Regulatory characteristics of amino acid transport in newborn rat renal cortex cells

The uptake of α-aminoisobutyric acid by slices of kidney cortex from newborn rats is enhanced by a preliminary incubation of the tissue in buffer at 37 °C. This effect is abolished by anaerobiosis, the presence of dinitrophenol or the removal of Na⁺ during the preliminary incubation. Cycloheximide (50 μM) and purimycin (1 mM) as well as α-aminoisobutyric acid, glycine and proline (5 mM) in the pre- incubation buffer also abolish the effect, while actinomycin D (0.8 μM) partially inhibits the enhancement due to preliminary incubation. A kinetic examination of the phenomenon indicates that the enhanced uptake is due to an increased entry rate into the cells without a change in efflux. There is no alteration in the apparent transport $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ but an increase in the $\text{V}$ for entry. The effect is dependent on tissue age being observed between birth and 22 days, after which there is a decrease in response to preliminary incubation with no effect seen in adult tissues.     

Mercury inhibition of avian fatty acid synthetase complex.

(1) Subcutaneous or intra-abdominal injections of 8 mg of HgCl2/100 g body weight markedly depressed hepatic fatty acid synthetase activity of chicks at 1 h post-injection. The depression occurred despite the fact that the chicks continued to eat up until the time they were killed. Under these same conditions, the hepatic activity of acetyl-CoA carboxylase (EC 6.4.1.2) was not affected by HgCl2, while the activity of the mitochondrial system of fatty acid elongation was stimulated. (2) When 2-mercaptoethanol was included in the incubation medium for a highly purified preparation of fatty acid synthetase, 500 muM HgCl2 was required to show definite inhibition of the enzyme. When 2-mercaptoethanol was omitted, 50 muM HgCl2 was inhibitory and 100 muM HgCl2 abolished enzyme activity. (3) 2 mM dithiothreitol completely protected the purified fatty acid synthetase preparation from inhibition by 100 muM HgCl2. When dithiothreitol was added after the addition of enzyme to the mercury-containing medium, protection of the enzyme was not complete. (4) Dialysis of cytosol fractions from chicks injected with HgCl2 against 500 vol. of 0.2 M potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 10 mM dithiothreitol for 4 h at 4 degrees stimulated the fatty acid synthetase activity of the fractions. Dialysis of cytosol fractions from noninjected chicks under the same conditions was without effect on fatty acid synthetase activity. (5) These data support the hypothesis that the inhibitory effect of HgCl2 administered in vivo on hepatic fatty acid synthetase activity in chicks is mediated through the interaction of mercury with the sulfhydryl groups of the enzyme.     

Amino acid transport in isolated rat thymocytes. Effects of divalent cations and ethanol.

In dispersed rat thymocytes neither basal alpha-aminoisobutyric acid influx nor influx stimulated by insulin, prostaglandin theophylline, or butyryl adenosine 3':5'-monophosphate (cyclic AMP) depended on extracellular calcium or magnesium. The divalent cation ionophore A23187 inhibited both basal and stimulated alpha-aminoisobutyric acid influx. The extent to which influx was inhibited depended on ionophore concentration, extracellular calcium concentration, and time but did not depend on extracellular magnesium. Significant inhibition could be detected at an ionophore concentration of 1 muM and maximal inhibition occurred with 6 muM A23187. A23187 increased cellular uptake of calcium and there was good agred calcium uptake and that for ionophore inhibition of alpha-aminoisobutyric acid influx. Incubating cells with A23187 and then adding ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid completely reversed ionophore-stimulated cellular calcum uptake but did not reverse inhibition of alpha-aminoisobutyric acid influx. Thus, A23187 produces irreversible inhibition of alpha-aminoisobutyric acid transport in dispersed rat thymocytes. Ethanol abolished insulin-stimulated alpha-aminoisobutyric acid influx but did not alter basal influx or that stimulated by prostaglandin E1, theophylline, or N6,O2'-dibutyryl adenosine 3':5'-monophosphate. Inhibition could be detected with 0.2% (v/v) ethanol and insulin-stimulated alpha-aminoisobutyric influx was abolished with 1% ethanol. The effect of ethanol occurred immediately and could be reversed completely. This ability of ethanol to inhibit selectively insulin-stimulated alpha-aminoisobutyric acid influx indicates that the mechanism through which insulin stimulates alpha-aminoisobutyric acid influx is functionally distinct from the stimulation produced by cyclic AMP.     

Induction of carcinoembryonic-antigen-gene expression in human colorectal carcinoma by sodium butyrate.

The ability of the insulin-induced phospho-oligosaccharide to stimulate amino acid transport was studied in isolated rat hepatocytes. At low alpha-aminoisobutyric acid concentrations (0.1 mM), both 100 nM-insulin and 10 microM-phospho-oligosaccharide doubled amino acid uptake after 2 h of incubation. This stimulation was prevented by 0.1 mM-cycloheximide or 5 micrograms of actinomycin D/ml, indicating that the phospho-oligosaccharide, like insulin, was acting via the synthesis of a high-affinity transport component. The effects of the phospho-oligosaccharide and of insulin were blocked by Ins2P (2.5 mM), but not by myo-inositol, inositol hexaphosphoric acid or several monosaccharides such as mannose, glucosamine and galactose. Both the temporal effect on amino acid entry and the extent of stimulation of this process by the phospho-oligosaccharide indicate that this molecule mimics, and may mediate, some of the long-term actions of insulin. However, the effects of phospho-oligosaccharide and insulin were not exactly the same, since the effect of insulin, but not of the phospho-oligosaccharide, was additive with that of glucagon.     

Effect of valine on the control of fatty acid synthesis in white adipose tissue of the rat.

In adipocytes from fed rats, the rate of fatty acid synthesis in the presence of glucose and insulin was inhibited 40% by valine (5 mm). tthis inhibition was largely abolished by the addition to the incubation medium of the transaminase inhibitor aminooxy acetate, and of pyruvate and agents which raise the intracellular pyruvate levels such as N,N,N1,N1-tetramethyl-p-phenylenediamine. Pyruvate output into the incubation medium from fat pads obtained from fed rats and incubated with glucose and insulin was decreased significantly by the addition of valine. When adipocytes were incubated under similar conditions, the final concentration of pyruvate in the incubation medium was 42 +/- 1.6 muM under control conditions and approximately one third of this value in the presence of 2.5 mM valine. Valine had no significant effect on pyruvate dehydrogenase (lipoate) (EC 1.2.4.1) activity when assayed in homogenates prepared from adipose tissue previously incubated for 60 min with the amino acid. Although the ketoacid analogue of valine alpha-ketoisovaleric acid, is a competitive inhibitor of pyruvate dehydrogenase (lipoate) (K1 = 1.4 mM), this cannot solely account for the valine-induced reduced rate of lipogenesis. Rather, the mechanism involves a reduction in pyruvate concentration and thereby a diminished flow through pyruvate dehydrogenase (lipoate). Details of the possible mechanism are discussed.     

Adenylate cyclase from rat-liver plasma membrane: inhibition by mersalyl and other mercurial derivatives.

The adenylate cyclase activity from a rat liver plasma membrane preparation was inhibited by low concentrations (1-10 muM) of the mercurial diuretic mersalyl. Complete inhibition was obtained with 0.1 mM mersalyl. Similar effects were observed whether the adenylate cyclase preparation was assayed in the presence of 10 muM GTP, 0.1 muM glucagon, 10 mM NaF or without any addition. The effect of mersalyl was not due to inhibition of the regenerating system present in the incubation medium, since the effect of mersalyl was preserved and even enhanced in its absence. The inhibition brought about by mersalyl was due to both a decrease of the maximal velocity of the reaction and of the affinity of the enzyme for the substrate. It was immediate, and irreversible spontaneously, but it was reversed by the simultaneous additions of 2-mercaptoethanol, in a dose-dependent fashion. Other -SH reagents were found to have an effect equal to, or lower than, that of mersalyl. Mersalyl had no effect upon Mg2+-ATPase, although it inhibited the (Na+-K+) activated ATPase. Since mersalyl is known to be a 'non-penetrant' reagent, it is postulated that a catalytically important, mercurial-sensitive, part of adenylate cyclase is at the surface of the plasma membrane. This view is supported by the following facts: (a) mersalyl acted with a similar dose-response curve upon an intact as well as a detergent-dispersed cyclase preparation while no effect was observed upon a solubilized Mg2+-ATPase preparation; (b) a covalent p-chloromercuribenzoate-Sephadex preparation (but not its supernatant) inhibited the cyclase from intact membranes. It is proposed that mercurial derivatives, by their relative specificity of action (no effect on Mg2+-ATPase), can serve as useful probes in the elucidation of the multicomponent structure of the cyclase system.     

Amino acid uptake in the developing chick embryo heart. The effect of insulin on α-aminoisobutyric acid accumulation

1. The uptake of (14)C-labelled alpha-aminoisobutyric acid by 5-day-old chick embryo hearts was investigated in vitro, together with the effect of insulin thereon. 2. At equilibrium the distribution ratio of this amino acid analogue between intracellular and extracellular water attained values greater than unity. Insulin enhanced the rate of alpha-aminoisobutyric acid accumulation and increased the value of its final concentration in the cell water. 3. The rate of alpha-aminoisobutyric acid accumulation and the effect of insulin on it were independent of the presence of glucose in the incubation medium. Bovine and chicken insulin were equally effective, and the action of the hormone was specifically prevented by an anti-insulin serum but not by puromycin. 4. A linear relationship was observed between the intracellular accumulation of the analogue and the logarithm of the insulin concentration in the range 50muunits-100m-units/ml. of incubation medium. 5. Evidence was obtained for the occurrence of two different transport processes for alpha-aminoisobutyric acid in the chick embryo heart: one subject to saturation and one that was not saturated by reasonable concentrations of the analogue. Insulin increased the effectiveness of the saturable component, increasing the maximal velocity of transport without altering the concentration for half-maximal velocity of transport, and decreased the contribution of the non-saturable component.     

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.     

L-Leucine activates branched chain alpha-keto acid dehydrogenase in rat adipose tissue

The activity of branched chain alpha-keto acid dehydrogenase in extracts of adipose tissue was elevated after homogenization of tissue segments which had been incubated in buffer containing 0.3 mM leucine. A maximum increase (4-fold) was observed in extracts of tissues incubated in buffer containing 2.5 mM leucine, alpha-Ketoisocaproate and leucine caused maximum increases which were of similar magnitude and which required the same length of incubation of the tissue segments (5 to 15 min). The effect of leucine on branched chain alpha-keto acid dehydrogenase activity was observed both in the presence and absence of insulin, which also increased the activity of the enzyme in tissue extracts. Intact adipose tissue segments oxidized [I-14C]leucine at a maximum rate approximately 4 times that of [1-(14)C]valine. The rate of valine oxidation by intact tissue segments was doubled by addition of 0.2 to 0.5 mM unlabeled leucine, but not isoleucine, to medium containing 2 mM [1-(14)C]valine. Leucine, but not valine, also stimulated the rate of oxidation of 2 mM [U-14C]isoleucine by intact tissue segments. These results suggest that branched chain alpha-keto acid dehydrogenase activity, which is thought to limit the rate of branched chain amino acid oxidation in adipose tissue, may be sensitive to changes in the concentration of leucine in rat blood.     

Metabolic characteristics of pancreatic beta-cells exposed to calcium-transporting ionophores.

The effects of the ionophores A-23187 and X-537 A on glucose metabolism, ATP content and sucrose permeability in pancreatic islets microdissected from obese-hyperglycemic mice were studied. The formation of 14CO2 from 10 mM D-[U-14C] GLUCOSE WAS INHIBITED BY OMISSION OF Ca2+ from the medium. A-23187 (10 muM) induced a further decrease of 14CO2 formation whereas X-537 A (10 muM) had no effect. At 20 mM glucose both A-23187 (48 muM) and X-537 A (43 muM) decreased the 14CO2 formation in the absence of Ca2+ whereas only X-537 A inhibited in the presence of Ca2+. X-537 A (43 muM) also decreased the formation of 3H2O from 20 mM D-[5-3H] glucose. The islet content of ATP was not changed after incubation in media deficient in either Mg2+ or Ca2+. However, omission of both Mg2+ and Ca2+ resulted in about 50% decrease of the ATP content. A-23187 and X-537 A induced dose-dependent decreases of the islet ATP content. X-537 A was much more potent than A-23187. Both ionophores induced stronger depression of the ATP content when Ca2+ was omitted. X-537 A (43 muM) but not A-23187 (48 muM) increased the beta-cell membrane permeability as indicated by an increased sucrose space in relation to the urea space of islets. Such an effect was not obtained with X-537 A at 1 muM or by omission of Ca2+. It is suggested that the marked metabolic effects of the ionophores reflect an impaired mitochondrial metabolism. These metabolic changes should be considered in interpretations of ionophore action on insulin secretion.     

Calcium enhances the hemolytic action of bile salts

The lysis of human erythrocytes by bile salts in buffer containing isotonic saline was dramatically enhanced by the addition of 5-10 mM calcium chloride. All bile acids tested showed this effect, with a marked increase in lysis occurring at 0.75 mM for deoxycholate, 1 mM for chenodeoxycholate, 2.5 mM for ursodeoxycholate and 5.5 mM with cholate in the presence of 10 mM calcium chloride. The effect appeared to be specific for calcium; strontium chloride and magnesium chloride gave no stimulatory effect. The increased lysis of the erythrocytes in the presence of 1 mM deoxycholate and 1-10 mM calcium chloride was not associated with increased uptake of the bile salt by the cells (measured with [14C]deoxycholate). Using erythrocytes previously labelled with [3H]cholesterol, there was no evidence of an enhanced removal of that membrane component in the presence of calcium and deoxycholate, compared to deoxycholate alone. The sensitivity of the cells to the effect of calcium in the presence of 1 mM deoxycholate increased with the length of time of their storage at 4 degrees C. The sensitivity returned to that of fresh cells after incubation at 37 degrees C with 30 mM adenosine plus 25 mM glucose, but this treatment did not further diminish the lysis. Lysis in the presence of 10 mM calcium chloride and 1 mM deoxycholate was partially blocked by increasing the KCl concentration at the expense of NaCl. The maximum effect occurred with a buffer comprising 100 mM KCl/50 mM NaCl. A more dramatic reduction in the lysis followed the incorporation of the calcium chelator, quin2, into the cells. The lysis induced by 1 mM deoxycholate in the presence of calcium was reduced by 80% in quin-2-loaded cells compared to controls. The data suggest that bile acids can promote the influx of calcium into erythrocytes, leading to lysis as a result of the efflux of intracellular potassium and/or the uptake of sodium from the incubation medium. The data further suggest that cellular effects may occur at lower bile acid concentrations than that thought to be required for detergent damage.     

Insulin-induced insulin resistance of alpha-aminoisobutyric acid transport in cultured human skin fibroblasts.

Cultured fibroblasts derived from skin biopsies were used to develop a system for studying insulin resistance in human tissue in vitro. Uptake of alpha-aminoisobutyric acid by cultured human skin fibroblasts was found to occur by a combination of saturable and nonsaturable processes. Insulin stimulated uptake by decreasing the Km of the saturable transport system from 0.58 mM to 0.26 mM. The maximal velocity of saturable uptake was 16.6 nmol/10(7) cells/min in both the presence and absence of insulin. Uptake of alpha-aminoisobutyric acid at 0.2 mM was studied in human skin fibroblasts with and without chronic exposure to insulin for 4 days at an initial concentration of 10 micrograms/ml. Unstimulated uptake was increased from 17 to 20 nmol/10(8) cells/min, and the increase in uptake due to maximal stimulation by insulin was unchanged at 16 nmol/10(8) cells/min in the cells exposed chronically to insulin. The apparent Km for insulin was increased from 80 microunits/ml to 2400 microunits/ml in the insulin-exposed cells. Thus, chronic exposure to insulin induces resistance of alpha-aminoisobutyric acid uptake by decreasing the apparent affinity for insulin.     

Simultaneous determination of l- and d-lactic acid in plasma by capillary electrophoresis

A novel method for simultaneous determination of d- and l-lactic acids in plasma was presented by capillary electrophoresis with photodiode array detection at 195nm. The separation was performed in an uncoated fused-silica capillary. The parameters influencing the resolution and the migration time of lactic acids were optimized. When 150mM phosphate-Tris buffer (pH 7.0) consisting of 220mM 2-hydroxypropyl-beta-cyclodextrin and 0.2mM tetradecyltrimethylammonium bromide was utilized as the running buffer, highly effective chiral separation of d- and l-lactic acids was achieved at about 42min at an effective voltage of -25kV. The resolution of lactic acid enantiomers was >/=1.25. The limits of detection of d- and l-lactic acids in standard solution without any pretreatment were 80 and 50muM (S/N=3), respectively. Sample pretreatment was preceded by protein-removal procedure with acetonitrile. With a pre-concentration procedure by 10 times, the limits of detection of d- and l-lactic acids were 20 and 15muM (S/N=10), respectively. The satisfactory analytical performance of the proposed method was validated.     

The effect of intracellular calcium ions on adrenaline-stimulated adenosine 3'':5''-cyclic monophosphate concentrations in pigeon erythrocytes, studied by using the ionophore A23187.

1. The bivalent cation ionophore A23187 was used to increase the intracellular concentration of Ca2+ in pigeon erythrocytes to investigate whether the increase in cyclic AMP content caused by adrenaline might be influenced by a change in intracellular Ca2+ in intact cells. 2. Incubation of cells with adrenaline, in the concentration range 0.55--55 muM, resulted in an increase in the concentration of cyclic AMP over a period of 60 min. The effect of adrenaline was inhibited by more than 90% with ionophore A23187 (1.9 muM) in the presence of 1 mM-Ca2+. This inhibition could be decreased by decreasing either the concentration of the ionophore or the concentration of extracellular Ca2+, and was independent of the concentration of adrenaline. 3. The effect of ionophore A23187 depended on the time of incubation. Time-course studies showed that maximum inhibition by ionophore A23187 was only observed when the cells were incubated with the ionophore for at least 15 min before the addition of adrenaline. 4. The inhibition by ionophore A23187 depended on the concentration of extracellular Ca2+. In the absence of Mg2+, ionophore A23187 (1.9 muM) inhibited the effect of adrenaline by approx. 30% without added Ca2+, by approx. 66% with 10 muM-Ca2+ and by more than 90% with concentrations of added Ca2+ greater than 30 muM. However, even in the presence of EGTA [ethanedioxybis(ethylamine)tetra-acetate](0.1--10 mM), ionophore A23187 caused an inhibition of the cyclic AMP response of at least 30%, which may have been due to a decrease in cell Mg2+ concentration. 5. The addition of EGTA after incubation of cells with ionophore A23187 resulted in a partial reversal of the inhibition of the effect of adrenaline. 6. Inclusion of Mg2+ (2 mM) in the incubation medium antagonized the inhibitory action of ionophore A23187. This effect was most marked when the ionophore A23187 was added to medium containing Mg2+ before the addition of the cells. 7. The cellular content of Mg2+ was decreased by approx. 50% after 20 min incubation with ionophore A23187 (1.9 muM) in the presence of Ca2+ (1 mM) but no Mg2+. When Mg2+ (2 mM) was also present in the medium, ionophore A23187 caused an increase of approx. 80% in cell Mg2+ content. Ionophore A23187 had no significant effect on cell K+ content. 8. Ionophore A23187 caused a decrease in cell ATP content under some conditions. Since effects on cyclic AMP content could also be shown when ATP was not significanlty lowered, it appeared that a decrease in ATP in the cells could not explain the effect of ionophore A23187 on cyclic AMP. 9. Ionophore A23187 (1.9 muM), with 1 mM-Ca2+, did not enhance cyclic AMP degradation in intact cells, suggesting that the effect of ionophore A23187 on cyclic AMP content was mediated through an inhibition of adenylate cyclase rather than a stimulation of cyclic AMP phosphodiesterase. 10. It was concluded that in intact pigeon erythrocytes adenylate cyclase may be inhibited by intracellular concentrations of Ca2+ in the range 1-10 muM.     

6-Phosphogluconate dehydrogenase. Purification and kinetics.

A method is described for the isolation and purification of 6-phosphogluconate dehydrogenase from pig liver. The molecular weight is estimated at 83,000 and that of the subunits is 42,000 as determined by gel electrophoresis. The pH maximum is 8.5 in 50 mM glycine/NaOH buffer and from 7.5 to 10 in 50 mM phosphate buffer at 30 degrees. Magnesium ion is not required for activity and acts as an inhibitor at concentrations above 20 mM. A cellular fractionation study indicates that this enzyme is located almost entirely within the soluble portion of the cytoplasm. Kinetic studies have been done in 50 mM glycine buffer, pH 8.5, at 30 degrees. The data are consistent with a sequential mechanism in which NADP+ is added first, followed by 6-phosphogluconate, and the products are released in the order, CO2, ribulose 5-phosphate, and NADPH. The Michaelis constant is 13.5 muM for 6-phosphogluconate. Dissociation constants are 4.8 muM for NADP+ and 5.1 muM for NADPH.     

The prostaglandins system and insulin release. Studies with the isolated perfused rat pancreas.

Using the isolated perfused rat pancreas PGE2 (1 MUM and 10 muM) had no effect on basal or glucose (10 and 20 mM)-induced insulin release (IR). PGF2 alpha stimulated basal IR at 1 muM and inhibited IR at 10 muM. The glucose-induced IR was unaffected by this PG. Furosemide (5 and 10 mM) led to a monophastic IR at low glucose (glu) and to a potentiation of IR at high glu. Only high indomethacin (Indo) (50 microgram/ml) inhibited glu-induced IR. The stimulatory effect of furosemide on IR could not be inhibited by indomethacin. However mepacrine (0.1 mM) abolished the furosemide effect. Also glu-induced IR was inhibited by mepacrine. Acetylsalicylic acid (30 mg/100 ml) had no significant influence on glu-induced IR. These findings provide evidence that phospholipase activation rather than increased PG synthesis might primarily be involved in the secretory process of insulin.     

Effect of glucocorticoids on the glucose transport system of isolated fat cells.

Glucocorticoids inhibit glucose utilization by fat cells. The possibility that this effect results from altered glucose transport was investigated using an oil-centrifugation technique which allows a rapid (within 45 s) estimation of glucose or 3-O-methylglucose uptake by isolated fat cells. At high concentration (greater than 25 muM), dexamethasone inhibited glucose uptake within 1 min of its addition to fat cells. Efflux of 3-O-methylglucose was also impaired by 0.1 mM dexamethasone. However, diminished glucose uptake was not a specific effect of glucocorticoids; high concentrations (0.1 mM) of 17beta-estradiol, progesterone, and deoxycorticosterone produced a similar response in adipocytes. At a more physiologic steroid concentration (0.1 muM), glucocorticoids inhibited glucose uptake in a time-dependent manner (maximum effect in 1 to 2 hours). This effect was specific for glucocorticoids since, under these conditions, glucose uptake was not changed by the non-glucocorticoid steroids. Lineweaver-Burk analysis showed that 0.1 muM dexamethasone treatment produced a decrease in Vmax for glucose uptake but did not change the Ku. Hexokinase activity and ATP levels were not altered by this treatment, suggesting that processes involved in glucose phosphorylation were not affected. Dexamethasone treatment also caused a reduction in uptake of 3-O-methylglucose when assayed using a low sugar concentration (0.1 mM). At a high concentration (10 mM), uptake of the methyl sugar was only slightly less than normal in treated cells. Stimulation by insulin markedly enhanced uptake of glucose and 3-O-methylglucose by both treated and untreated cells. At a low hexose concentration (0.1 mM) and in the presence of insulin, sugar uptake by dexamethasone-treated cells was slightly less than control cells. Stimulation by insulin did however completely overcome the alteration in hexose uptake when larger concentrations of sugars (greater than 5 mM) were used. There was no detectable change in total protein synthesis during incubation of fat cells with dexamethasone. However, actinomycin C blocked the inhibitory effect of dexamethasone on glucose uptake. Cycloheximide, which caused a small inhibition in glucose uptake, prevented the full expression of the inhibitory effect of dexamethasone on glucose transport. These results indicate that dexamethasone alters the facilitated transport of glucose and, secondly, suggest that synthesis of RNA and protein is needed for glucocorticoid action.     

Influence of aerobic energy deficit, ouabain and harmaline on the phenylalanine and galactose active transport by snail intestine

The effect of anaerobiosis (N2 bubbling of the medium) or 10(-4) M dinitrophenol on the penetration of 0.5 mM Phe in snail and rat everted intestine, in 2 min and 30 min incubation periods, has been studied. The aerobic energy deficit inhibits the amino acid net entry in both species, but whereas the active transport is annulled in rat, snail intestine is capable of continuing to accumulate Phe against a gradient. The prolonged action (30 min of preincubation) of 1 mM ouabain inhibits 0.1 mM Phe and 0.1 mM galactose entry in snail intestine. Amino acid uptake is far higher than the one obtained in the absence of Na+, in which condition Phe keeps accumulating against a gradient in the tissue water. Galactose active transport, instead, becomes null in the presence of the glucoside or in the absence of Na+. One mM harmaline is able to inhibit the initial entry of galactose into the tissue, while higher than 5 mM concentrations are required to inhibit that of Phe. Results confirm that snail intestine is capable of easily carrying out active transport processes with energy from anaerobic origin. On the other hand Phe transport is less sensitive to the absence of Na+, presence of ouabain or harmaline than that of galactose, so that contrary to what has been observed for the sugar, the active transport of the amino acid is not annulled in any of the three conditions.     

D-galactose uptake by snail intestine: competitive inhibition by phlorizin and sodium dependence

The net entry of galactose into the tissue of snail everted intestinal rings with 2 or 15 minute long incubation periods has been measured. With 10(-4) M phlorizin, the mediated transport is completely blocked while only the passive entry of sugar is produced. Lower concentrations of the glycoside partially inhibit transport according to competitive inhibition kinetics (K1 = 10(-7) M). The transport of galactose is Na+ dependent. In the absence of Na+, transport ceases and the sugar entry can be explained through simple diffusion. With 15 mM Na+ (control 71,4 mM) transport diminishes and a marked increase in the apparent Km with no changes in the Vmax is observed. One mM harmaline completely blocks galactose (0.5 mM) transport. One mM ouabain also makes transport null, but only after tissue preincubation with the inhibitor on the serosal side.     

Cholesterol esterase in rat adipose tissue and its activation by cyclic adenosine 3':5'-monophosphate-dependent protein kinase.

A high level of cholesterol esterase activity, comparable to that of hormone-sensitive triglyceridase, has been demonstrated in rad adipose tissue. Essentially all of the activity was in the isolated adipocytes, primarily in the 100,000 times g supernatant fraction of the adipocytes. Cholesterol esterase activity in the 100,000 times g supernatant fraction was increased 40 plus or minus 16% by incubation with ATP (0.5 mM), Mg-2+ (1.25 mM), and cyclic adenosine 3':5'-monophosphate (cyclic AMP) (10 muM), conditions which also activated hormone-sensitive triglyceridase. Protein kinase inhibitor (rabbit skeletal muscle) blocked activation, and activation was restored by the addition of excess protein kinase (bovine skeletal muscle). In extracts prepared from adipocytes first incubated for 5 min with 10 muM epinephrine and 1 mM theophylline, there was no cyclic AMP-dependent cholesterol esterase activation, implying that the enzyme had been activated by a similar mechanism in the intact cell. The physiological role of this high level of cholesterol esterase activity in adipose tissue is unclear. Its relationship to hormone-sensitive triglyceride lipase, with which it extensively co-fractionates, and its possible involvement in fat mobilization remain to be determined.