Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets

Graf, J.; Henderson, Ronald W.; Krumpholz, Birgit; Boyer, James L.

doi:10.1007/bf01869486

Cited by 83 publications

(44 citation statements)

References 37 publications

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“…Both the latter as well as the failure of C1-omission to lower pHi exclude the possibility that a Na+ coupled Cl-/ HCO3 exchanger functions as an acid extruder in BDE cells in contrast to some other cell types (20,24,25). Finally, a C1-/ HCO3 exchanger is the major acid loading mechanism in BDE (37). However, DIDS completely inhibited (Fig.…”

Section: Isolation Of Bile Duct Epithelial Cells Male Sprague-dawleymentioning

confidence: 88%

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

et al. 1993

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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-

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Section: Isolation Of Bile Duct Epithelial Cells Male Sprague-dawleymentioning

confidence: 88%

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

et al. 1993

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“…43). In hepatocytes, under physiological conditions, intracellular chloride is at electrochemical equilibrium due to the high chloride conductance of the hepatocellular plasma membrane (44). Increases of the intracellular chloride concentration are expected to occur in response to a depolarization of the plasma membrane potential.…”

Section: Discussionmentioning

confidence: 99%

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Reinehr

Sommerfeld

Keitel

et al. 2008

Journal of Biological Chemistry

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Although in rat hepatocytes CD95 is predominantly located inside the cell with almost undetectable immunostaining at the plasma membrane, the addition of CD95-ligand (CD95L) induces hepatocyte apoptosis, which is preceded by a targeting and activation of intracellularly localized CD95 to the plasma membrane including formation of the death-inducing signaling complex. This process involves an NADPH oxidase-dependent generation of reactive oxygen species (ROS) through a ceramide-and protein kinase C-dependent pathway, which leads to an activating phosphorylation of p47 phox . The mechanisms underlying CD95L-induced ceramide formation were addressed in the present study. It was found that CD95L lowered within seconds the apparent vesicular pH from 6.0 to 5.7 in a fluorescein isothiocyanate-dextran-accessible endosomal compartment, which was previously shown to contain acidic sphingomyelinase, and decreased N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide fluorescence, suggestive for an increase of cytosolic [Cl ؊ ]. Bafilomycin or 4,4-diisothiocyanostilbene-2,2-disulfonic acid disodium salt largely abolished the CD95L-induced endosomal acidification, ceramide formation, and downstream events, such as p47 phox phosphorylation, ROS formation, CD95 activation, and apoptosis. These responses were also abolished after knock-down of acidic sphingomyelinase in rat hepatocytes. Interestingly, caspase 8 inhibitors abolished these CD95L-induced signaling events, including the increase in cytosolic [Cl ؊ ], endosomal acidification, ceramide formation, and ROS generation as well as CD95 targeting to the plasma membrane and CD95 activation. The data suggest that CD95L initiates a rapid caspase 8-dependent endosomal acidification, which triggers ceramide-dependent ROS formation as an upstream event of trafficking of intracellularly stored CD95 to the plasma membrane. It is concluded that a rapid caspase 8 activation in response to CD95L signals to intracellularly stored CD95, which becomes activated and targeted to the plasma membrane. This autoamplification of CD95-activation is required for apoptosis induction. CD952 (Apo-1/Fas) belongs to the death receptor family and plays an important role in apoptosis induction in many cell types. In hepatocytes, CD95 is mainly located within the cellular interior and can either be activated after ligation with its natural ligand (CD95L) or in a ligand-independent way by hydrophobic bile salts or hyperosmotic cell shrinkage (Refs. 1-4; for review, see Ref. 5). Ligand-dependent and -independent CD95 activation in hepatocytes is a complex process that finally results in CD95 trafficking from the cellular interior to the plasma membrane, subsequent formation of the death-inducing signaling complex (DISC), and eventually apoptosis (1-12).In hepatocytes, proapoptotic stimuli such as CD95 ligand (CD95L), hydrophobic bile salts, or hyperosmotic cell shrinkage induce a rapid oxidative stress (ROS) response (2, 4), which triggers a Yes-dependent but ligand-independent activation of the epiderm...

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“…It could thus be assumed that in the polarized IRHC, the CP-/HCO-exchanger is located on the apical side and the two acid extruders are located on the basolateral side. Electrophysiological studies on IRHC have demonstrated that the resistances of tight junctions that seal the canalicular lumen from the bathing media are comparable with those of leaky epithelia and allow rapid ion equilibration between these two compartments (38). This implies that the behavior of the Cl-/HCO3 in response to Cl -removal/readmission from the bathing media should be the same whether the exchanger is restricted to the canalicular domain of IRHC or not.…”

Section: Discussionmentioning

confidence: 99%

Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

Alvaro¹,

Guardia²,

Bini³

et al. 1995

J. Clin. Invest.

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To elucidate mechanisms of glucagon-induced bicarbonaterich choleresis, we investigated the effect of glucagon on ion transport processes involved in the regulation of intracellular pH (pH1) in isolated rat hepatocyte couplets. It was found that glucagon (200 nM), without influencing resting pH1, significantly stimulates the Cl-/HCO3 exchange activity.The effect of glucagon was associated with a sevenfold increase in cAMP levels in rat hepatocytes. The activity of the Cl-/HCO3-exchanger was also stimulated by DBcAMP + forskolin. The effect of glucagon on the Cl-/HCO3 exchange was individually blocked by two specific and selective inhibitors of protein kinase A, uM) and H-89 (30 ,uM), the latter having no influence on the glucagon-induced cAMP accumulation in isolated rat hepatocytes. The Cl -channel blocker, NPPB (10 pmM), showed no effect on either the basal or the glucagon-stimulated Cl-/ HCO3 exchange. In contrast, the protein kinase C agonist, PMA (10 jpM), completely blocked the glucagon stimulation of the C1-/HCO3 exchange; however, this effect was achieved through a significant inhibition of the glucagonstimulated cAMP accumulation in rat hepatocytes. Colchicine pretreatment inhibited the basal as well as the glucagon-stimulated Cl-/HCO3 exchange activity. The Na+/H+ exchanger was unaffected by glucagon either at basal pH1 or at acid pH, values. In contrast, glucagon, at basal pH1, stimulated the Na+-HCO-symport. The main findings of this study indicate that glucagon, through the cAMP-dependent protein kinase A pathway, stimulates the activity of the Cl-/HCO3 exchanger in isolated rat hepatocyte couplets, a mechanism which could account for the in vivo induced bicarbonate-rich choleresis. (J. Clin. Invest. 1995.

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Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets

Cited by 83 publications

References 37 publications

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

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

Amplification of CD95 Activation by Caspase 8-induced Endosomal Acidification in Rat Hepatocytes

Effect of glucagon on intracellular pH regulation in isolated rat hepatocyte couplets.

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