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 ...