1Department of Ophthalmology and Visual Science and 2Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan
Submitted 4 August 2004 ; accepted in final form 18 October 2004
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ABSTRACT |
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calcium channels; capillaries; purinoceptors; vasotoxicity
In addition to a putative physiological role in the regulation of blood flow, P2X7 receptor activation can cause cell death in retinal microvessels (25). The activation of P2X7 receptors not only opens the associated ligand-gated channels but also, as reported for a number of other types (5, 7, 10, 20, 26), results in the formation of large transmembrane pores and apoptotic cell death (13, 25). Of potential clinical importance, pore formation and cell death during P2X7 receptor activation are enhanced in diabetic retinal microvessels (13, 25). Thus P2X7 receptors may play a role in the sight-threatening retinopathy caused by diabetes. On the other hand, it seems likely that under physiological conditions, the lethal effects of P2X7 receptor activation must be blocked because microvascular cell death has dire consequences for visual function. Consistent with prevention of purinergic vasotoxicity, cell death is rare in the microvasculature of the normal adult retina (1, 18).
To test hypotheses concerning the mechanisms by which the lethality of P2X7 receptor activation is held in abeyance, we performed experiments on pericyte-containing microvessels freshly isolated from the adult rat retina. In the present article, we report that there are potent mechanisms by which to minimize the vasotoxicity of extracellular ATP, an endogenous agonist for P2X7 receptors. One protective mechanism involves P2Y4 purinoceptors whose activation triggers events that inhibit P2X7 pore formation. Because ATP is a ligand for both P2Y4 receptors and P2X7 receptors, cell death due to the opening of pores is minimized during exposure of retinal microvessels to this endogenous nucleotide.
However, even though P2X7 pore formation is prevented during ATP exposure, we found that this nucleotide can kill microvascular cells. Our experiments indicate that ATP's lethality is dependent on the activation of voltage-dependent Ca2+ channels (VDCC). Of potential importance is that the VDCC-mediated vasotoxicity of ATP can be prevented by nitric oxide (NO), which is known to inhibit these Ca2+ channels in retinal microvessels (22). On the basis of our experimental findings, we propose that the NO-mediated inhibition of VDCC and the P2Y4-mediated inhibition of P2X7 pore formation allow ATP to function as a nonlethal vasoactive signal. On the other hand, dysfunction of these protective mechanisms may result in purinergic vasotoxicity.
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MATERIALS AND METHODS |
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Microvessel isolation. As detailed previously (13), 6- to 8-wk-old Long-Evans rats (Harlan Sprague-Dawley, Indianapolis, IN; Charles River Laboratories, Cambridge, MA) were killed by administration of a rising concentration of CO2, and their retinas were rapidly removed and incubated in 2.5 ml of Earle's balanced salt solution, which was supplemented with 0.5 mM EDTA, 20 mM glucose, 15 U of papain (Worthington Biochemicals, Freehold, NJ), and 2 mM cysteine for 30 min at 30°C and bubbled with 95% O2-5% CO2 to maintain pH and oxygenation. After transfer to solution A (in mM: 140 NaCl, 3 KCl, 1.8 CaCl2, 0.8 MgCl2, 10 Na+-HEPES, 15 mannitol, and 5 glucose, pH 7.4, with osmolarity adjusted to 310 mosM), each retina was gently sandwiched between two glass coverslips (15-mm diameter; Warner Instrument, Hamden, CT). Vessels adhered to the coverslip that contacted the vitreal side of the retina. By repeating this tissue print step, several coverslips containing microvessels could be obtained from a retina.
YO-PRO-1 uptake. A coverslip with freshly isolated pericyte-containing microvessels was positioned in a chamber located on the stage of a Nikon Eclipse TE300 or E800 microscope (Nikon, Tokyo, Japan) equipped for fluorescence with a x20 objective. Microvessels were exposed for a total of 90 min to solution A supplemented with 5 µM YO-PRO-1 (Molecular Probes, Eugene, OR), which is a 629-Da propidium diiodide dye that results in detectable fluorescence when it binds to nucleic acids after its entry into a cell. In experimental groups with ATP or benzoylbenzoyl-ATP (BzATP), these agonists were present in the YO-PRO-1-containing solution for the final 60 min of incubation. When UTP was used, it was present for the entire 90 min of exposure to the YO-PRO-1-containing solution. In experiments using 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM) or bromoenol lactone (BEL), these chemicals were present for the entire 90 min of YO-PRO-1 exposure plus an additional 30-min preincubation period in solution A. For each group, the percentage of YO-PRO-1-positive cells was determined after 30 min and again after a total of 90 min of exposure to a solution containing YO-PRO-1. Differential interference contrast optics facilitated detection of cells lacking fluorescence; more than 70 microvascular cells per coverslip were examined. In control experiments in which microvessels were exposed to solution A with YO-PRO-1 and no other additives, the percentage of cells positive for this dye increased from 7.3% (SD 0.6, n = 10) at 30 min to 11.2% (SD 0.6) at 90 min. In the experimental groups, the induced YO-PRO-1 uptake was calculated by subtracting 3.9%, i.e., the increase in controls, from the difference between the 30- and 90-min values.
Electrophysiology.
The perforated patch configuration of the patch-clamp technique was used to monitor ionic currents from pericytes located on microvessels that had been isolated from a retina within 3 h. A coverslip with microvessels was placed in a recording chamber (volume = 1 ml), which was perfused at 1.5 ml/min. Initially, the perfusate was solution A. Vessels were examined under x300 magnification with an inverted microscope equipped with phase-contrast optics. Pericytes were identified by their characteristic "bump on a log" location on the abluminal wall of microvessels (16, 24). Pipettes were filled with 50 mM KCl, 64 mM K2SO4, 6 mM MgCl2, 10 mM K+-HEPES, 240 µg/ml amphotericin, and 240 µg/ml nystatin at pH 7.4, with the osmolarity adjusted to 280 mosmol/l. When measured in solution A, the pipette resistances were typically 5 M
. After a pipette was mounted in the holder of a patch-clamp amplifier (Dagan, Minneapolis, MN), its tip was sealed onto the cell body of phase-bright pericyte. As amphotericin/nystatin perforated the patch, the access resistance to the pericytes studied decreased to <25 M
within 5 min. Pericytes were voltage clamped, and currents were filtered at 1 kHz with a four-pole Bessel filter, digitally sampled every 250 µs using a DigiData 1200B acquisition system (Axon Instruments, Union City, CA), and stored in a computer equipped with pCLAMP 8 (Axon Instruments) and Origin software (version 7; OriginLab, Northampton, MA) for data analysis and graphics display. As detailed previously (13), the peak amplitude of the inward current induced during
3 min of exposure to a perfusate containing ATP or BzATP was quantified from continuous recordings of pericytes that were voltage clamped at 103 mV, the equilibrium potential for K+. The zero-current potential was defined as the membrane potential. Adjustment for the calculated liquid junction potential (2) was made after data collection.
Cell viability assay.
Microvascular cells that did not exclude Trypan blue (0.04%) were classified as dead. The percentage of cells stained with Trypan blue was determined by examining isolated microvessels at x100 magnification with an inverted microscope equipped with bright-field optics. As detailed previously (25), the validity of this vital stain (961 Da) was not compromised by the formation of P2X7 pores, which are permeable to molecules of 900 Da. Because we could not establish with certainty whether a Trypan blue-positive cell was an endothelial cell or a pericyte, subclassification of microvascular cells into these two types was not feasible. At least 150 microvascular cells per coverslip were counted. Freshly isolated microvessels were exposed to purinergic agonists for 24 h, and then cell viability was quantified. UTP, nifedipine, sodium nitroprusside, or brilliant blue was present starting
30 min before the addition of ATP or BzATP. In experiments using oxidized ATP, freshly isolated microvessels were incubated for 2 h at 37°C with this antagonist before the addition of ATP. In protocols using BEL, this chemical was present for 60 min before the addition of BzATP. In protocols using BAPTA-AM, microvessels were exposed to this Ca2+ chelator for 60 min before being switched to a BzATP-containing, BAPTA-AM-free solution. In control experiments in which microvessels were maintained in solution A for 1 day, cell death was 12% (SD 4, n = 18). In experimental groups, the induced cell death was calculated by subtracting this value, i.e., 12%, from the percentage of microvascular cells that were trypan blue positive after 24-h exposure to experimental conditions.
Ca2+ imaging.
Freshly isolated microvessels were loaded with 1 µM fura-2 AM (Molecular Probes, Eugene, OR) at 37°C for 30 min. Subsequently, the extracellular fura-2 AM was washed out with solution A for 30 min to provide time for the AM to be cleaved. A coverslip containing fura-2-loaded microvessels was positioned in a perfusion chamber (volume = 1 ml), which was perfused at
1.5 ml/min. Digital imaging of fluorescence was performed at room temperature using an intensified charge-coupled device camera with a 12-bit dynamic range (Sensicam; Cooke, Auburn Hills, MI). The light source used was a high-intensity Hg2+ lamp coupled to a monochromator (Cairn Research, Faversham, UK). Imaging Workbench 5 (Indec BioSystems, Mountain View, CA) was used to control the imaging equipment and collect data. Using a Nikon Eclipse TE300 microscope at x400 magnification using a x40 oil-immersion lens objective, we measured fluorescence intensities at 340 and 380 nm from pericyte somas, which were defined as regions of interest using the imaging software. Subsequently, the 340/380 fluorescence ratio was calculated. Conversion to intracellular Ca2+ concentration was achieved by using the equation of Grynkiewicz et al. (9), in which Rmin and Rmax were determined by measuring the fluorescence ratio in fura-2-loaded pericytes located on freshly isolated microvessels exposed to Ca2+ calibration buffer solutions (C-3009; Sigma, St. Louis, MO) containing 0 and 39 µM free Ca2+ and supplemented with the Ca2+ ionophore ionomycin (5 µM). To determine the nifedipine sensitivity of BzATP- and ATP-induced Ca2+ increases, the bathing solution was supplemented for
1 min with 10 µM nifedipine 35 min after the onset of exposure to the purinergic agonist. In microvessels not loaded with fura-2, autofluorescence was not detected.
Chemicals. Unless noted otherwise, chemicals were obtained from Sigma.
Statistics. Unless noted otherwise, probability was evaluated using Student's t-test. Error bars in the figures represent SE.
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RESULTS |
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Hypothesizing that the microvascular lethality of ATP (25), which is an endogenous P2X7 ligand, is caused by the formation of lethal P2X7 pores, we began the present study by assessing the effect of this nucleotide on the uptake of YO-PRO-1 into cells of the pericyte-containing retinal microvasculature. Unexpectedly, we found that <4% of the microvascular cells became positive for YO-PRO-1 during exposure to 3 mM ATP (Fig. 1), which is the maximally effective concentration of this nucleotide for inducing P2X7 currents in retinal pericytes (Fig. 2). In contrast, markedly (P < 0.001) more microvascular cells, i.e., 50%, became YO-PRO-1 positive during exposure to 100 µM BzATP (Fig. 1), even though this is a submaximally effective dose for inducing P2X7 currents (Fig. 2). The relative paucity of pores opened during exposure to ATP suggested that although this nucleotide activates P2X7 receptor channels, the subsequent formation of pores is prevented.
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To further support our conclusion that exposure to UTP inhibits the opening of P2X7 pores, we performed electrophysiological assays in which extracellular Na+ was replaced by N-methyl-D-glucamine (NMDG+), which is a 195-Da cation that passes through P2X7 pores but not through P2X7 receptor channels (27). Thus, under these conditions, an inward current generated by the influx of NMDG+ indicates that pores are opened. As illustrated in Fig. 3A, the amplitude of the NMDG+ current induced in pericytes by 100 µM BzATP was markedly smaller when the perfusate contained UTP. This inhibitory effect of UTP was reversible, although a washout for >20 min was required. We obtained four recordings similar to the one illustrated in Fig. 3A. In this series of recordings, the NMDG+ current induced by 100 µM BzATP was inhibited by 88% (SD 8) (P = 0.038) when 30 µM UTP was in the perfusate. The number of pericytes that we successfully monitored during exposure to BzATP in the absence as well as in the presence of UTP was limited because prolonged recordings were required due to the >20 min needed to reverse BzATP's effects on microvascular currents (13). Thus, in another series of experiments, we used a protocol that allowed us to confirm in a larger number of sampled pericytes that UTP inhibits P2X7 pore formation. Specifically, we measured the peak amplitude of the BzATP-induced current in pericytes exposed to an NMDG+-containing perfusate that either lacked (n = 18) or contained (n = 10) 30 µM UTP. As shown in Fig. 3B, the amplitude of the NMDG+ current induced by 100 µM BzATP was 83% smaller (SD 6) (P = 0.002) if the pericyte-containing microvessels were exposed to UTP.
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In other experiments, we considered the possibility that the activation of phospholipase A2 (PLA2) is a step in the sequence of events linking UTP-sensitive receptor activation with the inhibition to pore formation. This putative mechanism was of interest because this enzyme appears to regulate the formation of P2X7 pores in submandibular acinar cells (4). As shown in Fig. 1, the inhibitory effect of UTP on the BzATP-induced uptake of YO-PRO-1 was reduced by 85% (SD 20) (P = 0.009) when microvascular cells were exposed to the PLA2 inhibitor BEL. Our experiments using BAPTA and BEL suggest that the activation of UTP-sensitive receptors inhibits the formation of P2X7 pores by a mechanism involving Ca2+ and PLA2.
Inhibition of P2X7-induced cell death. We asked whether the UTP-induced inhibition of pore formation would affect the lethality of 100 µM BzATP. As shown in Fig. 5, exposure to UTP markedly diminished the microvascular cell death induced by this concentration of BzATP. Conversely, substantial BzATP-induced death occurred when BAPTA or BEL blocked the UTP-mediated inhibition of pore formation (Fig. 5). Thus the formation of pores correlated with the induction of cell death in retinal microvessels exposed to 100 µM BzATP.
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High-dose BzATP-induced cell death: role of pores and VDCC. In another series of experiments, we assessed mechanisms by which 500 rather than 100 µM BzATP causes microvascular cell death. The higher concentration was of interest because it is the maximally effective BzATP dose for killing cells in retinal microvessels (25) and activating an inward current in pericytes (Fig. 2). We found that, similar to the situation with 100 µM BzATP, the uptake of YO-PRO-1 during exposure to 500 µM BzATP was significantly (P = 0.016) diminished in the presence of 30 µM UTP (Fig. 6A). However, unlike our findings with 100 µM BzATP, UTP did not significantly (P = 0.36) inhibit the cell death induced by 500 µM BzATP (Fig. 6B), even though the P2Y4 agonist inhibited pore formation. This finding suggests that 500 µM BzATP, unlike 100 µM BzATP, can trigger microvascular cell death by multiple mechanisms; one of which does not require the opening of P2X7 pores.
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In agreement with the hypothesis that VDCC play a role in the cell death induced by 500 µM BzATP, nifedipine (10 µM) in the presence of 30 µM UTP significantly (P = 0.022) diminished the lethality of this relative high BzATP concentration (Fig. 6B). Consistent with 500 µM BzATP activating multiple lethal pathways, we found that nifedipine in the absence of UTP did not significantly (P = 0.48) affect cell death induced by this concentration of BzATP (Fig. 6B). Because inhibition of VDCC alone is insufficient to block cell death induced by 500 µM BzATP, we not unexpectedly found that exposure of microvessels to a donor of NO, which inhibits the microvascular VDCC (22), failed to affect BzATP's lethality (Fig. 6B). On the basis of these experiments, we concluded that two lethal mechanisms are triggered by 500 µM BzATP. One mechanism is dependent on the formation of P2X7 pores, and the other is a VDCC-dependent process. Both mechanisms must be blocked to prevent 500 µM BzATP from causing microvascular cells to die.
ATP-induced microvascular cell death.
In another series of experiments, we assessed the role of the pore-dependent and VDCC-dependent pathways in mediating the cell death induced by the endogenous purinergic agonist ATP. As shown in Fig. 7A, this nucleotide caused microvascular cell death in a dose-dependent manner. Consistent with the importance of P2X7 receptors, ATP's lethality was significantly (P 0.008) decreased by the P2X7 receptor blockers 2',3'-dialdehyde-ATP (oxidized ATP) and brilliant blue (Fig. 7B). However, even though P2X7 receptors play an important role, YO-PRO-1 uptake was detected in relatively few microvascular cells exposed to a high concentration of extracellular ATP (Fig. 1). Thus we suspected that the activation of VDCC rather than the opening of P2X7 pores was the critical event leading to ATP-induced cell death. This seemed to be a reasonable possibility because our electrophysiological recordings demonstrated that 3 mM ATP caused the membrane potential of pericytes to decrease to from 42 mV (SD 6) to 19 mV (SD 2) (n = 4), which is similar (P = 0.77) to the effect on pericyte voltage of 500 µM BzATP. Consistent with VDCC being activated during exposure to ATP, the intracellular Ca2+ concentration during the plateau phase of pericytes' response to 3 mM ATP was decreased significantly (n = 18; P < 0.001) by 10 µM nifedipine (Figs. 6C and 8A). Supporting a role for VDCC in the microvascular cell death caused by 3 mM ATP, we observed that nifedipine (10 µM) decreased ATP-induced cell death by 96% (SD 21) (P < 0.00) (Fig. 8B). Taken together, our experiments indicate that ATP cytotoxicity in retinal microvessels is not due to the formation of P2X7 pores but rather to VDCC that open when nearly maximal activation of microvascular P2X7 receptor channels causes profound depolarization.
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DISCUSSION |
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Rather than killing microvascular cells by causing P2X7 pores to form, the vasotoxicity of ATP is mediated by the activation of VDCC. These Ca2+ channels open when nearly maximal activation of a microvessel's P2X7 receptor channels causes a profound depolarization that shifts the membrane potential into the "window of current" (12) in which VDCC are activated but not completely inactivated (24). As a result, a pathway for a sustained influx of Ca2+ is opened. Other studies of retinal microvessels (14, 23) have shown that an influx of Ca2+ via VDCC can result in cytotoxicity within the retinal microvasculature, although future studies are needed to determine the minimal duration of ATP-induced VDCC activation required for microvascular cells to become mortally damaged.
Consistent with the scenario in which VDCC mediate the lethal effect of ATP, the vasotoxicity of this endogenous nucleotide was prevented by exposure of retinal microvessels to the VDCC blocker nifedipine or to a NO donor known to inhibit these microvascular channels (22). Although it remains uncertain whether the inhibition of VDCC is the only mechanism by which nifedipine and NO protect against the vasotoxicity of ATP, it seems likely that these Ca2+ channels are key players. A parsimonious explanation of our experimental observations is that the NO-mediated inhibition of VDCC and the P2Y4-mediated inhibition of P2X7 pore formation prevent extracellular ATP from killing microvascular cells (Fig. 9A) and thereby allow this nucleotide to serve as a nonlethal vasoactive signal in the retina (Fig. 9B). On the other hand, dysfunction of either of these protective pathways could result in purinergic vasotoxicity and sight-threatening damage to the retina.
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Even though the release of stored Ca2+ during P2Y4 receptor activation is associated with a profound inhibition of P2X7 pore formation, it is uncertain why a seemingly similar rise in cytosolic Ca2+ caused by influx of this cation through P2X7 receptor channels (13) is less effective in blocking the formation of pores (Fig. 1). One likely explanation for this apparent discrepancy is that by measuring the average concentration of free Ca2+ throughout pericyte somas, we did not detect localized subcellular Ca2+ increases that may be of functional significance. Perhaps Ca2+ released from intracellular stores, compared with Ca2+ entering via P2X7 receptor channels, more effectively increases Ca2+ levels adjacent to sites where pore formation is regulated. Alternatively, the emptying of Ca2+ stored in the endoplasmic reticulum may be a signal to inhibit the formation of P2X7 pores within the plasma membrane. Our experiments also do not exclude a P2Y4-triggered signal, in addition to the release of Ca2+, that is necessary for the inhibition of P2X7 pore formation.
Suggestive of an isoform of PLA2 linking P2Y4 receptor activation with P2X7 pore formation, we found that the opening of pores was markedly inhibited by BEL, which is an inhibitor of Ca2+-insensitive PLA2 (iPLA2) (11). At present, it is uncertain whether there is a causal relationship between the P2Y4-mediated release of Ca2+ and the activation of this PLA2 isoform. A link is possible because although cell-free assays suggest that iPLA2 activation is independent of Ca2+, experimental evidence suggests that this isoform may be regulated within cells by Ca2+ (15). However, although our experiments with BEL point to a role for iPLA2 in regulating pore formation, a caution is that BEL also can inhibit other enzymes (15). If iPLA2 is important, then a goal for the future is to elucidate the mechanism by which its activation blocks the opening of P2X7 pores. One possibility is that iPLA2-induced changes in the lipid composition of the cell membrane interfere with the formation of pores. However, until the sequence of events linking the activation of P2X7 receptor channels with the opening of transmembrane pores is more completely elucidated, it will not be feasible to fully characterize the mechanism by which the activation of P2Y4 receptors inhibits the formation of P2X7 pores.
We postulate that to warrant the potential risk of triggering apoptosis, the capability of microvascular cells to form P2X7 pores must provide a considerable adaptive advantage for the retina. However, the physiological role of P2X7 pores in the normal functioning of microvessels is unclear. We speculate that although the concomitant activation of P2Y4 receptors markedly limits the number of P2X7 pores formed during exposure to extracellular ATP, a few pores do open during exposure of retinal microvessels to extracellular ATP. Consistent with this, we observed that ATP (1.5 mM) evoked an 8-pA (SD 4) (n = 4) inward current carried by NMDG+, which passes through P2X7 pores but not through P2X7 channels (20, 27).
A goal for the future is to elucidate the role of P2X7 pores in retinal microvascular physiology. We propose that one of the roles for the pores that do open, despite P2Y4 activation, is to provide Ca2+ influx pathways that functionally complement the other ATP-activated Ca2+-permeable pathways, i.e., P2X7 receptor channels (13) and the VDCC (Fig. 9A). In addition, pore formation is likely to allow molecules of up to 900 Da to enter and leave microvascular cells; however, at present, the functional implications of this exchange are unknown. On the basis of our studies, it appears that the number of pores formed and VDCC opened are tightly regulated in the normal retinal microvasculature by mechanisms such as the P2Y4-mediated suppression of P2X7 pore formation and the NO-induced inhibition of VDCC. As a result, pores and VDCC can play a role in mediating the purinergic regulation of capillary function without triggering the apoptosis of microvascular cells.
There are a number of potential sources of extracellular ATP that could activate the purinoceptors in retinal microvessels. It seems likely that only locally released ATP activates this microvasculature's purinergic receptors, because blood vessels in the retina lack extrinsic autonomic input (28), which in other vascular beds is a source of ATP (21). One local source of ATP is the glia whose processes ensheath retinal vessels and release this nucleotide (19), which may reach high concentrations in the limited space between the glial processes and the abluminal vascular wall. Other potential sources of extracellular ATP include the vascular endothelium, activated platelets, and hypoxic red blood cells (3, 8). In addition, relatively high concentrations of ATP can leak from damaged cells (17). Thus, under both physiological and pathophysiological conditions, it seems likely that microvascular cells of the retina can be exposed to ATP.
Our conclusions concerning the mechanism by which ATP induces microvascular cell death are based on experiments using freshly isolated retinal microvessels. One benefit of studying microvessels in isolation is that confounding effects mediated by nonvascular retinal cells are eliminated. Also, it is possible to precisely control the concentrations of agonists and inhibitors as well as the duration of exposure to these chemicals. On the other hand, it remains to be demonstrated that potentially vasotoxic concentrations of this nucleotide can occur adjacent to retinal microvascular cells in vivo. Also, the roles of P2Y4 receptors and NO in minimizing the vasotoxicity of ATP await confirmation in vivo. Clearly, an in vivo application of the viability, imaging, and electrophysiological techniques used in this study would be ideal. However, this does not appear to be feasible at present. Hence, despite some caveats, microvessels freshly isolated from the retina provide a useful experimental preparation with which to make new observations that can lead to new hypotheses concerning the physiology and pathobiology of the pericyte-containing microvasculature.
In summary, experiments using isolated microvessels indicate that extracellular ATP not only serves as a vasoactive signal but also can kill cells in the pericyte-containing microvasculature of the retina. Our recent demonstration that the formation P2X7 pores is enhanced in retinal microvessels early in the course of experimental diabetes (25) suggests that purinergic vasotoxicity may play a role in microvascular cell death, which is a hallmark of diabetic retinopathy (6, 18). However, because microvascular cell death would be detrimental to visual function, it seems essential that the lethal effects of extracellular ATP be prevented in the normal adult retina. Our findings that the activation of P2Y4 receptors inhibits P2X7 pore formation and that NO blocks the VDCC-dependent lethality of ATP support the concept that there are effective mechanisms by which to prevent purinergic vasotoxicity in microvessels of the normal retina. We speculate that, under physiological conditions, modulation of VDCC function and P2X7 pore formation by the NO- and P2Y4-dependent pathways plays a role in regulating pericyte Ca2+ levels and thereby the contractility of these mural cells. However, if these regulatory mechanisms become dysfunctional, then purinergic vasotoxicity may result in retinal dysfunction (Fig. 9).
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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