Biomedical subjects

Claire H Mitchell

Publications and source records attributed to Claire H Mitchell.

12 recordsLinked to original sources

PubMed2006

Identification of the A3 adenosine receptor in rat retinal ganglion cells.

PURPOSE: Adenosine can protect retinal ganglion cells from the death that accompanies a general ischemic challenge as well as excitotoxic death. In other tissues, both A1 and A3 adenosine receptor subtypes can mediate protection. While a role for the A1 adenosine receptor in ganglion cell protection has been established, a potential for the A3 receptor has only recently been proposed. Although the pharmacology is promising, the molecular identity of the responsible receptor is unclear as previous studies were unable to detect message for the A3 receptor in retinal ganglion cells. We combined laser capture microdisection (LCM) and immunopurification with traditional and real-time PCR to unequivocally demonstrate the presence of the A3 receptor message in rat retinal ganglion cells. METHODS: Retinal ganglion cells of Long-Evans rat pups were retrograde labeled with aminostilbamidine. Eyeballs were enucleated, embedded, frozen, sectioned, and fluorescent cells in the ganglion cell layer were collected with LCM. Purified ganglion cells were also isolated with a two-step panning procedure. cDNA for the A3 receptor obtained from the microdissected ganglion cell layer, immunopurified ganglion cells, whole retina and testis was amplified using RT-PCR, confirmed by DNA sequencing and compared with published sequences. A3 receptor message was also amplified using real-time PCR. Ca2+ levels in immunopanned ganglion cells were measured ratiometrically with fura-2. RESULTS: RNA from immunopurified ganglion cells and from dye-loaded cells in the ganglion cell layer contained message for the A3 receptor when amplified with either traditional RT-PCR or real-time PCR. The entire encoding region was sequenced and found to be 99% identical to the published code. The sequence closely resembled the consensus form of the gene, with other sequences deviating from this default code. Molecular identification was functionally confirmed in purified ganglion cells as the A3 receptor agonist Cl-IB-MECA prevented the excessive Ca2+ rise triggered by P2X7 agonist BzATP. CONCLUSIONS: Retinal ganglion cells express A3 adenosine receptor mRNA. Stimulation of this receptor can reduce the Ca2+ overload following excessive activation of P2X7 receptors.

Adenosine↗

PubMed2006

Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP.

The photoreceptors lie between the inner retina and the retinal pigment epithelium (RPE). The release of glutamate by the phototoreceptors can signal changes in light levels to inner retinal neurons, but the role of glutamate in communicating with the RPE is unknown. Since RPE cells are known to release ATP, we asked whether glutamate could trigger ATP release from RPE cells and whether this altered cell signalling. Stimulation of the apical face of fresh bovine RPE eyecups with 100 mum NMDA increased ATP levels more than threefold, indicating that both receptors for NMDA and release of ATP occurred across the apical membrane of fresh RPE cells. NMDA increased ATP levels bathing cultured human ARPE-19 cells more than twofold, with NMDA receptor inhibitors MK-801 and d-AP5 preventing this release. Blocking the glycine site of the NMDA receptor with 5,7-dichlorokynurenic acid prevented ATP release from ARPE-19 cells. Release was also blocked by channel blocker NPPB and Ca(2+) chelator BAPTA, but not by cystic fibrosis transmembrane conductance regulator (CFTR) blocker glibenclamide or vesicular release inhibitor brefeldin A. Glutamate produced a dose-dependent release of ATP from ARPE-19 cells that was substantially inhibited by MK-801. NMDA triggered a rise in cell Ca(2+) that was blocked by MK-801, by the ATPase apyrase, by the P2Y(1) receptor antagonist MRS2179 and by depletion of intracellular Ca(2+) stores with thapsigargin. These results suggest that glutamate stimulates NMDA receptors on the apical membrane of RPE cells to release ATP. This secondary release can amplify the glutaminergic signal by increasing Ca(2+) inside RPE cells, and might activate Ca(2+)-dependent conductances. The interplay between glutaminergic and purinergic systems may thus be important for light-dependent interactions between photoreceptors and the RPE.

Adenosine Triphosphate↗

PubMed2006

Balance of purines may determine life or death of retinal ganglion cells as A3 adenosine receptors prevent loss following P2X7 receptor stimulation.

The purines ATP and adenosine can act as a coordinated team of transmitters. As extracellular adenosine is frequently derived from the enzymatic dephosphorylation of released ATP, the distinct actions of the two purines can be synchronized. In retinal ganglion cells (RGCs), stimulation of the P2X7 receptor for ATP leads to increased intracellular Ca2+ and death. Here we define the contrasting effects of adenosine and identify protective actions mediated by the A3 receptor. Adenosine attenuated the rise in Ca2+ produced by the P2X7 agonist 3'-O-(4-benzoylbenzoyl)ATP (BzATP). Adenosine was also neuroprotective, increasing the survival of ganglion cells exposed to BzATP. The A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronimide (Cl-IB-MECA) mimicked the inhibition of the Ca2+ rise, whereas the A3 antagonist 3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191) reduced the protective effects of adenosine. Both Cl-IB-MECA and a second A3 receptor agonist IB-MECA reduced the cell loss triggered by BzATP. The actions of BzATP were mimicked by ATPgammaS, but not by ATP. In summary, adenosine can stop the rise in Ca2+ and cell death resulting from stimulation of the P2X7 receptor on RGCs, with the A3 adenosine receptor contributing to this protection. Hydrolysis of ATP into adenosine and perhaps inosine shifts the balance of purinergic action from that of death to the preservation of life.

Adenosine Triphosphate↗

PubMed2005

Paracrine stimulation of surfactant secretion by extracellular ATP in response to mechanical deformation.

We developed a heterologous system to study the effect of mechanical deformation on alveolar epithelial cells. First, isolated primary rat alveolar type II (ATII) cells were plated onto silastic substrata coated with fibronectin and maintained in culture under conditions where they become alveolar type I-like (ATI) cells. This was followed by a second set of ATII cells labeled with the nontransferable, vital fluorescent stain 5-chloromethylfluorescein diacetate to distinguish them from ATI cells. By morphometric analysis, equibiaxial deformation (stretch) of the silastic substratum induced comparable changes in cell surface area for both ATII and ATI cells. Surfactant lipid secretion was measured using cells metabolically labeled with [(3)H]choline. In response to 21% tonic stretch for 15 min, ATII cells seeded with ATI cells secreted nearly threefold more surfactant lipid compared with ATII cells seeded alone. ATI cells did not secrete lipid in response to stretch. The enhanced lipid secretion by ATII plus ATI cocultures was inhibited by treatment with apyrase and adenosine deaminase, suggesting that ATP release by ATI cells enhanced surfactant lipid secretion at 21% stretch. This was confirmed using a luciferase assay where, in response to 21% stretch, ATI cells released fourfold more ATP than ATII cells. Because ATI cells release significantly more ATP at a lower level of stretch than ATII cells, this supports the hypothesis that ATI cells are mechanosensors in the lung and that paracrine stimulation of ATII cells by extracellular ATP released from ATI cells plays a role in regulating surfactant secretion.

Adenosine Triphosphate↗

PubMed2005

Degradation of extracellular ATP by the retinal pigment epithelium.

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and betagamma-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca(2+). Both responses were inhibited by thapsigargin and P2Y(1) receptor inhibitor MRS 2179. Message for both P2Y(1) and P2Y(12) receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca(2+) signaling in the RPE.

Adenosine Diphosphate↗

PubMed2005

Inhibition of swelling-activated Cl- currents by functional anti-ClC-3 antibody in native bovine non-pigmented ciliary epithelial cells.

PURPOSE: To determine the potential role of ClC-3, a PKC-inhibitable Cl(-) channel, in mediating the swelling-activated Cl(-) current (I(Cl,swell)) of native bovine nonpigmented ciliary epithelial (NPE) cells. METHODS: Native bovine NPE cells were freshly harvested by enzymatic digestion. Whole-cell currents were recorded by patch-clamp measurements either in the presence or absence of a functional anti-ClC-3 antibody. RESULTS: Baseline whole-cell currents were small under isotonic conditions. Hypotonic cell swelling stimulated outwardly rectifying I(Cl,swell), which was reversibly inhibited by the Cl(-) channel blockers, phloretin (300 microM) or 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 100 microM). Intracellular dialysis with anti-ClC-3 C(670-687) antibody did not affect baseline currents, but significantly delayed and inhibited hypotonic stimulation of I(Cl,swell). Preabsorption of the antibody with its antigen prevented the inhibition of I(Cl,swell) by antibody. In addition, intracellular dialysis with control Ex(133-148) antibody did not affect the I(Cl,swell). Moreover, activation of PKC by pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited the initial stimulation of I(Cl,swell), but had no effects on the steady state currents. CONCLUSIONS: The results suggest that endogenous ClC-3 is involved in mediating I(Cl,swell) of native bovine NPE cells. The delayed stimulation of I(Cl,swell) by PDBu may reflect upregulation of swelling-activated Cl(-) channels of different subtypes, especially when the function of ClC-3 is blocked. This information will be useful in understanding the mechanisms controlling aqueous humor formation and thereby intraocular pressure.

Animals↗

PubMed2005

Stimulation of P2X7 receptors elevates Ca2+ and kills retinal ganglion cells.

PURPOSE: Retinal ganglion cells are known to express ionotropic P2X(7) receptors for ATP. Stimulation of these receptors in other cells can elevate Ca(2+) and sometimes lead to cell death. This study asked whether P2X(7) receptor stimulation alters the Ca(2+) levels and viability of retinal ganglion cells. METHODS: P2X(7) agonists were applied to retinal ganglion cells from neonatal rats loaded with fura-2 to examine their effect on intracellular Ca(2+) levels. The effect of P2X(7) receptor stimulation on cell viability was examined in rat retinal ganglion cells back-labeled with aminostilbamidine. RESULTS: The P2X(7) agonist benzoylbenzoyl adenosine triphosphate (BzATP) led to a large, sustained increase in Ca(2+). BzATP was >100-fold more effective than ATP at raising intracellular Ca(2+), when both agonists were applied at 10 microM. The response to BzATP was enhanced threefold by removal of extracellular Mg(2+), was dependent on extracellular Ca(2+), and was prevented by brilliant blue G (BBG). BzATP led to a concentration-dependent reduction in the number of cells with a median lethal dose (LD(50)) of 35 muM. Cell death was prevented by the P2X(7) antagonists BBG and oxidized ATP, but not by 30 microM suramin, consistent with the actions of the P2X(7) receptor. BzATP activated caspases in ganglion cells, but did not lead to membrane blebbing or increased permeability to Yo-Pro-1. The L-type Ca(2+) channel blocker nifedipine attenuated cell death, suggesting excessive Ca(2+) influx contributes to the lethal effects of BzATP. CONCLUSION: Short-term stimulation of the P2X(7) receptor can raise Ca(2+) in rat retinal ganglion cells, whereas sustained stimulation of the receptor can kill them.

Adenosine Triphosphate↗

PubMed2004

Differential P1-purinergic modulation of human Schlemm's canal inner-wall cells.

Intraocular pressure is directly dependent on aqueous humor flow into, and resistance to flow out of, the eye. Adenosine has complex effects on intraocular pressure. Stimulation of A1 and A2A adenosine receptors changes intraocular pressure oppositely, likely through opposing actions on the outflow of aqueous humor. While the cellular sites regulating outflow resistance are unknown, the cells lining the inner wall of Schlemm's canal (SC) are a likely regulatory site. We applied selective adenosine receptor agonists to SC cells in vitro to compare the responses to A1 and A2A stimulation. Parallel studies were conducted with human inner-wall SC cells isolated by a novel enzyme-assisted technique and with cannula-derived mixed inner- and outer-wall SC cells. A1 agonists increased whole cell currents of both inner-wall and cannula-derived SC cells. An A2A agonist reduced currents most consistently in specifically inner-wall SC cells. Those currents were also increased by A2B, but not consistently affected by A3, stimulation. A1, A2A, and A3 agonists all increased SC-cell intracellular Ca2+. The electrophysiological results are consistent with the possibility that inner-wall SC cells may mediate the previously reported modulatory effects of adenosine on outflow resistance. The results are also consistent with the presence of functional A2B, as well as A1, A2A, and A3 adenosine receptors in SC cells.

Anterior Eye Segment↗

PubMed2004

Release of ATP from retinal pigment epithelial cells involves both CFTR and vesicular transport.

The retinal pigment epithelium (RPE) faces the photoreceptor outer segments and regulates the composition of the interstitial subretinal space. ATP enhances fluid movement from the subretinal space across the RPE. RPE cells can themselves release ATP, but the mechanisms and polarity of this release are unknown. The RPE expresses the cystic fibrosis transmembrane conductance regulator (CFTR), and CFTR is associated with ATP release in other epithelial cells. However, an increasing number of reports have suggested that the exocytotic pathway contributes to release. In the present study, we examined the involvement of CFTR and the vesicular pathway in ATP release from RPE cells. Release from cultured human ARPE-19 cells and across the apical membrane of fresh bovine RPE cells in an eyecup was studied. A cAMP cocktail to activate CFTR triggered ATP release from fresh and cultured RPE cells. Release from both RPE preparations was largely prevented by the broad-acting blocker glibenclamide and the specific thiazolidinone CFTR inhibitor CFTR-172. The block by CFTR-172 was enhanced by preincubation and prevented ATP release with 3.5 microM IC(50). The rise in intracellular Ca(2+) accompanying hypotonic challenge was prevented by CFTR-172. The vesicular transport inhibitor brefeldin A prevented ATP release after stimulation with both hypotonic and cAMP conditions, suggesting vesicular insertion was also involved. These results show an intimate involvement of CFTR in ATP release from RPE cells which can autostimulate receptors on the apical membrane to modify Ca(2+) signaling. The requirement for both CFTR and vesicular transport pathways suggests vesicular insertion of CFTR may underlie the release of ATP.

Adenosine Triphosphate↗

PubMed2004

cAMP-activated maxi-Cl(-) channels in native bovine pigmented ciliary epithelial cells.

The eye's aqueous humor is secreted by a bilayered ciliary epithelium comprising pigmented (PE) and nonpigmented (NPE) epithelial cell layers. Stromal Cl(-) enters the PE cells and crosses gap junctions to the NPE cells for release into the aqueous humor. Maxi-Cl(-) channels are expressed in PE cells, but their physiological significance is unclear. To address this question, excised patches and whole native bovine PE cells were patch clamped, and volume was monitored by calcein fluorescence. In symmetrical 130 mM NaCl, cAMP at the cytoplasmic surface of inside-out patches produced concentration-dependent activation of maxi-Cl(-) channels with a unitary conductance of 272 +/- 2 pS (n = 80). Voltage steps from 0 to +/-80 mV, but not to +/-40 mV, produced rapid channel inactivation consistent with the typical characteristics of maxi-Cl(-) channels. cAMP also activated the maxi-Cl(-) channels in outside-out patches. In both cases, maxi-Cl(-) channels were reversibly inhibited by SITS and 5-nitro-2-(phenylpropylamino)benzoate (NPPB). Decreasing cytoplasmic Cl(-) concentration reduced both open-channel probability and unitary conductance. Similarly, the membrane-permeant 8-bromo-cAMP stimulated outward and inward whole cell currents; the stimulation was larger at higher intracellular Cl(-) concentration. As with unitary currents, cAMP-triggered whole cell currents displayed inactivation at +/-80 but not at +/-40 mV. Moreover, cAMP triggered NPPB-sensitive shrinkage of PE cells. The results suggest that cAMP directly activates maxi-Cl(-) channels of native PE cells that contribute to Cl(-) release particularly from Cl(-)-loaded cells. These cAMP-activated channels provide a potential mechanism for reducing and modulating net aqueous humor secretion by facilitating Cl(-) reabsorption into the ciliary stroma.

Animals↗

PubMed2003

Noninvasive assessment of aqueous humor turnover in the mouse eye.

PURPOSE: To develop a noninvasive test for monitoring changes in aqueous humor turnover in the mouse eye. METHODS: After topical instillation of fluorescein, the rate of decay of fluorescence from aqueous humor and cornea was monitored in Black Swiss, C57 Bl6, and DBA 2J mice with a microscope equipped with epifluorescence and a charge-coupled device (CCD) camera. RESULTS: The rate of decay of fluorescence was identical in right and left eyes over an approximately 70-minute measurement period. The rate of decay was similar in normal mice aged 2 and 18 months. Pilocarpine and latanoprost, known to enhance aqueous humor outflow in humans, accelerated the decay of fluorescence. Levobunolol, known to inhibit aqueous humor inflow in humans, slowed decay. Dimethylamiloride, an inhibitor of the Na(+),H(+) exchanger that is known to act on cultured cells of both the ciliary epithelium and trabecular meshwork and to lower mouse intraocular pressure (IOP), enhanced decay. DBA 2J mice, in which secondary glaucoma develops, displayed a slower decay of fluorescence at 18 months of age than age-matched unaffected animals. CONCLUSIONS: Monitoring decay of fluorescence provides a noninvasive index of aqueous humor dynamics in the mouse eye that facilitates study of ocular hypotensive drugs and mouse models of glaucoma. Coupled with knowledge of IOP, it permits semiquantitative conclusions about the relative roles of aqueous humor inflow and outflow in conditions with altered IOP. Based on this approach, dimethylamiloride appears to lower mouse IOP primarily by enhancing outflow of aqueous humor.

Amiloride↗

PubMed2002

Human trabecular meshwork cell volume regulation.

The volume of certain subpopulations of trabecular meshwork (TM) cells may modify outflow resistance of aqueous humor, thereby altering intraocular pressure. This study examines the contribution that Na+/H+, Cl-/HCO exchange, and K+-Cl- efflux mechanisms have on the volume of TM cells. Volume, Cl- currents, and intracellular Ca2+ activity of cultured human TM cells were studied with calcein fluorescence, whole cell patch clamping, and fura 2 fluorescence, respectively. At physiological bicarbonate concentration, the selective Na+/H+ antiport inhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicity triggered a regulatory volume decrease (RVD), which could be inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K+ channel blockers Ba2+ and tetraethylammonium, and the K+-Cl- symport blocker [(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism in isotonic conditions was dependent on bicarbonate; at physiological levels, the Na+/H+ exchange inhibitor dimethylamiloride reduced cell volume, whereas at low levels the Na+-K+-2Cl- symport inhibitor bumetanide had the predominant effect. Patch-clamp measurements showed that hypotonicity activated an outwardly rectifying, NPPB-sensitive Cl- channel displaying the permeability ranking Cl- > methylsulfonate > aspartate. 2,3-Butanedione 2-monoxime antagonized actomyosin activity and both increased baseline [Ca2+] and abolished swelling-activated increase in [Ca2+], but it did not affect RVD. Results indicate that human TM cells display a Ca2+-independent RVD and that volume is regulated by swelling-activated K+ and Cl- channels, Na+/H+ antiports, and possibly K+-Cl- symports in addition to Na+-K+-2Cl- symports.

Bicarbonates↗