Synthesis of silver nanoparticles using Emilia sonchifolia plant for treatment of bloodstream diseases caused by Escherichia coli.

OBJECTIVES: Among infectious diseases, bloodstream infection (BSI) caused by gram-negative bacteria (E. coli) is the leading cause of death worldwide. However, the bacteria have produced resistance to many of these antibiotics. Thus, the present study aimed to develop silver nanoparticles (AgNPs) loaded with Emilia sonchifolia (ES) extract (ES-AgNPs) to treat BSI efficiently. METHODS: AgNPs were synthesized by reduction of silver nitrate (AgNO3) solution by ES extract. Furthermore, these ES-AgNPs were characterized for particle size and zeta potential, crystallinity by powder X-ray diffraction (P-XRD) technique, in vitro antibacterial activity, time-kill assay, film bio adhesion, and fluorescence assay. RESULTS: Surface plasmon resonance (SPR) has been used to confirm the formation of AgNPs by seeing a shift in colour to dark-brown. The ES-AgNPs displayed a mean particle size of 137±3nm (PDI of 0.168±0.02) and zeta potential of 18.2±0.8mV. Furthermore, according to P-XRD results, the developed AgNPs are highly crystalline. The ES-AgNPs showed effective antibacterial action against E. coli with minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of 0.4±0.02µg/mL and 0.8±0.03µg/mL, respectively. In addition, ES-AgNPs inhibited biofilm formation and bacterial adhesion in a dose-dependent manner with 100% inhibition obtained in 48h at MBC. CONCLUSIONS: Present research work revealed that the ES-AgNPs obtained by green synthesis holds a prominent antibacterial activity in the treatment of BSIs caused by E. coli and they may be used as a competent substitute for current treatments. However, further, in vivo antibacterial studies are required to establish its efficacy in the treatment of BSIs.

Remusatia vivipara lectin and Sclerotium rolfsii lectin interfere with the development and gall formation activity of Meloidogyne incognita in transgenic tomato.

Root knot nematodes are serious threats to growth and yield of solaneous crops including tomato. In this study, a binary vector carrying Remusatia vivipara (rvl1) and Sclerotium rolfsii (srl1) lectin genes were introduced independently into Lycopersicon esculentum cv. Pusa Ruby via Agrobacterium tumefaciens for resistance against root knot nematode, Meloidogyne incognita. In total, one hundred and one rvl1 and srl1-transformed plants exhibiting kanamycin resistance were confirmed to carry transgenes as detected by polymerase chain reaction (PCR) with 4.59% transformation efficiency. Genetic analysis of T1 progeny confirmed Mendelian segregation of the introduced genes. Three events each of rvl1 and srl1 transgenic tomato were randomly selected for further confirmation by Southern and TAIL-PCR analyses. All three events of srl1 transgenics showed single copy transgene, whereas two rvl1 transgenic events showed single copy of transgene, while remaining event showed two copies of transgenes. Site of integration obtained for rvl1 and srl1 transgenic events by TAIL-PCR revealed that all the three events of rvl1 and srl1 transgenics differed for their site of integration and insertion sites did not contain any predicted gene. Moreover, expression of the rvl1 and srl1 transgenes was detected by haemagglutination assay in all three events of rvl1 and srl1, but not in non-transgenic tomato plant. Homozygous progenies of these events were grown and inoculated with M. incognita. Development and reproduction of M. incognita was severely affected in transgenic tomato plants expressing RVL1 and SRL1 exhibiting the high levels of resistance compared to non-transgenic plants. Therefore, these transgenic lines demonstrate a promising potential for variety development of tomato lines with enhanced resistance against M. incognita.

Rho-Rho kinase pathway in the actomyosin contraction and cell-matrix adhesion in immortalized human trabecular meshwork cells.

PURPOSE: The outflow facility for aqueous humor across the trabecular meshwork (TM) is enhanced by agents that oppose the actomyosin contraction of its resident cells. Phosphorylation of MYPT1 (myosin light chain [MLC] phosphatase complex of Type 1) at Thr853 and Thr696 inhibits dephosphorylation of MLC, leading to an increase in actomyosin contraction. In this study, we examined the effects of Rho kinase (ROCK) inhibitors on the relative dephosphorylation of the two sites of MYPT1 using human TM cells (GTM3). METHODS: Dephosphorylation of MYPT1 at Thr853 and Thr696 was determined by western blot analysis following exposure to selective inhibitors of ROCK, namely Y-27632 and Y-39983. Consequent dephosphorylation of MLC and decreases in actomyosin contraction were assessed by western blot analysis and collagen gel contraction assay, respectively. Changes in the cell-matrix adhesion were measured in real time by electric cell-substrate impedance sensing and also assessed by staining for paxillin, vinculin, and focal adhesion kinase (FAK). RESULTS: Both ROCK inhibitors produced a concentration-dependent dephosphorylation at Thr853 and Thr696 of MYPT1 in adherent GTM3 cells. IC50 values for Y-39983 were 15 nM and 177 nM for dephosphorylation at Thr853 and Thr696, respectively. Corresponding values for Y-27632 were 658 nM and 2270 nM. Analysis of the same samples showed a decrease in MLC phosphorylation with IC50 values of 14 nM and 1065 nM for Y-39983 and Y-27632, respectively. Consistent with these changes, both inhibitors opposed contraction of collagen gels induced by TM cells. Exposure of cells to the inhibitors led to a decrease in the electrical cell-substrate resistance, with the effect of Y-39983 being more pronounced than Y-27632. Treatment with these ROCK inhibitors also showed a loss of stress fibers and a concomitant decrease in tyrosine phosphorylation of paxillin and FAK. CONCLUSIONS: Y-39983 and Y-27632 oppose ROCK-dependent phosphorylation of MYPT1 predominantly at Thr853 with a corresponding decrease in MLC phosphorylation. A relatively low effect of both ROCK inhibitors at Thr696 suggests a role for other Ser/Thr kinases at this site. Y-39983 was several-fold more potent when compared with Y-27632 at inhibiting the phosphorylation of MYPT1 at either Thr853 or Thr696 commensurate with its greater potency at inhibiting the activity of human ROCK-I and ROCK-II enzymes.

Immunoprecipitation of A1 adenosine receptor-GTP-binding protein complexes in ciliary epithelial cells.

PURPOSE: To examine the interaction of alpha subunits of guanine nucleotide binding proteins with A1 adenosine receptors from the SV40-transformed bovine-derived pigmented (PE) and human-derived nonpigmented (NPE) ciliary epithelial cell lines using an immunoprecipitation approach and [3H]DPCPX, a selective radioligand to adenosine receptor. METHODS: Solubilized preparations of adenosine receptors from PE and NPE cell lines were immunoprecipitated with G protein specific antisera 8730 (anti-Gi alpha), 3646 (anti-Gi alpha 1), 1521 (anti-Gi alpha 2), and 1518 (anti-Gi alpha 3), and of adenosine receptor-G protein complexes were detected by the binding of radioactive [3H]DPCPX. RESULTS: Data indicate that [3H]DPCPX forms high-affinity complex with membrane-bound and solubilized forms of adenosine receptors from PE and NPE cells. Peptide-directed antisera against various G protein alpha subunits indicate that the A1 adenosine receptors from these cells are specifically coupled to Gi alpha complexes. The results further indicate that the A1 adenosine receptors are predominantly associated to Gi alpha-3. CONCLUSION: The findings document a selective interaction between the alpha subunits of the inhibitory guanine nucleotide binding protein (Gi) and A1 adenosine receptors in ocular ciliary epithelial cells in culture. The results suggest that adenosine receptors coupled to Gi alpha-3 may provide a site at which modulation of aqueous humor production in the ciliary epithelium occurs via the G protein-adenylyl cyclase pathway.

TNF-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes.

PURPOSE: To determine the expression and localization of tumor necrosis factor (TNF)-alpha and TNF-alpha receptor-1 in the retina of normal and glaucomatous eyes. METHODS: Using immunohistochemistry and in situ hybridization, retinal expression and localization of TNF-alpha and TNF-alpha receptor-1 were studied in retina sections from 20 eyes of donors with glaucoma, and 20 eyes of age-matched normal donors. RESULTS: According to immunohistochemistry, the intensity of the immunostaining and the number of labeled cells for TNF-alpha or its receptor were greater in retina sections of glaucomatous eyes than in control eyes of age-matched normal donors. In situ hybridization showed that mRNA signals for TNF-alpha or TNF-alpha receptor-1 were similarly more intense in glaucomatous eyes than in age-matched control eyes. Both protein and mRNA of TNF-alpha or TNF-alpha receptor-1 were predominantly localized to the inner retinal layers. Double-immunofluorescence labeling demonstrated that retinal immunostaining for TNF-alpha was predominantly positive in the glial cells, whereas immunostaining for TNF-alpha receptor-1 was mainly positive in the retinal ganglion cells. CONCLUSIONS: Upregulation of TNF-alpha and its receptor-1 in glaucomatous retina suggest that TNF-alpha-mediated cell death is involved in the neurodegeneration process of glaucoma.

Serum autoantibody against glutathione S-transferase in patients with glaucoma.

PURPOSE: To identify retinal proteins that are the targets of serum autoantibodies in patients with glaucoma. METHODS: To identify retinal antigens that are recognized by the sera of patients with glaucoma, immunoreactive bands were separated, by using two-dimensional gel electrophoresis of the bovine retinal soluble fraction. A 29-kDa band was then selected for further analysis. Tryptic peptides of the 29-kDa band were analyzed using electrospray mass spectrometry to identify the protein. After protein identification, immunoreactivity against this newly identified protein was studied by Western blot analysis using sera from 65 patients with glaucoma (25 with primary open-angle glaucoma [POAG]; 40 with normal-pressure glaucoma [NPG]) and 25 age-matched healthy subjects. In addition, serum antibody titers were compared in these groups, by using a specific enzyme-linked immunosorbent assay (ELISA). RESULTS: The 29-kDa band was identified as glutathione S-transferase (GST). Western blot analysis revealed that serum antibodies against GST antigen were recognized in 34 (52%) of 65 patients with glaucoma (22 of NPG and 12 of POAG) and 5 (20%) of 25 age-matched control subjects (chi(2) test, P < 0.05). By ELISA, it was also found that patients with glaucoma had higher titers of anti-GST antibody, compared with the control group (Mann-Whitney test; NPG versus control, P = 0.013; POAG versus control, P = 0.0006). CONCLUSIONS: These findings indicate that GST is one of the retinal antigens targeted by the serum antibodies detected in some patients with glaucoma.

Induction of HLA-DR expression in human lamina cribrosa astrocytes by cytokines and simulated ischemia.

PURPOSE: Recent evidence strongly suggests that activated immunity occurs during the neurodegenerative process of glaucomatous optic neuropathy. Although activation of lamina cribrosa astrocytes has been identified in glaucomatous optic nerve head, their role on the activated immune responses seen in glaucoma patients is unknown. Here, the authors aimed to study the potential role of lamina cribrosa astrocytes as a component of activated immune responses seen in glaucoma patients. METHODS: Expression of HLA-DR in optic nerve head astrocytes was studied using immunohistochemistry in postmortem eyes of patients with glaucoma and normal donors. Serum cytokine levels of patients with glaucoma and control subjects were measured using enzyme-linked, immunosorbent assay. In addition, in vitro experiments were performed using astrocyte cultures derived from human optic nerve head or fetal human brain. The cultured astrocytes were incubated under selected stress conditions such as exposure to cytokines, IFN-gamma and IL-10, or simulated ischemia for up to 48 hours. The expression of HLA-DR was studied in these cells using flow cytometry and immunocytochemistry. RESULTS: Immunohistochemistry demonstrated an upregulation of the HLA-DR expression in the optic nerve head astrocytes in glaucoma. In addition, serum levels of IL-10 was higher in the patients with normal pressure glaucoma compared to age-matched control subjects (P: = 0.001). Regarding in vitro experiments, unlike brain astrocytes, the percentage of cells expressing HLA-DR was approximately 3 times higher in the cultures of optic nerve head astrocytes exposed to simulated ischemia compared to cultures incubated under normal conditions (P: = 0.09). Incubation with IFN-gamma induced HLA-DR expression in brain and lamina cribrosa astrocytes, up to 25-fold, (P < 0.001) either in the absence or presence of simulated ischemia. Induction of HLA-DR expression by IL-10 was approximately 6 times higher in lamina cribrosa astrocytes incubated under simulated ischemia compared to that incubated under normal condition (P: = 0.004) and was not prominent in brain astrocytes. CONCLUSIONS: These findings suggest that optic nerve head astrocytes function as antigen-presenting cells and that their immunogenic capacity is more sensitive to ischemia than brain astrocytes. Taken together, these findings provide novel evidence that regulation of immunogenic capacity of optic nerve head astrocytes by cytokines or ischemic stress may have a role during the neurodegeneration process in patients with glaucoma.

Detection of EP2, EP4, and FP receptors in human ciliary epithelial and ciliary muscle cells.

We have examined the expression of three prostaglandin (PG) receptors, EP2, EP4, and FP, in a nonpigmented ciliary epithelial cell line (ODMCl-2) and in human ciliary muscle (HCM) cells. Total RNA preparations from either ODMCl-2 or HCM cells were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with sense and antisense primers for each of the three PG receptors. The RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors in both ODMCl-2 and HCM cells. PCR products corresponding to each receptor were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. Pharmacological studies with the EP2 receptor-selective agonist butaprost resulted in a significant increase in the cyclic AMP level in ODMCl-2 cells. The stimulation of cyclic AMP in ODMCl-2 cells by PGE2 and 11-deoxy PGE1, the respective EP1/EP2/EP3/EP4 and EP2/EP3/EP4 receptor agonists, was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. These results conclusively demonstrate the presence of both mRNA and protein for EP2, EP4, and FP receptors in ODMCl-2 and HCM cells.

T cell subsets and sIL-2R/IL-2 levels in patients with glaucoma.

PURPOSE: We hypothesize that cellular immunity may have a previously unrecognized role in glaucomatous optic neuropathy. The purpose of this study is to analyze subsets of T cells and the levels of cytokine IL-2 and the soluble IL-2 receptor in peripheral blood from patients with normal pressure glaucoma (NPG) or primary open angle glaucoma (POAG) in comparison to age-matched control subjects. METHODS: In this study, 38 patients (20 NPG; 18 POAG) and 19 controls were included. sIL-2R and IL-2 were assayed by ELISA. T cell subsets were analyzed by flow cytometry and lymphocyte proliferation was used to measure the reactive ability of T cells to phytohemagglutinin (PHA). RESULTS: The frequency of CD8(+)HLA-DR(+) lymphocytes were increased in patients with NPG (P = 0.008), and CD3(+)CD8(+) lymphocytes increased in both NPG (P = 0.03) and POAG patients (P = 0.0004). CD5(+) lymphocytes were higher only in POAG patients (P = 0.0012). In comparison to controls, the ratio of CD4(+)/CD8(+) lymphocytes was similar in both groups. The mean concentrations of sIL-2R in NPG (P = 0.011) and POAG (P = 0.0023) patients were higher than that found in control subjects although IL-2 concentrations were similar in these groups. In addition, the reactive ability of T lymphocytes to the non-specific reagent (PHA) was reduced significantly in NPG (P = 0.02) and POAG patients (P=0.04). CONCLUSION: The alterations of the cellular immune system in patients with glaucoma support our hypothesis that the immune system may play an important role in the initiation and/or sustainment of glaucomatous optic neuropathy in some patients.

Flow cytometry for quantification of retrogradely labeled retinal ganglion cells by Fluoro-Gold.

PURPOSE: To count retrogradely labeled retinal ganglion cells by Fluoro-Gold. METHODS: Retinal ganglion cells were retrogradely labeled using bilateral injections of Fluoro-Gold into the superior colliculus. One week after injections, retinas were dissociated and immunolabeled using specific antibody against Fluoro-Gold. The Fluoro-Gold labeled cells were then counted using flow cytometry. RESULTS: Flow cytometry revealed that approximately 7% of the dissociated retinal cells were ganglion cells retrogradely labeled by Fluoro-Gold (Fig. 1B). Based on the total count of retinal cells per eye, the total number of retinal ganglion cells was estimated at approximately 131,250 +/- 2,542 per rat eye. The coefficient of variation of counts was calculated as 1.98%. CONCLUSIONS: The use of flow cytometry facilitates simple, reproducible and rapid quantification of virtually all of the retinal ganglion cells labeled by Fluoro-Gold in a single rat eye.

Molecular identification of functional water channel protein in cultured human nonpigmented ciliary epithelial cells.

PURPOSE: Water channel proteins are important pathways for water movements across cell membranes, including those in the ciliary epithelium, which is the major site of aqueous humor secretion. In this study, we aimed to demonstrate the expression of functionally active aquaporin-1 (AQP1) water channels in cultured human ciliary epithelial cells. METHODS: Poly A(+) RNA was isolated from cell cultures of Simian Virus 40 (SV-40) transformed human nonpigmented ciliary epithelium (NPE) subjected to RT-PCR reaction using primers specific to AQP1. Northern analysis was used to define the expression of AQP1 in NPE cells. Western immunoblotting with polyclonal antibody raised against AQP1 was used to evaluate the AQP1 protein expression in the plasma membranes of human NPE cells. Light scattering method was used to determine the osmotic water permeability in the suspension of NPE cells. RESULTS: RT-PCR using specific primers for AQP1, Northern analysis and Western immunoblot using AQP1 specific antibody demonstrated the expression of AQP1 in the plasma membranes of NPE cells. Osmotic water permeability (P( f)) measurements confirmed that functional AQP1 water channels are expressed in human NPE cells and the P(f) for these cells was 9.8 x 10( -3) cm/s at 10 degrees C. CONCLUSIONS: The presence of AQP1 in human NPE cells suggests that it may have a role in the fluid flow across epithelial membranes. In addition, the existence of AQP1 in the human NPE cells provide an excellent in vivo model to study the regulation of aquaporins and their possible role in the aqueous humor secretion.

Cloning, nucleotide sequence, overexpression, and inactivation of the Escherichia coli 2-keto-4-hydroxyglutarate aldolase gene.

Having previously determined the complete amino acid sequence of 2-keto-4-hydroxyglutarate aldolase from Escherichia coli (C. J. Vlahos and E. E. Dekker, J. Biol. Chem. 263:11683-11691, 1988), we amplified the gene that codes for this enzyme by the polymerase chain reaction using synthetic degenerate deoxyoligonucleotide primers. The amplified DNA was sequenced by subcloning the polymerase chain reaction products into bacteriophage M13; the nucleotide sequence of the gene was found to be in exact agreement with the amino acid sequence of the gene product. Overexpression of the gene was accomplished by cloning it into the pKK223.3 expression vector so that it was under control of the tac promoter and then using the resultant plasmid, pDP6, to transform E. coli DH5 alpha F'IQ. When this strain was grown in the presence of isopropyl beta-D-thiogalactopyranoside, aldolase specific activity in crude extracts was 80-fold higher than that in wild-type cells and the enzyme constituted approximately 30% of the total cellular protein. All properties of the purified, cloned gene product, including cross-reactivity with antibodies elicited against the wild-type enzyme, were identical with the aldolase previously isolated and characterized. A strain of E. coli in which this gene is inactivated was prepared for the first time by insertion of the kanamycin resistance gene cartridge into the aldolase chromosomal gene.

Photoaffinity labeling of the allosteric AMP site of biodegradative threonine dehydratase of Escherichia coli with 8-azido-AMP.

The photoreactive AMP analog, 8-azido-AMP, stimulated the activity of biodegradative threonine dehydratase of Escherichia coli in a reversible manner and, like AMP, decreased the Km for threonine. The concentrations required for half-maximal stimulation by AMP and 8-azido-AMP were 40 microM and 1.5 microM, respectively, and the maximum stimulation by 8-azido-AMP was 25% of that seen with AMP. Gel-filtration experiments revealed that 8-azido-AMP stabilized a dimeric form of the enzyme, whereas AMP promoted a tetrameric species. When present together, AMP and 8-azido-AMP showed mutual competition in influencing catalytic activity as well as the conformational state of the protein. Photolabeling of AMP-free dehydratase with 8-azido-[2-3H]AMP resulted in a time and concentration-dependent enzyme inactivation and concomitant incorporation of 8-azido-AMP into protein. At low 8-azido-AMP concentrations, incorporation of about 1 mol 8-azido-AMP/mol dehydratase tetramer was correlated with almost complete inactivation of the enzyme. The presence of AMP in the photolabeling reaction greatly reduced the extent of enzyme inactivation and 8-azido-AMP binding. Ultraviolet irradiation with 20 microM 3H-labeled 8-azido-AMP revealed one tryptic peptide, Thr230-Thr-Gly-Thr-Leu-Ala-Asp-Gly-Cys-Asp-Val-Ser-Arg242, with bound radioactivity. This peptide, labeled at low concentration of 8-azido-AMP, most likely represents the AMP-binding region on the dehydratase molecule.

Amino acid sequence of the regulatory-site glyoxylate peptide of biodegradative threonine dehydratase of Escherichia coli.

Incubation of purified Escherichia coli biodegradative threonine dehydratase with glyoxylate resulted in covalent binding of 1 mol of glyoxylate per mol of protein with concomitant loss of enzyme activity. The glyoxylate-binding site was identified as a heptapeptide representing amino acid residues Ser-33-Asn-Tyr-Phe-Ser-Glu-Arg-39 in the protein primary structure. Addition of glyoxylate to a culture of E. coli cells led to time-dependent enzyme inactivation. Immunoprecipitation with anti-dehydratase antibody of extract from [14C]glyoxylate-treated cells revealed labeled dehydratase polypeptide. These results are interpreted to mean that enzyme inactivation by glyoxylate in E. coli cells is associated with covalent protein modification.

Protein kinase A-dependent phosphorylation of aquaporin-1.

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. Previous results from our laboratory demonstrated that water channel activity of AQP1 was significantly increased by protein kinase A (PKA) activators such as cyclic-AMP (cAMP) and forskolin. The purpose of this study is to determine whether PKA-dependent protein phosphorylation is involved in the regulation of water channel activity of AQP1. Results presented here suggest that catalytic subunit of protein kinase A significantly increased the amount of phosphorylated AQP1 protein. In addition, these results indicated that cAMP-responsive redistribution of AQP1 may be regulated by phosphorylation of AQP1. Moreover, they provide new insights on the molecular mechanisms for regulating water balance in several tissues involving rapid water transport such as ciliary epithelium. In addition, they suggest important potential roles for AQP1 in several clinical disorders involving rapid water transport such as glaucoma.

Catabolite gene activator protein and integration host factor act in concert to regulate tdc operon expression in Escherichia coli.

Anaerobic expression of the tdcABC operon of Escherichia coli requires cyclic AMP and the catabolite gene activator protein (CAP). Purified CAP binds to a 30-bp sequence in the tdc promoter between positions -55 and -26, and a mutant CAP site with base substitutions at positions -48, -47, and -45 failed to bind CAP and also drastically reduced the beta-galactosidase expression from a tdcB'-'lacZ fusion plasmid. Recently, we showed that efficient expression of the tdc operon also requires a functional integration host factor (IHF) and an IHF-binding site in the tdc promoter between positions -118 and -88. The levels of beta-galactosidase activity from the tdcB'-'lacZ fusion plasmids were also reduced in an IHF-deficient strain with the wild-type or mutant plasmid CAP sequence. In vitro footprinting experiments revealed that CAP and IHF occupy their specific binding sites on tdc DNA when they are present separately or together. These regulatory proteins also induced significant bending of the tdc promoter DNA. Our results suggest that CAP and IHF act in concert as positive transcription factors for tdc operon expression in vivo.

Regulation of aquaporin-4 water channels by phorbol ester-dependent protein phosphorylation.

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 microM) 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.