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Ethylcholine mustard aziridinium ion: a cholinotoxin of the retina in vivo
265
TIPS - July 1986
Willadenosine induce nonspecificbronchialhyperreactivity?
Adenosine and asthma The recent article in TiPS 1 b y Drs Church and Holgate on ' A d e n o sine and asthma' is interesting but somewhat misleading. Asthmatics appear to be hyperreactive to inhalation of adenosine but this observation per se does not suggest a role for this substance in the pathogenesis of asthma, for if it did then sulphur dioxide or even 'fog' should also be considered as mediators of asthma since asthmatics are hyperreactive to a diverse range of chemical and noxious stimuli. One of the crucial criteria for the identification of a mediator p r o p o s e d b y Dale 2 is the ability of the putative mediator to mimic the features of the disease. In the case of bronchial asthma, nonspecific bronchial hyperreactivity is the most characteristic, if not diagnostic, feature 3. It would therefore seem more p r u d e n t to determine the effect of adenosine (or any other putative mediator) for its
ability to alter airway reactivity to other spasmogens in either normal or asthmatic individuals. Furthermore, xanthines lacking adenosine antagonism (e.g. enprophylline) 4 have comparable profiles of clinical activity to classical xanthines (e.g. theophylline) in preventing late-onset airways obstruction in allergic asthmatics u n d e r g o i n g bronchial provocation s . Such observations not only suggest that the therapeutic benefit afforded b y the xanthines in the m a n a g e m e n t of bronchial asthma is unlikely to be secondary to adenosine antagonism b u t also questions a central role for adenosine in bronchial asthma. Adenosine released under the conditions cited b y Drs Church and Holgate would seem to provide yet another means of inducing bronchoconstriction in hyperreactive subjects; but this does not provide evidence of this mediator playing a central
ECMAz can cause more effective and equally selective retinal lesions than 6-hydroxydopamine
Ethylcholine mustard aziridinium ion: a cholinotoxin of the retina/n vivo Colhoun and Rylett ~ raise some important issues concerning the uses and abuses of nitrogen mustard analogues of choline, in vitro and in vivo, most of which we endorse. We accept their reading of the literature on the use of these c o m p o u n d s as cholinotoxins in vivo, namely that the evidence for effective and selective destruction of cholinergic cells is weak. However, we have had considerable success with ethylcholine mustard a z i r i d i n i u m ion (ECMAz) as an effective and selective cholinergic neurotoxin in the retina in vivo. O u r results (Ref. 2 and u n p u b lished observations) show that after intravitreal injection of a nominal 50 nmol of ECMAz in y o u n g chickens, there is a permanent reduction in the levels of choline acetyltransferase activity of 70--80%. Light and electron microscopic i m m u n o h i s t o c h e m i s try have unequivocally demonstrated that this loss is due to
destruction of the cholinergic cells. Side-effects of this treatment are minimal: we have examined biochemically and morphologically the serotoninergic, dopaminergic, GABAergic, glycinergic and enkephalin-containing cells of the retina, and have observed no significant effects on them. Somatostatin immunoreactive cells seem to be severely, b u t transiently (1-2 weeks), affected by exposure to ECMAz. More prolonged, b u t still reversible, effects were observed on photoreceptors. We have not found the rigorous quantification of yields of ECMAz to be as crucial for studies in vivo as Colhoun and Rylett 1 suggest: our yields routinely fall within the range 40-50% as quantified b y the iodine thiosulphate m e t h o d 3 reco m m e n d e d b y Colhoun and Rylett 1. O u r approach has been empirical, first standardizing a preparative technique then determ i n i n g effective doses. It seems
role in the induction of the nonspecific bronchial hyperreactivity. In conclusion, it would seem necessary to u n d e r s t a n d the aetiology of bronchial hyperreactivity rather than persevering with the identification of further spasmogens such as adenosine; these p r e s u m a b l y can only cause bronchoconstriction in asthmatics as a consequence of the underlying bronchial hyperreactivity. C . P. P A G E
Department of Pharmacology, King's College, London SW3 6LX, UK.
References 1 Church, M. K. and Holgate, S.T. (1986) Trends Pharmacol. Sci. 7, 49-50 2 Dale, H. H. (1933) Johns Hopkins Med. J. 53, 297-347 5 Scadding, J. G. (1985) in Bronchial asthma, mechanisms and therapeutics (Weiss, E. B., Segal, M.S. and Stein, M., eds), Little Brown 4 Persson, C. G. A., Karlsson, J.A. and Erjefalt, I. (1982) Life Sci. 30, 2181-2189 5 Pauwels, R., Van Renterghem, D., van der Straeten, M., Johannesson, N. and Persson, C. G. A. (1985) J. Allergy Clin. lmmunol. 76, 583-590
likely that quantification of the yields of the toxin will be important in reproducing our results in other laboratories. But in our hands, results have been reliably reproducible, and with appropriate calibration of effective doses, they should be transposable to other laboratories. We do not believe that the sideeffects observed in our experiments are due to the problems in preparing the toxin described b y Colhoun and Rylett 1. Hemicholinium-3 has been shown to affect cholinergic cells and photoreceptors of the rabbit retina 4, and this suggests to us that choline deprivation can affect more than just cholinergic cells, if p u s h e d far enough. Thus, the selectivity of the cholinotoxin may d e p e n d on the choline balance of the different cell types exposed to ECMAz. Another surprising feature of our results is that ECMAz only kills two of the three types of cholinergic cells found in the chicken retina s . We have not been able to find doses of ECMAz which eliminate the third type of cholinergic cell without causing nonspecific damage to the retina. Here we believe that Colhoun and Rylett ~ have pointed to the major
1986, Elsevier Science Publishers B.V., A m s t e r d a m
0165 - 6147/86/$02.00
266 problem with studies in vivoo The dose-toxicity curves for ECMAz are steep (Millar, T. J. et al., unpublished observations) and the zone distinguishing specific from nonspecific effects may be narrow (for example the Ki for inhibition by choline mustard aziridinium ion of high and low affinity choline uptake differs by only a factor of thirty6). Finally, the sensitivity of different cell types may vary depending upon their demand for choline, with overlap between metabolically inactive cholinergic cells and metabolically active non-cholinergic cells. As a result, the practice of injecting small volumes of highly concentrated ECMAz solutions in vivo 7"8 may lead to focal necrosis surrounded by a small zone of specific lesion, surrounded by a zone of ineffectiveness. Obviously the relatively free diffusion of ECMAz across the retinal surface after intravitreal injection has enabled
TIPS -]uly 1986 us to avoid some of these problems, and to obtain an effective, and selective, cholinergic lesion across the retinal surface. It may require complicated experiments involving multiple injections of small volumes of low concentrations of ECMAz centrally to produce effective and selective cholinergic lesions. But in our hands ECMAz gives a more effective and equally selective retinal lesion for cholinergic cells than does the classical neurotoxin, 6hydroxydopamine for dopaminergic cells. If what is at stake is a useful animal model of Alzheimer's disease 9, not to mention a tool for manipulai~qg cholinergic pathways in vivo with the potential that 6-hydroxydopamine has realised on dopaminergic and noradrenergic pathways, then it is certainly worth preserving. I. G. MORGAN AND T. J. MILLAR
Is formation of reactive oxygen by cytochrome P-450 perilous and predictable? Aalt Bast In protecting the organism, cytochrome P-450 also potentially endangers the organism by causing 'activation" of oxygen. Active oxygen production is increased by P-450 inducers such as phenobarbital and benzene, and various xenobiotics stimulate formation of NADPH-dependent microsomal H202 (e.g. hexobarbital) which in turn is responsible for oxygen and hydroxyl radical formation. Aalt Bast discusses the significance of radical formation to cellular defence mechanisms and various considerations regarding the rational design of safer drugs. The enormous amount of current literature devoted to the toxicity of oxygen almost seems to imply that aerobic life forms are possible despite and not thanks to the presence of oxygen! This statement could also be extended to the haemoprotein cytochrome P-450. Aalt Bast is Head of the Molecular Pharmacology Section of the Department of Pharmacochemistry at the Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
In the 1960s emphasis was placed on the favourable function of cytochrome P-450 in the metabolism of lipid soluble xenobiotics to more water soluble, rapidly excreted compounds, to avoid accumulation of xenobiotics to toxic concentrations. In order to adequately cope with the wide variety of xenobiotics, the low specificity and the ubiquitous presence of the cytochrome in the organism was thought to be
1986,Elsevier Science Publishers B.V.,Amsterdam 0165- 6147/86/$02.00
Department of Behavioural Biology, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra City, A.C.T. 2601, Australia.
References
1 Colhoun, E. H. and Rylett, R.J. (1986) Trends Pharmacol. Sci. 7, 55-58 2 Morgan, I. G., Ishimoto, I. and Boelen, M. (1984) Invest. Ophthalmol. Vis. Sci. 25, suppl., 104 3 Golumbic, C., Fruton, J.S. and Bergmann, M. (1946) J. Org. Chem. 11, 518-535 4 Pu, G. A. W. and Masland, R. H. (1984) J. Neurosci. 4, 1559--1576 5 Millar, T. J., Ishimoto, I., Johnson, C. D., Epstein, M.L., Chubb, I.W. and Morgan, I.G. (1985) Neurosci. Lett. 61, 311-316 6 Rylett, R. J. and Colhoun, E. H. (1984) J. Neurochem. 43, 787-794 7 Mantione, C.R., Zigmond, M. J., Fisher, A. and Hanin, I. (1983) J. Neurochem. 41, 251-255 8 Sandberg, K., Hanin, L, Fisher, A. and Coyle, J. T. (1984) Brain Res. 293, 49-55 9 Hanin, I., De Groat, W. C., Mantione, C. R., Coyle, J. T. and Fisher, A. (1983) Banbury Report 15: Biological Aspects of Alzheimer's Disease pp. 243-253
essential. Currently, research is focused on the formation of (toxic) metabolic intermediates and on bioactivation processes by cytochrome P-450. It has been established that several 'activated' states of oxygen are implicated within the cytochrome P-450 system. Is this again a perilous feature of cytochrome P-450, or is it essential for the enhancement of the efficacy of the system? Do the activated forms of oxygen merely constitute a trifle in cytochrome P450 activity or are they of importance? Is it necessary (and possible) to predict reactive oxygen production from the cytochrome in relation to substrate characteristics? Reactive oxygen from the cytochrome P-450 reaction cycle The reactivity of O 2 is largely explained by its diradical nature; it comprises two unpaired electrons. The diatomic oxygen molecule can be reduced to H20 by four electrons in a stepwise fashion (Fig. 1). The addition of the first electron (el) produces the superoxide anion radical (09. Addition of a second electron (e2) to O5 yields the peroxide anion (O20, which has no unpaired electrons. 022- in its protonated form, which occurs at physiological pH, yields hydrogen peroxide ( H 2 0 2 ) . Little is
TIPS - July 1986
Willadenosine induce nonspecificbronchialhyperreactivity?
Adenosine and asthma The recent article in TiPS 1 b y Drs Church and Holgate on ' A d e n o sine and asthma' is interesting but somewhat misleading. Asthmatics appear to be hyperreactive to inhalation of adenosine but this observation per se does not suggest a role for this substance in the pathogenesis of asthma, for if it did then sulphur dioxide or even 'fog' should also be considered as mediators of asthma since asthmatics are hyperreactive to a diverse range of chemical and noxious stimuli. One of the crucial criteria for the identification of a mediator p r o p o s e d b y Dale 2 is the ability of the putative mediator to mimic the features of the disease. In the case of bronchial asthma, nonspecific bronchial hyperreactivity is the most characteristic, if not diagnostic, feature 3. It would therefore seem more p r u d e n t to determine the effect of adenosine (or any other putative mediator) for its
ability to alter airway reactivity to other spasmogens in either normal or asthmatic individuals. Furthermore, xanthines lacking adenosine antagonism (e.g. enprophylline) 4 have comparable profiles of clinical activity to classical xanthines (e.g. theophylline) in preventing late-onset airways obstruction in allergic asthmatics u n d e r g o i n g bronchial provocation s . Such observations not only suggest that the therapeutic benefit afforded b y the xanthines in the m a n a g e m e n t of bronchial asthma is unlikely to be secondary to adenosine antagonism b u t also questions a central role for adenosine in bronchial asthma. Adenosine released under the conditions cited b y Drs Church and Holgate would seem to provide yet another means of inducing bronchoconstriction in hyperreactive subjects; but this does not provide evidence of this mediator playing a central
ECMAz can cause more effective and equally selective retinal lesions than 6-hydroxydopamine
Ethylcholine mustard aziridinium ion: a cholinotoxin of the retina/n vivo Colhoun and Rylett ~ raise some important issues concerning the uses and abuses of nitrogen mustard analogues of choline, in vitro and in vivo, most of which we endorse. We accept their reading of the literature on the use of these c o m p o u n d s as cholinotoxins in vivo, namely that the evidence for effective and selective destruction of cholinergic cells is weak. However, we have had considerable success with ethylcholine mustard a z i r i d i n i u m ion (ECMAz) as an effective and selective cholinergic neurotoxin in the retina in vivo. O u r results (Ref. 2 and u n p u b lished observations) show that after intravitreal injection of a nominal 50 nmol of ECMAz in y o u n g chickens, there is a permanent reduction in the levels of choline acetyltransferase activity of 70--80%. Light and electron microscopic i m m u n o h i s t o c h e m i s try have unequivocally demonstrated that this loss is due to
destruction of the cholinergic cells. Side-effects of this treatment are minimal: we have examined biochemically and morphologically the serotoninergic, dopaminergic, GABAergic, glycinergic and enkephalin-containing cells of the retina, and have observed no significant effects on them. Somatostatin immunoreactive cells seem to be severely, b u t transiently (1-2 weeks), affected by exposure to ECMAz. More prolonged, b u t still reversible, effects were observed on photoreceptors. We have not found the rigorous quantification of yields of ECMAz to be as crucial for studies in vivo as Colhoun and Rylett 1 suggest: our yields routinely fall within the range 40-50% as quantified b y the iodine thiosulphate m e t h o d 3 reco m m e n d e d b y Colhoun and Rylett 1. O u r approach has been empirical, first standardizing a preparative technique then determ i n i n g effective doses. It seems
role in the induction of the nonspecific bronchial hyperreactivity. In conclusion, it would seem necessary to u n d e r s t a n d the aetiology of bronchial hyperreactivity rather than persevering with the identification of further spasmogens such as adenosine; these p r e s u m a b l y can only cause bronchoconstriction in asthmatics as a consequence of the underlying bronchial hyperreactivity. C . P. P A G E
Department of Pharmacology, King's College, London SW3 6LX, UK.
References 1 Church, M. K. and Holgate, S.T. (1986) Trends Pharmacol. Sci. 7, 49-50 2 Dale, H. H. (1933) Johns Hopkins Med. J. 53, 297-347 5 Scadding, J. G. (1985) in Bronchial asthma, mechanisms and therapeutics (Weiss, E. B., Segal, M.S. and Stein, M., eds), Little Brown 4 Persson, C. G. A., Karlsson, J.A. and Erjefalt, I. (1982) Life Sci. 30, 2181-2189 5 Pauwels, R., Van Renterghem, D., van der Straeten, M., Johannesson, N. and Persson, C. G. A. (1985) J. Allergy Clin. lmmunol. 76, 583-590
likely that quantification of the yields of the toxin will be important in reproducing our results in other laboratories. But in our hands, results have been reliably reproducible, and with appropriate calibration of effective doses, they should be transposable to other laboratories. We do not believe that the sideeffects observed in our experiments are due to the problems in preparing the toxin described b y Colhoun and Rylett 1. Hemicholinium-3 has been shown to affect cholinergic cells and photoreceptors of the rabbit retina 4, and this suggests to us that choline deprivation can affect more than just cholinergic cells, if p u s h e d far enough. Thus, the selectivity of the cholinotoxin may d e p e n d on the choline balance of the different cell types exposed to ECMAz. Another surprising feature of our results is that ECMAz only kills two of the three types of cholinergic cells found in the chicken retina s . We have not been able to find doses of ECMAz which eliminate the third type of cholinergic cell without causing nonspecific damage to the retina. Here we believe that Colhoun and Rylett ~ have pointed to the major
1986, Elsevier Science Publishers B.V., A m s t e r d a m
0165 - 6147/86/$02.00
266 problem with studies in vivoo The dose-toxicity curves for ECMAz are steep (Millar, T. J. et al., unpublished observations) and the zone distinguishing specific from nonspecific effects may be narrow (for example the Ki for inhibition by choline mustard aziridinium ion of high and low affinity choline uptake differs by only a factor of thirty6). Finally, the sensitivity of different cell types may vary depending upon their demand for choline, with overlap between metabolically inactive cholinergic cells and metabolically active non-cholinergic cells. As a result, the practice of injecting small volumes of highly concentrated ECMAz solutions in vivo 7"8 may lead to focal necrosis surrounded by a small zone of specific lesion, surrounded by a zone of ineffectiveness. Obviously the relatively free diffusion of ECMAz across the retinal surface after intravitreal injection has enabled
TIPS -]uly 1986 us to avoid some of these problems, and to obtain an effective, and selective, cholinergic lesion across the retinal surface. It may require complicated experiments involving multiple injections of small volumes of low concentrations of ECMAz centrally to produce effective and selective cholinergic lesions. But in our hands ECMAz gives a more effective and equally selective retinal lesion for cholinergic cells than does the classical neurotoxin, 6hydroxydopamine for dopaminergic cells. If what is at stake is a useful animal model of Alzheimer's disease 9, not to mention a tool for manipulai~qg cholinergic pathways in vivo with the potential that 6-hydroxydopamine has realised on dopaminergic and noradrenergic pathways, then it is certainly worth preserving. I. G. MORGAN AND T. J. MILLAR
Is formation of reactive oxygen by cytochrome P-450 perilous and predictable? Aalt Bast In protecting the organism, cytochrome P-450 also potentially endangers the organism by causing 'activation" of oxygen. Active oxygen production is increased by P-450 inducers such as phenobarbital and benzene, and various xenobiotics stimulate formation of NADPH-dependent microsomal H202 (e.g. hexobarbital) which in turn is responsible for oxygen and hydroxyl radical formation. Aalt Bast discusses the significance of radical formation to cellular defence mechanisms and various considerations regarding the rational design of safer drugs. The enormous amount of current literature devoted to the toxicity of oxygen almost seems to imply that aerobic life forms are possible despite and not thanks to the presence of oxygen! This statement could also be extended to the haemoprotein cytochrome P-450. Aalt Bast is Head of the Molecular Pharmacology Section of the Department of Pharmacochemistry at the Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
In the 1960s emphasis was placed on the favourable function of cytochrome P-450 in the metabolism of lipid soluble xenobiotics to more water soluble, rapidly excreted compounds, to avoid accumulation of xenobiotics to toxic concentrations. In order to adequately cope with the wide variety of xenobiotics, the low specificity and the ubiquitous presence of the cytochrome in the organism was thought to be
1986,Elsevier Science Publishers B.V.,Amsterdam 0165- 6147/86/$02.00
Department of Behavioural Biology, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra City, A.C.T. 2601, Australia.
References
1 Colhoun, E. H. and Rylett, R.J. (1986) Trends Pharmacol. Sci. 7, 55-58 2 Morgan, I. G., Ishimoto, I. and Boelen, M. (1984) Invest. Ophthalmol. Vis. Sci. 25, suppl., 104 3 Golumbic, C., Fruton, J.S. and Bergmann, M. (1946) J. Org. Chem. 11, 518-535 4 Pu, G. A. W. and Masland, R. H. (1984) J. Neurosci. 4, 1559--1576 5 Millar, T. J., Ishimoto, I., Johnson, C. D., Epstein, M.L., Chubb, I.W. and Morgan, I.G. (1985) Neurosci. Lett. 61, 311-316 6 Rylett, R. J. and Colhoun, E. H. (1984) J. Neurochem. 43, 787-794 7 Mantione, C.R., Zigmond, M. J., Fisher, A. and Hanin, I. (1983) J. Neurochem. 41, 251-255 8 Sandberg, K., Hanin, L, Fisher, A. and Coyle, J. T. (1984) Brain Res. 293, 49-55 9 Hanin, I., De Groat, W. C., Mantione, C. R., Coyle, J. T. and Fisher, A. (1983) Banbury Report 15: Biological Aspects of Alzheimer's Disease pp. 243-253
essential. Currently, research is focused on the formation of (toxic) metabolic intermediates and on bioactivation processes by cytochrome P-450. It has been established that several 'activated' states of oxygen are implicated within the cytochrome P-450 system. Is this again a perilous feature of cytochrome P-450, or is it essential for the enhancement of the efficacy of the system? Do the activated forms of oxygen merely constitute a trifle in cytochrome P450 activity or are they of importance? Is it necessary (and possible) to predict reactive oxygen production from the cytochrome in relation to substrate characteristics? Reactive oxygen from the cytochrome P-450 reaction cycle The reactivity of O 2 is largely explained by its diradical nature; it comprises two unpaired electrons. The diatomic oxygen molecule can be reduced to H20 by four electrons in a stepwise fashion (Fig. 1). The addition of the first electron (el) produces the superoxide anion radical (09. Addition of a second electron (e2) to O5 yields the peroxide anion (O20, which has no unpaired electrons. 022- in its protonated form, which occurs at physiological pH, yields hydrogen peroxide ( H 2 0 2 ) . Little is