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United States Department of Agriculture

Agricultural Research Service

Title: Muscarinic Receptor Subtype Determines Vulnerability to Oxidative Stress in Cos-7 Cells

Authors
item Joseph, James
item Joseph, James
item Fisher, Derek
item Fisher, Derek
item Strain, Joy - FORMER ARS EMPLOYEE
item Strain, Joy - FORMER ARS EMPLOYEE

Submitted to: Free Radicals in Biology and Medicine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 31, 2001
Publication Date: January 15, 2002
Citation: Joseph, J.A., Fisher, D.R., and Strain, J. Muscarinic receptor subtype determines vulnerability to oxidative stress in COS-7 cells. Free Rad. Biol. Med. 2002, 32: 153-161.

Interpretive Summary: A great deal of information suggests that aging may be the result of our increasing inability to counteract the effect of very reactive molecules called free radicals. This appears to be especially true in the brain where some regions show decreasing function as we age. We make the free- radicals as part of our metabolic processes, and it appears that there may be regions of the brain that are more sensitive to these compounds than other regions and that may be why they show differential effects of aging. Since there are various receptors in the brain that help transmit signals, and they are unevenly distributed in the brain, we wondered if they might show different sensitivities to oxidative stress. Therefore, using molecular biological techniques we placed different subtypes of one of these receptors in cells that normally don't have them and looked at their sensitivities to oxidative stress from exposure to a neurotransmitter called dopamine. The results showed that indeed there were some subtypes that were more sensitive to oxidative stress than others suggesting that these receptors may be partially responsible for the brain regional effects of aging.

Technical Abstract: Research has suggested that there may be increased vulnerability to oxidative stress in aging that is brain region specific, and that this vulnerability may be important in determining regional differences in brain aging. We assessed whether one factor determining vulnerability might involve qualitative/quantitative differences in receptor subtypes in the brain. Therefore, cells transfected with one of five muscarinic receptor subtypes (M1-M5 AChR) were exposed to dopamine (DA, an oxidative stress, for 4 hours) and intracellular calcium levels (fluorescent imaging analysis) examined prior to and following stimulation. Results indicated that the ability of the cells to clear excess calcium (i.e., calcium Recovery) following stimulation varied as a function of transfected mAChR subtype, with DA-treated M1, M2, or M 4 cells showing greater decrements in Recovery than those transfected with M3 or M5 AChR. A similar pattern of results in M1- or M3-transfected DA-exposed cells was seen with respect to Viability (cell death). Viability of the untransfected cells was unaffected by DA. Pretreatment with Trolox (a Vitamin E analog) or PBN (a nitrone trapping agent) did not alter the DA effects on cell Recovery in the M1-transfected cells, but were effective in preventing the decrements in Viability. The calcium channel antagonists Nifedipine and Conotoxin (L and N respectively) prevented both the DA-induced deficits in Recovery and Viability. Results are discussed in terms of receptor involvement in the regional differences in vulnerability with age, and that loss of brain function may not inevitably lead to cell death.

Submitted to: Free Radicals in Biology and Medicine
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 31, 2001
Publication Date: January 15, 2002
Citation: Joseph, J.A., Fisher, D.R., and Strain, J. Muscarinic receptor subtype determines vulnerability to oxidative stress in COS-7 cells. Free Rad. Biol. Med. 2002, 32: 153-161.

Interpretive Summary: A great deal of information suggests that aging may be the result of our increasing inability to counteract the effect of very reactive molecules called free radicals. This appears to be especially true in the brain where some regions show decreasing function as we age. We make the free- radicals as part of our metabolic processes, and it appears that there may be regions of the brain that are more sensitive to these compounds than other regions and that may be why they show differential effects of aging. Since there are various receptors in the brain that help transmit signals, and they are unevenly distributed in the brain, we wondered if they might show different sensitivities to oxidative stress. Therefore, using molecular biological techniques we placed different subtypes of one of these receptors in cells that normally don't have them and looked at their sensitivities to oxidative stress from exposure to a neurotransmitter called dopamine. The results showed that indeed there were some subtypes that were more sensitive to oxidative stress than others suggesting that these receptors may be partially responsible for the brain regional effects of aging.

Technical Abstract: Research has suggested that there may be increased vulnerability to oxidative stress in aging that is brain region specific, and that this vulnerability may be important in determining regional differences in brain aging. We assessed whether one factor determining vulnerability might involve qualitative/quantitative differences in receptor subtypes in the brain. Therefore, cells transfected with one of five muscarinic receptor subtypes (M1-M5 AChR) were exposed to dopamine (DA, an oxidative stress, for 4 hours) and intracellular calcium levels (fluorescent imaging analysis) examined prior to and following stimulation. Results indicated that the ability of the cells to clear excess calcium (i.e., calcium Recovery) following stimulation varied as a function of transfected mAChR subtype, with DA-treated M1, M2, or M 4 cells showing greater decrements in Recovery than those transfected with M3 or M5 AChR. A similar pattern of results in M1- or M3-transfected DA-exposed cells was seen with respect to Viability (cell death). Viability of the untransfected cells was unaffected by DA. Pretreatment with Trolox (a Vitamin E analog) or PBN (a nitrone trapping agent) did not alter the DA effects on cell Recovery in the M1-transfected cells, but were effective in preventing the decrements in Viability. The calcium channel antagonists Nifedipine and Conotoxin (L and N respectively) prevented both the DA-induced deficits in Recovery and Viability. Results are discussed in terms of receptor involvement in the regional differences in vulnerability with age, and that loss of brain function may not inevitably lead to cell death.

Last Modified: 10/22/2014
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