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Different biochemical and pharmacological responses in two substrains of the wistar rat
TRANSGENIC MODELS AND EXPRESSION SYSTEMS FOR STUDYING THE FUNCTION OF CONJUGATING ENZYMES C.R. W o l f (i), T. J o w e t t (2), J. B i s h o p (3), T. simula (4), B. Burchell (5) & T. Friedberg (i).
DIFFERENT BIOCHEMICAL AND PHARMACOLOGICAL RESPONSES IN TWO SUBSTRA1NS OF THE WISTAR RAT
.M. Marselos, P. Pa[gpas, P. Stephanou and V. Vasiliou Department of Pharmacology, Medical School, University of Ioannina, 451 10 Ioannina, Greece.
(i) ICRF, M o l e c u l a r P h a r m a c o l o g y Unit, B i o m e d i c a l R e s e a r c h Centre, N i n e w e l l s H o s p i t a l and M e d i c a l school, Dundee DDI 9SY. (2) U n i v e r s i t y of N e w c a s t l e - u p o n - T y n e , Dept. of Biochem. & G e n e t i c s , C a t h e r i n e C o o k s o n B u i l d i n g , Medical School, Newcastle-upon-Tyne, NE2 4HH. (3) U n i v e r s i t y of E d i n b u r g h , C e n t r e for G e n o m e Research, King's Buildings, Edinburgh, EH9 3JQ. (4) U n i v e r s i t y of Adelaide, Dept. of O b s t e t r i c s & Gynaecology, The Queen Elisabeth Hospital, Adelaide. (5) U n i v e r s i t y of Dundee, B i o c h e m i c a l M e d i c i n e , Ninewells Hospital & Medical School, Dundee, DDI 9SY.
Two rat substrains have been isolated according to a different induction of the hepatic aldehyde dehydrogenase (ALDH) after treatment with phenobarbital (PB) (lmg/ml drinking water, 10 days). Animals responding to the treatment (RR), a,s well as the non-responding (rr) have been inbred in two different homogenous groups: These animals represent an ideal experimental model, due to their common origin. Apart from the dramatic induction (x30) of the cylosolic isozyme ALDH1 of aldehyde dehydrogenase, PB did not produce significant dilIcrences in other enzymes of drug metabolism, such a~ ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferases (GST), or the microsomal isozyme ALDH3m, between these two groups. 3-Methylcholanthrene (3MC) greatly increased (x300) the total ALDH activity in both substrains. However, the induction was slightly more pronounced in the rr rats, when measured either ~s cytosolic ALDH (ALDH3c) or as total cytosolic ALDH. A similar trend was noticed also in the EROD and GST activities, although for these enzFmes the differences were not significant between the two substrains. The in vivo effects of zoxazolaminc in RR andrr animals were also tested, either without pretreatment or alter administration of PB or 3MC. At the constitutive level, paralysis produced by zoxazolamine (i.p:, 75mg/kg) la~gtedfor a longer time in rr than in RR rats (about 355rain vs. 245min ). Alter pretreatment with PB (lmg/ml drinking water, for 10 days); paralysis time was greatly reduced, but the differences between the two substrains remained (rr 58rain, RR 12rain). Pretreatment with 3MC (i.p., 10 mgJkg, once) induced microsomal metabolism of zoxazolamine to such an extent that no animal of either substrai~awas paralyzed.
The ability to manipulate genes in the mouse genome and in t h e g e n o m e of o t h e r o r g a n i s m s s u c h as drosophila now allows a detailed understanding of the p r o t e c t i v e s y s t e m s that d e t e r m i n e t o x i c o l o g i c a l response to be identified as well as identifying the enzymes w h i c h are r e s p o n s i b l e for m e d i a t i n g the activation of c e r t a i n classes of t o x i n and carcinogen. It also allows greater insight into the potentially complex pathways of toxicological events to be i d e n t i f i e d . In r e c e n t y e a r s we h a v e b e e n interested in t h e e x p r e s s i o n of g l u t a t h i o n e tranferases and more recently glucuronosyl transferases in heterologous expression systems and in transgenic animals to establish their role in the above effects. We have, for example g e n e r a t e d mice which express specific glutathione S-transferases in the skin under control of the keratin pro'motor. We have also expressed glutathione S-tranferases in Ecoli, in Salmonella and yeast systems, to e s t a b l i ~ the role of these proteins in both the detoxification and the a c t i v a t i o n of certain chemical toxins and carcinogens. Our current work relating to this theme will be described in this presentation.
CALCIUM AND AGING. W.H. Gispen, Rudolf Magnus Institute for Neurosciences, Universiteitsweg 110, 3584 CG Utrecht, The Netherlands.
D O W N - R E G U L A T I O N OF FREE INTRACELLULAR CALCIUM IN THE AGING BRAIN
N e u r o n a l plasticity is defined as the capacity of neurons to adapt to a changing internal and external environment, to previous experience or to trauma, Plasticity is an essential feature of neuronal function and it has long been recognized that its loss may contribute to the pathogenesis of neurodegeneration and age-related brain disorders., The "calcium-hypothesis" of plasticity suggests t h a t neurite outgrowth during development and repair can only proceed when intracellular Ca 2+ levels lie within a specific outgrowthpermissive range. In a d d i t i o n , it has been ' p r o p o s e d by Kachaturian that age-related brain deficits m a y originate from a relativelY small disturbance of intrane0ronal Ca 2+ homeostatisis that persists for a long period of time. Such a disturbance may h a v e a variety of causes including dysfunction of voltage sensitive Ca 2+ channels or intracellular Ca 2+ buffering systems. One line of research focuses on the putative efficacy of so Called Ca 2+ antagonists that block voltage sensitive Ca 2+ channels of the L-type i.e. the 1,4dihydropyridenes. As neural tissue contains both vascular and neuronal receptors for t h e s e drugs, they are of particular potential significance in influencing neuronal plasticity or providing neuronal protection in the aged nervous system. Some o f the research that addresses these issues in animal models of brain aging and functional deficits will be discussed.
Age-related changes in resting levels of the free intraeellular calcium coneentration ([Ca2+ ]i) as well as alterations of the rise in [Ca2+]i following depolarization have been investigated in acutely isolated neurons of the mouse brain and of various regions of the rat brain. Characterization of the Ca2+ responses following KCI depolarization in the hippocampus, cortex, striatum, and cerebellum of young rats revealed significant regional differences in the basal [Ca2+]i level as well as in the KCl-induced rise in [Ca2+]i. Resting [Ca2+]i as well as Ca2+ responses after depolarization were lower brain neurons of aged mice and in the hippocampus and cortex neurons of aged rats, but not in the striatum or cerebellum. It is concluded that the Ca2+ homeostasis in some brain regions is specially susceptible to the aging process, resulting in a down regulation of [Ca2+]i probably as a consequence of an enhanced sensitivity of mechanisms regulating [Ca2+] i. This speculation was confirmed by an enhanced sensitivity oi: Ca2+stimulated Phospholipase C activity in the aging mouse brain. The alterations of the central Ca2+ homeostasis in the mouse and the rat were accompanied by paralle 1 changes of [Ca2+ ]i in spleenoeytes of both species in aging. The riSe of [Ca2+]i alter stimulation with the mitogen phytohemagglutinin (PHA) was significantly reduced in the plateau phase, which is maintained by Ca2+ influx mechanisms. Moreover, a reduced Ca2+ response was also found after stimulation of the cells with the Ca2+ ionophore A23187. The data indicate that comparable disturbances of the Ca2+ homeostasis occur in central neurons and peripheral cells and that these alterations mainly affect transmembraneous Ca2+ fluxes rather than Ca2+ release from inttacellular stores. These alterations may be compensated under normal conditions. However, in situations of additional stress like ischemia or hypoglycemia, these alterations of Ca2+ regulating mechanisms may result in a reduced capacity for adaptation. This assumption was supported by observations indicating that the reduction of [Ca2+]i after subchronic treatment with nimodipin (20 mg/kg, 4 days) was less in aged than in young mice.
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W.E. Miiller, H. Hartmann, A. Eckert, M. Rewsin and K. Velbinger, Department of Psychopharmacology, Central Institute of Mental Health, D-68159 ~annheim
171
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DIFFERENT BIOCHEMICAL AND PHARMACOLOGICAL RESPONSES IN TWO SUBSTRA1NS OF THE WISTAR RAT
.M. Marselos, P. Pa[gpas, P. Stephanou and V. Vasiliou Department of Pharmacology, Medical School, University of Ioannina, 451 10 Ioannina, Greece.
(i) ICRF, M o l e c u l a r P h a r m a c o l o g y Unit, B i o m e d i c a l R e s e a r c h Centre, N i n e w e l l s H o s p i t a l and M e d i c a l school, Dundee DDI 9SY. (2) U n i v e r s i t y of N e w c a s t l e - u p o n - T y n e , Dept. of Biochem. & G e n e t i c s , C a t h e r i n e C o o k s o n B u i l d i n g , Medical School, Newcastle-upon-Tyne, NE2 4HH. (3) U n i v e r s i t y of E d i n b u r g h , C e n t r e for G e n o m e Research, King's Buildings, Edinburgh, EH9 3JQ. (4) U n i v e r s i t y of Adelaide, Dept. of O b s t e t r i c s & Gynaecology, The Queen Elisabeth Hospital, Adelaide. (5) U n i v e r s i t y of Dundee, B i o c h e m i c a l M e d i c i n e , Ninewells Hospital & Medical School, Dundee, DDI 9SY.
Two rat substrains have been isolated according to a different induction of the hepatic aldehyde dehydrogenase (ALDH) after treatment with phenobarbital (PB) (lmg/ml drinking water, 10 days). Animals responding to the treatment (RR), a,s well as the non-responding (rr) have been inbred in two different homogenous groups: These animals represent an ideal experimental model, due to their common origin. Apart from the dramatic induction (x30) of the cylosolic isozyme ALDH1 of aldehyde dehydrogenase, PB did not produce significant dilIcrences in other enzymes of drug metabolism, such a~ ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferases (GST), or the microsomal isozyme ALDH3m, between these two groups. 3-Methylcholanthrene (3MC) greatly increased (x300) the total ALDH activity in both substrains. However, the induction was slightly more pronounced in the rr rats, when measured either ~s cytosolic ALDH (ALDH3c) or as total cytosolic ALDH. A similar trend was noticed also in the EROD and GST activities, although for these enzFmes the differences were not significant between the two substrains. The in vivo effects of zoxazolaminc in RR andrr animals were also tested, either without pretreatment or alter administration of PB or 3MC. At the constitutive level, paralysis produced by zoxazolamine (i.p:, 75mg/kg) la~gtedfor a longer time in rr than in RR rats (about 355rain vs. 245min ). Alter pretreatment with PB (lmg/ml drinking water, for 10 days); paralysis time was greatly reduced, but the differences between the two substrains remained (rr 58rain, RR 12rain). Pretreatment with 3MC (i.p., 10 mgJkg, once) induced microsomal metabolism of zoxazolamine to such an extent that no animal of either substrai~awas paralyzed.
The ability to manipulate genes in the mouse genome and in t h e g e n o m e of o t h e r o r g a n i s m s s u c h as drosophila now allows a detailed understanding of the p r o t e c t i v e s y s t e m s that d e t e r m i n e t o x i c o l o g i c a l response to be identified as well as identifying the enzymes w h i c h are r e s p o n s i b l e for m e d i a t i n g the activation of c e r t a i n classes of t o x i n and carcinogen. It also allows greater insight into the potentially complex pathways of toxicological events to be i d e n t i f i e d . In r e c e n t y e a r s we h a v e b e e n interested in t h e e x p r e s s i o n of g l u t a t h i o n e tranferases and more recently glucuronosyl transferases in heterologous expression systems and in transgenic animals to establish their role in the above effects. We have, for example g e n e r a t e d mice which express specific glutathione S-transferases in the skin under control of the keratin pro'motor. We have also expressed glutathione S-tranferases in Ecoli, in Salmonella and yeast systems, to e s t a b l i ~ the role of these proteins in both the detoxification and the a c t i v a t i o n of certain chemical toxins and carcinogens. Our current work relating to this theme will be described in this presentation.
CALCIUM AND AGING. W.H. Gispen, Rudolf Magnus Institute for Neurosciences, Universiteitsweg 110, 3584 CG Utrecht, The Netherlands.
D O W N - R E G U L A T I O N OF FREE INTRACELLULAR CALCIUM IN THE AGING BRAIN
N e u r o n a l plasticity is defined as the capacity of neurons to adapt to a changing internal and external environment, to previous experience or to trauma, Plasticity is an essential feature of neuronal function and it has long been recognized that its loss may contribute to the pathogenesis of neurodegeneration and age-related brain disorders., The "calcium-hypothesis" of plasticity suggests t h a t neurite outgrowth during development and repair can only proceed when intracellular Ca 2+ levels lie within a specific outgrowthpermissive range. In a d d i t i o n , it has been ' p r o p o s e d by Kachaturian that age-related brain deficits m a y originate from a relativelY small disturbance of intrane0ronal Ca 2+ homeostatisis that persists for a long period of time. Such a disturbance may h a v e a variety of causes including dysfunction of voltage sensitive Ca 2+ channels or intracellular Ca 2+ buffering systems. One line of research focuses on the putative efficacy of so Called Ca 2+ antagonists that block voltage sensitive Ca 2+ channels of the L-type i.e. the 1,4dihydropyridenes. As neural tissue contains both vascular and neuronal receptors for t h e s e drugs, they are of particular potential significance in influencing neuronal plasticity or providing neuronal protection in the aged nervous system. Some o f the research that addresses these issues in animal models of brain aging and functional deficits will be discussed.
Age-related changes in resting levels of the free intraeellular calcium coneentration ([Ca2+ ]i) as well as alterations of the rise in [Ca2+]i following depolarization have been investigated in acutely isolated neurons of the mouse brain and of various regions of the rat brain. Characterization of the Ca2+ responses following KCI depolarization in the hippocampus, cortex, striatum, and cerebellum of young rats revealed significant regional differences in the basal [Ca2+]i level as well as in the KCl-induced rise in [Ca2+]i. Resting [Ca2+]i as well as Ca2+ responses after depolarization were lower brain neurons of aged mice and in the hippocampus and cortex neurons of aged rats, but not in the striatum or cerebellum. It is concluded that the Ca2+ homeostasis in some brain regions is specially susceptible to the aging process, resulting in a down regulation of [Ca2+]i probably as a consequence of an enhanced sensitivity of mechanisms regulating [Ca2+] i. This speculation was confirmed by an enhanced sensitivity oi: Ca2+stimulated Phospholipase C activity in the aging mouse brain. The alterations of the central Ca2+ homeostasis in the mouse and the rat were accompanied by paralle 1 changes of [Ca2+ ]i in spleenoeytes of both species in aging. The riSe of [Ca2+]i alter stimulation with the mitogen phytohemagglutinin (PHA) was significantly reduced in the plateau phase, which is maintained by Ca2+ influx mechanisms. Moreover, a reduced Ca2+ response was also found after stimulation of the cells with the Ca2+ ionophore A23187. The data indicate that comparable disturbances of the Ca2+ homeostasis occur in central neurons and peripheral cells and that these alterations mainly affect transmembraneous Ca2+ fluxes rather than Ca2+ release from inttacellular stores. These alterations may be compensated under normal conditions. However, in situations of additional stress like ischemia or hypoglycemia, these alterations of Ca2+ regulating mechanisms may result in a reduced capacity for adaptation. This assumption was supported by observations indicating that the reduction of [Ca2+]i after subchronic treatment with nimodipin (20 mg/kg, 4 days) was less in aged than in young mice.
- -
W.E. Miiller, H. Hartmann, A. Eckert, M. Rewsin and K. Velbinger, Department of Psychopharmacology, Central Institute of Mental Health, D-68159 ~annheim
171
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