Effect of secretion on intracellular pH regulation in isolated rat bile duct epithelial cells.

Abstract

The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pH1) and HCO3 excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pHi was measured with 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECF-AM) using a microfluorimetric method. Basal pHi of BDE was 7.04±0.06 in Hepes and 7.16±0.10 in KRB and was unaffected by secretin (50-200 nM). Recovery rates from an acid load in Hepes or in KRB media (with and without amiloride) were also not altered by secretin, indicating that Na+/H+ exchange and Na /HCO-cotransport were not affected by this hormone. After acute Cl-removal, pH1 rose 0.24±0.08 pHU at a maximal rate of 0.125±0.06 pHU/min (H' flux rates = 6.02±3.27 mM/min) and recovered after Cl-readmission (0.188±0.08 pHU/min; H' flux rates = 11.82±5.34 mM/min). Pretreatment with 1 mM DIDS inhibited the effects of Cl-removal, while valinomycin, which induces cell depolarization, enhanced these effects, probably by stimulating electrogenic HCO3 influx. Secretin significantly increased both the maximal rate of alkalinization after Cl-removal (P < 0.012) and of pH1 recovery after Cl-readmission (P < 0.025), indicating stimulation of Cl-/HCO3 exchange activity. These findings were reproducedwithN',2'-O-Dibutyryladenosine-3'-5'-cyclicmonophosphate (DBcAMP). The Cl-channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 AM) significantly decreased the effects of secretin and DBcAMP on the pH1 changes promoted by acute Cl-removal/readmission. These findings establish that secretin stimulates the activity of the Cl-/HCO3 exchanger in BDE cells, probably by activating Cl-channels via the intracellular messenger cAMP. This in turn depolarizes the cell, stimulating electrogenic Na /HCO3 symport. The cell depolarization induced by C1-channel activation should enhance HCO3 entrance through electrogenic Na+/HCO-symport, which in turn stimulates the Cl-/ HCO3 exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile. (J. Clin. Invest. 1993. 92:1314-1325.) Key words: bile duct epithelium. intra-

Shared apical sorting of anion exchanger isoforms AE2a, AE2b1, and AE2b2 in primary hepatocytes

“…This study showed AE2 immunoreactivity restricted to the lumenal surface of the hepatobiliary tree [12]. Such an apical location of AE2 in liver cells is compatible with the reported apical AE activity seemingly involved in the biliary bicarbonate secretion [3,[13][14][15][16]. Although in baseline conditions, expression of alternative transcripts AE2b 1 and AE2b 2 each accounts for about 10% of the full-length AE2a message in the human liver [9], our recent finding that HNF1a may transactivate alternative expression [17] suggests that the physiological significance of alternative AE2 messages might be related to the possibility for liver cells to increase their expression when needed for bicarbonate secretion.…”

Decreased Anion Exchanger 2 Immunoreactivity in the Liver of Patients With Primary Biliary Cirrhosis

“…Additional statistical evaluation was also carried out, in which immunostaining data luminal membrane of intrahepatic bile ducts is consistent for each liver structure were categorized as dicotomic qualitative with a possible role for AE2 protein in the generation of canavariables (presence/absence, traces) and analyzed by Fisher's Exact licular and ductular bile through bicarbonate secretion. [6][7][8]14 Test. A final analysis was performed on data from samples having Once the liver specimens included in the present study scores for the three liver structures (a high background sometimes were processed for AE2 immunoperoxidase, stainings were precluded reliable scoring of canaliculi).…”

Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger

“…Normal rats express secretin receptors in the biliary epithelium, 8 and isolated normal rat bile duct units and cholangiocytes both respond to secretin with a cAMPdependent Cl Ϫ efflux and Cl Ϫ /HCO 3 Ϫ exchange activity. 10,11,13,15,16,20 However, in vivo response to secretin could not be shown in normal rats, although these animals effectively respond to the hormone after induction of bile ductular cell hyperplasia. 1,19,[21][22][23][24] In the model of isolated bivascularly perfused normal rat liver (IPRL), 25 secretin (via the hepatic artery) had no influence on the net bile flow, but it did increase biliary concentration of bicarbonate.…”

“…[1][2][3][4][5][6][7] Its interaction with a G-protein-coupled receptor selectively localized to the epithelial bile duct cells 8 results in increased intracellular levels of cyclic adenosine monophosphate (cAMP) [cAMP] i 7,9,10 and protein kinase A activation. 11,12 Phosphorylation and opening of a cAMP-dependent Cl Ϫ channel, the cystic fibrosis transmembrane conductance regulator (CFTR), 13 causes Cl Ϫ efflux to the ductular lumen. This appears to stimulate an apical Na ϩ -independent Cl Ϫ /HCO 3 Ϫ anion exchange (AE), 14 with HCO 3 Ϫ efflux and Cl Ϫ influx, that is facilitated by the outside to inside transmembrane gradient of Cl Ϫ at relatively high intracellular HCO 3 Ϫ concentration.…”

“…This appears to stimulate an apical Na ϩ -independent Cl Ϫ /HCO 3 Ϫ anion exchange (AE), 14 with HCO 3 Ϫ efflux and Cl Ϫ influx, that is facilitated by the outside to inside transmembrane gradient of Cl Ϫ at relatively high intracellular HCO 3 Ϫ concentration. [10][11][12]15,16 Several bicarbonate transporters, most of them encoded by the SLC4 and SLC26 gene families, 17 have been described to exert AE activity. A decade ago, we localized one of those polypeptides, the SLC4A2 or AE2, 18 to the apical membrane in cholangicytes.…”