Uridine uptake by isolated intestinal epithelial cells of guinea pig

Uptake of uridine was studied in isolated intestinal epithelial cells of guinea pig. Uptake was not severely influenced by metabolism. Free uridine was accumulated within cells 13-fold. Uptake was saturable with an apparent K_m value of 46 μM and a V_max of 0.9 nmol/mg protein per min. Uracil inhibited uptake only slightly; adenosine, guanosine and cytosine inhibited strongly. Antimycin A and ouabain inhibited almost 90%. If the extracellular Na⁺ concentration was decreased to 5 mM, the rate of uptake decreased 6.5-fold. The stimulatory effect of Na⁺ was related to the transmembraneous Na⁺-gradient. Cells from jejunum transported about 30% faster than cells from ileum. In conclusion, isolated enterocytes of guinea pig posses an active transport system for uridine.     

Calcium Signaling in <Emphasis Type="Italic">Carassius</Emphasis> Cerebellar Neurons: Role of the Mitochondria

We studied the involvement of the mitochondria playing the role of a calcium store in the control of calcium exchange in cerebellar neurons of a fish species tolerant to hypoxia, crucian (Carassius gibelio). In our experiments we used an ionophore, CCCP, that blocked accumulation of calcium by the above organelles. The intracellular concentration of free Ca²⁺ ([Ca²⁺] _і ) was measured using a calcium-sensitive dye, Fura-2AM, and the microfluorescent technique. We found that cerebellar neurons of Carassius gibelio possess a well-expressed system clearing the cytoplasm from excessive Ca²⁺, and the mitochondria are actively involved in this process. Under conditions of suppression of the process of accumulation of calcium by the mitochondria under the action of CCCP, the amplitude of calcium transients increased by about 50%. In addition, the decay phase of depolarization-induced intracellular calcium transients was slowed down considerably. Therefore, our experiments are indicative of the significant role of the mitochondria in the control of calcium dynamics in cerebellar neurons of Carassius gibelio in the course of functional activity of these cells.     

UPTAKE OF METHYLAMINE (AN AMMONIUM ANALOGUE) BY MACROCYSTIS PYRIFERA (PHAEOPHYTA)1

The ammonium analogue, methylamine, is taken up rapidly from dilute solution by Macrocystis pyrifera (L.) C. A. Agardh. ¹⁴C-methylamine was used to characterize the transport system, with respect to dependence on external concentration, temperature, pH and substrate specificity. The results suggest that methylamine enters the algal tissue via a specific mediated transport system. Uptake of methylamine showed no consistent relation to the N content of the plant tissue, but was highly dependent on the portion of plant sampled and severely affected by cutting the tissue. The strong inhibition of methylamine uptake by ammonium and lesser inhibition by other alkylamines suggests that the uptake system functions as an “ammonium permease”. Uptake of ¹⁴C-methylamine can be used as a highly sensitive measure of NH₄⁺ uptake activity and should be a useful tool for studying NH₄⁺ uptake in the laboratory and field.     

Uptake of Amino Acids by Barley Leaf Slices: Kinetics, Specificity, and Energetics

Uptake of ¹⁴C-labelled _L-lysine. _L-arginine, _L-glutamic acid, _L-aspartic acid, and glycine was studied in 0.75 mm wide barley (Hordeum vulgare L. cv. Lise) leaf slices. After an initial period (10 min) of rapid accumulation amino acid uptake proceeded at a steady, lower rate for several hours. Uptake was stimulated by 10^?4M Ca^?2+ ions. Uptake was strongly _pH dependent with the following optima: aspartic acid _pH 3.5. glutamic acid _pH 4.1. glycine _pH 5.8, lysine _pH 6–7, and arginine _pH 5–8 (a broad plateau). The optimal temperature was about 30°C. and the temperature coefficient in the range 0–20gGC was 2.3–2.5. Concentration-dependence data gave uptake isotherms which appeared to be multiphasic for all the amino acids used. The amino acids inhibited each other in a competitive fashion, indicating that they were all transported by a single carrier system. Uptake of lysinc was strongly inhibited by 10^?4M 2.4-dinitrophenol. Lysine uptake was not stimulated by light under aerobic conditions. However, it was much reduced in the dark under anaerobic conditions. This reduction was almost compensated for by light. The light-stimulation of uptake under anaerobic conditions was abolished by 10^?5M 3-(3,4-dichlorophenyl) 1.1-dimethylurea.     

Interactions of intracellular pH and intracellular calcium in primary cultures of rabbit corneal epithelial cells

Summary Homeostasis of intracellular calcium ([Ca⁺⁺]_i) and pH (pH_i) is important in the cell's ability to respond to growth factors, to initiate differentiation and proliferation, and to maintain normal metabolic pathways. Because of the importance of these ions to cellular functions, we investigated the effects of changes of [Ca⁺⁺]_i and pH_i on each other in primary cultures of rabbit corneal epithelial cells. Digitized fluorescence imaging was used to measure [Ca⁺⁺]_i with fura-2 and pH_i with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Resting pH_i in these cells was 7.37±0.05 (n=20 cells) and resting [Ca⁺⁺]_i was 129±10 nM (n=35 cells) using a nominally bicarbonate-free Krebs Ringer HEPES buffer (KRHB), pH 7.4. On exposure to 20 mM NH₄Cl, which rapidly alkalinized cells by 0.45 pH units, an increase in [Ca⁺⁺]_i to 215±14 nM occurred. Pretreatment of the cells with 100 μM verapamil or exposure to 1 mM ethylene bis-(oxyethylenenitrilo)-tetraacetic acid (EGTA) without extracellular calcium before addition of 20 mM NH₄Cl did not abolish the calcium increase, suggesting that the source of the calcium transient was from intracellular calcium stores. On removal of NH₄Cl or addition of 20 mM sodium lactate, there were minimal changes in calcium even though pH_i decreased. Treatment of CE cells with the calcium ionophores, ionomycin and 4-bromo A23187, increased [Ca⁺⁺]_i, but produced a biphasic change in pH_i. Initially, there was an acidification of the cytosol, and then an alkalinization of 0.10 to 0.11 pH units above initial values. When [Ca⁺⁺]_i was decreased by treating the cells with 5 mM EGTA and 20 μM ionomycin, pH_i decreased by 0.35±0.02 units. We conclude that an increase in pH_i leads to an increase in [Ca⁺⁺]_i in rabbit corneal epithelial cells; however, a decrease in pH_i leads to minor changes in [Ca⁺⁺]_i. The ability of CE cells to maintain proper calcium homeostasis when pH_i is decreased may represent an adaptive mechanism to maintain physiological calcium levels during periods of acidification, which occur during prolonged eye closure.     

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.     

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (R _LIS), phase I response, followed by an increase in tight junctional resistance (R _TJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC₅₀ of 6 μM). The objective of the study was to determine the degree to which the changes in [Ca²⁺]_i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La³⁺, and Mn²⁺ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease inR _LIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La³⁺ also inhibited the ATP-induced phase II response, but Mn²⁺ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase inR _TJ. KCl-induced membrane depolarization stimulated an acute decrease inR _LIS and a late increase inR _TJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca²⁺]_i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases inR _TJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic ATP-like changes inR _TE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease inR _LIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase inR _TJ.     

Sulphate ion influx and efflux inhydrodictyon reticulatum

Sulphate ion is accumulated in the cells of the algaHydrodidyon reticulatum to a concentration approximately 30 mmol 1^-1. The rate of SO₄ ^2- uptake is increased markedly in the light, however, the effect is observable only after the cells have been kept in the light for about one hour. Uptake of sulphate is strongly inhibited by phosphorylation uncouplers and also by DIDS. The two-phase sulphate uptake kinetics, reported earlier was confirmed in experiments in which competition with phosphate anion was tested. Phosphate competes with sulphate only in the range of higher substrate concentrations (from 0.2 mmol 1^-1) and does not affect the system which works at low substrate concentrations. Efflux of SO₄ ^2- is very slow; the reason of the flux hindrance is not yet known.     

Muscarinic Activation of BK Channels Induces Membrane Oscillations in Glioma Cells and Leads to Inhibition of Cell Migration

Patients with cerebral tumors often present with elevated levels of acetylcholine (ACh) in their cerebrospinal fluid. This motivated us to investigate physiological effects of ACh on cultured human astrocytoma cells (U373) using a combination of videomicroscopy, calcium microspectrofluorimetry and perforated patch-clamp recording. Astrocytoma cells exhibited the typical morphological changes associated with cell migration; polarized cells displayed prominent lamellipodia and associated membrane ruffling at the anterior of the cell, and a long tail region that periodically contracted into the cell body as the cell moved forward. Bath application of the ACh receptor agonist, muscarine, reversibly inhibited cell migration. In conjunction with this inhibition, ACh induced a dose-dependent, biphasic increase in resting intracellular free calcium concentration ([Ca²⁺] _i ) associated with periodic Ca²⁺ oscillations during prolonged ACh applications. The early transient rise in [Ca²⁺] _i was abolished by ionomycin and thapsigargin but was insensitive to caffeine and ryanodine while the plateau phase was strictly dependent on external calcium. The Ca²⁺ response to ACh was mimicked by muscarine and abolished by the muscarinic antagonists, atropine or 4-DAMP, but not by pirenzepine. Using perforated patch-clamp recordings combined with fluorescent imaging, we demonstrated that ACh-induced [Ca²⁺] _i oscillations triggered membrane voltage oscillations that were due to the activation of voltage-dependent, Ca²⁺-sensitive K⁺ currents. These K⁺ currents were blocked by intracellular injection of EGTA, or by extracellular application of TEA, quinine, or charybdotoxin, but not by apamin. These studies suggest that activation of muscarinic receptors on glioma cells induce the release of Ca²⁺ from intracellular stores which in turn activate Ca²⁺-dependent (BK-type) K⁺ channels. Furthermore, this effect was associated with inhibition of cell migration, suggesting an interaction of this pathway with glioma cell migration. Received: 17 December/Revised: 17 March 2000     

Ammonium uptake by Chlorella

The preincubation of Chlorella cells with glucose caused a tenfold increase of the maximal uptake rate of ammonium without change in the K _m (2 M). A similar stimulation of ammonium uptake was found when the cells were transferred to nitrogen-free growth medium. The time-course of uptake stimulation by glucose revealed a lag period of 10–20 min. The turnover of the ammonium transport system is characterized by a half-life time of 5–10 h, but in the presence of light 30% of uptake activity stayed even after 50 h. 6-Deoxyglucose was not able to increase the ammonium uptake rate. These data together were interpreted as evidence for induction of an ammonium transport system by a metabolite of glucose. Mechanistic studies of the ammonium transport system provided evidence for the electrogenic uptake of the ammonium ion. The charge compensation for NH ₄ ⁺ entry was achieved by immediate K⁺ efflux from the cells, and this was followed after 1 min by H⁺ extrusion. Ammonium accumulated in the cells; the rate of uptake was sensitive to p-trifluoromethoxy-carbonylcyanide-phenylhydrazon and insensitive to methionine-sulfoxime. Uptake studies with methylamine revealed that methylamine transport is obviously catalyzed by the ammonium transport system and, therefore, also increased in glucose-treated Chlorella cells.Abbreviation p.c. packed cells     

Calcium Homeostasis in Digitonin-Permeabilized Bovine Chromaffin Cells

The regulation of cytosolic calcium was studied in digitonin-permeabilized chromaffin cells. Accumulation of ⁴⁵Ca²⁺ by permeabilized cells was measured at various Ca²⁺ concentrations in the incubation solutions. In the absence of ATP, there was a small (10–15% of total uptake) but significant increase in accumulation of Ca²⁺ into both the vesicular and nonvesicular pools. In the presence of ATP, the permeabilized cells accumulated Ca²⁺ into carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-sensitive and -insensitive pools. The CCCP-sensitive pool—mainly mitochondria—was active when the calcium concentration was > 1 μM and was not saturated at 25 μM. The Ca²⁺ sequestered by the CCCP-insensitive pool could be inhibited by vanadate and released by inositol trisphosphate, a combination suggesting that this pool was the endoplasmic reticulum. The CCCP-insensitive pool had a high affinity for calcium, with an EC₅₀ of ～1 μM. When the Ca²⁺ concentration was adjusted to the level in the cytoplasm of resting cells (0.1 μM), the presumed endoplasmic reticulum pool was responsible for ～90% of the ATP-stimulated calcium uptake. At a calcium level similar to the acetylcholine-stimulated level in intact cells (5–10 μM), most of the Ca²⁺ (>95%) went into the CCCP-sensitive pool.     

The antiproliferative role of ERG K<Superscript>+</Superscript> channels in rat osteoblastic cells

We report on the role of K⁺ currents in the mechanisms regulating the proliferation of UMR 106-01 osteoblastic osteosarcoma cells. Electrophysiological analysis showed that UMR 106-01 cells produce robust K⁺ currents that can be pharmacologically separated into two major components: a E-4031-susceptible current, I _E-4031, and a tetraethylammonium (TEA)-susceptible component, I _TEA. Western blot and RT-PCR analysis showed that I _E-4031 is produced by ether a go-go (eag)-related channels (ERG). Incubation of the cells with E-4031 enhanced their proliferation by 80%. Application of E-4031 in the bath solution did not induce instantaneous changes in the membrane resting potential or in the level of cytosolic calcium; however, the cells were slightly depolarized and the calcium content was significantly increased upon prolonged incubation with the compound. Taken together these findings indicate that ERG channels can impair cell proliferation. This is a novel finding that underscores new modes of regulation of mitosis by voltage-gated K⁺ channels and provides an unexpected insight into the current view of the mechanisms governing bone tissue proliferation.     

Hysteretic activation of the Ca pump revealed by calcium transients in human red cells

The enzymatic basis for the Ca²⁺ pump in human red cells is an ATPase with hysteretic properties. The Ca²⁺-ATPase shifts slowly between a ground state deficient in calmodulin and an active state saturated with calmodulin, and rate constants for the reversible shifts of state were recently determined at different Ca²⁺ concentrations (Scharff, O. and Foder, B. (1982) Biochim. Biophys. Acta 691, 133–143). In order to study whether the Ca²⁺ pump in intact red cells also exhibits hysteretic properties we have analysed transient increases of intracellular calcium concentrations (Ca_i), induced by the divalent cation ionophore A23187. The time-dependent changes of Ca_i were measured by use of radioactive calcium (⁴⁵Ca²⁺) and analysed with the aid of a mathematical model, based partly on the Ca²⁺-dependent parameters obtained from Ca²⁺-ATPase experiments, partly on the A23187-induced Ca²⁺ fluxes determined in experiments with intact red cells. According to the model a delay in the activation of the Ca²⁺ pump is a prerequisite for the occurrence of A23187-induced calcium transients in the red cells, and we conclude that the Ca²⁺ pump in human red cells responds hysteretically. It is suggested that Ca²⁺ pumps in other types of cell also have hysteretic properties.     

Release of Calcium from Mitochondrial and Nonmitochondrial Intracellular Stores in Mouse Pancreatic Acinar Cells by Hydrogen Peroxide

In the present study we have studied how [Ca²⁺] _i is influenced by H₂O₂ in collagenase-dispersed mouse pancreatic acinar cells and the mechanism underlying this effect by using a digital microspectrofluorimetric system. In the presence of normal extracellular calcium concentration, perfusion of pancreatic acinar cells with 1 mm H₂O₂ caused a slow sustained [Ca²⁺] _i increase, reaching a stable plateau after 10–15 min of perfusion. This increase induced by H₂O₂ was also observed in a nominally calcium-free medium, reflecting the release of calcium from intracellular store(s). Application of 1 mm H₂O₂ to acinar cells, in which nonmitochondrial agonist-releasable calcium pools had been previously depleted by a maximal concentration of CCK-8 (1 nm) or thapsigargin (0.5 μm) was still able to induce calcium release. Similar results were observed when thapsigargin was substituted for the mitochondrial uncoupler FCCP (0.5 μm). By contrast, simultaneous addition of thapsigargin and FCCP clearly abolished the H₂O₂-induced calcium increase. Interestingly, co-incubation of intact pancreatic acinar cells with CCK-8 plus thapsigargin and FCCP in the presence of H₂O₂ did not significantly affect the transient calcium spike induced by the depletion of nonmitochondrial and mitochondrial agonist-releasable calcium pools, but was followed by a sustained increase of [Ca²⁺] _i . In addition, H₂O₂ was able to block calcium efflux evoked by CCK and thapsigargin. Finally, the transient increase in [Ca²⁺] _i induced by H₂O₂ was abolished by an addition of 2 mm dithiothreitol (DTT), a sulfhydryl reducing agent. Our results show that H₂O₂ releases calcium from CCK-8- and thapsigargin-sensitive intracellular stores and from mitochondria. The action of H₂O₂ is likely mediated by oxidation of sulfhydryl groups of calcium-ATPases. Received: 15 May 2000/Revised: 4 October 2000     

Nitrate and nitrite reduction by liquid membrane-encapsulated whole cells.

Purified enzymes and cell-free homogenates encapsulated by liquid-surfactant membrane have been shown to retain their catalytic activity (see previously published literature). This paper describes the preparation and properties of liquid-surfactant membrane-encapsulated whole cells of Micrococcus denitrificansATCC 21909. Batch and continuous studies with this model system have demonstrated that encapsulated viable cells reduce nitrates and retain their catalytic activity over anextended period of time. In batch operation, the reactivity of the encapsulated whole cells has been investigated under a variety of experimental conditions. The system is capable of reducing NO₃^? or NO₂^?. Data obtained indicate that encapsulated live cells have a broad pH and temperature optimum range. The encapsulated cells remain viable and do not “escape” into the external aqueous phase, even after five days of constant stirring with nitrate-containing simulated wastewater. Pulsed substrate addition experiments have demonstrated that the encapsulated cells also effectively reduce NO₂^? with no significant reduction in activity, even after 5.5 days of incubation at 30°C. The membrane selectivity for ion transfer has been achieved by incorporating oil-soluble ion exchangers in the membrane. Because of the protection of the liquid membranes, the catalytic reduction of NO₂^? by the encapsulated whole cells is not inhibited by 1 × 10^?4 M mercuric chloride, which is otherwise extremely toxic to the cells, when present in the external aqueous phase. Continuous reduction of 20 ppm of NO₂^? by liquid membrane-encapsulated whole cells has been demonstrated in a constantly stirred reactor over a test period of about one week. In this paper we will discuss the reduction of NO₃^?and NO₂^? by the liquid membrane-encapsulated whole cells of M. denitrificansATCC 21909 mainly in batch runs undera variety of experimental conditions, such as cell and substrate concentrations, product and inhibitor permeation, pH and temperature, effect of oil-soluble ion exchangers on the substrate diffusion, etc.     

Manganese transport by caco-2 cells

The uptake and transport kinetics of manganese (Mn) were investigated in the human intestinal Caco-2 cell line both from the absorption side (apical to basolateral) and from the exsorption side (basolateral to apical). With regard to the former, transport versus time revealed (as uptake) a biphasic pattern with an initial transient phase followed by steady-state conditions. Uptake versus Mn concentrations showed saturation-type kinetics with a 100% increase of Mn binding capacity when measurements were made from 0.5 to 2 h of incubation. The transport characteristics in steady-state conditions exhibited two components, saturable (V_max = 3.70 ± 0.07 nmol/cm²/h, K_m = 32.2 ± 3.4 μM) and nonsaturable (slope = [1.4 ± 0.2] x 10^-6 cm^-2/h) usually presumed to reflect transcellular (carrier mediated) and paracellular (diffusional) pathways, respectively. Mn fluxes were decreased by calcium and calcium antagonists, almost 100% inhibited at 4°C, and affected by quinacrine and ouabain. The inhibition of ATP synthesis was apparently ineffective. From the exsorption side, the Mn fluxes, without a transient period, had an approx 20-fold smaller rate than in the absorptive direction and showed mainly a nonsaturable route (slope = [0.6 ± 0.1] x 10^-6 cm^-2/h). The mechanisms participating in the Mn movements through the monolayer are discussed and proposed to be in common, at least partly, with other divalent cations such as calcium, zinc, or iron.     

Transport Pathways for Therapeutic Concentrations of Lithium in Rat Liver

Although both amiloride- and phloretin-sensitive Na⁺/Li⁺ exchange activities have been reported in mammalian red blood cells, it is still unclear whether or not the two are mediated by the same pathway. Also, little is known about the relative contribution of these transport mechanisms to the entry of therapeutic concentrations of Li⁺ (0.2–2 mm) into cells other than erythrocytes. Here, we describe characteristics of these transport systems in rat isolated hepatocytes in suspension. Uptake of Li⁺ by hepatocytes, preloaded with Na⁺ and incubated in the presence of ouabain and bumetanide, comprised three components. (a) An amiloride-sensitive component, with apparent K _m 1.2 mm Li⁺, V _max 40 μmol · (kg dry wt · min)⁻¹, showed increased activity at low intracellular pH. The relationship of this component to the concentration of intracellular H⁺ was curvilinear suggesting a modifier role of [H⁺] _i . This system persisted in Na⁺-depleted cells, although with apparent K _m 3.8 mm. (b) A phloretin-sensitive component, with K _m 1.2 mm, V _max 21 μmol · (kg · min)⁻¹, was unaffected by pH but was inactive in Na⁺-depleted cells. Phloretin inhibited Li⁺ uptake and Na⁺ efflux in parallel. (c) A residual uptake increased linearly with the external Li⁺ concentration and represented an increasing proportion of the total uptake. The results strongly suggest that the amiloride-sensitive and the phloretin-sensitive Li⁺ uptake in rat liver are mediated by two separate pathways which can be distinguished by their sensitivity to inhibitors and intracellular [H⁺]. Received: 8 April 1999/Revised: 19 July 1999     

Characterization of the weak calcium binding of trimeric globular adiponectin

Adiponectin is secreted from adipose tissue and functions as a protein hormone in regulating glucose metabolism and fatty acid catabolism. Adiponectin plays an important role as a novel risk factor and potential diagnostic and prognostic biomarker in cancer. Crystal structures of globular adiponectin have been resolved with three calcium‐binding sites on the top of its central tunnel. However, the calcium‐binding property of adiponectin remains elusive. Mouse globular adiponectin was cloned into pET11a and expressed in Escherichia coli. The folding of adiponectin was indicated by the spread of resonances in HSQC spectrum. Luminescence resonance energy transfer was used to obtain the binding constant (K_d) of Tb³⁺ and the inhibitor constant (K_i) of Ca²⁺ for globular adiponectin. The obtained calcium‐binding affinity to adiponectin is relatively low (~2 mM), which indicates that the high concentration of adiponectin in circulating system may function as calcium storage bank and buffer the free calcium concentration. Copyright © 2012 John Wiley & Sons, Ltd.     

The Characterization of [3H] Adenosine Uptake into Rat Cerebral Cortical Synaptosomes

: Uptake of adenosine, a putative inhibitory transmitter or modulator, was investigated in rat cerebral cortical synaptosomes. The accumulation of [³H]adenosine into synaptosomes, using an adenosine concentration of 10 μ.m , was linear for 30 min at 37°C. The uptake appeared to be mediated by kinetically saturable processes with apparent K_m's of 1 μam (“high-affinity A”) and 5 μm (“high-affinity B”), both of which were partially sensitive to the presence of external sodium and calcium ions. Both uptake processes were partially inhibited by 2,4-dinitrophenol, implying the presence of active uptake and diffusional components. A study of the metabolites of adenosine taken up by the two uptake systems indicates that the major metabolites were adenosine and nucleotides. However, adenosine incorporated by the high-affinity A uptake system is more likely to form deaminated metabolites, such as hypoxanthine and inosine, indicating a possible functional difference between the two uptake processes. A detailed comparison of the inhibitory properties of certain adenosine analogues and other pharmacological agents has revealed differences between the two adenosine uptake systems. Since the glial contamination in synaptosomal preparations is well established, one of the uptake systems we observed in the present study might be of glial origin. This notion is supported by the findings that the K_m values and kinetic properties of papaverine action in the synaptosomal high-affinity A uptake system are similar to those of astrocytes reported in the literature. In conclusion, the uptake processes of synaptosomal preparations show that accumulation of adenosine into neuronal (and possibly glial) elements may play a major role in regulating the extracellular adenosine concentration. Uptake inhibitors, such as diazepam, may exert, at least in part, their pharmacological actions by interfering with the regulation of extracellular adenosine concentrations.