Department of Physiology, University of Würzburg, 97070 Würzburg, Germany
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ABSTRACT |
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The mineralocorticoid aldosterone is the most important hormone for the regulation of Na+ and K+ homeostasis in mammals and is thereby involved in the regulation of extracellular volume and blood pressure. Because aldosterone is a steroid hormone, the classical way of action involves transcription, translation, and protein synthesis. We previously reported a rapid, nongenomic, and Zn2+-sensitive action of aldosterone on Na+/H+ exchange in renal epithelial [Madin-Darby canine kidney (MDCK)] cells (M. Gekle, N. Golenhofen, H. Oberleithner, and S. Silbernagl. Proc. Natl. Acad. Sci. 93: 10500-10504, 1996). Here we show that, in the absence of Na+ (i.e., with inactive Na+/H+ exchange), aldosterone induces a membrane potential-dependent and Zn2+-sensitive cytoplasmic acidification in MDCK cells within 2-4 min. This aldosterone-induced activation of a proton conductance is insensitive to the inhibitor of the classical genomic pathway, spironolactone. Furthermore, the inhibitor of serine/threonine kinases and staurosporine, as well as the specific inhibitor of protein kinase C (PKC), calphostin C, prevented proton conductance activation. Activation of PKC by phorbol esters mimicked the effect of aldosterone. Furthermore, preincubation of the cells with pertussis toxin reduced the effect of aldosterone significantly. We propose a new nongenomic mechanism of action for aldosterone, independently of the intracellular type 1 mineralocorticoid receptor: G protein-dependent stimulation of PKC by aldosterone leads to the activation of a plasma membrane proton conductance that enhances the activity of Na+/H+ exchange. This rapid nongenomic effect could explain the observation that aldosterone may alter renal Na+ and K+ excretion within 5-10 min.
Madin-Darby canine kidney cells; cytoplasmic pH; zinc ion
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INTRODUCTION |
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THE CLASSICAL GENOMIC MECHANISM of steroid hormone action involves binding to intracellular receptors, transcription, and translation. Thus the aldosterone-induced response following genomic activation should be preceded by a latent period in the range of several minutes to hours. Yet, it was shown as long as four decades ago that there is a rapid action of aldosterone, with a latency of ~5 min, on renal electrolyte excretion (10). This and other findings (25) indicate that aldosterone is responsible not only for chronic but also for acute regulation of water and electrolyte balance. However, the underlying mechanisms are not completely understood.
Aldosterone can induce a so-called early response by interfering with mechanisms of regulation of intracellular pH or Ca2+ (7, 11, 20, 24), intracellular generation of inositol 1,4,5-trisphosphate (IP3) (3), and protein kinase C (PKC) activation (7) with a latency of <15 min. These evidently nongenomic actions of aldosterone are thought to be mediated by a plasma membrane receptor with high affinity for aldosterone but low affinity for glucocorticoids (26). Former studies also revealed that aldosterone acts within several minutes on plasma membrane K+ conductance of different cells (20, 23). One important target of aldosterone during this early response is plasma membrane Na+/H+ exchange (20, 24). Recently, we have shown that an elevation of cytosolic Ca2+ serves as a second messenger in the signal transduction pathway initiated by aldosterone for rapid Na+/H+ exchange activation in Madin-Darby canine kidney C11 (MDCK-C11) cells (11). This cell clone shares many properties with renal collecting duct epithelium, including genomic sensitivity to aldosterone (12, 30). However, the observed aldosterone-induced activation of Na+/H+ exchange was also a Zn2+-sensitive mechanism (11), indicating that there was crucial cross talk between Na+/H+ exchange and plasma membrane proton conductance. Proton conductance would then be a prerequisite for the proper function and activation of Na+/H+ exchange, as also proposed in macrophages (6). In the present study we investigated the nature of the aldosterone-induced Zn2+-sensitive pH changes in detail and identified PKC as a downstream effector for aldosterone.
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METHODS |
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Cell culture. MDCK cells were obtained at passage 53 from the American Type Culture Collection. In this study we used a subtype denominated C11 (MDCK-C11) that has been cloned recently in our laboratory (12). The C11 subtype resembles the intercalated cells of the collecting duct. This cell type has been shown to be aldosterone sensitive in the classical way (12). Cells were seeded in plastic culture dishes (growth area = 75 cm2; Nunc, Wiesbaden, Germany) in 10 ml of minimum essential medium with Earle's salts, nonessential amino acids, and L-glutamine (MEM medium; Biochrom, Berlin, Germany) and were cultured under standard cell culture conditions (37°C, 5% CO2). The MEM medium was supplemented with 10% fetal calf serum (Biochrom) and 26 mM NaHCO3. The medium was changed three times a week and the cells were split once a week. For the measurement of cytoplasmic pH, cells were seeded on thin glass microscope coverslips pretreated with poly(L-Lys) (0.1 g/l; Serva, Heidelberg, Germany). All experiments were performed at 37°C under bicarbonate-free conditions.
Measurements of intracellular pH. Intracellular pH of single cells was determined using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF; Molecular Probes, Eugene, OR) as described elsewhere (9, 22). In brief, cells were incubated with MEM containing BCECF at a final concentration of 3 µM for 15 min. The coverslips were then rinsed four times with superfusion solution to remove the BCECF-containing medium. The coverslips were transferred to the stage of the microscope described above. The excitation light source was a 100-W mercury lamp. The excitation wavelengths were 460 and 488 nm, and the emitted light was filtered through a band-pass filter (515-565 nm). The data acquisition rate was one fluorescence intensity ratio (488/460 nm) every 2 s. Images were digitized on-line using video-imaging software (Attofluor; Zeiss, Oberkochen, Germany). After background subtraction, fluorescence intensity ratios (488/460 nm) were calculated. Calibration was performed after each experiment by the nigericin (Sigma, St. Louis, MO) technique (22), using at least three calibration solutions in the range from pH 6.8 to 7.8. The calibration solutions contained 115 mM KCl and 30 mM NaCl.
Cytoplasmic acidification was achieved by exposing the cells for 3-5 min to 20 mM NH4Cl in the buffer described below. The amount of N-methyl-D-glucamine (NMDG)-Cl was reduced to maintain constant osmolality. Removal of NH4Cl led to an instantaneous acidification.
Materials. Aldosterone, staurosporine, phorbol 12-myristate 13-acetate (PMA), ZnCl2, spironolactone, valinomycin, calphostin C, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7), pertussis toxin (PTX), cycloheximide, and nigericin were purchased from Sigma (Munich, Germany). The acetoxymethyl ester of fura 2 and BCECF were obtained from Molecular Probes. Bafilomycin A1 was purchased from Dr. Altendorf (Osnabrück, Germany), and SCH-28080 was kindly provided by Schering (Berlin, Germany). All other applied chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany).
Ringer solution was composed of (in mM) 141.0 NaCl, 4.0 KCl, 1.0 CaCl2, 1.0 MgCl2, 3.2 Na2HPO4, 0.8 NaH2PO4, and 5 glucose (pH 7.4 at 37°C). In Na+-free solutions, Na+ was replaced by NMDG. In high-K+ solutions, NaCl was replaced by KCl.
Statistics. The data are presented as means ± SE, and n represents the number of cells examined. Four to five cells were examined per coverslip. Significance of difference was tested by paired or unpaired t-test as applicable. Differences were considered significant if P < 0.05.
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RESULTS |
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Aldosterone induces a
Zn2+-sensitive
but spironolactone-insensitive acidification.
Cytoplasmic pH of MDCK-C11 cells in the presence of
Na+ was 7.22 ± 0.01 (n = 97). Removal of
Na+ led to a cytoplasmic
acidification to 6.99 ± 0.02 (n = 67; P < 0.05). In the absence of
Na+,
108 M aldosterone induced a
further cytoplasmic acidification in MDCK-C11 cells by 0.16 ± 0.03 pH units (n = 60;
P < 0.05) to pH 6.83 ± 0.02 (Fig. 1, A
and B). In the presence of
10
4 M
Zn2+, an inhibitor of proton
conductance (16), the aldosterone-induced acidification
was only 0.04 ± 0.01 pH units (n = 30; P < 0.05 vs. aldosterone alone;
Fig. 1, A and
B). Spironolactone
(10
5 M), the inhibitor of
the genomic pathway, did not prevent the aldosterone-induced
acidification (
0.17 ± 0.03 pH units,
n = 25; Fig.
1B). Inhibition of protein synthesis
by 10
5 M cycloheximide (20 min preincubation) did not prevent the aldosterone-induced acidification as shown in Fig. 1B.
Hence aldosterone induced a rapid and nongenomic activation of plasma
membrane proton conductance, independently of type 1 mineralocorticoid
receptors.
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DISCUSSION |
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Over the past 10 years several studies showed a rapid action of aldosterone on electrolyte handling in different cell types (11, 23, 27, 29). This rapid effect of aldosterone has been shown in animal as well as in human cells, emphasizing its importance also for human physiology (3, 8, 27). The onset of this rapid action was within 1-2 min and could not be prevented by inhibitors of transcription or translation. Thus there is a nongenomic pathway with high affinity for aldosterone and low affinity for hydrocortisone in different cell types (7, 11, 25). Furthermore, this nongenomic pathway is operative in classical aldosterone target cells, like collecting duct cells (11) or colon cells (7). An important cellular target of this rapid, nongenomic action of aldosterone is the Na+/H+ exchanger (11, 23). The resulting changes in intracellular pH modulate plasma membrane K+ and Na+ conductance and thus, e.g., transepithelial electrolyte transport (13, 23). The question arising now is about the nature of the downstream effectors of aldosterone for Na+/H+ exchanger activation. In vascular smooth muscle cells and in collecting duct-derived cells it has been shown that elevation of cytoplasmic Ca2+ is involved in the rapid action of aldosterone (11, 29). Aldosterone has also been shown to increase cytoplasmic Ca2+ in colonic epithelial cells (8). Furthermore, in vascular smooth muscle cells and in colonic epithelial cells, aldosterone activated PKC (4, 7). Thus Ca2+ and PKC are downstream effectors of aldosterone. However, it has also been shown that aldosterone led to a Zn2+-sensitive acidification and that Zn2+ could inhibit the aldosterone-induced rapid activation of Na+/H+ exchange in MDCK-C11 cells (11). Thus the activation of a plasma membrane proton conductance (16) seems to be another downstream effector of aldosterone.
In this study we show the membrane potential-dependent and Zn2+-sensitive acidification of MDCK-C11 cells by aldosterone. Furthermore, reversing the driving force for protons from inwardly directed to outwardly directed also reversed the effect of aldosterone, which now led to alkalinization. Because all other proton-extruding transporters were blocked in this experiment, the aldosterone-induced alkalinization was also due to the activation of a plasma proton conductance. The classical type 1 mineralocorticoid receptor is not involved in this rapid action of aldosterone as shown by the lack of effect of spironolactone. Another receptor, mediating the rapid effects of aldosterone, has not yet been identified. However, Wehling et al. (26) presented evidence for specific aldosterone binding sites in the plasma membrane of lymphocytes. These binding sites possibly represent a plasma membrane receptor for aldosterone that mediates the rapid effects.
We also investigated possible mediators for the proton conductance activation. As already reported earlier, Ca2+ seems not to be involved (11). Activation of proton conductance by PKC (2) and by tyrosine kinase (18) has been demonstrated. Our data suggest that the aldosterone-induced activation of a proton conductance is mediated by PKC, because staurosporine and calphostin C inhibited the effect of aldosterone to a similar extent as Zn2+. Furthermore, activation of PKC by PMA also induced a Zn2+-sensitive acidification in the absence of Na+. Thus PKC seems to be a downstream effector of aldosterone for proton conductance activation. These results give additional support to the hypothesis of a novel rapid pathway for aldosterone action via PKC activation (4, 7).
There are also data indicating the involvement of calmodulin when the aldosterone-induced Na+/H+ exchange activation was studied 60 min after hormone application (21). The lack of effect of the calmodulin antagonist W-7 shows that calmodulin does not act as a downstream effector for the rapid activation of proton conductance. This is in good agreement with the observation that aldosterone-induced cytoplasmic acidification was independent of the aldosterone-evoked increase of cytoplasmic Ca2+ concentration.
On the basis of recent publications and the data of the present study, it is evident that aldosterone exerts a rapid and nongenomic action in classical steroid hormone target epithelia, resulting in acute modulation of transmembrane and possibly transepithelial electrolyte transport (5, 7, 11, 23). Na+/H+ exchange activity seems to be the final target of aldosterone, and the subsequent changes in cytoplasmic pH serve as modulators of plasma membrane ion channels (13, 19, 23). Rapid activation of Na+/H+ exchange by aldosterone involves cytoplasmic Ca2+, PKC, and a Zn2+-sensitive plasma membrane proton conductance (4, 11). Beginning with the results of the present study, we propose that the action of PKC might not be a direct one on the exchanger protein but might be indirect via the activation of a proton conductance. Accordingly, aldosterone possibly binds to a plasma membrane receptor (26), thereby stimulating PKC, possibly via an activation of phospholipase C (4). This activation involves at least partially PTX-sensitive heterotrimeric G proteins, as was the case in vascular smooth muscle cells where PTX inhibited an aldosterone-induced increase of IP3 and cytoplasmic Ca2+ concentration (25, 29). Subsequent stimulation of PKC leads to the activation of a plasma membrane proton conductance, as also shown for direct PKC stimulation in enterocytes (2). Subsequently, the activity of Na+/H+ exchange increases (11). We cannot exclude that PKC also acts directly on the exchanger protein. However, its interaction with the proton conductance seems to be a requirement for Na+/H+ exchange activation because Zn2+ inhibited this activation.
The exact nature of the link between proton conductance and Na+/H+ exchange has still to be elucidated. However, cross talk between these two systems has also been described in other preparations (6). Recently, a dissociation of Na+/H+ exchange activity and bulk cytoplasmic pH has been described for rat colon cells, and a proton-tight submembrane domain was postulated (5). Such a compartment would allow the proton conductance to create an acidic microenvironment in the vicinity of the exchanger and thereby keep pH within the working range, despite acid extrusion (11, 14). In this case the Na+/H+ exchanger would serve to control cell volume: because the proton conductance "recycles" the protons extruded, the exchanger imports Na+ without limitation by cytoplasmic pH, leading to volume increase. In human lymphocytes aldosterone induces such a volume increase via activation of Na+/H+ exchange (28). Furthermore, this mechanism could be of special importance for Na+ absorption along the distal colon, which is mainly due to the activities of Na+/H+ exchange and Na+-K+-ATPase (5). Reabsorption of Na+ via the Na+/H+ exchanger across the apical membrane would lead to a dramatic local alkalinization in the vicinity of the exchanger, thereby reducing its activity. In this case the proton conductance could serve to create an "acidic" microenvironment in the vicinity of the exchanger and thus maintain Na+ reabsorption.
In conclusion, our data show that the rapid, nongenomic action of aldosterone on Na+/H+ exchange in mammalian epithelial cells is independent of the classical type 1 mineralocorticoid receptor but involves the activation of a plasma membrane proton conductance, probably via PKC. These results could explain the mechanism by which aldosterone alters renal Na+ and K+ excretion within 5-10 min, acting as an acute-phase hormone (10).
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ACKNOWLEDGEMENTS |
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The excellent technical assistance of R. Freudinger and S. Mildenberger is greatfully acknowledged.
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft (SFB 176/A6).
Address for reprint requests: M. Gekle, Physiologisches Institut, Universität Würzburg, Röntgenring 9, 97070 Würzburg, Germany.
Received 18 February 1997; accepted in final form 24 July 1997.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Boron, W. F.
Control of intracellular pH.
In: The Kidney, edited by D. W. Seldin,
and G. Giebisch. New York: Raven, 1992, p. 219-263.
2.
Calonge, M. L.,
and
A. A. Ilundain.
PKC activators stimulate H+ conductance in chicken enterocytes.
Pflügers Arch.
431:
594-598,
1996[Medline].
3.
Christ, M.,
C. Eisen,
J. Aktas,
K. Theisen,
and
M. Wehling.
The inositol-1,4,5-trisphosphate system is involved in rapid effects of aldosterone in human mononuclear leukocytes.
J. Clin. Endocrinol. Metab.
77:
1452-1457,
1993[Abstract].
4.
Christ, M.,
C. Meyer,
K. Sippel,
and
M. Wehling.
Rapid aldosterone signaling in vascular smooth muscle cells: involvement of phospholipase C, diacylglycerol and protein kinase C.
Biochem. Biophys. Res. Commun.
213:
123-129,
1995[Medline].
5.
Dagher, P. C.,
T. Behm,
A. Taglietta-Kohlbrecher,
R. W. Egnor,
and
A. N. Charney.
Dissociation of colonic apical Na/H exchange activity from bulk cytoplasmic pH.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1799-C1806,
1996
6.
Demaurex, N.,
J. Orlowski,
G. Brisseau,
M. Woodside,
and
S. Grinstein.
The mammalian Na+/H+ antiporters NHE-1, NHE-2 and NHE-3 are electroneutral and voltage independent, but can couple to an H+ conductance.
J. Gen. Physiol.
106:
85-111,
1995[Abstract].
7.
Doolan, C. M.,
and
B. J. Harvey.
Modulation of cytosolic protein kinase C and calcium ion activity by steroid hormones in rat distal colon.
J. Biol. Chem.
271:
8763-8767,
1996
8.
Doolan, C. M.,
and
B. J. Harvey.
Rapid effects of steroid hormones on free intracellular calcium in T84 colonic epithelial cells.
Am. J. Physiol.
271 (Cell Physiol. 40):
C1935-C1941,
1996
9.
Dreher, D.,
and
T. Rochat.
Hyperoxia induces alkalinization and dome formation in MDCK epithelial cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C358-C364,
1992
10.
Ganong, W. F.,
and
P. J. Mulrow.
Rate of change in sodium and potassium excretion after injection of aldosterone into the aorta and renal artery of the dog.
Am. J. Physiol.
195:
337-341,
1958.
11.
Gekle, M.,
N. Golenhofen,
H. Oberleithner,
and
S. Silbernagl.
Rapid activation of Na+/H+-exchange by aldosterone in renal epithelial cells requires Ca2+ and stimulation of a plasma membrane proton conductance.
Proc. Natl. Acad. Sci. USA
93:
10500-10504,
1996
12.
Gekle, M.,
S. Wünsch,
H. Oberleithner,
and
S. Silbernagl.
Characterization of two MDCK-cell subtypes as a model system to study principal and intercalated cell properties.
Pflügers Arch.
428:
157-162,
1994[Medline].
13.
Harvey, B. J.,
and
J. Ehrenfeld.
Role of Na+/H+ exchange in the control of intracellular pH and cell membrane conductances in frog skin epithelium.
J. Gen. Physiol.
92:
793-809,
1988[Abstract].
14.
Kaila, K.,
and
R. D. Vaughan-Jones.
Influence of sodium-hydrogen exchange on intracellular pH, sodium and tension in sheep cardiac Purkinje fibres.
J. Physiol. (Lond.)
390:
93-118,
1987[Abstract].
15.
Khalil, R. A.,
and
K. G. Morgan.
Protein kinase C: a second E-C coupling pathway in vascular smooth muscle?
News Physiol. Sci.
7:
10-15,
1992.
16.
Lukacs, G. L.,
A. Kapus,
A. Nanda,
R. Romanek,
and
S. Grinstein.
Proton conductance of the plasma membrane: properties, regulation, and functional role.
Am. J. Physiol.
265 (Cell Physiol. 34):
C3-C14,
1993
17.
Meggio, F.,
A. Donella Deana,
M. Ruzzene,
A. M. Brunati,
L. Cesaro,
B. Guerra,
T. Meyer,
H. Mett,
D. Fabbro,
P. Furet,
G. Dobrowolska,
and
L. A. Pinna.
Different susceptibility of protein kinases to staurosporine inhibition.
Eur. J. Biochem.
234:
317-322,
1995[Abstract].
18.
Nanda, A.,
and
S. Grinstein.
Chemoattractant-induced activation of vacuolar H+ pumps and of an H+-selective conductance in neutrophils.
J. Cell. Physiol.
165:
588-599,
1995[Medline].
19.
Oberleithner, H.,
U. Kersting,
and
M. Hunter.
Cytoplasmic pH determines K+ conductance in fused renal epithelial cells.
Proc. Natl. Acad. Sci. USA
85:
8345-8349,
1988[Abstract].
20.
Oberleithner, H.,
M. Weigt,
H.-J. Westphale,
and
W. Wang.
Aldosterone activates Na+/H+exchange and raises cytoplasmic pH in target cells of the amphibian kidney.
Proc. Natl. Acad. Sci. USA
84:
1464-1468,
1987[Abstract].
21.
Petzel, D.,
M. B. Ganz,
E. J. Nestler,
J. J. Lewis,
J. Goldenring,
F. Akcicek,
and
J. P. Hayslett.
Correlates of aldosterone-induced increases in Ca2+i and ISC suggest that Ca2+i is the second messenger for stimulation of apical membrane conductance.
J. Clin. Invest.
89:
150-156,
1992[Medline].
22.
Thomas, J. A.,
R. N. Buchsbaum,
A. Zimniak,
and
E. Racker.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[Medline].
23.
Urbach, V.,
E. Van Kerkhove,
D. Maguire,
and
B. J. Harvey.
Rapid activation of KATP channels by aldosterone in principal cells of frog skin.
J. Physiol. (Lond.)
491:
111-120,
1996[Abstract].
24.
Vilella, S.,
L. Guerra,
C. Helmle-Kolb,
and
H. Murer.
Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells.
Pflügers Arch.
422:
9-15,
1992[Medline].
25.
Wehling, M.
Nongenomic actions of steroid hormones.
Trends Endocrinol. Metab.
5:
347-353,
1994.
26.
Wehling, M.,
M. Christ,
and
K. Theisen.
Membrane receptors for aldosterone: a novel pathway for mineralocorticoid action.
Am. J. Physiol.
263 (Endocrinol. Metab. 26):
E974-E979,
1992[Medline].
27.
Wehling, M.,
J. Käsmayr,
and
K. Theisen.
Aldosterone influences free intracellular calcium in human mononuclear leukocytes in vitro.
Cell Calcium
11:
565-571,
1990[Medline].
28.
Wehling, M.,
J. Käsmayr,
and
K. Theisen.
Rapid effects of mineralocorticoids on sodium-proton exchanger: genomic or nongenomic pathway?
Am. J. Physiol.
260 (Endocrinol. Metab. 23):
E719-E726,
1991
29.
Wehling, M.,
C. B. Neylon,
M. Fullerton,
A. Bobik,
and
J. W. Funder.
Nongenomic effects of aldosterone on intracellular Ca2+ in vascular smooth muscle cells.
Circ. Res.
76:
973-979,
1995
30.
Wünsch, S.,
M. Gekle,
U. Kersting,
B. Schuricht,
and
H. Oberleithner.
Phenotypically and karyotypically distinct Madin-Darby canine kidney cell clones respond differently to alkaline stress.
J. Cell. Physiol.
164:
164-171,
1995[Medline].