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Letters to the Editor
Figure 3. TRPV2 protein and function in human red blood cells. (A) Western blot of protein lysates of COS-7 cells transfected with human TRPV2 cDNA, the cDNA of green fluorescent protein (GFP) as a control, and human red blood cells (RBC) using anti-human TRPV2. (B, C) Representative traces of cytosolic Ca2+ changes, detected as Fluo-4 fluorescence (F/F0), in human RBC challenged by the application of 100 mM cannabidiol (CBD) (B) or 30 mM Δ9-tetrahydrocannabinol (Δ9- THC) (C). (D) Summary of the peak amplitudes in (B) and (C) as mean ± standard error of mean (SEM) (78 and 82 cells measured in 3 independent experiments each). (E) In- and outward currents at -80 and +80 mV in the absence and presence of Δ9-THC (black line) recorded from human RBC and plotted versus time. The corresponding current-voltage relationships (IV) of the basic current (Imin) and the peak net current in Δ9-THC (Imax net) are depicted in (F) and (G). Data are shown as mean ± SEM (number of cells indicated in brackets). (H) Confocal microscopic images of human RBC not treated (control, left) or treated with 100 mM cannabidiol (CBD, middle) or 30 mM Δ9-THC (right). (I) Bar graphs showing the percentage of discocytes (black), stomatocytes (red) and spherocytes (blue) as mean ± SEM, from three independent healthy donors, treated as in (H), with P-values calculated by one-way analysis of variance (ANOVA), followed by the Bonferroni multiple comparison. The classification was done with 3-D stacks of confocal images. (J) Hemolysis (%) of human RBC treated with the vehicle (control, black), 30 mM Δ9-THC (gray open circle) or 30 mM Δ9-THC in the presence of 100 nM CB1- and CB2-receptor antagonists AM251 and JTE907 (Δ9-THC + AM + JTE, blue) plotted versus the extracellular NaCl concentration (mM) and respective tonicity (%), with extracellular [Ca2+] kept at 76 mM as described in Figure 1F. (K) Tonicity at which 50% lysis occurred (C50), calculated by sigmoidal fitting of the individual experiments in (J). (L) Hemolysis (%) of human RBC in buffer A (149 mM NaCl, 2 mM CaCl2, 4 mM KCl, 2 mM HEPES, pH 7.4), treated with vehicle (control, black), 30 mM Δ9-THC (gray open circle), 2 mM TRAM-34 (green) or 30 mM Δ9-THC plus TRAM-34 (red) for 30 min, after 26-fold dilution in buffer B (0-149 mM NaCl, 2 mM CaCl2, 4 mM KCl, 2 mM HEPES, pH 7.4); extra- cellular [Ca2+] was kept at 2 mM. (M) Tonicity at which 50% lysis occurred (C50), calculated by sigmoidal fitting of the individual experiments in (L). Data in (K) and (M) are shown as means ± SEM from two independent experiments performed in triplicate with the P-value calculated by one-way ANOVA, followed by the Bonferroni multiple comparison. (N) Working model for how TRPV2, activated by Δ9-THC, modulates the TRAM-34 sensitive KCa3.1 activity in a human RBC. Note that TRPC6 was not detectable in human RBC.
or 2 mM extracellular Ca2+ (Figure 3L, M). The data indi- cate that TRPV2, like Piezo1 and, maybe TRPC6, enables an influx of cations including Ca2+. The increase of intra- cellular Ca2+ by TRPV2 activates KCa3.1 which allows K+ efflux, resulting in the shift of the hemolysis curve. This shift does not occur in the presence of the KCa3.1 antago- nist TRAM-34 (Figure 3L-N).
Stabilization of the RBC membrane against hypotonic hemolysis by Δ9-THC and CBD has been described10 and it has been shown that in the presence of Δ9-THC at con- centrations of >15 mM almost all RBC assume a stomato- cyte-like concave shape.11 Some of those results were attributed to interactions between the hydrophobic, natu- rally occurring cannabinoids and the membrane lipids of the RBC. However, membrane partitioning experiments, electron spin resonance spectrometry and experiments with artificial liposomes of different compositions which were tested for the release of trapped markers in the pres- ence of Δ9-THC, suggested additional mechanisms.12-14
The data described in our study point to TRPV2 being a specific molecular target for Δ9-THC and CBD in RBC. Activation of the TRPV2 channel by the compounds pres- ent in the Cannabis sativa plant makes RBC more resistant to lysis in response to hypotonic solutions. Whether our data explain why hemp products have been used in folk medicine to treat malaria since ancient times15 needs to be shown by further studies.
Anouar Belkacemi,1 Claudia Fecher-Trost,1 René Tinschert,1 Daniel Flormann,2 Mahsa Malihpour,1 Christian Wagner,2,3 Markus R. Meyer,1 Andreas Beck1 and Veit Flockerzi1
1Experimentelle und Klinische Pharmakologie und Toxikologie und Präklinisches Zentrum für Molekulare Signalverarbeitung (PZMS), Universität des Saarlandes, Homburg, Germany; 2Experimentalphysik, Universität des Saarlandes, Saarbrücken, Germany and 3University of Luxembourg, Physics and Materials Science Research Unit, Esch-sur-Alzette, Luxembourg
Correspondence: VEIT FLOCKERZI - veit.flockerzi@uks.eu doi:10.3324/haematol.2020.274951
Received: October 26, 2020.
Accepted: February 8, 2021.
Pre-published: February 18, 2021.
Disclosures: no conflicts of interest to disclose.
Contributions: ABelkacemi, CFT, RT, DF, MM, and ABeck per- formed experiments, ABelkacemi, CFT, DF, ABeck and VF analyzed data. MRM provided reagents. ABelkacemi, ABeck and VF con- ceived and supervised the study. CFT, ABeck, CW and MRM edited the manuscript. ABelkacemi and VF wrote the manuscript.
Acknowledgments: we thank Dr. Petra Weissgerber and the Transgene Unit of the SPF animal facility (project P2 of SFB 894) of the Medical Faculty, Homburg, for taking care of the mice; Christine Wesely, Martin Simon-Thomas, Oliver Glaser and Armin Weber for excellent technical assistance; Prof. Dr. Michael J. Caterina, the Johns Hopkins University (Baltimore) and the University of California San Francisco (UCSF), for providing the Trpv2 KO mouse strain.
Funding: the study was funded by the Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Center 894 Project A3 (to ABelkacemi, VF) and A14 (to ABeck, VF) and FE 629/2-1 (to CFT).
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