Heterogeneous distribution of the sodium-dependent alanine transport activity in the rat hepatocyte plasma membrane

The localization of the sodium-dependent alanine uptake activity in rat liver cells was studied. Fractions representative of the canalicular, the contiguous (lateral) and the blood-sinusoidal surface of the hepatocyte were isolated by means of centrifugal fractionation and density gradient centrifugation. The distribution of various marker-enzyme activities in conjunction with the occurrence of alanine transport activity was studied both in fractions obtained after zonal density gradient centrifugation, and in the subcellular fractions mentioned above. It is concluded that the sodium-dependent alanine transport activity is primarily located in the blood-sinusoidal plasma membrane of the hepatocyte.     

Identification of a high-affinity glycine betaine transport system in Staphylococcus aureus

Staphylococcus aureus accumulates proline and glycine betaine when cells are grown at low water activity. In the present study, we have identified a high-affinity glycine betaine transport system in this bacterium. Optimal activity for this transport system was measured in the presence of high NaCl concentrations, but transport activity was not stimulated by high concentrations of other solutes.     

Glucocorticoids upregulate high-affinity, high-density lipoprotein binding sites in rat hepatocytes

Glucocorticoid hormones (GL) regulate high-density lipoprotein (HDL) plasma concentrations by increasing synthesis and secretion of HDL by the liver. However, little is known about the effect of GL on the uptake and processing of HDL by hepatocytes (HEP). To investigate this question, we studied the effects of dexamethasone (DEX) on the expression of high-affinity HDL-binding sites via the specific binding and internalization of iodine-labeled apolipoprotein E (apo E)-free HDL3 in a culture of rat HEP. Specific binding and internalization of HDL3 decreased by 60% in cells cultured in the absence of DEX for 48 hours. At concentrations of 10(-7) and 10(-5) mol/L, DEX prevented the decrease, maintaining specific binding and internalization versus the control level (at 24 hours). HDL-binding sites with a Kd of 20 micrograms/mL were revealed on the surface of cultured HEP. HEP demonstrated a greater binding capacity in the presence of DEX at concentrations of 10(-7) and 10(-5) mol/L (125 v 45 ng/mg cell protein). The effect of the hormone has demonstrated to be dose-dependent at concentrations between 10(-9) and 10(-7) mol/L, leveling off at 10(-7). Higher concentrations did not induce a further increase in specific binding and internalization. Withdrawal of the hormone from culture medium was associated with a decrease in specific binding of the ligand by 60% in the following 24 hours. To investigate the effect of glucocorticoid deficiency on liver uptake of HDL in vivo, specific binding and internalization were studied in a culture of HEP isolated from adrenalectomized rats (AER) at 2 hours after seeding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The distribution of omega-conotoxin MVIICnle-binding sites in rat brain measured by autoradiography

An analogue of omega-conotoxin MVIIC, [125I]omega-MVIICnle, has been employed in an autoradiographic assay to define the distribution of binding sites in rat brain of this neuronal calcium channel antagonist. In comparison with N-type channels (labeled by [125I]omega conotoxin GVIA), omega-MVIICnle sites are much denser in cerebellum (molecular layer) than in forebrain. Binding in thalamus is also comparatively high for omega-MVIICnle. Under these conditions, [125I]omega-MVIICnle binding to rat brain sections is not displaceable by the N-channel antagonist, omega-conotoxin GVIA. The calcium channel blocker [125I]omega-conotoxin MVIICnle labels a unique set of binding sites in mammalian brain.     

Neuronal high-affinity glutamate transport in the rat central nervous system

EAAC1 is a neuronal and epithelial high affinity glutamate transporter previously cloned from rabbit intestine. Here we report the isolation of EAAC 1 from rat brain* and its expression in the central nervous system based on in situ hybridization. Strong signals were detected in brain, spinal cord and retina. Expression of EAAC1 was particularly strong in pyramidal cells of the cerebral cortex, pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, various thalamic nuclei and cells of certain retinal layers. EAAC1 was also expressed in non-glutamatergic neurons such as GABAergic cerebellar Purkinje cells and alpha-motor neurons of the spinal cord. We propose that EAAC1 is not only involved in the sequestration of glutamate at glutamatergic synapses and in protecting neurons from glutamate excitotoxicity, but also in the cellular metabolism involving glutamate.     

Uptake of glucose analogues by rat brain cortex slices: Na+-independent membrane transport

Canine tracheal mucus was dissolved by a number of mucolytic agents, including disulfide bond reducing agents, hydrogen bond breaking agents, and chaotropic ions, and their effect on rheological properties was assessed. Sodium thiocyanate led to 85-100% dissolution with the maximum retention of elasticity. Thiocyanate exposure did not result in demonstrable alterations in the size or shape of the mucus glycoproteins. Sodium thiocyanate is therefore recommended as a suitable dispersing agent for physiochemical studies of glycoprotein secretions.     

Cyclic AMP-mediated enhancement of high-affinity choline transport and acetylcholine synthesis in brain

Intracerebroventricular administration of N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cyclic AMP) to mice increased high-affinity choline transport (HAChT) into synaptosomal preparations from the hippocampus, striatum, and frontal cortex in a time- dose-, and brain region-dependent manner. Similar observations were made when the cyclic AMP analogue 8-bromo-cyclic AMP, the adenylyl cyclase activator forskolin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine were administered. Inhibition of phosphatase 1 and 2A, with okadaic acid, increased basal choline transport and enhanced the response to db-cyclic AMP. The early increase of HAChT activity induced by db-cyclic AMP was blocked by H-7 and H-89, protein kinase A inhibitors, but not by cycloheximide, a protein synthesis inhibitor. Kinetic analysis of the early changes of HAChT revealed an increase in the apparent Vmax without a change of the Km for choline. Hemicholinium-3 (HC-3) binding was not altered when studied 1 h after db-cyclic AMP administration. In contrast, HC-3 binding and HAChT activity were both elevated when estimated 3 h after the treatment, and pretreatment with cycloheximide partially prevented the db-cyclic AMP-induced HAChT rise. As evidence that enhanced HAChT is associated with a direct action of cyclic AMP-dependent pathways on the cholinergic nerve terminals, addition of 8-bromocyclic AMP to isolated hippocampal synaptosomes induced an increase of HAChT that was prevented by H-89. Choline acetyltransferase activity was not affected at any time during the studies. The synthesis of acetylcholine, however, was enhanced 1 h after db-cyclic AMP addition. Our studies show that cyclic AMP-mimetic compounds appear to modulate the choline carrier by a dual mode: an early increase of the maximal velocity without a change of the number of HC-3 binding sites and a late rise of transport that is accompanied by an increase of HC-3 binding. We postulate that HAChT and consequently acetylcholine synthesis in vivo is modulated, in part, by protein kinase A.     

Regulation of sodium-dependent phosphate transport in osteoclasts

Osteoclasts are the primary cells responsible for bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the process of bone resorption and they possess specific Pi-transport system(s) capable of taking up Pi released by bone resorption. By immunochemical studies and PCR, we confirmed previous studies suggesting the presence of an Na-dependent Pi transporter related to the renal tubular "NaPi" proteins in the osteoclast. Using polyclonal antibodies to NaPi-2 (the rat variant), an approximately 95-kD protein was detected, localized in discrete vesicles in unpolarized osteoclasts cultured on glass coverslips. However, in polarized osteoclasts cultured on bone, immunofluorescence studies demonstrated the protein to be localized exclusively on the basolateral membrane, where it colocalizes with an Na-H exchanger but opposite to localization of the vacuolar H-ATPase. An inhibitor of phosphatidylinositol 3-kinase, wortmannin, and an inhibitor of actin cytoskeletal organization, cytochalasin D, blocked the bone-stimulated increase in Pi uptake. Phosphonoformic acid (PFA), an inhibitor of the renal NaPi-cotransporter, reduced NaPi uptake in the osteoclast. PFA also elicited a dose-dependent inhibition of bone resorption. PFA limited ATP production in osteoclasts attached to bone particles. Our results suggest that Pi transport in the osteoclast is a process critical to the resorption of bone through provision of necessary energy substrates.     

Pharmacology and subcellular distribution of [3H]rilmenidine binding sites in rat brain

We have previously reported that in rat brain membranes, [3H]rilmenidine, in addition to labelling alpha2-adrenoceptors and the I2B-subtype of imidazoline receptor binding site (I2B-RBS), may label an additional I-RBS population, distinct from previously classified I1-RBS and I2-RBS. In this study, using crude or fractionated rat brain membranes we examined the possible association of [3H]rilmenidine-labelled I-RBS with the A- and B-isoforms of monoamine oxidase (MAO) by studying the inhibition of [3H]rilmenidine binding by a number of MAO inhibitors; and comparing the maximal binding density (Bmax) and subcellular distribution of [3H]rilmenidine binding sites with that of MAO-A and MAO-B catalytic sites labelled by [3H]RO41-1049 and [3H]RO19-6327 and 12-RBS labelled by [3H]2-BFI. Inhibition of [3H]rilmenidine binding by all MAO inhibitors tested produced very shallow curves (slope 0.29-0.56). Clorgyline and moclobemide (selective MAO-A inhibitors) displayed moderate affinities (60-140 nM), while pargyline (non-selective MAO-inhibitor), RO41-1049 (selective MAO-A inhibitor) and RO19-6327 (selective MAO-B inhibitor) exhibited very low affinities (> 2 microM) for 50-75% of [3H]rilmenidine-labelled I-RBS in crude brain membranes and even lower affinity for the remaining binding. Under identical buffer conditions, the Bmax of [3H]rilmenidine-labelled I-RBS (1.45+/-0.14 pmol/mg protein) was considerably lower than those of MAO-A (13.10+/-0.15 pmol/mg) and MAO-B (10.35+/-0.50 pmol/mg) sites. These results suggest that [3H]rilmenidine does not interact directly with the active catalytic site of either MAO enzyme and could at best only associate with a subpopulation of MAO molecules. Binding studies on five fractions of rat cortex homogenates-nuclear (N), heavy (M) and light (L) mitochondrial, microsomal non-mitochondrial (P), and soluble cytosolic (S) fractions-revealed that 45% of total [3H]rilmenidine binding was present in the P fraction cf. 20 and 23% in the M and L fractions, in contrast to [3H]RO19-6327 and [3H]2-BFI which bound 11-13% in the P fraction and 36-38% and 35-44% in the M and L fractions, respectively. Binding of all ligands in the N fraction was 6-15% of total. These studies reveal that [3H]rilmenidine-labelled I-RBS, unlike the I2-RBS, are not predominantly associated with mitochondrial fractions containing the MAO enzymes (and cytochrome oxidase activity), but appear to be distributed in both the mitochondrial and plasma membrane fractions in rat cerebral cortex.     

Stereoselective S-nitrosocysteine recognition sites in rat brain

We examined the effects of intracisternal (i.c.) injections (10-250 nmol) of the L- and D-isomers of S-nitrosocysteine (L- and D-S-nitrosocysteine) on the mean arterial blood pressure and heart rate of conscious rats, and the decomposition of L- and D-S-nitrosocysteine to nitric oxide (NO) upon addition to brain homogenates. The i.c. injection of L-S-nitrosocysteine produced initial falls in mean arterial blood pressure and heart rate which were followed by increases in these parameters. The i.c. injection of D-S-nitrosocysteine did not produce initial falls in mean arterial blood pressure or heart rate but produced the subsequent increases in these parameters. L- and D-S-nitrosocysteine decomposed equally to NO. These results suggest that the initial effects of L-S-nitrosocysteine may be due to the activation of stereoselective recognition sites on brain neurons.     

Clinical significance of sodium-dependent lithium transport in affective psychoses

The steady state red blood cell/plasma lithium (Li) ratios were determined simultaneously with the in vitro rates of sodium-dependent Li transport of erythrocytes during prophylactic Li therapy in 30 unipolar, 52 bipolar forms of manic-depressive patients and in 58 nonmanic-depressive psychiatric patients as a control group. A reciprocal correlation was found between the Li ratios and the values of Na-dependent Li transport. These transport rates were in positive correlation with the steady state Li contents of red blood cells and that of the plasma. Significant differences were revealed between the different nosologic subgroups in the respect of in vitro rates of Na-dependent Li countertransport. Results are discussed in the light of clinical and pharmacogenetic aspects.     

The high-affinity sulphonylurea receptor regulates KATP channels in nerve terminals of the rat motor cortex

The coexpression of sulphonylurea binding sites and ATP-sensitive K+(KATP) channels was examined in the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. These channels were not detected on neuronal cell bodies, but sulphonylurea-sensitive KATP channels and charybdotoxin-sensitive, large-conductance calcium-activated K+ BKCa channels were detected by patch clamping of fused nerve terminals from the motor cortex. Subcellular fractionation revealed that high-affinity sulphonylurea binding sites were enriched in the nerve terminal fraction, whereas glibenclamide increased calcium-independent glutamate efflux from isolated nerve terminals. It is concluded that neuronal sulphonylurea receptors and KATP channels are functionally linked in the motor cortex and that they are both selectively expressed in nerve terminals, where the KATP channel may serve to limit glutamate release under conditions of metabolic stress.     

Identification and partial characterization of the high-affinity choline carrier from rat brain striatum

[3H]Choline mustard aziridinium ion binds irreversibly to the sodium-coupled high-affinity choline transport protein in a sodium-dependent and hemicholinium-sensitive manner, and thus is a useful affinity ligand. In rat striatal synaptosomal membranes, it radiolabels two polypeptides with apparent molecular masses of 58 and 35 kDa. Based upon the use of two different experimental approaches, it appears that neither of these polypeptides is glycosylated.     

Manganese poisoning reduces strychnine-insensitive glycine binding sites in the globus pallidus of the mouse brain

Manganese (Mn) poisoning is characterized by central nervous system manifestations, including psychiatric disturbances and extrapyramidal disorders. This metal is thought to produce neuronal degeneration due to cytotoxic products originated by oxidative stress and through an indirect excitotoxic process. In previous studies, we have found a reduction in the density of N-methyl-D-aspartate (NMDA) recognition sites in some brain areas of Mn-treated mice. Due to the close relationship between NMDA sites and strychnine-insensitive glycine (Gly) modulatory sites in the NMDA receptor complex, the [3H]-glycine ([3H]-Gly) binding was analyzed by autoradiographic methods in the brain of mice treated with manganese chloride for 8 weeks. Among all analyzed areas, only the globus pallidus showed a significant reduction in [3H]-Gly binding (27-28%). The Gly binding decrease, focalized in the globus pallidus, could reflect a degeneration of structures containing strychnine-insensitive Gly receptors, since this area is the most frequently reported damaged brain region in Mn intoxication. However, it might also be due to a Gly receptor down-regulation to control NMDA complex activation during Mn poisoning.     

Effects of K+-depolarization, arachidonic acid, ethanol, and aging on the high-affinity choline transport in rat hippocampus

The Na+-dependent high-affinity choline uptake (HACU) transport and the [3H]hemicholinium-3 ([3H]HC-3) specific binding were measured on hippocampal synaptosomes of young (3-6 months) and old (22 months) Wistar rats. In vitro effects of 100-300 microM arachidonic acid (AA) and of 5% ethanol were tested under basal as well as stimulated (55 mM KCl) conditions. The influence of AA (an irreversible decrease of HACU and a reversible increase of [3H]HC-3 binding) was more marked under stimulated rather than basal conditions in brain tissue of young rats. The increased K+-depolarization effect on HACU and the decreased influence of AA on [3H]HC-3 binding were estimated in brain tissue of old compared to young rats. Results suggest the involvement of different pools of the high-affinity choline carrier and marked changes due to aging in the regulation of the HACU transport.     

Increases in strychnine-insensitive glycine binding sites in cerebral cortex of chronic schizophrenics: evidence for glutamate hypothesis

Strychnine-insensitive glycine binding sites, an absolute requirement of the responses mediated by N-methyl-D-aspartate (NMDA) receptors, were measured in the postmortem brains of 13 chronic schizophrenics and 10 controls, using a radiolabeled receptor assay. Specific [3H]glycine binding was significantly increased in six of the 16 areas of the cerebral cortex that were investigated. Scatchard analysis performed in these areas showed a significant increase in the maximum number of binding sites, with no change in the affinity of binding. Multiple regression analysis confirmed that the increase was not due to age at death or interval from death to freezing. The increase was also observed in the off-drug cases of schizophrenics who had not taken antipsychotics for more than 40 days before death. These results suggest that the increases in NMDA-associated glycine binding sites, possibly ascribed to the postsynaptic compensation for impaired glutamatergic neurotransmission, might be implicated in the pathophysiology of schizophrenia.