It does not exclude that singular functions are also required to regulate specific ripening pathways in each type of fruits. This is the case of RIN, NOR, CNR and HB1 genes in tomato. Since then, it has been shown that this type of activation is associated with washresistant binding and allosteric modulation of the M1 receptor. There is evidence that xanomeline interacts reversibly with the orthosteric site, while it binds persistently to the receptor at a different secondary binding domain. While the unique short-term effects of xanomeline have been studied extensively, the long-term consequences of its persistent binding remain relatively unknown. The unique ability of xanomeline to persistently bind to and activate the M1 receptor may similarly result in downregulation/desensitization of the receptor. Alternatively, persistently-bound xanomeline may induce modification of the receptor conformation over time. We undertook the current study to Ginsenoside-Ro Further evaluate the possible mechanisms involved in the long-term changes observed in receptor binding and function by the washresistant component of xanomeline binding to the M1 receptor. We have shown 
that the increase in basal receptor activity observed following pretreatment with xanomeline for 1 h followed by washing is reversed when cells are allowed to incubate for 23 h in the absence of free ligand. Further experiments were designed to determine the time course of this reversal process. CHO hM1 cells were pretreated with 300 nM xanomeline for 1 h, washed, then allowed to incubate for various time periods in ligand-free media. Alternatively, cells were pretreated continuously with xanomeline for various time periods, from 30 minutes to 24 h, prior to washing and immediate use. As shown in Fig. 7A, a significant increase in basal receptor activation of PI hydrolysis was observed when cells were used immediately following 1 h xanomeline pretreatment and washing. However, receptor stimulation elicited by wash-resistant xanomeline binding quickly subsided when cells were allowed to incubate in the absence of free xanomeline, reaching control basal levels within 5 h. While continuous treatment with xanomeline for up to 24 h also resulted in a time-dependent reversal of persistent xanomeline receptor activation, it occurred at a much slower rate. In this case, xanomeline-induced stimulation of PI hydrolysis remained elevated for more than 10 h. ive cells. Maximal inhibition of binding of either radioligand was incomplete. Incubation of pretreated and washed cells for 23 h in the absence of free xanomeline resulted in marked enhancement of the apparent potency of xanomeline in decreasing binding of both radioligands. These changes in potency were approximately 2.3 and 3.5 orders of magnitude greater than those observed following washing off xanomeline, but prior to prolonged waiting, in the case of NMS and QNB, respectively. Again, maximal inhibition of binding of either radioligand was incomplete. Continuous incubation of cells with xanomeline for 24 h followed by washing away free drug immediately prior to Mechlorethamine hydrochloride conducting the binding assay resulted in further increase in xanomeline potency in decreasing binding of both radioligands. In all instances, radioligand binding was best described by a one-site model. As previously noted, binding data were adjusted to account for decreases in protein content following long-term pretreatments with xanomeline. Results are summarized in Table 4. We have previously shown that while xanomeline wash-resistant binding to the M1 receptor takes place at an allosteric domain on the receptor, receptor activation by this mode of xanomeline binding is sensitive to blockade by atropine and therefore involves the receptor orthosteric site. Therefore, additional binding experiments were designed todeterminewhether receptor activationis requiredfor the induction of the observed long-term effects of xanomeline in CHO hM1 cells. To this end, a receptor-saturating concentration of the muscarinic antagonist atropine was added.