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Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: Aquaporins in the Brain
Journal of Pharmacological Sciences
J Pharmacol Sci 96, 264 – 270 (2004)
©2004 The Japanese Pharmacological Society
Forum Minireview
Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: Aquaporins in the Brain Hideyuki Kobayashi1,*, Toshihiko Yanagita1, Hiroki Yokoo1, and Akihiko Wada1 1
Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan
Received September 29, 2004; Accepted October 13, 2004
Abstract. Water homeostasis of the brain is essential for its neuronal activity. Changes in water content in the intra- and extra-cellular space affect ionic concentrations and therefore modify neuronal activity. Aquaporin (AQP) water channels may have a central role in keeping water homeostasis in the brain. Among AQP subtypes cloned in mammalian, only AQP1, AQP4, and AQP9 were identified in the brain. Changes in AQP expression may be correlated with edema formation of the brain. In this review, we describe the physiological function of AQPs and the regulatory mechanism of their expression in the brain. Keywords: aquaporin, brain, edema, glucocorticoid, lipid raft
plexus which may play a role in cerebrospinal fluid (CSF) formation, AQP4 in astrocyte endfeet that face blood vessels and in ependymal cells, and AQP9 in astrocytes. Recently, low levels of AQP1 and 4 were found in the microvascular endothelial cells in the brain (5 – 8). For the normal function of membrane proteins, they must be expressed at the appropriate location at the appropriate level. Accumulating evidence suggests that selection and trafficking of membrane proteins are regulated by those enzymatic modifications and interaction with lipids. Lipid rafts are discrete membrane microdomains enriched in cholesterol and sphingolipids (9, 10), which may serve as a platform for protein selection and trafficking. Recent data have shown that some AQP subtypes are located on lipid rafts, and the intracellular trafficking of AQPs is regulated by lipid rafts. In this review, we describe the possible physiological function of AQPs in the brain and the mechanisms of the regulation of their expression and trafficking.
Introduction Regulation of water permeability across microvessels between blood and brain is essential for brain function, and its disruption by various brain disorders such as stroke, trauma, infection, and metabolic disorders causes brain edema. However, the molecular mechanisms for the regulation of water permeability in the brain are poorly understood. Aquaporins (AQPs) are composed of a family of proteins identified as water channels (1). At least 11 AQP subtypes have been identified in mammals: they share six transmembrane-spanning domains and AsnPro-Ala (NPA) sequences which form a water channel pore (2, 3). They have similar molecular weights approximately 30 kDa with amino acid similarity from 20% to 50%. The AQP family is subdivided according to characteristics: water selective channels (AQP1, 2, 4, 5, and 8) and channels transporting glycerol and other small solutes also called aquagryceroporins (AQP3, 7, 9, and 10). AQP6 may transport chloride at low pH (4). Each subtype has its own cellular distribution and distinct regulatory mechanisms of their expression. In the brain, AQP1 is mainly expressed in the choroid
Localization and physiological function of AQPs in the brain In the brain, AQP1, AQP4, and AQP9 are expressed with different distribution (Fig. 1). The existence of
*Corresponding author. FAX: +81-985-84-2776 E-mail: [email protected]
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and chemical (capsaicin injection) stimuli (16), suggesting a role for AQP1 in neural signal transduction, and in rapid water recycling.
Fig. 1. Localization of AQPs in the brain. AQP1 (rectangles) is located on the apical surface of the choroid plexus; AQP4 (triangles), in endfeet of astrocytes facing microvessels; and AQP9 (circles), in astrocyte membrane. AQP1 and AQP4 are also expressed in microvascular endothelial cells at low level.
AQP3, AQP5, and AQP8 was reported (11), but their physiological role has not been well analyzed. AQP1 AQP1 is expressed in the apical surface of the epithelium of the choroid plexus, the cell producing CSF (5). AQP1 has been proposed to facilitate cerebrospinal fluid formation. In fact, intraventricular pressure was reduced by 50% in mice lacking AQP1 after intraventricular cannulation, and in vitro production of CSF of AQP1 deleted mice was reduced by 25% (12). It has been believed that AQP1 is expressed in capillary endothelial cells throughout the body except in the brain. However, we have recently found that low level of AQP1 is expressed in the microvessels prepared from rat brain and in the cultured brain microvascular endothelial cells (H. Kobayashi et al., unpublished data). In addition, AQP1 immunoreactivity was present in the endothelium of a few microvessels in the normal human brain (13). AQP1 was also detected in microvessel endothelia in human astrocytoma and metastatic carcinomas (13). AQP1 expression was also observed in endothelial cells in glioblastoma transplanted into mouse brain (14), indicating that the endothelial cells in the brain are able to express AQP1. In the case of the spinal cord, AQP1 is expressed in peripheral nerve fibers that projects to the dorsal horn, which is involved in pain sensation. Osmotically induced swelling of the spinal cord was reduced in AQP1deleted mice (15) and markedly impaired pain sensation was demonstrated in response to thermal (tail flick test)
AQP4 AQP4 is the predominant subtype present in the brain (17). The most abundant site of AQP4 expression in the brain is the perivascular glial processes (18). Additionally, AQP4 was localized in the Prukinje layer of the cerebellum, as well as in the supraoptic and paraventricular nuclei of the hypothalamus (18). AQP4 is also expressed in ependymal cells, but was absent from neurons, oligodendrocytes, and microglia. We have found that AQP4 mRNA and protein were present in the microvessels prepared from rat cerebral cortex (6). In addition, the microvessels were immunochemically stained with anti-AQP4 antibody. Recent study at the electron microscopy level has confirmed that AQP4 is expressed in the microvascular endothelial cells (19). AQP4 level of the abluminal membrane of the brain endothelial cells was 1 / 5 of that of the endfeet of astrocytes. Luminal membrane also expresses AQP4 but its level was 1 / 2 of that of the abluminal membrane. AQP4 is anchored by a -syntrophin (an adapter protein associated with dystrophin), and the interaction with this molecule is essential for localization of AQP4 (20). In mice deleted a -syntrophin, most of the AQP4 disappeared from the endfoot membrane facing blood vessels, whereas the AQP4 density of the membrane of the neuropil side was increased (19). The distribution of AQP4 of endothelial cells was not changed by a syntrophin deletion, indicating that localization of AQP4 in the endfoot of astrocyte, but not that in the endothelial cell, is regulated by its interaction with a -syntrophin. AQP4 forms a characteristic structure of square arrays of intramembrane particles (also referred as assemblies or orthogonally arranged particles) (21). AQP4 exists as two isoforms, differing at their Ntermini, because of translation initiation at the first methionine (M1, 323 amino acids) or the second methionine (M23, 301 amino acids) (22). Endogenous AQP4 is a tetramer usually containing M1 and M23 subunits. In the brain, M23 is at least threefold more abundant (23). Furman et al. (24) have analyzed the difference of M1 and M23 in the formation of square arrays. They transfected Chinese hamster ovary cells with M1, M23, or M1+M23 isoforms and performed freeze-fracture analysis. M23 homotetramers produce large square lattices with inter-intramembrane particles cross-bridges, whereas M1 homotetramers produce dispersed 4- to 6-nm intramembrane particles with few incipient square arrays. When M1 and M23 were coexpressed to form hetero- and homotetramers, square
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arrays were of intermediate sizes, similar to square arrays in astrocyte endfeet. Thus, factors affecting the selection of translation initiation sites may regulate the size of square arrays, and, therefore, may regulate local water permeability of the cells. Water transport via AQP4 is essential for normal neuronal activity. Binder et al. (25) have shown that seizure susceptivity in increased in AQP4 deleted mice. They have systemically injected pentylentetrazole into the mice. At low dose of the drug, population of mice exhibited seizure activity was much lower in AQP4 deleted mice than wild type mice. At higher dose of pentylentetrazole, both groups exhibited seizure activity; however, the latency to generalized (tonic-clonic) seizures was significantly lower in wild type than AQP4 deleted mice. These results suggest that glial water channels may modulate brain excitability and the initiation and generalization of seizure activity. In the hypothalamus, AQP4 is expressed not in magnocellular neurons that produce the antidiuretic hormone, arginine vasopressin (AVP), but is expressed in adjacent glial cells. Although these glial cells have an indirect role in osmosensing (18), the release of AVP was not modified in an AQP4 deleted mouse, suggesting that the role of AQP4 in the osmoreceptor is minimum (26). AQP9 AQP9 is a subtype mainly expressed in liver and testis having characteristics of being permeable to
Table 1.
various solutes, including glycerol and lactate, and this subtype might have a role in energy metabolism. AQP9 is present in the cells surrounding the cerebral ventricles, including ependymal cells and the tanycytes of the mediobasal hypothalamus (27), whereas in situ hybridization indicates expression of AQP9 mRNA also in astrocytes and endothelial cells (28). The blood-brain barrier is absent in the mediobasal hypothalamus, and the tanycytes, whose elongated processes extend between the third ventricle and the external layer of the median eminence with endfeet surrounding the infudibular portal capillary plexus, have been proposed to participate in the regulated transmission of endocrinological signals between the extra cellular milieu of the CNS and the systemic circulation. The presence of AQP9 in the ependymal lining of the cerebral ventricles raises the possibility that this water channel may be involved in the extrachroideal production and the extraarachnoid reabsorption of the cerebrospinal fluid. Role of AQPs in edema formation and fluid clearance Several lines of evidence suggest that AQPs are involved in formation and resolution of brain edema. These are as follows: 1) changes in AQP4 expression in edematous regions after brain injury or by tumor, 2) changes in edema formation in AQP4 and a -syntrophin deleted mice, and 3) changes in AQP1 and AQP9 expression in pathological conditions.
Changes in brain AQP expression in pathological conditions Changes
Reference
AQP1 endothelial cells in implanted glioblastoma, rat endothelial cells and neoplastic astrocytes in astrocytoma, human endothelial cells and reactive astrocytes in metastatic carcinoma, human glioblastoma, human
+ + + +
(14) (13) (13) (45)
AQP4 astrocytes, infarction, human astrocytes in contused brain, bacterial meningitis and tumor, human astrocytoma, reactive astrocytes in metastatic adenocartinoma, human perivascular membrane, ischemia, rat trauma + hyponatremia, rat (mRNA) reactive astrocytes, injection of ringer, quinolinic acid or 6-OHDA, axotomy, rat (mRNA) focal ischemia, rat (mRNA) cultured astrocytes, hypoxia, rat (mRNA)
+ + + + + -
(46) (47) (30) (33) (48) (17) (49) (11)
AQP9 astrocytes, transient focal ischemia, mouse
+
(37)
Changes in AQP expression in pathological conditions of human or experimental animals and in cultured cells as indicated as + (increase) or - (decrease).
Aquaporins in the Brain
1) Changes in AQP4 expression in edematous regions after brain injury or by tumor Expression of AQP4 changes in various pathological conditions (Table 1). Up-regulation of AQP4 was observed in edematous regions of contused brain, brain with bacterial meningitis, and brain tumors (malignant astrocytoma as well as reactive astrocytes in metastatic adenocarcinomas) in humans (29, 30). AQP4 is also upregulated by brain injury caused by intrastriatal injection of quinolic acid or axotomy of the medial forebrain bundle in the rats (17). Up-regulation of AQP4 may be correlated with edema formation, but it is not known whether the increase has a causal role of edema or is a compensatory mechanism to resolve edema. On the contrary, acute global ischemia caused downregulation of AQP4 with concomitant disintegration of square arrays (21, 31) in perivascular astrocytic processes in the rats (32), suggesting that the reduction of AQP4 expression may be involved in the edema formation by reducing the water transport across the perivascular membrane into the vascular lumen (33). 2) Changes edema formation in AQP4 and a -syntrophin deleted mice Mice with a deletion of AQP4 or a -syntrophin (a molecule interacts with and regulates location of AQP4) revealed the role of AQP4 in the formation and solution of brain edema (Table 2). In AQP4-deleted mice, brain water content and swelling of pericapillary astrocytic foot processes were reduced. These AQP4-deleted mice
Table 2.
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showed reduced cerebral edema in response to water intoxication and stroke and showed improved survival and neurological status, indicaing that AQP4 plays a role in cerebral water balance in response to stress that are associated with the development of brain edema (34). AQP4 deletion would also have the opposite effect (increased brain swelling) in vasogenic (noncellular) edema because of impaired removal of excess brain water through glial limitans and ependymal barriers (35). AQP4-deleted mice had higher intracranial pressure and brain water content after continuous intraparenchymal fluid infusion. In a freeze-injury model of vasogenic brain edema, AQP4-deleted mice had remarkably worse clinical outcome, higher intracranial pressure, and greater brain water content. In addition, AQP4-deleted mice had higher intracranial pressure and corresponding accelerated neurological deterioration after implantation of melanoma cells into the brain. Thus, AQP4-mediated transcellular water movement is crucial not only for the development of brain edema after intoxication and ischemic stroke, but also for fluid clearance in vasogenic brain edema. Localization and function of AQP4 are regulated by its interaction with a-syntrophin (19). In the a -syntrophin-deleted mice, perivascular and subpial astroglial end-feet were swollen in the basal state, suggesting reduced clearance of water generated by brain metabolism (36). After transient cerebral ischemia, brain edema was attenuated in a-syntrophin deleted mice, indicating
Phenotype of mouse with the deletion of AQP or a-syntrophin and of AQP suppressed cells Change
Reference
AQP1 deleted mouse intracranial pressure CSF production
-
(12) (12)
AQP4 deleted mouse square arrays edema formation water intoxication seizure threshold
+
(50) (34) (34) (25)
a-Syntrophin deleted mouse AQP4 expression in astrocyte endfeet water clearance ischemia induced edema K+ clearance
-
(20) (20) (36) (51)
AQP4 suppressed astrocytes by RNAi water transport ischemia-induced gene expression (Glut 1, hexokinase)
-
(52) (52)
Changes in physiological parameters are indicated as + (increase) or - (decrease).
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the reduction of water influx. Therefore, a-syntrophinmediated AQP4 localization is essential for its function of bidirectional transport of water across the brain-blood interface (36). 3) Changes in AQP1 and AQP9 expression in pathological conditions AQP1 and AQP9 may also be involved in edema formation or compensatory mechanism against brain edema. AQP1 was induced in capillaries in implanted carcinoma of glioblastoma into the cranial window. The levels of AQP1 tended to decrease in the center, compared to the periphery, of tumor tissue, which may correlate with water transport within tumors (14). AQP9 has been hypothesized to play a role in extracellular water homeostasis and edema formation similar to AQP4 (37). After focal transient ischemia, AQP9 expression was increased on astrocytes in periinfarct areas, suggesting that AQP9 may play a role in the regulation of post-ischemic edema and in the clearance of lactate from the ischemic focus in view of its permeability to monocarbooxylates. Regulation of expression of AQPs It is an intriguing issue how the expression of AQPs is modified by drugs used for the treatment of brain edema. Gu et al. (38) have examined how the expression of AQP4 is regulated by steroid hormones in cultured rat astrocytes. They have found that testosterone upregulated AQP4 expression, but dexamethasone or 17bestradiol, did not. Testosterone also ameliorated the osmotic fragility of astrocytes from hypoosmotic stress, suggesting that the increased levels of AQP4 facilitated the testosterone function. Glucocorticoids are used for the treatment of brain edema caused by trauma, tumor, and cerebral hemorrhage, but its mechanism of action is not known (29). We have examined the effects of glucocorticoid on AQP1 expression in cultured brain endothelial cells and have found that AQP1 expression was increased by dexamethasone via increase in its transcription without change in the degradation rate of the message (H. Kobayashi et al., unpublished data). Since dexamethasone had no apparent effect on AQP4 expression in non-neoplastic human brain (30), beneficial effects of glucocorticoids in brain edema may be correlated with the induction of AQP1 in the brain microvascular endothelial cells.
Is AQP function in the brain microvessels regulated by adrenergic innervation? Several lines of evidence suggest that brain microvessel function is regulated by adrenergic innervation (for review, see (39)). It has been shown that the noradrenaline synthesizing enzyme, dopamine-b-hydroxylase, and adrenergic varicosities are located around the brain microvessels. In addition, a- and b-adrenergic receptors are present in the brain microvessels. Physiological experiments have shown that the stimulation of the locus coeruleus or the blockade of the neuronal uptake of norepinephrine by tricyclic antidepressants increased the permeability for water from blood to brain. In addition, in the porcine model of systemic sepsis, the b 2-adrenergic agonist dopexamine protected against sepsis-induced perimicrovessel edema (40). In contrast, the a 1-adrenergic agonist methoxamine caused perimicrovessel edema in non-septic and septic pig. These findings suggest the possibility that the AQP function in the brain microvessels is regulated by adrenergic innervation. The elucidation of the regulatory mechanisms of adrenergic innervation on the AQPs’ function in the brain microvessels may give us new insight into the regulation of water homeostasis in the brain. Localization of AQP1 in lipid rafts Lipid rafts are discrete membrane microdomains enriched in cholesterol and sphingolipids (9, 10). These microdomains have indispensable function in selection and trafficking of membrane proteins (41). Caveolae are a subset of lipid raft microdomains characterized by flask-shaped invagination of 50 – 100 m m in diameter in the plasma membrane (42), which are formed from lipid rafts by polymerization of caveolins, hairpinlike palmitoylated integral membrane proteins. Caveolae have a variety of function such as signal transduction, cholesterol transport, and endocytosis. Some AQPs are known to interact with lipid rafts and are regulated their trafficking by these domains. For example, AQP5 present in lipid rafts in rat parotid glands is translocated to the apical plasma membrane upon the stimulation of acetylcholine receptors of the glands (43). In the rat cardiac myocytes, AQP1 colocalizes with caveolin under the isotonic condition (44). Upon exposure of myocytes to hypertonic medium, AQP1 is dissociated from caveolin and internalized into the intracellular compartments. Lipid raft microdomains have an unique property of detergent-insolubility and low density and can be isolated by using density gradient centrifugation. We have separated the detergent-treated membrane fraction
Aquaporins in the Brain
of the cultured rat brain endothelial cells (H. Kobayashi et al., unpublished data). We have found that most of the AQP1 was recovered from the light floating fractions, suggesting that trafficking of AQP1 is regulated by the interaction with lipid rafts. Since the distribution of AQP1 in lipid rafts was not changed by the treatment of the cells with dexamethasone, glucocorticoids are unlikely to modify the intracellular trafficking of AQP1 (H. Kobayashi et al., unpublished data). Conclusion Accumulating data have clearly indicated that AQP4 and AQP9 in astrocytes are involved in the formation and amelioration of brain edema, but their pathophysiological role and the mechanism of action remain to be elucidated. It has been recently found that AQP1 and AQP4 are also present in the brain microvascular endothelial cells. The expression and localization of these AQPs are modified by a variety of pathophysiological states and therapeutic drugs. Intracellular trafficking of AQPs are likely to be regulated by interaction with lipid rafts. The elucidation of the regulatory mechanism of expression an intracellular localization of AQPs in the brain may provide clues for a better therapeutic strategy for the treatment of brain edema.
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Acknowledgments The work of our laboratory cited in this review was supported in part by the Grants-in-Aid for Scientific Research (C) (to HK 13675394 and 1550263) and Grants-in-Aid for Scientific Research Priority Areas, 21st Century COE (Centers of Excellence) program (Life Science) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT); a grant from Kimura Research foundation (to HK); and a Grant for Special Research Project from Miyazaki Medical College.
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J Pharmacol Sci 96, 264 – 270 (2004)
©2004 The Japanese Pharmacological Society
Forum Minireview
Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: Aquaporins in the Brain Hideyuki Kobayashi1,*, Toshihiko Yanagita1, Hiroki Yokoo1, and Akihiko Wada1 1
Department of Pharmacology, Miyazaki Medical College, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan
Received September 29, 2004; Accepted October 13, 2004
Abstract. Water homeostasis of the brain is essential for its neuronal activity. Changes in water content in the intra- and extra-cellular space affect ionic concentrations and therefore modify neuronal activity. Aquaporin (AQP) water channels may have a central role in keeping water homeostasis in the brain. Among AQP subtypes cloned in mammalian, only AQP1, AQP4, and AQP9 were identified in the brain. Changes in AQP expression may be correlated with edema formation of the brain. In this review, we describe the physiological function of AQPs and the regulatory mechanism of their expression in the brain. Keywords: aquaporin, brain, edema, glucocorticoid, lipid raft
plexus which may play a role in cerebrospinal fluid (CSF) formation, AQP4 in astrocyte endfeet that face blood vessels and in ependymal cells, and AQP9 in astrocytes. Recently, low levels of AQP1 and 4 were found in the microvascular endothelial cells in the brain (5 – 8). For the normal function of membrane proteins, they must be expressed at the appropriate location at the appropriate level. Accumulating evidence suggests that selection and trafficking of membrane proteins are regulated by those enzymatic modifications and interaction with lipids. Lipid rafts are discrete membrane microdomains enriched in cholesterol and sphingolipids (9, 10), which may serve as a platform for protein selection and trafficking. Recent data have shown that some AQP subtypes are located on lipid rafts, and the intracellular trafficking of AQPs is regulated by lipid rafts. In this review, we describe the possible physiological function of AQPs in the brain and the mechanisms of the regulation of their expression and trafficking.
Introduction Regulation of water permeability across microvessels between blood and brain is essential for brain function, and its disruption by various brain disorders such as stroke, trauma, infection, and metabolic disorders causes brain edema. However, the molecular mechanisms for the regulation of water permeability in the brain are poorly understood. Aquaporins (AQPs) are composed of a family of proteins identified as water channels (1). At least 11 AQP subtypes have been identified in mammals: they share six transmembrane-spanning domains and AsnPro-Ala (NPA) sequences which form a water channel pore (2, 3). They have similar molecular weights approximately 30 kDa with amino acid similarity from 20% to 50%. The AQP family is subdivided according to characteristics: water selective channels (AQP1, 2, 4, 5, and 8) and channels transporting glycerol and other small solutes also called aquagryceroporins (AQP3, 7, 9, and 10). AQP6 may transport chloride at low pH (4). Each subtype has its own cellular distribution and distinct regulatory mechanisms of their expression. In the brain, AQP1 is mainly expressed in the choroid
Localization and physiological function of AQPs in the brain In the brain, AQP1, AQP4, and AQP9 are expressed with different distribution (Fig. 1). The existence of
*Corresponding author. FAX: +81-985-84-2776 E-mail: [email protected]
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and chemical (capsaicin injection) stimuli (16), suggesting a role for AQP1 in neural signal transduction, and in rapid water recycling.
Fig. 1. Localization of AQPs in the brain. AQP1 (rectangles) is located on the apical surface of the choroid plexus; AQP4 (triangles), in endfeet of astrocytes facing microvessels; and AQP9 (circles), in astrocyte membrane. AQP1 and AQP4 are also expressed in microvascular endothelial cells at low level.
AQP3, AQP5, and AQP8 was reported (11), but their physiological role has not been well analyzed. AQP1 AQP1 is expressed in the apical surface of the epithelium of the choroid plexus, the cell producing CSF (5). AQP1 has been proposed to facilitate cerebrospinal fluid formation. In fact, intraventricular pressure was reduced by 50% in mice lacking AQP1 after intraventricular cannulation, and in vitro production of CSF of AQP1 deleted mice was reduced by 25% (12). It has been believed that AQP1 is expressed in capillary endothelial cells throughout the body except in the brain. However, we have recently found that low level of AQP1 is expressed in the microvessels prepared from rat brain and in the cultured brain microvascular endothelial cells (H. Kobayashi et al., unpublished data). In addition, AQP1 immunoreactivity was present in the endothelium of a few microvessels in the normal human brain (13). AQP1 was also detected in microvessel endothelia in human astrocytoma and metastatic carcinomas (13). AQP1 expression was also observed in endothelial cells in glioblastoma transplanted into mouse brain (14), indicating that the endothelial cells in the brain are able to express AQP1. In the case of the spinal cord, AQP1 is expressed in peripheral nerve fibers that projects to the dorsal horn, which is involved in pain sensation. Osmotically induced swelling of the spinal cord was reduced in AQP1deleted mice (15) and markedly impaired pain sensation was demonstrated in response to thermal (tail flick test)
AQP4 AQP4 is the predominant subtype present in the brain (17). The most abundant site of AQP4 expression in the brain is the perivascular glial processes (18). Additionally, AQP4 was localized in the Prukinje layer of the cerebellum, as well as in the supraoptic and paraventricular nuclei of the hypothalamus (18). AQP4 is also expressed in ependymal cells, but was absent from neurons, oligodendrocytes, and microglia. We have found that AQP4 mRNA and protein were present in the microvessels prepared from rat cerebral cortex (6). In addition, the microvessels were immunochemically stained with anti-AQP4 antibody. Recent study at the electron microscopy level has confirmed that AQP4 is expressed in the microvascular endothelial cells (19). AQP4 level of the abluminal membrane of the brain endothelial cells was 1 / 5 of that of the endfeet of astrocytes. Luminal membrane also expresses AQP4 but its level was 1 / 2 of that of the abluminal membrane. AQP4 is anchored by a -syntrophin (an adapter protein associated with dystrophin), and the interaction with this molecule is essential for localization of AQP4 (20). In mice deleted a -syntrophin, most of the AQP4 disappeared from the endfoot membrane facing blood vessels, whereas the AQP4 density of the membrane of the neuropil side was increased (19). The distribution of AQP4 of endothelial cells was not changed by a syntrophin deletion, indicating that localization of AQP4 in the endfoot of astrocyte, but not that in the endothelial cell, is regulated by its interaction with a -syntrophin. AQP4 forms a characteristic structure of square arrays of intramembrane particles (also referred as assemblies or orthogonally arranged particles) (21). AQP4 exists as two isoforms, differing at their Ntermini, because of translation initiation at the first methionine (M1, 323 amino acids) or the second methionine (M23, 301 amino acids) (22). Endogenous AQP4 is a tetramer usually containing M1 and M23 subunits. In the brain, M23 is at least threefold more abundant (23). Furman et al. (24) have analyzed the difference of M1 and M23 in the formation of square arrays. They transfected Chinese hamster ovary cells with M1, M23, or M1+M23 isoforms and performed freeze-fracture analysis. M23 homotetramers produce large square lattices with inter-intramembrane particles cross-bridges, whereas M1 homotetramers produce dispersed 4- to 6-nm intramembrane particles with few incipient square arrays. When M1 and M23 were coexpressed to form hetero- and homotetramers, square
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arrays were of intermediate sizes, similar to square arrays in astrocyte endfeet. Thus, factors affecting the selection of translation initiation sites may regulate the size of square arrays, and, therefore, may regulate local water permeability of the cells. Water transport via AQP4 is essential for normal neuronal activity. Binder et al. (25) have shown that seizure susceptivity in increased in AQP4 deleted mice. They have systemically injected pentylentetrazole into the mice. At low dose of the drug, population of mice exhibited seizure activity was much lower in AQP4 deleted mice than wild type mice. At higher dose of pentylentetrazole, both groups exhibited seizure activity; however, the latency to generalized (tonic-clonic) seizures was significantly lower in wild type than AQP4 deleted mice. These results suggest that glial water channels may modulate brain excitability and the initiation and generalization of seizure activity. In the hypothalamus, AQP4 is expressed not in magnocellular neurons that produce the antidiuretic hormone, arginine vasopressin (AVP), but is expressed in adjacent glial cells. Although these glial cells have an indirect role in osmosensing (18), the release of AVP was not modified in an AQP4 deleted mouse, suggesting that the role of AQP4 in the osmoreceptor is minimum (26). AQP9 AQP9 is a subtype mainly expressed in liver and testis having characteristics of being permeable to
Table 1.
various solutes, including glycerol and lactate, and this subtype might have a role in energy metabolism. AQP9 is present in the cells surrounding the cerebral ventricles, including ependymal cells and the tanycytes of the mediobasal hypothalamus (27), whereas in situ hybridization indicates expression of AQP9 mRNA also in astrocytes and endothelial cells (28). The blood-brain barrier is absent in the mediobasal hypothalamus, and the tanycytes, whose elongated processes extend between the third ventricle and the external layer of the median eminence with endfeet surrounding the infudibular portal capillary plexus, have been proposed to participate in the regulated transmission of endocrinological signals between the extra cellular milieu of the CNS and the systemic circulation. The presence of AQP9 in the ependymal lining of the cerebral ventricles raises the possibility that this water channel may be involved in the extrachroideal production and the extraarachnoid reabsorption of the cerebrospinal fluid. Role of AQPs in edema formation and fluid clearance Several lines of evidence suggest that AQPs are involved in formation and resolution of brain edema. These are as follows: 1) changes in AQP4 expression in edematous regions after brain injury or by tumor, 2) changes in edema formation in AQP4 and a -syntrophin deleted mice, and 3) changes in AQP1 and AQP9 expression in pathological conditions.
Changes in brain AQP expression in pathological conditions Changes
Reference
AQP1 endothelial cells in implanted glioblastoma, rat endothelial cells and neoplastic astrocytes in astrocytoma, human endothelial cells and reactive astrocytes in metastatic carcinoma, human glioblastoma, human
+ + + +
(14) (13) (13) (45)
AQP4 astrocytes, infarction, human astrocytes in contused brain, bacterial meningitis and tumor, human astrocytoma, reactive astrocytes in metastatic adenocartinoma, human perivascular membrane, ischemia, rat trauma + hyponatremia, rat (mRNA) reactive astrocytes, injection of ringer, quinolinic acid or 6-OHDA, axotomy, rat (mRNA) focal ischemia, rat (mRNA) cultured astrocytes, hypoxia, rat (mRNA)
+ + + + + -
(46) (47) (30) (33) (48) (17) (49) (11)
AQP9 astrocytes, transient focal ischemia, mouse
+
(37)
Changes in AQP expression in pathological conditions of human or experimental animals and in cultured cells as indicated as + (increase) or - (decrease).
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1) Changes in AQP4 expression in edematous regions after brain injury or by tumor Expression of AQP4 changes in various pathological conditions (Table 1). Up-regulation of AQP4 was observed in edematous regions of contused brain, brain with bacterial meningitis, and brain tumors (malignant astrocytoma as well as reactive astrocytes in metastatic adenocarcinomas) in humans (29, 30). AQP4 is also upregulated by brain injury caused by intrastriatal injection of quinolic acid or axotomy of the medial forebrain bundle in the rats (17). Up-regulation of AQP4 may be correlated with edema formation, but it is not known whether the increase has a causal role of edema or is a compensatory mechanism to resolve edema. On the contrary, acute global ischemia caused downregulation of AQP4 with concomitant disintegration of square arrays (21, 31) in perivascular astrocytic processes in the rats (32), suggesting that the reduction of AQP4 expression may be involved in the edema formation by reducing the water transport across the perivascular membrane into the vascular lumen (33). 2) Changes edema formation in AQP4 and a -syntrophin deleted mice Mice with a deletion of AQP4 or a -syntrophin (a molecule interacts with and regulates location of AQP4) revealed the role of AQP4 in the formation and solution of brain edema (Table 2). In AQP4-deleted mice, brain water content and swelling of pericapillary astrocytic foot processes were reduced. These AQP4-deleted mice
Table 2.
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showed reduced cerebral edema in response to water intoxication and stroke and showed improved survival and neurological status, indicaing that AQP4 plays a role in cerebral water balance in response to stress that are associated with the development of brain edema (34). AQP4 deletion would also have the opposite effect (increased brain swelling) in vasogenic (noncellular) edema because of impaired removal of excess brain water through glial limitans and ependymal barriers (35). AQP4-deleted mice had higher intracranial pressure and brain water content after continuous intraparenchymal fluid infusion. In a freeze-injury model of vasogenic brain edema, AQP4-deleted mice had remarkably worse clinical outcome, higher intracranial pressure, and greater brain water content. In addition, AQP4-deleted mice had higher intracranial pressure and corresponding accelerated neurological deterioration after implantation of melanoma cells into the brain. Thus, AQP4-mediated transcellular water movement is crucial not only for the development of brain edema after intoxication and ischemic stroke, but also for fluid clearance in vasogenic brain edema. Localization and function of AQP4 are regulated by its interaction with a-syntrophin (19). In the a -syntrophin-deleted mice, perivascular and subpial astroglial end-feet were swollen in the basal state, suggesting reduced clearance of water generated by brain metabolism (36). After transient cerebral ischemia, brain edema was attenuated in a-syntrophin deleted mice, indicating
Phenotype of mouse with the deletion of AQP or a-syntrophin and of AQP suppressed cells Change
Reference
AQP1 deleted mouse intracranial pressure CSF production
-
(12) (12)
AQP4 deleted mouse square arrays edema formation water intoxication seizure threshold
+
(50) (34) (34) (25)
a-Syntrophin deleted mouse AQP4 expression in astrocyte endfeet water clearance ischemia induced edema K+ clearance
-
(20) (20) (36) (51)
AQP4 suppressed astrocytes by RNAi water transport ischemia-induced gene expression (Glut 1, hexokinase)
-
(52) (52)
Changes in physiological parameters are indicated as + (increase) or - (decrease).
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the reduction of water influx. Therefore, a-syntrophinmediated AQP4 localization is essential for its function of bidirectional transport of water across the brain-blood interface (36). 3) Changes in AQP1 and AQP9 expression in pathological conditions AQP1 and AQP9 may also be involved in edema formation or compensatory mechanism against brain edema. AQP1 was induced in capillaries in implanted carcinoma of glioblastoma into the cranial window. The levels of AQP1 tended to decrease in the center, compared to the periphery, of tumor tissue, which may correlate with water transport within tumors (14). AQP9 has been hypothesized to play a role in extracellular water homeostasis and edema formation similar to AQP4 (37). After focal transient ischemia, AQP9 expression was increased on astrocytes in periinfarct areas, suggesting that AQP9 may play a role in the regulation of post-ischemic edema and in the clearance of lactate from the ischemic focus in view of its permeability to monocarbooxylates. Regulation of expression of AQPs It is an intriguing issue how the expression of AQPs is modified by drugs used for the treatment of brain edema. Gu et al. (38) have examined how the expression of AQP4 is regulated by steroid hormones in cultured rat astrocytes. They have found that testosterone upregulated AQP4 expression, but dexamethasone or 17bestradiol, did not. Testosterone also ameliorated the osmotic fragility of astrocytes from hypoosmotic stress, suggesting that the increased levels of AQP4 facilitated the testosterone function. Glucocorticoids are used for the treatment of brain edema caused by trauma, tumor, and cerebral hemorrhage, but its mechanism of action is not known (29). We have examined the effects of glucocorticoid on AQP1 expression in cultured brain endothelial cells and have found that AQP1 expression was increased by dexamethasone via increase in its transcription without change in the degradation rate of the message (H. Kobayashi et al., unpublished data). Since dexamethasone had no apparent effect on AQP4 expression in non-neoplastic human brain (30), beneficial effects of glucocorticoids in brain edema may be correlated with the induction of AQP1 in the brain microvascular endothelial cells.
Is AQP function in the brain microvessels regulated by adrenergic innervation? Several lines of evidence suggest that brain microvessel function is regulated by adrenergic innervation (for review, see (39)). It has been shown that the noradrenaline synthesizing enzyme, dopamine-b-hydroxylase, and adrenergic varicosities are located around the brain microvessels. In addition, a- and b-adrenergic receptors are present in the brain microvessels. Physiological experiments have shown that the stimulation of the locus coeruleus or the blockade of the neuronal uptake of norepinephrine by tricyclic antidepressants increased the permeability for water from blood to brain. In addition, in the porcine model of systemic sepsis, the b 2-adrenergic agonist dopexamine protected against sepsis-induced perimicrovessel edema (40). In contrast, the a 1-adrenergic agonist methoxamine caused perimicrovessel edema in non-septic and septic pig. These findings suggest the possibility that the AQP function in the brain microvessels is regulated by adrenergic innervation. The elucidation of the regulatory mechanisms of adrenergic innervation on the AQPs’ function in the brain microvessels may give us new insight into the regulation of water homeostasis in the brain. Localization of AQP1 in lipid rafts Lipid rafts are discrete membrane microdomains enriched in cholesterol and sphingolipids (9, 10). These microdomains have indispensable function in selection and trafficking of membrane proteins (41). Caveolae are a subset of lipid raft microdomains characterized by flask-shaped invagination of 50 – 100 m m in diameter in the plasma membrane (42), which are formed from lipid rafts by polymerization of caveolins, hairpinlike palmitoylated integral membrane proteins. Caveolae have a variety of function such as signal transduction, cholesterol transport, and endocytosis. Some AQPs are known to interact with lipid rafts and are regulated their trafficking by these domains. For example, AQP5 present in lipid rafts in rat parotid glands is translocated to the apical plasma membrane upon the stimulation of acetylcholine receptors of the glands (43). In the rat cardiac myocytes, AQP1 colocalizes with caveolin under the isotonic condition (44). Upon exposure of myocytes to hypertonic medium, AQP1 is dissociated from caveolin and internalized into the intracellular compartments. Lipid raft microdomains have an unique property of detergent-insolubility and low density and can be isolated by using density gradient centrifugation. We have separated the detergent-treated membrane fraction
Aquaporins in the Brain
of the cultured rat brain endothelial cells (H. Kobayashi et al., unpublished data). We have found that most of the AQP1 was recovered from the light floating fractions, suggesting that trafficking of AQP1 is regulated by the interaction with lipid rafts. Since the distribution of AQP1 in lipid rafts was not changed by the treatment of the cells with dexamethasone, glucocorticoids are unlikely to modify the intracellular trafficking of AQP1 (H. Kobayashi et al., unpublished data). Conclusion Accumulating data have clearly indicated that AQP4 and AQP9 in astrocytes are involved in the formation and amelioration of brain edema, but their pathophysiological role and the mechanism of action remain to be elucidated. It has been recently found that AQP1 and AQP4 are also present in the brain microvascular endothelial cells. The expression and localization of these AQPs are modified by a variety of pathophysiological states and therapeutic drugs. Intracellular trafficking of AQPs are likely to be regulated by interaction with lipid rafts. The elucidation of the regulatory mechanism of expression an intracellular localization of AQPs in the brain may provide clues for a better therapeutic strategy for the treatment of brain edema.
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Acknowledgments The work of our laboratory cited in this review was supported in part by the Grants-in-Aid for Scientific Research (C) (to HK 13675394 and 1550263) and Grants-in-Aid for Scientific Research Priority Areas, 21st Century COE (Centers of Excellence) program (Life Science) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT); a grant from Kimura Research foundation (to HK); and a Grant for Special Research Project from Miyazaki Medical College.
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