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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2002 Apr 30;99(10):7154–7159. doi: 10.1073/pnas.092140799

Aldosterone signaling pathway across the nuclear envelope

C Schäfer 1,*, V Shahin 1,*, L Albermann 1, M J Hug 1, J Reinhardt 1, H Schillers 1, S W Schneider 1, H Oberleithner 1,†
PMCID: PMC124544  PMID: 11983859

Abstract

We describe the route by which aldosterone-triggered macromolecules enter and exit the cell nucleus of Xenopus laevis oocyte. Oocytes were microinjected with 50 fmol aldosterone and then enucleated 2–30 min after injection. After isolation, nuclear envelope electrical resistance (NEER) was measured in the intact cell nuclei by using the nuclear hourglass technique. We observed three NEER stages: an early peak 2 min after injection, a sustained depression after 5–15 min, and a final late peak 20 min after injection. Because NEER reflects the passive electrical permeability of nuclear pores, we investigated with atomic force microscopy aldosterone-induced conformational changes of individual nuclear pore complexes (NPCs). At the early peak we observed small (≅100 kDa) molecules (flags) attached to the NPC surface. At the sustained depression NPCs were found free of flags. At the late peak large (≅800 kDa) molecules (plugs) were detected inside the central channels. Ribonuclease or actinomycin D treatment prevented the late NEER peak. Coinjection of aldosterone (50 fmol) and its competitive inhibitor spironolactone (500 fmol) eliminated the electrical changes as well as flag and plug formation. We conclude: (i) The genomic response of aldosterone can be electrically measured in intact oocyte nuclei. (ii) Flags represent aldosterone receptors on their way into the cell nucleus whereas plugs represent ribonucleoproteins carrying aldosterone-induced mRNA from the nucleoplasm into the cytoplasm. (iii) Because plugs can be mechanically harvested with the atomic force microscopy stylus, oocytes could serve as a bioassay system for identifying aldosterone-induced early genes.

Keywords: nuclear pore complex‖atomic force microscopy‖RNA export‖ mineralocorticoid receptor

Signals such as an increase in intracellular calcium concentration or intracellular alkalinization are the early responses of target cells to the mineralocorticoid hormone aldosterone. The intracellular calcium signal can be detected a few seconds after hormone exposure (1–3), whereas the pH signal occurs later but usually lasts longer (4). Although there are no doubts about the existence of these early hormone responses, the physiological relevance is still unknown. For almost 40 years it has been postulated that inorganic ions could play a crucial role in steroid hormone-induced gene activation (5). This suggestion has been supported by more recent findings in our own laboratory (6, 7). The signaling pathway of steroid hormones involving intracellular macromolecules is well established. Aldosterone is known to bind to mineralocorticoid receptors located in the cytosol. Hormone binding changes protein conformation and translocates the receptor into the nucleus where it attaches to specific DNA. Transcription occurs and finally mRNA is exported to be translated into aldosterone-induced proteins at the ribosomes. Although this macromolecular signal pathway has been described in detail, there are no data available that directly show both functionally and structurally import/export of macromolecules across the nuclear envelope (NE). Nuclear pore complexes (NPCs) mediate macromolecule transport serving as selective barriers in the control of gene activation (8–10). Electrical current through NPCs can be measured by patch–clamp techniques applied to cell nuclei (11–13) and related to macromolecule translocation (14). Whole NE electrical conductance can be measured by the recently developed nuclear hourglass technique (NHT) (15) and related to NPC function.

By means of atomic force microscopy (AFM) individual NPCs can be visualized and NPC conformation can be studied (16–19). Therefore, we considered that a combination of electrical tools with AFM should allow us to obtain both functional and structural information on NPCs during hormone stimulation. Indeed, we found dramatic shifts in passive electrical NE permeability during aldosterone stimulation that could be explained by the structural changes of individual NPCs. We visualized steroid hormone-induced protein import across nuclear pores and mRNA export from the nucleus to the cytoplasm at the molecular level.

Methods

Oocyte Injection and Preparation of Cell Nuclei.

Female Xenopus laevis were anaesthetized with 0.1% ethyl m-aminobenzoate methanesulfonate (Serva), and a portion of their ovaries was removed. Stage VI oocytes were dissected from ovary clusters and stored in modified Ringer's solution (87 mM NaCl/3 mM KCl/1.5 mM CaCl2/1 mM MgCl2/10 mM Hepes/100 units/100 μg penicillin/streptomycin, pH 7.4) before use.

For the aldosterone and spironolactone experiments oocytes were injected with 50 nl (<10% of total oocyte volume) 10−6 M aldosterone (aldosterone stock solution dissolved in ethanol at a concentration of 10−3 M; Sigma) or 50 nl 10−6 M aldosterone and 10−5 M spironolactone, a specific mineralocorticoid receptor antagonist (spironolactone stock solution dissolved in ethanol at a concentration of 10−3 M; Sigma). Then, nuclei were isolated 2–30 min after injection. For the ribonuclease experiments, 50 nl 10−6 M aldosterone or 50 nl solvent (0.1% ethanol in water) was injected. Then, nuclei were isolated 18–20 min after injection and used for further RNase treatment. To test for aldosterone-induced transcription, 10−5 M actinomycin D was coinjected with 10−6 M aldosterone (both dissolved in 50 nl solvent). For nuclear isolation, oocytes were transferred into nuclear isolation medium (NIM) composed of 10 mM NaCl, 90 mM KCl, 2.0 mM MgCl2, 1.1 mM EGTA, 10 mM Hepes titrated to pH 7.4. No ATP was present in the NIM to stop (“freeze”) nucleocytoplasmic transport immediately after nuclear isolation. In addition, NIM contained 1.5% polyvinylpyrrolidone (PVP, Mr = 40,000; Sigma) to compensate for the lack of macromolecules. The presence of PVP prevents nuclear swelling that instantaneously occurs during isolation in NIM in the absence of macromolecules (20).

NHT and Experimental Protocol.

The technical aspects of the method and its application in isolated cell nuclei have been described in detail (15). In short, the method uses a tapered glass tube, which narrows in its middle part to two-thirds of the diameter of the nucleus. A current of up to 1 mA is injected by two massive Ag/AgCl electrodes through either end of the glass tube. The voltage drops across the cell nucleus are measured with two conventional microelectrodes. Because current and voltage are simultaneously measured, the resistance can be continuously calculated. At the start of the experiment the nucleus is sucked into the tapered part of the capillary by gentle fluid movement. Thus, the whole current now flows through the accessible parts of the NE. The resulting rise in total electrical resistance indicates the NE electrical resistance (NEER).

In the aldosterone/spironolactone experiments, nuclei isolated after different time lags (2–30 min) after the hormone injection were brought into the NHT capillary filled with NIM. Twenty seconds later NEER measurements were started. In the RNase experiments (RNase A is an enzyme that catalyses depolymerization of single-stranded RNA) isolated nuclei were brought into the NHT capillary filled with NIM plus 0.2 μg mRNase A/ml (DNase-free mRNase A Tris-EGTA stock solution with a concentration of 1 mg/ml; Roth, Karlsruhe, Germany). In this series of experiments NEER was measured 20 s (first measurement) and 20 min (second measurement) after RNase exposure (paired study; nuclei remained in NHT capillary over the whole time of the measurement). In actinomycin D experiments NEER measurements were performed 19 min after coinjection of the transcription blocker and aldosterone.

NE Preparation and AFM.

NE preparation and AFM application to spread NEs has been described in detail (18). In short, nuclei were manually isolated in NIM by piercing the oocyte with two pincers. Individual intact nuclei were picked up with a Pasteur pipette and transferred to a glass coverslip placed under a stereomicroscope. Then, the chromatin was carefully removed by using sharp needles and the NE was spread on poly l-lysine-coated glass, with the nucleoplasmic side facing downward. Finally, the NE was rinsed with deionized water and dried.

We used a Multimode (with a NanoScope IIIa controller, Digital Instruments, Santa Barbara, CA) equipped with an optical microscope, a video camera, and a monitor to visualize the NE and the AFM tip on the AFM head stage. V-shaped 200-μm long silicon nitride cantilevers with spring constants of 0.06 M/m and pyramidal tips with an estimated tip diameter of 10 nm (Digital Instruments) were used. The images were recorded with 512 lines per screen, at constant force (height mode) in contact mode with a scan rate of 3–10 Hz. The forces applied during the scanning procedure were minimized by, in a first step, retracting the AFM tip until it lost contact with the sample surface and, in a second step, by reengaging the tip at a set point (i.e., force value) minimally above the liftoff value. Applying this approach, we usually could obtain scanning forces below 3 nN. Experiments were performed with a fluid cell. Although the AFM tip physically interacts with the NE we usually could perform multiple scans without damaging the preparation. Scanning at low forces (3 nN or less) left no visible marks on the surface.

AFM analysis of NPCs is explained in Fig. 1. We analyzed NPCs decorated with macromolecules in the ring periphery (so-called flags; Fig. 1 A and B) or with macromolecules in the ring center (so-called plugs; Fig. 1 C and D). About 100 μm2 per NE harvested from individual nuclei of aldosterone-injected oocytes was scanned by AFM and analyzed. The number of individual nuclei studied is indicated in Fig. 1. Flagged NPCs and plugged NPCs were related to total NPC number. Flags and plugs can be distinguished by size and location. Molecular size can be estimated according to methods described for a variety of proteins bound to mica surface (21, 22) or bound to biological substrates as DNA (23, 24). In short, we measured the diameter at half-maximal height of the individual molecules, which sufficiently compensates for the artificially induced overestimation of the protein width. After obtaining the dimensions of an individual protein (diameter and height) the molecular volume was calculated. Then molecular masses were derived from the respective molecular volumes. It turned out that an individual flag (diameter = 24 nm at half-maximal height; maximal height = 3 nm) has a molecular mass of about 100 kDa (range: 70 to 130 kDa). The plug (diameter = 40 nm at half-maximal height; maximal height = 8 nm) has an estimated molecular mass of about 800 kDa (range: 600 to 1,000 kDa). Because flags “sit” on the NPC surface their volume can be estimated directly from the images. Plugs in contrast “are stuck” in the central channel and their volume can be measured only after “dragging” individual plugs by means of mechanical force by the AFM tip from the depth of the central channel toward the NE surface. Furthermore, flags usually decorate the cytoplasmic ring in its periphery (see Fig. 1A) whereas plugs are found in the NPC center (see Fig. 1C). Finally, flags can be easily removed from the rings by increasing the lateral force (tip-sample interaction force: about 10 nN) as shown in Fig. 1B. Plugs can also be removed, but 10 times higher forces (about 100 nN) are necessary (plug removal shown in Fig. 1D). The latter technique (so-called plug harvesting) has been described in detail recently (25). In short, after imaging pores with plugs by using low loading forces we gradually increased force until plugs became loose. Blurred images indicated that plugs were displaced. Only 1–2 scans were necessary to obtain this result. Usually, the first scan removed the plug from the central channel and moved it toward the outer rim of the individual NPC. A subsequent sweep cleaned the nuclear pore ring so that some of the material got stuck on the AFM tip. Force curves performed subsequently indicated that “sticky material” contaminated the AFM tip (i.e., adhesion forces increased about 5- to 10-fold as compared with initial values before force application). Immediately loading forces were again lowered. This process resulted in clear images of NPCs deprived of plugs. Movie 1 illustrating this harvesting technique is available as supporting information on the PNAS web site, www.pnas.org.

Figure 1.

Figure 1

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AFM flag analysis: 100-kDa macromolecules (flags) docking to the NPC rings (A) were removed by increasing the scan force between AFM tip and NE (B). AFM plug analysis: 800-kDa macromolecules (plugs) exiting through NPC central channels (C) were removed by increasing the scan force between AFM tip and NE (D).

Results

Fig. 2 shows representative original tracings of two individual NEER measurements by using NHT. When nuclei are gently sucked into the tapered part of the glass capillary NEER can be measured as a sudden increase in electrical resistance followed by a new steady-state value. As long as the nucleus remains in place, NEER remains constant. The tracings show that aldosterone affects NEER. Twenty minutes after injection of aldosterone NEER is found increased by nearly 20%. To test the specificity of the aldosterone response we coinjected the aldosterone receptor antagonist spironolactone (at a 10 times higher concentration as compared with aldosterone). The data (aldosterone and aldosterone/spironolactone injections) are summarized in Fig. 3. Two minutes after aldosterone injection we observed an early NEER peak (2-min value: 119.6 ± 5.07%; mean ± SE, n = 17), followed by a sustained NEER depression (11-min value: 75.9 ± 4.45%; mean ± SE, n = 5). Eighteen to 20 min after aldosterone injection another late NEER peak appeared (18-min value: 121.3 ± 4.32%; mean ± SE, n = 10) and finally dissipated (30-min value: 102.9 ± 2.45%; mean ± SE, n = 4). Simultaneous injection of aldosterone and its inhibitor spironolactone completely prevented the aldosterone response. NEER remained virtually unaffected.

Figure 2.

Figure 2

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Two original tracings of NEER (given in Ω) from either H2O/ethanol- (solvent) or aldosterone-injected oocytes. Measurements were performed 20 min after injection. (Insets) Shown is the nuclear hourglass with a cell nucleus moving into the tapered part of the glass capillary corresponding to the individual phases of the original recording. NEER is measured when the nucleus is located in the narrow center of the glass tube.

Figure 3.

Figure 3

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NEER related to the corresponding control values (control = injection of solvent) is shown as a function of time after either aldosterone or aldosterone plus spironolactone injection. Number of cell nuclei studied is given adjacent to the respective mean value ± SE.

When we realized that application of aldosterone transiently affected NEER we searched for a structural correlate in the NE. The most likely supramolecular candidates for transmitting molecular signals between nucleus and cytoplasm were the NPCs, structures that undergo dramatic conformational changes when stimulated (18, 26–28). Therefore, we analyzed by AFM NEs isolated from aldosterone-treated oocytes. A representative example is given in Fig. 4. Before aldosterone injection, NPCs appear as smooth rings at the NE surface. Two minutes after hormone injection, NPCs are decorated with macromolecules. We call such macromolecules attached to the ring periphery flags. Eight minutes after injection, NPCs again appear as smooth rings. This is caused by the lack of flags at this stage of hormone stimulation. Nineteen minutes after aldosterone injection we detect rather large masses in the NPC central channels. We call such macromolecules located in the NPC centers plugs. In an extensive series of experiments we quantified flag and plug formation (for details see Methods) over a period of about 30 min comparable to the NEER measurements. As indicated in Fig. 5 we found about 25% of NPCs occupied with flags only 2 min after hormone injection. Flags rapidly disappeared thereafter. Coinjection of the competitive aldosterone antagonist spironolactone completely prevented flag formation. Fig. 6 shows the pattern of plug formation. Plugs appear 10 min after hormone injection. About 25% of NPCs are found plugged close to 20 min after aldosterone injection. A later small peak occurs about 30 min after hormone injection. Then plugs disappear.

Figure 4.

Figure 4

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AFM images of NE cytoplasmic surfaces before and after aldosterone injection. Before aldosterone injection: NPCs are clearly visible. Two minutes after aldosterone: most NPCs are flagged. Eight minutes after aldosterone: NPCs are again free of flags. Nineteen minutes after aldosterone: NPCs are plugged.

Figure 5.

Figure 5

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NPC flag analysis shown as a function of time after either aldosterone or aldosterone plus spironolactone injection. Mean values ± SE are given. n = number of individual cell nuclei studied. * indicates that the mean values of the aldosterone experiments were significantly different from the corresponding mean values of the aldosterone plus spironolactone experiments (P < 0.05). Because the mean values of the two series of experiments did not completely match in terms of time we statistically compared individual mean values within the closest time window.

Figure 6.

Figure 6

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NPC plug analysis shown as a function of time after either aldosterone or aldosterone plus spironolactone injection. For further details see Fig. 5.

Because plugs most likely represent ribonucleoproteins exported by the NPCs in response to aldosterone stimulation we applied NEER measurements to test this hypothesis. We performed two sets of experiments: In a first series we stimulated the oocytes with aldosterone, then measured NEER in the isolated nuclei 19 min after hormone injection and finally exposed them for 20 min to mRNase. In a second series we coinjected oocytes with aldosterone plus actinomycin D and performed NEER measurements 19 min later. As indicated in Fig. 7 actinomycin D injection or RNase exposure eliminated the late NEER peak, indicating that formation and export of mRNA are responsible for the electrical phenomena.

Figure 7.

Figure 7

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NEER related to the corresponding control values (control = injection of solvent) is shown after aldosterone injection, after aldosterone injection and subsequent RNase treatment, and after aldosterone plus actinomycin D coinjection. * indicates a significant difference between the mean data of the aldosterone experiments and those of the control experiments. § indicates a significant difference between the mean data of the aldosterone experiments and those of the RNase and actinomycin D experiments (P < 0.05).

Discussion

In kidney cells NEs respond to aldosterone by increasing the total number of pores per nucleus (29) and by facilitating macromolecule transport through individual NPCs (30). These responses occur within 24 h. In the present study we investigated the route of macromolecules through the nuclear pores immediately after hormone exposure. We took advantage of the fact that stage VI oocytes of X. laevis respond to aldosterone with an intracellular signal cascade that involves the cell nucleus.

AFM detected 100-kDa macromolecules (flags) attached to the cytoplasmic rings of individual NPCs. Flags are detectable only within the first few minutes after aldosterone injection. These flags are likely to be mineralocorticoid receptors because (i) the competitive receptor antagonist spironolactone inhibited NPC flagging and (ii) the molecular weight of the individual flags estimated by AFM matches the molecular mass of the mineralocorticoid receptor (31). As described, the proteins first bind to the NPC surface before, in a second step, translocation occurs through the NPC central channels (32). Receptor translocation explains the AFM observation that NPCs were again free of flags several minutes after hormone treatment. The existence of mineralocorticoid receptors in mature metaphase II-arrested oocytes has been recently shown although their physiological relevance has not yet been addressed (33). It is interesting to note that NE resistance was found to increase at the time when NPCs were flagged by the 100-kDa macromolecules. In the light of recent experiments we interpret this electrical conductance change as follows: the putative mineralocorticoid receptors dock to NPCs and occlude the so-called peripheral channels. The existence of such channels was postulated almost 10 years ago on the basis of electron microscopy (34) and was confirmed recently by visualization with AFM (35).

Using the NHT alone it is not possible to distinguish whether virtually all NPCs undergo a 20% increase in electrical resistance or whether 20% of the total number of NPCs transiently lose all of their electrical conductivity. However, quantitative comparison of structural AFM data with the functional NEER experiments indicates that most likely a certain percentage of NPCs (about 20%) become electrically tight for a short period (a few minutes) and then are again open for inorganic ion movements. A similar model was developed by Bustamante and coworkers (14, 36) applying patch–clamp techniques to the NE. The early NEER peak was followed by a marked decrease in the envelope resistance. This occurred at a time when NPCs were unflagged. Why NEER fell below the initial control values is currently a matter of speculation: Steroid hormone-induced transcription occurs over this time period and is known to be associated with chromatin decondensation and nuclear swelling (7). This nuclear event could be paralleled by an increased NPC ion permeability. Possibly, NPC rings expand and ATP and Ca2+-sensitive peripheral channels become conductive. Whether such a permeability change occurs in “hot spots” or affects the NE in general is still unknown. From the physiological point of view, it is more likely that the increase in ion permeability is strictly localized, allowing inorganic ions (particularly Ca2+) to gain access to the specific sites of transcription. It has been shown that Ca2+ spikes at defined concentrations over defined periods of time are prerequisites for successful gene expression (37–40). Locally restricted NPC permeability changes could allow for such mechanisms.

The late NEER peak coincides with NPC plugging. There is strong evidence that plugs are ribonucleoproteins exported by NPCs. First, the dimensions measured with AFM match with those reported for ribonucleoproteins estimated previously by electron microscopy (41, 42). Second, application of RNase decreases NEER in hormone-stimulated oocytes but does not decrease in nonstimulated oocytes. Third, plugs appear in the central NPC channels with a lag period of about 19 min after hormone injection consistent with export of early mRNA. A likely candidate is the serum- and glucocorticoid-dependent kinase sgk (see ref. 43 for review). It is believed that this ancient enzyme was important in controlling epithelial Na+ transport when early vertebrates made the transition from marine to a freshwater environment (44). Recently, it has been shown that the sgk is indeed a mineralocorticoid-induced regulator of epithelial sodium channels activity (45–48). Plug formation is paralleled by a sharp increase in NEER, indicating that NPC electrical conductance decreases. Because plugging visualized by AFM and the NEER increase also match quantitatively we assume that plugged NPCs are electrically tight. This could be caused by NPC ring dilation (see Fig. 1) with parallel occlusion of the peripheral channels. Indeed, the thickness of the cytoplasmic ring decreased by 10–30% when plugs were present in the central channels. This likely affects peripheral channel conductance. Similar conformational changes as NPC contraction in response to ATP (18) and NPC central channel occlusion in response to cisternal calcium store depletion (49) have been observed by AFM in the past. In nuclei of salivary gland cells of Chironomus thummi Ito and Loewenstein (50) showed as long ago as 1965 that the steroid hormone 20-OH ecdysone induces changes in NEER. This phenomenon could be explained by the ecdysone-induced transcriptional activity of the cell nucleus.

We recently described a method for plug harvesting from NPCs by using the AFM as a nanosurgical tool (25). Using this technique it should be feasible to harvest plugs and decipher their mRNA by using molecular biological techniques. This approach could lead to the sequential identification of aldosterone-induced early genes. One of the advantages of the oocyte system is that mature stage VI oocytes are inactive in terms of transcription (51, 52) but can be activated by exogenous stimuli (53). Here we showed that stage VI oocytes respond to aldosterone by triggering a fast signal cascade involving the cell nucleus.

In Fig. 8 we have modeled individual NPCs as supramolecular structures with high plasticity. In response to aldosterone the activated intracellular receptor proteins dock to the individual NPC rings, occluding the peripheral channels and causing NPC ion conductances to decrease. Then, the receptor is translocated and transcription occurs. This process is indicated by nuclear swelling, peripheral channel expansion, and concomitant high NPC ion conductance. Inorganic ions can easily move between cytosolic and nucleoplasmic compartments to the sites of transcription. The final step in steroid-induced NPC function is mRNA export (indicated by plug movement through the NPC central channel). NPC rings are dilated, which causes peripheral channels to collapse. Consequently, NPC ion conductance is low, which prevents further decondensation of the chromatin.

Figure 8.

Figure 8

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Activation cycle of an individual NPC in response to the steroid hormone aldosterone. Transport of macromolecules induces conformational changes in NPC structure reflected in NPC ion permeability.

In conclusion, these technical approaches open the way to further investigation of the components involved in hormonal signaling.

Supplementary Material

Supporting Movie
pnas_092140799_index.html (917B, html)

Acknowledgments

The excellent technical assistance of Marianne Wilhelmi and Barbara Windoffer is gratefully acknowledged. We thank our medical students Andrea Schlune, Ilsa Buchholz, and Karoline Enss for many helpful discussions during the course of the experiments and Prof. Hatt, Department of Physiology, University of Bochum, for his constant support of our work. The study was supported by the Interdisziplinäre Zentrum für Klinische Forschung (IZKF, TP A9) and the VW-Stiftung (AZ I/77299).

Abbreviations

NE

nuclear envelope

NEER

NE electrical resistance

NPC

nuclear pore complex

AFM

atomic force microscopy

NIM

nuclear isolation medium

NHT

nuclear hourglass technique

References

  • 1.Gekle M, Golenhofen N, Oberleithner H, Silbernagl S. Proc Natl Acad Sci USA. 1996;93:10500–10504. doi: 10.1073/pnas.93.19.10500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Harvey B J, Higgins M. Kidney Int. 2000;57:1395–1403. doi: 10.1046/j.1523-1755.2000.00981.x. [DOI] [PubMed] [Google Scholar]
  • 3.Wehling M, Ulsenheimer A, Schneider M, Neylon C, Christ M. Biochem Biophys Res Commun. 1994;204:475–481. doi: 10.1006/bbrc.1994.2484. [DOI] [PubMed] [Google Scholar]
  • 4.Oberleithner H, Weigt M, Westphale H-J, Wang W. Proc Natl Acad Sci USA. 1987;84:1464–1468. doi: 10.1073/pnas.84.5.1464. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Kroeger H. Exp Cell Res. 1966;41:64–80. doi: 10.1016/0014-4827(66)90547-7. [DOI] [PubMed] [Google Scholar]
  • 6.Schneider S, Wünsch S, Schwab A, Oberleithner H. Mol Cell Endocrinol. 1996;116:73–79. doi: 10.1016/0303-7207(95)03699-7. [DOI] [PubMed] [Google Scholar]
  • 7.Wünsch S, Schneider S, Schwab A, Oberleithner H. Cell Tissue Res. 1993;274:145–151. doi: 10.1007/BF00327995. [DOI] [PubMed] [Google Scholar]
  • 8.Allen T D, Cronshaw J M, Bagley S, Kiseleva E, Goldberg M W. J Cell Sci. 2000;113:1651–1659. doi: 10.1242/jcs.113.10.1651. [DOI] [PubMed] [Google Scholar]
  • 9.Görlich D, Mattaj I W. Science. 1996;271:1513–1518. doi: 10.1126/science.271.5255.1513. [DOI] [PubMed] [Google Scholar]
  • 10.Nigg E A. Nature (London) 1997;386:779–787. doi: 10.1038/386779a0. [DOI] [PubMed] [Google Scholar]
  • 11.Assandri R, Mazzanti M. J Membr Biol. 1997;157:301–309. doi: 10.1007/s002329900237. [DOI] [PubMed] [Google Scholar]
  • 12.Danker T, Mazzanti M, Tonini R, Rakowska A, Oberleithner H. Cell Biol Int. 1997;21:747–757. doi: 10.1006/cbir.1997.0219. [DOI] [PubMed] [Google Scholar]
  • 13.Mazzanti M, Bustamante J O, Oberleithner H. Physiol Rev. 2001;81:1–19. doi: 10.1152/physrev.2001.81.1.1. [DOI] [PubMed] [Google Scholar]
  • 14.Bustamante J O, Varanda W A. Braz J Med Biol Res. 1998;31:333–354. doi: 10.1590/s0100-879x1998000300003. [DOI] [PubMed] [Google Scholar]
  • 15.Danker T, Schillers H, Storck J, Shahin V, Krämer B, Wilhelmi M, Oberleithner H. Proc Natl Acad Sci USA. 1999;96:13531–13535. doi: 10.1073/pnas.96.23.13530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Oberleithner H. Steroids. 1999;64:42–50. doi: 10.1016/s0039-128x(98)00090-7. [DOI] [PubMed] [Google Scholar]
  • 17.Oberleithner H, Reinhardt J, Schillers H, Pagel P, Schneider S W. Cell Physiol Biochem. 2000;10:429–434. doi: 10.1159/000016379. [DOI] [PubMed] [Google Scholar]
  • 18.Rakowska A, Danker T, Schneider S W, Oberleithner H. J Membr Biol. 1998;163:129–136. doi: 10.1007/s002329900377. [DOI] [PubMed] [Google Scholar]
  • 19.Stoffler D, Fahrenkrog B, Aebi U. Curr Opin Cell Biol. 1999;11:391–401. doi: 10.1016/S0955-0674(99)80055-6. [DOI] [PubMed] [Google Scholar]
  • 20.Danker T, Shahin V, Schlune A, Schäfer C, Oberleithner H. J Membr Biol. 2001;184:91–99. doi: 10.1007/s00232-001-0078-1. [DOI] [PubMed] [Google Scholar]
  • 21.Henderson R M, Schneider S, Li Q, Hornby D, White S J, Oberleithner H. Proc Natl Acad Sci USA. 1996;93:8756–8760. doi: 10.1073/pnas.93.16.8756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Schneider S W, Lärmer J, Henderson R M, Oberleithner H. Pflügers Arch. 1998;435:362–367. doi: 10.1007/s004240050524. [DOI] [PubMed] [Google Scholar]
  • 23.Berge T, Ellis D J, Dryden D T, Edwardson J M, Henderson R M. Biophys J. 2000;79:479–484. doi: 10.1016/S0006-3495(00)76309-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Pietrasanta L I, Thrower D, Hsieh W, Rao S, Stemmann O, Lechner J, Carbon J, Hansma H. Proc Natl Acad Sci USA. 1999;96:3757–3762. doi: 10.1073/pnas.96.7.3757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Oberleithner H, Schäfer C, Shahin V, Schlune A, Schillers H, Reinhardt J. Single Mol. 2001;2:117–120. [Google Scholar]
  • 26.Oberleithner H, Schillers H, Wilhelmi M, Butzke D, Danker T. Pflügers Arch. 2000;439:251–255. doi: 10.1007/s004249900183. [DOI] [PubMed] [Google Scholar]
  • 27.Stoffler D, Goldie K N, Feja B, Aebi U. J Mol Biol. 1999;287:741–752. doi: 10.1006/jmbi.1999.2637. [DOI] [PubMed] [Google Scholar]
  • 28.Wang H, Clapham D E. Biophys J. 1999;77:241–247. doi: 10.1016/S0006-3495(99)76885-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Oberleithner H, Brinckmann E, Schwab A, Krohne G. Proc Natl Acad Sci USA. 1994;91:9784–9788. doi: 10.1073/pnas.91.21.9784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Folprecht G, Schneider S, Oberleithner H. Pflügers Arch. 1996;432:831–838. doi: 10.1007/s004240050205. [DOI] [PubMed] [Google Scholar]
  • 31.Arriza J L, Weinberger C, Cerelli G, Glaser T M, Handelin B L, Housman D E, Evans R M. Science. 1987;237:268–275. doi: 10.1126/science.3037703. [DOI] [PubMed] [Google Scholar]
  • 32.Newmeyer D D, Forbes D J. Cell. 1988;52:641–653. doi: 10.1016/0092-8674(88)90402-3. [DOI] [PubMed] [Google Scholar]
  • 33.Robert C, Barnes F L, Hue I, Sirard M A. Mol Reprod Dev. 2000;57:167–175. doi: 10.1002/1098-2795(200010)57:2<167::AID-MRD8>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  • 34.Hinshaw J E, Carragher B O, Milligan R A. Cell. 1992;69:1133–1141. doi: 10.1016/0092-8674(92)90635-p. [DOI] [PubMed] [Google Scholar]
  • 35.Shahin V, Danker T, Enss K, Ossig R, Oberleithner H. FASEB J. 2001;15:1895–1901. doi: 10.1096/fj.00-0838com. [DOI] [PubMed] [Google Scholar]
  • 36.Bustamante J O, Michelette E R, Geibel J P, Dean D A, Hanover J A, McDonnell T J. Pflügers Arch. 2000;439:433–444. doi: 10.1007/s004249900189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Bading H, Hardingham G E, Johnson C M, Chawla S. Biochem Biophys Res Commun. 1997;236:541–543. doi: 10.1006/bbrc.1997.7037. [DOI] [PubMed] [Google Scholar]
  • 38.Berridge M J. Nature (London) 1997;386:759–760. doi: 10.1038/386759a0. [DOI] [PubMed] [Google Scholar]
  • 39.Berridge M J, Bootman M D, Lipp P. Nature (London) 1998;395:645–648. doi: 10.1038/27094. [DOI] [PubMed] [Google Scholar]
  • 40.Hardingham G E, Chawla S, Johnson C M, Bading H. Nature (London) 1997;385:260–265. doi: 10.1038/385260a0. [DOI] [PubMed] [Google Scholar]
  • 41.Sperling R, Koster A J, Melamed-Bessudo C, Rubinstein A, Angenitzki M, Berkovitch-Yellin Z, Sperling J. J Mol Biol. 1997;267:570–583. doi: 10.1006/jmbi.1997.0898. [DOI] [PubMed] [Google Scholar]
  • 42.Stevens B J, Swift H. J Cell Biol. 1966;31:55–77. doi: 10.1083/jcb.31.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Lang F, Cohen P. Science's STKE. 2001. http://stke.sciencemag.org/cgi/content/full/OC_sigtrans , http://stke.sciencemag.org/cgi/content/full/OC_sigtrans;2001/108/re17. ;2001/108/re17. [DOI] [PubMed] [Google Scholar]
  • 44.Pearce D, Verrey F, Chen S Y, Mastroberardino L, Meijer O C, Wang J, Bhargava A. Kidney Int. 2000;57:1283–1289. doi: 10.1046/j.1523-1755.2000.00963.x. [DOI] [PubMed] [Google Scholar]
  • 45.Alvarez D L R, Zhang P, Naray-Fejes-Toth A, Fejes-Toth G, Canessa C M. J Biol Chem. 1999;274:37834–37839. doi: 10.1074/jbc.274.53.37834. [DOI] [PubMed] [Google Scholar]
  • 46.Naray-Fejes-Toth A, Fejes-Toth G. Kidney Int. 2000;57:1290–1294. doi: 10.1046/j.1523-1755.2000.00964.x. [DOI] [PubMed] [Google Scholar]
  • 47.Shigaev A, Asher C, Latter H, Garty H, Reuveny E. Am J Physiol. 2000;278:F613–F619. doi: 10.1152/ajprenal.2000.278.4.F613. [DOI] [PubMed] [Google Scholar]
  • 48.Verrey F. Am J Physiol. 1999;277:F319–F327. doi: 10.1152/ajprenal.1999.277.3.F319. [DOI] [PubMed] [Google Scholar]
  • 49.Perez-Terzic C, Pyle J, Jaconi M, Stehno-Bittel L, Clapham D E. Science. 1996;273:1875–1877. doi: 10.1126/science.273.5283.1875. [DOI] [PubMed] [Google Scholar]
  • 50.Ito S, Loewenstein W R. Science. 1965;150:909–910. doi: 10.1126/science.150.3698.909. [DOI] [PubMed] [Google Scholar]
  • 51.Dumont J N. J Morphol. 1972;136:153–179. doi: 10.1002/jmor.1051360203. [DOI] [PubMed] [Google Scholar]
  • 52.Golden L, Schafer U, Rosbash M. Cell. 1980;22:835–844. doi: 10.1016/0092-8674(80)90560-7. [DOI] [PubMed] [Google Scholar]
  • 53.Leonard D A, La Marca M J. Dev Biol. 1975;45:199–202. doi: 10.1016/0012-1606(75)90254-7. [DOI] [PubMed] [Google Scholar]

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