Table1_Cyclodextrin derivatives decrease Transient Receptor Potential vanilloid 1 and Ankyrin 1 ion channel activation via altering the surrounding membrane microenvironment by cholesterol depletion.DOCX

Transient Receptor Potential Vanilloid 1 (TRPV1) and Ankyrin 1 (TRPA1) are nonselective cation channels expressed in primary sensory neurons and several other non-neuronal structures such as immune cells, keratinocytes, and vascular smooth muscle cells. They play important roles in nociception, pain processing and their chanellopathies are associated with the development of several pathological conditions. They are located in cholesterol- and sphingolipid-rich membrane lipid raft regions serving as platforms to modulate their activations. We demonstrated earlier that disruption of these lipid rafts leads to decreased TRP channel activation and exerts analgesic effects. Cyclodextrins are macrocyclic molecules able to form host-guest complexes with cholesterol and deplete it from the membrane lipid rafts. The aim of this study was to investigate 8 structurally different (methylated and non-methylated) CD derivatives on cell viability, mitochondrial membrane potential, membrane composition and activation abilities of the TRPV1 and TRPA1 channels. We showed that non-methylated derivatives have preferable safety profiles compared to methylated ones. Furthermore, methylated derivatives reduced mitochondrial membrane potential. However, all investigated derivatives influence the ordered cell membrane structure depleting membrane cholesterol and inhibit the TRPV1 agonist capsaicin- and the TRPA1 agonist allyl isothiocyanate-induced Ca2+−influx. This mechanism of action might provide novel perspectives for the development of peripherally acting analgesics via indirectly decreasing the generation and transmission of nociceptive signals.</p

Confocal images of undifferentiated Caco-2 cells.

<p>Cells were treated with the solution of 0.05-RAMEB and 5 mM RAMEB (FRR). FITC-RAMEB (green) is localized in small vesicles (white arrows) under the CellMask labeled cell membrane (red) or in larger vesicles near the DAPI stained cell nucleus (light blue). Aggregated particles of FITC-RAMEB can be also seen outside the cell membrane (A). Nine consecutive confocal sections of a cluster of cells were recorded (B). Each section is one and half micrometer thick. FITC-RAMEB (green) is located in cytoplasmic granules. The granular bright particles are observed inside the cell membrane and outside of the cell nuclei.</p

Transepithelial electric resistance (TEER) of Caco-2 monolayers before and after 120 minutes permeability experiments.

<p>Cell layers were treated with 0.05-RAMEB (FR) alone or in the presence of 5 mM RAMEB (FRR). Untreated monolayers were kept in HBSS. Values are expressed as means ± SD, n = 9 for FR, n = 6 for FRR treatment and n = 6 for untreated samples. There were no significant differences between TEER values before and after the treatments (p>0.05) and among the groups (p>0.05).</p

Cyclodextrin enters differentiated Caco-2 cells of a high resistance Caco-2 cell layer.

<p>A confluent layer was treated with 0.05-RAMEB and imaged by confocal microscope in twelve two-micrometer thick sections, of which six are demonstrated in the panels on the left side (A–F). On the right, one middle section of the image shows the top view of the cell layer (H) at the level indicated by blue lines in side sections. Upper (G) and right (I) side images are appropriate sections from perpendicular directions at green and at red lines. Crosshair (green and red lines at the long white arrow) set to an intense FITC-RAMEB (green) granule (indicated by arrows), which is located at the nuclear (light blue DAPI stain) level of cells. Several smaller FITC-RAMEB green granules can be seen below the cell membrane marked by CellMask (dark blue).</p

Kinetics of FITC-RAMEB uptake in Caco-2 monolayers.

<p>Cell monolayers were treated with 0.05-RAMEB (FR) alone or in combination with 5 mM RAMEB (FRR) and in different time points the incubation was stopped. After washing cells were fixed with 3% paraformaldehyde solution and the accumulated FITC-RAMEB was determined by microplate reader. Cell nuclei were labeled with DAPI and fluorescence intensities of FITC-RAMEB were normalized for DAPI fluorescence intensities. Values are expressed as means ± SD, n = 3 for FR and FRR treatments.</p

Release of FITC-RAMEB from Caco-2 monolayers.

<p>Treatment with 0.5-RAMEB was carried out on Transwell® inserts for 120 minutes, then the monolayers were washed and FITC-RAMEB release was followed in the apical and basolateral chambers during the next 120 minutes. FITC fluorescence intensities were determined in the control group of the samples after the first 120 minutes and considered as 100% accumulation. The rate of release in the second group was compared to this value. Values are means ± SD, n = 4.</p