Figure 1.
TRPV1 activation as biosensor for the study of anandamide (AEA) cellular uptake.
(A) AEA-induced elevation of [Ca2+]i in intact HEK-293 cells over-expressing the human recombinant TRPV1 receptor was significantly reduced by I-RTX (a selective TRPV1 antagonist) as well by a series of compounds that selectively affect membrane transport processes such as bovine serum albumin) BSA (which sequesters AEA from the extracellular medium); OMDM-1 (a specific inhibitor of AEA cellular uptake with no effect on AEA enzymatic hydrolysis); MβCD (which interferes with AEA cellular uptake by inhibiting the formation of membrane lipid rafts) and oleic acid (OA) and a specific fatty acid binding protein 4-blocker (FABP4 inh) (two inhibitors of the intracellular trafficking of AEA via fatty acid binding proteins). ***p<0.001 compared to AEA+ vehicle (n = 3–5). (B) Capsaicin (CPS)-induced elevation of [Ca2+]i in intact HEK-293 cells over-expressing human TRPV1 receptors was significantly antagonized by I-RTX, but was insensitive to all other compounds that selectively affect membrane transport processes (BSA, OMDM1, MβCD, OA, FABP4 inhib). ***p<0.001 compared to CPS+ vehicle (n = 3–5). Data are expressed as percent of the maximal response observed with either AEA (1 µM, 72.0±1% of the effect of ionomycin, 4 µM, see also Fig. 6A) or CPS (76±8% of ionomycin, 4 µM). Each bar indicates means ± sem of at least 3 independent experiments.
Figure 2.
Use of digital holographic (DH) quantitative phase microscopy for the study of AEA uptake.
Representative phase images for the effects of anandamide (AEA, 1 µM) in non-transfected HEK-293 cells, and of AEA (1 µM), capsaicin (CPS, 0.5 µM) and PCL-NP-AEA (1 µM) on TRPV1-HEK-293 cell surface topology as observed with digital holographic (DH) quantitative phase microscopy. Apparent membrane extrusions, observed in a TRPV1- and extracellular Ca2+-dependent way (see Fig. 3), are shown at ∼200 sec after addition of the drugs (lower panels), whereas the cell surface topology of the same cells is also shown at time 0, prior to addition of the substances (upper panels). See also Videos S1 and S3.
Figure 3.
Effects of various drugs or combinations thereof on TRPV1-HEK-293 cell surface topology as observed with digital holographic (DH) quantitative phase microscopy.
Bars indicate the mean elapsed time (sec) from the addition of drugs required to observe the first apparent membrane extrusions, with 1500 s as maximum observed elapsed time. (A) Effect of anandamide (AEA, 1 µM), per se or with the selective TRPV1 antagonist BCTC (0.1 µM), or in the absence of extracellular Ca2+ in the medium; of capsaicin (CPS, 0.5 µM) per se or with the selective TRPV1 antagonist N-(4-Tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine -1(2H)-carbox-amide (BCTC, 0.1. µM), or in the absence of extracellular Ca2+ in the medium; the calcium ionophore, ionomycin (4 µM), with or without extracellular Ca2+ in the medium; and the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (Thaps, 1 µM). The lack of effect up to 1500 s of AEA and CPS in wild-type (WT) HEK-293 cells is also shown. (B) Effect of AEA (1 µM), per se or in the presence of the AEA uptake inhibitor OMDM1 (5 µM), or of fetal bovine serum (10%) in the medium; of PCL-NP-AEA (1 µM), per se or in the presence of the AEA uptake inhibitor OMDM1 (5 µM), or of fetal bovine serum (10%) in the medium; of capsaicin (CPS, 0.5 µM) per se or in the presence of the AEA uptake inhibitor OMDM1 (5 µM), or of fetal bovine serum (10%) in the medium. Each bar indicates means ± sem of 3 independent experiments. In (B) the effect of OMDM1 plus AEA on mean elapsed time was significantly different (P<0.01) from that of AEA alone. See also Videos S2 and S4.
Figure 4.
Characterization of anandamide release from PCL-NPs and evidence that PCL-NP rapidly enter HEK-293 cells over-expressing the human recombinant TRPV1 receptor.
(A) Anandamide release from PCL-NP-AEA at 37°C and at a 99.9% dilution in distilled water (0.5 µl stock suspension in 500 µl water). AEA was measured by HPLC. In a separate experiment, ∼80% of AEA was retained by PCL-NP-AEA incubated for 1 min with the assay buffer used for the experiments of Figs. 1,3. Each bar indicates means ± sem of at least 3 measurements. (B, C, D) Representative images of differential interference contrast (DIC) and fluorescence, and merged images, of cells treated for 1 min at 37°C with Rhodamine-123-loaded nanoparticles (PCL-NP-Rho, 1 µM) (B) or “free” Rhodamine-123 (Rho, 1 µM) (C). Representative images are also shown for cells treated with “empty” PCL-NP's (D).
Figure 5.
Representative kinetic experiments for the effects on [Ca2+]i in intact HEK-293 cells over-expressing the human recombinant TRPV1 receptor.
Effect of AEA (A) PCL-NP-AEA (B) and “empty” PCL-NP's (C). The effect of AEA (1 µM) in non-transfected HEK-293 cells is shown in (D).
Figure 6.
Effect of specific AEA cellular uptake inhibitors on PCL-NP-AEA mediated cellular uptake and [Ca2+]i elevation.
(A) PCL-NP-AEA (▪) exhibited similar potency and efficacy as “free” AEA (□) on [Ca2+]i in intact HEK-293 cells over-expressing the human recombinant TRPV1 receptor. The effect is expressed as percent of the effect on Ca2+ by ionomycin, 4 µM, and, in both cases, was dose-dependent and TRPV1-mediated because it was abolished by I-RTX (• and ○). (B) PCL-NP-AEA-induced elevation of [Ca2+]i in intact TRPV1-HEK-293 cells over-expressing the human TRPV1 receptor was significantly less sensitive to pharmacological tools previously used to specifically interfere with AEA cellular uptake or intracellular trafficking (BSA, OMDM1, MβCD, OA, FABP4 inhib). *p<0.05; ***p<0.001 compared to AEA-PCL-NP + vehicle (n = 3–5); #, ##, ### p<0.05, p<0.01 and p<0.005 compared with AEA+corresponding inhibitor (see Fig. 1A). (C) PCL-NP-AEA effect (full bars) on TRPV1-mediated [Ca2+]i was significantly less sensitive to concentrations of the selective AEA uptake inhibitors, OMDM-1 and AM1172, which instead strongly affected the effect of “free” AEA (dotted bars). *p<0.05; ***p<0.001 compared to the corresponding effect of the inhibitor observed using PCL-NP-AEA; # p<0.05 compared with PCL-NP-AEA + vehicle. In (B) data are expressed as percent of the maximal response observed with PCL-NP-AEA (1 µM) + vehicle (see panel (A)). In (C) data are expressed as percent of the maximal response observed with either PCL-NP-AEA (1 µM) + vehicle or AEA (1 µM) + vehicle (see panel (A)). Each bar indicates means ± sem of at least 3 independent experiments.
Figure 7.
Effect of the fatty acid binding protein-4 (FABP4) inhibitor [(2′-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)(1,1′-biphenyl)-3-yl)oxy]-acetic acid, 50 µM) on the cellular uptake of [14C]-anandamide in a “conventional “uptake assay carried out in TRPV1- HEK-293 cells.
The amount (cpm) of [14C]-AEA taken up by cells at 37°C subtracted of that taken up at 4° after a 90 sec incubation with the compound is shown on the y axis. Each bar indicates means ± sem of 3 independent experiments. The effect of the FABP4 inhibitor was significantly different (P<0.01) from that of vehicle (0.1% methanol).
Figure 8.
Examples of living cell images, obtained by a digital holographic microscopy (pseudo 3D rendering).
(A) In the image, each pixel grey-value is a measure of the light phase amount in the corresponding point of the sample; (B) codified-colours rendering allows for improved evaluation of phase values.