Structural Peculiarities of Na+,K+-ATPase Isozymes from the Calf Brain

Functionally active preparations of Na⁺,K⁺-ATPase isozymes from calf brain that contain catalytic subunits of three types (1, 2, and 3) were obtained using two approaches: a selective removal of contaminating proteins by the Jorgensen method and a selective solubilization of the enzyme with subsequent reconstitution of their membrane structure by the Esmann method. The ouabain inhibition constants were determined for the isozymes. The real isozyme composition of the Na⁺ pump from the grey matter containing glial cells and the brain stem containing neurons was determined. The plasma membranes of glial cells were shown to contain mainly Na⁺,K⁺-ATPase of the 11 type and minor amounts of isozymes of the 22(1) and the 31(2) type. The axolemma contains 21 and 31 isozymes. A carbohydrate analysis indicated that 11 enzyme preparations from the brain grey matter substantially differ from the renal enzymes of the same composition in the glycosylation of the 1 isoform. An enhanced sensitivity of the 3 catalytic subunit of Na⁺,K⁺-ATPase from neurons to endogenous proteolysis was found. A point of specific proteolysis in the amino acid sequence PNDNR⁴⁹² Y⁴⁹³ was localized (residue numbering is that of the human 3 subunit). This sequence corresponds to one of the regions of the greatest variability in 1-, 2-, 3-, and 4-subunits, but at the same time, it is characteristic of the 3 isoforms of various species. The presence of the 3 isoform of tubulin (cytoskeletal protein) was found for the first time in the high-molecular-mass Na⁺,K⁺-ATPase 31 isozyme complex isolated from the axolemma of brain stem neurons, and its binding to the 3 catalytic subunit was shown.     

Membrane transport ofp-nitrophenyl-α-galactoside by the melibiose carrier ofEscherichia coli

Summary p-Nitrophenyl--galactoside (-pNPG) was found to be a substrate for the melibiose transport system ofEscherichia coli. This sugar enters induced cells via the carrier and is split by -galactosidase to galactose andp-nitrophenol. In mutant cells lacking the -galactosidase [³H]--pNPG accumulated to concentrations 15 times higher than the external medium. The transport of -pNPG is inhibited by both Na⁺ and Li⁺. Na⁺ (10mm) reduced the K_m for -pNPG from 0.45 to 0.18mm and reduced theV _max from 6.7 nmoles/min/mg dry wt to a value of 3.0.     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

The mechanistic nature of the membrane potential dependence of sodium-sugar cotransport in small intestine

Summary Methods are described which demonstrate the use of unidirectional influx of¹⁴C-tetraphenylphosphonium (¹⁴C-TPP⁺) into isolated intestinal epithelial cells as a quantitative sensor of the magnitude of membrane potentials created by experimentally imposed ion gradients. Using this technique the quantitative relationship between membrane potential () and Na⁺-dependent sugar influx was determined for these cells at various Na⁺ and -methylglucoside (-MG) concentrations. The results show a high degree of dependence for the transport Michaelis constant but a maximum velocity for transport which is independent of . No transinhibition by intracellular sugar (40mm) can be detected. Sugar influx in the absence of Na⁺ is insensitive to 1.3mm phlorizin and independent of . The mechanistic implications of these results were evaluated using the quality of fit between calculated and experimentally observed kinetic constants for rate equations derived from several transport models. The analysis shows that for models in which translocation is the potential-dependent step the free carrier cannot be neutral. If it is anionic, the transporter must be functionally asymmetric. A model in which Na⁺ binding is the potential-dependent step (Na⁺ well concept) also provides an appropriate kinetic fit to the experimental data, and must be considered as a possible mechanistic basis for function of the system.     

Oligomerization reactions of deoxyribonucleotides on montmorillonite clay: The effect of mononucleotide structure on phosphodiester bond formation

Adenine deoxynucleotides bind more strongly to Na⁺-montmorillonite than do the corresponding ribonucleotides. Thymidine nucleotides binds less strongly to Na⁺-montmorillonite than do the corresponding adenine deoxynucleotides. Oligomers of 2-dpA up to the tetramer were detected in the reaction 2-d-5-AMP with EDAC (a water-soluble carbodiimide) in the presence of Na⁺-montmorillonite. Reaction of 3-d-5-AMP with EDAC on Na⁺-montmorillonite yields 3-d-2,5-pApA while the reaction of 2-d-3-AMP yields almost exclusively 3,5-cdAMP. The reaction of 5-TMP under the same reaction conditions give 3,5-cpTpT and 3,5-pTpT while 3-TMP gives mainly 3,5-cpT. The yield of dinucleotide products (dpNpN) containing the phosphodiester bond is 1% or less when Na⁺-montmorillonite is omitted from the reaction mixture.     

Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine

Summary The Na⁺/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed inXenopus oocytes. Injection of cloned RNA into oocytes increased Na⁺/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed inXenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of¹⁴C--methyl-d-glucopyranoside uptake into brush border membrane vesicles andXenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na⁺-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (K _i 6 and 9 m), and saturable (-methyl-d-glucopyranosideK _m 110 and 570 m). The sugar specificity was examined by competition experiments, and in both cases the selectivity wasd-glucose>-methyl-d-glucopyranoside>d-galactose>3-O-methyl-d-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na⁺/glucose cotransporter.     

Volume-sensitive taurine transport in fish erythrocytes

Summary Taurine plays an important role in cell volume regulation in both vertebrates and invertebrates. Erythrocytes from two euryhaline fish species, the eel (Anguilla japonica) and the starry flounder (Platichthys stellatus) were found to contain high intracellular concentrations of this amino acid ( 30 mmol per liter of cell water). Kinetic studies established that the cells possessed a saturable high-affinity Na⁺-dependent -amino-acid transport system which also required Cl^– for activity (apparentK _m (taurine) 75 and 80 m;V _max 0.85 and 0.29 mol/g Hb per hr for eel (20°C) and flounder cells (10°C), respectively. This -system operated with an apparent Na⁺/Cl^–/taurine coupling ratio of 211. A reduction in extracellular osmolarity, leading to an increase in cell volume, reversibly decreased the activity of the transporter. In contrast, low medium osmolarity stimulated the activity of a Na⁺-independent nonsaturable transport route selective for taurine, -amino-n-butyric acid and small neutral amino acids, producing a net efflux of taurine from the cells. Neither component of taurine transport was detected in human erythrocytes. It is suggested that these functionally distinct transport routes participate in the osmotic regulation of intracellular taurine levels and hence contribute to the homeostatic regulation of cell volume. Volume-induced increases in Na⁺-independent taurine transport activity were suppressed by noradrenaline and 8-bromoadenosine-3, 5-cyclic monophosphate, but unaffected by the anticalmodulin drug, pimozide.     

Na+-transport modulation induces isoform-specific expression of Na+,K+-ATPase α-subunit isoforms in C2C12 skeletal muscle cell

Changes in demands for Na⁺ transport alter expression of the Na⁺,K⁺-ATPase subunit isoforms. In skeletal muscle, the effects of these changes on expression the 2 isoform, the major isoform expressed in differentiated muscle cell, is not known. Therefore, this study examines regulation of the -subunit isoforms by Na⁺ in the C₂C₁₂ skeletal muscle cell that expresses the 1 and 2 isoforms. Western blot analysis showed that in differentiating C₂C₁₂ muscle cell, but not in undifferentiated myoblast, veratridine, a Na⁺ channel activator, greatly increased expression of the 2 isoform; expression of 1 was unaltered. Because the level of -actinin was unaltered, the data suggest that veratridine treatment did not significantly alter the progression of cell differentiation. Furthermore, a reduction in Na⁺ transport by tetrodotoxin again failed to alter expression of a1. Thus, in C₂C₁₂ skeletal muscle cell, changes in Na⁺ transport alters expression of the 2, but not the 1 isoform. These results differ from those observed previously in muscle cells that express only the 1 isoform. Because mammalian skeletal muscle expresses both the 1- and 2-subunit isoforms, the differential regulation that was observed may be physiologically relevant in these muscle cells in vivo.     

Structural analyses of lipid A from lipopolysaccharides of nodulating and non-nodulating Rhizobium trifolii

Structural studies have been performed on lipid A preparations derived from lipopolysaccharides of nodulating (nod⁺) Rhizobium trifolii wild type strains and non-nodulating (nod^-) mutants deprived of the medium size Nod plasmid.All preparations contain a lipid A backbone composed of a 1,6-linked d-glucosamine disaccharide (GlcN II-1,6-GlcN I) which is bis-phosphorylated at positions 1 and 4. The phosphate group at position 4 (GlcN II) is nonstoichiometrically substituted probably by a neutral glycosyl residue. Another substituent (probably also a neutral sugar) substitutes partly position 4 (GlcN I) of the disaccharide. The hydroxyl groups at positions 3 and 3 are likely to be esterified by fatty acyl residue. In lipid A of nod⁺ strains, 3-hydroxyhexa- and octadecanoic acid are linked to the amino group of GlcN I, and 3-hydroxyhexa- and tetradecanoic acid to the amino group of GlcN II. In lipid A of nod^- strains, mainly 3-hydroxyhexadecanoic acid is linked to the amino group of GlcN I, and 3-hydroxytetra- and hexadecanoic acid to that of GlcN II.The results show that rhizobial lipid A expresses many similarities to the lipid A of Enterobacteria. The structure shows, however, also peculiarities, especially regarding substituents of the lipid A backbone.This paper is dedicated to Professor Dr. Gerhart Drews on the occasion of his 60th birthday     

The small-intestinal Na+,d-glucose cotransporter: An asymmetric gated channel (or pore) responsive to ΔΨ

Summary At 0,d-glucose influx into, and efflux out of, membrane vesicles from small-intestinal brush borders are affected by trans Na⁺ and transd-glucose to different extents.d-glucose influx and efflux respond to (negative at the trans side) to different extents. The small-intestinal Na⁺,d-glucose cotransporter, is thus functionally asymmetric. This is not unexpected, in view of the structural asymmetry previously found. The characteristics of the of transinhibition byd-glucose are compatible with the mobile part of the cotransporter bearing a negative charge of at least 1 (in the substrate-free form). They are not compatible with its mobile part being electrically neutral. Pertinent equations are given in the Appendix. Partial Cleland's kinetic analysis and other criteria rule out (Iso) Ping Pong mechanisms, and makes likely a Preferred Ordered mechanism, with Na _out ⁺ binding to the cotransporter prior to the sugar_out. A likely model is proposed aimed at providing a mechanism of flux coupling and active accumulation.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Pump-leak sodium fluxes in low salt corn roots

Summary The influx and efflux of sodium from 4-hr washed, low salt corn roots (Zea mays L.) has been studied for characterization of passive and active components. Initial Na⁺ content of the roots is very low, 2.25±0.4 mol/g fresh weight. Na⁺ influx in the presence of 0.2mm Ca²⁺ and 0.002 to 20mm K⁺ is passive (a leak) based upon Goldman-type models, being determined by Na⁺ and cell potential (). Na⁺ was not transported by the K⁺ carrier and influx was unaffected by 50 m dicyclohexylcarbodiimide (DCCD). Permeability of the cells to Na⁺ was of the same order asP _k.Efflux of Na⁺ was by an efficient and rapid active transport system (a pump), thus accounting for the failure of these roots to accumulate high levels of Na⁺. In short-term loading and efflux experiments, internal Na⁺ turnover had a half-time of about 5 min. Sodium efflux was unaffected by DCCD. Net H⁺ flux was zero in the presence of DCCD regardless of sodium efflux, indicating absence of Na⁺/H⁺ antiport. Efflux of Na⁺ was equally rapid into medium containing no Na⁺ and only 0.002mm K⁺. K⁺ influx accounted for less than 4% of Na⁺ efflux, prompting the hypothesis that the Na⁺ (or cation?) efflux pump is the second electrogenic system previously defined based upon electrophysiological measurements.     

Selectivity of ATP-activated GTP-dependent Ca2+-permeable channels in rat macrophage plasma membrane

Outside-out configuration of the patch clamp technique was used to test whether an intracellular application of G protein activator (GTPS) affects ATP-activated Ca²⁺-permeable channels in rat macrophages without any agonist in the bath solution. With 145 mm K⁺ (pCa 8.0) in the pipette solution, activity of channels permeable to a variety of divalent cations and Na⁺ was observed and general channel characteristics were found to be identical to those of ATP-activated ones. Absence of extracellular ATP makes it possible to avoid the influence of ATP receptor desensitization and to study the channel selectivity using a number of divalent cations (105 mm) and Na⁺ (145 mm) as the charge carriers. Permeability sequence estimated by extrapolated reversal potential measurements was: Ca²⁺ Ba²⁺ Mn²⁺ Sr²⁺ Na⁺ K⁺ = 68 30 26 10 3.5 1. Slope conductances (in pS) for permeant ions rank as follows: Ca²⁺ Sr²⁺ Na⁺ Mn²⁺ Ba²⁺ = 19 18 14 12 10. Unitary Ca²⁺ currents display a tendency to saturate with the Ca²⁺ concentration increase with apparent dissociation constant (K _d ) of 10 mm. No block of Na⁺ permeation by extracellular Ca²⁺ in millimolar range was found. The data obtained suggest that (i) activation of some G protein is sufficient to gate the channels without the ATP receptor being occupied, (ii) the ATP receptor activation results in the gating of a special channel with the properties that differ markedly from those of the receptoroperated or voltage-gated Ca²⁺-permeable channels on the other cell types.DeceasedThe authors are grateful to K. Kiselyov and A. Mamin for technical assistance. The work was supported by the Russian Basic Research Foundation, Grant N 93-04-21722 and was made possible in part by Grant N R4A000 from the International Science Foundation.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

N-(indol-3-ylacetyl)amino acids as sources of auxin in plant tissue culture

N-(Indol-3-ylacetyl) derivatives (IAA conjugates) of aliphatic amino acids with a two- to six-carbon backbone including -l-amino acids, (-amino acids, and the ,-diamino acids ornithine and lysine were prepared, chemically characterized, and tested as sources of auxin in plant tissue culture. Stimulation of unorganized growth in Solanum nigrum L. callus and callus induction and developmental effects in tomato (Lycopersicon esculentum Mill. cv. Marglobe) hypocotyl explants were studied systematically. Relative auxin activities were estimated by comparing physiologically equivalent concentrations, in the optimal and suboptimal range, of the individual IAA conjugates. While the growth-promoting properties of some of the conjugates were species-dependent, those containing straight-chain two- to four-carbon -l-amino acid moieties were generally up to 100 times more active than those of their five- to six-carbon homologues. Branching of the amino acid backbone at C- (norvaline vs. valine and norleucine vs. isoleucine) and C- (norleucine vs. leucine) had a minor effect, but substitution of H- by a methyl group (-amino-l-butyric vs. -aminoisobutyric acids) almost completely blocked growth-promoting activity. IAA conjugates of -amino acids were, in most cases, nearly as active as those of their -amino-l-isomers. Among the conjugates of ,-diamino acids N -(IAA) ornithine was less active than N -(IAA)lysine. The activity of N -(IAA)lysine was less than for the -(IAA) isomer, and that of N ,N -(IAA)₂-lysine was different in tomato and Solanum nigrum. The l-alanine and -lysine conjugates were also found to be useful for induction and development of Oenothera leaf callus and in tomato cell-suspension culture, two systems which require highly active sources of auxin.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indol-3-ylacetic acid the abbreviations for N-(indol-3-ylacetyl)amino acids are listed in Table 1.     

Study of the involvement of osmotic adjustment and H<Superscript>+</Superscript>-ATPase activity in the resistance of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> suspension cells to salt stress

The salt-induced H⁺-ATPase activity and osmotic adjustment responses of Catharanthus roseus (L.) G. Don suspension cultures were studied. Cells were treated with 0, 50 or 100mM NaCl for 7days or were maintained for 8 months with 50 mM NaCl (50T cells). Growth, osmotic potential (), ions content, soluble sugars, proline and total amino acids were determined in the sap of control and salt-treated cells. Salinity reduced cell growth and . The higher decrease in the in salt-treated cells was due to higher accumulation of Na⁺ and Cl^–. The levels of organic solutes, such as soluble sugars, free proline and total amino acids, increased with salt treatment. These results suggest that salt-tolerant cells are able to osmotically adjust. Salinity treatments stimulated H⁺-ATPase activity. Immunodetection of the enzyme showed that the increased activity was due to an increased amount of protein in the plasmalemma. The induction by NaCl, especially at 100 mM NaCl and for 50T cells, could account for the K⁺ and Cl^– uptake but not for higher or lower tolerance.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

δ andk opiate receptors in primary astroglial cultures part II: Receptor sets in cultures from various brain regions and interactions with ß-receptor activated cyclic AMP

In a previous paper, and opiate receptors were shown to be co-localized on the same cell in enriched primary cultures of astroglia from neonatal rat cerebral cortex. Activation of the receptors inhibited adenylate cyclase. In this work, the presence of opiate receptors was investigated in astroglial primary cultures from neonatal rat striatum and brain stem. Cyclic adenosine 3, 5-monophosphate accumulation was quantified in the presence of different opioid receptor ligands after stimulation of the cyclic adenosine 3,5-monophosphate system with forskolin. Morphine was used as a receptor agonist. [d-Ala², D-Leu⁵]-enkephalin or[d-Pen²,d-Pen⁵]-enkephalin were used as receptor agonists and dynorphin 1–13 or U-50,488H were used as receptor agonists. Specific antagonists for the respective receptors were used. After striatum or brain stem cultures had been incubated in 10^–9–10^–5M of each [d-Ala²,d-Leu⁵]-enkephalin, [d-Pen², D-Pen⁵]-enkephalin and Dynorphin 1–13 or U-50,488H, dose related inhibitions of the 10^–5M rorskolin stimulated cyclic adenosine 3,5-monophosphate accumulation were observed. The changes were reversed to the forskolin-induced control level in the presence of the respective antagonists. 10^–9–10^–5M morphine did not significantly change the forskolin-induced accumulation of cyclic adenosine 3,5-monophosphate in the cultures studied. Furthermore, cultures from cerebral cortex, striatum or brain stem were incubated with isoproterenol alone or together with morphine or [d-Ala²,d-Leu⁵]-enkephalin. Isoproterenol stimulated cyclic adenosine 3,5-monophosphate accumulation more prominently in the cerebral cortex and striatum cultures than in the brain stem cultures. Morphine did not influence isoproterenol-induced cyclic adenosine 3,5-monophosphate accumulation, while [d-Ala²,d-Leu⁵]-enkephalin inhibited the accumulation. The results indicate that astroglial cells in primary cultures from striatum, brain stem and cerebral cortex express andk opioid receptors linked to the adenylate cyclase/cyclic adenosine 3,5-monophosphate system. No receptors were detected, however, in the present model. Aspects of the relation between the expression of opioid peptides and opioid receptors are discussed, while speculations are also made on the functional aspects of opioid receptors on astroglia.     

The regulation of total creatine content in a myoblast cell line

Total cellular creatine content is an important bioenergetic parameter in skeletal muscle. To understand its regulation we investigated creatine transport and accumulation in the G8 cultured skeletal myoblast line. Like other cell types, these contain a creatine transporter, whose activity, measured using a radiolabelling technique, was saturable (Km = 110 ± 25 M) and largely dependent on extracellular [Na⁺]. To study sustained influences on steady state creatine concentration we measured total cellular creatine content using a fluorimetric method in 48 h incubations. We found that the total cellular creatine content was relatively independent of extracellular creatine concentration, consistent with high affinity sodium-dependent uptake balanced by slow passive efflux. Accordingly, in creatine-free incubations net creatine efflux was slow ( 5 ± 1 % of basal creatine content per day over 6 days), while creatine content in 48 h incubations was reduced by 28 ± 13% of control by the Na⁺,K⁺-ATPase inhibitor ouabain. Creatine accumulation after 48 h was stimulated by treatment with the mixed - and -adrenergic agonist noradrenaline, the -adrenergic agonist isoproterenol, the ₂-agonist clenbuterol and the cAMP analogue N⁶,2-O-dibutyryladenosine 3,5-cyclic monophosphate, but was unaffected by the ₁ adrenergic agonist methoxamine. The noradrenaline enhancement of creatine accumulation at 48 h was inhibited by the mixed - and -antagonist labetalol and by the -antagonist propranolol, but was unaffected by the ₂ antagonist phentolamine; greater inhibition was caused by the ₂ antagonist butoxamine than the ₁ antagonist atenolol. Creatine accumulation at 48 h was increased to 230 ± 6% of control by insulin and by 140 ± 13% by IGF-I (both at 3 nM). Creatine accumulation at 48 h was also increased to 280 ± 40% of control by 3,3,5-triiodothyronine (at 70 M) and to 220 ± 35% of control by amylin (60 nM). As 3,3,5-triiodothyronine, amylin and isoproterenol all stimulate the Na⁺,K⁺-ATPase, we suggest that they stimulate Na⁺-creatine cotransport indirectly by increasing the transmembrane [Na⁺] concentration gradient and membrane potential.Abbreviations IGF-I insulin-like growth factor I - IGF-II insulin-like growth factor II - T₃ 3,3,5-triiodothyronine - CGRP calcitonin gene-related peptide