Cotransport of water by the Na+/glucose cotransporter

Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na+/glucose cotransporter. The Na+/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na+/Cl−/γ-aminobutyric acid, Na+/iodide and H+/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.

Transport of uncharged organic solutes in Xenopus oocytes expressing red cell anion exchangers (AE1s)

When expressed in Xenopus oocytes, the trout red cell anion exchanger tAE1, but not the mouse exchanger mAE1, elicited a transport of electroneutral solutes (sorbitol, urea) in addition to the expected anion exchange activity. Chimeras constructed from mAE1 and tAE1 allowed us to identify the tAE1 domains involved in the induction of these transports. Expression of tAE1 (but not mAE1) is known to generate an anion conductance associated with a taurine transport. The present data provide evidence that (i) the capacity of tAE1 and tAE1 chimeras to generate urea and sorbitol permeability also was associated with an anion conductance; (ii) the same inhibitors affected both the permeability of solutes and anion conductance; and (iii) no measurable water transport was associated with the tAE1-dependent conductance. These results support the view that fish red blood cells, to achieve cell volume regulation in response to hypotonic swelling, activate a tAE1-associated anion channel that can mediate the passive transport of taurine and electroneutral solutes.

Osmotic Water Permeability of Isolated Protoplasts. Modifications during Development1

A transference chamber was developed to measure the osmotic water permeability coefficient (Pos) in protoplasts 40 to 120 μm in diameter. The protoplast was held by a micropipette and submitted to a steep osmotic gradient created in the transference chamber. Pos was derived from the changes in protoplast dimensions, as measured using a light microscope. Permeabilities were in the range 1 to 1000 μm s−1 for the various types of protoplasts tested. The precision for Pos was ≤40%, and within this limit, no asymmetry in the water fluxes was observed. Measurements on protoplasts isolated from 2- to 5-d-old roots revealed a dramatic increase in Pos during root development. A shift in Pos from 10 to 500 μm s−1 occurred within less than 48 h. This phenomenon was found in maize (Zea mays), wheat (Triticum aestivum), and rape (Brassica napus) roots. These results show that early developmental processes modify water-transport properties of the plasma membrane, and that the transference chamber is adapted to the study of water-transport mechanisms in native membranes.

The human AQP4 gene: definition of the locus encoding two water channel polypeptides in brain.

The aquaporin family of membrane water transport proteins are expressed in diverse tissues, and in brain the predominant water channel protein is AQP4. Here we report the isolation and characterization of the human AQP4 cDNAs and genomic DNA. Two cDNAs were isolated corresponding to the two initiating methionines (M1 in a 323-aa polypeptide and M23 in a 301-aa polypeptide) previously identified in rat [Jung, J.S., Bhat, R.V., Preston, G.M., Guggino, W.B. & Agre, P. (1994) Proc. Natl. Acad. Sci. USA 91, 13052-13056]. Similar to other aquaporins, the AQP4 gene is composed of four exons encoding 127, 55, 27, and 92 amino acids separated by introns of 0.8, 0.3, and 5.2 kb. Unlike other aquaporins, an alternative coding initiation sequence (designated exon 0) was located 2.7 kb upstream of exon 1. When spliced together, M1 and the subsequent 10 amino acids are encoded by exon 0; the next 11 amino acids and M23 are encoded by exon 1. Transcription initiation sites have been mapped in the proximal promoters of exons 0 and 1. RNase protection revealed distinct transcripts corresponding to M1 and M23 mRNAs, and AQP4 immunoblots of cerebellum demonstrated reactive polypeptides of 31 and 34 kDa. Using a P1 and a lambda EMBL subclone, the chromosomal site of the human AQP4 gene was mapped to chromosome 18 at the junction of q11.2 and q12.1 by fluorescence in situ hybridization. These studies may now permit molecular characterization of AQP4 during human development and in clinical disorders.

Atrial natriuretic peptide-stimulated Ca2+ reabsorption in rabbit kidney requires membrane-targeted, cGMP-dependent protein kinase type II

Atrial natriuretic peptide (ANP) and nitric oxide (NO) are key regulators of ion and water transport in the kidney. Here, we report that these cGMP-elevating hormones stimulate Ca2+ reabsorption via a novel mechanism specifically involving type II cGMP-dependent protein kinase (cGK II). ANP and the NO donor, sodium nitroprusside (SNP), markedly increased Ca2+ uptake in freshly immunodissected rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). Although readily increasing cGMP, ANP and SNP did not affect Ca2+ and Na+ reabsorption in primary cultures of these segments. Immunoblot analysis demonstrated that cGK II, and not cGK I, was present in freshly isolated CNT and CCD but underwent a complete down-regulation during the primary cell culture. However, upon adenoviral reexpression of cGK II in primary cultures, ANP, SNP, and 8-Br-cGMP readily increased Ca2+ reabsorption. In contrast, no cGMP-dependent effect on electrogenic Na+ transport was observed. The membrane localization of cGK II proved to be crucial for its action, because a nonmyristoylated cGK II mutant that was shown to be localized in the cytosol failed to mediate ANP-stimulated Ca2+ transport. The Ca2+-regulatory function of cGK II appeared isotype-specific because no cGMP-mediated increase in Ca2+ transport was observed after expression of the cytosolic cGK Iβ or a membrane-bound cGK II/Iβ chimer. These results demonstrate that ANP- and NO-stimulated Ca2+ reabsorption requires membrane-targeted cGK II.

Characterization of a Maize Tonoplast Aquaporin Expressed in Zones of Cell Division and Elongation1

We studied aquaporins in maize (Zea mays), an important crop in which numerous studies on plant water relations have been carried out. A maize cDNA, ZmTIP1, was isolated by reverse transcription-coupled PCR using conserved motifs from plant aquaporins. The derived amino acid sequence of ZmTIP1 shows 76% sequence identity with the tonoplast aquaporin γ-TIP (tonoplast intrinsic protein) from Arabidopsis. Expression of ZmTIP1 in Xenopus laevis oocytes showed that it increased the osmotic water permeability of oocytes 5-fold; this water transport was inhibited by mercuric chloride. A cross-reacting antiserum made against bean α-TIP was used for immunocytochemical localization of ZmTIP1. These results indicate that this and/or other aquaporins is abundantly present in the small vacuoles of meristematic cells. Northern analysis demonstrated that ZmTIP1 is expressed in all plant organs. In situ hybridization showed a high ZmTIP1 expression in meristems and zones of cell enlargement: tips of primary and lateral roots, leaf primordia, and male and female inflorescence meristems. The high ZmTIP1 expression in meristems and expanding cells suggests that ZmTIP1 is needed (a) for vacuole biogenesis and (b) to support the rapid influx of water into vacuoles during cell expansion.

Visualization of Cavitated Vessels in Winter and Refilled Vessels in Spring in Diffuse-Porous Trees by Cryo-Scanning Electron Microscopy1

Xylem cavitation in winter and recovery from cavitation in the spring were visualized in two species of diffuse-porous trees, Betula platyphylla var. japonica Hara and Salix sachalinensis Fr. Schm., by cryo-scanning electron microscopy after freeze-fixation of living twigs. Water in the vessel lumina of the outer three annual rings of twigs of B. platyphylla var. japonica and of S. sachalinensis gradually disappeared during the period from January to March, an indication that cavitation occurs gradually in these species during the winter. In April, when no leaves had yet expanded, the lumina of most of the vessels of both species were filled with water. Many vessel lumina in twigs of both species were filled with water during the period from the subsequent growth season to the beginning of the next winter. These observations indicate that recovery in spring occurs before the onset of transpiration and that water transport through twigs occurs during the subsequent growing season. We found, moreover, that vessels repeat an annual cycle of winter cavitation and spring recovery from cavitation for several years until irreversible cavitation occurs.

Bronchiolar nonciliated secretory (Clara) cells: source of guanylin in the mammalian lung.

The peptide guanylin, which has recently been isolated from the intestine, is involved in the regulation of fluid secretion in the intestinal epithelium by activation of guanylate cyclase C, the putative guanylin receptor. Since the latter protein is also expressed in airway epithelia, we investigated the lung of three mammalian species for the presence and cellular localization of guanylin by immunoblot (Western blot) analyses and light and electron microscopical immunocytochemistry. In Western blots of bovine, guinea pig, and rat lung extracts, three different guanylin antisera directed against the midportion and against the C terminus of the precursor molecule identified a peptide band corresponding to the apparent molecular mass of guanylin. Localization studies in the lung revealed that guanylin is exclusively confined to nonciliated secretory (Clara) cells in the lining of distal conducting airways. The presence of guanylin in the lung and particularly its specific localization to Clara cells indicate that these cells may play a pivotal role in the local (paracrine) regulation of electrolyte/water transport in airway epithelia.