Summary
Regulation of urea transport by vasopressin in inner medullary collecting duct (IMCD) cells is thought to be important for the urinary concentrating mechanism. Isolated tubule perfusion studies suggest the existence of a saturable urea carrier. We have measured14C-urea efflux in IMCD cells which were freshly isolated and grown in primary culture. Cells were isolated from rat papilla by collagenase digestion and hypotonic shock. In suspended cells,14C-urea efflux (J urea from loaded cells was exponential with time constant 59±3 sec (sem,n=6, 23°C).J urea had an activation energy of 4.1 kcal/mole and was inhibited 42±7% by 0.25mm phloretin and 30–40% by the high affinity urea analogues dimethylurea and phenylurea.J urea was increased 40–60% by addition of vasopressin (10−8 m) or 8-bromo-cAMP (1mm); stimulatedJ urea was inhibited 55±8% by the kinase A inhibitor H-8. Phorbol esters and epidermal growth factor did not alterJ urea. IMCD cells grown in primary culture were homogeneous in appearance with>fivefold stimulation of cAMP by vasopressin. The exponential time constant for urea efflux was 610±20 sec (n=3).J urea was not altered by vasopressin, cAMP or phloretin. Another function of in vivo IMCD cells, vasopressin-dependent formation of endosomes containing water channels, was absent in the cultured cells. These results demonstrate presence of a urea transporter on suspended IMCD cells which is activated by cAMP and inhibited by phloretin and urea analogues. The urea transporter and its regulation by cAMP, and cAMP-dependent apical membrane endocytosis, are lost after growth in primary culture.
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References
Brown, D. 1989. Membrane recycling and epithelial cell function.Am. J. Physiol. 256:F1-F12
Chen, P.-Y., Pearce, D., Verkman, A.S. 1988. Membrane water and solute permeability determined quantitatively by self-quenching of an entrapped fluorophore.Biochemistry 27:5713–5719
Chen, P.-Y., Verkman, A.S. 1987. Non-electrolyte transport across renal proximal tubule cell membranes measured by tracer efflux and light scattering.Pfluegers Arch. 408:491–496
Chou, C.-L., Knepper, M.A. 1989. Inhibition of urea transport in inner medullary collecting duct by phloretin and urea analogues.Am. J. Physiol. 257:F359-F365
Clapp, W.L., Madsen, K.M., Verlander, J.W., Tisher, C.C. 1989. Morphological heterogeneity along the rat inner medullary collecting duct.Lab. Invest. 60:219–230
Dix, J.A., Ausiello, D.A., Jung, C.Y., Verkman, A.S. 1985. Target analysis studies of red cell water and urea transport.Biochim. Biophys. Acta 821:243–252
Gallucci, E., Micelli, S., Lippe, C. 1971. Non-electrolyte permeability across thin lipid membranes.Arch. Int. Physiol. Biochim. 79:881–887
Grenier, F.C., Rollins, T.E., Smith, W.L. 1981. Kinin-induced prostaglandin synthesis by renal papillary collecting tubule cells in culture.Am. J. Physiol. 241:F94-F104
Grupp, C., Pavenstadt-Grupp, I., Grunewald, R.W., Bevan, C., Stokes, J.B., Kinne, R.K.H. 1989. A Na−K−Cl cotransporter in isolated rat papillary collecting duct cells.Kidney Int. 36:201–209
Handler, J.S., Preston, A.S., Steele, R.E. 1984. Factors affecting the differentiation of epithelial transport and responsiveness to hormones.Fed. Proc. 43:2221–2224
Husted, R.F., Hayashi, M., Stokes, J.B. 1988. Characteristics of papillary collecting duct cells in primary culture.Am. J. Physiol. 255:F1160-F1169
Johnson, J.P., Steele, R.E., Perkins, F.M., Wade, J.B., Preston, A.S., Green, S.W., Handler, J.S. 1981. Epithelial organization and hormone sensitivity of toad urinary bladder cells in culture.Am. J. Physiol. 241:F129-F138
Knepper, M.A., Sands, J.M., Chou, C.-L. 1989. Independence of urea and water transport in rat inner medullary collecting duct.Am. J. Physiol. 256:F610-F621
Lencer, W.I., Weyer, P., Verkman, A.S., Ausiello, D.A., Brown, D. 1990. FITC-dextran as a probe for endosome function and localization in kidney.Am. J. Physiol. 258:C309-C317
Macey, R.I. 1984. Transport of water and urea in red blood cells.Am. J. Physiol. 246:C195-C203
Mayrand, R.R., Levitt, D.G. 1983. Urea and ethylene glycolfacilitated transport systems in the human red cell membrane.J. Gen. Physiol. 81:221–237
Meyer, M.M., Verkman, A.S. 1986. Human platelet osmotic water and nonelectrolyte transport.Am. J. Physiol. 251:C549-C557
Nord, E.P., Hart, D. 1989. Basolateral-equivalent pH i recovery pathways from an imposed acid load in cultured rat inner medullary collecting duct cells.Am. Soc. Nephrol. Meet. Abstr. 355a
O'Grady, S.M., DeJonge, H.R., Vaandrager, A.B., Field, M. 1988. Cyclic nucleotide-dependent protein kinase inhibition by H-8: Effect on ion transport.Am. J. Physiol. 254:C115-C121
Sands, J.M., Knepper, M.A. 1987. Urea permeability of mammalian inner medullary collecting duct system and papillary surface epithelium.J. Clin. Invest. 79:138–147
Sands, J.M., Nonoguchi, H., Knepper, M.A. 1987. Vasopressin effects on urea and H2O transport in inner medullary collecting duct segments.Am. J. Physiol. 25:F823-F832
Shi, L.-B., Brown, D., Verkman, A.S. 1990. Water, proton and urea permeability in endosomes from toad urinary bladder that contain the vasopressin-sensitive water channel.J. Gen. Physiol. 95:941–960
Shi, L.-B., Verkman, A.S. 1989. Very high water permeability in vasopressin-dependent endocytic vesicles in toad urinary bladder.J. Gen. Physiol. 94:1101–1115
Star, R.A. 1990. Apical membrane limits urea permeability across the rat inner medullary collecting duct.J. Clin. Invest. (in press)
Star, R.A., Nonoguchi, H., Balaban, R., Knepper, M.A. 1988. Calcium and cyclic adenosine monophosphate as second messengers for vasopressin in the rat inner medullary collecting duct.J. Clin. Invest. 81:1879–1888
Stokes, J.B., Grupp, C., Kinne, R.K.H. 1987. Purification of rat papillary collecting duct cells: Functional and metabolic assessment.Am. J. Physiol. 253:F251-F262
Teitelbaum, I., Berl, T. 1986. Effects of calcium on vasopressin-mediated cyclic adenosine monophosphate formation in cultured rat inner medullary collecting tubule cells. Evidence for the role of intracellular calcium.J. Clin. Invest. 77:1574–1583
Teitelbaum, I., Strasheim, A. 1989. Vasopressin-stimulated phospholipase C activity in the rat inner medullary collecting tubule is mediated by the oxytocin receptor.Am. Soc. Nephrol. Meet. Abstr. 15a
Verkman, A.S. 1989. Mechanisms and regulation of water permeability in renal epithelia.Am. J. Physiol. 257:C837-C850
Verkman, A.S., Lencer, W., Brown, D., Ausiello, D.A. 1988. Endosomes from kidney collecting tubule contain the vasopressin-sensitive water channel.Nature (London) 333:268–269
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Zhang, R., Verkman, A.S. Urea transport in freshly isolated and cultured cells from rat inner medullary collecting duct. J. Membrain Biol. 117, 253–261 (1990). https://doi.org/10.1007/BF01868455
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DOI: https://doi.org/10.1007/BF01868455