Submitted to: Journal of Alzheimer's Disease
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/1/2006
Publication Date: 12/1/2006
Citation: Joseph, J.A., Fisher, D.R., Carey, A.N., Bielinski, D.F. 2006. Dopamine-Induced Stress Signaling in COS-7 Cells Transfected With Selectively Vulnerable Muscarinic Receptor Subtypes is Partially Mediated Via the i3 Loop and Antagonized By Blueberry Extract. Journal of Alzheimer's Disease. 10:423-437. Interpretive Summary: Muscarinic receptors (MAChRs) are a type of signal receiver that are intimately involved in various aspects of both brain and vascular functioning. There is selective oxidative stress sensitivity (OSS) among the different MAChR types, with the M1, M2, and M4 type showing greating OSS than the M3 or M5 types. This OSS can be prevented by pretreatment with blueberry extract. Present studies were carried out with normal cells, cells with a shortened i3 loop (which is an important part of the receptor that allows communication with other areas of the cell), and chimeric, M1 cells with a M3 i3 loop and M3 cells with a M1 i3 loop, receptors. Blueberry treatment was given to determine if they would provide protection through alterations in chemical signaling. The findings suggested that the M1/M3 OSS differences may produce differences in communication (signaling) in signaling molecules such as pMAPK (phospho mitogen activated protein kinase) and pCREB (phosphor cyclic adenosine monophosphate response element binding protein), with M3 cells showing higher pMAPK and lower pCREB activation. These findings also suggest that blueberry may counteract OS effects by lowering activation of pCREB. In the shortened and chimeric receptors, results indicated that BB reduced OSS in M1 cells. However, blueberries were also effective in reducing OSS in cells with shortened M1 receptors, but only partially enhanced the protective effects of the M3 loop in the M1 cells. A similar partial effect of the blueberries was seen in the M3 cells with an M1 loop. It appears that antioxidants found in blueberries might be effecting additional areas on the chimerics to decrease OSS. Therefore the particular receptor configuration may determine the sensitivity to oxidative stress and to the site of action of the blueberry extract.
Technical Abstract: Muscarinic receptors (MAChRs) are intimately involved in various aspects of both neuronal and vascular functioning, and there is selective oxidative stress sensitivity (OSS) among MAChR subtypes, with M1, M2, and M4 showing > OSS as evidenced by the inability of the cell to extrude or sequester Ca2+ following oxotremorine-induced depolarization following exposure to dopamine (DA) (1mM/ 4 hrs) or A(beta) 25-35 (100 mM/ 24hrs)] than M3 or M5 subtypes in transfected COS-7 cells. This OSS can be prevented by pretreatment with blueberry (BB) extract. Present studies were carried out to determine BB treatment of the cells transfected with wild type, truncated or chimeric [where the i3 loop of one receptor was switched with the i3 loop of the other; i.e., M1(M3i3) and M3(M1i3)receptors would alter DA-induced changes in calcium buffering and would confer protection through alterations in pMAPK, pCREB or PKC signaling. The findings suggested that M1/M3 OSS differences may involve differential signaling in pMAPK and pCREB, under OS-treatment conditions, with M3 cells showing higher pMAPK and lower pCREB activation. These findings also suggest that BB may antagonize OS effects by lowering activation of pCREB and possibly PKCgamma induced by DA. In the truncated and chimeric receptors, results indicated that BB reduced OSS (i.e., Ca2+ buffering following depolarization) in response to DA in M1-transfected cells. However, BBs were also effective in preventing these Ca2+ buffering deficits in cells transfected with M1 receptors in which the i3 loop had been removed, but only partially enhanced the protective effects of the M3 i3 loop in the M1(M3i3) chimerics. A similar partial effect of BBs was seen in the M3(M1i3) chimerics which showed increased OSS in response to DA. It appears that antioxidants found in BBs might be targeting additional sites on these chimerics to decrease OSS.