Richard A. Weisiger

Cytoplasmic transport of lipids: Role of binding proteins

Abstract After entering the cell, small molecules must penetrate the cytoplasm before they are metabolized, excreted or can convey information to the cell nucleus. Without efficient cytoplasmic transport, most such molecules would efflux back from the cell before they could reach their targets. Conversely, intracellular lipids generated by hydrolysis of triglycerides, phospholipids and other esters must be transported away from their site of formation to prevent toxic accumulation. Intracellular movement of all molecules is slowed by molecular crowding, tortuosity, and the greater viscosity of the cytosol relative to water. However, lipids and other amphipathic molecules are further slowed by their tendency to bind to cytoplasmic membranes. Cytoplasmic binding proteins reduce membrane binding by increasing the aqueous solubility of their ligands. These aqueous carriers catalyze the transport of lipid molecules across hydrophilic water layers just as plasma membrane carriers catalyze the transport of hydrophilic molecules across the hydrophobic membrane core. They even display the principal features of carrier-mediated transport, including saturation, mutual competition, and countertransport. Higher concentrations of cytoplasmic binding proteins are associated with more rapid cytoplasmic transport of longchain fatty acids. Available data suggest that substantial intracellular concentration gradients of fatty acids should exist, and that these gradients may help determine which metabolic pathway the fatty acid enters. Thus, cytoplasmic carrier proteins may help regulate the uptake and metabolism of fatty acids and other lipid molecules.

Hepatic oleate uptake. Electrochemical driving forces in intact rat liver.

Recent observations suggest that the hepatic uptake of oleate may be sodium coupled. To assess the electrochemical forces driving fatty acid uptake, we used microelectrodes to monitor continuously the electrical potential difference across the plasma membrane in the perfused rat liver while simultaneously monitoring the rate of tracer [3H]oleate uptake from 1% albumin solutions. Isosmotic cation or anion substitution was used to vary the potential difference over the physiologic range. Depolarization of cells from -29 to -19 mV by substituting gluconate for chloride reduced steady-state oleate uptake by 34%. Conversely, hyperpolarization of cells to -52 mV by substituting nitrate for chloride increased uptake by 41%. Replacement of perfusate sodium with choline depolarized the cells to -18 mV and reduced uptake by 58%, an amount greater than expected from the degree of depolarization alone. Oleate in higher concentrations (1.5 mM in 2% albumin) depolarized cells by 3 mV in the presence of sodium, but had no effect in sodium-free buffer. These results suggest that a portion of oleate uptake in the intact liver occurs by electrogenic sodium cotransport. Uptake appears to be driven by both the electrical and sodium chemical gradients across the plasma membrane.

Role of cytoarchitecture in cytoplasmic transport

Abstract Cytoplasm is a thick viscoelastic gel consisting of a highly concentrated protein solution interspersed through a matrix of cytoskeletal filaments and organellar structures. Diffusion of soluble low-molecular-weight molecules through cytoplasm is slowed by a factor of 5–10 relative to a dilute aqueous solution, reflecting increased solvent viscosity, molecular crowding, tortuosity imposed by cytoplasmic membranes and organelles, and transient binding to immobile structures. Larger molecules are further slowed by cytoskeletal sieving. Messenger RNA and ribosomes may be prevented from entering certain parts of cytoplasm due to their size and may follow preferential channels as they exit the nucleus. Mechanisms also exist for energy-dependent transport of larger molecules and organelles

Specific and saturable binding of albumin to rat adipocytes: Modulation by epinephrine and possible role in free fatty acid transfer

Abstract Specific and saturable binding of 125I-bovine albumin to rat adipocytes in suspension was observed (apparent Kd 2.09 ± 0.52 × 10−6 M; 8.58 ± 2.49 × 106 sites per cell; mean ± SEM). The binding was rapid and reversible for at least 10 min, suggesting that endocytosis of albumin was minor under assay conditions. Pre-incubation of cells with epinephrine bitartrate caused an apparent increase in number and decrease in affinity of the adipocyte binding sites for albumin. These findings suggest that a specific and saturable interaction of albumin with the adipocyte surface may play a role in the cellular uptake and release of free fatty acids.

Extending the multiple indicator dilution method to include slow intracellular diffusion

The traditional multiple indicator dilution (MID) method is extended to incorporate cytoplasmic concentration gradients due to slow intracellular diffusion of the permeable molecule. The new model is governed by a system of partial differential equations that are solved using Laplace transformation. An analysis of the transformed solution shows that the traditional MID method is a special case of the extended model. We then use simulation analysis to show that the traditional MID model and the new diffusion model generate similar outflow curves. However, when the traditional MID equations were used to analyze outflow curves generated using a system in which intracellular diffusion is slow compared to other transport processes, the recovered rate constants for the transmembrane and excretion processes were incorrect. The diffusion model permits estimation of the rate of intracellular transport of amphipathic molecules from suitable indicator dilution data.

Sex differences in sulfobromophthalein-glutathione transport by perfused rat liver

Sex differences have been described in the hepatic transport of many organic anions. Proposed mechanisms include differences in the rate of metabolism, in the degree of binding to cytoplasmic proteins, and in the rate of membrane transport. To better define these factors, we used the perfused rat liver to study the hepatic transport of the glutathione conjugate of sulfobromophthalein (BSP-GSP), a model compound that does not require metabolism for excretion. Hepatic transport of BSP-GSH was saturable for both sexes. Clearance of BSP-GSH from 1% albumin solutions at steady-state was 35-52% greater in female livers than in male livers, and reflected a 47% larger apparent Vmax with no change in the apparent Km. Analysis of the rate of disappearance of BSP-GSH from recirculating perfusate and its appearance in bile using a simple two-compartment model indicated that the ratio of influx to efflux was greater in female livers. These findings are compatible with sex-related differences in the electrochemical driving forces for BSP-GSH uptake.

Uptake and metabolism of polymerized albumin by rat liver. Role of the scavenger receptor.

Hepatitis B virus binds avidly to albumin polymers, which in turn may mediate viral attachment to liver cells. This hypothesis is critically dependent on prior results obtained using glutaraldehyde-polymerized human serum albumin as a model for naturally occurring albumin species. We used the perfused rat liver to characterize the uptake, cellular distribution, and metabolism of glutaraldehyde-polymerized human albumin. 125I-glutaraldehyde-polymerized human albumin was efficiently removed from the perfusate by the liver (29% extraction). However, few autoradiographic grains were located over hepatic parenchymal cells (6%). Instead, most glutaraldehyde-polymerized human albumin appeared to be removed by endothelial (59%) or Kupffer (31%) cells. Hepatic uptake was strongly inhibited by formaldehyde-treated monomeric albumin, a known ligand of the endothelial scavenger receptor for chemically modified proteins. After uptake, most glutaraldehyde-polymerized human albumin was rapidly degraded and released into the perfusate (74% within 60 min). This process was blocked by chloroquine and leupeptin, suggesting that it involves lysosomal acid hydrolases. We conclude that glutaraldehyde-polymerized albumin is efficiently cleared and degraded by the endothelial scavenger pathway. Glutaraldehyde-polymerized albumin therefore appears to be a poor model for predicting the hepatic handling of naturally occurring albumin species bound to hepatitis B virions. Even if viral particles were to follow this pathway, few would enter parenchymal hepatocytes.

HCO3 --coupled Na+ influx is a major determinant of Na+ turnover and Na+/K+ pump activity in rat hepatocytes

SummaryRecent studies in hepatocytes indicate that Na+-coupled HCO3− transport contributes importantly, to regulation of intracellular pH and membrane HCO3− transport. However, the direction of net coupled Na+ and HCO3− movement and the effect of HCO3− on Na+ turnover and Na+/K+ pump activity are not known. In these studies, the effect of HCO3− on Na+ influx and turnover were measured in primary rat hepatocyte cultures with22Na+, and [Na+]i was measured in single hepatocytes using the Na+-sensitive fluorochrome SBFI. Na+/K+ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na+-dependent or ouabain-suppressible86Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K+ on membrane potential difference (PD) and [Na+]i. In hepatocyte monolayers, HCO3− increased22Na+ entry and turnover rates by 50–65%, without measurably altering22Na+ pool size or cell volume, and HCO3− also increased Na+/K+ pump activity by 70%. In single cells, exposure to HCO3− produced an abrupt and sustained rise in [Na+]i, from ≈8 to 12mm. Na+/K+ pump activity assessed in single cells by PD excursions during transient K+ removal increased ≃2.5-fold in the presence of HCO3−, and the rise in [Na+]i produced by inhibition of the Na+/K+ pump was similarly increased ≃2.5-fold in the presence of HCO3−. In intact perfused rat liver, HCO3− increased both Na+/K+ pump activity and O2 consumption. These findings indicate that, in hepatocytes, net coupled Na+ and HCO3− movement is inward and represents a major determinant of Na+ influx and Na+/K+ pump activity. About half of hepatic Na+/K+ pump activity appears dedicated to recycling Na+ entering in conjunction with HCO3− to maintain [Na+]i within the physiologic range.

Saturable stimulation of fatty acid transport through model cytoplasm by soluble binding protein

To better define the role of soluble binding proteins in the cytoplasmic transport of amphipathic molecules, we measured the diffusional mobility of a fluorescent long-chain fatty acid, 12- N-methyl-(7-nitrobenz-2-oxa-1,3-diazol)aminostearate (NBD-stearate), through model cytoplasm as a function of soluble binding protein concentration. Diffusional mobilities were correlated with the partition of the fatty acid between membrane and protein binding sites. Cytoplasm was modeled as a dense suspension of liposomes, and albumin was used as a model binding protein. Albumin saturably increased NBD-stearate mobility through the membrane suspension approximately eightfold. Fatty acid mobility in the absence of albumin was identical to the mobility of the membrane vesicles (1.99 ± 0.33 × 10-8cm2/s), whereas the mobility at saturating concentrations was identical to the mobility of albumin (1.65 ± 0.12 × 10-7cm2/s). The protein concentration producing half-maximal stimulation of NBD-stearate diffusion (42.8 ± 0.3 μM) was unexpectedly greater than that required to solubilize half of the NBD-stearate (17.9 ± 3.0 μM). These results support a proposed mechanism for cytoplasmic transport of small amphipathic molecules in which aqueous diffusion of the protein-bound form of the molecule largely determines the transport rate. However, slow interchange of fatty acid between the binding protein and membranes also appears to influence the transport rate in this model system.

Recovery of hepatic drug extraction after hypothermic preservation

Background To determine whether liver preservation before transplantation impairs hepatic drug metabolism, hepatic extraction of drugs with different metabolic pathways (fentanyl, morphine, and vecuronium) in isolated rat livers was measured either immediately or after 24 h of hypothermia at 4 degrees Celsius using a standard preservation‐reperfusion sequence. Methods Isolated rat livers were perfused via the portal vein for 30 min to document initial viability. Test livers (n = 5) were perfused with iced Belzer solution, stored for 24 h at 4 degrees Celsius, and flushed with 6% hetastarch. After hypothermic preservation for 24 h, or in control livers (n = 5) immediately after the 30‐min perfusion, livers were perfused single‐pass at a constant flow rate with solutions containing fentanyl, morphine, and vecuronium at 37 degrees Celsius. Perfusate and bile samples were obtained at regular intervals for 64 min, after which liver tissue was harvested for analysis. Drug concentrations were measured using radioimmunoassay and gas chromatography. Metabolic capacity of the liver was estimated from the extraction fraction of each drug at steady‐state. Results After warming to 37 degrees Celsius, preserved livers consumed oxygen and produced bile at rates similar to that of control livers. Hypothermic preservation did not affect extraction of fentanyl and morphine. Vecuronium extraction was initially less in preserved livers, but this difference disappeared as the preserved livers returned to 37 degrees Celsius (< 16 min). Biliary excretion and tissue concentrations of vecuronium were similar in each group. Conclusions Hypothermic preservation does not significantly impair extraction of these drugs in this liver preservation model. If these results apply to human liver transplantation, little danger of drug accumulation exists during the early postoperative period if hepatic function is normal.