scholarly journals Enantiomeric selectivity of adenosine transport systems in mouse erythrocytes and L1210 cells

1989 ◽  
Vol 263 (3) ◽  
pp. 957-960 ◽  
Author(s):  
W P Gati ◽  
L Dagnino ◽  
A R P Paterson
Keyword(s):  
Cell Types ◽  
Mirror Image ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Simple Diffusion ◽  
L1210 Cells ◽  
Marked Preference ◽  
Adenosine Transport ◽  
Mouse Leukaemia ◽  
Enantiomeric Selectivity

In mediating the entry of adenosine into mouse erythrocytes and mouse leukaemia L1210 cells, nucleoside transport systems were stereoselective, showing a marked preference for the D-enantiomer of adenosine (D-Ado). Inward zero-trans fluxes of the mirror-image isomer, L-adenosine (L-Ado), in those cells were slow relative to those of D-Ado. Contributing to L-Ado fluxes in both cell types were (i) a transporter-mediated process of high nitrobenzylthioinosine-sensitivity and (ii) simple diffusion.

Inhibition of glucose uptake in murine cardiomyocyte cell line HL-1 by cardioprotective drugs dilazep and dipyridamole

2004 ◽  
Vol 286 (2) ◽  
pp. H627-H632 ◽  
Author(s):  
Irina Shuralyova ◽  
Panteha Tajmir ◽  
Philip J. Bilan ◽  
Gary Sweeney ◽  
Imogen R. Coe
Keyword(s):  
Glucose Uptake ◽  
Cell Line ◽  
Glucose Transport ◽  
Cardiovascular Health ◽  
Cell Types ◽  
Nucleoside Transport ◽  
Dependent Inhibition ◽  
Nucleoside Transport Inhibitors ◽  
Adenosine Transport ◽  
High Doses

Inhibition of adenosine reuptake by nucleoside transport inhibitors, such as dipyridamole and dilazep, is proposed to increase extracellular levels of adenosine and thereby potentiate adenosine receptor-dependent pathways that promote cardiovascular health. Thus adenosine can act as a paracrine and/or autocrine hormone, which has been shown to regulate glucose uptake in some cell types. However, the role of adenosine in modulating glucose transport in cardiomyocytes is not clear. Therefore, we investigated whether exogenously applied adenosine or inhibition of adenosine transport by S-(4-nitrobenzyl)-6-thioinosine (NBTI), dipyridamole, or dilazep modulated basal and insulin-stimulated glucose uptake in the murine cardiomyocyte cell line HL-1. HL-1 cell lysates were subjected to SDS-PAGE and immunoblotting to determine which GLUT isoforms are present. Glucose uptake was measured in the presence of dipyridamole (3–300 μM), dilazep (1–100 μM), NBTI (10–500 nM), and adenosine (50–250 μM) or the nonmetabolizable adenosine analog 2-chloro-adenosine (250 μM). Our results demonstrated that HL-1 cells possess GLUT1 and GLUT4, the isoforms typically present in cardiomyocytes. We found no evidence for adenosine-dependent regulation of basal or insulin-stimulated glucose transport in HL-1 cardiomyocytes. However, we did observe a dose-dependent inhibition of glucose transport by dipyridamole (basal, IC50= 12.2 μM, insulin stimulated, IC50= 13.09 μM) and dilazep (basal, IC50= 5.7 μM, insulin stimulated, IC50= 19 μM) but not NBTI. Thus our data suggest that dipyridamole and dilazep, which are widely used to specifically inhibit nucleoside transport, have a broader spectrum of transport inhibition than previously described. Moreover, these data may explain previous observations, in which dipyridamole was noted to be proischemic at high doses.

Characterization and regulation of adenosine transport in T84 intestinal epithelial cells

1998 ◽  
Vol 274 (2) ◽  
pp. G261-G269 ◽  
Author(s):  
Edward C. Mun ◽  
Kevin J. Tally ◽  
Jeffrey B. Matthews
Keyword(s):  
Epithelial Cells ◽  
Binding Sites ◽  
Intestinal Epithelial Cells ◽  
Metabolic Inhibition ◽  
Nucleoside Transport ◽  
Facilitated Diffusion ◽  
Intestinal Epithelial ◽  
Binding Studies ◽  
Simple Diffusion ◽  
Adenosine Transport

Adenosine release from mucosal sources during inflammation and ischemia activates intestinal epithelial Cl−secretion. Previous data suggest that A2b receptor-mediated Cl− secretory responses may be dampened by epithelial cell nucleoside scavenging. The present study utilizes isotopic flux analysis and nucleoside analog binding assays to directly characterize the nucleoside transport system of cultured T84 human intestinal epithelial cells and to explore whether adenosine transport is regulated by secretory agonists, metabolic inhibition, or phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na+-independent, nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with low affinity but high capacity for adenosine. NBTI binding studies indicated a single population of high-affinity binding sites basolaterally. Neither forskolin, 5′-( N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both adenosine transport and the number of specific NBTI binding sites, suggesting that transporter number may be decreased through activation of protein kinase C. This basolateral facilitated adenosine transporter may serve a conventional function in nucleoside salvage and a novel function as a regulator of adenosine-dependent Cl− secretory responses and hence diarrheal disorders.

scholarly journals Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex

1989 ◽  
Vol 264 (1) ◽  
pp. 223-231 ◽  
Author(s):  
T C Williams ◽  
A J Doherty ◽  
D A Griffith ◽  
S M Jarvis
Keyword(s):  
Brush Border ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Facilitated Diffusion ◽  
Basolateral Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Outer Cortex ◽  
Overshoot Phenomenon

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.

Pharmacological Studies with Platelets and Cultured Vascular Cells

1979 ◽  
Author(s):  
J.L. Gordon ◽  
J.D. Pearson
Keyword(s):  
Platelet Aggregation ◽  
Cell Types ◽  
Luciferase Assay ◽  
Induce Platelet Aggregation ◽  
Aortic Endothelial Cells ◽  
Aortic Smooth Muscle ◽  
High Affinity ◽  
Adenosine Uptake ◽  
Adenosine Transport ◽  
Porcine Aortic Endothelial Cells

Suspensions of cultured porcine aortic smooth muscle cells (SMC) induce platelet aggregation, which is decreased by pretreating SMC with collagenase. In contrast, porcine aortic endothelial cells (EC) usually inhibit platelet aggregation. Radioimmunoassay of the culture medium demonstrates that both cell types secrete prostaglandins (PG); EC mainly PGI2 and SMC mostly PGE2. EC PG production is stimulated by cell-free plasma and inhibited by aspirin (IC50 ≃ 100μM) or sulphinpyrazone (IC50 = 50-100μM). Aspirin pretreated EC can induce platelet aggregation directly which is inhibited by 2-azido-AMP, an ADP analogue. [3H]-adenine and adenosine are incorporated into EC or SMC nucleotides, mainly ATP. EC and SMC possess a high affinity (Km < 1μM) system for adenine uptake. In contrast only EC have a high affinity (Km = 3μM) adenosine transport system, potently blocked by dipyridamole (IC50 < 0. 1μM at 10μM adenosine); EC and SMC have a lower affinity adenosine uptake site (Km = 250μM for EC, > 1mM for SMC) preferentially blocked by adenine (IC50 > 10μM at 10μM adenosine for both cell types) but not by dipyridamole. Treating EC or SMC with trypsin or thrombin stimulates the release of significant amounts of the cells’ ATP (measured by prelabelling or by luciferase assay) without detectable LDH release or vital dye uptake. This ATP is rapidly (t½ < 5 min) converted to ADP by an ectoATPase (Km ≃ 450μM for EC, 125μM for SMC), and subsequently to AMP and adenosine by other ectonucleotidases.

scholarly journals Structural, Molecular, and Functional Alterations of the Blood-Brain Barrier during Epileptogenesis and Epilepsy: A Cause, Consequence, or Both?

2020 ◽  
Vol 21 (2) ◽  
pp. 591 ◽  
Author(s):  
Wolfgang Löscher ◽  
Alon Friedman
Keyword(s):  
Endothelial Cells ◽  
Blood Brain Barrier ◽  
Defense Mechanism ◽  
Extracellular Fluid ◽  
Cell Types ◽  
Therapeutic Interventions ◽  
Brain Barrier ◽  
Transport Systems ◽  
Blood Brain ◽  
The Brain

The blood-brain barrier (BBB) is a dynamic, highly selective barrier primarily formed by endothelial cells connected by tight junctions that separate the circulating blood from the brain extracellular fluid. The endothelial cells lining the brain microvessels are under the inductive influence of neighboring cell types, including astrocytes and pericytes. In addition to the anatomical characteristics of the BBB, various specific transport systems, enzymes and receptors regulate molecular and cellular traffic across the BBB. While the intact BBB prevents many macromolecules and immune cells from entering the brain, following epileptogenic brain insults the BBB changes its properties. Among BBB alterations, albumin extravasation and diapedesis of leucocytes from blood into brain parenchyma occur, inducing or contributing to epileptogenesis. Furthermore, seizures themselves may modulate BBB functions, permitting albumin extravasation, leading to activation of astrocytes and the innate immune system, and eventually modifications of neuronal networks. BBB alterations following seizures are not necessarily associated with enhanced drug penetration into the brain. Increased expression of multidrug efflux transporters such as P-glycoprotein likely act as a ‘second line defense’ mechanism to protect the brain from toxins. A better understanding of the complex alterations in BBB structure and function following seizures and in epilepsy may lead to novel therapeutic interventions allowing the prevention and treatment of epilepsy as well as other detrimental neuro-psychiatric sequelae of brain injury.

P-glycoprotein-mediated secretion of a fluorescent cyclosporin analogue by teleost renal proximal tubules

1995 ◽  
Vol 268 (1) ◽  
pp. F46-F52 ◽  
Author(s):  
U. Schramm ◽  
G. Fricker ◽  
R. Wenger ◽  
D. S. Miller
Keyword(s):  
Steady State ◽  
Fluorescence Intensity ◽  
Fundulus Heteroclitus ◽  
Organic Anion ◽  
Proximal Tubules ◽  
Metabolic Inhibitors ◽  
Epifluorescence Microscopy ◽  
Transport Systems ◽  
Simple Diffusion ◽  
P Glycoprotein

The transport of a fluorescent cyclosporin analogue was measured in killifish (Fundulus heteroclitus) proximal tubules by means of epifluorescence microscopy and digital image analysis. Renal cells rapidly accumulated the cyclosporin analogue from the medium and attained steady state within 60 min; luminal fluorescence increased over the first 60-90 min. At steady state, luminal fluorescence intensity was two to three times higher than cellular. Cellular fluorescence intensity was a linear function of medium substrate concentration and was not affected by any treatment used. In contrast, luminal fluorescence exhibited a saturable component as the medium concentration of the cyclosporin was increased. Secretion into the lumen was blocked by metabolic inhibitors, vanadate, other cyclosporins, such as cyclosporin A and cyclosporin G, and substrates for P-glycoprotein (verapamil, vinblastine, and quinine) but not by substrates for the renal organic anion or organic cation transport systems, such as p-aminohippurate or tetraethylammonium. The data are consistent with the fluorescent cyclosporin analogue entering proximal tubule cells by simple diffusion and then being pumped into the tubular lumen by P-glycoprotein.

scholarly journals Uptake and intracellular activity of AM-1155 in phagocytic cells.

1996 ◽  
Vol 40 (12) ◽  
pp. 2756-2759 ◽  
Author(s):  
T Yamamoto ◽  
H Kusajima ◽  
M Hosaka ◽  
H Fukuda ◽  
Y Oomori ◽  
...  
Keyword(s):  
Cell Types ◽  
Intracellular Concentration ◽  
Nucleoside Transport ◽  
Metabolic Inhibitors ◽  
Phagocytic Cells ◽  
Penetration Process ◽  
Extracellular Concentration ◽  
Intracellular Activity ◽  
Human Polymorphonuclear Leukocytes

The uptake and intracellular activity of AM-1155 in murine J774.1 macrophages and human polymorphonuclear leukocytes were investigated. AM-1155 penetrated phagocytic cells rapidly and reversibly, although the penetration process was not affected by metabolic inhibitors such as sodium fluoride, cyanide m-chlorophenylhydrazone, or ouabain or by nucleoside transport system inhibitors such as adenosine. The intracellular concentration-to-extracellular concentration ratio of AM-1155 in both cell types of phagocytes ranged from 5 to 7. These ratios were almost equal to those for sparfloxacin. The intracellular activity of AM-1155 in J774.1 macrophages, examined with Staphylococcus aureus 209P as a test bacterium, was dependent on the extracellular concentration. AM-1155 at a concentration of 1 microgram/ml reduced the number of viable cells of S. aureus ingested by more than 90%. The intracellular activity of AM-1155 was more potent than those of sparfloxacin, ofloxacin, ciprofloxacin, flomoxef, and erythromycin. These results suggest that the potent intracellular activity of AM-1155 might mainly be due to the high intracellular concentration and its potent in vitro activity.

Rat lung type II cell and lamellar body: elemental composition in situ

1988 ◽  
Vol 254 (5) ◽  
pp. C614-C620 ◽  
Author(s):  
R. G. Eckenhoff ◽  
A. P. Somlyo
Keyword(s):  
Elemental Composition ◽  
Lamellar Body ◽  
Cell Types ◽  
Transport Systems ◽  
Alveolar Type ◽  
Type Ii ◽  
Freeze Dried ◽  
Type Ii Cell ◽  
The Difference

We determined the in situ elemental composition of alveolar type II cells (ATII) and lamellar bodies (LB) with electronprobe microanalysis (EPMA) of freeze-dried unstained cryosections (100-200 nm) obtained from lungs frozen in anesthetized rats. Twenty-nine ATII from seven rats were subjected to EPMA. Cytoplasmic (Cyto) composition was the following (in mmol/kg dry wt, mean +/- SE, n = 30): 136 +/- 14.1 Na, 60 +/- 2.8 Mg, 549 +/- 34.8 P, 278 +/- 10.5 S, 158 +/- 7.3 Cl, 525 +/- 26.4 K, and 6.6 +/- 0.9 Ca. LB composition was the following (n = 66): 44 +/- 4.0 Na, 7.9 +/- 0.8 Mg, 1,060 +/- 25.0 P, 79 +/- 4.8 S, 64 +/- 3.6 Cl, 114 +/- 4.1 K, and 30 +/- 0.9 Ca. P and S concentrations were consistent with previous biochemical determinations of phospholipid and protein content of isolated LBs. LBs contain significantly more Ca and less Mg than Cyto. Ca correlated significantly with LB P but not S concentration, and the reported low Ca binding affinity of similar phospholipid mixtures implies a high LB free Ca concentration. Ca was significantly higher in apical and exocytotic LBs compared with those in the perinuclear region. Differences between LB and Cyto monovalent ion concentrations are not entirely due to the difference in hydration revealed by significantly lower K-Cl ratios in LBs. The relative excess of Cl and Ca in LB suggests that these ions may be distributed by active transport systems known to be present in the Golgi apparatus and in Golgi-derived organelles of other cell types.

scholarly journals Stable intracellular acidification upon polyamine depletion induced by α-difluoromethylornithine or N1,N12-bis(ethyl)spermine in L1210 leukaemia cells

1995 ◽  
Vol 312 (3) ◽  
pp. 749-756 ◽  
Author(s):  
R Poulin ◽  
A E Pegg
Keyword(s):  
Steady State ◽  
Acid Stress ◽  
Weak Acid ◽  
Transport Processes ◽  
Set Point ◽  
L1210 Cells ◽  
Acid Load ◽  
Mouse Leukaemia ◽  
A Minor ◽  
Steady State Control

Polyamines play major roles in ionic and osmotic regulation, but their exact involvement in specific ion transport processes is poorly defined. Treatment of L1210 mouse leukaemia cells with either 5 mM alpha-difluoromethylornithine (DFMO), a suicide substrate of ornithine decarboxylase, or 25 microM N1,N12-bis(ethyl)spermine (BE-3-4-3), a dysfunctional polyamine analogue, caused a stable decreased in intracellular pH (pHi) by 0.1-0.4 unit from steady-state control values between 7.4 and 7.6, as measured either by partition of a weak acid or with a fluorescent pH-sensitive probe. This effect was not related to cell growth status or differences in metabolic acid generation, and was observed in either the presence or absence of HCO3-. Exogenous spermidine (10-25 microM) or putrescine (25-50 microM) fully reversed DFMO- or BE-3-4-3-induced acidification within 2 and 8 h respectively. Recovery of pHi in L1210 cells after a nigericin- or NH4(+)-mediated acid load in HCO3(-)-free buffers was mediated by Na+/H+ antiporter activity, in addition to a minor Na(+)-independent and amiloride-insensitive pathway. Decreased steady-state pHi was maintained in polyamine-depleted L1210 cells after recovery from acid stress. Moreover, the pHi-dependence of the rate of Na(+)-dependent H+ extrusion after an acid stress was altered by DFMO and BE-3-4-3, resulting in a set-point which was lower by 0.25-0.30 pH unit in polyamine-depleted cells. On the other hand, neither the rate nor the magnitude of Na+/H(+)-exchanger-mediated alkalinization induced by hypertonic shock was decreased by polyamine depletion. Thus polyamine depletion induces a persistent defect in pHi homeostasis which is due, at least in part, to a stable decrease in the pHi set-point of the Na+/H+ exchanger.

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