Cellular uptake of lithium via amino acid transport system A.

We now add to the agencies by which cells take up lithium the process of cotransport with neutral amino acids via System A. In the Ehrlich cell various natural and synthetic amino acids, depending on their structure, can cause substantial accelerations of Li+ uptake over a considerable range of levels of Na+, Li+ and H+. Half the maximal augmentation of uptake, namely 1.2 mequiv. Li/kg cell water per 15 min, was obtained for 5.4 mM alanine in a double-reciprocal plot. Alanine also stimulated the exodus of Li+ from the Ehrlich cell. The human red blood cell, lacking System A as it does, becomes an imperfect model for studying cellular uptake of Li+. Until the Li+ dependence of amino acid uptake in the reticulocyte is known, reticulocytosis can be suspected of contributing to the interpersonal variations seen in Li+-for-Na+ exchange.

Does the non-saturable cell entry apply to the charge-free form of amino acids?

The slow cellular entry shown by neutral alpha-amino acids at very high concentrations appears not to arise from diffusion of the totally uncharged species through the plasma membrane of the Ehrlich cell, judging from a similarity of the rates observed for the two conformational isomers of 1-amino-2-hydroxy-cyclohexane-carboxylic acid. One of these isomers provides in neutral solution 4 times as large a proportion of the charge-free species as the other, and 5 times the proportion calculated for alanine.

Substrate-dependent adaptive regulation and trans-inhibition of System A-mediated amino acid transport. Studies using rat hepatoma plasma membrane vesicles.

Substrate-dependent regulation of amino acid transport by System A occurs by both direct action at the carrier (trans-inhibition) and transcriptional control (adaptive regulation). While experiments with intact cells have led to working models that describe these regulatory phenomena, the use of subcellular approaches will serve to refine the present hypotheses. Adaptive induction of System A transport following amino acid starvation of cells was shown to be dependent on de novo RNA and protein synthesis, and the stimulated activity was shown to be retained in isolated plasma membrane vesicles. This stimulated transport activity was tightly associated with the plasma membrane, but could be solubilized by 4 M urea and 2.5% cholate, and recovered following reconstitution of the protein into artificial proteoliposomes. These data support the working hypothesis that adaptive induction of transport is the result of de novo synthesis and insertion into the plasma membrane of System A carrier protein. In contrast, the activity of System ASC in the vesicles from the amino acid starved cells was actually reduced by 2-5-fold when compared to amino acid-fed cells. A more rapid form of regulation of System A activity is trans-inhibition. The use of isolated plasma membrane vesicles demonstrated that trans-inhibition in whole cells did not survive membrane isolation. However, substrate loading of isolated membrane vesicles containing high levels of System A activity, produced trans-inhibition in a very specific manner in that System A substrates resulted in decreased transport activity, while those amino acids which are poor substrates for the System A carrier did not. Thus, trans-inhibition is not the result of a recycling process involving an intracellular pool of carriers, but rather can be accounted for by differences in the kinetics for amino acid binding and/or translocation on the two sides of the membrane.

Association of hepatic system A amino acid transporter with the membrane-cytoskeletal proteins ankyrin and fodrin.

System A activity is a highly regulated mechanism for the active transport of zwitterionic amino acids into mammalian cells. Monoclonal antibodies generated against a previously unidentified rat liver plasma membrane-associated protein were shown to immunoprecipitate solubilized System A transport activity. The immunoreactive protein was later determined by immunoblotting and peptide microsequencing to be rat liver alpha-fodrin (non-erythroid spectrin). Antibody against ankyrin, a protein that often serves as a bridge between integral membrane proteins and fodrin, also immunoprecipitated System A transport activity. Fractionation of solubilized plasma membrane proteins on sucrose gradients revealed that the System A transporter co-migrated as a complex with fodrin and ankyrin, even in the presence of detergent and urea. In contrast, the System N amino acid transporter does not co-migrate with ankyrin and fodrin, nor does the anti-fodrin antibody immunoprecipitate System N activity. The present data are the first to demonstrate an association between an organic solute transporter and the membranocytoskeletal proteins ankyrin and fodrin.

Inhibition of endothelial cell amino acid transport System y+ by arginine analogs that inhibit nitric oxide synthase.

A variety of N omega-monosubstituted L-arginine analogs are established inhibitors of nitric oxide synthase; in all cases, initial binding is competitive with the substrate L-arginine. The efficacy of such compounds in vivo will depend on their transport into the relevant nitric oxide synthase-containing cells; in fact, inhibition may actually be augmented if cellular uptake of L-arginine is also blocked by the analogs. Because vascular endothelial cells synthesize vasoactive nitric oxide under both physiological and pathophysiological conditions, we have performed inhibition analyses with novel arginine analogs to determine the substrate specificity of the primary L-arginine transport system. Na(+)-independent System y+, present in porcine pulmonary artery endothelial cells. As reported by others, no Na(+)-independent System bo,+ activity was detectable. For System y+. Dixon plots suggest competitive inhibition and apparent Ki values, which ranged between 0.1 and 0.8 mM, estimated for each inhibitor. Some influence of amino acid side chain structure could be detected, but in general, the data establish that this transport system accepts a broad range of arginine derivatives. Loading the cells with individual arginine analogs resulted in trans-stimulation of arginine uptake suggesting that they serve as substrates of System y+ as well as inhibitors. These results indicate that plasma membrane transport is unlikely to be a limiting factor in drug development for nitric oxide synthase inhibitors.

Synthesis and transport applications of 3-aminobicyclo[3.2.1] octane-3-carboxylic acids.

The isomeric 3-aminobicyclo[3.2.1]octane-3-carboxylic acids were synthesized and compared with the widely used (1R,2S,4S)-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid as to specificity to the Na+-independent membrane transport system L of the Ehrlich ascites tumor cell and of the rat hepatoma cell line HTC. The presence of an additional methylene group in the ring system leads to an optically symmetrical amino acid, with the advantages that the product is devoid of isomeric contamination. Hence, optical resolution is not necessary to secure a homogeneous test substrate for discrimination of amino acid transport systems. Through its inhibitory action on the cellular uptake of known system-specific amino acids, the bicyclo[3.2.1]octane amino acid proved more reactive than the bicycloheptane analogue with the Na+-independent amino acid transport system of the test cells and not perceptibly reactive with the accompanying Na+-dependent systems. Recent evidence of the presence of a second component of Na+-independent amino acid transport, beyond system L, increases the importance of securing a variety of possibly discriminatory model substrates.

Amino acid-dependent inactivation of glucagon-induced System A transport activity in cultured rat hepatocytes.

Hepatocytes isolated from glucagon-treated rats contain stimulated System A activity. If these cells are placed in primary culture, the enhanced transport decays rapidly provided the culture medium contains substrate amino acids. This amino acid-dependent inactivation can be composed of trans-inhibition (protein synthesis-independent), repression (protein synthesis-dependent), or both depending on the particular substrate tested. Repression was most prominently observed with a group of small neutral amino acids that are commonly found in proteins. A strong trans-inhibition response was induced by a variety of amino acid analogs. Amino acids showing no reactivity with System A produced neither trans-inhibition nor repression. Repression of System A activity in culture was blocked by inhibitors of both RNA and protein synthesis. In contrast to inhibitors of RNA biosynthesis such as actinomycin and alpha-amanitin, inhibitors of poly(A) polymerase (cordycepin and adenine-9-beta-D-arabinopyranoside) did not prevent the inactivation of the transport activity. These results demonstrate that both the stimulation of activity and the turnover of the hepatic System A activity are controlled at the transcriptional level.

Interaction between parallel transport systems examined with tryptophan and related amino acids.

Most of the neutral amino acids are transported across the plasma membrane by two or more parallel transport systems. In this study, we have limited transport interactions to System A and L by studying amino acids not transported by System ASC. Results are presented to emphasize that such amino acids as leucine, phenylalanine and tryptophan do enter the Ehrlich cell to substantial degrees by System A. We show how the phenomenon of competitive stimulation presents a strong argument that these systems operate between the same two compartments, extracellular and cellular. To discover what is needed to bring two amino acids into the transport relation shown by tryptophan and methionine, we arbitrarily set up two classes of amino acids: (1) those whose steady-state gradients will be increased when System L is deleted; (2) those whose gradients will instead be decreased. On blockading System L with 2-aminorbornane-2-carboxylic acid applied in symmetry to the two sides of the plasma membrane, we show as predicted that the gradient maintained for methionine is strongly increased, that for trypotphan decreased. The initial rate of uptake of all the amino acids mentioned is highly sensitive to the nature of the cellular amino acid pool. A high influx by exchange, relative to net influx, appears to characterize the amino acids that respond to competitive stimulation. Albumin-bound tryptophan appears not directly accessible to the tested carriers.

Induction and decay of amino acid transport in the liver. Turnover of transport activity in isolated hepatocytes after stimulation by diabetes or glucagon.

System A-mediated amino acid transport in liver tissue is stimulated by diabetes or by exogenous glucagon. The present report describes the decay process for stimulated System A activity in isolated rat hepatocytes. Transport induced by glucagon, insulin, or spontaneous diabetes (BB/G rats) decayed rapidly after initiation of primary cultures; the estimated half-life was about 1.5 h. In contrast, the stimulated activity in cultured hepatocytes from streptozotocin-diabetic rats had a half-life of about 2.5 h. It is not known if the loss of System A activity is the result of proteolysis or of another form of inactivation. The decay was blocked by either actinomycin or cycloheximide, but was unaffected by leupeptin, methylamine, chloroquine, dinitrophenol, rotenone, or tunicamycin. Studies with cycloheximide and actinomycin suggest the following: 1) within 30 min after initiation of cell cultures, synthesis of the corresponding mRNA for the transport-inactivating protein has begun; 2) the mRNA for transport-inactivating protein is relatively long-lived, but the inactivating protein itself has a half-life of less than 1 h; and 3) actinomycin blocks the decay through inhibition of transport-inactivating protein biosynthesis rather than by protection of the mRNA for the protein responsible for System A activity. A working model for the synthesis and decay of System A activity is presented. Cationic amino acid transport, System y+, was also stimulated severalfold after induction of diabetes or glucagon injection of rats. Systems ASC, X-, and N were enhanced to varying degrees in hepatocytes from diabetic or glucagon-injected rats, but the level of stimulation for each was not as great as that found for Systems A or y+.

Growth-dependent regulation of system A in SV40-transformed fetal rat hepatocytes.

Fetal RLA209-15 hepatocytes, transformed with a temperature-sensitive SV40 mutant, behave like fully differentiated cells at the growth-restrictive temperature of 40 degrees C. Conversely, incubation at the growth-permissive temperature of 33 degrees C results in a transformed phenotype characterized by rapid cell division and decreased production of liver-specific proteins. The results presented here demonstrate that the cells at 33 degrees C exhibited high rates of system A transport, but transfer to 40 degrees C reduced the activity greater than 50% within 24 h. This decline in transport was independent of cell density, although the basal rate of uptake was inversely proportional to cell density in rapidly dividing cells. Transfer of cells from 40 to 33 degrees C resulted in an enhancement of system A activity that was blocked by tunicamycin. Plasma membrane vesicles from cells maintained at either 33 or 40 degrees C retained uptake rates proportional to those in the intact cells; this difference in transport activity could also be demonstrated after detergent solubilization and reconstitution. Collectively, these data indicate that de novo synthesis of the system A carrier is regulated in conjunction with temperature-dependent cell growth in RLA209-15 hepatocytes.

A cycle of deprotonation and reprotonation energizing amino-acid transport?

Although lowering the pK2 of neutral amino acids only weakens their concentrative uptake by Ehrlich cells, the same change greatly enhances uptake of diamino acids. This effect does not arise merely from putting the distal amino group in its uncharged form, but depends on an enhanced deprotonation of the alpha-amino group. Parallel effects are seen for the transport system for basic amino acids, for which the assignment of pK values within the membrane is less ambiguous. To explain the paradoxical advantages of having the alpha-amino group protonated yet readily deprotonated, we propose that a proton withdrawn from that group is pumped over an intramembrane interval to energize amino-acid transport.

Induction of amino acid transport system A in rat hepatocytes is blocked by tunicamycin.

Primary cultures of rat hepatocytes respond to hormones or amino acid deprivation by increasing System A-mediated neutral amino acid transport. Previous reports have shown this stimulation to be dependent on RNA and protein synthesis, whereas the present report describes the inhibition of System A by tunicamycin (TM), an inhibitor of asparagine-linked glycoprotein biosynthesis. The basal System A activity, as monitored by Na+-dependent 2-aminoisobutyric acid uptake, was decreased by TM when hepatocytes were cultured for 24 h in the presence of the antibiotic. System Gly activity was also sensitive to TM, whereas the activities of Systems L1, L2, and N were relatively resistant and that of System ASC was only moderately affected. The increase in System A-mediated uptake after incubation of hepatocytes in the absence of amino acids (i.e. adaptive control) was almost completely abolished by including TM. Likewise, stimulation of hepatic 2-aminoisobutyric acid transport by glucagon, dexamethasone, insulin, or vasopressin was also blocked by the inhibitor. When glucagon alone or glucagon plus dexamethasone was added, the inhibition by TM was transient such that the degree of inhibition decreased with incubation time after the initial 2 h. Addition of TM to cells which had been treated previously for 2 h to 4 h with glucagon and dexamethasone blocked any further increase in transport indicating that the glycoprotein component of System A must be continually synthesized to sustain the increase in activity. Treatment of hepatocytes with various lectins did not inhibit 2-aminoisobutyric acid transport.

Na plus-facilitated reactions of neutral amino acids with a cationic amino acid transport system.

The predominant basis for transport interactions between neutral and cationic amino acids in the Ehrlich ascites-tumor cell and the rabbit reticulocyte has been identified as a reaction of the neutral amino acid plus Na(+) with the cationic amino acid transport system. This reaction is revealed both by a Na(+)-dependent transport inhibition by the neutral amino acid, and by mutual flux accelerations whereby the neutral amino acid and Na(+) exchange for the cationic amino acid.

Incomplete correspondence between repressive and substrate action by amino acids on transport systems A and N in monolayered rat hepatocytes.

Stimulation of System N transport of glutamine by amino acid starvation of the rat hepatocyte can be repressed by one of its substrates, histidine, but not by two others, glutamine or asparagine. Furthermore, 2-(methylamino)isobutyric acid is also repressive, although it is not perceptibly a substrate or inhibitor of that system. The repression of System A by glutamine proves in contrast not to be dissociated from transport: relatively slow System A uptake of glutamine has now been shown in this cell. System A transport of glutamine is conspicuous in the hepatoma cell HTC and is increased after amino acid starvation of both hepatocytes and the hepatoma cells. Differential repression of the systems could be shown, although lowering the pH prevented the derepression of one system as much as the other on amino acid starvation.

Characteristics of an amino acid transport system in rat liver for glutamine, asparagine, histidine, and closely related analogs.

In the rat hepatocyte, whether freshly separated or in primary culture, we do not find L-glutamine entry by Systems A and ASC as seen in cells previously studied. Instead the mediated entry of glutamine appears to occur exclusively by a Na+-dependent system ("N") apparently specific to amino acid amides and L-histidine; however, a portion of asparagine uptake occurs by System A. The simplest evidence for the separateness of the added system is the failure of model substrates for System A (e.g. N-methylalanine) to inhibit glutamine uptake significantly, and the failure of glutamine to inhibit the uptake of L-cysteine, model substrate for System ASC, at least in this cell. As is the case for cysteine, glutamine inhibits transport by System A (although not competitively), even though showing no transport by that system. Our finding confirms an earlier inference that glutamine uptake by this cell may follow a route not taken by alanine or serine, and explains the apparently erroneous companion inference that glutamine also shares a route with these two amino acids. Its uptake has now been characterized to show a series of differences from Systems A and ASC. Especially significant in view of the importance of glutamine metabolism are an insensitivity of the new system to stimulation by either insulin or glucagon, and its distinct enhancement (not as large as that for System A) on starvation of the cells with respect to amino acids. Hence, a second system has been found to show adaptive regulation.

The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway.

The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.