Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.     

Insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. Synthesis of a high affinity component of transport.

Insulin and glucagon stimulate amino acid transport in freshly prepared suspensions of isolated rat hepatocytes. The kinetic properties of alpha-amino[1-14C]isobutyric acid (AIB) transport were investigated in isolated hepatocytes following stimulation by either hormone in vitro. In nonhormonally treated cells (i.e. basal state), saturable transport occurred mainly through a low affinity (Km approximately equal to 40 mM) component. In insulin or glucagon-treated hepatocytes, saturable transport occurred through both a low affinity component (similar to that observed in the basal state) and a high affinity (Km approximately equal to 1 mM) component. At low AIB concentrations (less than 0.5 mM), insulin and glucagon at maximally stimulating doses increased AIB uptake about 2-fold and 5-fold, respectively. The high affinity component induced by either hormone exhibited the properties of the A (alanine preferring) mediation of amino acid transport. This component required 2 to 3 h for maximal expression, and its emergence was completely prevented by cycloheximide. Half-maximal stimulation was elicited by insulin at about 3 nM and by glucagon at about 1 nM. Dibutyryl cyclic AMP mimicked the glucagon effect and was not additive to it at maximal stimulation. Maximal effects of insulin and glucagon, or insulin and dibutyryl cyclic AMP, were additive. We conclude that insulin and glucagon can modulate amino acid entry in hepatocytes through the synthesis of a high affinity transport component.     

Effect of dibutyryl cyclic adenosine monophosphate on active amino acid transport in Streptomyces hydrogenans

The active uptake of 2-aminoisobutyric acid (AIB) and several other amino acids in resting cells of Streptomyces hydrogenans was found to be stimulated by exogenously added adenosine cyclic monophosphate (cAMP). The uptake of glycerol, sorbose, and pyrimidine nucleosides remained unaffected. Among the various cAMP derivatives tested, the dibutyryl derivative was found to be most effective, followed by monobutyryl cAMP, and cAMP. Dibutyryl cGMP was also found to stimulate AIB transport, and its effectivity was as good as that of dibutyryl cAMP. The effect of dibutyryl cAMP is time dependent and attains its maximum after 40–60 min of incubation at 30°C in K-Na-phosphate buffer. Dibutyryl cAMP-dependent transport stimulation has a high temperature coefficient and is prevented by rifamycin SV or chloramphenicol. The rate of leucine incorporation into protein was rapidly increased upon addition of dibutyryl cAMP. Kinetic studies reveal that the stimulation of AIB transport is characterized by an increase in maximum uptake rate and an unaltered apparent Michaelis constant. Analysis of the unidirectional fluxes show that both influx and efflux are enhanced by dibutyryl cAMP. It is concluded that exogenous dibutyryl cAMP stimulates de novo synthesis of certain protein including the transport catalysts for various amino acids.     

Functional and molecular characteristics of system L in human breast cancer cells

The functional and molecular properties of system L in human mammary cancer cells (MDA-MB-231 and MCF-7) have been examined. All transport experiments were conducted under Na(+)-free conditions. alpha-Aminoisobutyric acid (AIB) uptake by MDA-MB-231 and MCF-7 cells was almost abolished by BCH (2-amino-2-norbornane-carboxylic acid). AIB uptake by MDA-MB-231 cells was also inhibited by L-alanine (83.6%), L-lysine (75.6%) but not by L-proline. Similarly, L-lysine and L-alanine, respectively, reduced AIB influx into MCF-7 cells by 45.3% and 63.7%. The K(m) of AIB uptake into MDA-MB-231 and MCF-7 cells was, respectively, 1.6 and 8.8 mM, whereas the V(max) was, respectively, 9.7 and 110.0 nmol/mg protein/10 min. AIB efflux from MDA-MB-231 and MCF-7 cells was trans-stimulated by BCH, L-glutamine, L-alanine, L-leucine, L-lysine and AIB (all at 2 mM). In contrast, L-glutamate, L-proline, L-arginine and MeAIB had no effect. The interaction between L-lysine and AIB efflux was one of low affinity. The fractional release of AIB from MDA-MB-231 cells was trans-accelerated by D-leucine and D-tryptophan but not by D-alanine. MDA-MB-231 and MCF-7 cells expressed LAT1 and CD98 mRNA. MCF-7 cells also expressed LAT2 mRNA. The results suggest that AIB transport in mammary cancer cells under Na(+)-free conditions is predominantly via system L which acts as an exchange mechanism. The differences in the kinetics of AIB transport between MDA-MB-231 and MCF-7 cells may be due to the differential expression of LAT2.     

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat liver cells

The effect of dibutyryl cyclic AMP on the uptake of taurocholic acid by isolated rat hepatocytes was studied. In the presence of low levels (10–100 μM) of the cyclic nucleotide the initial rate of uptake was increased significantly, with a peak occurring at about 20 μM. In contrast, concentrations of dibutyryl cyclic AMP between 200 μM and 1 mM caused a significant decrease in the initial rate of uptake of the bile acid by the cells. Sodium-dependent transport of taurocholic acid was found to be enhanced by 20 μM dibutyryl cyclic AMP, but sodium-independent uptake appeared to be unaffected. Inhibition by 1 mM dibutyryl cyclic AMP, however, was found to occur in both the sodium-dependent and -independent components of the transport system. The initial rate of taurocholic acid uptake in hepatocytes incubated with 1.2 mM extracellular calcium was increased compared to that in calcium-depleted cells, and this increase was entirely due to enhanced sodium-dependent transport. 1.2 mM calcium and 20 μM dibutyryl cyclic AMP together did not stimulate the uptake rate to a greater extent either treatment alone. It is conclude that calcium and low levels of dibutyryl cyclic AMP alter the rate of taurocholic acid uptake by changing the flux of sodium in the hepatocytes. The inhibitory effect of 1 mM dibutyryl cyclic AMP was not relieved by the presence of 1.2 mM calcium in the cell incubation medium. The results show that dibutyryl cyclic AMP can affect the rate of transport of bile acid into liver cells, and suggest a possible regulatory role for cyclic AMP in this process.     

Energetic mechanism of system A amino acid transport in normal and transformed mouse fibroblasts

Ouabain treatment (0.4 mM) of normal and transformed C3H-10T1/2 cells caused a progressive increase in 2-aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 microM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t1/2 for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t1/2 in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl-phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.     

System A transport activity in normal rat hepatocytes and transformed liver cells: substrate protection from inactivation by sulfhydryl-modifying reagents

The transport of amino acids by normal rat hepatocytes and several hepatoma cell lines has been examined for inactivation by various protein-modifying reagents, including the sulfhydryl-preferring reagents N-ethylmaleimide (NEM) and p-chloromercuribenzene sulfonate (PCMBS). Uptake of 2-aminoisobutyric acid (AIB), a specific probe for hepatic System A-mediated transport, was equally sensitive to inhibition by the organic mercurial PCMBS in each of the cell types tested. In contrast, the sensitivity of System A to inactivation by NEM was substantially different among the five cell types. Normal hepatocytes showed the greatest sensitivity, while the hepatoma cells varied in their responsiveness from moderate to no inhibition. PCMBS inactivated greater than 85% of the System A activity in rat H4 hepatoma cells within 10 min (t1/2 = 3 min). The inhibition by PCMBS was rapidly reversed by treatment of the cells with dithiothreitol. Amino acids showing a high affinity for System A protected the transport system from inactivation, whereas non-substrates produced little or no protection. Amino acid-dependent protection was stereospecific and system-specific. L-norleucine competitively inhibited AIB uptake (Ki = 1.9 +/- 0.1 mM) in H4 cells and also protected System A from PCMBS-dependent inactivation (half-maximal protection occurred at an amino acid concentration of 0.6 +/- 0.1 mM). N-bromosuccinimide was completely ineffective as an inhibitor of System A activity in hepatocytes, whereas treatment of H4 rat hepatoma cells with this reagent resulted in greater than 95% inhibition.     

Membrane transport in synchronized Ehrlich ascites tumor cells: uptake of amino acids by the A and L system during the cell cycle.

Using the double thymidine block technique. Ehrlich ascites tumor cells (ELD) carried in continuous spinner culture have been synchronized. Simultaneous monitoring of 3H-thymidine incorporation, cell number and mitotic index yielded a cell cycle time of approximately 13.5 hours. This is composed of an S period of 3-4 hours. G2 of 6-8 hours and M of 1-2 hours. No appreciable G1 is present. Ehrlich cells synchronized in this manner were used to investigate the characteristics of two neutral amino acid transport systems during progression through the cell cycle. Unidirectional influx via the Na-dependent system A was studied using C14-alpha-aminoisobutyrate (AIB) as substrate. The Na-independent system L was monitored using 3H-leucine and 14C-cycloleucine as substrates. Transport by the A system was minimal in M and early S. It underwent a three-fold increase during late S and early G2. In mid G2 the transport via this system rapidly dropped and remained low again through M and early S. The intracellular/extracellular ratios of AIB indicate that the system is actively transporting AIB thoughout the cell cycle. The minimum ratios of approximately 3 were achieved during early M and the maximum ratios of approximately 9 were achieved in late S, early G2. The uptake of leucine and cycloleucine by the L system was quite different during the cell cycle. Maximal unidirectional influx by this system occurred during early and mid S period. Upon progression into G2 the transport rate dropped and remained reduced throughout M. Intracellular/extracellular ratios of leucine or cycloleucine were near unity at the peak of the transport activity (early S) and dropped to values of 0.5 to 0.6 throughout the remainder of the cycle. This result indicates that inward transport by the L system is, for the most part, non-active in growing cells.     

System A amino acid transport in a rat submandibular ductal cell line

1. Neutral amino acid transport was studied in an established cell line derived from rat submandibular glands, RSMTx. 2. The greatest portion of alpha-amino isobutyrate (AIB) transport is mediated by system A. This component is Na+ dependent, pH sensitive, markedly inhibited by methyl AIB and enhanced 2-5-fold by amino acid depletion. 3. Evidence for the presence of other neutral amino acid transport systems, presumably ASC and L, was also found in these cells.     

Neutral amino acid transport in human synovial cells: substrate specificity of adaptative regulation and transinhibition

Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2-aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of alpha-methyl-aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid-free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the depression of system A in these synovial cells was not serum-dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.     

Maintenance of glucagon-stimulated system A amino acid transport activity in rat liver plasma membrane vesicles

Plasma membrane vesicles prepared from intact rat liver or isolated hepatocytes retain transport activity by systems A, ASC, N, and Gly. Selective substrates for these systems showed a Na+-dependent overshoot indicative of energy-dependent transport, in this instance, driven by an artificially-imposed Na+ gradient. Greater than 85% of Na+-dependent 2-aminoisobutyric acid (AIB) uptake was blocked by an excess of 2-(methylamino)isobutyric acid (MeAIB) with an apparent Ki of 0.6 mM. Intact hepatocytes obtained from glucagon-treated rats exhibited a stimulation of system A activity and plasma membrane vesicles isolated from those same cells partially retained the elevated activity. Transport activity induced by substrate starvation of cultured hepatocytes was also evident in membrane vesicles prepared from those cells. The membrane-bound glucagon-stimulated system A activity decays rapidly during incubation of vesicles at 4 degrees C (t1/2 = 13 h), but not at -75 degrees C. Several different inhibitors of proteolysis were ineffective in blocking the decay of transport activity. Hepatic system N transport activity was also elevated in plasma membrane vesicles from glucagon-treated rats, whereas system ASC was essentially unchanged. The results indicate that both glucagon and adaptive regulation cause an induction of amino acid transport through a plasma membrane-associated protein.     

Neutral Amino Acid Transport in Astrocytes: Characterization of Na+-Dependent and Na+-Independent Components of α-Aminoisobutyric Acid Uptake

Neutral amino acid transport is largely unexplored in astrocytes, although a role for these cells in blood-brain barrier function is suggested by their close apposition to cerebrovascular endothelium. This study examined the uptake into mouse astrocyte cultures of alpha-aminoisobutyric acid (AIB), a synthetic model substrate for Na+-dependent system A transport. Na+-dependent uptake of AIB was characteristic of system A in its pH sensitivity, kinetic properties, regulatory control, and pattern of analog inhibition. The rate of system A transport declined markedly with increasing age of the astrocyte cultures. There was an unexpectedly active Na+-independent component of AIB uptake that declined less markedly than system A transport as culture age increased. Although the saturability of the Na+-independent component and its pattern of analog inhibition were consistent with system L transport, the following properties deviated: (1) virtually complete inhibition of Na+-independent AIB uptake by characteristic L system substrates, suggesting unusually high affinity of the transporter; (2) apparent absence of trans-stimulation of AIB influx; (3) unusually concentrative uptake at steady state (the estimated distribution ratio for 0.2 mM AIB was 55); and (4) susceptibility to inhibition by N-ethylmaleimide. Direct study of the uptake of system L substrates in astrocytes is needed to confirm the present indications of high affinity and concentrative Na+-independent transport.     

Cyclic AMP mediated increased amino acid transport: effect of isoproterenol administration.

A single injection of isoproterenol (IPR) stimulates cell proliferation in rodent salivary glands after a lag period of about 24 hrs. Among the many events occurring prior to stimulated DNA synthesis, there is an early increase in cAMP levels and elevated transport of amino acids into the parotid gland. Amino acid transport is also elevated in liver and pancreas, tissues not induced to proliferate by IPR. IPR-stimulated alpha-aminoisobutyric acid (AIB) transport in parotid, pancreas and liver is augmented by prior injection of theophylline and is mimicked by dibutyryl cAMP. In all three tissues, changes in cAMP levels and subsequent increases in AIB transport appear to be closely related events. Since only the parotid gland is stimulated to grow after IPR injection, amino acid transport and growth would not appear to be directly related.     

Derepression of amino acid transport by amino acid starvation in rat hepatoma cells.

Amino acid starvation causes an adaptive increase in the initial rate of transport of selected neutral amino acids in an established line of rat hepatoma cells in tissue culture. After a lag of 30 min, the initial rate of transport of alpha-aminoisobutyric acid (AIB) increases to a maximum after 4 to 6 h starvation of 2 to 3 times that seen in control cells. The increased rate of transport is accompanied by an increase in the Vmax and a modest decrease in the Km for this transport system, and is reversed by readdition of amino acids. The enhancement is specific for amino acids transported by the A or alanine-preferring system (AIB, glycine, proline); uptake of amino acids transported by the L or leucine-preferring system (threonine, phenylalanine, tyrosine, leucine) or the Ly+ system for dibasci amino acids (lysine) is decreased under these conditions. Amino acids which compete with AIB for transport also prevent the starvation-induced increase in AIB transport; amino acids which do not compete fail to prevent the enhancement. Paradoxically threonine, phenylalanine, tryptophan, and tyrosine, which do not compete with AIB for transport, block the enhancement of transport upon amino acid starvation. The starvation-induced enhancement of amino acid transport does not appear to be the result of a release from transinhibition. After 30 min of amino acid starvation, AIB transport is either unchanged or slightly decreased even though amino acid pools are already depleted. Furthermore, loading cells with high concentrations of a single amino acid following a period of amino acid starvation fails to prevent the enhancement of AIB transport, whereas incubation of the cells with the single amino acid for the entire duration of amino acid starvation prevents the enhancement; intracellular amino acid pools are similar under both conditions. The enhancement of amino acid transport requires concomitant RNA and protein synthesis, consistent with the view that the adaptive increase reflects an increased amount of a rate-limiting protein involved in the transport process. Dexamethasone, which dramatically inhibits AIB transport in cells incubated in amino acid-containing medium, both blocks the starvation-induced increase in AIB transport, and causes a time-dependent decrease in transport velocity in cells whose transport has previously been enhanced by starvation.     

Modification of rat thymocyte membrane properties by hyperthermia and ionizing radiation.

Thymocytes are one the most widely used cell models for the study of radiation-induced interphase death. This cell-type was chosen for the study of hyperthermic and radiation effects on two membrane-related processes implicated in the interphase death of cells: Na+-dependent 2-aminoisobutyric acid (AIB) transport and cyclic 3'-5' adenosine monophsophate formation. The response of AIB transport to heat is dose-dependent, but the biphasic thermal response curve (AIB uptake versus time) differs fom the sigmoidal radiation response curve. Heating thymocytes for 20-30 min at 43 degrees C stimulates AIB uptake. Additional heating at 43 degrees C, however, markedly reduces AIB uptake. Despite the immediate stimulating effect of heat (30 min at 43 degrees C), the thymocyte has already developed irrepairable impairments, as demonstrated by the fractionated heating experiments. The heat-induced impairment of AIB uptake is mainly on the Na+-dependent component of neutral amino-acid transport, affecting primarily the maximal rate of uptake, i.e. Vmax. Additional evidence for heat-induced plasma membrane damage is the alteration in cAMP levels. Heating thymocytes for 30 min or longer at 43 degrees C causes a massive rise in cAMP level within the cell. This differs from thymocytes exposed to radiation where no rise in cAMP is observed.     

Stimulation of the glucose transport system in isolated mouse pancreatic acini by cholecystokinin and analogues.

Cholecystokinin and analogues increased the uptake of 2-deoxy-D-glucose and 3-O-methylglucose into isolated mouse pancreatic acini. This uptake was mediated by a facilitated glucose transport system that was saturable, stereospecific, and was inhibited by both phloretin and cytochalasin B. In agreement with previous studies of acinar function, caerulein was more potent and pentagastrin less potent than cholecystokinin in increasing sugar transport. The cholinergic analogue carbachol mimicked the effect of caerulein; atropine completely abolished the effects of carbachol but was without influence on the effects of the polypeptide hormones. In contrast, secretion, as well as dibutyryl cyclic AMP and dibutyryl cyclic GMP, had no effect on 2-deoxy-D-glucose uptake. Two lines of evidence suggested that hormonal stimulation of this sugar transport system was related to mobilization of cellular Ca2+. First, depletion of cellular Ca2+ by incubation of acini with ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) reduced the effect of caerulein. Second, the Ca2+ ionophore A23187 mimicked the effects of caerulein on 2-deoxy-D-glucose uptake when Ca2+ was present in the medium.     

A reassessment of decreased amino acid accumulation by ehrlich ascites tumor cells in the presence of metabolic inhibitors

This study was undertaken to examine the mechanism by which metabolic inhibition reduces amino acid active transport in ehrlich ascites tumor cells. At 37 degrees C the metabolic inhibitor combination 0.1 mM 2,4-dinitrophenol (DNP) + 10 mM 2- deoxy-D-glucose (DOG) reduced the cell ATP concentration to 0.10- 0.15 mM in less than 5 min. This inhibition was associated with a 20.6 percent +/- 6.4 percent (SD) decrease in the initial influx of α-aminoisobutyric acid (AIB), and a two- to fourfold increase in the unidirectional efflux. These effects could be dissociated from changes in cell Na(+) or K(+) concentrations. Cells incubated to the steady state in 1.0-1.5 mM AIB showed an increased steady-state flux in the presence of DNP + DOG. Steady- state fluxes were consistent with trans-inhibition of AIB influx and trans-stimulation of efflux in control cells, but trans- stimulation of both fluxes in inhibited cells. In spite of the reduction of the cell ATP concentration to less than 0.15 mM and greatly reduced transmembrane concentration gradients of Na(+) and K(+), cells incubated to the steady state in the presence of the inhibitors still established an AIB distribution ration 13.8 +/- 2.6. The results are interpreted to indicate that a component of the reduction of AIB transport produced by metabolic inhibition is attributable to other actions in addition to the reduction of cation concentration gradients. Reduction of cell ATP alone is not responsible for the effects of metabolic inhibition, and both the transmembrane voltage and direct coupling to substrate oxidation via plasma-membrane-bound enzymes must be considered as possible energy sources for amino acid active transport.     

Acetylcholine synthesis in rat brain: dissimilar effects of clozapine and chlorpromazine.

Experiments have been conducted in which glucagon-induced stimulation of α-aminoisobutyric acid (AIB) transport in rat liver, presumably mediated by cyclic AMP, was markedly inhibited by actinomycin D, cycloheximide or puromycin. Inhibition of transport occurred despite the presence of high concentrations of cyclic AMP, suggesting that the nucleotide may act prior to, or at the same point as, the antibiotics. Kinetic studies showed that 1) the half-life of the glucagon-stimulated system(s) was less than 1 hour, and 2) glucagon treatment doubled V_max without having any effect on the apparent K_m for hepatic AIB transport. The results suggest that glucagon stimulates hepatic AIB transport by increasing the synthesis of some critical protein having a rapid turnover rate; this effect may be due to a prior increase in RNA synthesis.