Continuous prolonged incubation of cells with either xanomeline or carbachol reduced receptor sensitivity in responding to activation by agonists. As shown in Fig. 4, pretreatment with 300 nM xanomeline for 24 h resulted in antagonism of the response to carbachol, oxotremorine and xanomeline, as evidenced by a reduction in potency. This was accompanied by a marked decrease in the maximal response of only the latter two agonists. Nearly identical results were obtained when 10 mM carbachol was used for pretreatment. These effects are commensurate with the occurrence of comparable receptor internalization or down-regulation under these pretreatment conditions. However, it is interesting to note that pretreatment with either ligand for 24 h results in a greater effect on maximal PI hydrolysis elicited by oxotremorine or xanomeline than on that stimulated by carbachol. While we have currently shown that both oxotremorine and xanomeline appear as full agonists in our high receptor expression system, previous literature has suggested that these ligands may be partial agonists at the M1 receptor. This is supported by our observation that xanomeline and oxotremorine exhibit a lower maximal PI response than carbachol in rat wild-type M1 cells that express a lower number of Atropine sulfate receptors compared to human M1 cells. While the maximal response to the full agonist carbachol should not be affected by a reduction in receptor number in a high receptor expression system due to the presence of spare receptors, the response to partial agonists should be reduced, as full receptor occupancy is necessary for such agents to elicit a maximal response. The biphasic nature of the NMS binding displacement curve following long-term treatments with xanomeline may suggest that low and high concentrations of xanomeline result in differential modes of receptor regulation. At low concentrations of xanomeline, down-regulation or internalization may be the predominant mechanism occurring to explain the appearance of a high-potency phase of Lomitapide Mesylate inhibition of NMS binding following treatment with xanomeline for 24 h or 1-h pretreatment followed by washing and 23-h wait. Pretreatment with increasing concentrations of carbachol for 24 h results in highly potent, monophasic inhibition of 0.2 nM NMS binding. Additionally, NMS saturation binding experiments show that maximal receptor density is significantly reduced following both protocols of pretreatment with 300 nM xanomeline. As can be seen in Figs. 2A, 2B and 8, effects of xanomeline on receptor number is saturable. This may account for the inflection of the inhibition of NMS binding in cells pretreated with increasing concentrations of xanomeline for 24 h or for 1 h followed by washing and waiting for 23 h in the absence of free xanomeline. Saturation binding of NMS following 1-h pretreatment with an intermediate concentration of xanomeline, washing and waiting for 23 h or treatment for 24 h with this concentration results in an increase in NMS affinity. This concentration of xanomeline coincides with the end of the long plateau observed in displacement binding experiments. This increase in NMS affinity may mask further decreases in receptor availability occurring at concentrations within this range and contribute to the appearance of the plateau observed in Fig. 1A. The enhanced potency of xanomeline in decreasing binding of either radioligand observed after 24-h pretreatment or 1-h pretreatment followed by washing and waiting for 23 h supports the notion that the long-term effects of xanomeline are likely due to receptor degradation, where the receptors are no longer available to either radioligand. However, the observed similar incomplete inhibition of binding of either radioligand under the latter two conditions suggests that a portion of the cellsurface receptor population is not susceptible to regulation by xanomeline. In addition to down-regulation/internalization of the receptor, long-term pretreatment with high concentrations of xanomeline results in additional modifications of the receptor.