Regulation of Aquaporin-4 Water Channels by Phorbol Ester-dependent Protein Phosphorylation
1998; Elsevier BV; Volume: 273; Issue: 11 Linguagem: Inglês
10.1074/jbc.273.11.6001
ISSN1083-351X
AutoresZhiqiang Han, Martin B. Wax, Rajkumar V. Patil,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidney, the eye contains multiple water channel proteins (aquaporins) that transport water through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. However, because humans with defective AQP1 are phenotypically normal and because the ocular application of phorbol esters reduce intraocular pressure, we postulated that the water channel activity of AQP4 may be regulated by these agents. We now report that protein kinase C activators, phorbol 12,13-dibutyrate, and phorbol 12-myristate 13-acetate strongly stimulate the phosphorylation of AQP4 and inhibit its activity in a dose-dependent manner. Phorbol 12,13-dibutyrate (10 μm) and phorbol 12-myristate 13-acetate (10 nm) reduced the rate of AQP4-expressing oocyte swelling by 87 and 92%, respectively. Further, phorbol 12,13-dibutyrate significantly increased the amount of phosphorylated AQP4. These results demonstrate that protein kinase C can regulate the activity of AQP4 through a mechanism involving protein phosphorylation. Moreover, they suggest important potential roles for AQP4 in several clinical disorders involving rapid water transport such as glaucoma, brain edema, and swelling of premature infant lungs. The molecular mechanisms for regulating water balance in many tissues are unknown. Like the kidney, the eye contains multiple water channel proteins (aquaporins) that transport water through membranes, including two (AQP1 and AQP4) in the ciliary body, the site of aqueous humor production. However, because humans with defective AQP1 are phenotypically normal and because the ocular application of phorbol esters reduce intraocular pressure, we postulated that the water channel activity of AQP4 may be regulated by these agents. We now report that protein kinase C activators, phorbol 12,13-dibutyrate, and phorbol 12-myristate 13-acetate strongly stimulate the phosphorylation of AQP4 and inhibit its activity in a dose-dependent manner. Phorbol 12,13-dibutyrate (10 μm) and phorbol 12-myristate 13-acetate (10 nm) reduced the rate of AQP4-expressing oocyte swelling by 87 and 92%, respectively. Further, phorbol 12,13-dibutyrate significantly increased the amount of phosphorylated AQP4. These results demonstrate that protein kinase C can regulate the activity of AQP4 through a mechanism involving protein phosphorylation. Moreover, they suggest important potential roles for AQP4 in several clinical disorders involving rapid water transport such as glaucoma, brain edema, and swelling of premature infant lungs. Aquaporins are a rapidly growing family of water channel proteins found in animals, plant, and microorganisms (1Calamita G. Bishai W.R. Preston G.M. Guggino W.B. Agre P. J. Biol. Chem. 1995; 270: 29063-29066Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 2Chrispeels M.J. Agre P. Trend. Biochem. Sci. 1994; 19: 421-425Abstract Full Text PDF PubMed Scopus (255) Google Scholar). At least eight different aquaporins have been identified and cloned from mammals, including AQP1 from erythrocytes (3Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1649) Google Scholar, 4Agre P. Preston G.M. Smith B.L. Jung J.S. Raina S. Moon C. Guggino W.B. Nielsen S. Am. J. Physiol. 1993; 265: F463-F476Crossref PubMed Google Scholar), AQP2, AQP3, and AQP6 from kidney (5Fushimi K. Uchida S. Hara Y. Hirata Y. Marumo F. Sasaki S. Nature. 1993; 361: 549-552Crossref PubMed Scopus (858) Google Scholar, 6Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi Y. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (523) Google Scholar, 7Ma T. Yang B. Umenishi F. Verkman A.S. Genomics. 1997; 43: 387-389Crossref PubMed Scopus (30) Google Scholar), AQP4 from brain (8Jung J.S. Bhat R.V. Preston G.M. Guggino W.B. Baraban J.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13052-13056Crossref PubMed Scopus (613) Google Scholar, 9Hasegawa H. Ma T. Skach W. Matthay M.A. Verkman A.S. J. Biol. Chem. 1994; 269: 5497-5500Abstract Full Text PDF PubMed Google Scholar), AQP5 from salivary gland, (10Raina S. Preston G.M. Guggino W.B. Agre P. J. Biol. Chem. 1995; 270: 1908-1912Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar), AQP7 from testis (11Ishibashi K. Kuwahara M. Gu Y. Kageyama Y. Tohsaka A. Suzuki F. Marumo F. Sasaki S. J. Biol. Chem. 1997; 272: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), and AQP8 from testis and liver (12Ishibashi K. Kuwahara M. Kageyama Y. Tohsaka A. Marumo F. Sasaki S. Biochem. Biophys. Res. Commun. 1997; 237: 714-718Crossref PubMed Scopus (179) Google Scholar, 13Koyama Y. Yamamoto T. Kondo D. Funaki H. Yaoita E. Kawasaki K. Sato N. Hatakeyama K. Kihara I. J. Biol. Chem. 1997; 272: 30329-30333Crossref PubMed Scopus (150) Google Scholar). AQP1, AQP2, AQP3, and AQP7 have been shown to transport the nonionic small solutes such as urea and glycerol in addition to water (6Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi Y. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (523) Google Scholar, 11Ishibashi K. Kuwahara M. Gu Y. Kageyama Y. Tohsaka A. Suzuki F. Marumo F. Sasaki S. J. Biol. Chem. 1997; 272: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 14Abrami L. Berthonaud V. Deen P.M. Rousselet G. Tacnet F. Ripoche P. Pfl. Archiv. Eur. J. Physiol. 1996; 431: 408-414Crossref PubMed Scopus (34) Google Scholar), whereas AQP4, AQP5, AQP6, and AQP8 are highly selective to water permeation and exclude small solutes (8Jung J.S. Bhat R.V. Preston G.M. Guggino W.B. Baraban J.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13052-13056Crossref PubMed Scopus (613) Google Scholar, 9Hasegawa H. Ma T. Skach W. Matthay M.A. Verkman A.S. J. Biol. Chem. 1994; 269: 5497-5500Abstract Full Text PDF PubMed Google Scholar, 12Ishibashi K. Kuwahara M. Kageyama Y. Tohsaka A. Marumo F. Sasaki S. Biochem. Biophys. Res. Commun. 1997; 237: 714-718Crossref PubMed Scopus (179) Google Scholar). With the exception of kidney-specific, vasopressin-regulated AQP2, the aquaporins are thought to be constitutively active (15van Lieburg A.F. Knoers N.V. Deen P.M. Pediatr. Nephrol. 1995; 9: 228-234Crossref PubMed Scopus (19) Google Scholar, 16Agre P. Brown D. Nielsen S. Curr. Opin. Cell Biol. 1995; 7: 472-483Crossref PubMed Scopus (210) Google Scholar, 17Brown D. Katsura T. Kawashima M. Verkman A.S. Sabolic I. Histochem. Cell Biol. 1995; 104: 1-9Crossref PubMed Scopus (78) Google Scholar, 18King L.S. Agre P. Annu. Rev. Physiol. 1996; 58: 619-648Crossref PubMed Scopus (450) Google Scholar). The regulation of other aquaporins is controversial (see "Discussion") and is not well understood. The ciliary body expresses only two aquaporins (AQP1 and AQP4). Because humans with mutation defects in AQP1 are phenotypically normal (19Preston G.M. Smith B.L. Zeidel M.L. Moulds J.J. Agre P. Science. 1994; 265: 1585-1587Crossref PubMed Scopus (272) Google Scholar) and because the application of phorbol esters to the eye reduces intraocular pressure (20Mittag T.W. Yoshimura N. Podos S.M. Invest. Ophthalmol. Visual Sci. 1987; 28: 2057-2066PubMed Google Scholar), we postulated that phorbol ester regulation of AQP4 water channel activity may account for the observed reduction of intraocular pressure using these agents. AQP4 is unique because it encodes a water-selective channel that is not inhibited by high concentrations of mercurial compounds such as HgCl2 (9Hasegawa H. Ma T. Skach W. Matthay M.A. Verkman A.S. J. Biol. Chem. 1994; 269: 5497-5500Abstract Full Text PDF PubMed Google Scholar). Previous studies using immunocytochemistry, reverse transcription polymerase chain reaction, and Northern blotting with AQP4 confirmed its expression in kidney, brain, lung, and eye including retina and ciliary body (21Frigeri A. Gropper M.A. Turck C.W. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4328-4331Crossref PubMed Scopus (371) Google Scholar, 22Frigeri A. Gropper M.A. Umenishi F. Kawashima M. Brown D. Verkman A.S. J. Cell Sci. 1995; 108: 2993-3002Crossref PubMed Google Scholar, 23Patil R.V. Saito I. Yang X. Wax M.B. Exp. Eye Res. 1997; 64: 203-209Crossref PubMed Scopus (107) Google Scholar). Using an oocyte swelling assay and protein phosphorylation studies, we demonstrate here that water channel activity of AQP4 is regulated by phorbol ester-dependent protein phosphorylation via protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate 13-acetate; 4α-PDD, 4α-phorbol 12,13-didecanoate; PAGE, polyacrylamide gel electrophoresis. 1The abbreviations used are: PKC, protein kinase C; PDBu, phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate 13-acetate; 4α-PDD, 4α-phorbol 12,13-didecanoate; PAGE, polyacrylamide gel electrophoresis. pathway. AQP4 regulation by PKC suggests an important potential role for this aquaporin in several clinical disorders involving rapid water transport such as glaucoma, brain edema following stroke, and uncontrollable swelling of premature infant lungs. The plasmid containing rat AQP4 cDNA (8Jung J.S. Bhat R.V. Preston G.M. Guggino W.B. Baraban J.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13052-13056Crossref PubMed Scopus (613) Google Scholar) was purchased from ATCC (Rockville, MD). The EcoRI fragment of the plasmid containing entire AQP4 open reading frame was blunt-ligated into the BglII site of the Xenopus expression construct pXbG (3Preston G.M. Carroll T.P. Guggino W.B. Agre P. Science. 1992; 256: 385-387Crossref PubMed Scopus (1649) Google Scholar). Confirmation of the recombinant plasmid was made by nucleotide sequencing. Sense and antisense capped RNA transcripts of AQP4 were synthesized in vitro with T3 RNA polymerase using two recombinant plasmids with the AQP4 cDNA cloned in sense and antisense direction. Defolliculated stage V and VI oocytes from female Xenopus laevis (24Lu L. Montrose-Rafizadeh C. Hwang T.C. Guggino W.B. Biophys. J. 1990; 57: 1117-1123Abstract Full Text PDF PubMed Scopus (45) Google Scholar) were injected with 20 nl of water or cRNAs (1 mg/ml). After incubation in 200 mosmol modified Barth's buffer at 18 °C for 72 h, oocytes were transferred to 70 mosmol Barth's buffer diluted with distilled water, and the time course of osmotic volume increase was monitored at 20 °C. Because the time course of cell swelling was principally linear during the initial 40 s, osmotic water permeability (P f) of oocytes was calculated from this 40-s response as described previously (25Patil R.V. Han Z. Wax M.B. Biochem. Biophys. Res. Commun. 1997; 238: 392-396Crossref PubMed Scopus (56) Google Scholar). The effects of phorbol 12,13-dibutyrate (PDBu), phorbol 12-myristate 13-acetate (PMA), 4α-phorbol, 4α-phorbol 12,13-didecanoate (4α-PDD), (all from Calibiochem), and HgCl2 were examined by incubating oocytes in Barth's buffer containing appropriate concentrations of the reagent for 15 min prior to P f measurements. Rat brain homogenate (75 μg) was incubated at 25 °C for 30 min in the presence of 50 μm{γ-32P}ATP; 0.045 μg of PKC (Calibiochem) and PKC activators in phosphorylation buffer containing 20 mmTris-HCl (pH 7.4); 100 mm NaCl; 5 mmMgCl2; 5 mm NaH2PO4; 1.5 mm CaCl2; 0.2% (v/v) Triton X-100; 1 mm EDTA; 1 mm dithiothreitol; 1 mmphenylmethylsulfonyl fluoride; 5 μg/ml each of leupeptin, pepstatin, and antipain. At the end of incubation period the phosphorylation reaction was stopped by immunoprecipitation with AQP4 antibody as described below. Phosphorylated homogenate was incubated with 10 mg of preswollen protein A-Sepharose beads and incubated for 1 h at 4 °C. The Sepharose bead-associated, nonspecifically adsorbed proteins were removed by centrifugation for 10 s at 15,000 rpm in microcentrifuge. The supernatant was then mixed with 2 μl of AQP4 antibody (kindly provided by Dr. Verkman, University of California San Francisco), and the mixture was incubated for 12 h at 4 °C. The samples were then transferred to Eppendorf tube containing 10 mg of preswollen protein A-Sepharose beads and incubated for 1–2 h at 4 °C. The beads were collected by centrifugation and washed once with lysis buffer (20 mm Tris-HCl, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100, 0.2% bovine serum albumin (pH 8.0) containing 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml each of leupeptin, pepstatin, and antipain); three times with 1 ml buffer containing 20 mm Tris-HCl, 150 mmNaCl, 5 mm EDTA, 0.5% Triton X-100, 0.1% SDS, 0.2% bovine serum albumin (pH 8.0); and once with 1 ml of a buffer containing 50 mm Tris-HCl (pH 8.0). After the final wash, the beads were resuspended in 50 μl of SDS-PAGE sample buffer (50 mm Tris-HCl, 10% glycerol, 2% SDS, 10% 2-mercaptoethanol, 0.01% bromphenol blue (pH 6.8)), vortexed, and centrifuged. The recovered proteins were separated by SDS-PAGE (7.5%). Gels were dried and subjected to autoradiography. An expression construct was prepared by inserting the AQP4 coding sequences between 5′- and 3′-untranslated sequences of the Xenopus β-globin cDNA as described under "Experimental Procedures." Defolliculated oocytes were microinjected with 20 ng of in vitro transcribed AQP4 cRNA. Osmotic water permeability after transfer of oocytes from a 200 to a 70 mosmol solution was determined by monitoring changes in cell volume as described under "Experimental Procedures." Fig. 1 shows the effect of PDBu (PKC activator) on the osmotic water permeability of oocytes expressing AQP4. Oocytes incubated for 15 min in 200 mosmol Barth's buffer containing PDBu showed considerably decreased subsequent rate of swelling in 70 mosmol buffer compared with control oocytes, whereas oocytes incubated with 4α-phorbol (inactive phorbol) for 15 min in 200 mosmol Barth's buffer showed no effect on subsequent rate of swelling in 70 mosmol buffer. The effect of PDBu was dose-dependent; at 10 μm PDBu reduced the rate of oocyte swelling by 87% in 70 mosmol buffer. Similarly, oocytes incubated for 15 min in 200 mosmol Barth's buffer containing PMA showed a significantly lower subsequent rate of swelling in 70 mosmol buffer (Fig. 2) versus control oocytes. Oocytes incubated for 15 min in 200 mosmol Barth's buffer containing 4α-PDD (inactive PMA) showed no effect on subsequent rate of swelling in 70 mosmol buffer. The effect of PMA was also dose-dependent; at 10 nm PMA reduced the rate of oocyte swelling by 92% in 70 mosmol buffer. Ethanol (0.1%) was used to make 1 mm stock solutions of PDBu, 4α-phorbol, PMA, and 4α-PDD and had no effect alone on swelling. Swelling of oocytes expressing AQP4 was not blocked by 1 mmHgCl2 (data not shown), because AQP4 is a mercury-insensitive water channel (8Jung J.S. Bhat R.V. Preston G.M. Guggino W.B. Baraban J.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13052-13056Crossref PubMed Scopus (613) Google Scholar, 9Hasegawa H. Ma T. Skach W. Matthay M.A. Verkman A.S. J. Biol. Chem. 1994; 269: 5497-5500Abstract Full Text PDF PubMed Google Scholar). Oocytes injected with water or antisense cRNA showed a very low swelling rate that was unaffected by either PDBu or PMA treatment.Figure 2Effect of PMA on the osmotic water permeability of oocytes expressing AQP4 RNA. Oocytes were injected with 20 ng of AQP4 cRNA 72 h prior to experiments. Oocytes were incubated with PMA or 4α-PDD as described under "Experimental Procedures." After the treatments, osmotic swelling of the oocytes was monitored, and oocyte volume was calculated. Each point represents the mean ± S.E. of 8–12 oocytes. Similar results were obtained in three separate experiments with oocytes from different frogs.View Large Image Figure ViewerDownload (PPT) The coefficients of osmotic water permeability (P f) at 20 °C calculated from the rates of swelling were 32.4 ± 2 μm/s (mean ± S.E., n = 8) for untreated AQP4 injected oocytes and 4.3, 5.3, and 9.1 ± 1 μm/s (mean ± S.E., n = 12) for PDBu-treated oocytes at 10, 5, and 1 μmconcentrations, respectively (Fig. 3), whereas the P f values of oocytes incubated with 4α-phorbol were 31 ± 2 μm/s (mean ± S.E., n = 8), suggesting that the decrease in P f of AQP4 due to PDBu was specific. In another experiment, similar results were obtained using PMA. The P f values of oocytes incubated with PMA at 10, 5, and 1 nm, were 2.4, 8.2, and 13 ± 2 μm/s (mean ± S.E., n = 10), respectively, whereas the P f values of untreated oocytes and 4α-PDD-treated oocytes were 29 and 24.9 ± 3 μm/s (mean ± S.E., n = 10), respectively. The P f values of oocytes injected with water (data not shown) were 2.2 ± 1 μm/s (mean ± S.E., n = 8) suggesting that microinjection itself had no effect on the oocyte swelling. The phorbol ester-dependent decrease in water permeability suggests that AQP4 could participate in receptor-mediated regulation of water fluxes in a variety of tissues, such as kidney, heart, brain, lung, and eye, in which it is widely distributed (21Frigeri A. Gropper M.A. Turck C.W. Verkman A.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4328-4331Crossref PubMed Scopus (371) Google Scholar, 22Frigeri A. Gropper M.A. Umenishi F. Kawashima M. Brown D. Verkman A.S. J. Cell Sci. 1995; 108: 2993-3002Crossref PubMed Google Scholar). Activation of PKC by phorbol esters is known to stimulate the phosphorylation of several proteins, thereby modulating their function. Therefore, we tested whether phosphorylation of AQP4 could be achieved in vitro by PKC. For this purpose, we incubated equal aliquots of rat brain homogenate with or without PDBu in the presence of γ-32P and PKC. After separation of immunoprecipitated proteins using AQP4-specific antibody, we observed two bands with an apparent molecular masses of 31 and 40–45 kDa (Fig. 4). The 31-kDa band corresponds to unglycosylated protein, and the 40–45-kDa band corresponds to glycosylated proteins. The intensity of phosphorylated bands in the presence of PDBu was significantly higher than the bands in the absence of PDBu or in the presence of PKC inhibitor or 4α-phorbol. Analysis of Fig. 4 by densitometry showed that the density of the 31-kDa band in the presence of PDBu was 12–12.5 times higher than that in the absence of PDBu or in the presence of PKC inhibitor or 4α-phorbol. These results strongly suggested that both glycosylated and unglycosylated AQP4 peptides were phosphorylated by PKC in the presence of PDBu. Decreased oocyte P f in AQP4 expressing oocytes by PDBu and PMA but not by inactive phorbol esters and increased32P incorporation into AQP4 in in vitrophosphorylation in the presence of PDBu but not in the presence of inactive phorbol or PKC inhibitor suggest that the phorbol ester-dependent phosphorylation of AQP4 is involved in the regulation of its water channel activity. Further studies are necessary to identify the unique amino acid residue responsible for the phosphorylation of AQP4 and determine whether phosphorylation by phorbol ester-dependent PKC changes the activity or distribution pattern of AQP4 in native tissue. Thr-107, Ser-111, and Ser-180 (8Jung J.S. Bhat R.V. Preston G.M. Guggino W.B. Baraban J.M. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 13052-13056Crossref PubMed Scopus (613) Google Scholar) may be the potential phosphorylation sites for AQP4 because they are contained in the recognition motifs (S*/T*)X(R/K) or RXXS* used by serine/threonine protein kinases such as PKC (26Pearson R.B. Kemp B.E. Methods Enzymol. 1991; 200: 62-81Crossref PubMed Scopus (865) Google Scholar). The regulation of aquaporins is the subject of major controversy. Recently, it was reported that forskolin stimulated the water channel activity of AQP1 (27Yool A.J. Stamer W.D. Regan J.W. Science. 1996; 273: 1216-1218Crossref PubMed Scopus (141) Google Scholar). Although some investigators failed to reproduce these observations (28Agre P. Lee M.D. Devidas S. Guggino W.B. Science. 1997; 275: 1490-1491Crossref PubMed Scopus (84) Google Scholar), we were able to duplicate these results in our laboratory (25Patil R.V. Han Z. Wax M.B. Biochem. Biophys. Res. Commun. 1997; 238: 392-396Crossref PubMed Scopus (56) Google Scholar, 29Patil R.V. Han Z. Wax M.B. Science. 1997; 275: 1492PubMed Google Scholar). Further, we have shown recently that AQP1 is regulated by arginine vasopressin and atrial natriuretic peptide in oocytes (25Patil R.V. Han Z. Wax M.B. Biochem. Biophys. Res. Commun. 1997; 238: 392-396Crossref PubMed Scopus (56) Google Scholar). Previous studies have shown that water channel activity of AQP2 is stimulated by cAMP-dependent protein phosphorylation (30Kuwahara M. Fushimi K. Terada Y. Bai L. Marumo F. Sasaki S. J. Biol. Chem. 1995; 270: 10384-10387Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). These results have also been controversial in oocyte (31Ma T. Hasegawa H. Skach W.R. Frigeri A. Verkman A.S. Am. J. Physiol. 1994; 266: C189-C197Crossref PubMed Google Scholar) as well as in phosphorylation (32Lande M.B. Jo I. Zeidel M.L. Somers M. Harris H.J. J. Biol. Chem. 1996; 271: 5552-5557Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) studies using AQP2. Yet a recent report suggested that protein kinase activators such as forskolin and PMA had no effect on the water channel activity of AQP1, AQP2, AQP3, AQP4, or AQP5 expressing oocytes and concluded that phosphorylation is not involved in the regulation of these proteins (33Yang B. Verkman A.S. J. Biol. Chem. 1997; 272: 16140-16146Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Some of these discrepancies may be due to variation in oocyte batches undertaken for the studies. For example, studies with activation of ionic currents in Xenopus oocytes by arginine vasopressin showed that not all donor frogs are responsive to this peptide, and the response was variable between oocytes from a single donor (34Moriarty T.M. Gillo B. Sealfon S. Landau E.M. Brain Res. 1988; 464: 201-205PubMed Google Scholar). Furthermore, these studies indicated that there may be a seasonal variation in expression of the receptors for these neuropeptides. The regulation of AQP4 is likely significant clinically because it is a major water channel protein in brain and may therefore play an important role in the swelling that follows stroke. In lung, where there is a sharp increase in AQP4 expression just after birth (35Umenishi F. Carter E.P. Yang B. Oliver B. Matthay M.A. Verkman A.S. Am. J. Respir. Cell Mol. Biol. 1996; 15: 673-679Crossref PubMed Scopus (71) Google Scholar) it may play an important role in the clearance of fluid from the newborn lungs. In eye, its presence in the ciliary body (9Hasegawa H. Ma T. Skach W. Matthay M.A. Verkman A.S. J. Biol. Chem. 1994; 269: 5497-5500Abstract Full Text PDF PubMed Google Scholar, 23Patil R.V. Saito I. Yang X. Wax M.B. Exp. Eye Res. 1997; 64: 203-209Crossref PubMed Scopus (107) Google Scholar, 36Patil R.V. Yang X. Saito I. Coca P.M. Wax M.B. Biochem. Biophys. Res. Commun. 1994; 204: 861-866Crossref PubMed Scopus (13) Google Scholar) may contribute to aqueous humor production and elevated intraocular pressure as occurs in glaucoma. Furthermore, its presence in retinal Muller cells may contribute to visual function by its involvement in the light-dependent hydration of space-surrounding photoreceptors (see Ref. 37Lee M.D. King L.S. Agre P. Medicine. 1997; 76: 141-156Crossref PubMed Scopus (94) Google Scholar). In addition, recent evidence demonstrates that AQP4 knockout mice have impaired ability to concentrate urine, suggesting a functional role for this aquaporin in the kidney (38Ma T. Yang B. Gillespie A. Carlson E.J. Epstein C.J. Verkman A.S. J. Clin. Invest. 1997; 100: 957-962Crossref PubMed Scopus (389) Google Scholar). At the least, our results provide mounting evidence that in addition to AQP2, other aquaporins such as AQP4 are likely amenable to pharmacological regulation and furthermore such activity appears to be of physiologic relevance.
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