Cellular and subcellular localization of aquaporins 1, 3, 8, and 9 in amniotic membranes during pregnancy in mice

The amniotic membrane encloses the amniotic fluid and plays roles in the regulation of amniotic fluid flux through the intramembranous pathway during pregnancy. Aquaporins (AQPs) 1, 3, 8, and 9 are expressed in amniotic membranes. AQPs are water channel proteins that facilitate the rapid flux of water or small molecules across the plasma membrane. Recently, additional roles of AQPs in facilitating cell migration, proliferation, and apoptosis have been suggested, with AQPs being distributed in the appropriate subcellular regions for their functions. The cellular and subcellular distributions of AQPs in the amniotic membrane however remain unclear. We have examined the cellular and subcellular localization of AQPs in amniotic membranes during pregnancy in mice. After embryonic day 12 (E12), AQP1 was distributed in the plasma membrane of finely branched cell processes in the amniotic fibroblasts. AQP3 was present in both epithelial cells and fibroblasts between E10 and E12. The distribution of AQP3 in the epithelial cells dynamically changed as follows: at E14 in the lateral membrane and apical junction; at E16 in the lateral membrane alone; at E17 in the lateral membrane and cytoplasm. AQP8 was expressed in the epithelial cells and complementarily localized in the apical junction and the lateral membrane. AQP9 was detected only in the apoptotic cells of the epithelium. These cellular and subcellular localizations of amniotic AQPs indicate that each AQP plays distinct functional roles, such as in water and urea transport, cell migration, cell proliferation, and apoptosis, for amniotic fluid homeostasis or tissue remodeling of amniotic membranes.     

Volume regulation in cortical collecting duct cells: role of AQP2

BACKGROUND INFORMATION: The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell-volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell-volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells. RESULTS: In the present study, we have studied the regulatory cell-volume responses to hypo-osmotic or hyperosmotic challenges in two CCD cell lines: one not expressing AQPs and the other stably transfected with AQP2. We have used a fluorescent probe technique in which the acquisition of single-cell kinetic data can be simultaneously recorded with the intracellular pH. Experiments with hyperosmotic mannitol media demonstrated that, independent of AQP2 expression, CCD cells shrink but fail to show regulatory volume increase, at least under the studied conditions. In contrast, under hypo-osmotic shocks, regulatory volume decrease occurs and the activation of these mechanisms is more rapid in AQP2 transfected cells. This regulatory response takes place in parallel with intracellular acidification, which is faster in cells expressing AQP2. The acidification and the initial regulatory volume decrease response were inhibited by glibenclamide and BaCl2 only in AQP2 cells. CONCLUSIONS: These results suggest that increases in the osmotic water permeability due to the expression of AQP2 are critical for a rapid activation of regulatory volume decrease mechanisms, which would be linked to cystic fibrosis transmembrane conductance regulator and to barium-sensitive potassium channels.     

Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation

Mammalian blastocyst formation is dependent on establishment of trophectoderm (TE) ion and fluid transport mechanisms. We have examined the expression and function of aquaporin (AQP) water channels during murine preimplantation development. AQP 3, 8, and 9 proteins demonstrated cell margin-associated staining starting at the 8-cell (AQP 9) or compacted morula (AQP 3 and 8) stages. In blastocysts, AQP 3 and 8 were detected in the basolateral membrane domains of the trophectoderm, while AQP3 was also observed in cell margins of all inner cell mass (ICM) cells. In contrast, AQP 9 was predominantly observed within the apical membrane domains of the TE. Murine blastocysts exposed to hyperosmotic culture media (1800 mOsm; 10% glycerol) demonstrated a rapid volume decrease followed by recovery to approximately 80% of initial volume over 5 min. Treatment of blastocysts with p-chloromercuriphenylsulfonic acid (pCMPS, > or =100 microM) for 5 min significantly impaired (P < 0.05) volume recovery, indicating the involvement of AQPs in fluid transport across the TE. Blastocysts exposure to an 1800-mOsm sucrose/KSOMaa solution did not demonstrate volume recovery as observed following treatment with glycerol containing medium, indicating glycerol permeability via AQPs 3 and 9. These findings support the hypothesis that aquaporins mediate trans-trophectodermal water movements during cavitation.     

Hyperosmotic induction of aquaporin expression in rat astrocytes through a different MAPK pathway

Water homeostasis of the nervous system is important during neural signal transduction. Astrocytes are crucial in water transport in the central nervous system under both physiological and pathological conditions. To date, five aquaporins (AQP) have been found in rat brain astrocytes. Most studies have focused on AQP4 and AQP9, however, little is known about the expression of AQP3, ‐5, and ‐8 as well as their regulating mechanism in astrocytes. The expression patterns of AQP3, ‐5, and ‐8 in astrocytes exposed to hyperosmotic solutions were examined to clarify the roles of AQP3, ‐5, and ‐8 in astrocyte water movement. The expression of AQP4 and AQP9 under the same hyperosmotic conditions was also investigated. The AQP4 and AQP9 expressions continuously increased until 12 h after hyperosmotic solution exposure, whereas the AQP3, ‐5, and ‐8 expressions continued to increase until 6 h after hyperosmotic solution exposure. The different AQPs decreased at corresponding time points (24 h for AQP4 andAQP9; 12 h for AQP3, ‐5, and ‐8 after hyperosmotic solution exposure). The ERK inhibitor can attenuate the expression of AQP3, ‐5, and ‐8 after hyperosmotic solution exposure. The p38 inhibitor can inhibit the AQP4 and AQP9 expressions in cultured astrocytes. AQP expression is directly related to the extracellular hyperosmotic stimuli. Moreover, different AQPs can be regulated by a distinct MAPK signal transduction pathway. J. Cell. Biochem. 114: 111–119, 2012. © 2012 Wiley Periodicals, Inc.     

Reciprocity in the Developmental Regulation of Aquaporins 1, 3 and 5 during Pregnancy and Lactation in the Rat

Milk secretion involves significant flux of water, driven largely by synthesis of lactose within the Golgi apparatus. It has not been determined whether this flux is simply a passive consequence of the osmotic potential between cytosol and Golgi, or whether it involves regulated flow. Aquaporins (AQPs) are membrane water channels that regulate water flux. AQP1, AQP3 and AQP5 have previously been detected in mammary tissue, but evidence of developmental regulation (altered expression according to the developmental and physiological state of the mammary gland) is lacking and their cellular/subcellular location is not well understood. In this paper we present evidence of developmental regulation of all three of these AQPs. Further, there was evidence of reciprocity since expression of the rather abundant AQP3 and less abundant AQP1 increased significantly from pregnancy into lactation, whereas expression of the least abundant AQP5 decreased. It would be tempting to suggest that AQP3 and AQP1 are involved in the secretion of water into milk. Paradoxically, however, it was AQP5 that demonstrated most evidence of expression located at the apical (secretory) membrane. The possibility is discussed that AQP5 is synthesized during pregnancy as a stable protein that functions to regulate water secretion during lactation. AQP3 was identified primarily at the basal and lateral membranes of the secretory cells, suggesting a possible involvement in regulated uptake of water and glycerol. AQP1 was identified primarily at the capillary and secretory cell cytoplasmic level and may again be more concerned with uptake and hence milk synthesis, rather than secretion. The fact that expression was developmentally regulated supports, but does not prove, a regulatory involvement of AQPs in water flux through the milk secretory cell.     

Immunolocalization of Aquaporin-1, -5, and -7 in the Avian Testis and Vas Deferens

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been found in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs have been poorly investigated in the male reproductive system of birds. The localization of AQP subtypes (AQP1, 2, 3, 4, 5, 7, 8, 9, and 11) in the goose testis and vas deferens has been studied through immunohistochemistry and immunobloting. Interestingly, the testicular and deferential tissues were positive for AQP1, -5, and -7 but not the others. AQP1 immunoreactivity was detected in the capillary endothelial cells of testis and vas deferens. AQP5 was localized in the interstitial tissue of the testis, including Leydig cells, as well as in the basal cells of vas deferens. Double-labeling confocal microscopy revealed coexpression of AQP5 with capillary AQP1 in the testis. AQP7 was expressed in elongated spermatid and spermatozoa tails in the testis, as well as spermatozoa tails in the vas deferens. These results suggest that several subtypes of AQPs are involved in the regulation of water homeostasis in the goose male reproductive system. (J Histochem Cytochem 57:915–922, 2009)     

Immunolocalization of aquaporin 1, 5, and 9 in the female pig reproductive system

Thirteen mammalian aquaporin (AQP) isoforms have been identified, and they have a unique tissue-specific pattern of expression. AQPs have been documented in the reproductive system of both male and female humans, rats, and mice. However, tissue expression and cellular and subcellular localization of AQPs are unknown in the female reproductive system of pigs. In this study, AQP1 immunoreactivity was detected in the capillary endothelium of the ovary. Distinct immunolabeling of capillary endothelium was also observed in the oviduct and uterus. AQP5 was expressed in flattened follicle cells of primordial follicles, granulosa cells of developing ovarian follicles, and muscle cells of the oviduct and uterus. Staining of AQP5 was also observed in the epithelial cells of the oviduct and uterine epithelium. AQP9 immunoreactivity was observed in granulosa cells of developing follicles. AQP9 was also localized in the luminal epithelial cells of the oviduct and uterine epithelia cells. This is, to our knowledge, the first study that shows tissue expression and cellular and subcellular localization of AQPs in the reproductive system of the female pig. Moreover, these results suggest that several subtypes of the AQPs (AQP1, 5, and 9) are involved in regulation of water homeostasis in the reproductive system of gilts.     

Polarized sorting of aquaporins 5 and 8 in stable MDCK-II transfectants

Localization of aquaporin (AQP) water channels to either apical or basolateral membranes is important for various epithelial functions. We have established MDCK-II cell transfectants stably expressing AQP5 (RW5 cells) or AQP8 (RW8 cells). The expression of both AQPs was confirmed by the results of immunofluorescence microscopy and immunoblot analysis. When grown on polycarbonate filters, osmotically-obliged transepithelial water flow across RW5 and RW8 monolayers was approximately 3-fold greater than that occurring across a monolayer of the parental cell line. Importantly, results of confocal immunofluorescence microscopy studies showed that AQP5 sorted to the apical membranes of RW5 cells. In contrast, AQP8 sorted to the basolateral membranes of RW8 cells. This is the first report of (i) stable epithelial cell cultures exhibiting a functional, polarized distribution of AQPs 5 and 8, and (ii) a basolateral localization of AQP8 in a polarized epithelial cell.     

Osteoclast differentiation and function in aquaglyceroporin AQP9-null mice

Background information. Osteoclasts are cells specialized for bone resorption and play important roles in bone growth and calcium homoeostasis. Differentiation of osteoclasts involves fusion of bone marrow macrophage mononuclear precursors in response to extracellular signals. A dramatic increase in osteoclast cell volume occurs during osteoclast biogenesis and is believed to be mediated by AQP9 (aquaporin 9), a membrane protein that can rapidly transport water and other small neutral solutes across cell membranes. Results. In the present study we report an increase in expression of AQP9 during differentiation of a mouse macrophage cell line into osteoclasts. Bone marrow macrophages from wild‐type and AQP9‐null mice differentiate into osteoclasts that have similar morphology, contain comparable numbers of nuclei, and digest synthetic bone to the same extent. Bones from wild‐type and AQP9‐null mice contain similar numbers of osteoclasts and have comparable density and structure as measured by X‐ray absorptiometry and microcomputed tomography. Conclusions. Our results confirm that AQP9 expression rises during osteoclast biogenesis, but indicate that AQP9 is not essential for osteoclast function or differentiation under normal physiological conditions.     

The NPC motif of aquaporin-11, unlike the NPA motif of known aquaporins, is essential for full expression of molecular function

The recently identified molecule aquaporin-11 (AQP11) has a unique amino acid sequence pattern that includes an Asn-Pro-Cys (NPC) motif, corresponding to the N-terminal Asn-Pro-Ala (NPA) signature motif of conventional AQPs. In this study, we examined the effect of the mutation of the NPC motif on the subcellular localization, oligomerization, and water permeability of AQP11 in transfected mammalian cells. Furthermore, the effect was also assessed using zebrafish. Site-directed mutation at the NPC motif did not affect the subcellular localization of AQP11 but reduced its oligomerization. A cell swelling assay revealed that cells expressing AQP11 with a mutated NPC motif had significantly lower osmotic water permeability than cells expressing wild-type AQP11. Zebrafish deficient in endogenous AQP11 showed a deformity in the tail region at an early stage of development. This phenotype was dramatically rescued by injection of human wild-type AQP11 mRNA, whereas the effect of mRNA for AQP11 with a mutated NPC motif was less marked. Although the NPA motif is known to be important for formation of water-permeable pores by conventional AQPs, our observations suggest that the corresponding NPC motif of AQP11 is essential for full expression of molecular function.     

Cellular localization of aquaporin mRNA in hybrid poplar stems

? Premise of the study: Aquaporins (AQPs) are channel proteins, and their function is mostly associated with transmembrane water transport. While aquaporin genes are known to be expressed in woody poplar stems, little is known about AQP expression at the cellular level. Localization of AQP expression to particular cell and tissue types is a necessary prerequisite in understanding the biological role of these genes. ? Methods: Subsets of plants were subjected to 6 wk of high nitrogen fertilization (high N plants) or to a controlled drought. Experimental treatments affected cambial activity and wood anatomy. RNA in situ hybridization was used to characterize spatial expression of three AQP genes in stem cross sections. ? Key results: The strongest labeling consistently occurred in the cambial region and in adjacent xylem and phloem cells. Expression was also detected in rays. Contact cells exhibited high expression, while expression in other ray cells was more variable. High N plants exhibited a broader band of expression in the cambial region than plants receiving only adequate N fertilization (control plants) and plants subjected to drought. ? Conclusions: Water channels in stems were expressed in a manner that allows hydraulic coupling between xylem and other tissues that may serve as water reservoirs, including phloem and pith parenchyma. Expression of AQPs in rays may increase radial flow of water from xylem and phloem to the cambial region where AQPs may help sustain rapid cell division and expansion of developing vessel elements.     

Roles of aquaporins in the brain

It is now over 10 years ago that aquaporin 1 (AQP1) was discovered and cloned from the red blood cells, and in 2003 the Nobel price in Chemistry was awarded to Pr. Peter Agre for his work on AQPs, highlighting the importance of these proteins in life sciences. AQPs are water channels. To date this protein family is composed of 11 sub-types in mammalians. Three main AQPs described in the mammalian brain are AQP1, AQP4 and AQP9. Several recent studies have shown that these channels are implicated in numerous physiological functions. AQP1 has a role in cerebrospinal fluid formation, whereas AQP4 is involved in water homeostasis and extracellular osmotic pressure in brain parenchyma. AQP4 seems also to have an important function in oedema formation after brain trauma or brain ischemia. AQP9 is implicated in brain energy metabolism. The level of expression of each AQP is highly regulated. After a trauma or an ischemia perturbation of the central nervous system, the level of expression of each AQP is differentially modified, resulting in facilitating oedema formation. At present, the exact role of each AQP is not yet determined. A better understanding of the mechanisms of AQP regulation should permit the development of new pharmacological strategies to prevent oedema formation. AQP9 has been recently specifically detected in the catecholaminergic neurons of the brain. This new result strengthens the hypothesis that the AQPs are not only water channels, but that some AQPs may play a role in energy metabolism as metabolite channels.     

The evolutionary aspects of aquaporin family

Aquaporins (AQPs) were originally identified as channels facilitating water transport across the plasma membrane. They have a pair of highly conserved signature sequences, asparagine-proline-alanine (NPA) boxes, to form a pore. However, some have little conserved amino acid sequences around the NPA boxes unclassifiable to two previous AQP subfamilies, classical AQPs and aquaglyceroporins. These will be called unorthodox AQPs in this review. Interestingly, these unorthodox AQPs have a highly conserved cysteine residue downstream of the second NPA box. AQPs also have a diversity of functions: some related to water transport such as fluid secretion, fluid absorption, and cell volume regulation, and the others not directly related to water transport such as cell adhesion, cell migration, cell proliferation, and cell differentiation. Some AQPs even permeate nonionic small molecules, ions, metals, and possibly gasses. AQP gene disruption studies have revealed their physiological roles: water transport in the kidney and exocrine glands, glycerol transport in fat metabolism and in skin moisture, and nutrient uptakes in plants. Furthermore, AQPs are also present at intracellular organelles, including tonoplasts, mitochondria, and the endoplasmic reticulum. This review focuses on the evolutionary aspects of AQPs from bacteria to humans in view of the structural and functional diversities of AQPs.     

Hyperosmolar mannitol simulates expression of aquaporins 4 and 9 through a p38 mitogen-activated protein kinase-dependent pathway in rat astrocytes

The membrane pore proteins, aquaporins (AQPs), facilitate the osmotically driven passage of water and, in some instances, small solutes. Under hyperosmotic conditions, the expression of some AQPs changes, and some studies have shown that the expression of AQP1 and AQP5 is regulated by MAPKs. However, the mechanisms regulating the expression of AQP4 and AQP9 induced by hyperosmotic stress are poorly understood. In this study, we observed that hyperosmotic stress induced by mannitol increased the expression of AQP4 and AQP9 in cultured rat astrocytes, and intraperitoneal infusion of mannitol increased AQP4 and AQP9 in the rat brain cortex. In addition, a p38 MAPK inhibitor, but not ERK and JNK inhibitors, suppressed their expression in cultured astrocytes. AQPs play important roles in maintaining brain homeostasis. The expression of AQP4 and AQP9 in astrocytes changes after brain ischemia or traumatic injury, and some studies have shown that p38 MAPK in astrocytes is activated under similar conditions. Since mannitol is commonly used to reduce brain edema, understanding the regulation of AQPs and p38 MAPK in astrocytes under hyperosmotic conditions induced with mannitol may lead to a control of water movements and a new treatment for brain edema.     

The potential role of caveolin-1 in inhibition of aquaporins during the AVD

BACKGROUND INFORMATION: During apoptosis, the first morphological change is a distinct cell shrinkage known as the AVD (apoptotic volume decrease). This event is driven by a loss of intracellular K(+), which creates an osmotic gradient, drawing water out of the cell through AQPs (aquaporins). Loss of water in balance with K(+) would create a shrunken cell with an equivalent intracellular concentration of K(+) ([K(+)](i) = 140 mM). However, we have previously shown that the [K(+)](i) of the shrunken apoptotic cell is 35 mM, and this level is absolutely essential for the activation of apoptotic enzymes. We have recently found that AQPs are inactivated following the AVD, so that continued loss of K(+) will reduce the intracellular concentration to this critical level. Using thymocytes, we have investigated the expression profile and regulation of the AQP family members. RESULTS: In the present study, we have found that AQP1, AQP8 and AQP9 are present in non-apoptotic thymocytes and localized primarily to the plasma membrane. Expression and localization did not change when these cells were induced to undergo apoptosis by growth factor withdrawal for 24 h. To explore other possible mechanisms by which these water channels are inactivated, we investigated their association with CAV-1 (caveolin-1), binding to which is known to inactivate a variety of proteins. We found that CAV-1 is present in thymocytes and that this protein co-localizes with a portion of AQP1 in normal (non-apoptotic) thymocytes. However, thymocytes induced to undergo apoptosis greatly increase their AQP1/CAV-1 association. CONCLUSIONS: Taken together, these results indicate that AQPs are localized to the plasma membrane of shrunken apoptotic thymocytes where increased binding to CAV-1 potentially inactivates them. AQP inactivation, coupled with continued K(+) efflux, then allows the [K(+)](i) to decrease to levels conducive for the activation of downstream apoptotic enzymes and the completion of the apoptotic cascade.