ROLE AND MECHANISMS OF ACTION OF ACETYLCHOLINE IN THE REGULATION OF RAT CHOLANGIOCYTE SECRETORY FUNCTIONS

We investigated, in isolated bile duct units (IBDU) and cholangiocytes isolated from normal rat liver, the occurrence of acetylcholine (ACh) receptors, and the role and mechanisms of ACh in the regulation of th e Cl-/HCO3- exchanger activity. The Cl-/HCO3- exchanger activity was e valuated measuring changes in intracellular pH induced by acute Cl- re moval/readmission. M3 subtype ACh receptors were detected in IBDU and isolated cholangiocytes by immunofluorescence, immunoelectron microsco py, and reverse transcriptase PCR. M1 subtype ACh receptor mRNA was no t detected by reverse transcriptase PCR and M2 subtype was negative by immunofluorescence. ACh (10 mu M) showed no effect on the basal activ ity of the Cl-/HCO3- exchanger. When IBDU were exposed to ACh plus sec retin, ACh significantly (P < 0.03) increased the maximal rate of alka linization after Cl- removal and the maximal rate of recovery after Cl - readmission compared with secretin alone (50 nM), indicating that AC h potentiates the stimulatory effect of secretin on the Cl-/HCO3- exch anger activity. This effect of ACh was blocked by the M3 ACh receptor antagonist, 4-diphenyl-acetoxy-N-(2-chloroethyl)-piperidine (40 nM), b y the intracellular Ca2+ chelator, 1,2-bis (2-Aminophenoxy)-ethane-N,N ,N',N'-tetraacetic acid acetoxymethylester (50 mu M), but not by the p rotein kinase C antagonist, staurosporine (0.1 mu M). Intracellular cA MP levels, in isolated rat cholangiocytes, were unaffected by ACh alon e, but were markedly higher after exposure to secretin plus ACh compar ed with secretin alone (P < 0.01). The ACh-induced potentiation of the secretin effect on both intracellular cAMP levels and the Cl-/HCO3- e xchanger activity was individually abolished by two calcineurin inhibi tors, FK-506 and cyclosporin A (100 nM). Conclusions: M3 ACh receptors are markedly and diffusively represented in rat cholangiocytes. ACh d id not influence the basal activity of the Cl-/HCO3- exchanger, but en hanced the stimulation by secretin of this anion exchanger by a Ca2+-d ependent, protein kinase C-insensitive pathway that potentiates the se cretin stimulation of adenylyl cyclase. Calcineurin most likely mediat es the cross-talk between the calcium and adenylyl cyclase pathways. S ince secretin targets cholangiocytes during parasympathetic predominan ce, coordinated regulation of Cl-/HCO3- exchanger by secretin (cAMP) a nd ACh (Ca2+) could play a major role in the regulation of ductal bica rbonate excretion in bile just when the bicarbonate requirement in the intestine is maximal.