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(Investigative Ophthalmology and Visual Science. 2002;43:3223-3230.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Effects of Endothelin-1 on Components of Anterograde Axonal Transport in Optic Nerve

Martha E. Stokely1, Scott T. Brady2 and Thomas Yorio1,3

1 From the Departments of Biomedical Sciences and 3 Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, Texas; and the 2 Department of Cell Biology, University of Texas Southwestern Medical Center, Houston, Texas.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. Increased levels of endothelins (ETs) are associated with glaucoma and have been said to contribute to the development of glaucomatous optic neuropathy. In glaucoma, movement of selected components of anterograde axonal transport essential in ganglion cell survival is impaired—specifically, the transport of mitochondria. This study evaluates the effect(s) of a single administration of intravitreous ET-1 on anterograde axonal transport in the rat optic nerve.

METHODS. Proteins for anterograde axonal transport were pulse labeled by intravitreous injection of 35S-methionine plus or minus ET-1 (2 nmol) in HEPES buffer (pH 7.4). At appropriate time intervals, optic nerves were dissected, sectioned while frozen, and homogenized in denaturing buffer, and transported protein was quantitated by liquid scintillation counting. Counts corrected for efficiency, quench, background, and decay were statistically evaluated (ANOVA, n = 7).

RESULTS. Effects of treatment with intravitreous ET-1 on anterograde axonal transport were significant, biphasic, and prolonged (4 hours to 21 days). The initial phase was a significant enhancement of transport at times normally associated with small, fast-moving tubulovesicles (4 and 24 hours), followed by significant impairments at times normally associated with transport of mitochondria (28–36 hours), cytoplasmic matrix (4 days), and cytoskeletal proteins (21 days). The most pronounced effect of ET-1 was decreased axonal transport at times associated with normal anterograde transport of mitochondrial proteins (28, 32, and 36 hours, P = < 0.001, P < 0.015, and P < 0.001, respectively). This was mimicked by ET-3 at 28 hours.

CONCLUSIONS. Effects of intravitreous ET-1 are consistent with a receptor-mediated role for elevated ETs in pathologic misregulation(s) of anterograde axonal transport.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Glaucoma is a stereotypic optic neuropathy involving loss of retinal ganglion cells with axons that leave the retina to form the optic nerve.1 One characteristic of glaucoma is a dysfunction in the regulated delivery of mitochondria and uncharacterized tubulovesicles from their sites of synthesis and assembly in the cell body to sites of proper function that are situated along the axon and in terminals.2 The observed disruption in anterograde axonal transport may result from nerve compression, as a consequence of elevated intraocular pressure,3 or it may result from anoxic conditions4 5 during ischemia.6 The effect of direct compression on axonal transport appears to involve the loss of linear microtubule arrays in exposed axons,7 a mechanism also suggested to result from ischemia.5 However, experimental neuropathies known to act by this mechanism8 exhibit large proximal swellings in regions with unmyelinated axons, disorganized microtubules, and prominent accumulations of neurofilaments.9 In contrast, accumulations in glaucomatous human donor tissue appear to be predominantly mitochondrial and vesicular.2 This suggests that glaucomatous disease represents a selective misregulation of axonal transport, rather than an indiscriminate inhibition resulting from loss of microtubular arrays. Some other mechanism(s) could be involved in the full development of glaucomatous pathophysiology, which would contribute to glaucoma by subtly perturbing important physiological functions in the proximal optic nerve. One candidate agent to affect anterograde axonal transport is ET-1, a dually neuroactive10 and vasoactive11 peptide that is present in the normal eye12 and is reported to increase in experimental animal models of glaucoma with elevated intraocular pressure.13 14

ET-1, its cognate ET-3, and their receptors are normally present in the appropriate ocular regions to affect axonal compartments of retinal ganglion cells.12 15 16 17 ET-1, the most-studied member of the ET family of isopeptides,11 18 acts through the G-protein–coupled receptors ETA and ETB with specificities of ET-1 > ET-3 and ET-1 {cong} ET-3, respectively.19 These receptors are located at the optic nerve head,20 as well as within the ganglion cell and nerve fiber layers of the retina.21 Because of its vasoconstrictive properties, ET-1 has been extensively used experimentally to model retinal ischemia.22 23 24 However, indications from other regions of the central nervous system (CNS) are that either ischemic events or mechanical injury can induce the secretion of ET-1 from resident astrocytes25 26 and alter the expression of ET receptors in CNS tissues,27 28 29 30 including the retina.30 In addition, nonselective ET receptor antagonists can block secondary axonal degeneration in long-fiber tracts,31 a mechanism believed to contribute to retinal ganglion cell loss in some experimental models of glaucoma.32 These data suggest additional roles for ETs that may include alterations in anterograde axonal transport in such CNS tissues as the optic nerve.

Anterograde axonal transport is a complex and tightly regulated process by which neurons supply the axons of their long-fiber tracts with protein elements required for maintenance and survival. Because axons do not have the machinery required for protein synthesis, the timely delivery of specific cargoes to their functional domains, represents a complex logistic burden for the neuron. Axonal transport comprises multiple distinct rate components that deliver specific types of cargo to axonal domains.33 34 Fast anterograde axonal transport delivers a variety of membrane-bound organelle (MBO) cargoes and may be divided into several subcomponents, including very fast, small tubulovesicles moving with synaptic vesicle precursor proteins, and slower-moving MBO cargoes with mitochondrial proteins.35 Regulation of fast anterograde axonal transport is only partially understood.36 37 38

In glaucoma, the anterograde axonal transport of mitochondria is seriously compromised as axons traverse the perilaminar region of the optic nerve head,2 presumably leading to bioenergetic perturbations as a consequence of inappropriate supply. The specific type(s) of tubulovesicles affected in glaucoma are unknown,2 making it difficult to assess their contribution(s) to development of the neuropathy. In the present study, elevated ET-1 induced a complex pattern of aberrations, affecting all the distinct rate components of anterograde axonal transport, with its most pronounced effect(s) on the mitochondrial subcomponent.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Rats
Young adult male Sprague-Dawley rats (n = 7 control and n = 7 experimental rats for each time point or injection–sacrifice interval, ISI), weighing 200 to 250 g, were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and acclimatized to the animal facility for 2 weeks before use in this study. Rats were selected for use in because their retinal ganglion cell axons, as in humans, are unmyelinated before leaving the retina, and anterograde axonal transport in the optic nerve has been extensively characterized in this species in work previously published from this laboratory.39 40 41 42 43 All studies were conducted in accordance with NIH guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Intravitreous Injection of Radiolabeled Precursors, with or without ET
Newly synthesized proteins undergoing anterograde axonal transport in the optic nerve were pulse-labeled in the presence or absence of ET, as modified from previously published methods.34 35 44 (Modifications were minimal and involved the replacement of a distilled water vehicle for resuspension of radiolabeled precursors with either HEPES-buffered ET-1 or HEPES vehicle buffer alone.) 35S-methionine (Easytag Express Protein Labeling Mix; DuPont-NEN Life Sciences, Boston, MA) was lyophilized and resuspended in either vehicle alone (10 mM HEPES [pH7.4]; Sigma Chemical Co., St. Louis, MO) or in vehicle containing 500 µM ET-1 (Bachem, Belmont, CA). Rats were anesthetized by methoxyflurane inhalation, and 0.8 mCi (4 µL) of radiolabel in vehicle, with our without ET-1 (final dose 2 nmol), was injected into the vitreous of the left eye with a 30-gauge needle attached to a syringe (microliter 710, 22s gauge; Hamilton Co., Reno, NV) by polyethylene tubing (PE-20, Clay Adams Brand; BD Biosciences, Sparks, MD).34 44 In one experiment, ET-3 was substituted for ET-1, with the same methods used (2-nmol dose, 28-hour ISI, n = 7 control and n = 7 experimental animals). During intravitreous injections, retinas were observed through the pupil with a surgical microscope (model Stiffuss S; Carl Zeiss, Thornwood, NY). During introduction of the resuspended label into the vitreous, a transient blanching of the retina was observed in all animals, both control and experimental, that did not appear noticeably greater in the ET-1–treated animals and began to recover immediately after the injection was complete. One minute after injection, all retinas appeared normal in color. (Based on these initial observations, further observations of the retinas were not performed). Information on the dose-related effect(s) of intravitreous ET-1 in this species (rat) were unavailable, and physiological-pathologic concentrations of ET in the optic nerve head’s microenvironment are generally unknown. Therefore, dose selection was made on the basis of a small pilot study, using these methods and measuring the total pulse-labeled protein axonally transported into the rat optic nerve. (Three rats in every group was used only for the pilot study, 4-hour ISI, data not shown). The pilot study evaluated 0.3-, 0.4-, and 2-nmol doses of intravitreous ET-1 and the results showed a trend of increasingly enhanced axonal transport, compared with control, as the dose of ET-1 increased. However, significant effects on axonal transport (4-hour ISI) were seen only in the pilot study for the 2-nmol dose. The combination of a nonsignificant trend at lower doses with a large variance at the lowest significantly effective dose (2 nmol) was interpreted to mean that the 2-nmol dose was centrally located within the effective pharmacologic dose range, for anterograde axonal transport in rat optic nerve, at the 4-hour ISI. Possible effect(s) on nonassayed ocular tissues were not considered in dose selection, because data on these were unavailable for either acute or chronic intravitreous administration of ET-1 in rats.

Harvest and Preparation of Pulse-Labeled Optic Nerves
Animals were anesthetized with methoxyflurane at specified times after injection and then killed by decapitation. ISIs were selected based on the published characterizations of anterograde axonal transport in rat optic nerve for specific marker proteins associated with specific classes of axonally transported materials (Table 1) .35 36 39 40 45 46 47 48 49 50 Seven vehicle-treated animals and seven ET-treated animals were killed at each of the specified times (4, 24, 28, 32, and 36 hours and 4 and 21 days). Optic nerves were removed, flash frozen with crushed dry ice, and sectioned while frozen. Nerves were sectioned to aid complete homogenization and to provide additional data for future studies. The frozen sections (2 mm in length) were numbered as segments 1 to 4 (from proximal to distal), and glass-on-glass homogenized in 100 µL of BUST sample buffer (2% ß-mercaptoethanol, 8 M urea, 1% SDS, 0.1M Tris, 0.02% phenol red [pH 7.4]).51 A 25% aliquot of each homogenized segment was counted in a liquid scintillation counter, and counts were corrected for decay, quench, and counting efficiency.


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  		Table 1. Summary and Significance Effect of ET-1 on Anterograde Axonal Transport

 
Statistical Analysis
Corrected disintegrations per minute (dpm) were analyzed by ANOVA (n = 7) performed on computer (Systat 5; SPSS Sciences, Inc., Chicago, IL). All statistical analyses included data from the whole optic nerve (the sum of all four segments from an individual optic nerve). For the comparison of the effect of ET-3 with ET-1 at the 28-hour ISI, corrected dpm for the ET-1 group (n = 7) were normalized to the group of control rats simultaneously treated (n = 7), and the same method was used in the ET-3 group (n = 7) and its simultaneously treated group of control rats (n = 7). Normalized data for ET-1 and ET-3 were compared (ANOVA, n = 7) by computer (Systat 5; SPSS Sciences, Inc.).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
ET-1: Alterations in All Components of Anterograde Axonal Transport
The effects of intravitreous ET-1 treatment were significant, biphasic, and prolonged (Table 1 , P < 0.05; Figs. 1 2 3 4 ; n = 7 control and n = 7 experimental animals, at each time point). The most profound effect of ET-1 occurred at 28 hours. At the 28-hour ISI, this effect was mimicked by the ETB-receptor–selective agonist ET-3 (no significant difference between ET-1 and ET-3, P > 0.999, ANOVA, n = 7, Fig. 5 ) and suggests a receptor-mediated phenomenon, with similar effects for ET-1 and ET-3.



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  		Figure 1. Intravitreous injection of ET-1 altered delivery of axonally transported proteins to the optic nerve for an extended period. Plotting the amount of radiolabeled proteins detectable in the proximal 2 mm of the optic nerve provides a "window-on-the-nerve" view of axonal transport. Such a window shows the timed movements of radiolabeled materials through the most proximal 2-mm segment of the rat optic nerve (adjacent to the eye, designated segment 1 in the text and in Figs. 2 and 3 ) in vehicle-treated control and ET-1–treated experimental animals. A single injection produced a significant increase in the amount of material delivered by axonal transport in the first 24 hours after injection, followed by a dramatic decline in transported material delivered to the nerve at subsequent time points. The most dramatic decline was between 28 and 36 hours, an interval previously shown to include transport of mitochondrial protein markers. Data shown are the mean of seven results obtained in seven different animals, for each point plotted (n = 7 for both control and experimental rats). Error bars: SEM.

 


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  		Figure 2. A single intravitreous ET-1 injection produced significant alterations in the distribution of radiolabel within the optic nerve at all times evaluated. The changes were biphasic and may reflect changes in both the amount of material transported (amount of radiolabel protein at each time point) and in the delivery of transported material to the nerve (changes in distribution at different times after labeling). This is a graphical representation of the raw data set (n = 7 for both control and experimental rats, at every time point), because the only corrections made were for radioactive decay and calibration of the liquid scintillation counting technique. Optic nerve segments, 2 mm in length, are numbered consecutively from immediately behind the eye (segment 1). Evaluation for statistical significance of effects of ET-1 on individual nerve segments was not considered appropriate because of their physical continuity at the time of treatment. Statistical comparisons for whole optic nerve are shown in Figure 4 . Error bars: SEM.

 


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  		Figure 3. Biphasic effect(s) of intravitreous ET-1s on anterograde axonal transport were detected in all regions of the optic nerve (segments 1–4, 2 mm in length, numbered from the eye, n = 7 for both control experimental rats, for every time point). The differential response of different subcomponents of axonal transport suggests a complex response to elevated vitreous ETs. Magnitude of the effect (mean ET-treated minus mean vehicle-treated control) is expressed in corrected dpm. Direction of the effect is positive if the ET-treated value exceeded the control value (at 4 and 24 hours) and negative if the ET-treated value was less than the control value (28, 32, and 36 hours and 4 and 21 days). It should be noted that (unlike in Fig. 1 ) the x-axis is a category axis and is not to scale. SEMs for individual nerve segments are provided in Figure 2 . Evaluation for statistical significance of effects of ET-1 on individual nerve segments was not considered appropriate because of their physical continuity at the time of treatment.

 


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  		Figure 4. Elevated intravitreous ET-1 significantly increased the amount of material in transport at early times after injection, but profoundly decreased delivery of transported material to the nerve at times consistent with the transport of mitochondria. Effects of intravitreous ET-1 on anterograde axonal transport in rat optic nerve for each time interval examined are expressed as corrected dpm (n = 7 for both control experimental rats, at every time point. Because the x-axis is a category axis and is not to scale, the large time difference, inherent in presenting slow transport data with fast transport data, is marked by the appearance of white dots on the solid bars for the ET-1 treatment group, at the slow-component time intervals. Error bars: SEM. *Statistical significance at P < 0.05; **statistical significance at P < 0.01.

 


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  		Figure 5. No significant difference was noted between the effects of intravitreous ET-3 and intravitreous ET-1 on anterograde axonal transport at the 28-hour ISI (P > 0.999, ANOVA, normalized data, n = 7). The similar effects of ET-1, which activates both ETA and ETB receptors, and ET-3, which selectively activates ETB receptors, indicate that changes in axonal transport were largely ETB-receptor–mediated. Data for ET-1 and ET-3 groups were normalized to their appropriate simultaneously treated and processed vehicle-treated negative control groups’ data (n = 7 for the vehicle-treated control group that was treated and processed simultaneously with the ET-1 group, n = 7 for the ET-1-treated group; n = 7 for the vehicle-treated control group that was treated and processed simultaneously with the ET-3 group, n = 7 for the ET-3-treated group). Data were for the whole optic nerve (sum of all segments 1–4 for each optic nerve). Error bars: SEM. No SEM could be calculated for the control value, because all data displayed were normalized to control.

 
The direction and magnitude of the effects of ET-1 varied with time and the transported cargo (Table 1 , Figs. 3 4 ), suggesting a selective misregulation, as opposed to an indiscriminate inhibition, of anterograde axonal transport. Distributions of radiolabel within optic nerves were monitored (Figs. 2 3) but all statistical comparisons (Table 1 ; Fig. 4 ) were made on data from the whole optic nerve (n = 7 for each time point).

Effect of ET-1 on Axonal Transport of Some Small, Fast Tubulovesicles
There was a moderate but significant enhancement of axonal transport into the optic nerve at times normally associated with small, fast-moving tubulovesicles, but little or no mitochondrial marker proteins (4- and 24-hour ISIs; Table 1 , Figs. 1 2 3 4 ).35 The magnitude of enhancement of ET-1 was greater at the 4-hour ISI than the 24-hour ISI (Fig. 1) . The 4- and 24-hour ISIs were selected for use in the study because they are normally associated with similar amounts of total anterogradely transported material,35 but the chemical compositions of transported material are different.39 Typically, a single form of the kinesin motor52 is associated with transport at the 4-hour ISI, whereas multiple isoforms of the motor are associated with the 24-hour ISI.35 39 The possibility of a differential regulation in the transport of various classes of tubulovesicles during this subcomponent were the basis for our use of the 4- and 24-hour ISIs. In this study, effects of ET-1 on anterograde axonal transport at the 4- and 24-hour ISIs were consistent with a hypothesized differential misregulation in the transport of various classes of small, fast-transported tubulovesicles.

Effect of ET-1 and ET-3 on Axonal Transport in the Mitochondrial Subcomponent
Intravitreous effects of ET-1 were most severe within a 28- to 36-hour window (Table 1 ; Figs. 1 3 4 ). In this interval, a large reduction in transport occurred. At these times, a large pulse (Fig. 1) of mitochondrial proteins normally moves through the rat optic nerve.35 A closely related ET, ET-3, was also tested for an effect on transport at 28 hours. ET-3 has 1000-fold less affinity than ET-1 for vasoconstrictive ETA receptors,53 but has comparable affinity for ETB receptors.54 The ETB-selective agonist ET-3 had effects on axonal transport at 28 hours that were comparable to those of ET-1 (Fig. 5) .

Effect of ET-1 on Axonal Transport in the Slow Components
At times associated with both slow components of axonal transport, the effect(s) of ET-1 remained significant (Table 1) but were more moderate (Figs. 2 4) than those noted during the mitochondrial subcomponent of fast transport (Fig. 2) . This suggests a mechanism that is either somewhat component specific or partially reversible and could result in less accumulation of cytoplasmic matrix and cytoskeletal proteins35 39 45 46 47 48 49 than might be expected from a generalized loss of transport.8 9

Biphasic Effects of Intravitreous ET-1
The effects of intravitreous treatment with ET-1 on anterograde axonal transport were biphasic (Table 1 ; Figs. 1 3 4 ). The initial, rapid effect of treatment with ET-1 was a significant enhancement of anterograde axonal transport into the optic nerve at 4 and 24 hours (Figs. 1 4) . The slower but more prolonged effect of ET-1 was a significant reduction of anterograde axonal transport into the optic nerve at 28, 32, and 36 hours and 4 and 21 days (Figs. 1 4) .

Prolonged Effects of ET-1
Intravitreous ET-1, administered as a single bolus, exerted significant effects (Table 1) on anterograde axonal transport as early as 4 hours after treatment (Figs. 1 3) and as late as 21 days after treatment (Fig. 4) , inducing an extended period of aberrant axonal transport within the retinal ganglion cell axons of the optic nerve (Fig. 4) . Chronic administration was not required to achieve this effect.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
A single intravitreous application of ET-1 had a complex and profound effect on anterograde fast axonal transport (Fig. 1) . Changes occurred in both the very fastest moving material, thought to represent movement of small tubulovesicular structures including synaptic vesicle precursors, and the slower-moving subcomponents of fast anterograde transport, which contain mitochondrial markers.35 The most pronounced effect of intravitreous ET-1 was a sharp reduction in the mitochondrial subcomponent of anterograde transport (Figs. 1 3) . This sharp reduction in the mitochondrial subcomponent contrasted with a modest increase in material transported at the fastest rate (Figs. 1 3) .

Although, ischemia inhibits axonal transport,55 ETA-mediated vasoconstriction is apparently not a prerequisite for the effect of ET on axonal transport. The effects of the ETB receptor–selective agonist ET-3 on the mitochondrial subcomponent of axonal transport at 28 hours were comparable to the effects obtained with ET-1 suggesting that ET’s actions on axonal transport (Fig. 5) are an ETB-mediated effect (Fig. 5) . This suggests the possibility that activation of ETB lies in a direct mechanistic line downstream from ischemia, perhaps shortening a pathologic ischemic circuit.

The complexity of the effects of ET-1, with early increases in transported material followed by later decreases in transport (Figs. 1 3) , appears to reflect a mechanism that perturbs the neuron’s ability to regulate the timely delivery of specific cargoes to their functional domains. The early increases, which were greater at the 4-hour than at the 24-hour ISI (Fig. 1) , may reflect the markedly increased transport of a subset of the fastest tubulovesicles, highly enriched at the 4-hour ISI but less well represented at the 24-hour ISI. Markedly increased transport for a subset of vesicles, may partially obscure decreased transport for other types of vesicles that happen to be simultaneously transitioning through the optic nerve at the 24-hour ISI. This interpretation would not be inconsistent with the previously published neurochemical analyses on the composition of transported materials in the optic nerves of normal rats at these ISIs.35 39 40 Those studies have indicated that multiple distinct MBO cargoes move in fast axonal transport, with differences in protein content, motor isoforms, and destination.35 36 39 40 Targeted delivery of MBO cargoes to different destinations implies a different regulatory control. Such an explanation is compatible with a receptor-mediated event,36 56 and reconciles the early effects of ET-1 with observed glaucomatous disease.2

The effects of ET-1 and ET-3 on the subcomponent that contains mitochondrial proteins35 39 (Figs. 3 4) appears to be consistent with evidence that a reduced percentage of mitochondria successfully transition through the perilaminar region in glaucoma.2 The perilaminar region is both adjacent to the vitreous site of ET-1 and ET-3 applications and within the primary site of glaucomatous disease—the optic nerve head.32 The results in this study suggest the hypothesis that increases in vitreous ETs could contribute to the development of glaucomatous optic neuropathy,13 through pathologic misregulation of anterograde axonal transport.

All components of anterograde axonal transport were significantly affected by treatment with ET-1, including the transport of cytoskeletal materials moved in slow component a (SCa; Table 1 , Figs. 2 4 ). This appears to be consistent with the occasionally observed fibrillary changes reported in human glaucomatous donor tissue.2 That intravitreous ET had lesser effects on the slow components of axonal transport than were seen in the earlier mitochondria-associated subcomponent may be explained by the continued reductions in axonal energy supply due to reduced mitochondrial transport, reductions in synthesis of cytoskeletal proteins, or a subcomponent-specific action.

The ability of a single intravitreous administration of ET-1 to elicit a biphasic response (Figs. 1 3) may reflect activation of two separate signal transduction pathways. Activation of multiple ET receptors present on the retinal ganglion cell soma and/or axons15 21 or of a single class of receptors able to activate dual signaling pathways57 58 could produce a biphasic effect. Less-direct mechanism(s) may also contribute by activating ET receptors on retinal vasculature15 and resident glia,59 60 initiating processes that selectively alter either anterograde transport alone36 61 or the coordinated synthesis and anterograde transport of specific neuronal proteins.62 63 One type of resident glia, optic nerve head astrocytes, share intimate contact with retinal ganglion cell axons in the perilaminar region,59 60 express ET receptors,20 and undergo both morphologic and secretory changes in glaucoma.64

These results demonstrate that a single exposure to elevated levels of ET-1 in the vitreous can produce an extended series of effects (Fig. 2) that may impinge on neuronal physiology and energy supply for retinal ganglion cell axons in the optic nerve. The simplest interpretive model suggests that ET-1 may act directly on receptors that may be located on retinal ganglion cell axons in the perilaminar region, locally affecting anterograde axonal transport, perhaps inducing cytoskeletal modification,65 and/or aberrant phosphorylation of proteins36 56 66 67 that are critical to transport. However, the effects of ETs in other regions of the CNS are generally not simple.

A simple but more probable model suggests that both direct and indirect effects of elevated vitreous ETs contribute to the prolonged, multiple-component dysfunction in anterograde transport observed in this study. In many regions of the CNS, the synthesis and secretion of ETs have a dynamic and complex interrelationship with both ischemia25 68 and mechanical injury.69 70 By analogy to other CNS regions, ET-1 synthesis and release from astrocytes may be stimulated by either mechanical injury, possibly from elevated intraocular pressure, or retinal ischemia. Elevated ET could then induce a pathologic dysregulation of the mitochondrial subcomponent of anterograde axonal transport, presumably resulting in energy perturbations within retinal ganglion cell axons. ET-1 could stimulate astrocytic ETB-mediated responses, including increased ET synthesis and release,26 enhanced secretion of cytokines71 and efflux of glutamate.72 Vascular responses to elevated ET could include ETA-mediated rapid vasoconstriction (with a possible subsequent ischemic event and reiterative astrocytic responses), and ETB-induced vasodilation mediated by nitric oxide and possibly TNF-{alpha}.11 71 73 This series of events appears much like the vasospasms reported in some patients with glaucoma74 as elevated ETs and nitric oxide alternate with diminished retinal perfusion and glucose-oxygen deprivation.

The demonstration that elevated levels of ET-1 in the vitreous can produce an extended period of aberrant anterograde axonal transport (Table 1 , Figs. 1 4 ) within the optic nerve has a number of implications for the pathogenesis of glaucoma. ET-sensitive sites may be accessed by diffusion through the vitreous and/or local secretion of the peptide with similar pathologic consequences. Elevated ETs may exert direct receptor-mediated effects on retinal ganglion cells, resident glial cells, and retinal vasculature simultaneously or may affect a subset of these targets. In either case, increased activation of ET pathways initiates a shower of cascading events that interact to produce the stereotypic neuropathology of glaucoma. If accurate, this model predicts an important place for the regulation of ET and its receptors in glaucoma therapy.


   Acknowledgements
 
The authors thank Ganesh Prasanna, PhD, and Raghu Krishnamoorthy, PhD, for consultation and Christina Hulet for technical support.


   Footnotes
 
Supported in part by National Eye Institute Grant EY11979 and by grants from the National Institute of Neurological Disorders and Stroke (STB), the Juvenile Diabetes Foundation (STB), and the Welch Foundation (STB).

Submitted for publication April 12, 2002; accepted June 11, 2002.

Commercial relationships policy: N.

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: Thomas Yorio, Graduate School of Biomedical Sciences, UNT Health Science Center, 3500 Camp Bowie Boulevard, Fort Worth, TX 76107-2699; yoriot{at}hsc.unt.edu.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Quigley, HA. (1999) Neuronal death in glaucoma Prog Retinal Eye Res 18,39-57[Medline][Order article via Infotrieve]
  2. Hollander, H, Makarov, F, Stefani, FH, Stone, J. (1995) Evidence of constriction of optic nerve axons at the lamina cribrosa in the normotensive eye in humans and other mammals Ophthalmic Res 27,296-309[Medline][Order article via Infotrieve]
  3. Minckler, DS, Bunt, AH, Johanson, GW. (1977) Orthograde and retrograde axoplasmic transport during acute ocular hypertension in the monkey Invest Ophthalmol Vis Sci 16,426-441[Abstract/Free Full Text]
  4. Sabri, MI, Ochs, S. (1972) Relation of ATP and creatine phosphate to fast axoplasmic transport in mammalian nerve J Neurochem 12,2821-2828
  5. Siesjo, BK. (1985) Oxygen deficiency and brain damage: localization, evolution in time, and mechanisms of damage J Toxicol Clin Toxicol 23,267-280[Medline][Order article via Infotrieve]
  6. Cioffi, GA, Sullivan, P. (1999) The effect of chronic ischemia on the primate optic nerve Eur J Ophthalmol 9,S34-S36
  7. Gallant, PE. (1992) The direct effects of graded axonal compression on axoplasm and fast axonal transport J Neuropathol Exp Neurol 51,220-230[Medline][Order article via Infotrieve]
  8. Sahenk, Z, Brady, ST, Mendell, JR. (1987) Studies on the pathogenesis of vincristine-induced neuropathy Muscle Nerve 10,80-84[Medline][Order article via Infotrieve]
  9. Topp, KS, Tanner, KD, Levine, JD. (2000) Damage to the cytoskeleton of large diameter sensory neurons and myelinated axons in vincristine-induced painful peripheral neuropathy in the rat J Comp Neurol 424,563-576[Medline][Order article via Infotrieve]
  10. Shihara, M, Hirooka, Y, Hori, N, et al (1998) Endothelin-1 increases the neuronal activity and augments the responses to glutamate in the NTS Am J Physiol 275,R658-R665[Abstract/Free Full Text]
  11. Yanagisawa, M, Kurihara, H, Kimura, S, et al (1988) A. novel potent vasoconstrictor peptide produced by vascular endothelial cells Nature 332,411-415[Medline][Order article via Infotrieve]
  12. MacCumber, MW, Jampel, HD, Snyder, SH. (1991) Ocular effects of endothelins: abundant peptides in the eye Arch Ophthalmol 109,705-709[Abstract]
  13. Yorio, T, Krishnamoorthy, R, Prasanna, G. (2002) Endothelin: is it a contributor to glaucoma pathophysiology? J Glaucoma 11,259-270[Medline][Order article via Infotrieve]
  14. Kallberg, ME, Brooks, DE, Garcia-Sanchez, GA, Komaromy, AM, Szabo, NJ. (2002) Endothelin 1 levels in the aqueous humor of dogs with glaucoma J Glaucoma 11,105-109[Medline][Order article via Infotrieve]
  15. Stitt, AW, Chakravarthy, U, Gardiner, TA, Archer, DB. (1995) Endothelin-like immunoreactivity and receptor binding in the choroid and retina Curr Eye Res 15,111-117
  16. Ripodas, A, De Juan, JA, Roldan-Pallares, M, et al (2001) Evidence for endothelin-1 expression in astrocytes Brain Res 912,137-143[Medline][Order article via Infotrieve]
  17. De Juan, JA, Moya, FJ, Ripodas, A, Bernal, R, Fernandez-Cruz, A, Fernandez-Durango, R. (2000) Changes in the density and localisation of endothelin receptors in the early stages of rat diabetic retinopathy and the effect of insulin treatment Diabetologia 43,773-785[Medline][Order article via Infotrieve]
  18. Inoue, A, Yanagisawa, M, Kimura, S, et al (1989) The human endothelin family: Three structurally and pharmacologically distinct isopeptides predicted by three separate genes Proc Natl Acad Sci USA 86,2863-2867[Abstract/Free Full Text]
  19. Sakurai, T, Yanagisawa, M, Masaki, T. (1992) Molecular characterisation of endothelin receptor Trends Pharmacol Sci 13,103-108[Medline][Order article via Infotrieve]
  20. Yorio, T, Narayan, S, Lambert, W, et al (2000) Regulation of endothelin receptor expression in ocular tissues [ARVO Abstract] Invest Ophthalmol Vis Sci 41(4),S251Abstract nr 1322
  21. MacCumber, MW, D’Anna, SA. (1994) Endothelin receptor-binding subtypes in the human retina and choroid Arch Ophthalmol 112,1231-1235[Abstract]
  22. Cioffi, GA, Sullivan, P. (1999) The effect of chronic ischemia on the primate optic nerve Eur J Ophthalmol 9(suppl 1),S34-S36
  23. Orgul, S, Cioffi, GA, Wilson, DJ, Bacon, DR, Van Buskirk, EM. (1996) An endothelin-1 induced model of optic nerve ischemia in the rabbit Invest Ophthalmol Vis Sci 37,1860-1869[Abstract/Free Full Text]
  24. Cioffi, GA, Orgul, S, Bhandari, A, Bacon, DR. (1996) An endothelin-1 induced model of chronic optic nerve ischemia in primates [ARVO Abstract] Invest Ophthalmol Vis Sci 37(3),S463Abstract nr 2102
  25. Ehrenreich, H, Costa, T, Clouse, KA, et al (1993) Thrombin is a regulator of astrocytic endothelin-1 Brain Res 600,201-207[Medline][Order article via Infotrieve]
  26. Ehrenreich, H, Anderson, RW, Ogino, Y, et al (1991) Selective autoregulation of endothelins in primary astrocyte cultures: endothelin receptor-mediated potentiation of endothelin-1 secretion New Biol 3,135-141[Medline][Order article via Infotrieve]
  27. Kohzuki, M, Onodera, H, Yasujima, M, et al (1995) Endothelin receptors in ischemic rat brain and Alzheimer brain J Cardiovasc Pharmacol 26,S329-S331
  28. Ho, MC, Lo, AC, Kurihara, H, Yu, AC, Chung, SS, Chung, SK. (2001) Endothelin-1 protects astrocytes from hypoxic/ischemic injury FASEB J 15,618-626[Abstract/Free Full Text]
  29. Yamashita, K, Niwa, M, Kataoka, Y, et al (1994) Microglia with an endothelin ETB receptor aggregate in rat hippocampus CA1 subfields following transient forebrain ischemia J Neurochem 63,1042-1051[Medline][Order article via Infotrieve]
  30. Rogers, SD, Demaster, E, Catton, M, et al (1997) Expression of endothelin-B receptors by glia in vivo is increased after CNS injury in rats, rabbits, and humans Exp Neurol 145,180-195[Medline][Order article via Infotrieve]
  31. Uesugi, M, Kasuya, Y, Hayashi, K, Goto, K. (1998) SB209670, a potent endothelin receptor antagonist, prevents or delays axonal degeneration after spinal cord injury Brain Res 786,235-239[Medline][Order article via Infotrieve]
  32. Levkovitch-Verbin, H, Quigley, HA, Kerrigan-Baumrind, LA, D’Anna, SA, Kerrigan, D, Pease, ME. (2001) Optic nerve transection in monkeys may result in secondary degeneration of retinal ganglion cells Invest Ophthalmol Vis Sci 42,975-982
  33. Tytell, M, Black, MM, Garner, JA, Lasek, RJ. (1981) Axonal transport: each major rate component reflects the movement of distinct macromolecular complexes Science 214,179-181[Abstract/Free Full Text]
  34. Brady, ST, Lasek, RJ. (1982) The slow components of axonal transport: movements compositions and organization Weiss, D eds. Axoplasmic Transport ,206-217 Spring-Verlag New York.
  35. Elluru, RG. (1994) Characterization of the Axonal Transport of Kinesin and Type 1 Hexokinase in the Rat Visual System University of Texas Southwestern Medical Center Dallas, TX. Thesis
  36. Morfini, G, Szebenyi, G, Elluru, R, Ratner, N, Brady, ST. (2002) Glycogen synthase kinase 3 phosphorylates kinesin light chains and negatively regulates kinesin-based motility EMBO J 21,281-293[Medline][Order article via Infotrieve]
  37. Ratner, N, Bloom, GS, Brady, STA. (1998) role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein J Neurosci 18,7717-7726[Abstract/Free Full Text]
  38. Tsai, MY, Morfini, G, Szebenyi, G, Brady, ST. (2000) Release of kinesin from vesicles by hsc70 and regulation of fast axonal transport Mol Biol Cell 11,2161-2173[Abstract/Free Full Text]
  39. Elluru, RG, Bloom, GS, Brady, ST. (1995) Fast axonal transport of kinesin in the rat visual system: functionality of kinesin heavy chain isoforms Mol Biol Cell 6,21-40[Abstract]
  40. Elluru, RG, Bloom, GS, Brady, ST. (1995) Axonal transport of kinesin in the rat optic nerve/tract (Abstract) J Cell Biol 111,417
  41. McQuarrie, IG, Brady, ST, Lasek, RJ. (1986) Diversity in the axonal transport of structural proteins: major differences between optic and spinal axons in the rat J Neurosci 6,1593-1605[Abstract]
  42. McQuarrie, IG, Brady, ST, Lasek, RJ. (1989) Retardation in the slow axonal transport of cytoskeletal elements during maturation and aging Neurobiol Aging 10,359-365[Medline][Order article via Infotrieve]
  43. Oblinger, MM, Brady, ST, McQuarrie, IG, Lasek, RJ. (1987) Cytotypic differences in the protein composition of the axonally transported cytoskeleton in mammalian neurons J Neurosci 7,453-462[Abstract]
  44. Brady, ST. (1985) Axonal transport methods and applications Boulton, A Baker, G eds. Neuromethods ,419-476 Humana Press Clifton, NJ.
  45. Jahn, RW, Schiebler, W, Greengard, P, DeCamilli, PA. (1985) 38,000-dalton membrane protein (p38) present in synaptic vesicles Proc Natl Acad Sci USA 82,4137-4141[Abstract/Free Full Text]
  46. Wilson, JE. (1985) Regulation of mammalian hexokinase activity Bietner, IR eds. Regulation of Carbohydrate Metabolism ,46-103 CRC Press, Inc Boca Raton, FL.
  47. Garner, JA, Lasek, RJ. (1982) Cohesive axonal transport of the slow component b complex of polypeptides J Neurosci 2,1824-1835[Medline][Order article via Infotrieve]
  48. Garner, JA, Lasek, RJ. (1981) Clathrin is axonally transported as part of slow component b complex of polypeptides J Cell Biol 88,172-178[Abstract/Free Full Text]
  49. Brady, ST, Black, MM. (1986) Axonal transport of microtubule proteins: cytotypic variations of tubulin and MAPS in neurons Ann NY Acad Sci 466,199-217[Medline][Order article via Infotrieve]
  50. Hirokawa, N. (1998) Kinesin and dynein superfamily proteins and the mechanism of organelle transport Science 279,519-526[Abstract/Free Full Text]
  51. Brady, ST, Witt, AS, Kirkpatrick, LL, et al (1999) Formation of compact myelin is required for maturation of the axonal cytoskeleton J Neurosci 19,7276-7288
  52. Brady, ST. (1985) A novel brain ATPase with properties expected for the fast axonal transport motor Nature 317,73-75[Medline][Order article via Infotrieve]
  53. Pachter, JA, Mayer-Ezell, R, Cleven, RM, Fawzi, AB. (1993) Endothelin (ETA) receptor number and calcium signalling are up-regulated by protein kinase C-beta 1 overexpression Biochem J 294,153-158
  54. Pang, IH, Yorio, T. (1997) Ocular actions of endothelins Proc Soc Exp Biol Med 215,21-34[Medline][Order article via Infotrieve]
  55. Radius, RL. (1980) Optic nerve fast axonal transport abnormalities in primates: occurrence after short posterior ciliary artery occlusion Arch Ophthalmol 98,2018-2022[Abstract]
  56. Fang, X, Yu, SX, Lu, Y, Bast, RC, Jr, Woodgett, JR, Mills, GB. (2000) Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A Proc Natl Acad Sci USA 97,11960-11965[Abstract/Free Full Text]
  57. van Biesen, T, Hawes, BE, Luttrell, DK, et al (1995) Receptor-tyrosine-kinase- and G beta gamma-mediated MAP kinase activation by a common signalling pathway Nature 376,781-784[Medline][Order article via Infotrieve]
  58. van Biesen, T, Hawes, BE, Raymond, JR, Luttrell, LM, Koch, WJ, Lefkowitz, RJG. (1996) (o)-protein alpha-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism J Biol Chem 271,1266-1269[Abstract/Free Full Text]
  59. Hollander, H, Makarov, F, Dreher, Z, van Driel, D, Chan-Ling, TL, Stone, J. (1991) Structure of the macroglia of the retina: sharing and division of labour between astrocytes and Müller cells J Comp Neurol 313,587-603[Medline][Order article via Infotrieve]
  60. Stone, J, Makarov, F, Hollander, H. (1995) The glial ensheathment of the soma and axon hillock of retinal ganglion cells Vis Neurosci 12,273-279[Medline][Order article via Infotrieve]
  61. Sanchez, S, Sayas, CL, Lim, F, Diaz-Nido, J, Avila, J, Wandosell, F. (2001) The inhibition of phosphatidylinositol-3-kinase induces neurite retraction and activates GSK3 J Neurochem 78,468-481[Medline][Order article via Infotrieve]
  62. Kokaia, Z, Andsberg, G, Yan, Q, Lindvall, O. (1998) Rapid alterations of BDNF protein levels in the rat brain after focal ischemia: evidence for increased synthesis and anterograde axonal transport Exp Neurol 154,289-301[Medline][Order article via Infotrieve]
  63. Tonra, JR, Curtis, R, Wong, V, et al (1998) Axotomy upregulates the anterograde transport and expression of brain-derived neurotrophic factor by sensory neurons J Neurosci 18,4374-4383[Abstract/Free Full Text]
  64. Pena, JD, Taylor, AW, Ricard, CS, Vidal, I, Hernandez, MR. (1999) Transforming growth factor beta isoforms in human optic nerve heads Br J Ophthalmol 83,209-218[Abstract/Free Full Text]
  65. Koyama, Y, Baba, A. (1994) Endothelins are extracellular signals modulating cytoskeletal actin organization in rat cultured astrocytes Neuroscience 61,1007-1016[Medline][Order article via Infotrieve]
  66. Koyama, Y, Baba, A. (1999) Endothelin-induced protein tyrosine phosphorylation of cultured astrocytes: its relationship to cytoskeletal actin organization Glia 24,324-332
  67. Koyama, Y, Yoshioka, Y, Hashimoto, H, Matsuda, T, Baba, A. (2000) Endothelins increase tyrosine phosphorylation of astrocytic focal adhesion kinase and paxillin accompanied by their association with cytoskeletal components Neuroscience 101,219-227[Medline][Order article via Infotrieve]
  68. Smith-Swintosky, VL, Zimmer, S, Fenton, JW, II, Mattson, MP. (1995) Protease nexin-1 and thrombin modulate neuronal Ca2+ homeostasis and sensitivity to glucose deprivation-induced injury J Neurosci 15,5840-5850[Abstract]
  69. Hama, H, Kasuya, Y, Sakurai, T, et al (1997) Role of endothelin-1 in astrocyte responses after acute brain damage J Neurosci Res 47,590-602[Medline][Order article via Infotrieve]
  70. Ostrow, LW, Langan, TJ, Sachs, F. (2000) Stretch-induced endothelin-1 production by astrocytes J Cardiovasc Pharmacol 36,S272-S274
  71. Oda, H, Murayama, T, Sasaki, Y, Okada, T, Nomura, Y. (1997) Endothelin enhances lipopolysaccharide-induced expression of inducible nitric oxide synthase in rat glial cells Eur J Pharmacol 339,253-260[Medline][Order article via Infotrieve]
  72. Sasaki, Y, Takimoto, M, Oda, K, et al (1997) Endothelin evokes efflux of glutamate in cultures of rat astrocytes J Neurochem 68,2194-2200[Medline][Order article via Infotrieve]
  73. Yamashita, K, Sakurai-Yamashita, Y, Niwa, M, Taniyama, K. (1998) The glial endothelin-nitric oxide system in ischemia-related neuronal cell death Nippon Yakurigaku Zasshi 111,29-36[Medline][Order article via Infotrieve]
  74. Flammer, J, Orgul, S. (1998) Optic nerve blood-flow abnormalities in glaucoma Prog Retinal Eye Res 17,267-289[Medline][Order article via Infotrieve]



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