Lysosomal Amino Acid Efflux Assay

Volume changes and whole cell ionic currents activated by gradual osmolarity reductions (GOR) of 1.8 mosM/min were characterized in C6 glioma cells. Cells swell less in GOR than after sudden osmolarity reductions (SOR), the extent of swelling being partly Ca2+ dependent. In nominally Ca2+-free conditions, GOR activated predominantly whole cell outward currents. Cells depolarized from the initial −79 mV to a steady state of −54 mV reached at 18% osmolarity reduction [hyposmolarity of −18% (H-18%)]. Recordings of Cl− and K+ currents showed activation at H-3% of an outwardly rectifying Cl− current, with conductance of 1.6 nS, sensitive to niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid, followed at H-18% by an outwardly rectifying K+ current with conductance of 4.1 nS, inhibited by clofilium but insensitive to the typical K+ channel blockers. With 200 nM Ca2+ in the patch pipette, whole cell currents activated at H-3% and at H-13% cells depolarized from −77 to −63 mV. A K+ current activated at H-1%, showing a rapid increase in conductance, suppressed by charybdotoxin and insensitive to clofilium. These results show the operation of two different K+ channels in response to GOR in the same cell type, activated by Ca2+ and osmolarity and with different osmolarity activation thresholds. Taurine and glutamate efflux, monitored by labeled tracers, showed delayed osmolarity thresholds of H-39 and H-33%, respectively. This observation clearly separates the Cl− and amino acid osmosensitive pathways. The delayed amino acid efflux may contribute to counteract swelling at more stringent osmolarity reductions.

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Specificity of the volume-activated amino acid efflux pathway in cultured human breast cancer cells

General Physiology and Biophysics ◽

10.4149/gpb_2011_01_45 ◽

2011 ◽

Vol 30 (1) ◽

pp. 45-51

Author(s):

D. Shennan ◽

J. Thomson

Keyword(s):

Breast Cancer ◽

Amino Acid ◽

Cancer Cells ◽

Human Breast Cancer ◽

Breast Cancer Cells ◽

Human Breast Cancer Cells ◽

Human Breast ◽

Amino Acid Efflux ◽

Efflux Pathway

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Carotid Artery Injection Technique: Bounds for Bolus Mixing by Plasma and by Brain

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.1985.86 ◽

1985 ◽

Vol 5 (4) ◽

pp. 576-583 ◽

Cited By ~ 51

Author(s):

William M. Pardridge ◽

Elliot M. Landaw ◽

Leonard P. Miller ◽

Leon D. Braun ◽

William H. Oldendorf

Keyword(s):

Amino Acid ◽

Carotid Artery ◽

Kinetic Parameters ◽

Upper Bound ◽

Rat Plasma ◽

Transport Processes ◽

Injection Technique ◽

Conscious Rat ◽

Mixing Effects ◽

Amino Acid Efflux

Estimation of Michaelis-Menten kinetic parameters ( Km, Vmax) of blood–brain barrier (BBB) transport processes with the carotid artery single injection technique assumes that mixing of the bolus with unlabeled substrate either from (a) circulating plasma or (b) amino acid efflux from brain, is minimal. The maximum extent to which the bolus could mix by these two sources is quantified in the present studies by measuring 14C-phenylalanine extraction in pentobarbital-anesthetized and conscious rats after the addition of 0–80% rat serum to the arterial injection solution. An upper bound (±SE) of bolus mixing due to mixing from both sources, expressed in terms of percentage of rat plasma, is 8.8 ± 1.9 and 7.0 ± 2.1% for the anesthetized and conscious rat, respectively. The estimated contribution to bolus mixing due to amino acid efflux from brain is 3.3 and 2.1% for the anesthetized and conscious rat, respectively. Based on these estimates, the upper bound for bolus mixing with circulating rat plasma is only 5.5 and 4.9%, respectively, for the anesthetized and conscious catheterized rat. Thus, any bolus mixing after rapid carotid injection is relatively small and is comparable to the mixing effects observed with the carotid artery infusion technique. Mixing effects on the order of 5% are shown to have no significant effect on the estimation of kinetic parameters of BBB nutrient transport, except for neutral and basic amino acid transport, which are characterized by very low Km values relative to the usual amino acid plasma concentrations. In the rat, a 5% mixing results in an enrichment of the bolus concentration of unlabeled amino acid that approximates the Km of the transport process, and this results in an overestimation of the absolute Km value. However, mixing effects are shown to have little, if any, impact on the estimation of the transport Vmax, KD, or apparent Km. Thus, amino acid influx rates predicted from kinetic constants obtained with the carotid injection technique are reliable, even if bolus mixing effects with the carotid injection technique are as high as 7–9%.

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