Transient receptor potential channel vanilloid type 2 in red cells of cannabis consumer
Authors: Daniel Flormann, Min Qiao, Nicoletta Murciano, Giulia Iacono, Alexis Darras, Sebastian Hof, Steffen M. Recktenwald, Maria Giustina Rotordam, Nadine Becker, Jürgen Geisel, Christian Wagner, Marieke von Lindern, Emile van den Akker, Lars Kaestner
Journal: American Journal of Hematology (2022)

The abundance of the transient receptor potential channel vanilloid type 2 (TRPV2) in red blood cells (RBCs) was recently discovered,1 inciting immediate discussion on its potential physiological importance.2 TRPV2 is a reportedly mechanosensitive nonselective cation channel with numerous properties similar to those of Piezo1.2 It has been proposed that TRPV2 may cause the induction of storage lesions in RBCs.2, 3 Furthermore, TRPV2 channels can be activated by cannabidiol or Δ9-tetrahydrocannabinol (Δ9-THC) and vice versa; thus, the changes induced in RBC by the application of Δ9-THC can be attributed to TRPV2 channel activity, which was first reported in RBCs of TRPV2-KO mice.1 Figure 1A,B shows confocal images of the RBCs of a 29-year-old man who participated in an initial study.1 Under control conditions (HEPES-buffered solution), the RBCs were predominantly discocytes, as expected for healthy donors (Figure 1A). The addition of 30 μM Δ9-THC led to a large fraction of super-hydrated spherocytes (Figure 1B). Another representative healthy donor, under control conditions and with Δ9-THC stimulation, showed a significantly milder response, as depicted in Figure 1C,D, respectively. This was substantiated by the quantitative comparison of healthy controls (Figure 1E) with the proband under investigation (Figure 1F). The medical history of the donor revealed no significant findings other than regular cannabis consumption (smoking 2–3 g marijuana daily for several months). Further studies were conducted in the proband and in two additional marijuana consumers with very similar smoking habits. Blood was collected to further investigate increased RBC sensitivity to Δ9-THC stimulation. First, we confirmed the initial result of the super-hydrated RBCs by comparing confocal images of the three male marijuana smokers (MS) with three age-matched male nonsmokers (NS) (35.7 ± 1.2 vs. 36.3 ± 6.4 years; p = .9), as outlined in Figure S1. Next, we performed a complete RBC count, in the central clinical laboratory of Saarland University Hospital, to compare MS to NS. Based on the confocal recordings, we expected, but did not find, differences in the mean cellular volume (MCV) upon application of 30 μM Δ9-THC for all probands (Figure S2A). However, we found a slight MCV increase in the MS group compared with the NS group (Figure 1G), although, with a sample size of N = 3, this increase did not reach the p < .05 significance level. In contrast, the RBC distribution width (RDW) was significantly different between the MS and NS groups (Figure 1H). To explain the discrepancy between the confocal measurements and the RBC count, we identified experimental timing as a confounding parameter. While the cell swelling is a transient process that is undetectable after 45 min of acute Δ9-THC stimulation (Figure S2B), this time limit passed within the routine handling at the central laboratory. Furthermore, clinical cell counters are optimized devices that provide accurate and reproducible measures, that is, the methodological contribution to the variations in the measurements was minimal and we are able to detect more subtle changes than with other methods for the steady-state conditions (Figure 1G,H). Additionally, the RBC Ca2+ response was investigated in terms of Fluo-4 fluorescence intensity with acute 30 μM Δ9-THC stimulation using flow cytometry (LSRFortessa, BD Biosciences, Franklin Lakes, NJ), and it showed an increase in the high Ca2+ RBC fraction upon Δ9-THC stimulation (Figure 1I,J). Whether the heightened sensitivity of MS versus NS RBCs was caused by hypersensitizing TRPV2 or by modulation of its expression level during erythropoiesis was addressed by comparing MS and NS RBCs using functional patch-clamp measurements and western blotting. Figure 1K depicts a representative example of whole-cell RBCs currents of an MS that were measured in an automated patch-clamp robot (SynchroPatch 384, Nanion Technologies, Munich, Germany), both with and without 30 μM Δ9-THC application. Figure S3 shows the current changes of all responding cells, which were plotted separately for each proband. The average differential current (difference before and after Δ9-THC application) of the MS versus NS is plotted in Figure 1L. These numbers present the recordings at a clamped potential (−100 mV) and the cellular conductance under the experimental (artificial but highly reproducible) patch-clamp conditions, not reflecting the channel properties in intact RBCs at an unclamped membrane potential. This indicates a similar cellular conductance for MS and NS RBCs, and thus similarity in TRPV2 expression levels. Furthermore, the percentage of responding cells (Figure 1M) was in the same order of magnitude as the percentage of high Ca2+ cells outlined in Figure 1J. The western blots based on two different TRPV2 antibodies (Figure 1N,O and Figure S4) were consistent with the patch-clamp data, showing no significant differences in expression levels between MS and NS. Notably, unaltered TRPV2 expression levels in RBC suggest that TRPV2 expression regulation was likely not perturbed during erythropoiesis. However, when compared with discocytes, the increased cross section and the decreased deformability of overhydrated RBCs are likely to impair the capillary flow of RBCs. Therefore, we performed two sets of experiments. The RBC filterability4 decreased (insignificantly) for MS compared with NS, and 30 μM Δ9-THC significantly affected the filterability (Figure 1P). However, measurements of the flow properties of MS and NS RBCs, both with and without the additional application of 30 μM Δ9-THC, showed no detectable differences (Figure S5). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. The blood donors provided informed consent for the investigation. Panel Q of Figure 1 is modified from Egée and Kaestner, 2021 (doi: 10.3389/fphys.2021.677573), which is published under the terms of the Creative Commons Attribution License (CC BY). I (L.K.) am the copyright owner and the reference is cited. The data supporting the findings of this study are available from the corresponding author upon reasonable request. The data supporting the findings of this study are available from the corresponding author upon reasonable request. Appendix S1. Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.