Differentially expressed adenylyl cyclase isoforms mediate secretory functions in cholangiocyte subpopulation

Abstract

Cyclic adenosine monophosphate (cAMP) is generated by adenylyl cyclases (ACs), a group of enzymes with different tissue specificity and regulation. We hypothesized that AC isoforms are heterogeneously expressed along the biliary tree, are associated with specific secretory stimuli, and are differentially modulated in cholestasis. Small duct and large duct cholangiocytes were isolated from controls and from lipopolysaccharide-treated or alpha-naphthylisothiocyanate-treated rats. AC isoform expression was assessed via real-time polymerase chain reaction. Secretion and cAMP levels were measured in intrahepatic bile duct units after stimulation with secretin, forskolin, HCO(3)(-)/CO(2), cholinergic agonists, and beta-adrenergic agonists, with or without selected inhibitors or after silencing of AC8 or soluble adenylyl cyclase (sAC) with small interfering RNA. Gene expression of the Ca(2+)-insensitive isoforms (AC4, AC7) was higher in small duct cholangiocytes, whereas that of the Ca(2+)-inhibitable (AC5, AC6, AC9), the Ca(2+)/calmodulin-stimulated AC8, and the soluble sAC was higher in large duct cholangiocytes. Ca(2+)/calmodulin inhibitors and AC8 gene silencing inhibited choleresis and cAMP production stimulated by secretin and acetylcholine, but not by forskolin. Secretion stimulated by isoproterenol and calcineurin inibitors was cAMP-dependent and gamma-aminobutyric acid-inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO(3)(-)](i) was cAMP-dependent and inhibited by sAC inhibitor and sAC gene silencing. Treatment with lipopolysaccharide or alpha-naphthylisothiocyanate increased expression of AC7 and sAC but decreased expression of the other ACs.

Conclusion: These studies demonstrate a previously unrecognized role of ACs in biliary pathophysiology. In fact: (1) AC isoforms are differentially expressed in cholangiocyte subpopulations; (2) AC8, AC9, and sAC mediate cholangiocyte secretion in response to secretin, beta-adrenergic agonists, or changes in [HCO(3)(-)](i), respectively; and (3) AC gene expression is modulated in experimental cholestasis.

Figure 1. Calmodulin inhibitors diminish secretin and acetylcholine but not forskolin stimulated fluid secretion and intracellular cAMP levels in normal rat IBDU

(A) Secretion was measured as luminal expansion over time. S=secretin; A=acetylcholine; F=forskolin; H89 is an inhibitor of PKA. (*P<0.001 vs controls; #P<0.001 vs secretin; §P<0.001 vs acetylcholine; ▲ P<0.001 vs forskolin). (B) Controls or ophiobolin-A(1μM)-pretreated cells or W7(1μM)-pretreated cells were stimulated with secretin (50 nM) for 30 minutes. Ophiobolin-A and W7 inhibited secretin-stimulated increase in cAMP, but not basal levels of cAMP. Columns represent the mean±SD of indicated replicas normalized to controls (*P<0.05 vs. Secretin 50 nM; **P<0.01vs. Secretin 50 nM).

Figure 2. (A) Evaluation of AC8 message expression in NRC following AC8 silencing

AC8 siRNA results in a greater than 90% reduction in the fold-expression in AC8 mRNA. (mean±SEM of 3 experiments). *P<0.05 vs. scramble SiRNA. (B) Effect of forskolin and secretin on cAMP levels in AC8-silenced. Contrary to NRC exposed to scramble siRNA, secretin did not increase cAMP levels in AC8 silenced cells. Forskolin increased cAMP levels of both silenced and non-silenced NRC (mean±SEM of 7 experiments; *P<0.05 vs. the corresponding basal value). (C) Effects of AC8 silencing on secretin- stimulated and forskolin-stimulated secretion in IBDU. A significant reduction in secretin but not forskolin luminal secretion in AC8 silenced IBDU was found (*P<0.001 vs controls; ▲P< 0.001 vs secretin+scramble siRNA).

Figure 3. Evidence for beta-adrenergic activated calcineurin-inhibitable AC9

Secretion was measured as luminal expansion in IBDU as in Figure 1 and 2A. H89-dependent stimulation of secretion by calcineurin inhibitors cyclosporin-A or FK508 (p<0.001) is consistent with the presence of AC9. Secretion was also stimulated by administration of isoproterenol, a β-adrenergic agonists known to stimulate AC9 in airways(32)(p<0.001). Co-administration of GABA inhibited the isoproterenol-stimulated secretion (*P<0.001 vs controls; ▲P<0.001 vs cyclosporinA; #P<0.001 vs FK506; § P< 0.05 vs isoproterenol).

Figure 4. Increases in intracellular bicarbonate concentrations stimulated fluid secretion in normal rat IBDU

Secretion was measured as in Figure 1 and 3. To avoid changes in pHi, these manoeuvres were conducted in isohydric conditions buffering with 5% CO₂ (in KRB 25 mM) or 10 % CO₂ (in KRB 50 mM). HCO₃⁻-stimulated fluid secretion was inhibited by H89 (10μM), by the carbonic anhydrase inhibitor acetazolamide (100 μM), by the specific sAC inhibitor KH7 (30 μM) and by gene silencing of sAC with siRNA (50nM). (*P < 0.001vs Hepes; § P<0.001 vs. Hepes to KRB 25 mM; ▲ P<0.001 vs KRB 25 mM to KRB 50 mM; ■P < 0.01 vs KRB 25 mM to KRB 50 mM; # P<0.001 vs KRB 25 mM to KRB 50 mM+scramble siRNA).

Figure 5. Expression levels of different ACs isoforms in small and large cholangiocytes isolated from LPS-treated and ANIT-fed rats

Plot A shows the results for SDC, while the results for LDC are shown in plot B. Colums represent averages ± standard deviations of the ratio between AC copy number/β-actin copy number in ANIT or LPS-treated animals vs AC copy number/β-actin copy number in untreated controls (cholangiocyte subpopulations isolated from normal rats) on two different isolation. Each isolation represent the pools of n=16 rats for normal cholangiocytes and n=8 rats for ANIT or LPS treatments.

Figure 6. Cartoon showing ACs isoforms expression in large duct cholangiocytes subpopulations, with the proposed integration of cAMP signaling

(see discussion for details). ACs are color-coded to indicate their mechanisms of activation/inhibition. Membrane topography of AC is hypothetical except for the ciliary location of AC6, AC4 and AC8(12, 18). AC8 is also functionally linked to stimulation of the basolateral secretin receptor (SR) and cholinergic stimuli. Stimulation of secretin receptor and activation of AC8 increases [cAMP] and apical CFTR-dependent secretion, including vesicular transport and AE2 activity. Increased bile flow will bend cholangiocyte primary cilia, triggering Ca²⁺ influx via Policystin 1 and 2 (PC1, PC2)(12). Ca²⁺ influx will in turn inhibit AC6, counteracting the effect of secretin(12). β-adrenergic agonist (isoproterenol) stimulates the calcineurin-inhibitable AC9, while modulated by calcineurin. sAC transduces the intracellular HCO3 concentration into cAMP levels. All these mechanisms concur to determine the level of cellular cAMP responsible for CFTR-dependent secretion.