Subsequently, NMS saturation binding isotherms were established. As shown in Fig. 9A and Table 5, long-term changes in receptor density were still evident following blockade of the orthosteric site with atropine only during the initial pretreatment period. Furthermore, atropine did not prevent persistent binding of xanomeline to the receptor, supporting the notion that this 
mode of binding occurs at a secondary site on the receptor. In contrast, when atropine was present only during the 23-h incubation after xanomeline pretreatment and washing, the long-term effects of xanomeline were completely obliterated. We also used specific receptor mutants to further prove a role of receptor activation in Butenafine hydrochloride xanomeline-mediated receptor regulation. Our laboratory has previously shown that point mutation of arginine-123 in the sequence of the rat M1 receptor results in nearly complete loss of receptor responsiveness to agonists without significant changes in receptor binding Pimozide properties. We utilized this receptor mutant expressed in CHO cells to determine if a functional receptor is necessary to elicit the longterm effects of xanomeline on radioligand binding. In agreement with previous findings, we have shown that xanomeline binds to and activates the hM1 acetylcholine receptor in a wash-resistant manner. Our current results also indicate that persistent binding of xanomeline to the M1 muscarinic receptor elicits additional long-term alterations in radioligand binding to the M1 receptor in the absence of free drug. Understanding of these effects is of prime importance in relation to the chronic use of xanomeline in the treatment of schizophrenia. Long-term exposure of cells to xanomeline was accompanied by loss of persistent activation of hydrolysis of inositol phosphates by xanomeline in conjunction with attenuation of receptor activation by other agonists. Possible interpretations of these observations include decreased receptor availability, modifications in receptor conformation, or blockade of the receptor by persistently-bound xanomeline. Any of these effects would result in diminishing radioligand binding in addition to suppressing agonist-mediated activation of the M1 receptor. We have currently shown that acute, as well as chronic, pretreatment with xanomeline results in long-term changes in NMS binding to M1 receptors. Previous reports have indicated that similar long-term changes can occur following exposure to xanomeline for as little as 1 minute. Comparisons with carbachol were made in the current study in order to assess whether these effects are unique to xanomeline. As can be seen in Figs. 1A and 1B, xanomeline pretreatments resulted in changes in radioligand binding very distinct from those induced by carbachol. Exposure of cells to xanomeline for 1 h followed by washing resulted in a concentration-dependent decrease in NMS binding with a slightly lower potency than that seen in untreated cells subjected to radioligand binding in the presence of xanomeline. This is in contrast to results obtained using carbachol for pretreatment followed by washing, where no change in radioligand binding was observed. Receptor internalization and down-regulation induced by sustained exposure to conventional reversible agonists are well-documented phenomena. In accordance with these findings, pretreatment with carbachol for 24 h resulted in a marked decrease in NMS binding. The resultant single high-potency binding profile following carbachol long-term treatment was in sharp contrast to the biphasic curve obtained following 24-h xanomeline pretreatment. Interestingly, a similar biphasic curve resulted following pretreatment with xanomeline for 1 h followed by washing and waiting 23 h in xanomelinefree media. Again, this is unlike results obtained using carbachol for pretreatment, where no effect on radioligand binding was observed under these conditions. In fact, previous literature has shown that the marked decrease in binding elicited by 12-h carbachol pretreatment is fully reversed following washing and incubation in carbachol-free media for 24 h.