Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca

Vasopressin plays an essential role for the regulation of water balance by activating the collecting duct-specific water channel, aquaporin-2 (AQP2). Here we present evidence that vasopressin may also act as a long-term, transcriptional regulator of AQP2. The studies were performed on LLC-PK1 cells, which normally express V2 receptor (V2R) and which were transfected with a fragment of the human AQP2 promoter. Activation of the adenylate cyclase-coupled V2R in LLC-PK1 cells induced phosphorylation of adenosine 3',5'-cyclic monophosphate (cAMP) responsive element binding protein (CREB) and expression of c-Fos. Binding of these factors to the CRE and AP1 site did, in combination, lead to AQP2 promoter activation. These results establish the role of vasopressin as a regulator of transcription and are the first example of how a message from a highly specific receptor is, via a dual effect of the cAMP signal on CREB and immediate early gene expression, transduced to the transcription of a final target protein with known biological effects.

Astrocytes play a key role for maintenance of brain water homeostasis, but little is known about mechanisms of short-term regulation of astrocyte water permeability. Here, we report that glutamate increases astrocyte water permeability and that the molecular target for this effect is the aquaporin-4 (AQP4) serine 111 residue, which is in a strategic position for control of the water channel gating. The glutamate effect involves activation of group I metabotropic glutamate receptors (mGluR), intracellular calcium release, and activation of calcium/calmodulin-dependent protein kinase II (CaMKII) and nitric oxide synthase (NOS). The physiological impact of our results is underlined by the finding that mGluR activation increases the rate of hypoosmotic tissue swelling in acute rat hippocampal slices. Cerebral ischemia is associated with an excessive release of glutamate, and in postischemic cerebral edema ablation of AQP4 attenuates the degree of damage. Thus, we have identified AQP4 as the molecular target for drugs that may attenuate the development of brain edema.

(AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability (P f) was measured in individual LLC-PK 1 cells that were transiently transfected with AQP4. To identify which cells were transfected, AQP4 was tagged at the NH 2 terminus with green fluorescent protein.Transfected cells showed a strong fluorescent signal in basolateral membrane and a low-to-negligible signal in the cytosol and apical membrane. Activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) significantly decreased P f of cells expressing AQP4 but had no effect on neighboring untransfected cells. No redistribution of AQP4 in response to PDBu was detected. Dopamine also decreased the P f in transfected cells. The effect was abolished by the PKC inhibitor Ro 31-8220. Reduction of AQP4 water permeability by PDBu and dopamine was abolished by point mutation of Ser 180 , a consensus site for PKC phosphorylation. We conclude that PKC and dopamine decrease AQP4 water permeability via phosphorylation at Ser 180 and that the effect is likely mediated by gating of the channel. water channels; protein kinase C phosphorylation; LLC-PK 1 cells; green fluorescent protein; water transport CELLS THAT REQUIRE A HIGH water permeability, such as kidney epithelial cells, express specific water channels, aquaporins (AQPs; recently reviewed in Refs. 3 and 23). Because regulation of cell and total body water content is an essential homeostatic function, the question has been raised of whether the activity of AQPs is dynamically regulated by G protein-coupled receptors and intracellular messengers. Short-term regulation of the activity of AQPs by G protein-coupled receptors has until now mostly been studied for aquaporin-2 (AQP2) (18), the water channel that is expressed in kidney collecting duct and that is regulated by vasopressin (AVP) (6,8,9,17,27,28,40,45,48,50).Aquaporin-4 (AQP4) (22, 25) is expressed in collecting duct principal cells (13,14,55) and is important for concentration of urine (5). It has been shown that AQP4 can be phosphorylated by protein kinase C (PKC) in vitro, and, when expressed in Xenopus laevis oocytes, the water permeability of AQP4 is decreased by PKC activation (21). The physiological significance of these findings has not yet been revealed. Studies of the dynamic regulation of water permeability in welldifferentiated mammalian cells have so far been associated with a number of methodological problems. Here we employ a method that allows simultaneous studies of water permeability of renal epithelial cells that do or do not express AQP4 tagged with green fluorescent protein (GFP). By using this method, we show that the water permeability of AQP4 is downregulated by PKC activation and by dopamine. To examine whether the effect of PKC was direct, or mediated via an intermediary protein, studies were also performed with AQP4, where the consensus site for PKC phosphorylation, Ser 180

Nickel is a common cause of pneumoconiosis. Here, we show that nickel inactivates aquaporin (AQP)-3, the water channel expressed apically in epithelial cells of human terminal airways. Human AQP3 was transiently transfected into human lung cells, and water permeability was measured in transfected and neighboring untransfected cells. Incubation with NiCl 2 rapidly, dosedependently, and reversibly decreased water permeability in AQP3-expressing cells. Acidification of the extracellular medium also caused rapid, dose-dependent, and reversible inhibition of AQP3. Sensitivity of AQP3 to nickel was lower at alkaline pH than at neutral and acidic pH. Cells transfected with human AQP4 and AQP5, which are also expressed in airway epithelia, were insensitive to nickel and extracellular acidification. Zinc and cadmium, other common causes of pneumoconiosis, had no effect on the water permeability of AQP3. were also involved in regulation of AQP3 by extracellular pH. In addition, the aromatic side chain of His 154 was shown to be important for the water permeability of AQP3. Our results imply that nickel and extracellular pH may modulate lung water clearance and that defective water clearance may be an early component of nickel-induced lung disease.Nonenzymatic regulation of ion channels by Ni 2ϩ and other divalent cations or by pH is a well established phenomenon with many important physiological and pathophysiological implications. Less is known about nonenzymatic regulation of water channels, aquaporins (AQPs) 1 (1). Mercury inhibits most mammalian water channels via binding to cysteine residues (2-4) and has been an important tool in studies of AQPs. Gold and silver were recently reported to inhibit a water channel from human erythrocytes, presumably AQP1, but a molecular basis for this inhibition has not been revealed (5). The question of whether Ni 2ϩ and other divalent ions known to regulate the activity of ion channels modulate the activity of AQPs has, to our knowledge, not yet been addressed.Nickel is widely used in modern industry (reviewed in Ref. 6). Inhalation is the primary route of occupational exposure to nickel and other heavy metals, and inhalation of nickel compounds is a common cause of pneumoconiosis (6, 7). AQP3, AQP4, and AQP5 are expressed in the airway epithelia (8 -10). AQP3 is located at the apical membrane of human lung epithelium (10). Here, we have examined the effects of Ni 2ϩ on the water permeability of human AQP3, AQP4, and AQP5 expressed in a human lung cell line. Since AQP3 has, when expressed in oocytes, been reported to be pH-sensitive (11), we also examined the effect of extracellular acidification. We show that Ni 2ϩ and pH regulate the water permeability of human AQP3, but not of human AQP4 and AQP5. We also address the question of whether Ni 2ϩ and pH may interact in the regulation of human AQP3.Identification of the molecular sites responsible for the Ni 2ϩ and pH sensitivity of AQP3 is important for future development of therapeutic agents. Histidine, with a pK a of ϳ6.5, is the ...

Aquaporins are a family of water channels found in animals, plants, and microorganisms. A subfamily of aquaporins, the aquaglyceroporins, are permeable for water as well as certain solutes such as glycerol, lactate, and urea. Here we show that the brain contains two isoforms of AQP9--an aquaglyceroporin with a particularly broad substrate specificity--and that the more prevalent of these isoforms is expressed in brain mitochondria. The mitochondrial AQP9 isoform is detected as an approximately 25 kDa band in immunoblots. This isoform is likely to correspond to a new AQP9 mRNA that is obtained by alternative splicing and has a shorter ORF than the liver isoform. Subfractionation experiments and high-resolution immunogold analyses revealed that this novel AQP9 isoform is enriched in mitochondrial inner membranes. AQP9 immunopositive mitochondria occurred in astrocytes throughout the brain and in a subpopulation of neurons in the substantia nigra, ventral tegmental area, and arcuate nucleus. In the latter structures, the AQP9 immunopositive mitochondria were located in neurons that were also immunopositive for tyrosine hydroxylase, as demonstrated by double labeling immunogold electron microscopy. Our findings suggest that mitochondrial AQP9 is a hallmark of astrocytes and midbrain dopaminergic neurons. In physiological conditions, the flux of lactate and other metabolites through AQP9 may confer an advantage by allowing the mitochondria to adjust to the metabolic status of the extramitochondrial cytoplasm. We hypothesize that the complement of mitochondrial AQP9 in dopaminergic neurons may relate to the vulnerability of these neurons in Parkinson's disease.

Aquaporin-3 (AQP3) is an aquaglyceroporin expressed in erythrocytes and several other tissues. Erythrocytes are, together with kidney and liver, the main targets for copper toxicity. Here we report that both water and glycerol permeability of human AQP3 is inhibited by copper. Inhibition is fast, dose-dependent, and reversible. If copper is dissolved in carbonic acid-bicarbonate buffer, the natural buffer system in our body, doses in the range of those observed in Wilson disease and in copper poisoning caused significant inhibition. AQP7, another aquaglyceroporin, was insensitive to copper. Copper is an essential trace element in the human body. It is present in the blood and is incorporated in several vital proteins, such as Cu,Zn-SOD, dopamine ␤-hydroxylase, ceruloplasmin, cytochrome c oxidase, etc. (recently reviewed in Ref. 1). Blood levels of copper are increased in several pathological conditions, such as Wilson disease, Indian childhood cirrhosis, juvenile rheumatoid arthritis, thalassemia, and sickle cell anemia (1-5).Aquaporin-3 (AQP3) 1 is an aquaglyceroporin expressed in several mammalian tissues including erythrocytes (6 -12). It is well established that erythrocytes are permeable for glycerol, but the molecular mechanism for this effect is still obscure. It is also well established that glycerol permeability of erythrocytes is extremely sensitive to copper. We have recently shown that AQP3 water permeability is sensitive to nickel (13). Here we report that copper is a potent inhibitor of AQP3 permeability for glycerol and water. We expressed AQP3 in a human cell line and tested the effect of copper on glycerol and water permeability of the transfected cells. Three extracellular amino acid residues were found to be responsible for the copper sensitivity. We modeled the situation when AQP3 is co-expressed with another AQP, not permeable for glycerol, and found that only glycerol but not water permeability will be inhibited by copper in such cells. The results of this study provide a better understanding of processes that occur in severe copper metabolism defects and copper poisoning. MATERIALS AND METHODScDNA Constructs-The constructs for expression of human AQP3 (wild type and mutants) and the long form of AQP4 have been described previously (13). cDNA fragments encoding full-length AQP3 and AQP4 were obtained by amplification from the human lung QUICK-Clone cDNA library (Clontech). cDNA encoding mouse AQP7 was a kind gift from J. M. Carbrey and P. Agre (Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD). The cDNA fragments were subcloned in-frame into the pEGFP-N2 vector for AQP3 tagged with green fluorescent protein (GFP) at the COOH terminus into pIRES2-EGFP vector (Clontech) for AQP3 and GFP expressed in the same cell as separate proteins and into the pEGFP-C2 vector for AQP4 and AQP7 tagged with GFP at NH 2 termini. The point mutations in extracellular loops of human AQP3 were generated by PCR-based mutagenesis using wild-type cDNA as a template and c...

It now generally is agreed that Na,K-ATPase, in addition to its role in the maintenance of Na ؉ and K ؉ gradients across the cell membrane, plays a role in communicating information from the extracellular environment to intracellular signaling pathways. It was reported recently that interaction between ouabain-bound Na,K-ATPase and the 1,4,5-trisphosphate receptor (IP 3 R) triggers slow calcium oscillations and activation of NF-B. Here it is demonstrated that this signaling pathway can serve to prevent cell death and promote cell growth. Rat renal proximal tubular cells in primary culture first were grown in the presence of 10% serum and then exposed to 0.2% serum for 24 h to induce apoptosis. Serum starvation increased the apoptotic index from 1.21 ؎ 0.26 to 14.01 ؎ 1.17%. Ouabain in concentrations that did not inhibit Na,K-ATPase activity (1 to 10 nM) completely abolished the apoptotic effect of serum starvation. Ouabain protection from apoptosis was not observed when release of calcium from intracellular stores via the IP 3 R was prevented. It was shown that the NH 2 terminal tail of the Na,K-ATPase ␣ subunit plays a key role in ouabain-triggered calcium oscillations. It was found that ouabain did not protect from apoptosis in serum-deprived cells that expressed a mutant Na,K-ATPase ␣ subunit with deletion of the NH 2 terminal tail. . Recent studies suggest that Na,K-ATPase, in addition to being the major determinant of intracellular ion composition, may act as a signal transducer (2-5).Ouabain, a steroid derivative, is a specific ligand of Na,KATPase that dose-dependently inhibits the activity of Na,KATPase (6). Noninhibitory doses of ouabain activate the signaling function of Na,K-ATPase. The signaling cascade that is triggered by Na,K-ATPase is complex, and several different pathways have been implicated (7-9). We recently reported that ouabain can trigger an interaction between Na,K-ATPase and the inositol 1,4,5-trisphosphate (IP 3 ) receptor, which results in low-frequency Ca 2ϩ oscillations and activation of the transcription factor NF-B. This phenomenon was observed in rat renal proximal tubular cells in primary culture and in a kidney cell line (10).A number of recent studies have demonstrated that ouabain in noninhibitory doses can promote cell proliferation (11-13). Because NF-B has an antiapoptotic effect (14), we speculated that ouabain also might act to protect from apoptosis. Here we demonstrate that noninhibitory doses of ouabain can protect rat renal proximal tubular cells from serum deprivation-triggered apoptosis, albeit not from pharmacologically staurosporinetriggered apoptosis. We show that protection from serum-triggered apoptosis depends on NF-B activation. Normal kidney development is critically dependent on a well-controlled balance between cell proliferation and apoptosis (15). Accumulating evidence suggests that ouabain is a mammalian hormone, produced in the adrenal glands and the hypothalamus (16,17). Therefore, ouabain might play an important role as a modulator of kidney growth ...