By testing the effects of selective agonists/antagonists of cannabinoid receptors (CBRs) on salivation, produced by the submandibular gland in vivo, we showed an involvement of both functional CB1R and CB2R subtypes in the down-regulation of saliva outflow. By mimicking the conditions of single or repetitive cannabis intake, we found that a reduction of saliva outflow reached its maximum level in 10 min and recovered briefly after single administration of cannabinoid but was reduced for prolonged periods if the agonist was applied repetitively (every 5 min). A prolonged specific inhibition of CB1R by AM251 leads to increased saliva outflow, which might indicate a regulatory role for CBRs in continuous secretion of saliva fluid under basal conditions. A similar effect was not clearly observed with the CB2R antagonist. We did not found any significant changes in K+, Na+ and Phosphorus concentrations in the final saliva collected from the gland ducts in response to repetitive administration of cannabinoid in vivo. However, we found a significant increase of total protein concentration, which started 10-15 minutes after WIN55,212 administration (time-point of maximal inhibition of saliva outflow) and maintained for all period if the agonist was applied repetitively (every 5 min). Also, WIN55212-2 leads to increase ?-amylase activity. The cannabinoid-induced increase of protein concentration was accompanied with an increase of total Ca2+ concentration in the final saliva supporting the hypothesis for a role of Ca2+ in CBRs-induced secretion of salivary proteins. In the case of single administration of cannabinoids, any significant changes in both total protein and Ca2+ concentrations were found in final saliva after 10 minutes of drugs action. An administration of the antagonists of CB1Rs and CB2Rs alone did not show any effects on either basal total protein or Ca2+ concentrations in final saliva during all periods tested (30 minutes). Cannabinoids dramatically altered the ionic content of the primary saliva. WIN55,212-2 application to the basal pole of the acini produced a transient drop of [Na+]e in acinar lumen reflecting inhibited transport of Na+ ions, that may, in turn, lead to the suppressed saliva flow rate observed in vivo. The inhibited Na+ transport is mediated by cannabinoid-induced decrease of Na+/K+-ATPase activity in the submandibular gland acinii. The fact that observed WIN55,212-2-induced drop of [Na+]e was abolished by selective inhibition of CB1R indicates that CB1Rs are also functional in acinar cells, where they are mainly responsible for regulating ion transport. In vitro application to the isolated acinar cells of rat submandibular salivary gland of WIN55,212-2 (5 µM), virodhamine (30 µM) or (R)-(+)-Methanandamide (5 µM) in Ca2+-free extracellular solution (1 mM EGTA) and in the presence of SERCA inhibitor - thapsigargin (0,1 µM) decreased total cellular calcium content. CB1Rs and CB2Rs agonist WIN55212-2 induced a dose-dependent (ЕС50=1.75?10-7 M) increase of [Ca2+]cyt in isolated acinar cells: we observed a steady-state slow [Ca2+]cyt rise, which persists even after the removal of an agonist. In isolated acinar cells, (R)-(+)-methanandamine and ACPA (CB1R selective agonists) induced a significant increase of [Ca2+]cyt, evidently confirming a presence of functional CB1Rs coupled with Ca2+ signaling cascades in submandibular acinar cells. Our data suggest that activation CB1Rs and CB2Rs in submandibular acinar cells triggers [Ca2+]cyt signaling by distinct intracellular mechanisms: CB1Rs trigger intracellular Ca2+ mobilization from the ER, while CB2Rs may initially induce Ca2+ release but preferentially activate SOCE. We also showed that WIN55,212-2 (5 µM) inhibit activity of endoplasmic reticulum Са2+-АТPase of submandibular acinar cells.
