Hepatocyte [3H]-palmitate uptake: effect of albumin surface charge modification.

The role of plasma proteins on the cellular uptake of lipophilic substrates has perplexed investigators for many years. We tested the hypothesis that an ionic interaction between the protein-ligand complex and hepatocyte surface may be responsible for supplying more ligand to the cell for uptake. The surface-charged groups on albumin were modified to yield proteins having a range of isoelectric points (ALB, ALBs, ALBm, ALBe had values of 4.8-5.0, 4.5-4.7, 3.0-3.5, 8.4-8.6, respectively). [3H]-Palmitate uptake studies were performed with adult rat hepatocyte suspensions using similar unbound ligand fractions in the presence of the different binding proteins. Mass spectrometry, isoelectric focusing (pI), and heptane:water partitioning were used to determine protein molecular weight, pI, and protein-palmitate equilibrium binding constant, respectively. Hepatocyte [3H]-palmitate clearance in the presence of ALBs and ALBm were significantly lower (p < 0.05) than ALB, whereas [3H]-palmitate clearance in the presence of ALBe was significantly higher (p < 0.05) than ALB. The data were consistent with the notion that ionic interactions between extracellular protein-ligand complexes and the hepatocyte surface facilitate the uptake of long-chain fatty acids.     

Cytosolic fatty acid binding protein enhances rat hepatocyte [3H]palmitate uptake

Liver cytosolic fatty acid binding protein (FABP) represents the intracellular equivalent to extracellular serum albumin, participating in the intracellular transport of long-chain fatty acids. In this study we observed the effect of increasing and decreasing FABP levels on hepatocyte [3H]palmitate uptake in male Sprague-Dawley rats. We also were interested to determine whether uptake, from either the unbound or unbound and protein-bound fractions, was fundamentally different at the different FABP levels. FABP levels were modified by hypophysectomy and clofibrate treatment (50 mg/100 g body weight for 10 days). Results showed that the [3H]palmitate clearance rates paralleled the 54% decrease and 73% increase in FABP levels in hypophysectomy and clofibrate-treated animals, respectively. In the presence of 2 and 20 microM albumin, hepatocyte clearance rates of unbound [3H]palmitate from hypophysectomized animals (0.16+/-0.01 and 0.64+/-0.01 mL x s(-1) x 10(-6) cells, respectively) were significantly lower (p<0.01) than those of the sham group (0.30+/-0.02 and 1.00+/-0.06 mL x s(-1) x 10(-6) cells, respectively). However, the unbound [3H]palmitate clearance rates from the clofibrate-treated group (0.39+/-0.04 and 1.18+/-0.12 mL x s(-1) x 10(-6) cells) were significantly higher (p<0.01) than the control group (0.29+/-0.02 and 0.81+/-0.05 mL x s(-1) x 10(-6) cells) for 2 and 20 microM albumin, respectively. To investigate whether uptake was fundamentally different between the hypophysectomized and clofibrate-treated groups, we expressed the clearance rates as enhancement factors, i.e., EF = CL20 microM/CL2microM. No statistical difference was observed between EF of the hypophsectomized (3.8+/-0.4) and EF of the clofibrate-treated (3.1+/-0.3) groups, suggesting that the extracted ligand originated from similar fractions.     

Studies on the role of insulin in the regulation of glyceride synthesis in rat epididymal adipose tissue.

1. When rat isolated fat-cells were incubated with fructose and palmitate, insulin significantly stimulated glyceride synthesis as measured by either [14C]fructose incorporation into the glycerol moiety or of [3H]palmitate incorporation into the acyl moiety of tissue glycerides. Under certain conditions the effect of insulin on glyceride synthesis was greater than the effect of insulin on fructose uptake. 2. In the presence of palmitate, insulin slightly stimulated (a) [14C]pyruvate incorporation into glyceride glycerol of fat-cells and (b) 3H2O incorporation into glyceride glycerol of incubated fat-pads. 3. At low extracellular total concentrations of fatty acids (in the presence of albumin), insulin stimulated [14C]fructose, [14C]pyruvate and 3H2O incorporation into fat-cell fatty acids. Increasing the extracellular fatty acid concentration greatly inhibited fatty acid synthesis from these precursors and also greatly decreased the extent of apparent stimulation of fatty acid synthesis by insulin. 4. These results are discussed in relation to the suggestion [A.P. Halestrap & R.M.Denton (1974) Biochem. J. 142, 365-377] that the tissue may contain a specific acyl-binding protein which is subject to regulation. It is suggested that an insulin-sensitive enzyme component of the glyceride-synthesis process may play such a role.     

Transport of fatty acid is obligatory coupled with H+ entry in spheroplasts of Escherichia coli K12

Transport of palmitate by spheroplasts of Escherichia coli K12 was studied. [14C]Palmitate was accumulated in spheroplasts approximately 1700-fold over the extracellular concentration of unbound [14C]palmitate. Uptake of [14C]palmitate was inhibited to 13% by addition of H+ uncoupler carbonyl cyanide-m-chlorophenylhydrazone (CCCP). Spheroplasts exhibited the uptake of 9-aminoacridine depending on the addition of palmitate to the incubation mixture. The rate of [14C]palmitate uptake by the spheroplasts pre-equilibrated in a buffer at pH 7.5 or 8.0 significantly increased in comparison with the spheroplasts pre-equilibrated in a buffer at pH 7.0 when the spheroplasts were incubated at an external pH of 7.0.     

Perturbation of N-linked oligosaccharide structure results in an altered incorporation of [3H]palmitate into specific proteins in Chinese hamster ovary cells

Increased [3H]palmitate incorporation into specific cellular proteins has been reported to occur in Chinese hamster ovary (Wellner, R. B., Ray, B., Ghosh, P. C., and Wu, H. C. (1984) J. Biol. Chem. 259, 12788-12793) and yeast (Wen, D., and Schlesinger, M. J. (1984) Mol. Cell. Biol. 4, 688-694) mutant cells. In this paper we report studies concerning the relationship between N-linked oligosaccharide structure and [3H]palmitate incorporation into proteins of Chinese hamster ovary (CHO) cells. We have compared the incorporation of [3H]palmitate into proteins of wild-type and four different mutant CHO cell lines defective in various steps of N-linked protein glycosylation. Sodium dodecyl sulfate-gel electrophoretic analysis showed that three of the mutants exhibited increased [3H]palmitate incorporation into several CHO cellular proteins (approximately 30,000-38,000 molecular weight) as compared to the wild-type cells. One of the affected mutants which accumulates the Man5Gn2Asn intermediate structure was examined in detail. In agreement with earlier reports, virtually all of the [3H] palmitate-labeled proteins of both wild-type and mutant cell lines are membrane-bound. Pretreatment of the mutant cell line with tunicamycin blocked the increased [3H]palmitate incorporation into the two specific proteins (both of approximately 30,000 molecular weight) observed in untreated cells; the decreased incorporation of [3H]palmitate into the 30,000 molecular weight species was accompanied by a concomitant increase in the incorporation of [3H]palmitate into two proteins of approximately 20,000 molecular weight. Pretreatment of wild-type cells with tunicamycin also caused increased [3H]palmitate incorporation into the 20,000 molecular weight species. Endoglycosidase H treatment of [3H]palmitate-labeled extracts from the mutant cell line resulted in the disappearance of the heavily labeled 30,000 molecular weight species and the appearance of intensely labeled 20,000 molecular weight species. Pretreatment of the mutant cell line with either castanospermine or deoxynojirimycin reduced the [3H]palmitate incorporation in to the 30,000 molecular weight species increased in untreated cells, but did not cause increased [3H]palmitate incorporation into the 20,000 molecular weight species. Our results indicate that perturbation of N-linked oligosaccharide structure results in altered incorporation of [3H]palmitate into specific proteins in CHO cells.     

Proteolipid formation in Mycoplasma capricolum. Influence of cholesterol on unsaturated fatty acid acylation of membrane proteins

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph was grown on media supplemented with [3H]palmitate or [3H]oleate. The isolated bacterial membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Of the more than 50 membrane polypeptides revealed by Coomassie blue staining, approximately 25 were labeled with [3H]palmitate and only about 6 were labeled with [3H]oleate. Exhaustive delipidation of the membranes with chloroform:methanol did not alter the labeling pattern. Treatment of delipidated membranes by mild alkaline hydrolysis released up to 71% of the [3H]palmitate and 93% of the [3H]oleate. The data suggest that numerous membrane proteins of M. capricolum are covalently modified by acylation with saturated and unsaturated fatty acids. Cerulenin, a specific inhibitor of fatty acid synthesis had no effect on the labeling of mycoplasma membrane proteins by either [3H]palmitate or [3H]oleate. A small amount of membrane-associated cholesterol previously shown to stimulate sequentially the synthesis of unsaturated phospholipid, RNA, and protein (Dahl, J. S., and Dahl, C. E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 692-696) specifically enhances the acylation of certain proteolipids by oleate but not by palmitate.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Biochemical aspects of cardiac muscle differentiation. Possible control of deoxyribonucleic acid synthesis and cell differentiation by adrenergic innervation and cyclic adenosine 3':5'-monophosphate.

A single injection of either isoproternol or N6, O2'-dibutyryl adenosine 3':5'-monophosphate (dibutyryl cyclic AMP) results in an inhibition in the rate of [3H]thymidine incorporation into DNA of differentiating cardiac muscle of the neonatal rat. This inhibition is not due to substantially altered cellular uptake or catabolism of [3H]thymidine. Inhibition of [3H]thymidine incorporation by isoproterenol or dibutyryl cyclic AMP is potentiated by theophylline. Maximal inhibition (95%) is observed 24 h after administration of isoproterenol, and the rate of incorporation returns to a value 80% of control by 72 h. Norepinephrine also inhibits [3H]thymidine incorporation whereas cyclic GMP, N2, 02-Dibutyryl guanosine 3':5'-monophosphate (dibutyryl cyclic GMP), and phenylephrine have little effect. Equilibrium sedimentation analysis of cardiac muscle DNA in neutral and alkaline cesium chloride gradients using bromodeoxyuridine as a density label indicate that isoproterenol and dibutyryl cyclic AMP inhibit [3H]thymidine incorporation into DNA that is replicating semiconservatively. Administration of isoproterenol or dibutyryl cyclic AMP to neonatal rats inhibits by approximately 60% the incorporation of [3H]thymidine into DNA of tissue slices of cardiac muscle prepared 16 h later. [3H]Thymidine incorporation into DNA of tissue slices is into chains that were growing in vivo. This incorporation is linear for at least 4 h of incubation and is inhibited by isoproterenol and dibutyryl cyclic AMP. Inhibition is not due to altered cellular uptake of [3H]thymidine nor is it due to a cytotoxic action. Several other compounds which elevate intracellular levels of cyclic AMP (epinephrine, norepinephrine, glucagon, and prostaglandin E1) also inhibit [3H]thymidine incorporation into DNA or cardiac muscle tissue slices. Cyclic GMP, dibutyryl cyclic GMP, sodium butyrate, and phenylephrine have little effect. Isoproterenol administered together with theophylline to neonatal rats signficantly stimulates the in corporation of [3H]phenylalanine into total cardiac muscle protein and into myosin. This enhanced incorporation may be due in part to an increase in the cellular uptake of [3H]phenylalanine. DNA synthesis decreases progressively in differentiating cardiac muscle of the rat during postnatal development and essentially ceases by the middle of the third week (Claycomb, W. C. (1975) J. Biol. Chem. 250, 3229-3235). In reviewing the literature it was found that this decline in synthetic activity correlates temporally with a progressive increase in tissue concentrations of norepinephrine and cyclic AMP and with the anatomical and physiological development of the adrenergic nerves in this tissue. Because of these facts and data presented in this report it is proposed that cell proliferation and cell differentiation in cardiac muscle may be controlled by adrenergic innervation with norepinephrine and cyclic AMP serving as chemical mediators.     

Fatty acid-acylated proteins in secretory mutants of Saccharomyces cerevisiae.

Yeast secretory (sec) mutants that are blocked in the transport of secretory proteins and accumulate membrane organelles were used to study the biosynthesis of fatty acid-acylated proteins. Four proteins were labeled with [3H]palmitate in sec mutants accumulating endoplasmic reticulum membranes. Three of these (molecular weights approximately equal to 20,000, 50,000, and 120,000) were N-linked glycoproteins, based on their ability to be labeled with [3H]mannose and their sensitivity to endoglycosidase H. The fourth protein (molecular weight approximately equal to 30,000) also was labeled with [3H]mannose but was insensitive to endoglycosidase H; it appeared to contain O-linked sugars. In sec mutants accumulating Golgi membranes or post-Golgi vesicles, a 35-kilodalton protein was labeled with [3H]palmitate. Analysis of Staphylococcus aureus protease V8 digests and pulse-chase experiments indicated that the 30-kilodalton protein was a precursor of 35 kilodaltons. None of these proteins was labeled with [3H]palmitate in a sec mutant that blocked the penetration of nascent polypeptides into endoplasmic reticulum; thus, acylation occurred in endoplasmic reticulum. All four proteins could be recovered from fractions enriched for yeast membranes. Fatty acids were not released from proteins by boiling in sodium dodecyl sulfate or extraction with organic solvents but were recovered as methyl esters after proteins were treated with KOH-methanol, a reaction characteristic of an acyl ester linkage.     

Two isoforms of eIF-5A in chick embryo. Isolation, activity, and comparison of sequences of the hypusine-containing proteins.

Eukaryotic translation initiation factor 5A (eIF-5A) (older terminology, eIF-4D) is unique in that it contains the unusual amino acid hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine). Hypusine is formed by a post-translational event in which a specific lysine residue is modified by a structural contribution from spermidine. Metabolic labeling of chick embryo fibroblasts with [3H]spermidine or [3H]lysine gives rise to two distinct proteins, designated I (approximately 20 kDa and pI 5.6) and II (approximately 18 kDa and pI 5.35), that contain [3H]hypusine. Upon incubation with [3H]lysine the labeling of the two proteins followed a similar time course and showed approximately the same ratio over the 6-h incubation period. [3H]Hypusine-containing proteins from cells which had been cultured with [3H]spermidine were employed as tracers for isolation of hypusine-containing proteins from whole chick embryos. Four such proteins were obtained. Two of these proteins, I and II, correspond to the two native proteins synthesized in chick embryo fibroblasts; the other two forms, Ia and IIa, displayed properties suggesting that they were derived from the native proteins, I and II, respectively, during purification. The amino acid compositions and the tryptic peptide maps of the 20-kDa protein (I) and the 18 kDa protein (II) suggest that they are closely related but distinct proteins. In fact, amino acid sequence analysis of the two major proteins revealed differences in the polypeptide backbone of the two proteins. In spite of structural differences, the two native forms (I and II), as well as the two altered forms (Ia and IIa), were effective in stimulating methionyl-puromycin synthesis, providing evidence that they are indeed functional isoforms of eIF-5A.     

Putrescine uptake and release by a normal rat small intestine crypt cell line, IEC-6

IEC-6 cells were cultured on permeable filter inserts with separate access to the apical and basolateral sides. [3H]Putrescine uptake favored the apical side and its release (in Earle's balanced salt solution containing 0.1% bovine serum albumin) was six times greater in the apical-to-basolateral than in the basolateral-to-apical direction. Release in DMEM did not show this preference. The uptake of [3H]putrescine was stimulated approximately 1.3 times the basal level by 10 mM asparagine (ASN) or 5% dialyzed fetal bovine serum whether the [3H]putrescine was added at a concentration of 1 or 100 nM. The increased uptake was maintained for up to 6 h. When [3H]putrescine was removed after 4 h of uptake, the cells continued to release it into the medium on both sides for up to 4 h. Stimulated cells released only 50% as much as unstimulated cells. Unlabeled putrescine reduced the uptake of [3H]putrescine with an IC50 of 1.81 x 10(-6) M (r = 0.9476) and 1.02 x 10(-6) M (r = 0.9967) for unstimulated and ASN-stimulated cells, respectively. When the intracellular putrescine was reduced by difluoromethylornithine, the uptake of [3H]-putrescine was not changed, but its release was inhibited. Sodium was not required for [3H]putrescine uptake or release. Although the stimulated cells attained intracellular levels of [3H]putrescine which, if expressed as concentration based on cell volume, were up to 500 times the original extracellular concentration, a true concentration gradient could not be proven because 85% of the [3H]putrescine was probably bound to polyanions as shown by butanol extraction.     

Use of a single-injection tissue-sampling technique to study steroid uptake by the decidualized endometrium of the mouse

A tissue-sampling paired-tracer method was used to investigate the effect of plasma proteins on uptake by the decidualized endometrium of [3H]progesterone, [3H]oestradiol and [3H]corticosterone. When injected arterially in protein-free Ringer, the extraction of progesterone and oestradiol was 100%, while that of corticosterone was only 60%. The addition of 4% albumin or injection in mouse plasma resulted in significant decreases in progesterone extraction to about 80% and 65% respectively. Injection in pregnant guinea-pig plasma reduced progesterone extraction further (to 33%). While neither 4% albumin nor mouse plasma had any significant effect on the uptake of oestradiol, neonatal rat plasma reduced oestradiol extraction to 40%. These results are consistent with high-affinity binding proteins having a limiting effect on the availability of steroids to target tissues.     

The subcellular compartmentation of fatty acid transporters is regulated differently by insulin and by AICAR

Cellular fatty acid uptake is facilitated by a number of fatty acid transporters, FAT/CD36, FABPpm and FATP1. It had been presumed that FABPpm, was confined to the plasma membrane and was not regulated. Here, we demonstrate for the first time that FABPpm and FATP1 are also present in intracellular depots in cardiac myocytes. While we confirmed previous work that insulin and AICAR each induced the translocation of FAT/CD36 from an intracellular depot to the PM, only AICAR, but not insulin, induced the translocation of FABPpm. Moreover, neither insulin nor AICAR induced the translocation of FATP1. Importantly, the increased plasmalemmal content of these LCFA transporters was associated with a concomitant increase in the initial rate of palmitate uptake into cardiac myocytes. Specifically, the insulin-stimulated increase in the rate of palmitate uptake (+60%) paralleled the insulin-stimulated increase in plasmalemmal FAT/CD36 (+34%). Similarly, the greater AICAR-stimulated increase in the rate of palmitate uptake (+90%) paralleled the AICAR-induced increase in both plasmalemmal proteins (FAT/CD36 (+40%)+FABPpm (+36%)). Inhibition of palmitate uptake with the specific FAT/CD36 inhibitor SSO indicated that FABPpm interacts with FAT/CD36 at the plasma membrane to facilitate the uptake of palmitate. In conclusion, (1) there appears to be tissue-specific sensitivity to insulin-induced FATP1 translocation, as it has been shown elsewhere that insulin induces FATP1 translocation in 3T3-L1 adipocytes, and (2) clearly, the subcellular distribution of FABPpm, as well as FAT/CD36, is acutely regulated in cardiac myocytes, although FABPpm and FAT/CD36 do not necessarily respond identically to the same stimuli.     

Transport and metabolism of palmitate in the rat liver. Net flux and unidirectional fluxes across the cell membrane.

The unidirectional fluxes of palmitate across the liver cell membrane and metabolic uptake rates were measured employing the multiple-indicator dilution technique. The following results were obtained: (1) Influx and net uptake rates do not vary proportionally to each other when albumin and palmitate concentrations are varied. (2) Efflux is significant for albumin concentrations in the range between 1.5 and 500 microM. (3) At 150 microM albumin net uptake rates are proportional to the total (bound plus free) extracellular palmitate concentration in the range from 10 to 600 microM; the dependence of influx rates on the palmitate concentration is rather concave up. (4) When albumin and palmitate are both varied at an equimolar ratio, pseudo-saturation appears in the net uptake rates; the influx rates also show pseudo-saturation, but with a declining tendency at the higher concentrations. (5) The intracellular palmitate concentration is strongly influenced by albumin. At very low concentrations of the protein (1.5 microM) the intracellular concentration is practically equal to the extracellular one; at physiological albumin concentrations, however, the intracellular palmitate concentration is less than 2% of the extracellular one. (6) Saturation of net uptake with respect to the intracellular palmitate concentration was not observed with concentrations up to 46 microM.     

Hyperthyroidism facilitates cardiac fatty acid oxidation through altered regulation of cardiac carnitine palmitoyltransferase: studies in vivo and with cardiac myocytes.

The aim was to establish whether increased cardiac fatty acid oxidation in hyperthyroidism is due to direct alterations in cardiac metabolism which favour fatty acid oxidation and/or whether normal regulatory links between changes in glucose supply and fatty acid oxidation are dysfunctional. Euthyroid rats were sampled in the absorptive state or after 48 h starvation. Rats were rendered hyperthyroid by injection of tri-iodothyronine (1000 microg/kg body wt. per day; 3 days). We evaluated the regulatory significance of direct effects of hyperthyroidism by measuring rates of palmitate oxidation in the absence or presence of glucose using cardiac myocytes. The results were examined in relation to the activity/regulatory characteristics of cardiac carnitine palmitoyltransferase (CPT) estimated by measuring rates of [3H]palmitoylcarnitine formation from [3H]carnitine and palmitoyl-CoA by isolated mitochondria. To define the involvement of other hormones, we examined whether hyperthyroidism altered basal or agonist-stimulated cardiac cAMP concentrations in cardiac myocytes and whether the effects of hyperthyroidism could be reversed by 24 h exposure to insulin infused subcutaneously (2 i. u. per day; Alzet osmotic pumps). Rates of 14C-palmitate oxidation (to 14CO2) by cardiac myocytes were significantly increased (1.6 fold; P< 0.05) by hyperthyroidism, whereas the percentage suppression of palmitate oxidation by glucose was greatly diminished. Cardiac CPT activities in mitochondria from hyperthyroid rats were 2-fold higher and the susceptibility of cardiac CPT activity to inhibition by malonyl-CoA was decreased. These effects were not mimicked by 48 h starvation. The decreased susceptibility of cardiac CPT activities to malonyl-CoA inhibition in hyperthyroid rats was normalised by 24 h exposure to elevated insulin concentration. Acute insulin addition did not influence the response to glucose in cardiac myocytes from euthyroid or hyperthyroid rats and basal and agonist-stimulated cAMP concentrations were unaffected by hyperthyroidism in vivo. The data provide insight into possible mechanisms by which hyperthyroidism facilitates fatty acid oxidation by the myocardium, identifying changes in cardiac CPT activity and malonyl-CoA sensitivity that would be predicted to render cardiac fatty acid oxidation less sensitive to external factors influencing malonyl-CoA content, and thereby to favour fatty acid oxidation. The increased CPT activity observed in response to hyperthyroidism may be a consequence of an impaired action of insulin but occurs through a cAMP-independent mechanism.     

Mechanism for binding of fatty acids to hepatocyte plasma membranes

The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.     

Oleate prevents palmitate-induced cytotoxic stress in cardiac myocytes

The cytotoxicity of saturated fatty acids has been implicated in the pathophysiology of cardiovascular disease, though their effects on cardiac myocytes are incompletely understood. We examined the effects of palmitate and the mono-unsaturated fatty acid oleate on neonatal rat ventricular myocyte cell biology. Palmitate (0.5mM) increased oxidative stress, as well as activation of the stress-associated protein kinases (SAPK) p38, Erk1/2, and JNK, following 18h and induced apoptosis in approximately 20% of cells after 24h. Neither antioxidants nor SAPK inhibitors prevented palmitate-induced apoptosis. Low concentrations of oleate (0.1mM) completely inhibited palmitate-induced oxidative stress, SAPK activation, and apoptosis. Increasing mitochondrial uptake of palmitate with l-carnitine decreased apoptosis, while decreasing uptake with the carnitine palmitoyl transferase-1 inhibitor perhexiline nearly doubled palmitate-induced apoptosis. These results support a model for palmitate-induced apoptosis, activation of SAPKs, and protein oxidative stress in myocytes that involves cytosolic accumulation of saturated fatty acids.     

Capillary transport of adenosine

We tested the hypothesis that capillary exchange of adenosine is influenced by the ability of endothelial cells (ECs) to take up adenosine. Triple-indicator diffusion experiments were performed by injecting [14C]adenosine, [3H]9-beta-D-arabinofuranosylhypoxanthine ( [3H]araH), and radioiodinated serum albumin (RISA) into the arterial perfusate of isolated nonworking guinea pig hearts. Tracer appearance in venous effluent was observed over time. The early extraction of [14C]adenosine was much higher than that of [3H]araH. Extracted [3H]araH returned to the vascular space, but [14C]adenosine did not. Quantitative analysis of the curves by using a mathematical model indicates that approximately half of the extracted adenosine enters ECs and is metabolized. The remainder enters the interstitium and is taken up by myocytes, ECs, or other cells and is metabolized. We conclude that uptake of adenosine by ECs represents a significant influence on the capillary exchange of adenosine.     

Glucagon effect on myocardial transport and utilization of energy-metabolites from the coronary microcirculation in the perfused rabbit heart

Effect of glucagon on energy-metabolite transport into cardiac muscle was studied during a single transit through the isolated rabbit heart using a rapid paired-tracer dilution method. Kinetic experiments revealed that 1.5 microM glucagon stimulated the influx of palmitate bound to 30 g/litre albumin, by increasing the V 2.3 times and increasing the Km for transport 2.4 times. Tracer uptake of D-glucose, as the only exogenous substrate provided, was increased by 80% by 1.5 microM glucagon. Myocardial utilization of [3H]-or [14C]-labelled short-chain monocarboxylic acids (L-lactate, pyruvate and acetate) was significantly reduced by glucagon, to the same degree as their unidirectional sarcolemmal transport. Inhibition of L-[14C]lactate uptake was dose-dependent and in positive correlation with myocardial lactate production. It is concluded that glucagon may regulate sarcolemmal permeability and myocardial utilization for energy-metabolites from the coronary circulation.     

Influence of collagen gel substrata on certain biochemical activities of hepatocytes in primary culture

In order to study the influence of cell shape as modulated by the extracellular matrix on the cellular activity, hepatocytes isolated from liver were maintained on collagen I coated plastic substrata and collage I gel substrata and certain hepatocyte specific functions were investigated. The incorporation of³[H]-leucine into total proteins and albumin secreted by cells maintained on collagen gel was found to be significantly higher compared to those maintained on a collagen coated plastic substrata, indicating that hepatocytes on collagen gel have an enhanced albumin synthesizing capacity. Increased incorporation of³⁵[S]-sulphate into total proteoglycans (PG) and a relatively higher fraction of the³⁵[S]-PG in the extracellular space showed an increased rate of synthesis and secretion of sulphated PGs by cells maintained on collagen gels. But in contrast to the above results, the incorporation of³[H]-leucine into cytokeratins C₈, C₁₈ and actin were significantly low in cells maintained on collagen gel. The tyrosine amino transferase activity exhibited by hepatocytes preincubated with dexamethasone on collagen gel was also significantly low. The different forms of collagen substrata appeared to have no effect on the amino acid transport by hepatocytes, further suggesting that the various hepatocyte specific functions are not uniformly altered when hepatocytes are maintained on three-dimensional collagen gel substrata. These results indicate that the shape of the cell as determined by the nature of the matrix substratum influences the synthetic activity of secretory proteins and those remaining intracellularly, differently.