Anion Transport Pathways Research Articles

Molecular mechanism underlying regulation of Arabidopsis CLCa transporter by nucleotides and phospholipids

Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels and anion/proton antiporters. Arabidopsis thaliana CLCa (AtCLCa) transporter localizes to the tonoplast which imports NO3− and to a less extent Cl− from cytoplasm. The activity of AtCLCa and many other CLCs is regulated by nucleotides and phospholipids, however, the molecular mechanism remains unclear. Here we determine the cryo-EM structures of AtCLCa bound with NO3− and Cl−, respectively. Both structures are captured in ATP and PI(4,5)P2 bound conformation. Structural and electrophysiological analyses reveal a previously unidentified N-terminal β-hairpin that is stabilized by ATP binding to block the anion transport pathway, thereby inhibiting the AtCLCa activity. While AMP loses the inhibition capacity due to lack of the β/γ- phosphates required for β-hairpin stabilization. This well explains how AtCLCa senses the ATP/AMP status to regulate the physiological nitrogen-carbon balance. Our data further show that PI(4,5)P2 or PI(3,5)P2 binds to the AtCLCa dimer interface and occupies the proton-exit pathway, which may help to understand the inhibition of AtCLCa by phospholipids to facilitate guard cell vacuole acidification and stomatal closure. In a word, our work suggests the regulatory mechanism of AtCLCa by nucleotides and phospholipids under certain physiological scenarios and provides new insights for future study of CLCs.

New Anionophores and Insights into Ion-Transport-Induced Cancer Cell Death

In this issue of Chem, Pellois, Gabbai, and colleagues introduce pnictogenium cations as useful transmembrane anion transporters, and Gale, Sessler, Injae, and co-workers provide insights into the cellular mechanism elicited by some of these compounds, leading to the induction of apoptosis in cancer cells.

91 - Unveil mitochondrial superoxide anion transport pathway in ischemia/reperfusion injury of the mouse liver via two-photon fluorescence imaging

Monitor dynamic changes in mitochondrial and cytoplasmic superoxide anion (O2•−) levels can unveil O2•− signalling pathway of hepatic ischemia/reperfusion (IR) injury and find relative therapeutic target. However, reversible and ratio two-photon fluorescence probe for mitochondrial O2•− hasn’t been presented. Therefore, we developed a reversible and ratio two-photon fluorescence probe for mitochondrial O2•−. With the aid of a cytoplasm O2•− probe and two-photon fluorescent microscopy, the crosstalk between mitochondrial O2•− and cytoplasmic O2•− has been explored. We found that mitochondrial O2•− transported to cytoplasm via VDAC channel in hepatic ischemia/reperfused mice. Furthermore, blocking VDAC channel and increasing mitochondrial Mn-SOD may reduce the risk of IR injury death. Our work uncovers mitochondria O2•− possible transport pathway and molecular mechanism of IR injury, also provides a new therapeutic strategy for IR injury.

Generation and Control of Electrohydrodynamic Flows in Aqueous Electrolyte Solutions.

To drive electrohydrodynamic (EHD) flows in aqueous solutions, the separation of cation and anion transport pathways is essential because a directed electric body force has to be induced by ionic motions in liquid. On the other hand, positive and negative charges attract each other, and electroneutrality is maintained everywhere in equilibrium conditions. Furthermore, an increase in an applied voltage has to be suppressed to avoid water electrolysis, which causes the solutions to become unstable. Usually, EHD flows can be induced in non-aqueous solutions by applying extremely high voltages, such as tens of kV, to inject electrical charges. In this study, two methods are introduced to generate EHD flows induced by electrical charge separations in aqueous solutions, where two liquid phases are separated by an ion-exchange membrane. Due to a difference in the ionic mobility in the membrane, ion concentration polarization is induced between both sides of the membrane. In this study, we demonstrate two methods. (i) The relaxation of ion concentration gradients occurs via a flow channel that penetrates an ion-exchange membrane, where the transport of the slower species in the membrane selectively becomes dominant in the flow channel. This is a driving force to generate an EHD flow in the liquid. (ii) A long waiting time for the diffusion of ions passing through the ion-exchange membrane enables the generation of an ion-dragged flow by externally applying an electric field. Ions concentrated in a flow channel of a 1 x 1 mm2 cross-section determine the direction of the liquid flow, corresponding to the electrophoretic transport pathways. In both methods, the electric voltage difference required for an EHD flow generation is drastically reduced to near 2 V by rectifying the ion transport pathways.

Oxygen vacancy localization and anisotropic oxygen anion transport in Sr1−xYxCoO3−δ (x = 0.1, 0.2) under solid oxide fuel cell cathode conditions

In-situ neutron diffraction was utilized to study the influence of Y-doping on the crystal structure, oxygen non-stoichiometry, and oxygen anion transport pathways in Sr1−xYxCoO3−δ at temperatures and oxygen partial pressures typical of SOFC cathode conditions, from 500 °C to 800 °C, and oxygen partial pressure, pO2, between 10−3 atm and 1 atm. The resulting diffraction patterns of Sr0.9Y0.1CoO3−δ and Sr0.8Y0.2CoO3−δ were fit to tetragonal space groups P4/mmm and I4/mmm, respectively. While prior reports have suggested a degree of superstructuring in these materials at room temperature, our elevated temperature data demonstrates that this is not present under SOFC conditions. Both materials show similar oxygen stoichiometry with preferential oxygen vacancy formation at the equatorial sites of the Co-centered octahedra. While these equatorial sites are equivalent in Sr0.9Y0.1CoO3−δ, further vacancy localization within Sr0.8Y0.2CoO3−δ leads to a structure consisting of vacancy rich and vacancy poor Co-centered octahedra alternating in the c-axis direction. Both structures suggest a sinusoidal oxygen anion transport pathway via the high vacancy sites, further localized to the alternating vacancy rich layers in Sr0.8Y0.2CoO3−δ. A secondary vacancy ordered Brownmillerite phase was observed at 600 °C and below at the lowest pO2 for Sr0.9Y0.1CoO3−δ.

Concentration dependence of cation-induced electrohydrodynamic flow passing through an anion exchange membrane

Electrohydrodynamic (EHD) flow is a type of liquid flow driven by an external electric force. In electrolyte solutions, anions and cations usually interact with each other to maintain electroneutrality. Under such a condition, it is difficult to drive a liquid flow by applying electric potentials on the order of 1 V; at least a few tens of volts is required to generate EHD flows, which may not be preferable for aqueous solutions. In this study, we propose a novel method of generating a liquid flow through a channel with cross-sectional dimensions of 1 × 1 mm2, which is placed in an ion exchange membrane to separate the cation and anion transport pathways. When the optimized design of the experimental apparatus was used, EHD flows were successfully generated in aqueous solutions by applying a relatively low electric potential of 2.2 V, and the flow velocity was measured over a wide range of electrolyte concentrations by particle image velocimetry. It was found that high concentration gradients caused the rapid discharge of ions passing through the channel and contributed to achieving a flow speed on the order of 1 mm/s. EHD flows were also theoretically explained using the Navier–Stokes equations to model an ion-drag flow driven by nonequilibrium ion transport in external electric fields. This flow generation method is practical only when ion transport pathways are well controlled and effectively rectified. The present findings will lead to the development of a promising technology to control liquid flows in multiscale fluidic channels.

Carbonate Dynamics and Opportunities With Low Temperature, Anion Exchange Membrane-Based Electrochemical Carbon Dioxide Separators

Fossil fuel power plants are responsible for a significant portion of anthropogenic atmospheric carbon dioxide (CO2) and due to concerns over global climate change, finding solutions that significantly reduce emissions at their source has become a vital concern. When oxygen (O2) is reduced along with CO2 at the cathode of an anion exchange membrane (AEM) electrochemical cell, carbonate and bicarbonate are formed which are transported through electrolyte by migration from the cathode to the anode where they are oxidized back to CO2 and O2. This behavior makes AEM-based devices scientifically interesting CO2 separation devices or “electrochemical CO2 pumps.” Electrochemical CO2 separation is a promising alternative to the state-of-the-art solvent-based methods because the cells operate at low temperatures and scale with surface area, not volume, suggesting that the industrial electrochemical systems could be more compact than amine sorption technologies. In this work, we investigate the impact of the CO2 separator cell potential on the CO2 flux, carbonate transport mechanism, and process costs. The applied electrical current and CO2 flux showed a strong correlation that was both stable and reversible. The dominant anion transport pathway, carbonate versus bicarbonate, undergoes a shift from carbonate to mixed carbonate/bicarbonate with increased potential. A preliminary techno-economic analysis shows that despite the limitations of present cells, there is a clear pathway to meet the U.S. Department of Energy (DOE) 2025 and 2035 targets for power plant retrofit CO2 capture systems through materials and systems-level advances.

Live tissue imaging of organic cation and anion transport at the blood‐CSF barrier reveals specific transporter function and distinct transcellular pathways

Formed by the choroid plexus (CP) epithelial cells, the blood‐cerebrospinal fluid (CSF) barrier plays an active role in removing neurotoxins, drugs, and metabolites from the brain. Several organic cation and anion transporters, including the plasma membrane monoamine transporter (PMAT), organic anion transporter 1 and 3 (OAT1/3), and multidrug resistance‐associated protein 1 and 4 (MRP1/4) have been reported to be expressed in CP, but their relative expression and specific function at the blood‐CSF barriers have not been well characterized at the tissue level. The goal of this study is to quantify the expression of organic cation and anion transporters in CP tissues and determine their specific roles in organic cation or anion transport by live tissue imaging using fluorescent substrates. In both human and mouse CPs, real‐time PCR analysis showed highest mRNA expression of PMAT followed by MRP1, OAT3 and MRP4. CP also expresses several drug metabolizing enzymes including the monoamine oxidases (MAOs), catechol‐O‐methyltransferase (COMT), UDP‐glucuronosyltransferases (UGTs) and sulfotransferases (SULT). In CP isolated from wildtype (wt) mouse, the fluorescent organic cation probe IDT307 is transported into CP cells at the apical (CSF‐facing) membrane and is highly accumulated in CP cells with a large storage in mitochondria. There was little efflux at the basolateral membrane. In contrast, IDT307 uptake and intracellular accumulation is attenuated by ~3–4 folds in CPs isolated from Pmat knockout mice. Interestingly, in wt CPs, the organic anion probe fluorescein‐methotrexate (FL‐MTX) is rapidly taken up and efficiently excreted into the capillary side with little intracellular accumulation. MK571, a potent inhibitor of MRP1/4, abolished basolateral efflux of FL‐MTX, resulting in high accumulation within CP cells. Together, our study in live CP tissue demonstrates an important role of PMAT in apical uptake of organic cations and a crucial function of MRP1/4 in basolateral efflux of organic anions. Based on these studies, we propose distinct molecular and cellular pathways for organic cation and anion transport at the blood‐CSF barrier.Support or Funding InformationThis study is supported by National Institutes of Health Grant GM066233.

Mutations associated with Dent's disease affect gating and voltage dependence of the human anion/proton exchanger ClC-5

Dent's disease is associated with impaired renal endocytosis and endosomal acidification. It is linked to mutations in the membrane chloride/proton exchanger ClC-5; however, a direct link between localization in the protein and functional phenotype of the mutants has not been established until now. Here, two Dent's disease mutations, G212A and E267A, were investigated using heterologous expression in HEK293T cells, patch-clamp measurements and confocal imaging. WT and mutant ClC-5 exhibited mixed cell membrane and vesicular distribution. Reduced ion currents were measured for both mutants and both exhibited reduced capability to support endosomal acidification. Functionally, mutation G212A was capable of mediating anion/proton antiport but dramatically shifted the activation of ClC-5 toward more depolarized potentials. The shift can be explained by impeded movements of the neighboring gating glutamate Gluext, a residue that confers major part of the voltage dependence of ClC-5 and serves as a gate at the extracellular entrance of the anion transport pathway. Cell surface abundance of E267A was reduced by ~50% but also dramatically increased gating currents were detected for this mutant and accordingly reduced probability to undergoing cycles associated with electrogenic ion transport. Structurally, the gating alternations correlate to the proximity of E267A to the proton glutamate Gluin that serves as intracellular gate in the proton transport pathway and regulates the open probability of ClC-5. Remarkably, two other mammalian isoforms, ClC-3 and ClC-4, also differ from ClC-5 in gating characteristics affected by the here investigated disease-causing mutations. This evolutionary specialization, together with the functional defects arising from mutations G212A and E267A, demonstrate that the complex gating behavior exhibited by most of the mammalian CLC transporters is an important determinant of their cellular function.

Visualizing oxygen anion transport pathways in NdBaCo2O5+δ by in situ neutron diffraction

The layered perovskite NdBaCo2O5+δ (NBCO) was characterized using neutron powder diffraction under in situ conditions from 577–852 °C and in 10−1 to 10−4 atm oxygen. The best fit to the data was obtained in the tetragonal (P4/mmm) space group. No oxygen atom vacancy ordering was observed that warranted a lowering of the symmetry to orthorhombic (Pmmm). Two P4/mmm structural models were investigated: Model 1 (no split sites) and Model 2 (split Nd and O2 sites). Transport of oxygen through the material via the vacancy hopping mechanism likely involves the nearest-neighbor oxygen atom sites in the Nd layer. Total oxygen stoichiometry values were in the range 5.51 ≤ δ ≤ 5.11. The tetragonal lattice parameters increased with temperature as expected. However, the a-axis expands while the c-axis contracts with decreasing pO2 at a given temperature due to increasing vacancy concentration in the Nd layer.

Organic anion transport pathways in antiviral handling in choroid plexus in Oat1 (Slc22a6) and Oat3 (Slc22a8) deficient tissue

Transporters in the choroid plexus (CP) regulate transport of numerous compounds of physiological and therapeutic interest between blood and CSF and thus likely play a key role in determining CNS levels of drugs, toxins and metabolites. Here, high CP expression was noted for the organic anion transporters, Oat1 (SLC22A6 or NKT) and Oat3 (SLC22A8) which are also the principal Oats in the renal proximal tubule, as well as SLC22A17, hypothesized to be involved in iron transport. Because Oat1 and Oat3 have overlapping substrate specificity, ex vivo preparations of CP from Oat1(−/−) and Oat3(−/−) mice were used to isolate the individual transport function of each, respectively. Tissue from either knockout mouse mediated the probenecid-inhibitable transport of the Oat substrate, 6-carboxyfluorescein (6CF), confirming the presence of Oat1 and Oat3 function. Because many antiviral medications are Oat substrates, including those crucial in the treatment of HIV infections, the interaction of the antivirals zidovudine, acyclovir, tenofovir, lamivudine, and stavudine, with Oat1 and Oat3 in CP, was investigated by determining the inhibition of 6CF uptake. All the antivirals tested manifested significant interaction with both Oat1 and Oat3, with the exception of stavudine which did not significantly affect Oat1 function. These results could have important implications for antiretroviral (and other drugs) penetration into or retention within the CNS, a major reservoir for virus during HIV infection. Apart from any effect at the blood brain barrier (BBB), designing specific inhibitors of Oat1 and Oat3 may be helpful in altering CNS drug levels by blocking organic anion transporters in the CP. The role of SLC22A17 in the CP deserves further exploration. The ability of Oats to regulate the movement of small molecules across the BBB, CP, proximal tubule and other tissues may also be important for their role in remote sensing and signaling [1,21]).

Expression and function of renal and hepatic organic anion transporters in extrahepatic cholestasis

Obstructive jaundice occurs in patients suffering from cholelithiasis and from neoplasms affecting the pancreas and the common bile duct. The absorption, distribution and elimination of drugs are impaired during this pathology. Prolonged cholestasis may alter both liver and kidney function. Lactam antibiotics, diuretics, non-steroidal anti-inflammatory drugs, several antiviral drugs as well as endogenous compounds are classified as organic anions. The hepatic and renal organic anion transport pathways play a key role in the pharmacokinetics of these compounds. It has been demonstrated that acute extrahepatic cholestasis is associated with increased renal elimination of organic anions. The present work describes the molecular mechanisms involved in the regulation of the expression and function of the renal and hepatic organic anion transporters in extrahepatic cholestasis, such as multidrug resistance-associated protein 2, organic anion transporting polypeptide 1, organic anion transporter 3, bilitranslocase, bromosulfophthalein/bilirubin binding protein, organic anion transporter 1 and sodium dependent bile salt transporter. The modulation in the expression of renal organic anion transporters constitutes a compensatory mechanism to overcome the hepatic dysfunction in the elimination of organic anions.

The Cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes

Lysosomes are the stomachs of the cell-terminal organelles on the endocytic pathway where internalized macromolecules are degraded. Containing a wide range of hydrolytic enzymes, lysosomes depend on maintaining acidic luminal pH values for efficient function. Although acidification is mediated by a V-type proton ATPase, a parallel anion pathway is essential to allow bulk proton transport. The molecular identity of this anion transporter remains unknown. Recent results of knockout experiments raise the possibility that ClC-7, a member of the CLC family of anion channels and transporters, is a contributor to this pathway in an osteoclast lysosome-like compartment, with loss of ClC-7 function causing osteopetrosis. Several mammalian members of the CLC family have been characterized in detail; some (including ClC-0, ClC-1 and ClC-2) function as Cl--conducting ion channels, whereas others act as Cl-/H+antiporters (ClC-4 and ClC-5). However, previous attempts at heterologous expression of ClC-7 have failed to yield evidence of functional protein, so it is unclear whether ClC-7 has an important function in lysosomal biology, and also whether this protein functions as a Cl- channel, a Cl-/H+ antiporter, or as something else entirely. Here we directly demonstrate an anion transport pathway in lysosomes that has the defining characteristics of a CLC Cl-/H+ antiporter and show that this transporter is the predominant route for Cl- through the lysosomal membrane. Furthermore, knockdown of ClC-7 expression by short interfering RNA can essentially ablate this lysosomal Cl-/H+ antiport activity and can strongly diminish the ability of lysosomes to acidify in vivo, demonstrating that ClC-7 is a Cl-/H+ antiporter, that it constitutes the major Cl- permeability of lysosomes, and that it is important in lysosomal acidification.

Anion transport pathways involved in PG E2 and acid-stimulated murine duodenal bicarbonate secretion in vivo

Anion transport pathways involved in PG E2 and acid-stimulated murine duodenal bicarbonate secretion in vivo

Identity and regulation of ion transport mechanisms in the corneal endothelium.

Corneal transparency is dependent on regulation of the hydration of the corneal stroma. Water is driven into the cornea across the epithelial and endothelial cell layers by the stromal swelling pressure. This fluid leak into the cornea is counterbalanced by the corneal fluid pump, which is predominantly attributed to the ion and fluid transport capacity of the endothelial cell layer. Primary and secondary active transport mechanisms are responsible for generating a net ion flux from the stromal to anterior chamber side of the endothelium; however, the identity and location of all the components of this transport system are not known. The endothelial fluid pump is dependent on the presence of Cl(-) and HCO(3)(-), and can be slowed by carbonic anhydrase inhibitors. A number of anion transport mechanisms have been identified and characterized in the endothelium, including basolateral Na(+)/2HCO(3)(-) cotransport, Na(+)/K(+)/2Cl(-) cotransport, Cl(-)/HCO(3)(-) exchange, and apical anion channels permeable to both Cl(-) and HCO(3)(-). Furthermore, there is evidence for a carbonic anhydrase mediated CO(2)-diffusive mode of apical HCO(3)(-) flux. These findings are incorporated into a new model of transendothelial anion transport, which suggests that there are a number of alternate pathways for anion transport. There have been few studies on activation of signal transduction pathways that could stimulate endothelial fluid transport. Interestingly, recent studies show that multiple autocrine signaling pathways are in place that could be upregulated during physical stimulation and may be responsible for maintaining basal levels of fluid secretion.

Molecular identification of the multispecific organic anion transporter family (the OAT family): the role in the pharmacokinetics and toxicokinetics

The multispecific organic anion transporters have been indicated to be involved in the transmembrane transport of various anionic substances. The kidney and liver possess the distinct organic anion transport pathways for the elimination of potentially toxic anionic drugs and metabolites. In the kidney, proximal tubular cells actively excrete organic anions of both endogenous and exogenous origin. We have isolated the renal multispecific organic anion transporter, OAT1 (organic anion transporter 1), from the rat kidney. OAT1 is a 551-amino acid residue protein with 12 putative membrane spanning domains. OAT1 mediates sodium-independent, anion exchange for a variety of organic anions including p-aminohippurate, cyclic nucleotides, prostanoides, dicarboxylates, and anionic drugs including beta-lactams, non-steroidal antiinflammatory drugs, diuretics and antiviral drugs. So far, three other isoforms have been identified. OATs comprise a new family of multispecific organic anion transporter, i.e., the OAT family. OATs show weak structural similarity to organic cation transporters (OCTs) and OCTN/carnitine transporters. All of the members of the OAT family are commonly expressed in the kidney, suggesting its significance in the renal organic anion excretion. In addition, OAT members appear to be responsible for the distribution/elimination of water soluble anionic drugs into/from the liver, brain and fetus.

Modes of control of insect Malpighian tubules: synergism, antagonism, cooperation and autonomous regulation

Rates of fluid and ion secretion by insect Malpighian tubules are controlled by peptides, including CRF-related peptides and kinins, and in some species by serotonin. It now appears to be a general rule that tubule secretion rate is controlled through the interaction of two or more haemolymph-borne factors. In this review we suggest that these interactions may be classified as synergistic, cooperative, or antagonistic. When presented together, two diuretic factors may act in synergism, so that fluid secretion is stimulated to a greater extent than the sum of their individual effects. Synergism may involve one or more second messenger systems. Alternatively, diuretic factors may act in cooperation, so that although their overall effects are additive, cation and anion transport pathways are controlled separately by distinct second messenger systems. There is also one example of antagonism between factors controlling tubule secretion and between their respective second messengers; one factor is stimulatory, the other is inhibitory. In addition to the complex control of fluid and ion transport by haemolymph-borne factors, sophisticated autonomous regulatory mechanisms have been identified in Malpighian tubules. When triggered by appropriate stimuli, these mechanisms play homeostatic roles, preserving haemolymph osmolality or ionic composition.

Ochratoxin A secretion in primary cultures of rabbit renal proximal tubule cells.

Primary cultures of rabbit renal proximal tubule cells grown under improved culture conditions were used to study the transepithelial transport of the nephrotoxic mycotoxin ochratoxin A. The basal-to-apical transepithelial flux, i.e., secretion, of this fluorescence organic acid was measured in primary cultures of rabbit renal proximal tubule cells. The basal-to-apical flux of ochratoxin A increased with time and reached a steady state after 12 h. On the other hand, the apical-to-basal flux, i.e., reabsorption, of ochratoxin A was minimal over time. The secretory flux of ochratoxin A was as much as eightfold greater than the reabsorptive flux, indicating that net secretion is the primary mechanism for ochratoxin A clearance by the proximal tubule. The kinetic analysis of ochratoxin A flux revealed secretion to be a saturable and very high-affinity process with an apparent K50 of 0.33 +/- 0.21 mM. A saturating concentration of the prototypical organic anion substrate para-aminohippurate (PAH) reduced ochratoxin A secretion by approximately 75%. The kinetic analysis of PAH inhibition of ochratoxin A secretion revealed an IC50 of 195 mM, which is similar to the IC50 for PAH inhibition of peritubular ochratoxin A uptake in tubule suspensions and the Km, values for peritubular PAH uptake. The organic anions probenecid, octanoate, and alpha-ketoglutarate reduced ochratoxin A excretion to the same degree as PAH, whereas the amino acid phenylalanine had a minimal effect on ochratoxin A secretion. Thus, collectively, these observations indicate that the secretion of ochratoxin A in primary cultures of rabbit renal proximal tubules is limited to the organic anion secretory pathway. The high affinity measured for the basal-to-apical flux of ochratoxin A suggests that at concentrations typical of naturally occurring exposures, transepithelial secretion by the organic anion transport pathway represents a significant avenue for excretion of this mycotoxin by the renal proximal tubule.

Human proximal duodenal alkaline secretion is mediated by Cl-/HCO3- exchange and HCO3- conductance.

The proximal duodenal epithelium secretes bicarbonate into an adherent mucus layer, thereby protecting the mucosa from injury by gastric acid and pepsin. While bicarbonate secretion is stimulated and inhibited by a number of agonists and antagonists, the apical anion transport pathways have not been addressed fully. The objective was to assess if apical Cl-/HCO3- exchange and Cl-:HCO3- conductance are involved in duodenal mucosal bicarbonate secretion (DMBS). In healthy volunteers, the proximal 4 cm of duodenum was isolated, perfused with either saline or 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS), and bicarbonate secretion and transepithelial potential difference (PD) were stimulated by either PGE2 or the phosphodiesterase inhibitor theophylline to increase cyclic AMP. Luminal DIDS abolished PGE2-stimulated DMBS, yet had no effect on the increase in PD and failed to significantly alter theophylline-induced DMBS and PD. Therefore, in human proximal duodenum, it appears that PGE2 and cAMP activate distinct HCO3- transport pathways likely involving a DIDS-sensitive Cl-/HCO3- exchanger and DIDS-insensitive HCO3- conductance.

Disruption of actin organization by cytochalasin D does not impair biliary secretion of organic anions in the rat.

The bile canaliculi of hepatocytes contract spontaneously, and it is hypothesized that this canalicular motility provides a propulsive force for normal intrahepatic bile flow. Cytochalasin disrupts actin polymerization, inhibits contraction, and decreases bile flow. We investigated whether this cholestasis was associated with impaired canalicular secretion. Isolated rat hepatocyte doublets, with and without incubation with 2 mumol/L cytochalasin D (cytD), were superfused, under first-order conditions, to steady state with fluorescein isothiocyanate-labeled glycocholic acid (FITC-GC) and carboxy-4',5'-dimethylfluorescein diacetate (CMFD), which are fluorescent substrates for the bile acid and the nonbile acid organic anion transport pathways, respectively. Fluorescent microscopic images were quantified and the data analyzed by noncompartmental and compartmental kinetic methods. cytD dilated the canalicular spaces fivefold but did not change the proportion of doublets that secreted either probe. Cytochalasin did not affect the mean cellular transit times of FITC-GC (2.8 and 2.5 minutes for control and cytochalasin-treated groups, respectively) and of carboxy-4',5'-dimethylfluorescein (3.8 and 3.7 minutes, respectively). Analysis with a three-compartment model gave estimates of the rate constants for canalicular secretion: 0.21 +/- 0.04 and 0.22 +/- 0.03 min-1 in control and treated cells, respectively, for FITC-GC, and 0.14 +/- 0.01 and 0.16 +/- 0.02 min-1, respectively, for carboxy-dimethylfluorescein. When kinetics are first-order, the canalicular secretion of organic anions is not altered by actin disruptive agents, suggesting that actin filaments do not modulate the function or distribution of these transporters. This suggests that impaired contractility rather than impaired canalicular secretion is the mechanism of cytD-induced cholestasis.