Marina Zelenina

Regulation of brain aquaporins

Emerging evidence suggests that brain aquaporins (AQP) play important roles for the dynamic regulation of brain water homeostasis and for the regulation of cerebrospinal fluid production. This review deals with the short- and long-term regulation of AQP4 and AQP9, both expressed in astrocytes, and of AQP1, expressed in the choroid plexus. AQP1 and 4 have in other cell types been shown to be regulated by phosphorylation. Phosphorylation affects the gating of AQP4 and the trafficking and insertion into membrane of AQP1. Mercury inhibits the water permeability of AQP1 and AQP9, but not AQP4. The permeability of AQP4 is increased by lead. AQP4 is also regulated by protein-protein interaction. The assembly between AQP4 and syntrophin is required for the proper localization of AQP4 in the astrocyte plasma membrane that faces capillaries. There is evidence from studies on peripheral tissues that steroid hormones regulate the expression of AQP1, AQP4 and AQP9. There is also evidence that the expression of AQP1 can be regulated by ubiquitination, and that osmolality can regulate the expression of AQP1, AQP4 and AQP9. Further insight into the mechanisms by which brain AQPs are regulated will be of utmost clinical importance, since perturbed water flow via brain AQPs has been implicated in many neurological diseases and since, in brain edema, water flow via AQP4 may have a harmful effect.

Arginine vasopressin stimulates phosphorylation of aquaporin-2 in rat renal tissue

Aquaporin-2 (AQP2), the protein that mediates arginine vasopressin (AVP)-regulated apical water transport in the renal collecting duct, possesses a single consensus phosphorylation site for cAMP-dependent protein kinase A (PKA) at Ser256. The aim of this study was to examine whether AVP, and other agents that increase cAMP levels, could stimulate the phosphorylation of AQP2 in intact rat renal tissue. Rat renal papillae were prelabeled with32P and incubated with vehicle or drugs, and then AQP2 was immunoprecipitated. Two polypeptides corresponding to nonglycosylated (29 kDa) and glycosylated (35-48 kDa) AQP2 were identified by SDS-PAGE. AVP caused a time- and dose-dependent increase in phosphorylation of both glycosylated and nonglycosylated AQP2. The threshold dose for a significant increase in phosphorylation was 10 pM, which corresponds to a physiological serum concentration of AVP. Maximal phosphorylation was reached within 1 min of AVP incubation. This effect on AQP2 phosphorylation was mimicked by the vasopressin (V2) agonist, 1-desamino-[8-d-arginine]vasopressin (DDAVP), or forskolin. Two-dimensional phosphopeptide mapping indicated that AVP and forskolin stimulated the phosphorylation of the same site in AQP2. Immunoblot analysis using a phosphorylation state-specific antiserum revealed an increase in phosphorylation of Ser256 after incubation of papillae with AVP. The results indicate that AVP stimulates phosphorylation of AQP2 at Ser256via activation of PKA, supporting the idea that this is one of the first steps leading to increased water permeability in collecting duct cells.Aquaporin-2 (AQP2), the protein that mediates arginine vasopressin (AVP)-regulated apical water transport in the renal collecting duct, possesses a single consensus phosphorylation site for cAMP-dependent protein kinase A (PKA) at Ser256. The aim of this study was to examine whether AVP, and other agents that increase cAMP levels, could stimulate the phosphorylation of AQP2 in intact rat renal tissue. Rat renal papillae were prelabeled with 32P and incubated with vehicle or drugs, and then AQP2 was immunoprecipitated. Two polypeptides corresponding to nonglycosylated (29 kDa) and glycosylated (35-48 kDa) AQP2 were identified by SDS-PAGE. AVP caused a time- and dose-dependent increase in phosphorylation of both glycosylated and nonglycosylated AQP2. The threshold dose for a significant increase in phosphorylation was 10 pM, which corresponds to a physiological serum concentration of AVP. Maximal phosphorylation was reached within 1 min of AVP incubation. This effect on AQP2 phosphorylation was mimicked by the vasopressin (V2) agonist, 1-desamino-[8-D-arginine]vasopressin (DDAVP), or forskolin. Two-dimensional phosphopeptide mapping indicated that AVP and forskolin stimulated the phosphorylation of the same site in AQP2. Immunoblot analysis using a phosphorylation state-specific antiserum revealed an increase in phosphorylation of Ser256 after incubation of papillae with AVP. The results indicate that AVP stimulates phosphorylation of AQP2 at Ser256 via activation of PKA, supporting the idea that this is one of the first steps leading to increased water permeability in collecting duct cells.

Identification of a molecular target for glutamate regulation of astrocyte water permeability

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.

Regulation of brain aquaporins.

Three aquaporins are expressed in the brain. AQP4, the predominant brain water channel, is expressed in astrocyte endfeet facing brain capillaries, perisynaptic spaces, and nodes of Ranvier. It is implicated in brain edema formation and resolution. It is also believed to assist clearance of K(+) released during neuronal activity. AQP1 is expressed in epithelial cells of choroid plexus and is implicated in cerebrospinal fluid formation. AQP9, which has been reported to be present in astrocytes and in subpopulations of neurons, is implicated in the brain energy metabolism. All three brain AQPs are strongly upregulated in brain tumors and in injured brain tissue. Water and solute transport via AQPs depends on concentration gradients across the membrane, but the magnitude of the transport is to a large extent determined by the single channel permeability of AQPs and by their abundance in the cell membrane. The future therapies will have to address not only the forces driving the water and solute transport (e.g. as mannitol infusion does in the treatment of brain edema), but also the regulation of AQPs, which provide the means for water entry to the brain, for water exit from the brain, and for redistribution of water and solutes within the brain compartments. This review summarizes the data concerning structure, permeability, role in the brain, short-term and long-term regulation of the three AQPs.

Erythropoietin modulation of astrocyte water permeability as a component of neuroprotection

Disturbed brain water homeostasis with swelling of astroglial cells is a common complication in stroke, trauma, and meningitis and is considered to be a major cause of permanent brain damage. Astroglial cells possess the water channel aquaporin 4 (AQP4). Recent studies from our laboratory have shown that glutamate, acting on group I metabotropic glutamate receptors (mGluRs), increases the permeability of astrocyte AQP4, which, in situations of hypoxia-ischemia, will increase astrocyte water uptake. Here we report that erythropoietin (EPO), which in recent years has emerged as a potent neuro-protective agent, antagonizes the effect of a group I mGluR agonist on astrocyte water permeability. Activation of group I mGluRs triggers fast and highly regular intracellular calcium oscillations and we show that EPO interferes with this signaling event by altering the frequency of the oscillations. These effects of EPO are immediate, in contrast to the neuroprotective effects of EPO that are known to depend upon gene activation. Our findings indicate that EPO may directly reduce the risk of astrocyte swelling in stroke and other brain insults. In support of this conclusion we found that EPO reduced the neurological symptoms in a mouse model of primary brain edema known to depend upon AQP4 water transport.

Osmotic water permeability measurements using confocal laser scanning microscopy

Abstract We have developed a method for measurement of plasma membrane water permeability (Pf) in intact cells using laser scanning confocal microscopy. The method is based on confocal recording of the fluorescence intensity emitted by calcein-loaded adherent cells during osmotic shock. Pf is calculated as a function of the time constant in the fluorescence intensity change, the cell surface-to-volume ratio and the fractional content of the osmotically active cell volume. The method has been applied to the measurement of water permeability in MDCK cells. The cells behaved as linear osmometers in the interval from 100 to 350 mosM. About 57% of the total cell volume was found to be osmotically inactive. Water movement across the plasma membrane in intact MDCK cells was highly temperature dependent. HgCl2 had no effect on water permeability, while amphotericin B and DMSO significantly increased Pf values. The water permeability in MDCK cells transfected with aquaporin 2 was an order of magnitude higher than in the intact MDCK cell line. The water permeability of the nuclear membrane in both cell lines was found to be unlimited. Thus the intranuclear fluid belongs to the osmotically active portion of the cell. We conclude that the use of confocal microscopy provides a sensitive and reproducible method for measurement of water permeability in different types of adherent cells and potentially for coverslip-attached tissue preparations.

Lead induces increased water permeability in astrocytes expressing aquaporin 4

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P(f)) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on P(f) in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in P(f). The effect of lead was attenuated in the presence of a calcium (Ca(2+))/calmodulin-dependent protein kinase II (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased P(f) in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on P(f) in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.

Water channels (aquaporins) and their role for postnatal adaptation.

Birth is a transition from an underwater life in the uterus to a terrestrial life in a milieu where supply of water is limited. Rapid adaptation to the new environment is crucial for survival and health of infants. The discovery of a family of molecules—aquaporin (AQP) water channels—that are responsible for regulated water transport across cell membranes has made it possible to identify the molecular mechanisms behind the postnatal homeostatic adaptation and to better understand water imbalance–related disorders in infancy and childhood. Thirteen mammalian AQP isoforms have been identified, most of them having a unique tissue-specific pattern of expression. Most mammalian AQPs can be dynamically regulated, which makes them potential targets for the development of new drugs for diseases associated with disturbances in water homeostasis. This review deals with AQP in kidney, lung, and brain. Evidence is presented that AQPs are expressed in a specific age-dependent manner and that the timed expression of AQPs may have a crucial role during the early postnatal period.

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.

Expression of water and ion transporters in tracheal aspirates from neonates with respiratory distress.

Aim:  The aim of the study was to determine whether neonatal respiratory distress is related to changes in water and ion transporter expression in lung epithelium.