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
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