Miotic Tolerance to Sarin Vapor Exposure: Role of the Sympathetic and Parasympathetic Nervous Systems
http://www.100md.com
《毒物学科学杂志》
National Academy of Sciences–National Research Council, Washington, D.C.
U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland
Geo-Centers, Inc., Gunpowder, Maryland
ABSTRACT
O-isopropyl methylphosphonofluoridate, also known as sarin or GB, is a highly toxic organophosphorous compound that exerts its effect by inhibiting the enzyme acetylcholinesterase. While the effects of a single exposure to GB vapor are well characterized, the effects of multiple exposures to GB vapor are less clear. Previous studies in the rat and guinea pig have demonstrated that multiple exposures result in tolerance to the miotic effect of nerve agents. The aim of the present study was to examine potential mechanisms responsible for tolerance to the miotic effect of GB vapor that has been observed in the rat after multiple exposures. Multiple whole-body inhalation exposures to GB vapor were conducted in a dynamic airflow chamber. Exposures lasted 60 min and each of the three exposures occurred at 24-h intervals. The results of the present study demonstrate that the -adrenergic antagonist phentolamine and the -adrenergic receptor antagonist propranolol did not affect the development of tolerance to the miotic effect of GB vapor, suggesting that enhanced sympathetic tone to the eye is not responsible for the observed tolerance. Administration of atropine before the first exposure prevented the tolerance to the miotic effect of GB vapor after the third exposure, suggesting that the tolerance is the result of muscarinic receptor desensitization secondary to receptor stimulation. The present study extends the findings of previous studies to strengthen the hypothesis that the miotic tolerance observed in the rat upon repeated exposure to nerve agents is due to desensitization of muscarinic acetylcholine receptors located on the pupillary sphincter.
Key Words: sarin; miosis; tolerance; parasympathetic nervous system; muscarinic receptors.
INTRODUCTION
O-isopropyl methylphosphonofluoridate, also known as sarin or GB, is a highly toxic organophosphorous (OP) compound that exerts its effect by inhibiting the enzyme acetylcholinesterase (AChE). Inhibition of AChE results in the accumulation of the neurotransmitter acetylcholine and excessive stimulation of cholinergic receptors. At higher doses, effects of exposure include salivation, muscle twitches, tremors, convulsions, seizures, and death, usually attributable to respiratory failure (Taylor, 1996). However, at lower doses, few if any of these severe toxic signs are observed. Miosis is one of the threshold clinical effects that can be observed following less-than-lethal of GB vapor exposures, and it occurs at concentrations of GB many times lower than those required to cause death (Mioduszewski et al., 2002a, 2002b).
Normally, both the sympathetic and parasympathetic branches of the autonomic nervous system play a role in the control of pupillary size. Stimulation of the sympathetic branch releases norepinephrine onto -adrenergic receptors located on the radial muscles of the iris, resulting in dilation of the pupil (Yu and Koss, 2002, 2003). Muscarinic acetylcholine receptors are present on the pupillary sphincter of the rat, and administration of muscarinic antagonists results in pupillary dilation, demonstrating the role for muscarinic receptors and the parasympathetic nervous system in the control of pupil diameter (Furuta et al., 1998; Smith et al., 1996). Nerve agent–induced miosis is due to excessive stimulation of the parasympathetic nervous system and stimulation of muscarinic acetylcholine receptors located on the pupillary sphincter muscle.
It has been demonstrated that repeated low-dose exposure to an OP compound can result in desensitization of muscarinic acetylcholine receptors in the retina (Tandon et al., 1994) and brain (Tang et al., 1999) of the rat. Additionally, repeated exposure to a nerve agent results in a reduced miotic response after multiple exposures (Dabisch et al., 2005; Soli et al., 1980). Similarly, repeated topical ocular exposure of monkeys to the cholinesterase inhibitors echothiophate and isoflurophate resulted in a gradual decrease over time in the miotic response to both compounds, and a decreased miotic response to the cholinomimetic carbachol (Bito and Banks, 1969).
While tolerance to the miotic effect of nerve agents has been observed previously, the mechanism responsible for the development of the tolerance remains unclear. Therefore, the aim of the present study was to examine potential mechanisms responsible for the tolerance to the miotic effect of GB vapor observed after repeated exposure. Our laboratory has shown previously that this miotic tolerance to GB vapor is not due to a decreased inhibitory effect of GB in the eye, or to a decrease in the amount of GB reaching the eye (Dabisch et al., 2005). It has also previously been shown that the tolerance is not likely due to internalization of muscarinic acetylcholine receptors in the pupillary sphincter muscle (Soli et al., 1980). The present study investigated two additional hypotheses aimed at elucidating the mechanisms mediating the tolerance to the miotic effect of GB vapor. The first experimental hypothesis considered the possibility that enhanced sympathetic outflow to the eye due to inhibition of ganglionic AChE opposes the miotic effect of GB vapor on the pupil, resulting in a lesser degree of miosis. The second hypothesis proposed that the tolerance is due to desensitization of muscarinic acetylcholine receptors located on the pupillary sphincter muscle.
MATERIALS AND METHODS
Animal use.
Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 200–250 g were used in this study. All experiments and procedures were approved by the U.S. Army Edgewood Chemical Biological Center Institutional Animal Care and Use Committee and were conducted in accordance with the requirements of Army Regulation 70–18 (1984) and the Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals.
GB vapor generation.
GB vapor generation was accomplished as described previously (Muse et al., 2000). Briefly, neat liquid GB was placed into a gas-tight syringe and mounted onto a variable-rate syringe drive (Model 22, Harvard Apparatus Inc., South Natick, MA). Once activated, the syringe drive delivered a constant flow of GB through a flexible plastic line into a spray-atomization system (Spray Atomization Nozzle J SS, Spraying Systems Co., Wheaton, IL). Compressed air (30–40 psi) flowing through the atomizer atomized the GB liquid into fine droplets, which, because of the volatility of GB, quickly evaporated. The GB vapor was drawn into a 750-l dynamic airflow inhalation chamber constructed of stainless-steel with Lexan windows. The interior of the exposure chamber was maintained under negative pressure (0.25'' H2O), which was monitored with a calibrated magnehelix (Dwyer, Michigan City, IN). A thermo-anemometer (Model 8565, Alnor, Skokie, IL) was used to monitor chamber airflow at the chamber outlet. At various times throughout the exposure, GB vapor concentration in the chamber was determined from a chamber air sample collected onto a solid sorbent tube. O-isopropyl methylphosphonofluoridate concentrations were determined using a gas chromatograph equipped with a flame ionization detector (Agilent Technologies Inc., Wilmington, DE). The target GB vapor concentration for each exposure in the study was 4.0 mg/m3.
Infrared photography.
The right eye of each rat was digitally photographed to determine the degree of miosis present after exposure to GB vapor. This technique has been described previously (Dabisch et al., 2005; Miller et al., 2003). Each animal was temporarily hand restrained (<30 s) to minimize movement of the head, and the pupil was illuminated with an infrared spotlight (model SL1236, Advanced Illumination, Rochester, VT). Images were digitally recorded with an infrared-capable video camera (model XC-ST50, Sony) equipped with a 75-mm/F2.7 lens (model LMV7527) and an image-acquisition PC card (model PCI-1411, National Instruments Corp., Austin, TX). All digital images were taken from a distance of 41 in. under controlled low-light conditions (<10 foot-candles) and were stored as .JPG files for further analysis. Pupil and iris radii were determined with custom image analysis software (Miller et al., 2003) written using the LabView software suite (Version 6, National Instruments Corp., Austin, TX). To correct for any variability in the distance from the camera to the animal's eye, the results were expressed as the ratio of the pupil radius to the iris radius.
Exposure protocol.
Rats were split into two groups and subjected to multiple whole-body inhalation exposures to either GB vapor or room air. Animals were exposed to either GB vapor or room air for 1 h on each of three consecutive days. Exposures occurred at 24-h intervals. As stated previously, the target GB vapor concentration for each exposure in the study was 4.0 mg/m3. The concentration of GB vapor in the air that causes miosis in 50% of exposed male rats (EC50) for a 60-min exposure is 0.030 mg/m3 (Mioduszewski et al., 2002a), whereas the concentration that results in death in 50% of exposed male rats (LC50) for a 60-min exposure is 7.55 mg/m3 (Mioduszewski et al., 2002b). In the present study, the average GB vapor concentration generated was 4.13 ± 0.07 mg/m3. This concentration is above the EC50 for miosis but below the LC50 for a 60-min exposure. Other than miosis, the rats did not show any overt clinical signs of nerve agent exposure, and no deaths occurred, confirming that the concentration generated was between the EC50 for miosis and the LC50 for a 60-min exposure. The GB-treated and air-treated groups were exposed simultaneously in separate chambers, and the exposures occurred at the same time each day. Baseline pupil images of all animals were recorded several days before exposure to GB vapor; 1 h before each exposure; 15 min, 3 h, and 5 h after each exposure; and at 24-h intervals for 1 week following the third exposure.
Effect of adrenergic receptor blockade on the development of miotic tolerance.
In the present study, tolerance to the miotic effect of GB vapor was present after 2 exposures. This tolerance was expected and has been reported previously (Dabisch et al., 2005; Soli et al., 1980). To determine whether the observed miotic tolerance to GB vapor is due to enhanced sympathetic nervous system activity, rats were treated with different antagonists, or combinations of antagonists, of the sympathetic nervous system. Rats were then exposed to either GB vapor or room air according the to exposure schedule described previously. If the miotic tolerance were due to enhanced sympathetic tone to the muscles controlling pupil diameter, it would be expected that antagonists of the sympathetic nervous system would prevent the tolerance. Two groups of four rats each (1 GB-exposed group and 1 air-exposed group) were injected with the -adrenergic receptor antagonist phentolamine (5 mg/kg im) 15 min before the third exposure. The dose of phentolamine used has been previously shown to block -adrenergic receptor-mediated activity in the heart (Barres et al., 2001). Another two groups of four rats each (1 GB-exposed group and 1 air-exposed group) were injected with the -adrenergic receptor antagonist propranolol (4 mg/kg im) 15 min before the third exposure. This dose of propranolol has previously been shown to block -adrenergic receptor-mediated activity in the heart (Towa et al., 2004). A final two groups of four rats each (1 GB-exposed group and 1 air-exposed group) were injected with a combination of propranolol (4 mg/kg im) and phentolamine (5 mg/kg im) 15 min before the third exposure. Pupil diameter was recorded before and after administration of drugs and at various time points throughout the exposure sequence. Whole blood AChE and butyrylcholinesterase (BChE) activities were determined in animals treated with the combination of phentolamine and propranolol according to the methodology described in detail previously (Dabisch et al., 2005).
Effect of muscarinic receptor blockade on the development of miotic tolerance.
As stated previously, tolerance to the miotic effect of GB vapor was present after two exposures. To determine if overstimulation and subsequent desensitization of muscarinic acetylcholine receptors was responsible for the observed miotic tolerance, two groups of four rats each were treated with the muscarinic receptor antagonist atropine (6 mg/kg im) before the first exposure to "protect" muscarinic receptors from overstimulation. One of the groups was exposed to GB vapor, and the other was exposed to room air only. Rats were given an additional dose of atropine (2 mg/kg im) 3 h after the first exposure. The animals were then subjected to the final two exposures in the sequence. Pupil diameter was recorded before and after the administration of atropine, and at various time points throughout the exposure sequence. Whole-blood AChE and BChE activities were determined in atropine-treated animals according to the methodology described in detail previously (Dabisch et al., 2005).
Data analysis.
Statistical analysis was done by analysis of variance (ANOVA) with a Scheffe post-test. A p-value < 0.05 was used as the criterion for statistical significance. All numerical values are reported as mean ± SEM.
RESULTS
Effect of Multiple GB Vapor Exposures on Pupil Size and Function
After the first exposure to GB vapor, all rats had pinpoint pupils (Fig. 1B), whereas animals exposed to air only did not show a change in pupil size (data not shown). The ratio of pupil radius to iris radius in GB-exposed animals was reduced by 99 ± 3%. After the second GB vapor exposure, all rats again had pinpoint pupils (Fig. 1C), whereas those exposed to air only did not show a change in pupil size (data not shown). The ratio of pupil radius to iris radius in GB-exposed animals was reduced by 97 ± 3%. Additionally, after the second GB vapor exposure, the rats showed a more rapid recovery from miosis than after the first exposure (Fig. 2). With the third GB vapor exposure, rats were tolerant to the miotic effect of GB vapor, as the ratio of pupil radius to iris radius was 55 ± 9% of baseline.
Effect of Adrenergic Receptor Blockade on the Development of Miotic Tolerance
Tolerance to the miotic effect of GB vapor was present after two exposures. To determine whether the observed miotic tolerance to GB vapor is due to enhanced sympathetic nervous system activity, rats were treated with phentolamine, propranolol, or the combination of phentolamine and propranolol 20 min before the third exposure. If the observed tolerance was due to enhanced sympathetic tone to the muscles controlling pupil diameter, it would be expected that administration of antagonists of sympathetic receptors before the third exposure would prevent the tolerance.
Immediately before the third exposure, both air- and GB-exposed animals had similar pupil sizes (Fig. 3). In a first group of animals, the -adrenergic antagonist phentolamine (5 mg/kg im) was administered to rats 20 min before the third exposure. Phentolamine administration produced a similar degree of pupil constriction in both air- and GB-exposed animals (Fig. 3), and it did not prevent the tolerance to the miotic effect of GB vapor that was observed in untreated animals (Fig. 4).
In another group of animals, the -adrenergic antagonist propranolol (4 mg/kg im) was administered to rats 20 min before the third exposure. Administration of propranolol did not significantly affect pupil size, and it also failed to prevent the tolerance to the miotic effect of GB vapor observed in untreated animals (data not shown).
In a final group of animals, the combination of phentolamine (5 mg/kg) and propranolol (4 mg/kg) was administered intramuscularly 20 min before the third exposure. Administration of this combination produced a change in pupil size similar to that seen with phentolamine alone, and, again, it failed to prevent the tolerance to the miotic effect of GB vapor observed in untreated animals (data not shown). Administration of the combination of phentolamine and propranolol also did not affect the degree of inhibition of whole-blood AChE by GB (data not shown).
Effect of Muscarinic Receptor Blockade on the Development of Miotic Tolerance
Tolerance to the miotic effect of GB vapor was present after two exposures. To determine whether overstimulation and subsequent desensitization of muscarinic acetylcholine receptors was responsible for the observed miotic tolerance, the muscarinic receptor antagonist atropine was administered before the first exposure to "protect" muscarinic receptors from overstimulation. Administration of atropine (6 mg/kg im) produced a slight increase in pupil size (data not shown), and it prevented the miosis associated with a single exposure to GB vapor (Fig. 5). Additionally, rats given atropine before the first exposure did not develop tolerance to the miotic effect of GB vapor after the third exposure (Figs. 6 and 7). Finally, administration of atropine did not affect inhibition of whole-blood AChE by GB (data not shown).
DISCUSSION
Development of tolerance to the miotic effects of repeated nerve agent exposure has been reported by Soli et al. (1980) for the nerve agent soman and previously by our laboratory (Dabisch et al., 2005) for rats repeatedly exposed to GB vapor. In the latter study, the tolerance to the miotic effect of GB vapor was found to persist for approximately 96 h. It is well established that both the duration of the exposure and the concentration of agent play a role in determining the degree of miosis observed (Mioduszewski et al., 2002a; Whalley et al., 2004). Thus, it is likely that varying both the duration of the exposure and the concentration of GB used will alter the time course of the development of miotic tolerance. Additionally, it is likely that varying the frequency of exposure will alter the time course of the development of miotic tolerance, because this would alter the time available for recovery between exposures. However, the present study investigated only a single concentration, duration, and frequency of exposure. Thus, it is not possible to determine the exact effect of each parameter on the development of miotic tolerance.
Although the development of tolerance to the miotic effect nerve agents has been demonstrated previously, the mechanism responsible for this phenomenon remains unclear. Our laboratory has shown previously that the miotic tolerance to GB vapor observed following repeated whole-body vapor exposures is not due to a decreased inhibitory effect of GB in the eye, or to a decrease in the amount of GB reaching the eye (Dabisch et al., 2005). It has also been shown previously that the tolerance is not likely due to internalization of muscarinic acetylcholine receptors in the pupillary sphincter muscle (Soli et al., 1980). The present study investigated two additional hypotheses that could explain the mechanisms mediating tolerance to the miotic effect of GB vapor.
It is possible that the miotic tolerance observed after multiple exposures to a nerve agent might be due to enhanced sympathetic tone in the eye. Over the course of the exposure sequence, it is possible that the AChE in the superior cervical ganglion is gradually inhibited, resulting in a gradual increase in sympathetic tone to the radial muscles of the iris. This enhanced sympathetic tone would oppose the increased parasympathetic tone that is already present as a result of inhibition of AChE at the pupillary sphincter muscle, resulting in the diminished miotic response to GB that is observed after multiple exposures. To test this hypothesis, the present study used the -adrenergic receptor antagonist phentolamine to block sympathetically mediated dilation of the eye. If enhanced sympathetic tone was responsible for the observed tolerance, then phentolamine would prevent or diminish the tolerance. However, animals treated with phentolamine before the third exposure still display tolerance to the miotic effect of GB vapor after exposure. Additionally, air- and GB-exposed animals showed a similar degree of pupillary constriction immediately following phentolamine administration, suggesting that the basal sympathetic tone in the eye was similar in both groups, and also demonstrating that the drug was reaching the eye. Thus, enhanced -adrenoreceptor-mediated sympathetic tone tone in the eye does not appear to occur after multiple GB vapor exposures, and thus it cannot explain the miotic tolerance that is observed.
In addition to stimulation of -adrenergic receptors on the radial muscle of the iris, sympathetic dilation of the pupil may also be mediated by stimulation of -adrenergic receptors located on the pupillary sphincter. -adrenergic receptor-mediated relaxation has been reported in bovine pupillary sphincters (Barilan et al., 2003; Geyer et al., 1998), and -adrenergic receptors are present on the pupillary sphincter muscle of the rat eye (Lahav et al., 1978). In the present study, the -adrenergic receptor antagonist propranolol was used to assess the role of enhanced sympathetic tone and stimulation of -adrenergic receptors in the tolerance to the miotic effect of GB vapor. If stimulation of -adrenergic receptors were playing a role in the observed tolerance, then administration of propranolol would, at least partially, prevent the tolerance. However, animals pretreated with propranolol before the third exposure still displayed tolerance to the miotic effect of GB vapor after the exposure, suggesting that stimulation of -adrenergic receptors is not a factor in the observed miotic tolerance to GB vapor. Additionally, administration of propranolol did not result in a change in pupil size. Because of the lack of effect, it is possible that propranolol did not reach the eye. However, several studies have demonstrated penetration of lipophilic -blockers such as propranolol into the central nervous system (Cruickshank and Neil-Dwyer, 1985; Schiff and Saxey, 1984). Thus, it is likely that propranolol is reaching the eye, but that -adrenergic receptors do not play a significant role in the control of rat pupil size at rest. These results suggest that sympathetic control of resting pupil size in the rat is mediated by -adrenergic and not -adrenergic receptors.
It has been demonstrated previously that muscarinic acetylcholine receptors desensitize as a result of either direct stimulation with muscarinic agonists or indirect stimulation after administration of organophosphorus compounds. This desensitization has been demonstrated in several tissues and species, including rabbit intestinal smooth muscle (Murthy and Makhlouf, 2000), rat atrial cells (Shiu et al., 2002), rat retina (Tandon et al., 1994), rat brain (Tang et al., 1999), and human neuroblastoma cells (Willets et al., 2002). Nerve agent vapor-induced miosis is likely due to a local effect on the eye, namely inhibition of AChE on the pupillary sphincter, resulting in excess cholinergic stimulation (Sim, 1956; Soli et al., 1980). Thus, it is possible that the miotic tolerance to GB vapor that is present after multiple exposures is due to desensitization of muscarinic receptors located on the pupillary sphincter muscle secondary to excessive cholinergic stimulation following inhibition of AChE. If this hypothesis is correct, it would be expected that blockade of muscarinic receptors with a muscarinic receptor antagonist such as atropine would prevent excessive stimulation and desensitization of the receptors, as well as the subsequent tolerance to the miotic effect of GB vapor.
In the present study, animals pretreated with atropine before the first exposure fail to develop tolerance to the miotic effect of GB vapor after the third exposure, suggesting that the mechanism responsible for the miotic tolerance involves stimulation and subsequent desensitization of muscarinic acetylcholine receptors. This is the first study to report this effect of atropine on the development of tolerance to nerve agents. Additionally, because nerve agent vapor–induced miosis is a local effect, it is likely that atropine is preventing the development of miotic tolerance by blocking muscarinic acetylcholine receptors located on the pupillary sphincter. It has been demonstrated previously that multiple exposures to GB vapor result in loss of the pupillary light reflex (Dabisch et al., 2005), a response mediated by muscarinic receptors located on the pupillary sphincter and often used as an indicator of parasympathetic pupillary function (Dutsch et al., 2004). The data from the present study support the results of the previous study, because the desensitization of pupillary muscarinic receptors explains the loss of the pupillary light reflex observed previously.
Desensitization of muscarinic acetylcholine receptors can involve any of several mechanisms. Activation of a protein kinase by the beta-gamma subunits of the muscarinic receptor–associated G protein can lead to receptor phosphorylation and desensitization (Haga and Haga,1992; Shiu et al., 2002). Another possibility is the binding of muscarinic receptor–associated G protein subunits to membrane caveolae, preventing the re-association of the subunits with the receptor and resulting in desensitization (Murthy and Makhlouf, 2000). Finally, stimulation of muscarinic receptors can lead to receptor endocytosis and desensitization (Krudewig et al., 2000). It is not possible to determine which mechanism is mediating the tolerance observed in the present study without further experimentation. However, it has been previously reported that administration of the nerve agent soman produced miotic tolerance without producing a decrease in the number of muscarinic receptors present on the pupillary sphincter (Soli et al., 1980). Thus, it is likely that a mechanism other than receptor endocytosis may be responsible for the tolerance to the nerve agent–induced miosis that is seen after multiple exposures.
The present study demonstrated that the -adrenergic antagonist phentolamine and the -adrenergic receptor antagonist propranolol did not affect the development of tolerance to the miotic effect of GB vapor in the rat, suggesting that enhanced sympathetic tone in the eye is not responsible for the observed tolerance. However, administration of atropine before the first exposure prevented the tolerance to the miotic effect of GB vapor after the third exposure, suggesting that the tolerance is the result of muscarinic receptor desensitization secondary to receptor stimulation. Additionally, the present study demonstrated that sympathetic control of resting pupil size in the rat appears to be mediated by -adrenergic and not -adrenergic receptors. The present study thus confirms and extends the findings of previous studies to strengthen the hypothesis that the miotic tolerance observed upon repeated exposure to nerve agents is due to desensitization of muscarinic acetylcholine receptors located on the pupillary sphincter.
ACKNOWLEDGMENTS
The authors are grateful to the following individuals: Jacqueline Scotto and Jill Jarvis for veterinary care; William Muse, Emily Davis, Kathy Matson, Charles Crouse, David Burnett, Bernardita Gaviola, David McCaskey, and Candice Krauthauser for technical assistance; and Dr. Stanley Hulet for editorial assistance.
REFERENCES
Army Regulation 70–18. (1984). "The Use of Animals in Department of Defense Programs."
Barilan A., Nachman-Rubinstein, R., Oron, Y., and Geyer, O. (2003). Muscarinic blockers potentiate beta-adrenergic relaxation of bovine iris sphincter. Graefes Arch. Clin. Exp. Ophthalmol. 241, 226–231.
Barres, C., Pereira de Souza Neta, E., and Julien, C. (2001). Effect of -adrenoreceptor blockade on the 0.4 Hz sympathetic rhythm in conscious rats. Clin. Exp. Pharmacol. Physiol. 28, 983–985.
Bito, L. Z., and Banks, N. (1969). Effects of chronic cholinesterase inhibitor treatment: The pharmacological and physiological behavior of the anti-ChE-treated monkey (Macaca mulatta) iris. Arch. Ophthalmol. 32, 681–686.
Cruickshank, J. M., and Neil-Dwyer, G. (1985). Beta-blocker brain concentrations in man. Eur. J. Clin. Pharmacol. 28S, 21–23.
Dabisch, P. A, Burnett, D. C., Miller, D. B., Jakubowski, E. M., Muse, W. T., Forster, J. S., Scotto, J. A., Jarvis, J. R., Davis, E. A., Hulet, S. W., et al. (2005). Tolerance to the miotic effect of sarin vapor in rats following multiple low-level exposures. J. Ocular Pharmacol. Ther. In press.
Dutsch, M., Marthol, H., Michelson, G., Neundorfer, B., and Hilz, M. J. (2004). Pupillography refines the diagnosis of diabetic autonomic neuropathy. J. Neurol. Sci. 222, 75–81.
Furuta, M., Ohya, S., Imaizumi, Y., and Watanabe, M. (1998). Molecular cloning of m3 muscarinic acetylcholine receptor in rat iris. J. Smooth Muscle Res. 34, 111–122.
Geyer, O., Bar-Ilan, A., Nachman, R., Lazar, M., and Oron, Y. (1998). 3-adrenergic relaxation of bovine iris sphincter. FEBS Lett. 429, 356–358.
Haga, H., and Haga, T. (1992). Activation by G protein - subunits of agonist- and light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin. J. Biol. Chem. 267, 2222–2227.
Institute of Laboratory Animal Resources, Commission of Life Sciences, National Research Council. (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C.
Krudewig, R., Langer, B., Vogler, O., Markschies, N., Erl, M., Jakobs, K. H., and van Koppen, C. J. (2000). Distinct internalization of M2 muscarinic acetylcholine receptors confers selective and long-lasting desensitization of signaling to phospholipase C. J. Neurochem. 74, 1721–1730.
Lahav, M., Melamed, E., Dafna, Z., and Atlas, D. (1978). Localization of beta receptors in the anterior segment of the rat eye by a fluorescent analogue of propranolol. Invest, Ophthalmol. Vis. Sci. 17, 645–651.
Miller, D. B., Benton, B. J., Hulet, S. J., Mioduszewski, R. J., Whalley, C. E., Carpin, J. C., and Thomson, S. A. (2003). An analysis method for quantifying elliptical and partially obstructed pupil areas in response to chemical agent vapor exposure. Proceedings of the 29th Annual Northeast Bioengineering Conference, Newark, NJ (Abstract).
Mioduszewski, R. J., Manthei, J. H., Way, R. A., Burnett, D. C., Gaviola, B. P., Muse, W. T., Thomson, S. A., Sommerville, D. R., Crosier, R. B., Scotto, J. A., et al. (2002a). ECBC Low Level Operational Toxicology Program: Phase I—Inhalation Toxicity of Sarin Vapor in Rats as a Function of Exposure Concentration and Duration. Technical Report ECBC-TR-235. Edgewood Chemical Biological Center, U.S. Army Soldier and Biological Chemical Command, Aberdeen Proving Ground, MD.
Mioduszewski, R., Manthei, J., Way, R., Burnett, D., Gaviola, B., Muse, W., Thomson, S., Sommerville, D., and Crosier, R. (2002b). Interaction of exposure concentration and duration in determining acute toxic effects of sarin vapor in rats. Toxicol. Sci. 66, 176–184.
Murthy, K. S., and Makhlouf, G. M. (2000). Heterologous desensitization mediated by G protein–specific binding to caveolin. J. Biol. Chem. 275, 30211–30219.
Muse, W., Anthony, S., Buettner, L., Crouse, C., Crouse, L., Mioduszewski, R., and Thomson, S. (2000). Generation, sampling, and analysis of sarin (gb) vapor for inhalation studies. Proceedings of the International Chemical Weapons Demilitarization Conference (CWD 2000), The Hague, Netherlands. (Abstract).
Schiff, A. A., and Saxey, A. (1984). Autoradiography of nadolol and propranolol in the rat. Xenobiotica 14, 687–691.
Shiu, Z., Khan, I. A., Tsuga, H., Dobrzynski, H., Haga, T., Henderson, Z., and Boyett, M. R. (2002). Role of receptor kinase in long-term desensitization of the cardiac muscarinic receptor-K+ channel system. A., J. Physiol. 283, H819–H828.
Sim, V. M. Effect on pupil size of exposure to GB vapour. (1956). Directorate of Chemical Defence Research and Development—Proton Technical Paper #531.
Smith, R. D., Grzelak, M. E., Belanger, B., and Morgan, C. A. (1996). The effects of tropicamide on mydriasis in young rats exhibiting a natural deficit in passive-avoidance responding. Life Sci. 59, 753–760.
Soli, N. E., Lund Karlsen, R., Opsahl, M., and Fonnum, F. (1980). Correlations between acetylcholinesterase activity in guinea-pig iris and papillary function: A biochemical and pupillographic study. J. Neurochem. 35, 723–728.
Tandon, P., Padilla, S., Barone, S., Pope, C. N., and Tilson, H. A. (1994). Fenthion produces a persistent decrease in muscarinic receptor function in the adult rat retina. Toxicol. Appl. Pharmacol. 125, 271–280.
Tang, J., Carr, R. L., and Chambers, J. E. (1999). Changes in rat brain cholinesterase activity and muscarinic receptor density during and after repeated oral exposure to chlorpyrifos in early postnatal development. Toxicol. Sci. 51, 265–272.
Taylor, P. (1996). Anticholinesterase agents. In The Pharmacological Basis of Therapeutics (Limbird, L. E., and Hardman, J. G., Eds.), 9th edition, pp.161–176. McGraw-Hill Medical Publishing Division, New York.
Towa, S., Kuwahara, M., and Tsubone, H. (2004). Characteristics of autonomic nervous function in Zucker-Fatty rats: Investigation by power spectral analysis of heart rate variability. Exp. Anim. 53, 137–144.
Whalley, C. E., Benton, B. J., Manthei, J. H., Way, R. A., Jakubowski, E. M., Burnett, D. C., Gaviola, B. I., Crosier, R. B., Sommerville, D. R., Muse, W. T. et al. (2004). Low-level cyclosarin (GF) vapor exposure in rats: Effect of exposure concentration and duration on pupil size. Technical Report ECBC-TR-407. Edgewood Chemical Biological Center, U.S. Army Soldier and Biological Chemical Command, Aberdeen Proving Ground, MD.
Willets, J. M., Challiss, R. A. J., and Nahorski, S. R. (2002). Endogenous G protein–coupled receptor kinase 6 regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells. J. Biol. Chem. 277, 15523–15529.
Yu, Y., and Koss, M. C. (2002). 1A-Adrenoceptors mediate sympathetically evoked pupillary dilation in rats. J. Pharmacol. Exp. Ther. 300, 521–525.
Yu, Y., and Koss, M. C. (2003). Functional characterization of alpha-adrenoceptors mediating pupillary dilation in rats. Eur. J. Pharmacol. 471, 135–140.(Paul A. Dabisch, Dennis B)
U.S. Army Edgewood Chemical Biological Center, Aberdeen Proving Ground, Maryland
Geo-Centers, Inc., Gunpowder, Maryland
ABSTRACT
O-isopropyl methylphosphonofluoridate, also known as sarin or GB, is a highly toxic organophosphorous compound that exerts its effect by inhibiting the enzyme acetylcholinesterase. While the effects of a single exposure to GB vapor are well characterized, the effects of multiple exposures to GB vapor are less clear. Previous studies in the rat and guinea pig have demonstrated that multiple exposures result in tolerance to the miotic effect of nerve agents. The aim of the present study was to examine potential mechanisms responsible for tolerance to the miotic effect of GB vapor that has been observed in the rat after multiple exposures. Multiple whole-body inhalation exposures to GB vapor were conducted in a dynamic airflow chamber. Exposures lasted 60 min and each of the three exposures occurred at 24-h intervals. The results of the present study demonstrate that the -adrenergic antagonist phentolamine and the -adrenergic receptor antagonist propranolol did not affect the development of tolerance to the miotic effect of GB vapor, suggesting that enhanced sympathetic tone to the eye is not responsible for the observed tolerance. Administration of atropine before the first exposure prevented the tolerance to the miotic effect of GB vapor after the third exposure, suggesting that the tolerance is the result of muscarinic receptor desensitization secondary to receptor stimulation. The present study extends the findings of previous studies to strengthen the hypothesis that the miotic tolerance observed in the rat upon repeated exposure to nerve agents is due to desensitization of muscarinic acetylcholine receptors located on the pupillary sphincter.
Key Words: sarin; miosis; tolerance; parasympathetic nervous system; muscarinic receptors.
INTRODUCTION
O-isopropyl methylphosphonofluoridate, also known as sarin or GB, is a highly toxic organophosphorous (OP) compound that exerts its effect by inhibiting the enzyme acetylcholinesterase (AChE). Inhibition of AChE results in the accumulation of the neurotransmitter acetylcholine and excessive stimulation of cholinergic receptors. At higher doses, effects of exposure include salivation, muscle twitches, tremors, convulsions, seizures, and death, usually attributable to respiratory failure (Taylor, 1996). However, at lower doses, few if any of these severe toxic signs are observed. Miosis is one of the threshold clinical effects that can be observed following less-than-lethal of GB vapor exposures, and it occurs at concentrations of GB many times lower than those required to cause death (Mioduszewski et al., 2002a, 2002b).
Normally, both the sympathetic and parasympathetic branches of the autonomic nervous system play a role in the control of pupillary size. Stimulation of the sympathetic branch releases norepinephrine onto -adrenergic receptors located on the radial muscles of the iris, resulting in dilation of the pupil (Yu and Koss, 2002, 2003). Muscarinic acetylcholine receptors are present on the pupillary sphincter of the rat, and administration of muscarinic antagonists results in pupillary dilation, demonstrating the role for muscarinic receptors and the parasympathetic nervous system in the control of pupil diameter (Furuta et al., 1998; Smith et al., 1996). Nerve agent–induced miosis is due to excessive stimulation of the parasympathetic nervous system and stimulation of muscarinic acetylcholine receptors located on the pupillary sphincter muscle.
It has been demonstrated that repeated low-dose exposure to an OP compound can result in desensitization of muscarinic acetylcholine receptors in the retina (Tandon et al., 1994) and brain (Tang et al., 1999) of the rat. Additionally, repeated exposure to a nerve agent results in a reduced miotic response after multiple exposures (Dabisch et al., 2005; Soli et al., 1980). Similarly, repeated topical ocular exposure of monkeys to the cholinesterase inhibitors echothiophate and isoflurophate resulted in a gradual decrease over time in the miotic response to both compounds, and a decreased miotic response to the cholinomimetic carbachol (Bito and Banks, 1969).
While tolerance to the miotic effect of nerve agents has been observed previously, the mechanism responsible for the development of the tolerance remains unclear. Therefore, the aim of the present study was to examine potential mechanisms responsible for the tolerance to the miotic effect of GB vapor observed after repeated exposure. Our laboratory has shown previously that this miotic tolerance to GB vapor is not due to a decreased inhibitory effect of GB in the eye, or to a decrease in the amount of GB reaching the eye (Dabisch et al., 2005). It has also previously been shown that the tolerance is not likely due to internalization of muscarinic acetylcholine receptors in the pupillary sphincter muscle (Soli et al., 1980). The present study investigated two additional hypotheses aimed at elucidating the mechanisms mediating the tolerance to the miotic effect of GB vapor. The first experimental hypothesis considered the possibility that enhanced sympathetic outflow to the eye due to inhibition of ganglionic AChE opposes the miotic effect of GB vapor on the pupil, resulting in a lesser degree of miosis. The second hypothesis proposed that the tolerance is due to desensitization of muscarinic acetylcholine receptors located on the pupillary sphincter muscle.
MATERIALS AND METHODS
Animal use.
Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighing 200–250 g were used in this study. All experiments and procedures were approved by the U.S. Army Edgewood Chemical Biological Center Institutional Animal Care and Use Committee and were conducted in accordance with the requirements of Army Regulation 70–18 (1984) and the Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals.
GB vapor generation.
GB vapor generation was accomplished as described previously (Muse et al., 2000). Briefly, neat liquid GB was placed into a gas-tight syringe and mounted onto a variable-rate syringe drive (Model 22, Harvard Apparatus Inc., South Natick, MA). Once activated, the syringe drive delivered a constant flow of GB through a flexible plastic line into a spray-atomization system (Spray Atomization Nozzle J SS, Spraying Systems Co., Wheaton, IL). Compressed air (30–40 psi) flowing through the atomizer atomized the GB liquid into fine droplets, which, because of the volatility of GB, quickly evaporated. The GB vapor was drawn into a 750-l dynamic airflow inhalation chamber constructed of stainless-steel with Lexan windows. The interior of the exposure chamber was maintained under negative pressure (0.25'' H2O), which was monitored with a calibrated magnehelix (Dwyer, Michigan City, IN). A thermo-anemometer (Model 8565, Alnor, Skokie, IL) was used to monitor chamber airflow at the chamber outlet. At various times throughout the exposure, GB vapor concentration in the chamber was determined from a chamber air sample collected onto a solid sorbent tube. O-isopropyl methylphosphonofluoridate concentrations were determined using a gas chromatograph equipped with a flame ionization detector (Agilent Technologies Inc., Wilmington, DE). The target GB vapor concentration for each exposure in the study was 4.0 mg/m3.
Infrared photography.
The right eye of each rat was digitally photographed to determine the degree of miosis present after exposure to GB vapor. This technique has been described previously (Dabisch et al., 2005; Miller et al., 2003). Each animal was temporarily hand restrained (<30 s) to minimize movement of the head, and the pupil was illuminated with an infrared spotlight (model SL1236, Advanced Illumination, Rochester, VT). Images were digitally recorded with an infrared-capable video camera (model XC-ST50, Sony) equipped with a 75-mm/F2.7 lens (model LMV7527) and an image-acquisition PC card (model PCI-1411, National Instruments Corp., Austin, TX). All digital images were taken from a distance of 41 in. under controlled low-light conditions (<10 foot-candles) and were stored as .JPG files for further analysis. Pupil and iris radii were determined with custom image analysis software (Miller et al., 2003) written using the LabView software suite (Version 6, National Instruments Corp., Austin, TX). To correct for any variability in the distance from the camera to the animal's eye, the results were expressed as the ratio of the pupil radius to the iris radius.
Exposure protocol.
Rats were split into two groups and subjected to multiple whole-body inhalation exposures to either GB vapor or room air. Animals were exposed to either GB vapor or room air for 1 h on each of three consecutive days. Exposures occurred at 24-h intervals. As stated previously, the target GB vapor concentration for each exposure in the study was 4.0 mg/m3. The concentration of GB vapor in the air that causes miosis in 50% of exposed male rats (EC50) for a 60-min exposure is 0.030 mg/m3 (Mioduszewski et al., 2002a), whereas the concentration that results in death in 50% of exposed male rats (LC50) for a 60-min exposure is 7.55 mg/m3 (Mioduszewski et al., 2002b). In the present study, the average GB vapor concentration generated was 4.13 ± 0.07 mg/m3. This concentration is above the EC50 for miosis but below the LC50 for a 60-min exposure. Other than miosis, the rats did not show any overt clinical signs of nerve agent exposure, and no deaths occurred, confirming that the concentration generated was between the EC50 for miosis and the LC50 for a 60-min exposure. The GB-treated and air-treated groups were exposed simultaneously in separate chambers, and the exposures occurred at the same time each day. Baseline pupil images of all animals were recorded several days before exposure to GB vapor; 1 h before each exposure; 15 min, 3 h, and 5 h after each exposure; and at 24-h intervals for 1 week following the third exposure.
Effect of adrenergic receptor blockade on the development of miotic tolerance.
In the present study, tolerance to the miotic effect of GB vapor was present after 2 exposures. This tolerance was expected and has been reported previously (Dabisch et al., 2005; Soli et al., 1980). To determine whether the observed miotic tolerance to GB vapor is due to enhanced sympathetic nervous system activity, rats were treated with different antagonists, or combinations of antagonists, of the sympathetic nervous system. Rats were then exposed to either GB vapor or room air according the to exposure schedule described previously. If the miotic tolerance were due to enhanced sympathetic tone to the muscles controlling pupil diameter, it would be expected that antagonists of the sympathetic nervous system would prevent the tolerance. Two groups of four rats each (1 GB-exposed group and 1 air-exposed group) were injected with the -adrenergic receptor antagonist phentolamine (5 mg/kg im) 15 min before the third exposure. The dose of phentolamine used has been previously shown to block -adrenergic receptor-mediated activity in the heart (Barres et al., 2001). Another two groups of four rats each (1 GB-exposed group and 1 air-exposed group) were injected with the -adrenergic receptor antagonist propranolol (4 mg/kg im) 15 min before the third exposure. This dose of propranolol has previously been shown to block -adrenergic receptor-mediated activity in the heart (Towa et al., 2004). A final two groups of four rats each (1 GB-exposed group and 1 air-exposed group) were injected with a combination of propranolol (4 mg/kg im) and phentolamine (5 mg/kg im) 15 min before the third exposure. Pupil diameter was recorded before and after administration of drugs and at various time points throughout the exposure sequence. Whole blood AChE and butyrylcholinesterase (BChE) activities were determined in animals treated with the combination of phentolamine and propranolol according to the methodology described in detail previously (Dabisch et al., 2005).
Effect of muscarinic receptor blockade on the development of miotic tolerance.
As stated previously, tolerance to the miotic effect of GB vapor was present after two exposures. To determine if overstimulation and subsequent desensitization of muscarinic acetylcholine receptors was responsible for the observed miotic tolerance, two groups of four rats each were treated with the muscarinic receptor antagonist atropine (6 mg/kg im) before the first exposure to "protect" muscarinic receptors from overstimulation. One of the groups was exposed to GB vapor, and the other was exposed to room air only. Rats were given an additional dose of atropine (2 mg/kg im) 3 h after the first exposure. The animals were then subjected to the final two exposures in the sequence. Pupil diameter was recorded before and after the administration of atropine, and at various time points throughout the exposure sequence. Whole-blood AChE and BChE activities were determined in atropine-treated animals according to the methodology described in detail previously (Dabisch et al., 2005).
Data analysis.
Statistical analysis was done by analysis of variance (ANOVA) with a Scheffe post-test. A p-value < 0.05 was used as the criterion for statistical significance. All numerical values are reported as mean ± SEM.
RESULTS
Effect of Multiple GB Vapor Exposures on Pupil Size and Function
After the first exposure to GB vapor, all rats had pinpoint pupils (Fig. 1B), whereas animals exposed to air only did not show a change in pupil size (data not shown). The ratio of pupil radius to iris radius in GB-exposed animals was reduced by 99 ± 3%. After the second GB vapor exposure, all rats again had pinpoint pupils (Fig. 1C), whereas those exposed to air only did not show a change in pupil size (data not shown). The ratio of pupil radius to iris radius in GB-exposed animals was reduced by 97 ± 3%. Additionally, after the second GB vapor exposure, the rats showed a more rapid recovery from miosis than after the first exposure (Fig. 2). With the third GB vapor exposure, rats were tolerant to the miotic effect of GB vapor, as the ratio of pupil radius to iris radius was 55 ± 9% of baseline.
Effect of Adrenergic Receptor Blockade on the Development of Miotic Tolerance
Tolerance to the miotic effect of GB vapor was present after two exposures. To determine whether the observed miotic tolerance to GB vapor is due to enhanced sympathetic nervous system activity, rats were treated with phentolamine, propranolol, or the combination of phentolamine and propranolol 20 min before the third exposure. If the observed tolerance was due to enhanced sympathetic tone to the muscles controlling pupil diameter, it would be expected that administration of antagonists of sympathetic receptors before the third exposure would prevent the tolerance.
Immediately before the third exposure, both air- and GB-exposed animals had similar pupil sizes (Fig. 3). In a first group of animals, the -adrenergic antagonist phentolamine (5 mg/kg im) was administered to rats 20 min before the third exposure. Phentolamine administration produced a similar degree of pupil constriction in both air- and GB-exposed animals (Fig. 3), and it did not prevent the tolerance to the miotic effect of GB vapor that was observed in untreated animals (Fig. 4).
In another group of animals, the -adrenergic antagonist propranolol (4 mg/kg im) was administered to rats 20 min before the third exposure. Administration of propranolol did not significantly affect pupil size, and it also failed to prevent the tolerance to the miotic effect of GB vapor observed in untreated animals (data not shown).
In a final group of animals, the combination of phentolamine (5 mg/kg) and propranolol (4 mg/kg) was administered intramuscularly 20 min before the third exposure. Administration of this combination produced a change in pupil size similar to that seen with phentolamine alone, and, again, it failed to prevent the tolerance to the miotic effect of GB vapor observed in untreated animals (data not shown). Administration of the combination of phentolamine and propranolol also did not affect the degree of inhibition of whole-blood AChE by GB (data not shown).
Effect of Muscarinic Receptor Blockade on the Development of Miotic Tolerance
Tolerance to the miotic effect of GB vapor was present after two exposures. To determine whether overstimulation and subsequent desensitization of muscarinic acetylcholine receptors was responsible for the observed miotic tolerance, the muscarinic receptor antagonist atropine was administered before the first exposure to "protect" muscarinic receptors from overstimulation. Administration of atropine (6 mg/kg im) produced a slight increase in pupil size (data not shown), and it prevented the miosis associated with a single exposure to GB vapor (Fig. 5). Additionally, rats given atropine before the first exposure did not develop tolerance to the miotic effect of GB vapor after the third exposure (Figs. 6 and 7). Finally, administration of atropine did not affect inhibition of whole-blood AChE by GB (data not shown).
DISCUSSION
Development of tolerance to the miotic effects of repeated nerve agent exposure has been reported by Soli et al. (1980) for the nerve agent soman and previously by our laboratory (Dabisch et al., 2005) for rats repeatedly exposed to GB vapor. In the latter study, the tolerance to the miotic effect of GB vapor was found to persist for approximately 96 h. It is well established that both the duration of the exposure and the concentration of agent play a role in determining the degree of miosis observed (Mioduszewski et al., 2002a; Whalley et al., 2004). Thus, it is likely that varying both the duration of the exposure and the concentration of GB used will alter the time course of the development of miotic tolerance. Additionally, it is likely that varying the frequency of exposure will alter the time course of the development of miotic tolerance, because this would alter the time available for recovery between exposures. However, the present study investigated only a single concentration, duration, and frequency of exposure. Thus, it is not possible to determine the exact effect of each parameter on the development of miotic tolerance.
Although the development of tolerance to the miotic effect nerve agents has been demonstrated previously, the mechanism responsible for this phenomenon remains unclear. Our laboratory has shown previously that the miotic tolerance to GB vapor observed following repeated whole-body vapor exposures is not due to a decreased inhibitory effect of GB in the eye, or to a decrease in the amount of GB reaching the eye (Dabisch et al., 2005). It has also been shown previously that the tolerance is not likely due to internalization of muscarinic acetylcholine receptors in the pupillary sphincter muscle (Soli et al., 1980). The present study investigated two additional hypotheses that could explain the mechanisms mediating tolerance to the miotic effect of GB vapor.
It is possible that the miotic tolerance observed after multiple exposures to a nerve agent might be due to enhanced sympathetic tone in the eye. Over the course of the exposure sequence, it is possible that the AChE in the superior cervical ganglion is gradually inhibited, resulting in a gradual increase in sympathetic tone to the radial muscles of the iris. This enhanced sympathetic tone would oppose the increased parasympathetic tone that is already present as a result of inhibition of AChE at the pupillary sphincter muscle, resulting in the diminished miotic response to GB that is observed after multiple exposures. To test this hypothesis, the present study used the -adrenergic receptor antagonist phentolamine to block sympathetically mediated dilation of the eye. If enhanced sympathetic tone was responsible for the observed tolerance, then phentolamine would prevent or diminish the tolerance. However, animals treated with phentolamine before the third exposure still display tolerance to the miotic effect of GB vapor after exposure. Additionally, air- and GB-exposed animals showed a similar degree of pupillary constriction immediately following phentolamine administration, suggesting that the basal sympathetic tone in the eye was similar in both groups, and also demonstrating that the drug was reaching the eye. Thus, enhanced -adrenoreceptor-mediated sympathetic tone tone in the eye does not appear to occur after multiple GB vapor exposures, and thus it cannot explain the miotic tolerance that is observed.
In addition to stimulation of -adrenergic receptors on the radial muscle of the iris, sympathetic dilation of the pupil may also be mediated by stimulation of -adrenergic receptors located on the pupillary sphincter. -adrenergic receptor-mediated relaxation has been reported in bovine pupillary sphincters (Barilan et al., 2003; Geyer et al., 1998), and -adrenergic receptors are present on the pupillary sphincter muscle of the rat eye (Lahav et al., 1978). In the present study, the -adrenergic receptor antagonist propranolol was used to assess the role of enhanced sympathetic tone and stimulation of -adrenergic receptors in the tolerance to the miotic effect of GB vapor. If stimulation of -adrenergic receptors were playing a role in the observed tolerance, then administration of propranolol would, at least partially, prevent the tolerance. However, animals pretreated with propranolol before the third exposure still displayed tolerance to the miotic effect of GB vapor after the exposure, suggesting that stimulation of -adrenergic receptors is not a factor in the observed miotic tolerance to GB vapor. Additionally, administration of propranolol did not result in a change in pupil size. Because of the lack of effect, it is possible that propranolol did not reach the eye. However, several studies have demonstrated penetration of lipophilic -blockers such as propranolol into the central nervous system (Cruickshank and Neil-Dwyer, 1985; Schiff and Saxey, 1984). Thus, it is likely that propranolol is reaching the eye, but that -adrenergic receptors do not play a significant role in the control of rat pupil size at rest. These results suggest that sympathetic control of resting pupil size in the rat is mediated by -adrenergic and not -adrenergic receptors.
It has been demonstrated previously that muscarinic acetylcholine receptors desensitize as a result of either direct stimulation with muscarinic agonists or indirect stimulation after administration of organophosphorus compounds. This desensitization has been demonstrated in several tissues and species, including rabbit intestinal smooth muscle (Murthy and Makhlouf, 2000), rat atrial cells (Shiu et al., 2002), rat retina (Tandon et al., 1994), rat brain (Tang et al., 1999), and human neuroblastoma cells (Willets et al., 2002). Nerve agent vapor-induced miosis is likely due to a local effect on the eye, namely inhibition of AChE on the pupillary sphincter, resulting in excess cholinergic stimulation (Sim, 1956; Soli et al., 1980). Thus, it is possible that the miotic tolerance to GB vapor that is present after multiple exposures is due to desensitization of muscarinic receptors located on the pupillary sphincter muscle secondary to excessive cholinergic stimulation following inhibition of AChE. If this hypothesis is correct, it would be expected that blockade of muscarinic receptors with a muscarinic receptor antagonist such as atropine would prevent excessive stimulation and desensitization of the receptors, as well as the subsequent tolerance to the miotic effect of GB vapor.
In the present study, animals pretreated with atropine before the first exposure fail to develop tolerance to the miotic effect of GB vapor after the third exposure, suggesting that the mechanism responsible for the miotic tolerance involves stimulation and subsequent desensitization of muscarinic acetylcholine receptors. This is the first study to report this effect of atropine on the development of tolerance to nerve agents. Additionally, because nerve agent vapor–induced miosis is a local effect, it is likely that atropine is preventing the development of miotic tolerance by blocking muscarinic acetylcholine receptors located on the pupillary sphincter. It has been demonstrated previously that multiple exposures to GB vapor result in loss of the pupillary light reflex (Dabisch et al., 2005), a response mediated by muscarinic receptors located on the pupillary sphincter and often used as an indicator of parasympathetic pupillary function (Dutsch et al., 2004). The data from the present study support the results of the previous study, because the desensitization of pupillary muscarinic receptors explains the loss of the pupillary light reflex observed previously.
Desensitization of muscarinic acetylcholine receptors can involve any of several mechanisms. Activation of a protein kinase by the beta-gamma subunits of the muscarinic receptor–associated G protein can lead to receptor phosphorylation and desensitization (Haga and Haga,1992; Shiu et al., 2002). Another possibility is the binding of muscarinic receptor–associated G protein subunits to membrane caveolae, preventing the re-association of the subunits with the receptor and resulting in desensitization (Murthy and Makhlouf, 2000). Finally, stimulation of muscarinic receptors can lead to receptor endocytosis and desensitization (Krudewig et al., 2000). It is not possible to determine which mechanism is mediating the tolerance observed in the present study without further experimentation. However, it has been previously reported that administration of the nerve agent soman produced miotic tolerance without producing a decrease in the number of muscarinic receptors present on the pupillary sphincter (Soli et al., 1980). Thus, it is likely that a mechanism other than receptor endocytosis may be responsible for the tolerance to the nerve agent–induced miosis that is seen after multiple exposures.
The present study demonstrated that the -adrenergic antagonist phentolamine and the -adrenergic receptor antagonist propranolol did not affect the development of tolerance to the miotic effect of GB vapor in the rat, suggesting that enhanced sympathetic tone in the eye is not responsible for the observed tolerance. However, administration of atropine before the first exposure prevented the tolerance to the miotic effect of GB vapor after the third exposure, suggesting that the tolerance is the result of muscarinic receptor desensitization secondary to receptor stimulation. Additionally, the present study demonstrated that sympathetic control of resting pupil size in the rat appears to be mediated by -adrenergic and not -adrenergic receptors. The present study thus confirms and extends the findings of previous studies to strengthen the hypothesis that the miotic tolerance observed upon repeated exposure to nerve agents is due to desensitization of muscarinic acetylcholine receptors located on the pupillary sphincter.
ACKNOWLEDGMENTS
The authors are grateful to the following individuals: Jacqueline Scotto and Jill Jarvis for veterinary care; William Muse, Emily Davis, Kathy Matson, Charles Crouse, David Burnett, Bernardita Gaviola, David McCaskey, and Candice Krauthauser for technical assistance; and Dr. Stanley Hulet for editorial assistance.
REFERENCES
Army Regulation 70–18. (1984). "The Use of Animals in Department of Defense Programs."
Barilan A., Nachman-Rubinstein, R., Oron, Y., and Geyer, O. (2003). Muscarinic blockers potentiate beta-adrenergic relaxation of bovine iris sphincter. Graefes Arch. Clin. Exp. Ophthalmol. 241, 226–231.
Barres, C., Pereira de Souza Neta, E., and Julien, C. (2001). Effect of -adrenoreceptor blockade on the 0.4 Hz sympathetic rhythm in conscious rats. Clin. Exp. Pharmacol. Physiol. 28, 983–985.
Bito, L. Z., and Banks, N. (1969). Effects of chronic cholinesterase inhibitor treatment: The pharmacological and physiological behavior of the anti-ChE-treated monkey (Macaca mulatta) iris. Arch. Ophthalmol. 32, 681–686.
Cruickshank, J. M., and Neil-Dwyer, G. (1985). Beta-blocker brain concentrations in man. Eur. J. Clin. Pharmacol. 28S, 21–23.
Dabisch, P. A, Burnett, D. C., Miller, D. B., Jakubowski, E. M., Muse, W. T., Forster, J. S., Scotto, J. A., Jarvis, J. R., Davis, E. A., Hulet, S. W., et al. (2005). Tolerance to the miotic effect of sarin vapor in rats following multiple low-level exposures. J. Ocular Pharmacol. Ther. In press.
Dutsch, M., Marthol, H., Michelson, G., Neundorfer, B., and Hilz, M. J. (2004). Pupillography refines the diagnosis of diabetic autonomic neuropathy. J. Neurol. Sci. 222, 75–81.
Furuta, M., Ohya, S., Imaizumi, Y., and Watanabe, M. (1998). Molecular cloning of m3 muscarinic acetylcholine receptor in rat iris. J. Smooth Muscle Res. 34, 111–122.
Geyer, O., Bar-Ilan, A., Nachman, R., Lazar, M., and Oron, Y. (1998). 3-adrenergic relaxation of bovine iris sphincter. FEBS Lett. 429, 356–358.
Haga, H., and Haga, T. (1992). Activation by G protein - subunits of agonist- and light-dependent phosphorylation of muscarinic acetylcholine receptors and rhodopsin. J. Biol. Chem. 267, 2222–2227.
Institute of Laboratory Animal Resources, Commission of Life Sciences, National Research Council. (1996). Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, D.C.
Krudewig, R., Langer, B., Vogler, O., Markschies, N., Erl, M., Jakobs, K. H., and van Koppen, C. J. (2000). Distinct internalization of M2 muscarinic acetylcholine receptors confers selective and long-lasting desensitization of signaling to phospholipase C. J. Neurochem. 74, 1721–1730.
Lahav, M., Melamed, E., Dafna, Z., and Atlas, D. (1978). Localization of beta receptors in the anterior segment of the rat eye by a fluorescent analogue of propranolol. Invest, Ophthalmol. Vis. Sci. 17, 645–651.
Miller, D. B., Benton, B. J., Hulet, S. J., Mioduszewski, R. J., Whalley, C. E., Carpin, J. C., and Thomson, S. A. (2003). An analysis method for quantifying elliptical and partially obstructed pupil areas in response to chemical agent vapor exposure. Proceedings of the 29th Annual Northeast Bioengineering Conference, Newark, NJ (Abstract).
Mioduszewski, R. J., Manthei, J. H., Way, R. A., Burnett, D. C., Gaviola, B. P., Muse, W. T., Thomson, S. A., Sommerville, D. R., Crosier, R. B., Scotto, J. A., et al. (2002a). ECBC Low Level Operational Toxicology Program: Phase I—Inhalation Toxicity of Sarin Vapor in Rats as a Function of Exposure Concentration and Duration. Technical Report ECBC-TR-235. Edgewood Chemical Biological Center, U.S. Army Soldier and Biological Chemical Command, Aberdeen Proving Ground, MD.
Mioduszewski, R., Manthei, J., Way, R., Burnett, D., Gaviola, B., Muse, W., Thomson, S., Sommerville, D., and Crosier, R. (2002b). Interaction of exposure concentration and duration in determining acute toxic effects of sarin vapor in rats. Toxicol. Sci. 66, 176–184.
Murthy, K. S., and Makhlouf, G. M. (2000). Heterologous desensitization mediated by G protein–specific binding to caveolin. J. Biol. Chem. 275, 30211–30219.
Muse, W., Anthony, S., Buettner, L., Crouse, C., Crouse, L., Mioduszewski, R., and Thomson, S. (2000). Generation, sampling, and analysis of sarin (gb) vapor for inhalation studies. Proceedings of the International Chemical Weapons Demilitarization Conference (CWD 2000), The Hague, Netherlands. (Abstract).
Schiff, A. A., and Saxey, A. (1984). Autoradiography of nadolol and propranolol in the rat. Xenobiotica 14, 687–691.
Shiu, Z., Khan, I. A., Tsuga, H., Dobrzynski, H., Haga, T., Henderson, Z., and Boyett, M. R. (2002). Role of receptor kinase in long-term desensitization of the cardiac muscarinic receptor-K+ channel system. A., J. Physiol. 283, H819–H828.
Sim, V. M. Effect on pupil size of exposure to GB vapour. (1956). Directorate of Chemical Defence Research and Development—Proton Technical Paper #531.
Smith, R. D., Grzelak, M. E., Belanger, B., and Morgan, C. A. (1996). The effects of tropicamide on mydriasis in young rats exhibiting a natural deficit in passive-avoidance responding. Life Sci. 59, 753–760.
Soli, N. E., Lund Karlsen, R., Opsahl, M., and Fonnum, F. (1980). Correlations between acetylcholinesterase activity in guinea-pig iris and papillary function: A biochemical and pupillographic study. J. Neurochem. 35, 723–728.
Tandon, P., Padilla, S., Barone, S., Pope, C. N., and Tilson, H. A. (1994). Fenthion produces a persistent decrease in muscarinic receptor function in the adult rat retina. Toxicol. Appl. Pharmacol. 125, 271–280.
Tang, J., Carr, R. L., and Chambers, J. E. (1999). Changes in rat brain cholinesterase activity and muscarinic receptor density during and after repeated oral exposure to chlorpyrifos in early postnatal development. Toxicol. Sci. 51, 265–272.
Taylor, P. (1996). Anticholinesterase agents. In The Pharmacological Basis of Therapeutics (Limbird, L. E., and Hardman, J. G., Eds.), 9th edition, pp.161–176. McGraw-Hill Medical Publishing Division, New York.
Towa, S., Kuwahara, M., and Tsubone, H. (2004). Characteristics of autonomic nervous function in Zucker-Fatty rats: Investigation by power spectral analysis of heart rate variability. Exp. Anim. 53, 137–144.
Whalley, C. E., Benton, B. J., Manthei, J. H., Way, R. A., Jakubowski, E. M., Burnett, D. C., Gaviola, B. I., Crosier, R. B., Sommerville, D. R., Muse, W. T. et al. (2004). Low-level cyclosarin (GF) vapor exposure in rats: Effect of exposure concentration and duration on pupil size. Technical Report ECBC-TR-407. Edgewood Chemical Biological Center, U.S. Army Soldier and Biological Chemical Command, Aberdeen Proving Ground, MD.
Willets, J. M., Challiss, R. A. J., and Nahorski, S. R. (2002). Endogenous G protein–coupled receptor kinase 6 regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells. J. Biol. Chem. 277, 15523–15529.
Yu, Y., and Koss, M. C. (2002). 1A-Adrenoceptors mediate sympathetically evoked pupillary dilation in rats. J. Pharmacol. Exp. Ther. 300, 521–525.
Yu, Y., and Koss, M. C. (2003). Functional characterization of alpha-adrenoceptors mediating pupillary dilation in rats. Eur. J. Pharmacol. 471, 135–140.(Paul A. Dabisch, Dennis B)