Long term regulation of nucleoside transport by thyroid hormone (T3) in cultured chromaffin cells

The adenosine transport in cultured chromaffin cells was increased by the presence of triiodo-l-thyronine (T₃) throughout the prolonged period studied. The V_max values of this transport obtained in absence and presence of 1 M T₃ were 36.21±2.1 and 44.17±3.5 (means±SD) pmol/10⁶cells/min respectively for 26 hours incubation-time with the hormone. The K_m values were not significantly modified. The number of adenosine transporters in cultured chromaffin cells, measured by [³H]nitrobenzylthioinosine (NBTI) binding, was increased by 1 M T₃ for 26 hours incubation-time. The values of binding sites per cell were 33,500±3,000 and 40,153±3,700 in absence and presence of T₃ respectively, without changing the K_d constant. When the transport studies were carried out in presence of cycloheximide, an inhibitor of protein synthesis, the adenosine transport capacity decreased with a half-life values of 23.9±2.8 and 24.3±2.1 hours both in the presence or absence of T₃ respectively. When cells were incubated in the presence of both T₃ and cycloheximide, not only the activatory effect of T₃ was completely abolished but also adenosine transport was decreased to the same extent as with cycloheximide alone. These results indicated that T₃ activation of adenosine transport in chromaffin cells required the protein-synthesizing mechanism.     

Adenosine transport and nitrobenzylthioinosine binding in human placental membrane vesicles from brush-border and basal sides of the trophoblast

Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK _m approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK _d 0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK _i values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK _d 1.05±0.13nM and apparentK _i values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [³H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM _r on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters.     

[3H]Histamine uptake and release by astrocytes from rat brain: Effects of sodium deprivation,high potassium,and potassium channel blockers

Histamine transport has been characterized in cultured astroglial cells of rat brain. The kinetics of [³H]-histamine uptake yielded a K_m of 0.19±0.03 M and a V_max of 3.12±0.75 pmol×mg protein^–1×min^–1. Transport system revealed high affinity for histamine and an approximately ten times higher capacity than that shown in cultured glial cells of chick embryonic brain. Ouabain which interferes with utilization of ATP to generate ion gradients, and the replacement of Na⁺ with choline inhibited the initial rate of uptake showing a strong Na⁺-dependency and suggesting the presence of a tightly coupled sodium/histamine symporter. Dissipation of K⁺-gradient (in>out) by high K⁺ or by K⁺-channel blockers, BaCl₂, (100 M), quinine (100 M) or Sparteine (20 M) produced also remarkable inhibitions in the uptake of [³H]-histamine. Impromidine, a structural histamine-analogue could inhibit the uptake non-competitively in a range of concentrations of 1 to 10 M with a K_i value of 2.8 M, indicating the specificity of the uptake. [³H]histamine uptake measurements carried out by using a suspension of dissociated hypothalamic cells, of rat brain showed a strong gliotoxin-sensitivity and yielded a Km of 0.33±0.08 M; and a V_max of 2.65±0.35 pmoles×mg protein^–1×min^–1. The uptake could be reversed by incubating the cells in histamine-free Krebs medium. The [³H]histamine efflux was sensitive to Na⁺ omission, ouabain treatment and high K⁺ or K⁺ channel blockers, resulting in marked elevations in the efflux. Data indicate that glial uptake of histamine is a high affinity, Na⁺-dependent and electrogenic, driven by an inward-oriented sodium ion gradient and an outward-oriented potassium ion gradient and functions as part of histamine inactivation, at least in a shunt mechanism.Abbreviations used HA histamine - [³H]HA [2.5-³H]-histamine - dl--aAA dl-alpha-aminoadipic acid - (Na⁺+K⁺) ATP-ase sodium and potassium activated adenosine triphosphatase - SAH S-Adenosyl-d-Homocysteine - HNMT histamine-N-methyltransferase     

Identification of nucleoside transport binding sites in the human myocardium

The role of nucleoside transport in ischemia-reperfusion injury and arrhythmias has been well documented in various animal models using selective blockers. However, clinical application of nucleoside transport inhibitors remains to be demonstrated in humans. It is not known whether human heart has nucleoside transport similar to that of animals. The aim of this study is to pharmacologically identify the presence of nucleoside transport binding sites in the human myocardium compared to animals.Myocardial tissue was obtained from guinea pig left and right ventricle, canine left ventricle, human intraoperative right atrium and human cadaveric right atrium and right and left ventricles. Myocardial preparations were obtained from tissue samples after homogenized and a differential centrifugation.Equilibrium binding assays were performed using [³H]-p-nitrobenzylthioinosine (NBMPR) at room temperature in the presence or absence of non-radioactive NBMPR or other nucleoside transport blockers such as p-nitrobenzylthioguanosine dipyridamole, lidoflazine, papaverin, adenosine and doxorubcine. From saturation curves and inhibition kinetics, we determined the relative maximal binding (B_max) and dissociation constant (K_d) of [³H]-NBMPR binding of human myocardial preparations.Results demonstrated that the fresh human myocardial preparations have a specific binding site for NBMPR with a B_max of 283 ± 32 fmol/mg protein and K_d of 0.56 ± 0.12 nM. These values are lower than those obtained from guinea pigs (B_max = 1440 ± 187 fmol/mg protein and K_d = 0.21 ± 0.03 nM) and canine atrium (B_max 594 ± 73 fmol/mg protein, and K_d = 1.12 ± 0.22 nM).Displacement kinetics studies revealed the relative potencies (of certain unrelated drugs as follow: p-nitrobenzylthioguanosine > dipyridamole > lidoflazine > pavaverine > Diltazam > adenosine > doxyrubicin. It is concluded that human myocardium contains an active nucleoside transport site which may play a crucial role in post-ischemic reperfusion-mediated injury in a wide spectrum of ischemic syndromes.     

HETEROGENEITY OF HISTAMINE Hi-RECEPTORS: SPECIES VARIATIONS IN [3H]MEPYRAMINE BINDING OF BRAIN MEMBRANES

[³H]Mepyramine binds with high affinity to membranes from brain of human, rat, guinea-pig, rabbit and mouse with drug specificity indicating an association with histamine H₁receptors. Considerable species differences occur in the affinity of [³H]mepyramine, with guinea-pig and human having 34 times greater affinity than rat, mouse or rabbit. The greater affinity of [³H]mepyramine in guinea-pig than in rat is attributable both to faster association and slower dissociation rates in guinea-pig. Species differences in affinity for H₁ receptor sites occur for some antihistamines but not for others. Some tricyclic antidepressant and neuroleptic drugs are extremely potent inhibitors of [³H]mepyramine binding, exceeding in potency any H₁ antihistamines examined. The tricyclic antidepressant doxepin and the neuroleptic clozapine are the most potent of all drugs examined in competing for [³H]mepyramine binding. The regional distribution of specific [³H]mepyramine binding differs considerably in the various species examined.     

β-Adrenoreceptors of multiple affinities in a clonal capillary endothelial cell line and its functional implication

-Adrenoreceptor has been studied in a clonal capillary endothelial cell line established from the vascular bed of the bovine adrenal medulla. [³H]Dihydroalprenolol ([³H]DHA) binding to the isolated plasma membranes from these cells has demonstrated the presence of -adrenoreceptors with two different affinities. the dissociation constants (K_d) have been found to be 0.27±0.09×10^–9 M and 2.96±0.31×10^–9 M, respectively with the corresponding B_max of 5.1±0.05 and 70.0±0.2 pmol/mg protein, respectively. Inhibition of [³H]DHA binding to the -receptor by atenolol (a ₁-antagonist) and ICI 118,551 (a ₁-antagonist) has suggested that the IC_50cor (=K_i) for atenolol and ICI 118,551 for high affinity site are 0.08±0.03×10^–12 M and 0.25±0.08×10^–12 M, respectively. This, therefore, indicates that both atenolol and ICI 118,551 are able to displace the bound ligand effectively but the ₁-selective antagonist atenolol is 3 times more potent than its ₂ counterpart, ICI 118,551. Displacement of [³H]DHA binding to the endothelial cell plasma membrane by the agonists isoproterenol, epinephrine and norephinephrine has established a relative order of K_i for these agents as isoproterenol (0.56±0.19×10^–9 M)–9 M)>-norepinephrine (0.71±0.24×10^–9 M) for the high affinity site. The corresponding values for the low affinity site, however, are 4.62±0.64×10^–9 M, 6.21±0.86×10^–9 M and 5.90±0.82×10^–9 M, respectively for the same agonists. Increased intracellular cAMP accompanied with cellular proliferation in the presence of isoproterenol has suggested not only the coupling of -adrenoreceptors to the adenylate cyclase system but also its involvement in endothelial cell proliferation.Abbreviations DHA Dihydroalprenolol - cAMP 3:5 cyclic adenosine monophosphate - DTT dithiothreitol - MEM minimal essential medium - 8Br-cAMP 8-bromo-adenosine 3:5 cyclic monophosphate     

Nitric Oxide Modulates Muscarinic Acetylcholine Receptor Binding in the Cerebral Cortex of Gerbils

To determine whether nitric oxide (NO) acts as a modulator of muscarinic acetylcholine receptor (mACh-R) function, we performed a radioligand receptor assay using [³H]quinuclidinyl benzylate ([³H]QNB), the NO radical (NO·) donor 3-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC7) and a gerbil brain cortical membrane preparation. NOC7 (at 10 M, 100 M or 1 mM concentrations) significantly reduced the [³H]QNB binding K_d values (from 0.196 ± 0.009 nM in the control, to 0.151 ± 0.013, 0.144 ± 0.012 and 0.153 ± 0.007 nM respectively). NOC7 did not alter the displacement curves of atropine or carbachol. Reduction of SH groups with dithiothreitol, in the presence of the NO donor, significantly increased [³H]QNB binding affinity whereas alkylation by N-ethylmaleimide markedly decreased it. The observed enhancing effect on mACh-R binding affinity for [³H]QNB, may reflect conformational changes in the receptors mediated by the NO generated, and these changes might be explained by NO reactions with such groups through conditions supporting redox reactions intrinsic to the NO molecule, similar to those occurring in redox regulatory sites reported for other neurotransmitter pathways in the CNS.     

Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump

A dual-wavelength fluorimeter was constructed, which used two light emitting diodes (LEDs) to excite the fluorescence dye RH 421 alternately with two different wavelengths. The ratio of the emissions at the two excitation wavelengths provided a drift-insensitive signal, which allowed detection of very small changes of the fluorescence intensity. Those small changes were induced by ion binding and release in conformation E₁ of the Na,K-ATPase. Titration experiments were performed to determine equilibrium dissociation constants (± standard deviation) for each step in the complete binding and release sequence: 0.12 ± 0.01 mM (E₂(K₂) KE₁), 0.08 ± 0.01 mM (KE₁ E₁), 3.0 ± 0.2 mM (NaE₁ E₁), 5.2 ± 0.4 mM (Na₂E₁ NaE₁) and 6.5 ± 0.4 m_M (Na₃E₁ Na₂E₁) at pH 7.2 and T=16°C. These numbers show that the affinities of the binding sites exposed to the cytoplasm, are higher for K⁺ than for Na⁺ ions, similar to what was found on the extracellular side. The physiological requirement for extrusion of Na⁺ from the cytoplasm, and for import of K⁺ from the extracellular medium seems to be facilitated not by favorable binding affinities in state E₁ but by the two ATP-driven reaction steps of the cycle, E₂(K₂) + ATP K₂E₁ · ATP and Na₃E₁ · ATP (Na₃) E_l-P, which border the ion exchange reactions at the binding sites in conformation E₁. Correspondence to: H.-J. Apell     

Differential effect of ethanol on muscarinic cholinergic binding to rat and locust neural membranes

We have compared the effect of ethanol, a membrane perturbant, on the muscarinic binding sites in neural membranes from a vertebrate (rat) and an insect (locust). The binding of the muscarinic antagonist [³H]quinuclidinyl benzilate ([³H]QNB) to both rat and locust neural membranes was inhibited by ethanol at 10–500 mM concentrations; but this inhibition was greater in the locust. Ethanol (500 mM) increased the apparent dissociation constant (K d) of [³H]QNB binding to rat membranes from 0.13±0.01 nM in control to 0.20±0.02 nM; there was also an small but significant reduction in the number of binding sitesB _max. In locust, 500 mM ethanol reduced theB _max of [³H]QNB binding from 590±30 in control to 320±40 pmol/g protein; no significant alteration in theK _D was detected. The dissociation rate constant (k _off) of [³H]QNB increased from 0.020±0.003 in controls to 0.031±0.004 (min^–1) in the presence of 500mM ethanol, the association rate constant (k _on) did not change significantly. In locust, 500 mM ethanol did not affect eitherk _on ork _off. Competition experiments revealed that the binding affinities of both the agonist carbamylcholine and the antagonist atropine to the rat membranes were reduced in the presence of ethanol. In contrast, ethanol caused no alteration in the binding affinities of these ligands to the locust membranes. This differential effect of ethanol on rat and locust muscarinic binding suggests a difference in the hydrophobic domains and/or the membrane interactions of the muscarinic receptors in the two species.     

Similarities of adenosine uptake systems in astrocytes and neurons in primary cultures

Uptake of extracellular adenosine was studied in primary cultures of astrocytes or neurons. Both cell types showed a high affinity uptake. TheK _m values were not significantly different (6.5±3.75 M in astrocytes and 6.1±1.86 M in neurons), but the intensity of the uptake was higher in astrocytes than in neurons (V _max values of 0.16±0.030 and 0.105±0.010 nmol×min^–1×mg^–1 protein, respectively). The temperature sensitivity was similar in the two cell types. Adenosine uptake inhibitors and benzodiazepines inhibited the adenosine uptake systems in both astrocytes and neurons with IC₅₀ values in the high nanomolar or the micromolar range and the rank order of potency was similar in the two cell types. In both cell types the (–) isomers of two sets of benzodiazepine stereoisomers were more potent than the (+) isomers. Dixon analysis showed that dipyridamole, papaverine, hexobendine and chlordiazepoxide inhibited the adenosine uptake competitively and clonazepam noncompetitively in both cell types.     

Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3

B7-H3 (CD276) is both an inhibitory ligand for natural killer cells and T cells and a tumor antigen that is widely expressed among human solid tumors. Anti-B7-H3 mouse monoclonal antibody 8H9 has been successfully used for radioimmunotherapy for patients with B7-H3(+) tumors. We present the humanization, affinity maturation, and epitope mapping of 8H9 based on structure determination, modeling, and yeast display methods. The crystal structure of ch8H9 Fab fragment was solved to 2.5-Å resolution and used as a template for humanization. By displaying the humanized 8H9 single chain Fv (scFv) on the surface of yeast, the affinity was matured by sequential random mutagenesis and fluorescence-activated cell sorting. Six mutations (three in the complementarity-determining region and three in the framework regions) were identified and incorporated into an affinity-matured humanized 8H9 construct (hu8H9-6m) and an affinity-matured chimeric 8H9 construct (ch8H9-6m). The hu8H9-6m scFv had a 160-fold improvement in affinity (0.9 nm K_D) compared with parental hu8H9 scFv (144 nm K_D). The IgG formats of ch8H9-6m and hu8H9-6m (nanomolar to subnanomolar K_D) had 2–9-fold enhancements in affinity compared with their parental forms, potent in vitro antibody-dependent cell-mediated cytotoxicity (0.1–0.3 μg/ml EC₅₀), and high tumor uptake in mouse xenografts. Based on in silico docking studies and experimental validation, the molecular epitope of 8H9 was determined to be dependent on the FG loop of B7-H3, a region critical to its function in immunologic blockade and unique among anti-B7-H3 antibodies published to date.     

GABA-agonists induce the formation of low-affinity GABA-receptors on cultured cerebellar granule cells via preexisting high affinity GABA receptors

The kinetics of specific GABA-binding to membranes isolated from cerebellar granule cells, cultured for 12 days from dissociated cerebella of 7-day-old rats was studied using [³H]GABA as the ligand. The granule cells were cultured in the presence of the specific GABA receptor agonist 4, 5, 6, 7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 150 M) or THIP plus the antagonist bicuculline methobromide (150 M of each) or in the absence of the agonist or antagonist. Membranes isolated from granule cells cultured in a medium without the GABA agonist revealed a single binding site for GABA with a binding constant (K _D) of 7.9±0.4 nM and aB _max of 3.42±0.08 pmol×mg^–1 protein. Membranes from cells cultured in the presence of THIP had two binding sites for GABA withK _D-values of 6.8±0.9 nM and 476±311 nM, respectively. The correspondingB _max values were 4.41±0.42 pmol×mg^–1 and 5.81±1.20 pmol×mg^–1. The effect of culturing the cells in THIP was antagonized by the simultaneous presence of bicuculline in the culture media, i.e. no significant low-affinity binding for GABA was found on the membranes from granule cells cultured in both THIP and bicuculline. TheK _D value (14.3±1.4 nM) for the high affinity binding site was, however, slightly increased compared to the non-treated cells. These findings suggest that the ability of THIP to induce formation of low-affinity GABA receptors is mediated by preexisting high-affinity GABA-receptors on the granule cells.     

Nucleoside transport in rat erythrocytes: two components with differences in sensitivity to inhibition by nitrobenzylthioinosine andp-chloromercuriphenyl sulfonate

Summary The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial,p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 M) by NBMPR was biphasic –35% of the transport activity was inhibited with an IC₅₀ value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 M. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparentK _m for uridine 50±18 and 163±28 M, respectively). The two transport systems also differed in their sensitivity topCMBS. NBMPR-insensitive uridine transport was inhibited bypCMBS with an IC₅₀ of 25M, while 1 mMpCMBS had little effect on NBMPR-sensitive transport by intact cells.pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by -mercaptoethanol, suggesting that thepCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [³H]NBMPR binding to the cell membrane (apparentK _d46±25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25±6 molecules/site per sec at 22°C.pCMBS had no effect on [³H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [³H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent M_r on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR andpCMBS.     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

Kinetics of sodiumd-glucose cotransport in bovine intestinal brush border vesicles

Summary Brush border membrane vesicles (BBMV) purified from steer jejunum were used to study the kinetics of sodiumd-glucose cotransport under voltage clamped, zero-trans conditions. When the initial rate of glucose transport (J _gluc) was measured over a wide range of glucose concentrations ([S]=0.01–20mm), curvature of the Woolf-Augustinsson-Hofstee plots was seen, compatible with a diffusional and one major, high capacity (maximal transport rateJ _max=5.8–8.8 nmol/mg·min) saturable system. Further studies indicated that changes incis [Na] altered theK _t , but not theJ _max, suggesting the presence of a rapid-equilibrium, ordered bireactant system with sodium adding first.Trans sodium inhibitedJ _gluc hyperbolically. KCl-valinomycin diffusion potentials, inner membrane face positive, loweredJ _gluc, while potentials of the opposite polarity raiseJ _gluc. At low glucose concentrations ([S]<0.05mm), a second, minor, high affinity transport system was indicated. Further evidence for this second saturable system was provided by sodium activation curves, which were hyperbolic when [S]=0.5mm, but were sigmoidal when [S]=.0.01mm. Simultaneous fluxes of²²Na and [³H]glucose at 1mm glucose and 30mm NaCl yielded a cotransport-dependent flux ratio of 21 sodium/glucose, suggestive of 11 (Na/glucose) high capacity, low affinity system and a 31 (Na/glucose) high affinity, low capacity system. Kinetic experiments with rabbit jejunal brush borders revealed two major Na-dependent saturable systems. Extravesicular (cis) Na changed theK _t , but not theJ _max of the major system.     

Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP,K+, Na+, and strophanthidin in vitro

The effect of lipid peroxidation on the affinity of specific active sites of Na⁺, K⁺-ATPase for ATP (substrate), K⁺ and Na⁺ (activators), and strophanthidin (a specific inhibitor) was investigated. Brain cell membranes were peroxidized in vitro in the presence of 100M ascorbate and 25M FeCl₂ at 37°C for time intervals from 0–20 min. The level of thiobarbituric acid reactive substances and the activity of Na⁺, K⁺-ATPase were determined. The enzyme activity decreased by 80% in the first min. from 42.0±3.8 to 8.8±0.9 mol Pi/mg protein/hr and remained unchanged thereafter. Lipid peroxidation products increased to a steady state level from 0.2±0.1 to 16.5 ±1.5 nmol malonaldehyde/mg protein by 3 min. In peroxidized membranes, the affinity for ATP and strophanthidin was increased (two and seven fold, respectively), whereas affinity for K⁺ and Na⁺ was decreased (to one tenth and one seventh of control values, respectively). Changes in the affinity of active sites will affect the phosphorylation and dephosphorylation mechanisms of Na⁺, K⁺-ATPase reaction. The increased affinity for ATP favors the phosphorylation of the enzyme at low ATP concentrations whereas, the decreased affinity for K⁺ will not favor the dephosphorylation of the enzyme-P complex resulting in unavailability of energy for transmembrane transport processes. The results demonstrate that lipid peroxidation alters Na⁺, K⁺-ATPase function by modification at specific active sites in a selective manner, rather than through a non-specific destructive process.     

Amino acid metabolism in the thermophilic phototroph,Chloroflexus aurantiacus: properties and metabolic role of two l-threonine (l-serine) dehydratases

Two l-threonine (l-serine) dehydratases (EC 4.2.1.16) of the thermophilic phototrophic bacterium Chloroflexus aurantiacus Ok-70-fl were purified to electrophoretic homogeneity by procedures involving anion exchange and hydrophobic interaction chromatography. Only one of the two enzymes was sensitive to inhibition by l-isoleucine (K _i=2 M) and activation by l-valine. The isoleucine-insensitive dehydratase was active with l-threonine (K _m=20 mM) as well as with l-serine (K _m=10 mM) whereas the other enzyme, which displayed much higher affinity to l-threonine (K _m=1.3 mM), was inactivated when acting on l-serine. Both dehydratases contained pyridoxal-5-phosphate as cofactor. When assayed by gel filtration techniques at 20 to 25° C, the molecular weights of both enzymes were found to be 106,000±6,000. In sodium dodecylsulfate-polyacrylamide gel electrophoresis, the two dehydratases yielded only one type of subunit with a molecular weight of 55,000±3,000. The isoleucine-insensitive enzyme was subject to a glucose-mediated catabolite repression.Abbreviations A absorbance - ile isoleucine - PLP pyridoxal-5-phosphate - SDS sodium dodecyl sulfate - TDH threonine dehydratase - U unit     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Growth kinetics of haloalkaliphilic,sulfur-oxidizing bacterium<Emphasis Type="Italic"> Thioalkalivibrio versutus</Emphasis> strain ALJ 15 in continuous culture

The chemolithoautotrophic, sulfur-oxidizing bacterium Thioalkalivibrio versutus strain ALJ 15, isolated from a soda lake in Kenya, was grown in a continuous culture, with thiosulfate or polysulfide as growth-limiting energy source and oxygen as electron acceptor, at pH 10 and at pH 0.6, 2 M and 4 M total sodium. The end product of the sulfur-compound oxidation was sulfate. Elemental sulfur and a cell-bound, polysulfide-like compound appeared as intermediates during substrate oxidation. In the thiosulfate-limited culture, the biomass yields and maximum specific growth rates decreased two and three times, respectively, with increasing sodium concentration. The apparent affinity constant measured for thiosulfate and polysulfide was in the micromolar range (K_s=6±3 M). The maintenance requirement (m_s=8±5 mmol S₂O₃²/g dry weight h^–1) was in the range of values found for other autotrophic sulfur-oxidizing bacteria. The organism had a comparable maximum specific rate of oxygen uptake with thiosulfate, polysulfide, and sulfide, while elemental sulfur was oxidized at a lower rate. Glycine betaine was the main organic compatible solute. The respiration rates with different species of polysulfides (S_n^2–) were tested. All polysulfide species were completely oxidized at high rates to sulfate. Overall data demonstrated efficient growth and sulfur compounds oxidation of haloalkaliphilic chemolithoautotrophic bacteria from soda lakes.Communicated by W.D. Grant