Isolation of pure cholinergic nerve endings from Torpedo electric organ. Evaluation of their metabolic properties

Pure cholinergic nerve endings (synaptosomes) were isolated from the electric organ of Torpedo by a rapid procedure. These synaptosomes are approximately 3 micron in diameter. They contain an occasional mitochondrion, numerous synaptic vesicles, and sometimes an active zone is observed. No postynaptic membrane attachment is found. This nerve ending fraction is extremely pure as shown by morphological controls and biochemical data. It is rich in choline acetyltransferase (450 nmol/h per mg protein) and acetylcholine (ACh) (130 nmol/mg protein). The isolated endings retain their cytoplasmic components and they synthesize ACh and are stable in vitro for several hours, as shown by biochemical measurements and morphological analysis.     

Osmoregulation in Rhodobacter sphaeroides.

Betaine (N,N,N-trimethylglycine) functioned most effectively as an osmoprotectant in osmotically stressed Rhodobacter sphaeroides cells during aerobic growth in the dark and during anaerobic growth in the light. The presence of the amino acids L-glutamate, L-alanine, or L-proline in the growth medium did not result in a significant increase in the growth rate at increased osmotic strengths. The addition of choline to the medium stimulated growth at increased osmolarities but only under aerobic conditions. Under these conditions choline was converted via an oxygen-dependent pathway to betaine, which was not further metabolized. The initial rates of choline uptake by cells grown in media with low and high osmolarities were measured over a wide range of concentrations (1.9 microM to 2.0 mM). Only one kinetically distinguishable choline transport system could be detected. Kt values of 2.4 and 3.0 microM and maximal rates of choline uptake (Vmax) of 5.4 and 4.2 nmol of choline/min.mg of protein were found in cells grown in the minimal medium without or with 0.3 M NaCl, respectively. Choline transport was not inhibited by a 25-fold excess of L-proline or betaine. Only one kinetically distinguishable betaine transport system was found in cells grown in the low-osmolarity minimal medium as well as in a high-osmolarity medium containing 0.3 M NaCl. In cells grown and assayed in the absence of NaCl, betaine transport occurred with a Kt of 15.1 microM and a Vmax of 3.2 nmol/min . mg of protein, whereas in cells that were grown and assayed in the presence of 0.3 M NaCl, the corresponding values were 18.2 microM and 9.2 nmol of betaine/min . mg of protein. This system was also able to transport L-proline, but with a lower affinity than that for betaine. The addition of choline of betaine to the growth medium did not result in the induction of additional transport systems.     

Uptake and metabolism of choline in the organotypic culture of newborn mouse cerebellum

The uptake and metabolism of [methyl-14C]choline in the organotypic culture of newborn mouse cerebellum was examined. Explants of 8 day in vitro (8 DIV) were incubated for 48 h under standard conditions with 21.0 microM [14C]choline at 35 degrees C. During the first hour of incubation, most of the [14C]choline incorporated was transferred to phosphocholine. The amount of [14C]phosphocholine increased gradually at the initial rate of 0.95 +/- 0.17 nmol/mg protein/h and saturated after 7 h (4.31 +/- 1.30 nmol/mg protein). The synthesis of [14C]phospholipids was observed after a distinct time lag. About 96% of the radioactivity in the lipids was incorporated into phosphatidylcholine. The amount of phosphatidylcholine increased linearly up to 48 h of incubation: 11.9 +/- 2.10 nmol/mg protein at 24 h and 21.9 +/- 2.43 nmol/mg protein at 48 h. From double-label studies it was found that phosphocholine was a precursor of phosphatidylcholine. The content of [14C]choline within explants remained nearly constant through the incubation period. Acetylcholine synthesis in mouse cerebellum culture was relatively low, and the content remained constant through the incubation period (0.006 +/- 0.003 nmol/mg protein). Activities of acetylcholine synthesis of cerebral and cerebellar homogenates were compared. Phosphatidylcholine synthesized in mouse cerebellum culture separated into two spots on thin layer chromatograph using silica gel G plates. Gas chromatographs suggested that the separation depends on the difference in fatty acid composition.     

Choline deficiency induced by Mycoplasma fermentans enhances apoptosis of rat astrocytes

A choline uptake system accumulating free choline in an energy-dependent process is described in Mycoplasma fermentans. The uptake system has a K(m) of 2.2x10(-5) M and a V(max) of 0.15 nmol 10 min(-1) mg(-1) cell protein and the choline incorporated could be recovered in the soluble fraction as free choline, phosphorylcholine and CDP-choline. Choline accumulation by M. fermentans resulted in a marked choline depletion of the growth medium. The choline depletion of an astrocyte cell culture induced by M. fermentans was associated with the apoptotic death of the cells. Apoptosis was not obtained with heat-inactivated mycoplasmas and could be reversed by the addition of free choline to the growth medium.     

Salt stress enhances choline uptake in the halotolerant cyanobacterium Aphanothece halophytica

The uptake of [14C]choline by a suspension of exponential-phase Aphanothece halophytica under various conditions has been studied. Salt stress was found to enhance the uptake of choline. The kinetics of choline transport followed the Michaelis-Menten relationship with apparent K(m) values of 272 and 286 microM, maximum rates of transport (V(max)) of 18 and 37 nmol/min/mg protein for unstressed and salt-stressed cells, respectively. Choline uptake under salt stress was significantly reduced in chloramphenicol-treated cells, suggesting that the activation by salt stress occurred via an inducible transport system. This was corroborated by the existence of the periplasmic choline binding protein, whose content was higher in cells grown under salt-stress condition. Exogenously provided choline significantly increased the growth rate of cells grown under salt stress, although less efficiently than glycine betaine. The presence of 1 mM choline in the growth medium conferred tolerance to high salinity on A. halophytica with the maintenance of high growth up to 1.5 M NaCl. The uptake of choline was Na(+)-dependent, sensitive to various metabolic inhibitors as well as thiol-reactive agents. The results of competition studies suggested that N-methyl on one end of molecule and on the other end either an aldehyde, an alcohol or a neutral group were important features for substrate recognition.     

Transport of cyclic adenosine 3'',5''-monophosphate across Escherichia coli vesicle membranes.

The uptake and efflux of cyclic adenosine 3',5'-monophosphate (3',5'-cAMP) by Escherichia coli membrane vesicles were studied. Metabolic energy was not required for the uptake process and was found to actually decrease the amount of 3',5'-cAMP found in the vesicles. 3',5'-cAMP uptake exhibits saturation kinetics (Km = 10 mM, Vmax = 2.8 nmol/mg of protein per min) and was competitively inhibited by a number of 3',5'-cAMP analogs. The uptake of 3',5'-cAMP was found to be sharply affected by a membrane phase transition. The excretion of 3',5'-cAMP was studied by using everted membrane vesicles. Efflux in this system was dependent upon metabolic energy and was reduced or abolished by uncouplers. Different energy sources powered efflux at different rates, showing a relationship between the degree of membrane energization and rate of excretion of 3',5'-cAMP. The efflux process also displayed saturation kinetics (Km = 10.0 mM, Vmax = 0.98 nmol/mg of protein per min) and was competitively inhibited by the same 3',5'-cAMP analogs and to the same degree as was the uptake process. 3',5'-cAMP was found to be chemically unaltered by both the uptake and excretion processes. These data are interpreted as showing that the uptake and excretion of 3',5'-cAMP in E. coli membrane vesicles are carrier-mediated phenomena, possibly employing the same carrier system. Uptake is by facilitated diffusion whereas efflux is via an energy-dependent, active transport process. Evidence is presented showing that cells can regulate the number of 3',5'-cAMP transport carriers. The rate of 3',5'-cAMP excretion is possibly regulated by both the degree of membrane energization and the number of carriers present per cells.     

Disposition and hepatoprotection by phosphatidyl choline liposomes in mouse liver

Small unilamellar liposomes with an average diameter of 80 nm were prepared from phosphatidyl choline of various sources using the dialysis method with cholate as a detergent. When 14C-labeled soybean liposomes were intravenously injected into male NMRI mice, up to 10% of the total label was found in the liver lipid. The uptake was dose-dependent and reached an apparent saturation 4 h after injection. The liver maintained a constant radioactivity corresponding to 1.9 +/- 0.13 mg phospholipid/g liver until ten hours after injection of 850 mg labeled phosphatidyl choline/kg body wt. Little radioactivity was taken up by the spleen. Analogous doses of liposomes prepared from egg yolk phosphatidyl choline led to a radioactivity corresponding to 1.3 +/- 0.4 mg lipid/g liver 4 h after injection. Liposomes with a similar size were prepared from hydrated, i.e., saturated phosphatidyl choline. After intravenous administration of these liposomes, an amount of 5.3 +/- 0.5 mg labeled lipid was found per g liver after 4 h. In contrast to unsaturated liposomes, 5.8 +/- 0.8 mg lipid per gram spleen was trapped by the spleen. The pharmacodynamic effect of these different liposomes was studied in benzo[a]pyrene-pretreated mice intoxicated with 400 mg/kg paracetamol. Animals which received paracetamol exhibited serum alanine aminotransferase activities of 4220 +/- 1140 units/l after 4 h and exhaled 120 +/- 19 nmol ethane kg-1 h-1. When pretreated with 850 mg soybean phosphatidyl choline/kg body wt. (i.v.) 2 h prior to paracetamol, the increase in serum transaminase activity was reduced to 117 +/- 104 units/l and ethane exhalation amounted to 18 +/- 8 nmol kg-1 h-1. In contrast, similar pretreatment with egg yolk phosphatidyl choline or hydrated phosphatidyl choline failed to protect against paracetamol-induced hepatotoxicity. The different pharmacodynamic effects of the two phosphatidyl cholines of plant or animal origin cannot be explained on the basis of their different pharmacokinetics. In the case of soybean phosphatidyl choline liposomes, the amount of radioactive lipid found in the liver correlated with the hepatoprotective potency.     

Characterisation of tolbutamide hydroxylase activity in the common brushtail possum, (Trichosurus vulpecula) and koala (Phascolarctos cinereus): inhibition by the eucalyptus terpene 1,8-cineole

Plant constituents such as terpenes are major constituents of the essential oil in Eucalyptus sp. 1,8-Cineole and p-cymene (Terpenes present in high amounts in Eucalyptus leaves) are potential substrates for the CYP family of enzymes. We have investigated tolbutamide hydroxylase as a probe substrate reaction in both koala and terpene pretreated and control brushtail possum liver microsomes and examined inhibition of this reaction by Eucalyptus terpenes. The specific activity determined for tolbutamide hydroxylase in the terpene treated brushtails was significantly higher than that for the control animals (1865+/-334 nmol/mg microsomal protein per min versus 895+/-27 nmol/mg microsomal protein per min). The activity determined in koala microsomes was 8159+/-370 nmol/mg microsomal protein per min. Vmax values and Km values for the terpene treated possum, control, possum and koala were 1932-2225 nmol/mg microsomal protein per min and 0.80 0.81 mM; 1406-1484 nmol/mg microsomal protein per min and 0.87-0.92 mM and 5895-6403 nmol/mg microsomal protein per min and 0.067-0.071 mM, respectively. Terpenes were examined as potential inhibitors of tolbutamide hydroxylase activity. 1,8-Cineole was found to be a competitive inhibitor for the enzyme responsible for tolbutamide hydroxylation (Ki 15 microM) in the possum. In koala liver microsomes stimulation of tolbutamide hydroxylase activity was observed when concentrations of cineole were increased. Therefore, although inhibition was observed, the type of inhibition could not be determined.     

Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high-choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (greater than 96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline.     

Calcium Transport by Primary Cultured Neuronal and Glial Cells from Chick Embryo Brain

The uptake of calcium was examined in primary cultures of pure neurons and of glial cells from dissociated hemispheres of chick embryo brain. Neuronal cultures took up calcium at a rate of 2.0 nmol per min per mg cell protein at medium concentrations of 1.2 mM-Ca²⁺ and 5.4 mM-K⁺. The rate of calcium entry into neurons was increased 2.7-fold by elevating medium potassium to 60 MM. The effect of high external potassium was to increase the V_max value for calcium transport from 5.5 to 13 nmol per min per mg; the Michaelis constant for calcium, 1.2 mM, was unchanged. The potassium-dependent component of calcium entry into the neuronal cultures was eliminated by addition of 0.1 mM-D-600 (a verapamil derivative) or by 1 mM-CoCl₂, but 0.5 μM-tet-rodotoxin had no significant effect. When choline replaced potassium in uptake medium no change in calcium transport was detected in neurons, nor was the entry of calcium increased when choline replaced sodium. Glial cultures took up calcium at 20% of the basal rate for neuronal cultures on a weight-of-protein basis. Uptake was not increased by potassium; during depolarization by potassium the calcium transport activity of glia was less than 10% that of neurons. It was concluded that cultured neurons contain a depolarization-sensitive, calcium-specific channel. A similar calcium transport activity was not detected in cultured glial cells.     

Vascular Cholinesterases and Choline Uptake in Isolated Rat Forebrain Microvessels: A Possible Link

The two parameters of the active [methyl-3H]choline uptake into isolated rat forebrain microvessels, Km and Vmax, were determined for 1-, 3-, 10-, and 24-month-old Charles River male rats and compared with the activities of the enzymes choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) in these microvessels over the same time course. The value of Km remained constant over the entire period, but that of Vmax increased from 8.5 +/- 1.0 to 80.6 +/- 16.4 nmol g-1 (mean +/- SEM) over the first 3 months of life. Over the same period, the increase in ChAT activity, from an initial value of 7.1 +/- 1.6 to 10.2 +/- 0.3 nmol g-1 min-1, was not proportional to that of choline uptake. Levels of BuChE activity (0.9-1.3 mumol g-1 min-1) were almost unchanged throughout the entire 24-month period, but those of AChE showed a steady and significant increase from 1 to 24 months, remaining relatively high at senescence (4.7 mumol g-1 min-1), when choline uptake had decreased to one-third of its optimal value. Selective inhibition of AChE with 1,5-bis(4-allyldimethylammonium-phenyl)pentan-3-one dibromide (0.5 microM) in unruptured capillaries from 3-month-old rats resulted in a decrease in Vmax of choline uptake from approximately 81 to 59 nmol g-1 min-1 or with 9-amino-1,2,3,4-tetrahydroacridine (10 microM) in capillaries from 2-month-old rats from approximately 30 to 15 nmol g-1 min-1. Selective inhibition of BuChE with tetraisopropyl pyrophosphoramide (100 microM) resulted in an increase in Vmax from approximately 81 to 96 nmol g-1 min-1. It is possible that the two vascular enzyme systems are coupled to a hypothetical endothelial choline transporter, but with an action opposite to each other.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (beta-alanyl-L-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na+, K+ or choline+ gradient conditions (extravesicular greater than intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na+-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a Km of 9.6 +/- 1.4 mM and a Vmax of 2.9 +/- 0.2 nmol/mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-L-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na+-independent process.     

Transport of 2-oxoisocaproate in isolated hepatocytes and liver mitochondria

The transport of 2-oxoisocaproate into isolated hepatocytes and liver mitochondria of rat was studied using [U-14C]2-oxoisocaproate and the silicone oil filtration procedure. 2-Oxoisocaproate uptake by hepatocytes was composed of: rapid adsorption, unmediated diffusion and carrier-mediated transport. The carrier-mediated transport was strongly inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulphonic acid and p-chloromercuribenzoate, was less sensitive to alpha-cyano-4-hydroxycinnamate and insensitive to p-chloromercuriphenylsulphonate. Other 2-oxo acids: pyruvate, 2-oxoisovalerate and 2-oxo-3-methylvalerate, were also inhibitory. The kinetic parameters of the carrier-mediated transport were Km 30.6 mM and Vmax 23.4 nmol/min per mg wet wt, at 37 degrees C. It is concluded that at its low, physiological, concentration, 2-oxoisocaproate penetrates the hepatocyte membrane mainly by unmediated diffusion. The uptake of 2-oxoisocaproate by isolated liver mitochondria was partly inhibited by alpha-cyano-4-hydroxycinnamate, the inhibitor of mitochondrial monocarboxylate carrier. The remaining uptake was linearly dependent on 2-oxoisocaproate concentration and represented unmediated diffusion. The carrier-mediated transport exhibited the following kinetic parameters: Km 0.47 mM, Vmax 1.0 nmol/min per mg protein at 6 degrees C; and Km 0.075 mM and Vmax about 8 nmol/min per mg protein at 37 degrees C.     

Effect of ethanolamine on choline uptake and incorporation into phosphatidylcholine in human Y79 retinoblastoma cells

The effect of physiological concentrations of ethanolamine on choline uptake and incorporation into phosphatidylcholine was investigated in human Y79 retinoblastoma cells, a multipotential, undifferentiated retinal cell line that has retained many neural characteristics. These cells have a high-affinity uptake system for choline, and the majority of the choline taken up was incorporated into phosphatidylcholine via the CDP-choline pathway. The presence of extracellular ethanolamine significantly decreased high-affinity choline uptake and, subsequently, the amount of choline incorporated into phosphatidylcholine. When 100 mumol/L ethanolamine was added, there was a decrease of about 8% in the phosphatidylcholine content. Ethanolamine had no effect on choline incorporation into phosphatidylcholine, however, once choline was taken up by the cell. The K'M and V'max for high-affinity choline uptake was increased from 0.93 to 9.74 microM and 19.60 to 79.25 pmol/min per mg protein, respectively, by the presence of 25 mumol/L ethanolamine. In contrast, 25 mumol/L choline had no effect on the kinetic parameters of high-affinity ethanolamine uptake. Therefore, the reduction in high-affinity choline transport by ethanolamine apparently is not simply due to competitive inhibition. 2,2-Dimethylethanolamine and 2-methylethanolamine both reduced choline uptake to a greater extent than ethanolamine. However, because these compounds exist at much lower concentrations than ethanolamine, they probably have little physiological influence. These results suggest that changes in ethanolamine concentration within the physiologic range can regulate the synthesis and content of phosphatidylcholine in a neural cell by influencing the uptake of choline.     

Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

Uptake of glycine betaine and its analogues by bacteroids of Rhizobium meliloti

Bacteroids isolated from alfalfa nodules induced by Rhizobium meliloti 102F34 transported glycine betaine at a constant rate for up to 30 min. Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect. The apparent Km for glycine betaine uptake was 8.3 microM and V was about 0.84 nmol min-1 (mg protein)-1 in the presence of 200 mM-NaCl which gave maximum stimulation of the transport. Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive. Glycine betaine transport showed considerable structural specificity: choline, proline betaine, gamma-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids. Both a high-affinity activity and a low-affinity activity were found for choline uptake. These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.     

Energy-dependent calcium uptake activity of microsomes from the aorta of normal and hypertensive rats.

Energy-dependent calcium uptake activity of microsomes isolated from the rat aorta has been characterized. The microsomes consist of smooth membrane vesicles which in the presence of MG-ATP as an energy source continuously sequester calcium over a 60-min period. This calcium uptake is greatly stimulated by oxalate anion which serves as a calcium trapping agent. Unlike the calcium uptake of mitochondria this uptake is not inhibited by sodium azide. Sucrose density gradient analysis of the microsomal calcium uptake suggests that the system is associated with the sarcoplasmic reticulum. In presence of 5 mM Mg-ATP and 20 muM calcium approximately 38 nmol of calcium per mg of microsomal protein are taken up in 20 min. In the absence of ATP, less than 2 nmol of calcium per mg of protein are taken up in the first 2 min with no further uptake of calcium in subsequent time periods. When calcium uptake activity is plotted against calcium or ATP concentration of the medium, half maximal activity is calculated for 24.3 muM calcium and for 1.6 mM ATP. The calcium uptake characteristics of the rat aorta microsomes are compatible with a postulated role in the relaxation of the vascular smooth muscle and the provision of an intracellular calcium store for muscle contraction. Aorta microsomes from SHR rats (a genetic strain that is spontaneously hypertensive) have a significantly reduced uptake when compared with the corresponding nonhypertensive control strain. The level of calcium and ATP for half maximal activity of the rat aorta microsomal calcium uptake system is approximately the same in the SHR and the control strain. The rate of release of calcium from rat aorta microsomes is apparently identical in SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR strain and control. The calcium uptake activity of kidney and liver microsomes isolated from the SHR rat appears to be identical to that found in the control strain.     

Choline transport into rat liver mitochondria. Characterization and kinetics of a specific transporter.

Rat liver mitochondria possess a specific choline transporter in the inner membrane. The transporter shows saturable kinetics at high membrane potential with a Km of 220 microM and a Vmax of 0.4 nmol/mg of protein/min at pH 7.0 and 25 degrees C. At physiological concentrations of choline, the rate of choline uptake by the transporter shows a linear dependence on membrane potential; uptake is distinct from the nonspecific cation diffusion process. Hemicholinium-3, hemicholinium-15, quinine, and quinidine, all analogues of choline, are high affinity competitive inhibitors of choline transport with Ki values of 17, 55, 15, and 127 microM, respectively. The choline transporter is distinct from other known mitochondrial transporters. Rat heart mitochondria do not appear to possess a choline transporter. Evidence suggests that the transporter is an electrophoretic uniporter. Analogue studies have shown that the hydroxyl and the quaternary ammonium groups of choline are necessary for binding to the transporter. A comparison of molecular models of choline and the high affinity inhibitors has provided evidence for the preferred conformation of choline for binding to the transporter. The presence of a choline transporter in the mitochondrial inner membrane provides a potential site for control of choline oxidation and hence supply of endogenous betaine.     

Dicarboxylate transport in human placental brush-border membrane vesicles

Pathways for transport of dicarboxylic acid metabolites by human placental epithelia were investigated using apical membrane vesicles isolated by divalent cation precipitation. The presence of Na+/dicarboxylate cotransport was assessed directly by [14C]succinate tracer flux measurements and indirectly by fluorescence determinations of voltage sensitive dye responses. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of succinate achieving levels approximately 5-fold greater than those observed at equilibrium. The increased succinate uptake was specific for Na+ as no stimulation was observed in the presence of Li+, K+ or choline+ gradients. In addition to concentrative accumulation of succinate, a direct coupling of Na+/succinate cotransport was suggested by the absence of a sizeable conductive pathway for succinate uptake and decreased succinate uptake levels associated with a more rapid decay of an imposed Na+ gradient. Na+ gradient-driven succinate uptake was not the result of parallel Na+/H+ and succinate/OH- exchange activities but was reduced by the Na+-coupled inhibitor harmaline. The voltage sensitivity of Na+ gradient-driven succinate uptake suggests Na+/succinate cotransport is electrogenic occurring with net transfer of positive charge. Substrate-specificity studies suggest the tricarboxylic acid cycle intermediates as candidates for transport by the Na+-coupled pathway. Decreasing pH increased the citrate-induced inhibition of succinate uptake suggesting divalent citrate as the preferred substrate for transport. Initial rate determinations of succinate uptake indicate succinate interacts with a single saturable site (Km 33 microM) with a maximal transport rate of 0.5 nmol/mg per min.