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

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. OSS was assessed by determining the loss of ability of the cell to extrude or sequester Ca²⁺ following oxotremorine-induced depolarization following exposure to dopamine (DA) 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. These findings also suggest that BB may antagonize OS effects by lowering activation of pCREB and possibly PKCγ induced by DA. In the truncated and chimeric receptors, results indicated that BB reduced OSS 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.