However, we showed that anti-M3R antibodies against these linear epitopes exactly influenced Ca influx via M3R in HSG cells. Therefore, we suggest that these linear peptides might consist of the conformational epitopes on the M3R. Several B cell epitopes were identified on the extracellular domains, and some SS patients were reactive to several extracellular domains other than the second extracellular loop. The second extracellular loop of M3R has been the focus of our interest in epitopes and function of anti-M3R antibodies [4,5,9,10]. Recently, Koo et al.[6] reported that the third extracellular loop represents a functional ZVADFMK epitope bound by SS-IgG. Selleckchem GSK3 inhibitor In contrast

to these results, we found in the present study that antibodies to the second extracellular loop of M3R inhibited the increase of (Ca2+)i induced by cevimeline hydrochloride in a functional assay using HSG cells. This inhibitory effect of anti-M3R antibodies might explain the reduction in salivary secretion in some SS patients. Our data also demonstrated that antibodies against the third extracellular loop did not have an effect on the increase in (Ca2+)i, while antibodies against the N-terminal and first extracellular

loop enhanced the increase in (Ca2+)i. These results indicate that the effects of anti-M3R antibodies on the secretion of saliva could be different from these epitopes, although further experiments using antibodies from more patients are necessary. Although the molecular mechanisms on the difference among individual B cell epitopes have not been elucidated, we could propose the following three possibilities. The first is that antibodies against the second extracellular domain GNAT2 of M3R directly inhibit

the intracellular signal pathway, resulting in the decrease of Ca2+ influx and reduction of saliva. In contrast, antibodies against N-terminal region and the first extracellular domain of M3R might enhance the intracellular signalling and increase of Ca2+ influx. The second is that anti-M3R antibodies binding to the second extracellular domain could inhibit the M3R agonist, and then antibodies suppress indirectly the stimulation of Ca2+ influx. The third is that anti-M3R antibodies influence the expression of M3R molecules on HSG. Some antibodies which target the N-terminal region or the first extracellular loop of M3R may be able to up-regulate expression of M3R and enhance Ca2+ influx, whereas the other antibodies against the second extracellular domain might down-regulate the expression of M3R on HSG, resulting in a reduction of Ca2+ influx. It has been reported that the expression of M3R in salivary glands could be affected by anti-M3R antibodies in patients with SS [1].