13
Toxicology Letters, 53 (1990) 13-17 Elsevier
TOXLET 02378
Signal pathway of Naf in fused renal epithelial cells
Hans Oberleithner Institutor Physiologic. Un~yersjt~t ~~rzb~g, Wiirzburg (F.R.G.1
Key words: MDCK-cell; Epithelial cell; Cell nucleus; Na+ channel; Gene activation
Intracellular sodium is involved in a variety of cellular processes including regulation of Na+/K+-ATPase activity, mitogen-induced cell growth and cell proliferation. In renal epithelial cells Na+ enters across the apical membrane, increases intracellular Na+ concentration and thereby initiates a cascade of cellular events f 11.Induction of cell differentiation depends on the activation of specific genes in the cell nucleus. Cation concentration in the cell nucleus may be crucial for the puffing pattern that indicates the activation of specific loci in chromosomes [2]. Na+ may be an important signal for such mechanisms because it counterbalances net negative charges of proteins accumulated in the nucleoplasm. Some years ago it was proposed that there is a direct communication pathway for Na+ between extracellular space and cell nucleus [3]. This conclusion was based on the observation that radioactive Na+ accumulates more rapidly in the cell nucleus than in the cytoplasm. We tested this hypothesis in renal epithelial cells. We performed electrophysiological experiments in MDCKcells, an established cell line from the collecting duct of dog kidney. We fused single epithelial cells to ‘giant’ cells and kept them in cell culture for at least 3 days [4]. The multika~ons (up to 20 nuclei per cell) reduce their number of nuclei due to nuclear fusion and/or nuclear degradation. They express a glycoprotein in the apical plasma membrane that binds specifically wheat-germ agglutinin characteristic for the Na+ transporting principal cell of the renal collecting duct [5]. Thus, fused ‘giant’ epithelial cells in culture offer at least two major advantages over single cells: they express properties of a Na+-transporting epithelium and they are large enough for multiple impalements. ‘Giant’ cells grown on glass were superfused with HEPES-buffered Ringer solution and impaled by conventional and Na+-sensitive microelectrodes (Fig. la). Under the Address for correspondence: H. O~rleithner, 9, D-8700 Wiirzburg F.R.G.
Institut fiir Physiolo~e, Unive~it~t Wiirzburg, R~ntgen~n~
0378-4274/90/$3.50 @ I990 Elsevier Science Publishers B.V. (Biomedical Division)
14
nucleus
cytoplasm
Na*
Nat
I
a
t
48 vrn
b
. i
$
1
*o 10 c
.
20 distance
30
40
SO
from nuclear CWitV4 tlrt’ll)
60 70
d
Fig. 1. (a) Measurement of the Na+ signal in nucleus and cytoplasm by Na*-sensitive microelectrodes. A conventional ~~r~l~tr~e measures the electrical potential (not shown). The intra~lluiar change of Na+ ~oocentration is induced by a step-change of extracellufar Na+. (b) The change in intracell~ar Na+ concentration is shown in nucleus and cytoplasm. The two recording sites are 48 pm apart. (c) Relationship between the delay time of the Na+ signal and the distance of the cytoplasmic recording site from the nucleus. (d) Na+-influx rates were obtained in nucleus and cytoplasm induced by a sudden increase of extracellular Na+ from 26 to 126 mmol/t.
control of high-resolution interference contrast microscopy (630 x magnification) a cell nucleus was impaled by a Na+-electrode and the Naf electrochemical potential was measured in response to a rapid concentration step of extraceil~ar Na+ from 26 to 126 mmol/l. Then, the electrode tip was placed in the cytoplasm at a given distance from the nucleus and the superfusion procedure was repeated. Over the whole course of this expe~ment a conventional microelectrode - its tip inserted in the cytoplasm - recorded the electrical potential difference that was subtracted from the Na+ signal. This procedure allows the continuous monitoring of free Na+ concentration in nucleus and cytoplasm in response to altered extra&Mar Na+ . The Na+ signal arrives first in the nucleoplasm and subsequently, with some delay,
15
in the cytoplasm (Fig. 1b). The delay time is a function of the distance between the nucleus and the site in the cytoplasm where the Na+ signal is measured (Fig. lc). This indicates that Na+ enters the nucleus first and then diffuses into the bulk phase of the cytoplasm. Na+ diffusion through the nuclear envelope is most probably mediated by the nuclear pores that represent a highly permeable pathway for ions [6]. From the initial slope of the Na+ concentration change induced by the rapid increase of extracellular Na+ we estimated the rate of Na+ influx in nucleus and cytoplasm. As shown in Figure Id, Na+ influx is about 3-fold higher in nucleoplasm than in cytoplasm. These results indicate that there is indeed a rapid communication pathway between extracellular space and cell nucleus. Obviously, Na+ passes through the nucleoplasm before it diffuses into the bulk phase of the cytoplasm. In a previous study it was suggested that the endoplasmic reticulum could serve as a functional channel network to span the distance between plasma membrane and nucleus [3]. An alternative explanation for the observed phenomenon could be an uneven distribution of Na+ transport molecules in the apical plasma membrane. To test for this hypothesis we measured local densities of Na+ currents at various sites of the apical plasma membrane. First, the overall resistance of the cell was evaluated by the intracellular injection of short current pulses (1 nA, 0.5 s) while the resulting voltage deflections were measured with another microelectrode inserted in the cytoplasm [7]. Then, the mouth of a patch pipette (tip diameter 4 pm) was placed in close proximity to the apical plasma membrane. The pipette was filled with a modified Ringer solution in which 100 mM Na+ was replaced by choline+. While the cell was superfused systemically by regular Ringer solution we performed localized perturbations (1 s duration) at various sites of the apical cell membrane and measured the resulting change of the plasma membrane potential with an intracellular microelectrode. The local reduction of Na+ concentration hyperpolarizes the membrane indicating the existence of Na+ channels. From these voltage changes and the membrane resistance we estimated the Na+ current (&+) at various loci of the apical plasma membrane. We videotaped the experiments and thus could determine the specific loci and relate them to INa. In Figure 2a, four examples are given. It is obvious that the density of INS+(pA/cm2) is high near the nucleus and small in the cell periphery. The data are summarized in Figure 2b. In addition, we measured in 7 ‘giant’ cells the total apical cell surface (4553 + 311 pm2) and the apical area just above the nucleus (240 f 29 pm2). Furthermore, we evaluated total INa+of the apical plasma membrane. From these data we calculated that in the apical supranuclear surface (Fig. 2b), which is only 5% of the total apical surface, about 24% of total INa+ is recovered. This is consistent with the view that Na+ channels are inserted into the apical plasma membrane in close proximity to the cell nucleus. Based on the steepness of the lateral mobility of the Na+ channel density we assume that the lateral mobility of the Na+ transport molecules is restricted and that they are anchored in the supranuclear surface of the apical plasma membrane. Wheat-germ agglutinin (WGA) binds specifically to glycoproteins of the apical
a
b
1
:lf
iTI
~
n=lZ 11-20
distance
21-30
from
314.0
nuclear
Ll-50
51-60
centre (pm)
Fig. 2. (a) Redrawing of videotaped ‘giant’ epi~eii~ cells. The numbers correspond to Na+ currents (pA/cmZ) at various sites of the apical plasma membrane. They were obtained in patches of 225 pm* by the local superfusion technique. (b) I Na+values are plotted versus distance from the cell nucleus. (c) Fluorescence-micrograph of a ‘giant’ MDCK cell. The apical plasma surface was labeled with wheat-germ agglutinin (WGA). Note that the fluorescence intensity is marked at the centre of the cell (where the nucleus is focalized) and low at the cell periphery.
plasma membrane of fused MDCK cells. We tested for the distribution pattern of this glycoprotein asssuming that there may be some correlation between INS+and the glycoprotein density. The apical membranes of ‘giant’ cells in culture were treated with fluorescence-labeled WGA, exposed to incident light of specific wavelength under high magnification ( x 630) and the images of the cells videotaped. Then we analysed the distribution pattern of WGA-binding sites of the apical membrane. At least, in some cells we found an accumulation of the fluorescence marker at the supranuclear surface (Fig. 2~). This could indicate that there is a close relationship between Naf channels and WGA-binding proteins. The uneven distribution of the apical Na+ transporters explains the phenomenon that an extra~llular-induced Na+ signal arrives first in the cell nucleus and then in the bulk cytoplasm. The major portion of Na+ enters the cell close to the nucleus and then, after nuclear passage, diffuses further into the bulk phase of the cytoplasm. The route of the Na+ signal could be of physiological relevance. Since the activation of genes could be regulated by the Na+ concentration in the nucleoplasm, a rapid and direct crosstalk between extracellular space and nucleus could be important in controlling basic processes such as cell growth and cell differentiation. ACKNOWLEDGEMENT
Supported by Deutsche Forschungsgemeinschaft
SFB 176 (A6).
REFERENCES I Stanton. B.A. and Kaisshng, B. (1989) Regulation of renal ion transport and cell growth by sodium. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26), FI-FlO. 2 Kroeger, H. (1963) Chemical nature of the system controlling gene activities in insect cells. Nature 200, 1234-1235. 3 Siebert, G. and Langendorf, H. (1970) Ionenhaushatt im Zellkem. Naturwissenschaften 57, 119-124. 4 Kersting, K., Joha, H., Steigner, W., Gassner, B., Gstraunthaler, G., Pfaller, W. and Oberleithner, H. (1989) Fusion of cultured dog kidney (MDCK) cells. I. Technique, fate of plasma membranes and cell nuclei. J. Membrane Bioi. 111,3748. 5 Minuth, W.W., Gilbert, P., Rudoiph, U. and Spielman, W.S. (1989) Successive hist~hemical differentiation steps during postnatal development of the collecting duct in rabbit kidney. Histochemistry 93, 19-25. 6 Paine, P.L., Moore, L.C. and Horowitz, S.B. (1975) Nuclear envelope permeability. Nature 254, 109114. 7 Oberleithner, H., Kersting, U. and Hunter, M. (1988) Cytoplasmic pH determines K+ conductance in fused renal epithelial cells. Proc. Natl. Acad. Sci. USA 85,8345-8349.
Toxicology Letters, 53 (1990) 13-17 Elsevier
TOXLET 02378
Signal pathway of Naf in fused renal epithelial cells
Hans Oberleithner Institutor Physiologic. Un~yersjt~t ~~rzb~g, Wiirzburg (F.R.G.1
Key words: MDCK-cell; Epithelial cell; Cell nucleus; Na+ channel; Gene activation
Intracellular sodium is involved in a variety of cellular processes including regulation of Na+/K+-ATPase activity, mitogen-induced cell growth and cell proliferation. In renal epithelial cells Na+ enters across the apical membrane, increases intracellular Na+ concentration and thereby initiates a cascade of cellular events f 11.Induction of cell differentiation depends on the activation of specific genes in the cell nucleus. Cation concentration in the cell nucleus may be crucial for the puffing pattern that indicates the activation of specific loci in chromosomes [2]. Na+ may be an important signal for such mechanisms because it counterbalances net negative charges of proteins accumulated in the nucleoplasm. Some years ago it was proposed that there is a direct communication pathway for Na+ between extracellular space and cell nucleus [3]. This conclusion was based on the observation that radioactive Na+ accumulates more rapidly in the cell nucleus than in the cytoplasm. We tested this hypothesis in renal epithelial cells. We performed electrophysiological experiments in MDCKcells, an established cell line from the collecting duct of dog kidney. We fused single epithelial cells to ‘giant’ cells and kept them in cell culture for at least 3 days [4]. The multika~ons (up to 20 nuclei per cell) reduce their number of nuclei due to nuclear fusion and/or nuclear degradation. They express a glycoprotein in the apical plasma membrane that binds specifically wheat-germ agglutinin characteristic for the Na+ transporting principal cell of the renal collecting duct [5]. Thus, fused ‘giant’ epithelial cells in culture offer at least two major advantages over single cells: they express properties of a Na+-transporting epithelium and they are large enough for multiple impalements. ‘Giant’ cells grown on glass were superfused with HEPES-buffered Ringer solution and impaled by conventional and Na+-sensitive microelectrodes (Fig. la). Under the Address for correspondence: H. O~rleithner, 9, D-8700 Wiirzburg F.R.G.
Institut fiir Physiolo~e, Unive~it~t Wiirzburg, R~ntgen~n~
0378-4274/90/$3.50 @ I990 Elsevier Science Publishers B.V. (Biomedical Division)
14
nucleus
cytoplasm
Na*
Nat
I
a
t
48 vrn
b
. i
$
1
*o 10 c
.
20 distance
30
40
SO
from nuclear CWitV4 tlrt’ll)
60 70
d
Fig. 1. (a) Measurement of the Na+ signal in nucleus and cytoplasm by Na*-sensitive microelectrodes. A conventional ~~r~l~tr~e measures the electrical potential (not shown). The intra~lluiar change of Na+ ~oocentration is induced by a step-change of extracellufar Na+. (b) The change in intracell~ar Na+ concentration is shown in nucleus and cytoplasm. The two recording sites are 48 pm apart. (c) Relationship between the delay time of the Na+ signal and the distance of the cytoplasmic recording site from the nucleus. (d) Na+-influx rates were obtained in nucleus and cytoplasm induced by a sudden increase of extracellular Na+ from 26 to 126 mmol/t.
control of high-resolution interference contrast microscopy (630 x magnification) a cell nucleus was impaled by a Na+-electrode and the Naf electrochemical potential was measured in response to a rapid concentration step of extraceil~ar Na+ from 26 to 126 mmol/l. Then, the electrode tip was placed in the cytoplasm at a given distance from the nucleus and the superfusion procedure was repeated. Over the whole course of this expe~ment a conventional microelectrode - its tip inserted in the cytoplasm - recorded the electrical potential difference that was subtracted from the Na+ signal. This procedure allows the continuous monitoring of free Na+ concentration in nucleus and cytoplasm in response to altered extra&Mar Na+ . The Na+ signal arrives first in the nucleoplasm and subsequently, with some delay,
15
in the cytoplasm (Fig. 1b). The delay time is a function of the distance between the nucleus and the site in the cytoplasm where the Na+ signal is measured (Fig. lc). This indicates that Na+ enters the nucleus first and then diffuses into the bulk phase of the cytoplasm. Na+ diffusion through the nuclear envelope is most probably mediated by the nuclear pores that represent a highly permeable pathway for ions [6]. From the initial slope of the Na+ concentration change induced by the rapid increase of extracellular Na+ we estimated the rate of Na+ influx in nucleus and cytoplasm. As shown in Figure Id, Na+ influx is about 3-fold higher in nucleoplasm than in cytoplasm. These results indicate that there is indeed a rapid communication pathway between extracellular space and cell nucleus. Obviously, Na+ passes through the nucleoplasm before it diffuses into the bulk phase of the cytoplasm. In a previous study it was suggested that the endoplasmic reticulum could serve as a functional channel network to span the distance between plasma membrane and nucleus [3]. An alternative explanation for the observed phenomenon could be an uneven distribution of Na+ transport molecules in the apical plasma membrane. To test for this hypothesis we measured local densities of Na+ currents at various sites of the apical plasma membrane. First, the overall resistance of the cell was evaluated by the intracellular injection of short current pulses (1 nA, 0.5 s) while the resulting voltage deflections were measured with another microelectrode inserted in the cytoplasm [7]. Then, the mouth of a patch pipette (tip diameter 4 pm) was placed in close proximity to the apical plasma membrane. The pipette was filled with a modified Ringer solution in which 100 mM Na+ was replaced by choline+. While the cell was superfused systemically by regular Ringer solution we performed localized perturbations (1 s duration) at various sites of the apical cell membrane and measured the resulting change of the plasma membrane potential with an intracellular microelectrode. The local reduction of Na+ concentration hyperpolarizes the membrane indicating the existence of Na+ channels. From these voltage changes and the membrane resistance we estimated the Na+ current (&+) at various loci of the apical plasma membrane. We videotaped the experiments and thus could determine the specific loci and relate them to INa. In Figure 2a, four examples are given. It is obvious that the density of INS+(pA/cm2) is high near the nucleus and small in the cell periphery. The data are summarized in Figure 2b. In addition, we measured in 7 ‘giant’ cells the total apical cell surface (4553 + 311 pm2) and the apical area just above the nucleus (240 f 29 pm2). Furthermore, we evaluated total INa+of the apical plasma membrane. From these data we calculated that in the apical supranuclear surface (Fig. 2b), which is only 5% of the total apical surface, about 24% of total INa+ is recovered. This is consistent with the view that Na+ channels are inserted into the apical plasma membrane in close proximity to the cell nucleus. Based on the steepness of the lateral mobility of the Na+ channel density we assume that the lateral mobility of the Na+ transport molecules is restricted and that they are anchored in the supranuclear surface of the apical plasma membrane. Wheat-germ agglutinin (WGA) binds specifically to glycoproteins of the apical
a
b
1
:lf
iTI
~
n=lZ 11-20
distance
21-30
from
314.0
nuclear
Ll-50
51-60
centre (pm)
Fig. 2. (a) Redrawing of videotaped ‘giant’ epi~eii~ cells. The numbers correspond to Na+ currents (pA/cmZ) at various sites of the apical plasma membrane. They were obtained in patches of 225 pm* by the local superfusion technique. (b) I Na+values are plotted versus distance from the cell nucleus. (c) Fluorescence-micrograph of a ‘giant’ MDCK cell. The apical plasma surface was labeled with wheat-germ agglutinin (WGA). Note that the fluorescence intensity is marked at the centre of the cell (where the nucleus is focalized) and low at the cell periphery.
plasma membrane of fused MDCK cells. We tested for the distribution pattern of this glycoprotein asssuming that there may be some correlation between INS+and the glycoprotein density. The apical membranes of ‘giant’ cells in culture were treated with fluorescence-labeled WGA, exposed to incident light of specific wavelength under high magnification ( x 630) and the images of the cells videotaped. Then we analysed the distribution pattern of WGA-binding sites of the apical membrane. At least, in some cells we found an accumulation of the fluorescence marker at the supranuclear surface (Fig. 2~). This could indicate that there is a close relationship between Naf channels and WGA-binding proteins. The uneven distribution of the apical Na+ transporters explains the phenomenon that an extra~llular-induced Na+ signal arrives first in the cell nucleus and then in the bulk cytoplasm. The major portion of Na+ enters the cell close to the nucleus and then, after nuclear passage, diffuses further into the bulk phase of the cytoplasm. The route of the Na+ signal could be of physiological relevance. Since the activation of genes could be regulated by the Na+ concentration in the nucleoplasm, a rapid and direct crosstalk between extracellular space and nucleus could be important in controlling basic processes such as cell growth and cell differentiation. ACKNOWLEDGEMENT
Supported by Deutsche Forschungsgemeinschaft
SFB 176 (A6).
REFERENCES I Stanton. B.A. and Kaisshng, B. (1989) Regulation of renal ion transport and cell growth by sodium. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26), FI-FlO. 2 Kroeger, H. (1963) Chemical nature of the system controlling gene activities in insect cells. Nature 200, 1234-1235. 3 Siebert, G. and Langendorf, H. (1970) Ionenhaushatt im Zellkem. Naturwissenschaften 57, 119-124. 4 Kersting, K., Joha, H., Steigner, W., Gassner, B., Gstraunthaler, G., Pfaller, W. and Oberleithner, H. (1989) Fusion of cultured dog kidney (MDCK) cells. I. Technique, fate of plasma membranes and cell nuclei. J. Membrane Bioi. 111,3748. 5 Minuth, W.W., Gilbert, P., Rudoiph, U. and Spielman, W.S. (1989) Successive hist~hemical differentiation steps during postnatal development of the collecting duct in rabbit kidney. Histochemistry 93, 19-25. 6 Paine, P.L., Moore, L.C. and Horowitz, S.B. (1975) Nuclear envelope permeability. Nature 254, 109114. 7 Oberleithner, H., Kersting, U. and Hunter, M. (1988) Cytoplasmic pH determines K+ conductance in fused renal epithelial cells. Proc. Natl. Acad. Sci. USA 85,8345-8349.
