© 2001 by The Society for Integrative and Comparative Biology
Organic Osmolyte Channels in the Renal Medulla: Their Properties and Regulation1
1 Max-Planck-Institut für molekulare Physiologie, Abteilung Epithelphysiologie, Otto-Hahn-Str. 11, 44227 Dortmund, Germany
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SYNOPSIS |
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 SYNOPSIS INTRODUCTIONCONTENT OF ORGANIC OSMOLYTES...PROPERTIES OF HYPOTONICITY...REGULATION OF ORGANIC OSMOLYTE...CONCLUDING REMARKSReferences |
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In the mammalian kidney renal medullary cells use organic osmolytes such as sorbitol, myo-inositol, glycerophosphorylcholine, betaine, and taurine to adjust their intracellular osmolarity (and thereby their volume) to rapid and drastic changes in extracellular osmolarity. Using an immortalized cell line derived from rabbit thick ascending limb of Henle's loop (TALH cells) and primary cultures of rat inner medullary collecting duct (IMCD cells) the membrane transport systems activated during exposure to hypotonicity were investigated. In TALH cells an increase in sorbitol permeability of the (luminal) plasma membrane occurs by activation of a channel-like transporter involving a calcium/calmodulin-dependent protein kinase. A similar system seems to operate in IMCD cells. In addition, the latter cells possess a swelling-activated anion channel that is also permeable for taurine and myo-inositol and inhibited by "anion channel" blockers, such as NPPB and DIDS. The sorbitol permeability of the plasma membrane appears to be furthermore regulated by a transient insertion of active transporters into the basolateral cell surface by a membrane recycling mechanism.
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INTRODUCTION |
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SYNOPSISINTRODUCTIONCONTENT OF ORGANIC OSMOLYTES...PROPERTIES OF HYPOTONICITY...REGULATION OF ORGANIC OSMOLYTE...CONCLUDING REMARKSReferences |
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Organic osmolytes play a major role in the volume regulation of renal cells located in the medulla and papilla of the mammalian kidney. Due to the operation of the countercurrent systems for urinary concentration and dilution the osmolarity of the extracellular space these cells are exposed to can vary widely and rapidly. The medullary cells, therefore, possess mechanisms that control their intracellular content of sodium, potassium, and chloride and use organic osmolytes to compensate for residual differences in osmolarity across the plasma membrane. This contribution compiles the knowledge about two renal medullary nephron segments accumulated thus far with regard to transport systems for organic osmolytes that are activated when the extracellular medium becomes hypotonic with regard to the intracellular medium. These segments are the thick ascending limb of Henle's loop and the papillary collecting duct. Here cell preparations are available which can be studied in vitro under well defined experimental conditions, thus enabling a delineation of their properties and their mode of regulation.
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CONTENT OF ORGANIC OSMOLYTES IN RENAL MEDULLARY CELLS |
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SYNOPSISINTRODUCTIONCONTENT OF ORGANIC OSMOLYTES...PROPERTIES OF HYPOTONICITY...REGULATION OF ORGANIC OSMOLYTE...CONCLUDING REMARKSReferences |
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As compiled in Table 1 immortalized thick ascending loop of Henle (TALH) cells derived from rabbit kidney when grown at different osmolarities contain considerably different amounts of organic osmolytes. At 600 mosmol/liter sorbitol, myo-inositol, and betaine are accumulated in the cell, contributing about 350 mosmol/liter to the intracellular osmolarity. When adapted to 300 mosmol/liter the largest changes are observed in the sorbitol and the myo-inositol content. The intracellular osmolarity contributed by organic osmolytes thereby drops to about 80 mosmol/liter. Thus, the change in extracellular osmolarity is completely compensated after a culture period of 5 days by organic osmolytes.
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In primary culture of rat inner medullary collecting duct (IMCD) cells taurine and glycerophosphorylcholine (GPC) accumulate, in addition to sorbitol, myo-inositol and betaine. At 600 mosmol extracellular osmolarity the total intracellular osmolarity of organic osmolytes amounts to about 250 mosmol/liter. When the osmolarity of the medium is reduced, again reduction of organic osmolyte content is a major mechanism for the adaptation of intracellular osmolarity.
There are several mechanisms by which renal cells increase their intracellular content of organic osmolytes during exposure to hypertonic media (for a review see Grunewald and Kinne, 1999
). Betaine, myoinositol and taurine are taken up by the cells via sodium (and chloride) dependent cotransport systems. These transporters are upregulated during the hypertonic stress. With regard to sorbitol the activity of aldose reductase increases in the cells which leads to an increase in the intracellular conversion of D-glucose to sorbitol. A third mechanism is the reduction of intracellular breakdown of the organic osmolyte. This appears to be the major route by which the intracellular content of glycerophosphorylcholine is augmented.
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PROPERTIES OF HYPOTONICITY-ACTIVATED ORGANIC OSMOLYTE RELEASE |
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When the osmolarity of the extracellular medium is acutely reduced, both, TALH cells and IMCD cells release organic osmolytes. In TALH cells mainly sorbitol is acutely released, it leaves cells grown on solid support across their luminal plasma membrane (Kinne-Saffran and Kinne, 1997
). Both efflux and influx of 3H-sorbitol are markedly stimulated under these conditions. Furthermore, as shown in Figure 1 the influx of labeled sorbitol is not altered when the concentration of nonlabeled substrate is increased—suggesting the activation of a channel-like transport system upon exposure to hypotonicity.
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In IMCD cells all cellular organic osmolytes leave the cells under hypotonic conditions (Grunewald and Kinne, 1999
; Ruhfus et al., 1998). In these cells electrophysiological measurements (Fig. 2) have revealed the presence of a hypotonicity-activated anion channel with an anion selectivity SCN– > I– > NO3– > Br– > Cl– > F– (Boese et al., 1996b; Sauer et al., 1995). Furthermore, the similarity in the time course of activation (Boese, 1996), an identical location exclusively at the basal pole of the cell (Ruhfus, 1996), and the very similar sensitivity to "anion channel" blockers, such as NPBB (5-nitro-2-(3-phenylpropylamino)benzoate) and DIDS (4,4'-diisothiocyanato-stilbene-2-2'-disulfonic acid) (Boese, 1996a) suggest that this channel is the major efflux pathway for taurine as well as myo-inositol (Ruhfus and Kinne, 1996).
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Studies on the efflux pathways for the other organic osmolytes in IMCD cells have revealed that there are marked differences in the osmotic threshold for activation, in the time course of activation, cellular location, and signal transduction pathways (Kinne, 1998
). Thus it appears that the efflux pathways for sorbitol, betaine and GPC are separate entities mediating the efflux of only one specific organic osmolyte. |
REGULATION OF ORGANIC OSMOLYTE CHANNELS |
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Modification of "resident" osmolyte channels
Studies on TALH cells demonstrated that the activation of the sorbitol efflux pathway was reduced when the calcium concentration in the extracellular medium was decreased (Kinne-Saffran and Kinne, 1997
). This result suggested the involvement of calcium-dependent kinases in the activation process. Indeed, as shown in Figure 3, it could be demonstrated in experiments employing a plasma membrane fraction enriched in luminal membranes that the sorbitol permeability of the plasma membranes could be markedly increased by the presence of ATP and calcium (in addition to magnesium and GTP) in the incubation medium. This ATP and calcium dependence could be shown to be most probably due to the modification (phosphorylation?) of the sorbitol channel by a calcium/calmodulin-dependent kinase since it could be achieved by prephosphorylation of the membranes and was attenuated by low concentrations of trifluoperazine (1 µM), high concentrations of staurosporine (200 µM) but not by the protein kinase C inhibitory peptide (Kinne-Saffran and Kinne, 1997).
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Also in the IMCD cells, there is a close correlation between the changes in intracellular calcium and the activation of the sorbitol efflux pathway as well as a decreased activation when the ATP content of the cells is reduced or the inhibitor trifluoperazine is used (Bevan et al., 1990
; Kinne et al., 1995, 1996; Mooren and Kinne, 1994; Ruhfus et al., 1996; Tinel et al., 1994, 1997). Thus, also in these cells a phosphorylation-dephosphorylation reaction appears to modulate sorbitol channels residing in the plasma membrane.
The membrane recycling mechanism
In epithelial cells stimulation of transport by transient insertion of membrane vesicles containing specific transport systems into the plasma membrane is a well established cellular principle (Brown, 1989). Such a principle seems to be also employed when regulating sorbitol permeability of the basal-lateral plasma membranes in IMCD cells.
Studies using fluid phase markers such as FITC-dextran proved that transient swelling of the cells leads—as shown in Figure 4—to an uptake of FITC-dextran exclusively at the basal-lateral cell pole (Czekay et al., 1994). During a second hypotonic stimulus FITC-dextran release and increase in sorbitol permeability are closely correlated with regard to the time of activation and their temperature sensitivity. In addition, both processes are calcium dependent and inhibited by cytochalasin D. Thus, in addition to the phosphorylation reaction postulated above, insertion and retrieval of sorbitol channels at the basal-lateral cell pole seem to play a major role in regulating the sorbitol permeability of the membrane.
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CONCLUDING REMARKS |
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SYNOPSISINTRODUCTIONCONTENT OF ORGANIC OSMOLYTES...PROPERTIES OF HYPOTONICITY...REGULATION OF ORGANIC OSMOLYTE...CONCLUDING REMARKSReferences |
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Organic osmolytes are almost universally used in nature when large differences in the intra- and extracellular osmolarity have to be compensated for (Yancey et al., 1982)
. As compatible and protective osmolytes they allow cells to regulate their cellular volume in such a way that inorganic osmolytes do not interfere with the cellular metabolism or cell membrane functions. The multiplicity of organic osmolytes and their diversity with regard to transport systems, in addition, makes it possible to fine tune the cellular responses and to be prepared for multiple (often unpredictable and random) disturbances of their volume.
Similarly osmolyte release channels are quite widely distributed within species and phyla (Kirk, 1997; Strange and Jackson, 1995). Their molecular nature is just beginning to be unraveled as are the signal transduction pathways and the molecular basis of regulation of their activity.
A comparative approach as taken in this article, in particular, by comparing two medullary cells which during organogenesis evolve from different tissues, and at this symposium, in general, will certainly contribute significantly to the in depth understanding of volume regulation. Besides the identification of molecular elements and events the integration of the various processes at the different levels of cellular organization poses a major challenge to future investigations dealing with one of the most basic phenomena in cell physiology.
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ACKNOWLEDGMENTS |
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The authors gratefully acknowledge the support by Christiane Pfaff, Sabine Hellwig, and Petra Glitz for cell culture and HPLC analysis and the excellent secretarial and expert editorial work of Daniela Mägdefessel and Brigitte Böhle.
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FOOTNOTES |
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1 From the Symposium Osmoregulation: An Integrated Approach presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, at Atlanta, Georgia.
2 E-mail: rolf.kinne{at}mpi-dortmund.mpg.de
3 Present address: Good Clinical Practices, Schering AG, Berlin, Germany
4 Present address: Department of Physiological Sciences, University of Newcastle Medical School, Newcastle upon Tyne, Great Britain
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SYNOPSISINTRODUCTIONCONTENT OF ORGANIC OSMOLYTES...PROPERTIES OF HYPOTONICITY...REGULATION OF ORGANIC OSMOLYTE...CONCLUDING REMARKSReferences |
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