M1 Muscarinic Receptor Activation Mediates Cell Death in M1-HEK293 Cells
Figure 6
M1 mAChR activation induces cleaved-caspase 3 but does not induce changes in proliferation.
HEK293-M1 and HEK293-Vec cells were stimulated across a 48 hour time course (2, 4, 8, 24, and 48 hours) with 100 µM carbachol (A). Induction of cleaved caspase-3 was evident following carbachol but only in the HEK293-M1 (M1) expressing cells. The induction was statistically significant at 24 hours and by 48 hours almost 50% of the cells expressed cleaved caspase 3. In contrast, there was no expression of cleaved caspase 3 in HEK-Vector (Vec) expressing cells. (B. To determine whether this expression was M1-mediated and MEK-mediated HEK293-M1 cells were incubated with the M1 antagonist MT7 or the MEK blocker U0126 and then challenged with carbachol. Induction of cleaved caspase 3 by carbachol was strongly inhibited by both drugs indicating that it was M1- and MEK-mediated. C. Photomicrographs of cleaved caspase 3 in HEK293-M1 cells 48 hours after vehicle (left image) or carbachol (right image) addition showing strong induction of cleaved caspase 3 in carbachol treated cells. (D) HEK293-Vec cells and HEK293-M1 cells were stimulated with 100 µM carbachol or vehicle for 24 hours, where BrdU was included during the final hour of treatment. BrdU incorporation revealed that the carbachol mediated reduction in cell numbers was not as a consequence of influencing proliferation. BrdU positive cells are as a percentage of the total cells counted to represent percentage proliferating cells. Data are representative of at least three independent experiments.