured rat glomerular mesangial cells. Such increasesin [Na+]i depend on their cellular second messenger[Ca”+]i. The hormonally mobilized [Na+]i is totally due to the cellular influx of Na+ from the extracellular space. The early and sustained phasesof [Na+]i mobilization are dependent on the activation of Na+/Ca*+ exchange and Na+/H’ exchange, respectively. The change in [Na+]i is closely related to that in pHi. Cellular alkalinization enhancescell contraction, cellular growth, etc., in glomerular mesangial cells (3, 8, 30). The present results indicate that AVP, ET-l, and angiotensin-II increase [Na+]i mediated via their cellular second messenger[Ca’+]i and that the change in [Na+]i is closely related to that in pHi in glomerular mesangialcells. References 1. Ausiello DA, KreisbergJI, Roy C, Karnovsky MJ 1980 Contraction of cultured rat glomerular cells of apparent mesangial origin after stimulation with angiotensin II and arginine vasopressin. J Clin Invest 65754-760 2. Tanaka T, Fujiwara Y, Orita Y, Sasaki E, Kitamura H, Abe H 1984 The functional characteristics of cultured rat mesangial cells. Jpn Circ J 48:1017-1029 3. Mene P, Simonson MS, Dunn MJ 1989 Physiology of the mesangial cell. Physiol Rev 69:1347-1424 4. Schor N, Ichikawa I, Brenner BM 1981 Mechanisms of action of various hormones and vasoactive substance on glomerular ultrafiltration in the rat. Kidney Int 20~442-451 5. Bonventre JV, Skorecki KL, Kreisberg JI, Cheung JY 1986 Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells. Am J Physiol251:F94-F102 H, Kim JK, Schrier RW 1988 AVP6. Takeda K, Meyer-Lehnert induced Ca’+ fluxes and contraction of rat glomerular mesangial cells. Am J Physiol 255:F142-F150 7 Okuda T, Yamashita N, Kurokawa K 1986 Angiotensin II and vasopressin stimulate calcium-activated chloride conductance in rat
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AVP, ET, AND mesangial
cells. J Clin Invest
[Na+]i IN GLOMERULI
781443-1448 1990 Effects of mitogens and cell proliferation, pH and Ca*+. Am J
8. Ganz MB, Perfetto MC, Boron WF other agents on rat mesangial Physiol259:F269-F278 9.
Cheung JY, Constantine JM 1987 Cytosolic
pH (pHc) regulation in mesanglal (GM) cells: effects of vasopressin and phorbol 13-acetate. Kidney Int 31:264 (Abstract) 10. Ganz MB, Boyarski G, Boron WF, Sterzel RB 1988 Effects of angiotensin II and vasopressin on intracellular pH of glomerular mesangial cells. Am J Physiol254:F787-F794 11. Hatori N, Fine BP, Nakamura A, Cragoe Jr EJ, Aviv A 1987 Angiotensin II effect on cytosolic pH in cultured rat vascular smooth muscle cells. J Biol Chem 262:5073-5078 12. Little PJ, Cragoe Jr EJ, Bobik A 1986 Na-H exchange is a major pathway for Na influx in rat vascular smooth muscle. Am J Physiol 251:C707-C712 13. Grinstein S, Rothstein A 1986 Mechanisms of regulation of the Na+/H+ exchange. J Membr Biol90:1-12 14. Okada K, Ishikawa S, Saito T 1990 Effect of vasopressin on Na+ kinetics in cultured rat vascular smooth muscle cells. Biochem Biophys Res Commun 173:224-230 15. Minta A, Tsien RY 1989 Fluorescent indicators for cytosolic sodium. J Biol Chem 264:19449-19457 16. Kreisberg JI, Karnovsky MJ 1983 Glomerular cells in culture. Kidney Int 23:439-447 vasopressin increases 17. Ishikawa S, Okada K, Saito T 1988 Arginme cellular free calcium concentration and adenosine 3’,5’-monophosphate production in rat renal papillary collecting tubule cells in culture. Endocrinoloev 123:1376-1384 Optimal concentration of cellular free 18. Ishikawa S, Saito F1990 calcium for AVP-induced CAMP in collecting tubules. Kidney Int 37:1060-1066 19. Grnkiewicz C, Poenie M, Tsien RY 1985 A new generation of Ca + indicators with greatly improved fluorescence properties. J Biol glomerular 12-myristate
Chem
260:3440-3450
S, Okada K, Saito T 1990 Prompt inhibition of arginine vasopressin-induced cellular adenosine 3’,5’-monophosphate production by extracellular sodium depletion in rat renal inner medullary collecting duct cells in culture. Endocrinology 127:560-566 21. Okada K, Tsai P, Caramel0 C, Schrier RW 1991 Effects of extraand intracellular pH on vascular action of arginine vasopressin. Am J Physiol260:F39-F45 22. Kruszynski M, Lammek B, Manning M, Seto J, Handar J, Sawyer WH 1980 [l-(Beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)2-(0-methyl)tyrosine]arginine vasopressin and [l-@etamercapto-beta,betacyclopentamethylenepropionic acid)JargInine vasopressin, two highly potent antagonists of the vasopressor response to arginine vasopressin. J Med Chem 23:364-368 23. Vavra I, Machova A, Holecek V, Cort JH, Zaoral M, Sorm F 1968 Effect of a synthetic analogue of vasopressin in animals and in patients with diabetes insipidus. Lancet 1:948-952 24. Simonson MS, Dunn MJ 1990 Endothelin. Pathways of transmembrane signaling. Hypertension [Suppl] 15:1-5-I-12 25. Simonson MS, Wann S, Mene P, Bubyak GR, Krester M, Dunn MJ 1989 Endothelin activates the phosphoinositide cascade in cultured elomerular mesantial cells. I Clin Invest 83708-712 smooth muscle and neuro26. Alturi MB, Altura BT”1977 Vascular hypophyseal hormones. Fed Proc 36:1853-1860 27. Meyer-Lehnert H, Schrier RW 1988 Cyclosporine A enhances vasopressin-induced Car+ mobilization and contraction in mesangial cells. Kidney Int 34:89-97 28. Nabel EG, Berk BC, Brock TA, Smith TW 1988 Na+-Ca2+ exchange in cultured vascular smooth muscle cells. Circ Res 62:486-493 Na+ dependence of changes 29. Smith JB, Smith L 1987 Extracellular in free Ca*+, a5Ca2+ efflux and total cell Ca*+ produced by angiotensin II in cultured arterial muscle cells. J Biol Chem 262:17455-17460 30. Ganz MB, Pekar SK, Perfetto MC, Sterzel RB 1988 Arginine vasopressin promotes growth of rat glomerular mesangial cells in culture. Am J Physiol255:F898-F906 20.
Ishikawa
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Vol. 131, No. 3 in U.S.A.
Printed
Society
Increases in Cellular Sodium Concentration by Arginine Vasopressin and Endothelin in Cultured Rat Glomerular Mesangial Cells* SAN-E
ISHIKAWA,
Division Japan
of
Endocrinology
KOJI
OKADA,
AND TOSHIKAZU
and Metabolism,
Department
SAITO of
Medicine,
ABSTRACT The present study was undertaken to determine whether arginine vasopressin (AVP), angiotensin-II, and endothelin (ET) increase the cellular sodium concentration ([Na+]i) in cultured rat glomerular mesangial cells. [Na+]i was measured using the fluorescence indicator dye sodium-binding benzofuran isophthalate. These three vasoconstrictor hormones increased cellular free calcium ([Ca*‘]i) and [Na+]i in a dosedependent manner ([Na+]i: basal, 11.5; 10e7 M AVP, 20.5; 10T7 M angiotensin-II, 13.8; and lo-’ M ET, 21.2 mM). The mobilization of [Caz+]i was faster than that of [Na+]i. The AVP-induced increase in [Na+ ]i was completely blunted by the potent V1 antagonist d(CH.&Tyr(Me)AVP. Vasoconstrictor hormones produced a biphasic cellular pH (pHi) change, characterized by a transient acidification, followed by a sustained alkalinization. The Ca*+-free condition mark-
V
ASOCONSTRICTOR hormones, such as arginine vasopressin (AVP) and angiotensin-II, produce cell contraction of glomerular mesangial cells (l-3). Schor et al. (4) demonstrated in a micropuncture study that the glomerular capillary ultrafiltration coefficient (Kf) is modified by AVP and angiotensin-II when injected intraarterially into rats. Recently, it has becomewell known that AVP and angiotensin-11act on glomerular mesangial cells mediated via their cellular second messenger,cellular free calcium ([Ca”]i) (57). In the absenceof bicarbonate, these hormones produce a biphasic celIular pH (pHi) change, characterized by a transient acidification, followed by a prolonged alkalinization that is both Na+ dependent and amiloride sensitive (8). The change in pHi is dependent on the Na+/H+ exchange across the plasma membrane, and the hormonally induced Na+/H+ exchange per seis controlled by [Ca’+]i (9, 10). Similar results were obtained in homologous cells of vascular smooth muscle (11-14). It is of great value to evaluate the role of cellular sodium ([Na+]i) in the cellular action of vasoconstrictor hormones in glomerular mesangial cells, becausecellular alkalinization is suggestedto depend on Na+ entry in exchange for H+ excretion, which is closely related to cell proliferation and cell contraction (3, 8). The fluorescence indicator dye Received January 28, 1992. Address requests for reprints to: San-e Ishikawa, M.D., Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School, 3311-l Yakushiji Minamikawachi-machi, Tochigi 329-04, Japan. * This work was supported by a grant from the Ministry of Education, Science, and Culture of Japan. The study will be presented at 9th International Congress of Endocrinology in Nice, France, August 1992.
Jichi Medical
School, Tochigi
329-04,
edly reduced AVP- and ET-induced increases in [Ca’+]i and [Na+]i and biphasic changes in pHi. In the Na+-free state, the hormonally mobilized [Ca”]i was significantly enhanced. Basal [Na+]i decreased to below 3 mM, and there was little increase in [Na+]i after the addition of vasoconstrictor hormones, suggesting that the source of [Na+]i is extracellular space. Only early acidification was obtained in the absence of a sustained alkalinization. The [Na+]i mobilization was closely related to the biphasic change in pHi. These results indicate that AVP, ET, and angiotensin-II increase [Na+]i in glomerular mesangial cells, and that the early mobilization of [Na+]i depends on Na+/Ca’+ exchange, and the sustained phase depends on Na+/H+ exchange. The hormonally mobilized [Ca”‘]i is essential for the activation of Na+/H+ exchange, and an increase in [Na+]i is suggested to play an important role in cellular alkalinization. (Endocrinology 131: 1429-1435,1992)
sodium-binding benzofuran isophthalate (SBFI) enables us to determine [Na’Ji in intact cells (15). The present study was undertaken to determine whether the vasoconstrictor hormones AVP, angiotensin-II, and endothelin (ET) affect [Na+]i in cultured rat glomerular mesangial cells. Whether Ca*+ and Na+ modulate the vasoconstrictor hormone-induced increasein [Na+]i was also examined. Materials and Methods Cell culture The experimental procedure was modified from the method of Kreisberg and Kamovsky (16). Male Sprague-Dawley rats, weighing 150-l 75 g, were used. Kidneys were removed under sterile conditions, and cortical tissues were cut away from the medulla. They were minced with physiological saline solution (PSS; 140 mu NaCl, 4.6 mu KCl, 1 mu MgC&, 2 mu CaC12, 10 mu glucose, and 10 mM HEPES, pH 7.4) by a sharp razor blade, and then passed through a series of steel sieves with decreasing pore sizes (60 and 200 mesh) with the glomeruli appearing on top of the 200-mesh sieve. ‘The minced renal cortical tissues were incubated with 3 ml collagenase (1 mg/ml; Worthington Biochemicals, Freehold, NJ) for 60 min at 37 C. After centrifuging the tubes at 500 x g for 4 n&at room temperature, the pellets we& resuspended with Dulbecco’s Modified Eagle’s Medium (Flow Laboratories, McLean, VA) supplemented with 20% fetal bovine serum, 100 U/ml penicillin, and lOb*pg/ml streptomycin. The dispersed glomeruli were harvested into 35 x IO-mm plastic dishes with the medium and kept in a humidified incubator at 37 C under 95% air and 5% COZ. After the culture cells were confluent; they were subcultured using CaZc- and Mg’+-free Hanks’ solution containing 0.025% trypsin and 0.01% EDTA. The dispersed cells were collected into culture tubes and centrifuged at 500 x g for 5 min at room temperature. The pellets were resuspended in Dulbecco’s Modified Eagle’s Medium containing 20%
1429
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AVP, ET, AND
1430 TABLE in cultured
1. AVP-
and ET-l-induced rat glomerular mesangial
increases cells
in [Ca”]i
[Ca*+]i (nM)
and [Na+]i
[Na+]i
(mM)
Control 1 X lo-’ M AVP lx10”~AVP 1 x 10-r M AVP
106.9 166.0 346.7 503.9
k + f f
6.1 (6) 6.6 (6)” 33.8 (6)” 30.8 (6)’
11.5 15.1 19.9 20.5
f f f f
0.4 0.4 1.0 1.0
(10) (6)” (6)” (10)’
Control 1 x 1O-g M ET-1 1 x lo-* M ET-1 1 x 10-r M ET-1
108.8 184.8 261.4 787.1
f f + +.
6.7 (6) 15.4 (6)” 35.1 (6)” 98.2 (6)”
9.9 16.3 21.2 18.7
+ f + f
0.8 2.3 2.0 1.1
(6) (6)b (6)” (6)”
Values are the mean parentheses. a P < 0.01 us. control. b P < 0.05 vs. control.
TABLE cultured
a SEM.
The
number
of observations
2. Effects of AVP analogs on [Ca”+]i rat glomerular mesa&al cells
[Na+li IN GLOMERULI
and [Na+]i
is in
in
Measurement
of
En&. Voll31.
1992 No 3
[Na+]i
The experimental procedure was similar to that described in our previous studies (14,20). The cells were rinsed twice with 1 ml PSS and loaded with 10 PM SBFI/AM (Molecular Probes, Inc., Eugene, OR) in a volume of 0.25 ml for 3 h at 37 C. SBFI/AM was dissolved in PSS containing 0.02% pluronic F-127, a nonionic detergent. After aspirating the SBFI/AM solution, the glass slides were rinsed and then placed in a 1 x l-cm quartz cuvette with the aid of a special holder in a fluorescence spectrophotometer (CAF-110). The dual wavelength excitation method for measurement of SBFI fluorescence was used. The fluorescence was monitored at 500 nm, with excitation wavelengths of 340 and 380 nm in the ratio mode, in a manner similar to the monitoring of the effect of calcium on fura-2. After a stable fluorescence signal was achieved, the effecters were added. Basal and hormone-stimulated peak values were determined. [Na+]i was calibrated by equilibrating [Na+]i with the extracellular Na+ concentration, using 1 X lo+’ M gramicidin. The reference standard solutions were made from appropriate mixtures of Na+ and KC solutions, based on the solution of PSS. The total concentrations of Na+ and K+ were adjusted to 135 mM. The [Na+]i was determined by the relation between the ratio and the authentic [Na’]i.
[Ca*+]i (nM) Basal Control 1 x 1o-B M d(CH&Tyr(Me)AVP 1 x lo-’ M dDAVP
1 X 1O-7M AVP 1 X lo-’
106.0 f 5.2
496.9 f 22.8”
107.6 + 7.9 104.6 f 14.7
106.1 f 6.4
M
dDAVP
104.5 + 14.7 [Na+]i (mM)
Basal Control
1 x lo-’
M
AVP 1 X lo-’ M dDAVP
11.7 f 0.5
21.5 f 0.8”
10.9 + 0.7 12.0 f 0.6
10.9 f 0.7
1Xlo”M
d(CH&Tyr(Me)AVP 1 X 10-r M dDAVP
12.8 + 1.2
Values are the mean f SEM (n = 6). a P < 0.01 us. the basal level. fetal bovine serum, penicillin, and streptomycin and cultured in a humidified incubator. The culture cells at 3-10 passages were subjected to the following studies on days 7-10 of the subculture. For measurements of [Ca”]i, [Na+]i and pHi, the cells were cultured on thin glass slides (13 mm in diameter; Matsunami Kogyo Co., Osaka,
Measurement
of
pHi
The experimental procedure was similar to that reported in our previous studies (20,21). The cells were rinsed twice with 1 ml PSS and loaded with 2 2’,7’-bis-(2-carboxymethyI)S(and W 6)carboxyfluorescein, acetoxymethyl ester (BCECF/AM; Molecular Probes), for 60 min at 37 C. BCECF/AM was dissolved in PSS. The complete intracellular hydrolysis of BCECF/AM to BCECF was judged by changes in the excitation and emission spectra. The fluorescence was monitored at 530 nm, with excitation wavelengths of 450 and 500 nm in the ratio mode. After measurement of the basal pHi level, the effecters were added. Since autofluorescence of unloaded cells was less than 1% of the total fluorescence of BCECF-loaded cells, autofluorescence did not affect the pHi calculations. The fluorescent signal was calibrated at several pH values (6.6, 7.0, and 7.4) in KC1 solution (140 nut KCl, 4.6 mu NaCl, 1 ~I-IM MgC&, 2 nut CaC&, 10 mu glucose, and 10 mu HEPES) containing the K+/H+ ionophore nigericin (10 pg/ml).
Statktics All values of [Ca’+]i, [Na+]i, and pHi were analyzed by an analysis of multiple variance, using Scheffe’s method and Student’s t test. P < 0.05 was considered significant.
Japan). Measurement
of
[Ca”+li
The experimental procedure was similar to that used in our previous studies (17, 18). The cells were rinsed twice with 1 ml PSS and loaded with 5 PM fura-Z/AM (Dojin Biochemicals, Kumamoto, Japan) in a volume of 250 ~1 for 60 min at 37 C. After aspiration of the fura-Z/AM solution, the glass slides were rinsed and then placed in a 1 X l-cm quartz cuvette with the aid of a special holder in a fluorescence spectrophotometer (CAF-110, Japan Spectroscopic Co., Tokyo, Japan). The dual wavelengh excitation method for measurement of furafluorescence was used. The fluorescence was monitored at 500 nm, with excitation wavelengths of 340 and 380 nm in the ratio mode. The effecters were added after a stable fluorescence signal (R) was achieved. AVP (grade VI, Sigma, St. Louis, MO), ET-1 (Peptides Institute, Osaka, Japan), angiotensin-II (Peptides Institute), and atial natriuretic peptide (Peptides Institute) were used. From the ratio of fluorescence at 340 and 380 nm, the [Ca’+]i was determined, as described by Grynkiewicz et al. (19), using the following expression: [Ca’+]i (nM) = & X [(R - R,,,&R,,,,, R)] x 8, where R is the ratio of fluorescence of the sample at 340 and 380 nm, and R max and R,, were determined by treating the cells with 5 X 10m5 M digitonin and I X 10e2 M MnC12, respectively. The term @ is the ratio of fluorescence of furaat 380 nm in zero and saturating Ca’+ concentrations. K,, is the dissociation constant of furafor Ca’+, assumed to be 224 nM at 37 C (19).
Results AVP caused an increase in [Ca’+]i in a dose-dependent manner in glomerular mesangial cells in culture (Table 1). AVP (1 X lo-’ M) increased [Ca’+]i to 503.9 + 30.8 from 106.9 f 6.1 nM (P < 0.01). The [Ca’+]i promptly rose and reached a peak level a few seconds after the addition of AVP. The sustained phase of [Ca’+]i gradually decreased, but remained high above the basal level during the 15min observation period. As shown in Table 1, AVP alsoincreased [Na’]i in glomerular mesangial cells. Such an increase was obtained with AVP in a dose-dependent manner (Table 1). An increase in [Na+]i reached a peak level approximately 1 min after exposure to AVP, followed by sustainedelevation of [Na+]i, which lasted at least 15 min. Quite similar results were obtained with ET-l (Table 1). It was clear that the AVPor ET-l-induced peak value of [Na+]i appeared later than that of [Ca’+]i. Table 2 shows the effects of AVP analogs on AVP-mobilized [Ca”+]i or [Na+]i to elucidate whether Vi or V2 receptors
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AVP, ET, AND [Na+]i IN GLOMERULI
Control
[Na+Jr
1431
6
.
p+]l
Control
Ml) 800
(mM) 20 -
.--I
-
---
400 200 looI
OmtvlCCa*~~~..
OmM [3=a*+lo .
,/ 50 -
16
L
lo-
10 i I
500 -
OmMCNa+lo v
I
:
\yy+
400 -
200 ----y-----!
--.-I I I
100 -
I
5min /
1 min The effects of Ca’+- and Na+-free conditions on 1 X lo-’ M AVP-induced increases in [Na+]i (A) and [Ca*+]i (B) in cultured rat glomerular mesa&al cells. Upper panel, Control; middle panel, Ca*+-free condition; lower panel, Na+-free condition. FIG.
1.
are involved in the cellular action of AVP in glomerular mesangial cells. The selective and potent AVP V, antagonist
[1-(/3-mercapto-/3,/3-cyclopentamethylenepropionic acid)2(0-methyl)tyrosine]AVP[ [l X 10m6M; d(CH&Tyr(Me)AVP] (22) completely blocked the AVP-induced increasesin [Ca’+] i and [Na+]i. In contrast, the Vz agonist l-deamino-8-o-AVP (1 x lo-’ M; dDAVP) (23) did not affect either [Ca’+]i or [Na+]i. Next, we examined the hormonally mobilized [Ca’+]i and [Na+]i under Ca*+-free conditions to clarify whether cellular free Ca*+mobilization is essentialfor the hormonally induced increase in [Na+]i. Cells were preincubated for lo-15 min with the Ca*+-free medium containing 1 mu EGTA. As
shown in Fig. 1, in the Ca*+-free state, the 1 X lo-’ M AVPmobilized [Ca’+]i was markedly diminished as [Ca’+]i increased from 29.1 f 4.1 to 76.7 + 11.4 nM (P < 0.01). The sustained elevation of [Ca’+]i disappeared. Similarly, the mobilization of [Na+]i by 1 X lo-’ M AVP was also remarkably blunted. [Na+]i was elevated to only 1.8 ITLM(mean value), which was significantly less than the value in the control group. Similar results were obtained with 1 X lo-’ M ET-l (Fig. 2). In addition, Ca*+ ionophore ionomycin (1 x 10m6M) increased [Na+]i from 10.2 f 1.0 to 24.1 + 2.3 mu (n = 6; P < 0.01). Such an increase by ionomycin was totally absent in cells pretreated with the Ca*+-free medium containing 1 mu EGTA.
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1432
AVP,
A
ET,
AND
[Na+]i
B
Control
~8?J126 (mM)20.
Endo. 1992 Voll31 l No 3
IN GLOMERULI
800
l
400 -
15 lo-
200 5loo20-
16lo-
200 -
5loo-
OmM ma+ Jo 50 -
lo-
! !
5.
10.
/ I
O-
5min 400 -
200 -
100.
FIG. 2. The effects of Ca*+- and Na+-free conditions on 1 X low7 M ET-l-induced increases in [Na+]i (A) and [Ca*+]i (B) in cultured rat glomerular mesangial cells. Upper panel, Control; middle panel, Ca*+-free condition; lower panel, Na+-free condition.
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AVP, ET, AND Fontrot
[Na+]i IN GLOMERULI
1433
PHI
co/
Control i ;
7.2 -
: ,..,..
,.. :
___.i. :. ._L
:.
:
I... .!.. 1::.
;. ..j.:.f. !
i . . .._ j-..
,I,_
;
i..;..
j;
j
;...
T.‘.
7.2. 7.2 -
7.1OmMCCa*+]o
7.2 -
I :
,.
OmM 1 -i--T-~-~-1
1
7.1.
HatJO
;.. :. .:
I [
:
:
:
lmin FIG. 4. Changes in pHi after the addition of 1 x lo-? M ET-l in cultured rat glomerular mesangial cells. Upper panel, Control; lower panel, Ca*+-free condition.
lmin
3. Changes in pHi after the addition of 1 x 10T7 M AVP in cultured rat glomerular mesangial cells. Upper panel, Control; middle panel, Ca*+-free state; lower panel, Na+-free state. FIG.
TABLE 3. The changes in pHi in response to AVP and ET-l in cultured rat glomerular mesangial cells pHi 1 X lo-’
Control Ca’+-free state Na+-free state
AVP
Initial acidification
(nadir)
beak)
7.16 f 0.01 7.17 3z 0.02
7.12 f 0.01” 7.15 f 0.01”
7.23 + 0.01” 7.18 f 0.02
7.19 f 0.02
7.12 + 0.02”
7.17 + 0.03
1 X lo-’ Initial acidification
Basal
(nadir) Control Ca*+-free state Na+-free state
M
Basal
7.16 f 0.02 7.17 f 0.01 7.19 + 0.02
7.13 f 0.01” 7.16 + 0.02 7.11 zk 0.03”
Sustained alkalinization
M
ET-1 Sustained alkalinization
beak) 7.25 + 0.03”
7.17 + 0.01 7.18 f 0.03
Values are the mean k SEM (n = 6). “P < 0.01 us. the basal level. A similar
under Na+-free conditions. AVP-mobilized [Ca”+]i was enhanced under
study was performed
The 1 X lo-’
M
Na+-free conditions. The peak value of [Ca’+]i was significantly greater in the Na+-free state than in the controls (Fig. 1). In the ET-1 study, the peak level of [Ca’+]i in the Na’free condition was almost equal to that in the controls, but the sustained phase of [Ca’+]i was higher in the Na+-free state than in the controls (Fig. 2). In contrast, exposure to the Na+-free solution decreasedbasal [Na+]i to 2.5 + 0.2 mM and enormously reduced the responseof [Na+]i to AVP and ET1 (Figs. 1 and 2). Another vasoactive hormone, 1 X 10e7M angiotensin-II, also increased [Ca’+]i to 198.7 + 16.1 from 91.7 f 7.4 nM (n = 6; P < O.Ol), significantly less than that produced by 1 x lo-’ M AVP or ET-l. A mild elevation of [Na+]i was found after the addition of 1 X lo-’ M angiotensin-II. The increases in [Na+]i and [Ca’+]i induced by angiotensin-II were 100 times less than those caused by AVP or ET-l. Atria1 natriuretic peptide had no effect on [Ca’+]i and [Na+]i in glomerular mesangial cells (data not shown). Figure 3 depicts the change in pHi after 1 X lo-’ M AVF was added. The typical alteration in pHi is shown in the upper panel. After exposure of cells to 1 X lo-’ M AVP, the initial acidification occurred during the 3-min observation period, followed by sustained alkalinization. The basal pHi was 7.16 + 0.01 (n = 6), and the minimal and maximal pHi values were 7.12 f 0.01 and 7.23 + 0.01, respectively (P < 0.01 us. the basal pHi). In the Ca2+-free condition, both the initial acidification and the sustained alkalinization were markedly blunted (Fig. 3 and Table 3). Lastly, the pHi study was carried out in cells treated with the Na+-free condition. AVP (1 X 10e7M) caused an initial acidification (7.19 -r- 0.02
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AVP, ET, AND
1434
[Na’li
to 7.12 + 0.02) without any elevation of pHi in the sustained phase. Similar results were obtained with 1 X 10m7M ET-l (Table 3 and Fig. 4). Discussion It is well known that [Ca’+]i is the cellular second messenger for AVP, ET-l, and angiotensin-II in glomerular mesangial cells, in which mobilization is dependent on the breakdown of phosphatidylinositol(5-7, 24, 25). As for AVP, the Vi receptors are involved in the cellular action of AVP (3,5). The biological activities of AVP and ET-1 to mobilize [Ca’+] i are 100 times greater than that of angiotensin-II. This finding confirms the study of contraction of isolated mesenteric artery by Altura and Altura (26). The study, using Ca’+free medium, suggestedthe sourceof Ca2+.A [Ca’+]i transient produced by AVP or ET-l was one seventh lessin the Ca2+free condition than in the control condition, and the sustained elevation of [Ca”]i disappeared in the Ca*‘-free state. The early mobilization of [Ca”]i is derived from both intraand extracellular Ca*+, and the sustained phase depends to a great extent on extracellular Ca*+. Such a hormonally mobilized [Ca”]i results in contraction of glomerular mesangial cells (6, 27). However, little is known about what mechanismsare involved in the cell contraction. The present study further evaluated the interaction of [Ca’+]i and [Na+]i to explore the role of [Na+]i in the cellular action of vasoconstrictor hormones. As shown in Figs. 3 and 4, AVP and ET-1 produced an initial cellular acidification, followed by a sustained cellular alkalinization in glomerular mesangial cells. Such a biphasic pHi change is based on ion exchanges across the plasma membrane. An initial cellular acidification depends on the activation of Ca*+-ATPase. Vasoconstrictor hormones cause a huge increase in [Ca2+]i, which produces efflux of 45Ca2+ (6). During this period, Na+ gets into the cells in exchange for Ca’+. The Na+/Ca’+ exchange promotes an initial increase in [Na+]i in glomerular mesangial cells (9, 10). The Na’/Ca2+ exchange was supported by studies in Ca2+-free and Na+free conditions. The hormonally induced mobilization of [Ca’+]i was remarkably attenuated in the Ca2+-free state, providing the reduction in Ca2+-ATPase activity. The vasoconstrictor hormone-produced cellular acidification and [Na+]i mobilization were markedly reduced. In the Na’-free state AVP- or ET-l-mobilized [Ca”]i was significantly augmented, which depends on the reversed exchange of Ca2+ and Na’ (9,10,14,28,29). The basallevel of [Na+]i decreased to below 3 mM, and there was little increment in [Na+]i after the addition of AVP or ET-1 in glomerular mesangial cells, As the changes in [Na+]i and [Ca”+]i are of millimolar and nanomolar magnitudes, respectively, the dependence on Na+/Ca’+ exchange cannot be biochemically interpreted. Further study will be necessary to explore the exact mechanism. For example, the possibility that there is any difference in the turnover of Ca2+ efflux and Na+ influx or that the vasoconstrictor hormones directly open the Na+ channel of plasma membrane could be evaluated. Also, it is possibile that the Ca2+-free condition decreases[Ca’+]i mobilization by AVP or ET-l, which reduces cellular signal transduction
IN GLOMERULI
Endo. Voll31.
1992 No 3
and, thus, attenuates the cellular action of hormones. Next, we focused on the relation between the sustained cellular alkalinization and the mobilization of [Ca’+]i in response to vasoconstrictor hormones in cultured glomerular mesangial cells. The sustained cellular alkalinization disappeared under the Na+-free condition. The Na+-free state abolished three different vasoconstrictor hormone-induced increases in [Na+]i, suggesting that the source of Na+ is extracellular fluid. As demonstrated previously (9, lo), the Na+/H+ exchange participates the hormonally induced cellular alkalinization. The depletion of exchangable Na+ blocks the Na+/H+ exchange and results in the absenceof cellular alkalinization. Therefore, the change in [Na+]i is cIosely related to that in pHi. Under the Ca2+-free condition, the reduced mobilization of [Ca’+]i by vasoconstrictor hormones blunted the hormonally induced increase in [Na+]i and cellular alkalinization. [Ca’+]i acts as a cellular secondmessenger and activates the Na+/H+ exchanger in glomerular mesangial cells. In addition, the Na+/H+ exchanger is independent of protein kinase-C, because the inhibitor of protein kinase-C, l-(5-isoquinolinesulfonyl)2-methyl-piperazine dihydrochloride (H-7), did not affect the AVP-induced cellular alkalinization (our unpublished observation). In summary, we demonstrated that three vasoconstrictor hormones, AVP, ET-l, and angiotensin-II, increase[Na+]i in a dose-dependent manner in cultured rat glomerular mesangial cells. Such increasesin [Na+]i depend on their cellular second messenger[Ca”+]i. The hormonally mobilized [Na+]i is totally due to the cellular influx of Na+ from the extracellular space. The early and sustained phasesof [Na+]i mobilization are dependent on the activation of Na+/Ca*+ exchange and Na+/H’ exchange, respectively. The change in [Na+]i is closely related to that in pHi. Cellular alkalinization enhancescell contraction, cellular growth, etc., in glomerular mesangial cells (3, 8, 30). The present results indicate that AVP, ET-l, and angiotensin-II increase [Na+]i mediated via their cellular second messenger[Ca’+]i and that the change in [Na+]i is closely related to that in pHi in glomerular mesangialcells. References 1. Ausiello DA, KreisbergJI, Roy C, Karnovsky MJ 1980 Contraction of cultured rat glomerular cells of apparent mesangial origin after stimulation with angiotensin II and arginine vasopressin. J Clin Invest 65754-760 2. Tanaka T, Fujiwara Y, Orita Y, Sasaki E, Kitamura H, Abe H 1984 The functional characteristics of cultured rat mesangial cells. Jpn Circ J 48:1017-1029 3. Mene P, Simonson MS, Dunn MJ 1989 Physiology of the mesangial cell. Physiol Rev 69:1347-1424 4. Schor N, Ichikawa I, Brenner BM 1981 Mechanisms of action of various hormones and vasoactive substance on glomerular ultrafiltration in the rat. Kidney Int 20~442-451 5. Bonventre JV, Skorecki KL, Kreisberg JI, Cheung JY 1986 Vasopressin increases cytosolic free calcium concentration in glomerular mesangial cells. Am J Physiol251:F94-F102 H, Kim JK, Schrier RW 1988 AVP6. Takeda K, Meyer-Lehnert induced Ca’+ fluxes and contraction of rat glomerular mesangial cells. Am J Physiol 255:F142-F150 7 Okuda T, Yamashita N, Kurokawa K 1986 Angiotensin II and vasopressin stimulate calcium-activated chloride conductance in rat
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AVP, ET, AND mesangial
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S, Okada K, Saito T 1990 Prompt inhibition of arginine vasopressin-induced cellular adenosine 3’,5’-monophosphate production by extracellular sodium depletion in rat renal inner medullary collecting duct cells in culture. Endocrinology 127:560-566 21. Okada K, Tsai P, Caramel0 C, Schrier RW 1991 Effects of extraand intracellular pH on vascular action of arginine vasopressin. Am J Physiol260:F39-F45 22. Kruszynski M, Lammek B, Manning M, Seto J, Handar J, Sawyer WH 1980 [l-(Beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)2-(0-methyl)tyrosine]arginine vasopressin and [l-@etamercapto-beta,betacyclopentamethylenepropionic acid)JargInine vasopressin, two highly potent antagonists of the vasopressor response to arginine vasopressin. J Med Chem 23:364-368 23. Vavra I, Machova A, Holecek V, Cort JH, Zaoral M, Sorm F 1968 Effect of a synthetic analogue of vasopressin in animals and in patients with diabetes insipidus. Lancet 1:948-952 24. Simonson MS, Dunn MJ 1990 Endothelin. Pathways of transmembrane signaling. Hypertension [Suppl] 15:1-5-I-12 25. Simonson MS, Wann S, Mene P, Bubyak GR, Krester M, Dunn MJ 1989 Endothelin activates the phosphoinositide cascade in cultured elomerular mesantial cells. I Clin Invest 83708-712 smooth muscle and neuro26. Alturi MB, Altura BT”1977 Vascular hypophyseal hormones. Fed Proc 36:1853-1860 27. Meyer-Lehnert H, Schrier RW 1988 Cyclosporine A enhances vasopressin-induced Car+ mobilization and contraction in mesangial cells. Kidney Int 34:89-97 28. Nabel EG, Berk BC, Brock TA, Smith TW 1988 Na+-Ca2+ exchange in cultured vascular smooth muscle cells. Circ Res 62:486-493 Na+ dependence of changes 29. Smith JB, Smith L 1987 Extracellular in free Ca*+, a5Ca2+ efflux and total cell Ca*+ produced by angiotensin II in cultured arterial muscle cells. J Biol Chem 262:17455-17460 30. Ganz MB, Pekar SK, Perfetto MC, Sterzel RB 1988 Arginine vasopressin promotes growth of rat glomerular mesangial cells in culture. Am J Physiol255:F898-F906 20.
Ishikawa
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