In this study we tested the hypothesis that extracellular ATP regulates the function of the pericyte-containing retinal microvessels. Pericytes, which are more numerous in the retina than in any other tissue, are abluminally located cells that may adjust capillary perfusion by contracting and relaxing. At present, knowledge of the vasoactive molecules that regulate pericyte function is limited. Here, we focused on the actions of extracellular ATP because this nucleotide is a putative glial-to-vascular signal, as well as being a substance released by activated platelets and injured cells. In microvessels freshly isolated from the adult rat retina, we monitored ionic currents via perforated-patch pipettes, measured intracellular calcium levels with the use of fura-2, and visualized microvascular contractions with the aid of time-lapse photography. We found that ATP induced depolarizing changes in the ionic currents, increased calcium levels and caused pericytes to contract. P2X7 receptors and UTP-activated receptors mediated these effects. Consistent with ATP serving as a vasoconstrictor for the pericyte-containing microvasculature of the retina, the microvascular lumen narrowed when an adjacent pericyte contracted. In addition, the sustained activation of P2X7 receptors inhibited cell-to-cell electrotonic transmission within the microvascular networks. Thus, ATP not only affects the contractility of individual pericytes, but also appears to regulate the spatial and temporal dynamics of the vasomotor response.

We tested the hypothesis that extracellular lactate regulates the function of pericyte-containing retinal microvessels. Although abluminally positioned pericytes appear to adjust capillary perfusion by contracting and relaxing, knowledge of the molecular signals that regulate the contractility of these mural cells is limited. Here, we focused on lactate because this metabolic product is in the retinal extracellular space under both physiological and pathophysiological conditions. In microvessels freshly isolated from the adult rat retina, we used perforated-patch pipettes to monitor ionic currents, fura-2 to measure calcium levels, and time-lapse photography to visualize changes in mural cell contractility and lumen diameter. During lactate exposure, pericyte calcium rose; these cells contracted, and lumens constricted. This contractile response appears to involve a cascade of events resulting in the inhibition of Na+/Ca2+ exchangers (NCXs), the decreased of which function causes pericyte calcium to increase and contraction to be triggered. On the basis of our observation that gap junction uncouplers minimized the lactate-induced rise in pericyte calcium, we propose that the NCXs inhibited by lactate are predominately located in the endothelium. Indicative of the importance of endothelial/pericyte gap junctions, uncouplers of these junctions switched the pericyte response to lactate from contraction to relaxation. In addition, we observed that hypoxia, which closes microvascular gap junctions, also switched lactate's effect from vasocontraction to vasorelaxation. Thus the response of pericyte-containing retinal microvessels to extracellular lactate is metabolically modulated. The ability of lactate to serve as a vasoconstrictor when energy supplies are ample and a vasodilator under hypoxic conditions may be an efficient mechanism to link capillary function with local metabolic need.

The aim of this study was to identify the mechanisms by which angiotensin II alters the physiology of the pericyte-containing microvasculature of the retina. Despite evidence that this vasoactive signal regulates capillary perfusion by inducing abluminal pericytes to contract and thereby microvascular lumens to constrict, little is known about the events linking angiotensin exposure with pericyte contraction. Here, using microvessels freshly isolated from the adult rat retina, we monitored pericyte currents via perforated-patch pipettes, measured pericyte calcium levels with fura-2 and visualized pericyte contractions and lumen constrictions by time-lapse photography. We found that angiotensin activates nonspecific cation (NSC) and calcium-activated chloride channels; the opening of these channels induces a depolarization that is sufficient to activate the voltage-dependent calcium channels (VDCCs) expressed in the retinal microvasculature. Associated with these changes in ion channel activity, intracellular calcium levels rise, pericytes contract and microvascular lumens narrow. Our experiments revealed that an influx of calcium through the NSC channels is an essential step linking the activation of AT 1 angiotensin receptors with pericyte contraction. Although not required in order for angiotensin to induce pericytes to contract, calcium entry via VDCCs serves to enhance the contractile response of these cells. In addition to activating nonspecific cation, calcium-activated chloride and voltage-dependent calcium channels, angiotensin II also causes the functional uncoupling of pericytes from their microvascular neighbours. This inhibition of gap junction-mediated intercellular communication suggests a previously unappreciated complexity in the spatiotemporal dynamics of the microvascular response to angiotensin II.

The purpose if this study was to elucidate how extracellular ATP causes cell death in the retinal microvasculature. Although ATP appears to serve as a vasoactive signal acting via P2X(7) and P2Y(4) purinoceptors, this nucleotide can kill microvascular cells of the retina. Because P2X(7) receptor activation causes transmembrane pores to form and microvascular cells to die, we initially surmised that pore formation accounted for ATP's lethality. To test this hypothesis, we isolated pericyte-containing microvessels from rat retinas, assessed cell viability using Trypan blue dye exclusion, detected pores by determining the uptake of the fluorescent dye YO-PRO-1, measured intracellular Ca(2+) with the use of fura-2, and monitored ionic currents via perforated patch pipettes. As predicted, ATP-induced cell death required P2X(7) receptor activation. However, we found that pore formation was minimal because ATP's activation of P2Y(4) receptors prevented P2X(7) pores from forming. Rather than opening lethal pores, ATP kills via a mechanism involving voltage-dependent Ca(2+) channels (VDCC). Our experiments suggest that when high concentrations of ATP caused nearly all microvascular P2X(7) receptor channels to open, the resulting profound depolarization opened VDCC. Consistent with lethal Ca(2+) influx via VDCC, ATP-induced cell death was markedly diminished by the VDCC blocker nifedipine or a nitric oxide (NO) donor that inhibited microvascular VDCC. We propose that purinergic vasotoxicity is normally prevented in the retina by NO-mediated inhibition of VDCC and P2Y(4)-mediated inhibition of P2X(7) pore formation. Conversely, dysfunction of these protective mechanisms may be a previously unrecognized cause of cell death within the retinal microvasculature.

PURPOSE.Although decentralized control of blood flow is particularly important in the retina, knowledge of the functional organization of the retinal microvasculature is limited. Here, the authors characterized the distribution and regulation of L-type voltage-dependent calcium channels (VDCCs) within the most decentralized operational complex of the retinal vasculature-the feeder vessel/capillary unit-which consists of a capillary network plus the vessel linking it with a myocyteencircled arteriole. METHODS. Perforated-patch recordings, calcium-imaging, and time-lapse photography were used to assess VDCC-dependent changes in ionic currents, intracellular calcium, abluminal cell contractility, and lumen diameter, in microvascular complexes freshly isolated from the rat retina. RESULTS. Topographical heterogeneity was found in the distribution of functional VDCCs; VDCC activity was markedly greater in feeder vessels than in capillaries. Experiments showed that this topographical distribution occurs, in large part, because of the inhibition of capillary VDCCs by a mechanism dependent on the endogenous polyamine spermine. An operational consequence of functional VDCCs predominantly located in the feeder vessels is that voltage-driven vasomotor responses are generated chiefly in this portion of the feeder vessel/capillary unit. However, early in the course of diabetes, this ability to generate voltage-driven vasomotor responses becomes profoundly impaired because of the inhibition of feeder vessel VDCCs by a spermine-dependent mechanism. CONCLUSIONS. The regulation of VDCCs by endogenous spermine not only plays a critical role in establishing the physiological organization of the feeder vessel/capillary unit, but also may contribute to dysfunction of this decentralized operational unit in the diabetic retina. (Invest Ophthalmol Vis Sci.

The vulnerability of neurons to ischaemia requires that blood flow in the nervous system be tightly coupled to metabolic needs. In the inner portion of the mammalian retina, the coupling of capillary perfusion to local needs must be particularly efficient due to the low density of capillaries (Funk, 1997). Although the sparseness of retinal microvessels minimizes vascular interference with light, it results in a tenuous link between the supply of energy substrates and the needs of neurons. For this reason, the retinal circulation is specialized for responsiveness to local demands (Anderson, 1996) and thus provides a useful system for testing hypotheses concerning the mechanisms by which locally derived signals regulate capillary function.In this study we tested the hypothesis that extracellular ATP regulates the function of the pericytecontaining retinal microvessels. Pericytes, which are more numerous in the retina than in any other tissue, are abluminally located cells that may adjust capillary perfusion by contracting and relaxing. At present, knowledge of the vasoactive molecules that regulate pericyte function is limited. Here, we focused on the actions of extracellular ATP because this nucleotide is a putative glial-to-vascular signal, as well as being a substance released by activated platelets and injured cells. In microvessels freshly isolated from the adult rat retina, we monitored ionic currents via perforated-patch pipettes, measured intracellular calcium levels with the use of fura-2, and visualized microvascular contractions with the aid of time-lapse photography. We found that ATP induced depolarizing changes in the ionic currents, increased calcium levels and caused pericytes to contract. P2X 7 receptors and UTP-activated receptors mediated these effects. Consistent with ATP serving as a vasoconstrictor for the pericyte-containing microvasculature of the retina, the microvascular lumen narrowed when an adjacent pericyte contracted. In addition, the sustained activation of P2X 7 receptors inhibited cell-to-cell electrotonic transmission within the microvascular networks. Thus, ATP not only affects the contractility of individual pericytes, but also appears to regulate the spatial and temporal dynamics of the vasomotor response.

Objectives: To investigate chymase involvement in idiopathic macular hole onset, the effects of chymase on monkey eyes and cultured Müller cells were investigated. Methods: Immunohistochemistry using antinestin and antiglial fibrillary acidic protein antibodies was performed in a normal monkey eye. After chymase was injected into the monkey vitreous, histological changes in the retina were evaluated using the TdT-mediated dUTP nick-end labeling (TUNEL) assay. Expression of c-kit, a stem cell factor receptor, and nestin was examined in porcine Müller cells cultured with basic fibroblast growth factor. The effects of chymase on proliferation and TUNEL staining in Müller cells were also examined. Results: The number of nestin and glial fibrillary acidic protein-positive cells was higher in the macula than in other regions. Thickening of the posterior hyaloid membrane and some apoptotic cells were found in the macula of chymase-treated eyes. The expression of c-kit and nestin in Müller cells was shown and enhanced when cultured with basic fibroblast growth factor. Exposure to chymase inhibited Müller cell proliferation and produced TUNEL-positive cells. Conclusions: There might be Müller cells possessing atypical properties near the macular region and chymase might cause fibrosis and apoptosis through these cells. These findings suggest that increased chymase activity may result in idiopathic macular hole onset.

PURPOSE.To determine the modification of the glutamate-induced death of retinal neurons by endothelin (ET)-1. METHODS. Cultured retinal neurons from fetal rats were exposed to glutamate (1.0 mM) alone or glutamate with ET-1 (10 Ϫ10-10 Ϫ7 M) for 10 minutes. Neuronal death was assessed by the trypan blue exclusion or TUNEL assays at 2, 6, and 24 hours after the exposure. The effects of adding BQ-123 or BQ-788, ET A , and ET B receptor antagonists, respectively, in combination with ET-1 was also assessed. RESULTS. Immunohistochemical analyses showed that the ETs as well as ET A and ET B receptors were expressed on cultured retinal neurons consisting mainly of amacrine cells. A brief exposure of the cultured retinal neurons to glutamate alone significantly increased the number of dead cells, and the addition of ET-1 with glutamate caused a further significant increase in retinal neuronal death compared with the cells exposed to glutamate alone. A significant increase in neuronal death was detected at doses of 10 nM of ET-1 and higher after a 24-hour exposure (P Ͻ 0.05, Dunnett), whereas brief exposure of neurons to up to 1 M ET-1 alone did not cause delayed cell death of neurons. BQ-123 (10 nM) suppressed the enhancement of retinal toxicity caused by ET-1 (10 nM), whereas BQ-788 had no significant effect. CONCLUSIONS. These results indicate that ET-1 enhances glutamate-induced retinal cell death, possibly through ET A receptors. ET-1 may act synergistically with glutamate to damage retinal neurons under hypoxic conditions. (Invest Ophthalmol Vis Sci. 2005;46:4684 -4690)