D. Alvaro et al., ROLE AND MECHANISMS OF ACTION OF ACETYLCHOLINE IN THE REGULATION OF RAT CHOLANGIOCYTE SECRETORY FUNCTIONS, The Journal of clinical investigation, 100(6), 1997, pp. 1349-1362
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.