Biomedical subjects

Paul J Donaldson

Publications and source records attributed to Paul J Donaldson.

12 recordsLinked to original sources

PubMed2006

Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens.

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH is maintained at unusually high concentrations as a result of direct GSH uptake and/or intracellular de novo synthesis from its precursor amino acids; cysteine, glycine and glutamine/glutamate. With increasing age, the levels of GSH, particularly in the core of the lens, are significantly reduced. It has been proposed that alterations in the transport of GSH and/or its precursor amino acids may contribute to the changes in GSH levels in older lenses. As considerable uncertainty exists about the molecular identity of GSH transporters in the lens, we have focused on identifying transporters involved in the uptake of the precursor amino acids required for GSH synthesis. Previously, we identified an uptake system for cyst(e)ine mediated by the Xc(-) exchanger and the Excitatory Amino Acid Transporters (EAATs) in the rat lens. In this current study, we have identified and localised additional uptake systems that contribute to GSH synthesis. Transcripts for GLYT1 (glycine transporter) and ASCT2 (glutamine/glutamate transporter) were detected in rat lens fiber cells using the reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of both GLYT1 and ASCT2 at the protein level. Immunocytochemistry revealed GLYT1 expression to be restricted to cortical regions of the lens. Labelling was predominantly cytoplasmic with some labelling of the membrane. In contrast, ASCT2 was expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, ASCT2 expression was predominantly cytoplasmic. However, with deeper distance into the lens, labelling became more membraneous indicating insertion of ASCT2 into the membranes of mature fiber cells of the lens core. The molecular identification and localisation of GLYT1 and ASCT2 in the lens suggests that these transporters may be responsible for the uptake of the precursor amino acids, glycine and glutamine, which are involved in GSH synthesis. Moreover, the presence of ASCT2 in the centre of the lens raises the possibility that ASCT2 may work with the Xc(-) exchanger to accumulate cysteine where it can potentially act as a low molecular mass antioxidant.

Amino Acid Transport System ASC↗

PubMed2006

Roles for KCC transporters in the maintenance of lens transparency.

PURPOSE: To determine whether the potassium chloride cotransporter (KCC) family is expressed in the rat lens and to ascertain whether the transporters are involved in the regulation of lens volume and transparency. METHODS: RT-PCR was performed on RNA extracted from fiber cells to identify members of the KCC family expressed in the lens. Western blot analysis and immunocytochemistry, using KCC isoform-specific antibodies, were used to verify expression at the protein level and to localize KCC isoform expression. Organ-cultured rat lenses were incubated in isotonic artificial aqueous humor (AAH) that contained either the KCC-specific inhibitor [(dihydronindenyl)oxy] alkanoic acid (DIOA), the KCC activator N-ethylmaleimide (NEM), or the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) for up to 18 hours. Lens wet weight was monitored, and lens transparency and tissue morphology were recorded with dark-field and confocal microscopy, respectively. RESULTS: Molecular experiments to characterize KCC isoform expression showed that KCC1, -3, and -4 were all expressed in the lens at both the transcript and protein levels and that KCC2 was not. Immunocytochemistry indicated that the three KCC isoforms exhibited distinct differentiation-dependent expression patterns, with KCC1 and -3 being restricted to the lens cortex, whereas KCC4 was found throughout the entire lens, including the lens core. In the lens cortex, most of the labeling for all KCC isoforms was cytoplasmic, whereas in the lens core, KCC4 labeling was associated with the membrane. Incubation of lenses in 100 microM DIOA for 18 hours caused lenses to increase their wet weight and induced a cortical opacity that was caused by extensive damage to peripheral fiber cells located up to 150 microm in from the lens capsule, whereas deeper fiber cells appeared unaffected by DIOA exposure. Lower concentrations of DIOA (10 microM) revealed that this damage was initiated primarily by the swelling of peripheral fiber cells. In contrast, NPPB-treated lenses exhibited a deeper zone (>100 microm) of cell damage that was initiated by the dilation of the extracellular space between fiber cells. Exposure of lenses to the KCC activator NEM caused cell shrinkage in peripheral fiber cells but extensive cell swelling in deeper fiber cells. Peripheral cell swelling caused a differential recruitment of KCC isoforms from a cytoplasmic pool to the plasma membrane. DIOA-induced cell swelling increased the association of KCC4 with membrane, whereas hypotonic cell swelling dramatically increased the association of KCC1 with the membrane. CONCLUSIONS: The rat lens expresses three KCC transporter isoforms (KCC1, -3, and -4) in a differentiation-dependent manner. Modulation of transporter activity and subcellular localization suggests that multiple KCC transporters mediate KCl efflux in peripheral fiber cells in a dynamic fashion. These results indicate that, in addition to Cl- channels, KCC transporters play a role in mediating a circulating flux of Cl- ions, which contributes to the maintenance of lens transparency through controlling the steady state volume of lens fiber cells.

Animals↗

PubMed2005

Molecular characterization of the cystine/glutamate exchanger and the excitatory amino acid transporters in the rat lens.

PURPOSE: To determine whether the cyst(e)ine/glutamate exchanger (XC-) and the excitatory amino acid transporters (EAAT1 to -5) are expressed in the rat lens. METHODS: A combination of molecular-based and immunocytochemical strategies was used to screen for the presence of the light-chain subunit of XC- (xCT) and the five known EAAT isoforms in the rat lens. An initial molecular profiling of xCT and EAAT1 to -5 expression was achieved by reverse transcription-polymerase chain reaction (RT-PCR). The presence of transporter proteins was verified by Western blot analysis and immunocytochemistry. RESULTS: Transcripts for xCT and EAAT1 to -5 were detected by RT-PCR in lens fiber cells. Western blot analysis confirmed the expression of xCT and all five EAAT isoforms at the protein level. Immunocytochemistry revealed xCT expression to be present throughout the lens. Notably, changes in the subcellular distribution of xCT were shown to occur as a function of fiber cell differentiation. In the outer cortex, xCT labeling was predominantly cytoplasmic but progressively became more membranous with distance into the lens, due to xCT insertion into the broad sides of fiber cells. In the core, xCT labeling was localized around the entire membrane of inner fiber cells suggesting a redistribution of the exchanger. In contrast, EAAT expression was restricted to the outer cortex of the lens, with EAAT4/5 shown to be the predominant isoforms in cortical fiber cells. Western blot analysis of crude fiber membranes dissected from the outer cortex, inner cortex, and core region of the lens confirmed the presence of xCT in all three of these regions and demonstrated that EAATs were absent from the core region. CONCLUSIONS: The molecular identification and localization of xCT and EAAT1 to -5 in the lens raises the possibility that in the outer cortex XC- and EAAT4/5 may work together to accumulate cysteine for GSH synthesis. The presence of xCT and the absence of the EAATs in the center of the lens suggest that XC- could operate with an alternative glutamate uptake pathway to accumulate cysteine where it can potentially act as a low-molecular-mass antioxidant.

Amino Acid Transport System X-AG↗

PubMed2004

Functional imaging: new views on lens structure and function.

1. We have developed an experimental imaging approach that allows the distribution of lens membrane proteins to be mapped with subcellular resolution over large distances as a function of fibre cell differentiation. 2. Using this approach in the rat lens, we have localized precisely histological sites of connexin 46 cleavage, quantitatively mapped changes in gap junction distribution and fibre cell morphology and correlated these changes to differences in intercellular dye transfer. 3. Profiling of glucose transporter isoform expression showed that lens epithelial cells express GLUT1, whereas deeper cortical fibre cells express the higher-affinity GLUT3 isoform. Near the lens periphery, GLUT3 was located in the cytoplasm of fibre cells, but it underwent a differentiation-dependent membrane insertion. 4. Similarly, the putative adhesion protein membrane protein 20 is inserted into fibre cell membranes at the stage when the cells lose their nuclei. This redistribution is strikingly rapid in terms of fibre cell differentiation and correlates with a barrier to extracellular diffusion. 5. Our imaging-orientated approach has facilitated new insights into the relationships between fibre cell differentiation and lens function. Taken together, our results indicate that a number of strategies are used by the lens during the course of normal differentiation to change the subcellular distribution, gross spatial location and functional properties of key membrane transport proteins.

Animals↗

PubMed2004

Gap junction processing and redistribution revealed by quantitative optical measurements of connexin46 epitopes in the lens.

PURPOSE: To map changes in the structure and function of fiber cell gap junctions that occur with lens differentiation. METHODS: Equatorial lens sections were fluorescently labeled with antibodies to the gap junction protein connexin (Cx)46, the membrane marker wheat germ agglutinin, and the nuclear stain propidium iodide. Two-photon microscopy and digital image analysis were used to quantify label and cell morphology as a function of radial distance (r/a) across the lens. Loop- and tail-specific Cx46 antibodies were used to identify regions of posttranslational modification. Local fiber cell coupling was imaged in situ using two-photon flash photolysis of caged fluorescein. RESULTS: Antibody labeling showed that the cytoplasmic tail of Cx46 was removed in two zones (r/a approximately 0.9 and r/a approximately 0.7). In addition, with increasing depth, the large radially aligned plaques of peripheral fiber cells became fragmented and dispersed around the cell membrane, and cells became more circular in cross section. Fluorescein transfer between peripheral fiber cells was highly anisotropic and occurred predominantly within a column of fiber cells, resulting in radially directed transport. In regions beyond the zone of nuclear loss, transport was more isotropic and occurred across columns of fiber cells. CONCLUSIONS: The cleavage of Cx46 is associated with a spatial redistribution of gap junction plaques. The distribution of gap junction plaques around the cell membrane can explain the observed directionality of intercellular solute transfer. The findings suggest that the processing and redistribution of gap junction proteins is central to controlling radial and circumferential solute gradients in different regions within the lens.

Animals↗

PubMed2004

Cl- influx into rat cortical lens fiber cells is mediated by a Cl- conductance that is not ClC-2 or -3.

PURPOSE: Exposure of organ-cultured lenses to Cl(-) channel blockers under isotonic conditions induces a localized cortical zone of extracellular space dilations. The purpose of this study was to investigate whether elongated lens fiber cells from this zone contain an anion conductance that mediates Cl(-) influx and whether two chloride channel isoforms known to be expressed in the lens (ClC-2 and -3) are responsible. METHODS: Fiber cells were isolated by enzymatic dissociation in the presence of Gd(3+) and Co(2+) and their electrical properties analyzed by whole-cell patch clamping. Cells from the zone of extracellular space dilations were selected for analysis on the basis of cell length. RT-PCR and immunocytochemistry were used to determine whether ClC-2 or -3 channel isoforms are expressed in fiber cells located in the zone of extracellular space dilations. RESULTS: Cells from the zone of extracellular space dilations were typically >120 microm in length and exhibited an outwardly rectifying Cl(-) conductance that was blocked by DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) and displayed an anion selectivity sequence of I(-) > Cl(-) >> gluconate. ClC-2 and -3 were found to be expressed at the transcript and protein level in lens fiber cells, but subsequent immunocytochemical studies indicated that expressed proteins did not colocalize with cell membranes in the zone of extracellular space dilations, being predominately cytoplasmic in nature. CONCLUSIONS: Taken together, the data indicate that extracellular space dilations are due to the inhibition of a Cl(-) channel(s) that normally mediates Cl(-) influx into cortical lens fiber cells under isotonic conditions. The molecular identity of this channel remains to be determined.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2003

Resolving morphology and antibody labeling over large distances in tissue sections.

Protein expression patterns are a primary determinant of tissue function and in this study we developed methods to study protein expression over macroscopic distances at subcellular levels of detail. Using the mammalian lens as a model tissue system, we show that by combining two-photon microscopy with novel image montage methods (fast beam blanking coupled with mathematical alignment tools) we have extended the limited field of view of laser scanning microscopes. To illustrate the utility of our approach, the distribution of connexin-46 was visualized across equatorial sections of the rat mammalian lens. By optimizing fixation protocols, good morphological preservation could be achieved over the thickness of the lens (approximately 4 mm) while preserving antigenicity of lens proteins. Using the same image data, changes in lens fiber cell morphology were mapped quantitatively by automatic image analysis routines. The methods presented should be generally applicable to any tissue system where changes in antibody labeling and tissue structure occur over large and small distances.

Animals↗

PubMed2003

Insertion of MP20 into lens fibre cell plasma membranes correlates with the formation of an extracellular diffusion barrier.

It is known that during lens differentiation a number of fibre cell specific membrane proteins change their expression profiles. In this study we have investigated how the profiles of the two most abundant fibre cell membrane proteins AQP0 (formerly known as Major Intrinsic Protein, MIP) and MP20 change as a function of fibre cell differentiation. While AQP0 was always found associated with fibre cell membranes, MP20 was initially found in the cytoplasm of peripheral fibre cells before becoming inserted into the membranes of deeper fibre cells. To determine at what stage in fibre cell differentiation MP20 becomes inserted into the membrane, sections were double-labelled with an antibody against MP20, and propidium iodide, a marker of cell nuclei. This showed that membrane insertion of MP20 occurs in a discrete transition zone that coincided with the degradation of cell nuclei. To test the significance of the membrane insertion of MP20 to overall lens function, whole lenses were incubated for varying times in a solution containing either Texas Red-dextran or Lucifer yellow as markers of extracellular space. Lenses were fixed and then processed for immunocytochemistry. Analysis of these sections showed that both tracer dyes were excluded from the extracellular space in an area that coincided with insertion of MP20 into the plasma membrane. Our results suggest that the insertion of MP20 into fibre cell membranes coincides with the creation of a barrier that restricts the diffusion of molecules into the lens core via the extracellular space.

Animals↗

PubMed2003

Connexin expression patterns in the rat cornea: molecular evidence for communication compartments.

PURPOSE: To identify and localize candidate connexin family members in adult rat cornea that may be important in coordinating corneal cell biology. METHODS: To identify candidate connexin family members in adult rat cornea, a RT-PCR-based screening approach was initially adopted. Fourteen pairs of connexin isoform-specific primers were used to amplify connexin transcripts from two populations of RNA isolated from either the central cornea or the whole cornea. Immunohistochemistry and confocal microscopy were then used to confirm the presence and localization of connexins. RESULTS: Eight connexin transcripts (Cxs 26, 30.3, 31, 31.1, 33, 37, 43, 50) are present in central cornea, and the peripheral cornea additionally expresses Cxs 30, 40, 45, and 46. No Cx32 or Cx36 transcripts were amplified. Immunohistochemistry revealed that Cxs 26, 30, 31.1, 37, and 43 are expressed in spatially distinct patterns within the cornea. Cx26 and Cx43 occur in basal cells of the whole corneal epithelium and between endothelial cells. Cx26 also immunolocalizes to the first layer of intermediate epithelial cells, and Cx43 antibody labels stromal keratocytes. Cx30 is expressed in the peripheral corneal epithelium and disappears toward the central cornea. Cx31.1 expression is restricted to superficial corneal epithelial cells, and Cx37 spans the intermediate corneal epithelium. CONCLUSION: The spatially distinct cellular expression patterns of Cxs 26, 30, 31.1, 37, and 43 in the corneal epithelium imply that gap junctions play important roles in controlling corneal epithelial proliferation and differentiation and overall corneal maintenance.

Animals↗

PubMed2003

Expression patterns for glucose transporters GLUT1 and GLUT3 in the normal rat lens and in models of diabetic cataract.

PURPOSE: To determine whether the expression levels and cellular distribution of the facilitative glucose transporters GLUT1 and -3 undergo changes in the hyperglycemic lens. METHODS: Hyperglycemia was induced in vivo by injecting rats with streptozotocin or in vitro by culturing lenses in the presence of 50 mM glucose. Northern blot analysis and quantitative RT-PCR were used to detect changes in GLUT1 and -3 transcript levels, and Western blot analysis was used to monitor changes in GLUT3 protein expression levels in diabetic rats. Immunocytochemistry was used to map the cellular distribution of GLUT3 in normal and hyperglycemic lenses. RESULTS: GLUT1 and -3 were found to be differentially expressed in the epithelial and fiber cells, respectively. In the fiber cells, the distribution of GLUT3 protein changed as a function of fiber cell differentiation. In young differentiating fiber cells, GLUT3 was mainly found in the cytoplasm, but with increasing depth into the lens became inserted into the narrow sides of older fiber cells, before becoming completely dispersed around the entire membrane of the oldest fiber cells. Hyperglycemia had similar effects on tissue damage and transporter expression in both the in vitro and in vivo models. Tissue damage was characterized by an initial local cell swelling that with prolonged insult gradually spread and resulted in the creation of large areas of tissue liquefaction. Northern blot analysis and quantitative RT-PCR showed that transcript for GLUT3 but not GLUT1 was upregulated under hyperglycemic conditions. This increase in GLUT3 expression was confirmed at the protein level by both Western blot analysis and immunocytochemistry. In hyperglycemic lenses, GLUT3 antibody labeling was localized to the region of tissue liquefaction. CONCLUSIONS: GLUT3 in the lens exhibits dynamic changes in expression levels and cellular localization as a function of fiber cell differentiation and hyperglycemia. In the lens cortex, regions of GLUT3 overexpression and hyperglycemic tissue damage overlap, suggesting a functional relationship.

Animals↗

PubMed2002

Molecular profiling and cellular localization of connexin isoforms in the rat ciliary epithelium.

The functionally distinct epithelial layers of the ciliary body act as a syncitium to produce the aqueous humour. Ultrastructural studies have shown that the pigmented (PE) and non-pigmented (NPE) cell layers of the ciliary epithelium are connected by gap junctions. However the molecular composition of gap junctions both between and within the two cell layers has not been comprehensively studied. To address this issue the authors have performed an extensive molecular screening of connexin (Cx) expression patterns in ciliary epithelium of the rat. Initially, mRNA was extracted from rat ciliary bodies, reverse-transcribed, and subjected to two rounds of PCR using primer sets designed against each of the 14 Cx isoforms known to be expressed in the rat. This initial screening protocol amplified eight candidate Cx isoforms (Cxs 26, 31, 33, 37, 40, 43, 45 and 46). The Cx isoforms identified in this initial screen were then first assigned to the ciliary epithelium itself (Cxs 26, 31, 40 and 43) or structures outside the epithelium (Cxs 37, 40, and 45) using immunohistochemistry performed on ciliary body whole mounts. No convincing evidence for either Cx 33 or 46 labelling was found in the ciliary body. Then the four Cx isoforms localized to the epithelium were further localized to specific membrane domains within the epithelial cell layers by performing high resolution imaging of the antibody labeling patterns obtained in cryosections. This enabled Cx26 and 31 to be specifically localized to spatially different gap junctions between NPE cells. Cx31 labeled gap junctions associated with an extensive network of membrane interdigitations found between NPE cells at their basal surfaces. In contrast Cx26 labeling in NPE cells was restricted to the basolateral membranes of adjacent NPE cells. Cx40 and Cx43 were both localized to the PE-NPE interface where they formed discrete homomeric/homotypic gap junction plaques. No convincing evidence was found for antibody labeling between PE cells. Thus it appears that intercellular communication, both within the NPE layer and between the PE and NPE cell layers, is mediated by gap junction channels that have distinctive permeability properties. In particular the results raise the possibility that the permeability of PE-NPE gap junctions can be modulated by changing the Cx43 : Cx40 expression ratio. Whether such a change in Cx expression ratios occurs and what effect it has on aqueous humour production and composition remains to be determined.

Animals↗

PubMed2002

Molecular identification of P-glycoprotein: a role in lens circulation?

PURPOSE: To determine whether P-glycoprotein is expressed in the rat lens and to assess what type of damage occurs when P-glycoprotein inhibitors are applied to organ-cultured lenses. METHODS: An initial screening for the P-glycoprotein isoforms multidrug resistance (mdr)1a, mdr1b, and mdr2 was performed by RT-PCR on RNA extracted from rat lens fiber cells. Northern blot analysis was used to determine whether transcript levels detected by RT-PCR were significant. The presence of P-glycoprotein in the lens was confirmed by Western blot analysis and immunocytochemistry. Organ-cultured lenses, maintained in isotonic artificial aqueous humor, were exposed to various concentrations of the P-glycoprotein inhibitor tamoxifen. Lens opacification was assessed by dark-field microscopy, and the underlying cellular changes were visualized by confocal microscopy of lens sections, using a fluorescent membrane marker. Initial cellular damage was assessed after a 6-hour exposure to 100 micro M tamoxifen. Other P-glycoprotein inhibitors, verapamil, and 1,9-dideoxyforskolin (DDFK) were assessed, and the damage phenotypes were compared with those seen for tamoxifen. RESULTS: Transcript for all three P-glycoprotein isoforms was detected with RT-PCR, but only mdr1a and mdr2 could be detected by Northern blot analysis. P-glycoprotein was localized in the plasma membrane of lens epithelial and fiber cells. Treatment of organ-cultured lenses with increasing doses of the P-glycoprotein inhibitor tamoxifen for 18 hours showed that two distinct damage phenotypes were evident. At a dose of 20 micro M tamoxifen, tissue damage was found in a discrete zone that initially started approximately 100 micro m from the capsule, whereas at higher doses (60-100 micro M tamoxifen), extensive vesiculation of fiber cell membranes occurred throughout the entire lens cortex. Decreasing tamoxifen (100 micro M) exposure to 6 hours showed that the inner zone of damage was caused by the dilation of extracellular space between fiber cells. The extracellular space dilution and fiber cell vesiculation could be reproduced by varying the concentrations of other P-glycoprotein inhibitors, verapamil and DDKF. CONCLUSIONS: The P-glycoproteins mdr1a and mdr2 are expressed in the lens and appear to be functional. The initial cellular damage phenotype of extracellular space dilations caused by the P-glycoprotein inhibitors was identical with that caused by chloride channel inhibitors, indicating that P-glycoprotein may play a role in regulating cell volume in the lens. Whether the secondary damage phenotype of fiber cell vesiculation, induced by high doses of P-glycoprotein inhibitors, was due to the inhibition of additional regulatory activities of P-glycoprotein or to nonspecific effects of the drugs remains to be determined. However, regardless of the precise mode of action, these results indicate that P-glycoprotein should be considered in the regulatory mechanisms associated with the control of lens volume and in the initiation of osmotic cataract.

ATP Binding Cassette Transporter, Subfamily B↗