Regulation of retinol uptake and esterification in MCF-7 and HepG2 cells by exogenous fatty acids.

The influence of extracellular fatty acids on the uptake and esterification of [3H]retinol bound to human retinol-binding protein (RBP), to RBP-transthyretin (TTR), or in dispersed form by the human hepatoma, HepG2, and human mammary epithelial carcinoma, MCF-7, cell lines was studied. The esterification of [3H]retinol was significantly increased in cells incubated with myristic, palmitic, stearic oleic, or linoleic acid-albumin complexes and was observed for all forms of [3H]retinol. Enhancement of [3H]retinol uptake was also observed in cells incubated with these fatty acids, but this increase was relatively small for the dispersed form as compared to that observed for [3H]retinol bound to RBP or RBP-TTR. Comparing equal concentrations of the [3H]retinol donors, cell uptake and esterification was greatest from the dispersed form and least from that bound to RBP-TTR. After preincubation of cells with oleate, uptake and esterification of [3H]retinol was increased but not to the extent observed when oleate and [3H]retinol donor were co-incubated. Incubation of cells with oleate resulted in rapid and correlated increases in the rates of [3H]retinol uptake and esterification which persisted until the steady state for [3H]retinol uptake was achieved. Beyond this time, net esterification of [3H]retinol continued in the presence of oleate. This kinetic pattern was observed for all [3H]retinol donors. These effects on [3H]retinol uptake and esterification were dose-dependent as the oleate to albumin ratio was varied from 0.5 to 3.0 and were observed across a physiological concentration range of RBP-3H-retinol. The data indicate that: 1) the fatty acid status of cells is a determinant of retinol uptake and esterification; and 2) the form of retinol presentation to cells is not qualitatively important for these processes.     

The mechanism of uptake of retinol by plasma-membrane vesicles.

The mechanism of retinol uptake by human placental brush-border membrane vesicles was investigated using initial-velocity studies of [3H]retinol uptake from the [3H]retinol-RBP (retinol-binding protein) complex. The process was rapid and time- and temperature-dependent. The uptake was specifically reversed by the addition of native or apo-RBP, but not by serum albumin. By contrast, uptake of free [3H]retinol was temperature-independent, partially reversible and showed no requirement for a specific protein for reversibility. Treatment of membrane vesicles with p-chloromercuribenzenesulphonate (PCMBS), which inhibited 125I-RBP binding, also inhibited the uptake of retinol from RBP, but the uptake of free retinol was unaffected. Addition of PCMBS after the attainment of steady-state uptake equilibrium abolished the binding of RBP, but did not affect the retinol already taken up from RBP. The results suggest that binding of RBP to its specific receptor is obligatory for the subsequent delivery of retinol to the membrane. Since the studies were carried out on isolated membrane vesicles, endocytosis of RBP is most unlikely to be involved in the placental transport of retinol. A double-reciprocal plot of initial velocity versus [3H]retinol-RBP concentration gave an apparent Km of 116 +/- 13 nM. Transthyretin decreased the rate of uptake of [3H]retinol from RBP without substantially altering the steady-state uptake levels, suggesting that membranes take up retinol from uncomplexed RBP. High-pressure gel-filtration chromatography showed that [3H]retinol is largely transferred to a membrane component with an apparent molecular mass of 125 kDa.     

Characteristics of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells

The uptake of retinol was examined in cultured Sertoli cells when retinol was provided as a complex with the transport protein retinol-binding protein (RBP). Sertoli cells accumulated [3H]retinol in a time- and temperature-dependent manner. At 32 degrees C, the rate of retinol accumulation was biphasic. Accumulation was linear for approximately 1 h, but then accumulation continued at a linear but decreased rate for 23 h. The change in rate of retinol accumulation occurred when the cells had accumulated approximately 0.53 pmol of retinol/micrograms of cellular DNA. This amount of retinol was approximately equal to the cellular content of cellular retinol-binding protein (CRBP). Extraction and HPLC analysis of the cell-associated radioactivity yielded retinol and retinyl esters, indicating that a significant proportion of the accumulated retinol was esterified. Excess unlabeled retinol-RBP competed with [3H]retinol-RBP for [3H]retinol delivery to the cells, indicating that RBP delivery of retinol was a saturable and competable process. However, free [3H]retinol associated with Sertoli cells in a noncompetable manner. The transport constant for specific retinol accumulation from RBP was 3.0 microM, suggesting that any change in the normal circulating retinol-RBP level (approximately 2 microM) would directly affect the rate of retinol accumulation. Neither iodinated nor reductively methylated RBP was accumulated by or tightly bound to Sertoli cells. In addition, energy inhibitors and lysosomal poisons had no effect on [3H]retinol accumulation, indicating that RBP delivery of retinol to Sertoli cells did not occur by endocytosis of the retinol-RBP complex. Competition studies indicated, however, that protein recognition is important in the retinol uptake process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Newly administered [3H]retinol is transferred from hepatocytes to stellate cells in liver for storage

We have recently shown that newly administered vitamin A (retinol) is initially taken up by the parenchymal cells of the liver, and subsequently (within 1-2 h) transferred to non-parenchymal liver cells (NPC) (Blomhoff et al., ref. [10]). In the present study we have separated the NPC by different methods to determine the cell type responsible for this uptake of [3H]retinol. When liver cells were prepared between 5 and 18 h after intraduodenal administration of [3H]retinol, the radioactive retinol was recovered mainly in the stellate cells. Other liver cells (i.e., hepatocytes, endothelial cells and Kupffer cells) contained only small amounts of [3H]retinol. Further, fluorescence microscopy studies indicated that stellate cells contain large quantities of retinol. Our results show that newly administered [3H]retinol, which is initially located in the hepatocytes, is transferred to the stellate cells and stored there.     

Transthyretin Blocks Retinol Uptake and Cell Signaling by the Holo-Retinol-Binding Protein Receptor STRA6

Vitamin A is secreted from cellular stores and circulates in blood bound to retinol-binding protein (RBP). In turn, holo-RBP associates in plasma with transthyretin (TTR) to form a ternary RBP-retinol-TTR complex. It is believed that binding to TTR prevents the loss of RBP by filtration in the kidney. At target cells, holo-RBP is recognized by STRA6, a plasma membrane protein that serves a dual role: it mediates uptake of retinol from extracellular RBP into cells, and it functions as a cytokine receptor that, upon binding holo-RBP, triggers a JAK/STAT signaling cascade. We previously showed that STRA6-mediated signaling underlies the ability of RBP to induce insulin resistance. However, the role that TTR, the binding partner of holo-RBP in blood, plays in STRA6-mediated activities remained unknown. Here we show that TTR blocks the ability of holo-RBP to associate with STRA6 and thereby effectively suppresses both STRA6-mediated retinol uptake and STRA6-initiated cell signaling. Consequently, TTR protects mice from RBP-induced insulin resistance, reflected by reduced phosphorylation of insulin receptor and glucose tolerance tests. The data indicate that STRA6 functions only under circumstances where the plasma RBP level exceeds that of TTR and demonstrate that, in addition to preventing the loss of RBP, TTR plays a central role in regulating holo-RBP/STRA6 signaling.     

Comparison of synaptosomal and glial uptake of pipecolic acid and GABA in rat brain

The active uptake of [³H]pipecolic acid increased with incubation time and its uptake at 3 min was half of that at 20 min. [¹⁴C]GABA uptake rose earlier, and its uptake at 3 min was almost 80% of that at 20 min. On the other hand, a ratio (pellet/medium) of [³H]pipecolic acid uptake into glial cell-enriched fractions, was much less (0.4–0.6) than that of [¹⁴C]GABA (25.8–74.1). GABA, 10^–4 M, and pipecolic acid, 10^–4 M, produced a significant inhibition of [³H]pipecolic acid uptake into P₂ fractions. Pipecolic acid, 10^–4 M, significantly reduced the synaptosomal and glial uptake of [¹⁴C]GABA. GABA, 10^–4 M, affected neither spontaneous nor high K⁺-induced release of [³H]pipecolic acid from brain slices. It is suggested that pipecolic acid is involved in either synaptic transmission or in its modulation at GABA synapses in the central nervous system.     

Binding study of riboflavin-binding protein with riboflavin and its analogues by differential scanning calorimetry

Thermal unfolding parameters of hens' egg-white riboflavin-binding-protein (RBP) were measured by differential scanning calorimetry. Thermal denaturation scans of apoRBP and RBP complexes with riboflavin and its analogues (FMN, N10 DL-glyceryl isoalloxazine, and N10 -hydroxypentyl isoalloxazine) have been measured. It was found that ligand binding causes increase of RBP thermal stability, as manifested by a change of denaturation temperature from 60.8°C for apoRBP to 72.8°C for RBP—Rf complex. For RBP—FMN complex, the denaturation temperature of 73.0°C was even higher than for the RBP—Rf complex. The other two flavin analogues showed transition temperatures in between 66.9°C and 68.8°C, respectively. Analysis of excess heat capacity data showed that the best fit was the sum of two independent thermal transitions. One of the transitions, which contributed 70% to the total heat effect, has transition temperature in the broad range of 60.5–73.2°C; the other transition temperature is in the narrower range of 65.4–71.1°C. The observed transitions can be related to RBP domains.     

Uptake and Incorporation of Docosahexaenoic Acid (DHA) into Neuronal Cell Body and Neurite/Nerve Growth Cone Lipids: Evidence of Compartmental DHA Metabolism in Nerve Growth Factor-Differentiated PC12 Cells

Docosahexaenoic acid (DHA) accumulates in nerve endings of the brain during development. It is released from the membrane during ischemia and electroconvulsive shock. DHA optimizes neurologic development, it is neuroprotective, and rat adrenopheochromocytoma (PC12) cells have decreased PLA₂ activity when DHA is present. To characterize DHA metabolism in PC12 cells, media were supplemented with [³H]DHA or [³H]glycerol. Fractions of nerve growth cone particles (NGC) and cell bodies were prepared and the metabolism of the radiolabeled substrates was determined by thin-layer chromatography. [³H]glycerol incorporation into phospholipids indicated de novo lipid synthesis. [³H]DHA uptake was more rapid in the cell bodies than in the NGC. [³H]DHA first esterified in neutral lipids and later in phospholipids (phosphatidylethanolamine). [³H]glycerol primarily labeled phosphatidylcholine. DHA uptake was compartmentalized between the cell body and the NGC. With metabolism similar to that seen in vivo, PC12 cells are an appropriate model to study DHA in neurons.     

In vitro interaction of fenretinide with plasma retinol-binding protein and its functional consequences.

The synthetic retinoid fenretinide (4-HPR; N-[4-hydroxyphenyl] all-trans-retinamide) interacts with plasma apo-retinol-binding protein (RBP) to form a tight complex (K'd approximately 0.2 microM) which does not exhibit binding affinity to transthyretin (TTR). Therefore, a substantial modification of the retinol hydroxyl group does not appear to affect the interaction with RBP but does drastically interfere with the protein-protein recognition. The remarkable early reduction in plasma retinol level induced by fenretinide administration may be associated with the high binding affinity of this retinoid to RBP and to its interference with the RBP-TTR complex formation.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

The structure of human retinol-binding protein (RBP) with its carrier protein transthyretin reveals an interaction with the carboxy terminus of RBP

Whether ultimately utilized as retinoic acid, retinal, or retinol, vitamin A is transported to the target cells as all-trans-retinol bound to retinol-binding protein (RBP). Circulating in the plasma, RBP itself is bound to transthyretin (TTR, previously referred to as thyroxine-binding prealbumin). In vitro one tetramer of TTR can bind two molecules of retinol-binding protein. However, the concentration of RBP in the plasma is limiting, and the complex isolated from serum is composed of TTR and RBP in a 1 to 1 stoichiometry. We report here the crystallographic structure at 3.2 A of the protein-protein complex of human RBP and TTR. RBP binds at a 2-fold axis of symmetry in the TTR tetramer, and consequently the recognition site itself has 2-fold symmetry: Four TTR amino acids (Arg-21, Val-20, Leu-82, and Ile-84) are contributed by two monomers. Amino acids Trp-67, Phe-96, and Leu-63 and -97 from RBP are flanked by the symmetry-related side chains from TTR. In addition, the structure reveals an interaction of the carboxy terminus of RBP at the protein-protein recognition interface. This interaction, which involves Leu-182 and Leu-183 of RBP, is consistent with the observation that naturally occurring truncated forms of the protein are more readily cleared from plasma than full-length RBP. Complex formation prevents extensive loss of RBP through glomerular filtration, and the loss of Leu-182 and Leu-183 would result in a decreased affinity of RBP for TTR.     

Studies on the Cellular Uptake of Retinol Binding Protein and Retinol

The uptake and release of (125)I-RBP and of holoRBP labeled with [(3)H]retinol ((3)H-ROH) were studied in two cell lines which synthesize and secrete RBP, the HepG2 hepatocarcinoma cell line and the Caki-1 kidney adenocarcinoma cell line, and in HeLa cells that do not express the endogenous RBP gene. In all three cell lines a part of endocytosed (125)I-RBP is recycled to the extracellular medium and part is degraded. Nonspecific endocytosis of (125)I-RBP was estimated to be approximately 10% of total endocytosed (125)I-RBP. In HepG2 cells the (3)H-ROH from the [(3)H]retinol-RBP complex ((3)H-ROH-RBP) is recycled bound to RBP into serum-free chase medium. This (3)H-ROH recycling is blocked in HepG2 cells by cyclohexymide and by brefeldin A, an inhibitor of protein export from the main secretory route, and is absent in HeLa cells, which do not synthesize RBP. These data suggest that at least part of retinol taken up from exogenous holoRBP is delivered to newly synthesized RBP. (3)H-ROH recycled by HeLa cells is bound to serum albumin, as is a portion of that recycled by HepG2 cells. Transfer of (3)H-ROH from RBP to serum albumin does not occur in the absence of cells. We conclude that RBP is endocytosed through a specific pathway and that the RBP-associated retinol is transferred to newly synthesized RBP or to serum albumin.     

Uptake,exchange, and release of GABA by cerebellar glomeruli

Glomerular particles were isolated from the bovine cerebellar vermis and studied in vitro to further assess the possibility that -aminobutyric acid (GABA) is utilized as a neurotransmitter in this synaptic complex. Cerebellar glomeruli accumulated [³H]GABA at two different high affinity sites, with affinities (K _T) of 2.2×10^–6M and 3.0×10^–5M. These uptake sites could not be distinguished on the basis of their temperature sensitivities, sodium dependence, substrate specificities or responses to metabolic inhibitors. Although an exchange process contributed to the uptake measured in these experiments, a considerable amount of the [³H]GABA accumulated by glomerular particles was stored in an osmotically-sensitive, nonexchangeable pool. Glomerular particles preloaded with [³H]GABA exhibited a Ca²⁺-independent release of this amino acid in response to membrane depolarization. However, when preloaded glomerular particles were exposed to unlabeled GABA, which presumably displaced [³H]GABA from the exchangeable pool, a K^–-evoked and Ca²⁺-dependent release of the remaining [³H]GABA occurred. The observed net uptake, together with the depolarization-induced and Ca²⁺-dependent release, of [³H]GABA from glomerular particles supports the suggestion that functionally active GABAergic synapses are present in these structures.     

Uptake and metabolism of retinol in cultured Sertoli cells: evidence for a kinetic model

When cultured Sertoli cells derived from 20-day-old weanling rats were supplied [3H]retinol bound to serum retinol binding protein-transthyretin complex, [3H]retinol was rapidly incorporated and [3H]retinyl esters were synthesized. Within 28 h after administration, 83% of the labeled retinoids were accounted for as retinyl esters (64% as retinyl palmitate). Sertoli cells derived from vitamin A deficient rats and supplied [3H]retinol in culture under identical conditions likewise incorporated [3H]retinol and synthesized retinyl esters. In contrast to normal Sertoli cells, vitamin A deficient Sertoli cells eventually metabolized virtually all of the cellular [3H]retinol to retinyl esters. The primary metabolic fate of retinol administered to Sertoli cell cultures was the synthesis of retinyl esters under all conditions tested. However, administration of [3H]retinol bound to serum retinol binding protein gave metabolic profiles having a higher proportion of retinyl esters and lower proportions of unresolved polar material than administration of [3H]retinol alone. The kinetics of retinol uptake and intracellular retinyl ester synthesis in cultured Sertoli cells was complex. An initial, rapid phase of [3H]retinol incorporation lasting 30 min was followed by a slower rate of incorporation and a concomitant decrease in the intracellular concentration of [3H]retinol. During the time course the specific activity of [3H]retinyl palmitate eventually exceeded that of intracellular [3H]retinol. These observations suggest that two intracellular pools of retinol may exist in Sertoli cells.     

Interactions of retinol with binding proteins: studies with retinol-binding protein and with transthyretin.

The interactions within the molecular complex in which retinol circulates in blood were studied. To monitor binding between retinol-binding protein (RBP) and transthyretin (TTR), TTR was labeled with a long-lived fluorescence probe (pyrene). Changes in the rotational volume of TTR following its association with RBP were monitored by fluorescence anisotropy of the probe. Titration of TTR with holo-RBP revealed the presence of 1.5 binding sites characterized by a dissociation constant Kd = 0.07 microM. At 0.15 M NaCl, binding of RBP to TTR showed an absolute requirement for the native ligand, retinol. At higher ionic strength (0.5 M NaCl), RBP complexed with retinal also bound to TTR with high affinity (Kd = 0.134 microM). RBP containing retinoic acid did not bind to TTR even at the high salt concentration. The data suggest that the TTR binding site on RBP is in close proximity to the retinoid binding site and that the head group of retinoic acid, when bound to RBP, presents steric hindrance for the interactions with TTR. The implications of the data for selectivity in retinoid transport in the circulation are discussed. The kinetics of the steps leading to complete dissociation of the retinol-RBP-TTR complex was also studied. The first step of this process was dissociation of retinol, which had a rate constant of 0.06/min. Following loss of retinol, the two proteins dissociate. The rate of dissociation is slow (k = 0.055/h), however, indicating that the complex apo-RBP-TTR will be an important factor in regulating serum levels of retinol.     

Specific binding of progesterone to the cell surface and its role in the meiotic divisions in Rana oocytes

Progesterone is believed to act at the cell surface to induce the resumption of the meiotic divisions in amphibian oocytes. Analysis of [³H]- and [¹⁴C]progesterone uptake and exchange by the plasma-vitelline membrane complex, nucleus and cytoplasm of the isolated Rana oocyte indicates that progesterone uptake by the plasma membrane is saturable, specific and temperature-dependent, and has a slow off-rate. Estradiol (a noninducer) did not compete with progesterone, whereas testosterone (an inducer) blocked progesterone uptake by the membrane complex. Scatchard-type plots indicate an apparent K_d of 5.1·10⁻⁷ M over the [progesterone]_o range of 0.01–1.0 μM with maximum binding at about 70 fmol per oocyte. Membrane uptake at higher [progesterone]_o (2–40 μM) indicates apparent cooperative binding, with saturation up to 10 pmol per oocyte. Cytoplasmic uptake was apparently nonspecific and less temperature-dependent than membrane uptake and steroid concentrations (progesterone and pregnanediones) exceeded water solubility by 30–60 min. Nuclear uptake was saturable and specific but uptake was independent of temperature. A comparison of membrane binding and a physiological response (nuclear breakdown) indicated only about 10% of the membrane sites need be filled to initiate a 50% response.     

Biochemical characterization of [3H]norepinephrine uptake in dissociated brain cell cultures from chick embryos

The uptake of [³H]norepinephrine ([³H]NE) was studied in dissociated brain cell cultures prepared from 8-day-old chick embryos using the whole brain (minus optic lobes). Uptake of [³H]NE, 5×10^–9 M, 10 min incubation, in freshly dissociated noncultured embryonic chick brain cells, was detected in 6-day-old embryos; it was temperature and drug (cocaine, metanephrine) sensitive and increased with brain development. In cultured cells, which were assayed at various days in culture, the increase in [³H]NE accumulation per culture was less than that seen in freshly dissociated noncultured embryonic cells. When [³H]NE uptake was expressed per mg protein, a decrease with days in culture was observed, reflecting perhaps a dilution of growth or proliferation of cells not accumulating NE. Metanephrine, 5×10^–6 M, an inhibitor of extraneuronal uptake, inhibited [³H]NE in 5-day-old cultures whereas desmethylimipramine, an inhibitor of neuronal uptake, inhibited [³H]NE uptake in 15- and 20-day-old cultures. Cocaine, another neuronal inhibitor, inhibited [³H]NE at 10 and 15 days only. We interpret these findings to suggest that during early growth in culture most neuroblasts accumulate NE nonspecifically and, as neuronal maturation proceeds, NE accumulation becomes specific.     

Retinol-binding protein,retinol, and modified-relative-dose response in Ugandan children aged 12–23 months and their non-pregnant caregivers

Retinol-binding protein (RBP), retinol, and modified-relative-dose response (MRDR) are used to assess vitamin A status. We describe vitamin A status in Ugandan children and women using dried blood spot (DBS) RBP, serum RBP, plasma retinol, and MRDR and compare DBS-RBP, serum RBP, and plasma retinol. Blood was collected from 39 children aged 12–23 months and 28 non-pregnant mothers aged 15–49 years as a subsample from a survey in Amuria district, Uganda, in 2016. DBS RBP was assessed using a commercial enzyme immunoassay kit, serum RBP using an in-house sandwich enzyme-linked immunosorbent assay, and plasma retinol/MRDR test using high-performance liquid chromatography. We examined (a) median concentration or value (Q1, Q3); (b) R² between DBS-RBP, serum RBP, and plasma retinol; and (c) Bland-Altman plots. Median (Q1, Q3) for children and mothers, respectively, were as follows: DBS-RBP 1.15 µmol/L (0.97, 1.42) and 1.73 (1.52, 1.96), serum RBP 0.95 µmol/L (0.78, 1.18) and 1.47 µmol/L (1.30, 1.79), plasma retinol 0.82 µmol/L (0.67, 0.99) and 1.33 µmol/L (1.22, 1.58), and MRDR 0.025 (0.014, 0.042) and 0.014 (0.009, 0.019). DBS RBP-serum RBP R² was 0.09 for both children and mothers. The mean biases were −0.19 µmol/L (95% limits of agreement [LOA] 0.62, −0.99) for children and −0.01 µmol/L (95% LOA −1.11, −1.31) for mothers. DBS RBP-plasma retinol R² was 0.11 for children and 0.13 for mothers. Mean biases were 0.33 µmol/L (95% LOA −0.37, 1.03) for children, and 0.29 µmol/L (95% LOA −0.69, 1.27) for mothers. Serum RBP-plasma retinol R² was 0.75 for children and 0.55 for mothers, with mean biases of 0.13 µmol/L (95% LOA −0.23, 0.49) for children and 0.18 µmol/L (95% LOA −0.61, 0.96) for mothers. Results varied by indicator and matrix. The serum RBP-retinol R² for children was moderate (0.75), but poor for other comparisons. Understanding the relationships among vitamin A indicators across contexts and population groups is needed.     

Spontaneous and evoked release of [3H]taurine from a P2 subcellular fraction of the rat retina

The effects of spontaneous and evoked [³H]taurine release from a P₂ fraction prepared from rat retinas were studied. The P₂ fraction was preloaded with [³H]taurine under conditions of high-affinity uptake and then examined for [³H]taurine efflux utilizing superfusion techniques. Exposure of the P₂ fraction to high K⁺ (56 mM) evoked a Ca²⁺-independent release of [³H]taurine. Li⁺ (56 mM) and veratridine (100 M) had significantly less effect (8–15% and 15–30%, respectively) on releasing [³H]taurine compared to the K⁺-evoked release. 4-Aminopyridine (1 mM) had no effect on the release of [³H]taurine. The spontaneous release of [³H]taurine was also Ca²⁺-independent. When Na⁺ was omitted from the incubation medium K⁺-evoked [³H]taurine release was inhibited by approximately 40% at the first 5 minute depolarization period but was not affected at a second subsequent 5 minute depolarization period. The spontaneous release of [³H]taurine was inhibited by 60% in the absence of Na⁺. Substitution of Br^– for Cl^– had no effect on the release of either spontaneous or K⁺-evoked [³H]taurine release. However, substitution of the Cl^– with acetate, isethionate, or gluconate decreased K⁺-evoked [³H]taurine release. Addition of taurine to the superfusion medium (homoexchange) resulted in no significant increase in [³H]taurine efflux. The taurine-transport inhibitor guanidinoethanesulfonic acid increased the spontaneous release of [³H]taurine by approximately 40%. These results suggest that the taurine release of [³H]taurine is not simply a reversal of the carrier-mediated uptake system. It also appears that taurine is not released from vesicles within the synaptosomes but does not rule out the possibility that taurine is a neurotransmitter. The data involving chloride substitution with permeant and impermeant anions support the concept that the major portion of [³H]taurine release is due to an osmoregulatory action of taurine while depolarization accounts for only a small portion of [³H]taurine release.     

Assessing the cellular transmembrane electrical potential difference on the hepatic uptake of palmitate

Understanding the driving forces for the hepatic uptake of endogenous and exogenous substrates in isolated cells and organs is fundamental to describing the underlying hepatic physiology/pharmacology. In this study we investigated whether uptake of plasma protein-bound [³H]-palmitate across the hepatocyte wall is governed by the transmembrane electrical potential difference (PD). Uptake was studied in isolated hepatocytes and isolated perfused rat livers (IPL). Protein-binding and vasoactive properties of the different perfusates were determined using in vitro heptane/buffer partitioning studies and the multiple indicator dilution (MID) technique in the IPL, respectively. Altering hepatocyte PD by perfusate ion substitution resulted in either a substantial depolarization (–14 ± 1 mV, n = 12, mean ± S.E., substituting choline for Na⁺) or hyperpolarization (–46 ± 3 mV, n = 12, mean ± S.E., substituting nitrate for Cl^–). Perfusate ion substitution also affected the equilibrium binding constant for the palmitate–albumin complex. IPL studies suggested that, other than with gluconate buffer, hepatic [³H]-palmitate extraction was not affected by the buffer used, implying PD was not a determinant of extraction. [³H]-Palmitate extraction was much lower (p < 0.05) when gluconate was substituted for Cl^– ion. This work contrasts with that for the extraction of [³H]-alanine where hepatic extraction fraction was significantly reduced during depolarization. Changing the albumin concentration did not affect hepatocyte PD, and [³H]-palmitate clearance into isolated hepatocytes was not affected by the buffers used. MID studies with vascular and extravascular references revealed that, with the gluconate substituted buffer, the extravascular volume possibly increased the diffusional path length thus explaining reduced [³H]-palmitate extraction fraction in the IPL. (Mol Cell Biochem 270: 115–124, 2005)