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1 From the School of Optometry and 2 Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California; and 3 Inspire Pharmaceuticals, Durham, North Carolina.
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Abstract |
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METHODS. Freshly isolated monolayers of bovine and human fetal RPE were mounted in Ussing chambers for measurements of cytosolic calcium levels ([Ca2+]i), membrane voltages and resistances, and transepithelial fluid transport. Retinal detachments were experimentally produced in Long-Evans rats by injecting modified phosphate-buffered saline into the subretinal space (SRS). Experimental or vehicle solutions were injected into the vitreous, and the size of blebs in the SRS was scored under masked conditions.
RESULTS. Addition of INS37217 to Ringer’s solution bathing the apical membrane transiently increased [Ca2+]i, altered membrane voltages and resistances and generally produced responses that were similar in magnitude to those of uridine triphosphate (UTP). In fluid transport experiments performed with the capacitance probe technique, INS37217 significantly increased fluid absorption across freshly isolated bovine and fetal human RPE monolayers. All in vitro results were blocked by apical 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), which has been shown to block P2Y2 receptors in the RPE. Intravitreal administration of INS37217, but not UTP, in the rat model of retinal detachment enhanced the removal of SRS fluid and facilitated retinal reattachment when compared with vehicle control.
CONCLUSIONS. These findings indicate that INS37217 stimulates the RPE fluid "pump" function in vitro by activating P2Y2 receptors at the apical membrane. In vivo INS37217 enhances the rates of subretinal fluid reabsorption in experimentally induced retinal detachments in rats and may be therapeutically useful for treating a variety of retinal diseases that result in fluid accumulation in the subretinal space.
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Introduction |
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The RPE passively and actively transports fluid in the subretinal-to-choroidal direction, and this RPE fluid "pump" function is thought to play a major role in reabsorption of subretinal fluid and in promoting and maintaining retinal attachment.12 The polarized distribution of ion channel and transporter proteins at apical and basolateral membranes allows the RPE to carry out net vectorial transport of ion and fluid between the subretinal space (SRS) and the blood.13 These proteins can be regulated by activation of surface receptors on apical and basolateral membranes through the action of a variety of paracrine, autocrine, and hormonal signaling molecules, which can influence both the directionality and magnitude of fluid transport across the RPE.14 15 16 17 18 19 20 For example, in freshly isolated monolayers of bovine RPE, activation of P2Y2 receptors by addition of uridine triphosphate (UTP) to Ringer’s solution bathing the apical membrane has been shown to stimulate fluid absorption transiently by approximately 150% above prestimulation levels.20 Stimulation of RPE fluid absorption in vivo is expected to enhance the net transport of fluid in the subretinal-to-choroidal direction. A major objective of the present study was to investigate the effects of natural and synthetic P2Y2 receptor agonists (UTP and INS37217, respectively) in vivo in stimulating reabsorption of subretinal fluid in an experimental model of retinal detachment. INS37217 is a synthetic P2Y2 receptor agonist engineered with enhanced metabolic stability and resistance to extracellular nucleotidase-mediated hydrolysis.21 22 In the present study, the effects of INS37217 on a variety of RPE cellular functions were investigated, including mobilization of cytosolic Ca2+ levels, ion transport in vitro, and fluid transport in vitro and in vivo. Our collective findings demonstrate that INS37217 stimulates intracellular Ca2+ [Ca2+]i-signaling and ion-coupled fluid transport in vitro and that intravitreally administered INS37217, but not UTP, stimulates subretinal fluid reabsorption in a rat model of retinal detachment.
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Methods |
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Solutions
Control Ringer’s solution contained the following (in mM): 120 NaCl, 5 KCl, 23 NaHCO3, 1 MgCl2, 1.8 CaCl2, 2.0 taurine, and 10 glucose. This solution was bubbled continuously with 8% CO2, 10% O2, balance N2 (pH 
7.4) and osmolarity of the Ringer’s solution was 295 ± 5 mOsM. Glutathione (1 mM) was added to solutions minutes before perfusion. UTP, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt (DIDS), and 1,2-bis (2-aminophenoxy)ethane-N,N,N'N'-tetra acetic acid (BAPTA) were obtained from Sigma Chemical Co. (St. Louis, MO). All solutions were made iso-osmotic. Fura-2 was obtained from Molecular Probes (Eugene, OR). INS37217 was obtained from Inspire Pharmaceuticals (Durham, NC).
Electrophysiology
Calomel electrodes in series with Ringer’s solutions and agar bridges were used to measure the transepithelial potential (TEP), and the intracellular microelectrode signals were referenced to either the apical or basal bath to measure the membrane potentials, VA and VB, where TEP = VB - VA. Conventional microelectrodes were made from borosilicate glass tubing of 0.5 mm inner diameter and 1 mm outer diameter with a filament (Sutter Instrument Co., Novato, CA), were back filled with 150 mM KCl, and had resistances of 80 to 200 M
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The total transepithelial resistance (RT), and the ratio of the apical to basolateral membrane resistance (RA/RB) were obtained by passing 4 µA current pulses (8 µA peak to peak) across the tissue and measuring the resultant changes in TEP, VA, and VB. Current pulses were bipolar, with a period of 3 seconds. RT is the resultant change in TEP divided by 4 µA, and RA/RB is the absolute value of the change in VA divided by the change in VB (RA/RB = 
VA/VB). The current-induced voltage deflections were digitally subtracted from the records for clarity. The control Ringer’s solution for measurements of TEP and RT contained (in mM): 120 NaCl, 5 KCl, 23 NaHCO3, 1 MgCl2, 1.8 CaCl2, and 5 glucose. In the electrophysiology experiments (see Figs. 3
4
), the black bar indicates a solution change at the manifold outside of the recording chamber. In some cases the response onset was variably delayed because of "dead space" in the fluid delivery system and because of thickness variations in the unstirred layer at the apical membrane.
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VA 
VB. In contrast, if RS >> RA then VA 0. The transepithelial (or total) resistance RT is expressed in terms of the membrane and shunt resistances as follows
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Calibration of [Ca2+]i was performed at the end of each experiment by first perfusing both membranes with a zero-calcium Ringer’s solution containing 10 mM EDTA, which chelates any residual-free calcium, and 10 µM ionomycin, which is a calcium ionophore that facilitates the equilibration [Ca2+]i and extracellular Ca2+ [Ca2+]o. After this zero calcium calibration, the tissue was then exposed to saturating (1.8 mM) concentration of calcium. Then [Ca2+]i was determined according to the equation [Ca2+]i = K(R - Rmin)/(Rmax - R), where Rmax is the maximum ratio of the fluorescence intensities at 350 and 385 nm, which was determined at the saturating Ca2+ signal; Rmin is the ratio of fluorescence intensities in the absence of [Ca2+]o; K is equal to Kd (Fmin/Fmax), where Kd is the dissociation constant for fura-2 AM (220 nM)28 ; and Fmin and Fmax are the fluorescence intensities at 385 nm in the absence and presence of saturating [Ca2+]o, respectively.
Fluid Transport
A modified capacitive probe technique, previously described,15
was used to determine the rate of fluid movement across the RPE. In brief, the RPE was mounted in a water-jacketed Ussing chamber and oriented vertically, with the apical and basolateral membranes separately exposed to Ringer’s solution held in the bathing reservoirs. Stainless steel probes (Accumeasure System 1000; MT Instruments, Latham, NY) were lowered into the apical and basolateral bathing wells to measure the capacitance of the air gap between the probe and fluid meniscus. The fluid transport rate, JV (in microliters per square centimeter per hour), was determined by monitoring the fluid movement-induced changes in the air gap capacitance at the apical and basolateral baths. The probes on both sides of the tissue were backed off from the surface of the Ringer’s solution during a bathing solution change. To check that the solution changes per se did not appreciably alter JV, a control-to-control Ringer’s solution change was performed near the beginning of each experiment and at appropriate intervals during the experiment. The capacitance probes were moved away from the bathing reservoirs, and fresh control Ringer’s solution was perfused into the chamber. The fluid transport apparatus also allows the experimenter to continuously monitor TEP and RT, but for technical reasons (solution perfusion rates, TEP, RT sampling rates, electrode stability) we can only compare the initial changes in TEP and RT (phase I, see the Results section) with those in the electrophysiology experiments. Experiments were continued only if JV, TEP, and RT were not appreciably altered by this control-to-control Ringer’s solution change. The water-jacketed Ussing chamber was placed in an incubator to maintain steady state control over temperature (pCO2) and humidity.
Human Fetal Tissue
The research adhered to the tenets of the Declaration of Helsinki. Research protocols were approved by the University of California Committee for the Protection of Human Subjects. Fetal eyes were obtained by an independent procurer (Advanced Bioscience Resources, Alameda, CA).
In Vivo Preparation: Rat Study Design
All animal experiments were conducted in compliance with the ARVO Statement for the use of Animals in Ophthalmic and Vision Research, and the protocol was approved by the Animal Care and Use Committee of the University of California at Berkeley. Retinal detachments were created in Long-Evans female rats by injecting 2 to 3 µL of modified phosphate-buffered saline (MPBS) Ringer solution into the SRS; only one eye per rat was used. With a charge-coupled device (CCD) camera, images of the subretinal blebs were obtained at 1-minute intervals for several hours. The acquisition of images is described in further detail later. In the control part of each experiment (at 0–30 minutes after creation of the retinal detachment), apparent bleb size reached a steady state size, which remained unchanged during the course of anesthesia (several hours). MPBS solutions, with or without INS37217 (5 mM) were formulated and injected (3 µL) into the vitreous of the rat eye under masked and randomized conditions. The vials and their contents were indistinguishable. After vitreous injection, the apparent bleb size either increased or decreased monotonically or was constant over the next 60 minutes, as judged by the experimenter using the seven rank scale (0 ± 3) illustrated in Figure 9A
. Ranks were assigned by observing the change in apparent bleb size between 30 and 90 minutes after drug or placebo vitreous injection. Animals were reanesthetized the next day, and a separate estimate of rank was obtained. A rank of -3 means that the retinal bleb was apparently flattened. A rank of +3 means that the bleb approximately doubled in size. A 0 rank means that the apparent bleb size was unchanged over time. After all the experiments were completed, the content of each vial was unmasked and compared with the experimenter’s conclusions based on the observation of images obtained between 30 and 90 minutes and at 1 day after administration of drug or placebo.
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Schematic Diagram of the Preparation
An anesthetized rat was immobilized with ear bars and a nose clip. Body temperature was kept at 35°C to 37°C by a water jacket. Before the vitreous injection procedures, the experimental eye was anesthetized with proparacaine 1% ophthalmic solution, and the iris was dilated with a 1% ophthalmic atropine solution. A specially designed double convex lens, lubricated with carboxymethyl-cellulose (Celluvisc; Allergan, Irvine, CA) eye drops, was placed on the cornea to prevent drying and to increase the visual angle of observation for internal eye structures. The eyelids of the contralateral eye were closed with microserrefine to prevent corneal drying. Using a stereotaxic micromanipulator under stereomicroscope guidance, a 26-gauge guidance needle was inserted into the vitreous chamber, carefully avoiding the lens (Fig. 2A)
. A 33-gauge blunt needle attached to a syringe (Hamilton, Reno, NV) was inserted into the 26-gauge guidance needle and used to inject drugs into the vitreous and create subretinal blebs. The blebs were created by the 33-gauge needle which was pushed through the guidance needle under visual control until it penetrated the retina while avoiding the RPE. In many cases it was possible to position the 33-gauge needle so that it first penetrated a small retinal blood vessel. Withdrawal of the 33-gauge needle caused a small blood clot that helped seal the retinal hole.
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Data Collection and Analysis
An air-cooled digital CCD camera, attached to a stereo microscope (SMZ800; Nikon, Melville, NY), was used to take series of time-lapse images (every minute) over the first 2 hours. The collected images were compared to estimate the changes in apparent bleb size. In Fig. 2B
, panel I illustrates the retinal configuration at the time of bleb creation (t = 0). Similarly, panels II and III illustrate the retinal configurations at t =30 and 90 minutes, respectively. The maximum observation time during the first day was limited by anesthesia to 4 hours. In the randomized, masked trial experiments, we observed the subretinal bleb for 30 minutes, to point II, before the vitreal injection of placebo or drug. During this initial period there was no leak of fluorescein and no change in apparent bleb size. At point II, after vitreous injection of either placebo or INS37217 we observed the bleb for another 60 minutes. Because the changes in apparent bleb size were monotonic we could use the two images at 30 and 90 minutes to estimate the change in apparent bleb size.
Changes in apparent bleb size were visualized by using a dotted line to mark the bleb border image and an "X" to mark constant reference structures such as the optic nerve or a blood vessel, (Fig. 2B ; t = 0). By transferring the dotted line along with the fiducial mark we could identify changes in apparent bleb size that occurred over the 90 minutes observation period (Fig. 2B) . In each experiment, we also obtained images the next day that were used to strengthen our conclusion about the direction of change in apparent bleb size.
Statistics
Results are expressed as means ± SD unless otherwise indicated. Given the relatively small sample size, we used the nonparametric, Mann-Whitney test for statistical comparisons of unpaired dated and the Wilcoxon signed rank test for paired data.29
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P < 0.05 is regarded as significant in all comparisons.
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Results |
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(cm2) and 0.7 ± 0.6, respectively.
After addition of UTP or INS37217, the TEP and membrane voltages underwent three distinct phases with onset times labeled I, II, and III.31
During phase I, the TEP increased because VB depolarized faster than VA; in addition RT decreased and RA/RB increased, consistent with an decrease in RB. The voltage and resistance changes during phase I are consistent with an increase in basolateral membrane Cl conductance.20
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In three experiments, similar to Fig. 3A
, the mean UTP-induced depolarization of VA was 22.5 ± 0.5 mV during phase I, which increased TEP by 2.5 ± 0.7 mV. Concomitantly, the decrease in RT was 17.7 ± 7.6 (cm2) while RA/RB increased by 2.3 ± 0.9.
The INS37217-induced changes in phase I (Fig. 3B)
were not significantly different in magnitude from the UTP-induced changes (P > 0.5, Mann-Whitney test). In five experiments, the mean INS37217-induced depolarization of VA was 16.0 ± 4.0 mV during phase I, which increased TEP by 3.8 ± 2.0 mV. Concomitantly, the decrease in RT was 10 ± 4 (cm2) while RA/RB increased by 1.6 ± 0.7. In six experiments, we compared the magnitude of the INS37217- and UTP-induced phase I changes in TEP as a function of concentration. At 1, 10, and 25 µM (n = 3), there was no significant difference (P > 0.5, Wilcoxon signed rank test) but at 50 and 100 µM (n = 6 and 4), respectively, the INS37217 responses were significantly (P < 0.01) larger than the comparable UTP responses.
The transition between phases I and II occurred when the TEP peaked and began to decrease. During phase II, RT and RA/RB increased, and VA depolarized faster than VB. These phase II changes are more clearly seen in Figure 3B and are consistent with a decrease in apical membrane conductance. Approximately 90% of this conductance is due to Ba2+-sensitive K channels,23 suggesting that the closure of K channels is a major contributor to the phase-II voltage changes.20 Phase III is operationally defined as the time point at which TEP began to increase again. During phase III, TEP increased because VA hyperpolarized at a faster rate than VB; in addition, RA/RB slowly decreased and RT first decreased and then increased. These phase III electrical effects were relatively small, variable in time course, and difficult to study.
Effect of Apical or Basolateral Application of DIDS on INS37217-Induced Electrical Responses
Previous work has shown that micromolar amounts of apical 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) inhibits P2Y receptors in a variety of cell types, including bovine RPE.20
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To determine whether the INS37217-mediated electrical effects can be inhibited by apical application of DIDS, INS37217 was added to apical Ringer’s solution in the absence or presence of DIDS. Figure 4A
shows that in the presence of 500 µM apical DIDS, the electrical effects of INS37217 were almost completely abolished. In the presence of DIDS, the mean ± SD of INS37217-induced changes in VA, TEP, RT, and RA/RB were all reduced by at least a factor of five compared to control values: 2.4 ± 3.1 and 0.2 ± 0.2mV, 0.2 ± 0.4 (cm2), and 0.3 ± 0.4, respectively (n = 5).
Previous work in bovine RPE has demonstrated that the addition of 500 µM DIDS to the basolateral bath specifically blocks basolateral membrane Cl conductance without altering K conductance.23 37 38 If phase I of the INS37217-induced electrical responses is due to an increase in basolateral membrane Cl conductance, as suggested earlier, then the effects of INS37217 should be blocked by addition of DIDS to the basolateral bath. This notion was confirmed in the experiment shown in Fig. 4B , in which 500 µM DIDS clearly inhibited the effects of apical INS37217. This result was confirmed in two other experiments. The findings shown in Figures 4A and 4B provide strong pharmacologic evidence that the effects of INS37217 are mediated by activation of the P2Y2 receptor at the apical membrane and a subsequent increase in basolateral membrane Cl conductance.
Role of [Ca2+]i
In many systems, agonist induced activation of P2Y2 receptors has been shown to increase IP3-mediated release of Ca2+ from the endoplasmic reticulum (ER).2
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40
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The downstream effects of activation of the P2Y2 receptor can be blocked by pharmacologically unloading Ca2+ from ER stores with inhibitors of sarco-endoplasmic reticulum Ca2+ adenosine triphosphatase (ATPase) (SERCA).20
Figure 5A
shows that 100 µM apical INS37217 transiently increased [Ca2+]i from 80 to 200 nM. In 10 experiments, INS37217 increased [Ca2+]i by 101 ± 14.4 nM (mean ± SEM) from a baseline of 89.5 ± 14.4 nM. These INS37217-induced effects on [Ca2+]i are consistent with P2Y2 receptor-mediated signaling through the PLC-ß pathway.
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To determine whether INS37217 produced long term, steady state increases in [Ca2+]i, we treated the apical membrane with Ringer’s solution containing 100 µM INS37217 for 12 minutes while continuously monitoring [Ca2+]i. Fig. 5C shows that INS37217 produced a typical transient increase in [Ca2+]i. A small elevation in baseline [Ca2+]i (0.4 ratio units or 23% of the transient increase) was maintained for as long as the agonist was present in the apical bath. This result was obtained in three experiments and suggests that apical INS37217 can cause sustained alterations in intracellular Ca2+ signaling in RPE.
INS37217-Induced Changes in Fluid Transport across Bovine and Human RPE
In these experiments TEP, RT, and net fluid transport (JV) were measured in the absence or presence of INS37217 in the apical bath. Fig. 6A
shows that before the addition of 50 µM INS37217, the RPE absorbed fluid at approximately 1.3 µL/cm2 per hour. TEP was 5.5 mV, and RT was 153 (cm2). The addition of INS37217 (50 µM) to the apical bath increased TEP to 8 mV and decreased RT to 141 (cm2). In addition, JV increased to approximately 2.0 µL/cm2 per hour. The TEP and JV changes were reversible and lasted as long as the agonist was applied. In 13 tissues, the mean values of TEP, RT, JV were 7.5 ± 0.4 mV, 173 ± 11 (cm2), and 1.6 ± 0.3 µL/cm2 per hour (mean ± SEM), respectively. After the addition of 50 µM INS37217 to the apical bath, TEP increased by 2 mV to 9.7 ± 0.7 mV (P < 0.005), RT decreased to 163 ± 9 (cm2) (P < 0.001) and mean fluid absorption increased by more than a factor of two, to 3.8 ± 0.6 µL/cm2 per hour (P < 0.02).
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(cm2), and a large increase in JV to approximately 40 µL/cm2 per hour that declined over the following 50 minutes back to the baseline of 5.0 µL/cm2 per hour. This result suggests that INS37217 can stimulate ion and fluid transport in human tissues. To test if the INS37217-induced increase in [Ca2+]i is associated with the observed stimulation of fluid transport, we evaluated the effects of INS37217 on JV in the absence and presence of CPA. Because CPA was shown to inhibit the INS37217-induced increase in [Ca2+]i,, we expect that pretreatment with CPA would also inhibit the INS37217-mediated increase in JV. This expectation was confirmed in the experiment summarized in Fig. 7A . Addition of CPA (5 µM) to the apical bath increased JV from 1 to approximately 3.8 µL/cm2 per hour, which may be attributed to a CPA-induced increase in [Ca2+]i (see Fig. 5B ), resulting in an increase in basolateral membrane Ca2+-sensitive Cl conductance and a concomitant increase in apical-to-basolateral fluid transport.26 44 45 In the presence of CPA, the addition (or removal) of 100 µM apical INS37217 produced no change in JV. In three experiments, in the presence of CPA plus INS37217, JV was 3.5 ± 1.2 µL/cm2 per hour, not significantly different from CPA alone (4.1 ± 1.4 µL/cm2 per hour; mean ± SEM). Apical DIDS, which is a putative antagonist for the P2Y2 receptor, was shown to block INS37217-mediated electrophysiological effects (Fig. 4A) . Therefore, we expected that apical DIDS should also block the INS37217-induced changes in JV. Fig. 7B is one of three similar experiments that compared the INS37217-induced changes in JV, in the absence and presence of apical DIDS (500 µM). In the control response, 100 µM INS37217 was added to the apical bath and transiently increased JV from approximately 1 to 4 µL/cm2 per hour. These responses were reversible. Apical DIDS (500 µM) produced no appreciable changes in JV. There was no effect of INS37217 on JV in the presence of 500 µM DIDS. In three experiments, in the presence of DIDS plus INS37217 JV was 1.9 ± 0.8 µL/cm2 per hour, not significantly different from DIDS alone (1.5 ± 0.1 µL/cm2 per hour). The results shown in Figures 7A and 7B strongly suggest that the INS37217-induced increases in net fluid absorption across RPE are mediated by activation of apical membrane P2 receptors and ER release of Ca2+.
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Discussion |
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Our results show that constitutive addition of INS37217 to Ringer’s solution bathing the apical membrane of bovine RPE produced a large, transient increase in [Ca2+]i, which was followed by a modest, sustained elevation of [Ca2+]i in the presence of the agonist. INS37217 elicited changes in membrane voltages and resistances were similar in characteristics to those elicited by UTP. INS37217 also increased fluid absorption in the apical-to-basolateral direction in bovine and human fetal RPE monolayers; and, in fluid-secreting bovine RPE, it reversed the direction of fluid transport. In addition, INS37217-induced increases in fluid transport were blocked by apical DIDS in vitro (Fig. 7B) and in vivo (n = 4, not shown), after its injection into the bleb. Both results indicate that INS37217 had its main effect at the RPE apical surface. In an experimental model of retinal detachment in rat, intravitreal injection of INS37217, but not UTP, enhanced subretinal fluid reabsorption and retinal reattachment. These in vivo findings strongly suggest that intravitreal UTP is degraded before diffusing to the RPE apical membrane and underscore the necessity of developing an intravitreally administered, hydrolysis-resistant P2 receptor agonist to stimulate subretinal fluid reabsorption in vivo.
Physiological Implications
Accumulation of fluid in the SRS is a hallmark of all clinical retinal detachments, and a mechanism to facilitate net fluid efflux out of this space may be clinically useful to reattach the retina.47
Pharmacologic enhancement of the RPE pump to reabsorb extraneous subretinal fluid therefore represents a potential approach for inducing reattachment of the retina in the clinic. From the present in vitro bovine RPE experiments we determined that 50 µM INS37217 increased JV by 2 µL/cm2 per hour. If this increase occurred over the entire surface of the RPE (approximately 5 cm2),48
it would result in the removal of approximately 0.25 mL/d, a clinically significant amount.49
In the in vitro experiment using native human RPE (Fig. 6C)
addition of 50 µM INS37217 to the apical bath kept JV elevated above baseline for more than 60 minutes. The area under this curve is the total volume transported per unit area. If this transport (approximately 17 µL/cm2) took place across the back of an adult human eye (approximately 5 cm2) it would cause the removal of 0.085 mL/h or 2 mL/d.
In addition, the in vivo data allowed us to make a similar calculation using the intact rat eye. Bleb volume changes were calculated by assuming that the bleb formed an ellipsoid of revolution, where V = 4/3 · 
· a · b · h, where a and b are semimajor and semiminor axes, respectively, h is a height of the bleb, and a = b (spread of fluid is isotropic in the plane of the retina). The percentage of volume change was calculated by measuring the number of pixels along the a and h dimensions at t = 0 and t = 60 minutes: V60/V0 = (2/3 · · a2 · h)60/(2/3 · · a2 · h)0. We used this ratio and the initially injected volume to estimate the mean INS37217-induced change, which was 1.04 ± 0.5 µL/h (mean ± SD, n =5). In a typical experiment, a 3 µL injection formed a circular cross-section on the retina of 2 mm diameter (0.03-cm2 area) The INS37217-induced efflux rate through this area would be 32 µL/cm2 per hour.
Vitreous injection of the hydrolysis resistant INS37217 could activate the entire surface of the RPE, which would cause the removal of an extra 3.8 mL/d, assuming an efflux rate of 32 µL/cm2 per hour over the entire area of the RPE. This could be an overestimate of the RPE contribution if the entire RPE area were not activated or if some of the extra fluid exited by a different pathway (e.g., the ciliary body). Nevertheless, based on these extrapolated analyses of the present in vitro and in vivo data, our findings strongly suggest that stimulation of RPE fluid pump function by INS37217 represents a reasonable approach for removing abnormally accumulated subretinal fluid associated with retinal detachments.
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Acknowledgements |
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Footnotes |
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Submitted for publication December 20, 2001; revised April 29, 2002; accepted June 18, 2002.
Commercial relationships policy: I, E (BRY, WMP); P (BRY); F, C (SSM); N (AM, SJ, SAB, JR).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Sheldon S. Miller, NIH/NEI, Building 31, Room 6A03, 31 Center Drive MSC 2510, Bethesda, MD 20892-2510; millers{at}nei.nih.gov.
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L. A. Voloboueva, J. Liu, J. H. Suh, B. N. Ames, and S. S. Miller (R)-{alpha}-Lipoic Acid Protects Retinal Pigment Epithelial Cells from Oxidative Damage Invest. Ophthalmol. Vis. Sci., November 1, 2005; 46(11): 4302 - 4310. [Abstract] [Full Text] [PDF]
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D. Soto, J. Pintor, A. Peral, A. Gual, and X. Gasull Effects of Dinucleoside Polyphosphates on Trabecular Meshwork Cells and Aqueous Humor Outflow Facility J. Pharmacol. Exp. Ther., September 1, 2005; 314(3): 1042 - 1051. [Abstract] [Full Text] [PDF]
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 D. Reigada, W. Lu, X. Zhang, C. Friedman, K. Pendrak, A. McGlinn, R. A. Stone, A. M. Laties, and C. H. Mitchell Degradation of extracellular ATP by the retinal pigment epithelium Am J Physiol Cell Physiol, September 1, 2005; 289(3): C617 - C624. [Abstract] [Full Text] [PDF]
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J. E. Fries, I. M. Goczalik, T. H. Wheeler-Schilling, K. Kohler, E. Guenther, S. Wolf, P. Wiedemann, A. Bringmann, A. Reichenbach, M. Francke, et al. Identification of P2Y Receptor Subtypes in Human Muller Glial Cells by Physiology, Single Cell RT-PCR, and Immunohistochemistry Invest. Ophthalmol. Vis. Sci., August 1, 2005; 46(8): 3000 - 3007. [Abstract] [Full Text] [PDF]
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O. Strauss The Retinal Pigment Epithelium in Visual Function Physiol Rev, July 1, 2005; 85(3): 845 - 881. [Abstract] [Full Text] [PDF]
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D. Reigada and C. H. Mitchell Release of ATP from retinal pigment epithelial cells involves both CFTR and vesicular transport Am J Physiol Cell Physiol, January 1, 2005; 288(1): C132 - C140. [Abstract] [Full Text] [PDF]
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J. E. Fries, T. H. Wheeler-Schilling, E. Guenther, and K. Kohler Expression of P2Y1, P2Y2, P2Y4, and P2Y6 Receptor Subtypes in the Rat Retina Invest. Ophthalmol. Vis. Sci., October 1, 2004; 45(10): 3410 - 3417. [Abstract] [Full Text] [PDF]
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M. Nour, A. B. Quiambao, W. M. Peterson, M. R. Al-Ubaidi, and M. I. Naash P2Y2 Receptor Agonist INS37217 Enhances Functional Recovery after Detachment Caused by Subretinal Injection in Normal and rds Mice Invest. Ophthalmol. Vis. Sci., October 1, 2003; 44(10): 4505 - 4514. [Abstract] [Full Text] [PDF]
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F. Wang, K. G. Rendahl, W. C. Manning, D. Quiroz, M. Coyne, and S. S. Miller AAV-Mediated Expression of Vascular Endothelial Growth Factor Induces Choroidal Neovascularization in Rat Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 781 - 790. [Abstract] [Full Text] [PDF]
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C. H. Meyer, K. Hotta, W. M. Peterson, C. A. Toth, and G. J. Jaffe Effect of INS37217, a P2Y2 Receptor Agonist, on Experimental Retinal Detachment and Electroretinogram in Adult Rabbits Invest. Ophthalmol. Vis. Sci., November 1, 2002; 43(11): 3567 - 3574. [Abstract] [Full Text] [PDF]
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