Differentiation dependent expression of TRPA1 and TRPM8 channels in IMR-32 human neuroblastoma cells.
TRPA1 and TRPM8 are transient receptor potential (TRP) channels involved in sensory perception. TRPA1 is a non-selective calcium permeable channel activated by irritants and proalgesic agents. TRPM8 reacts to chemical cooling agents such as menthol. The human neuroblastoma cell line IMR-32 undergoes a remarkable differentiation in response to treatment with 5-bromo-2-deoxyuridine. The cells acquire a neuronal morphology with increased expression of N-type voltage gated calcium channels and neurotransmitters. Here we show using RT-PCR, that mRNA for TRPA1 and TRPM8 are strongly upregulated in differentiating IMR-32 cells. Using whole cell patch clamp recordings, we demonstrate that activators of these channels, wasabi, allyl-isothiocyanate (AITC) and menthol activate membrane currents in differentiated cells. Calcium imaging experiments demonstrated that AITC mediated elevation of intracellular calcium levels were attenuated by ruthenium red, spermine, and HC-030031 as well as by siRNA directed against the channel. This indicates that the detected mRNA level correlate with the presence of functional channels of both types in the membrane of differentiated cells. Although the differentiated IMR-32 cells responded to cooling many of the cells showing this response did not respond to TRPA1/TRPM8 channel activators (60% and 90% for AITC and menthol respectively). Conversely many of the cells responding to these activators did not respond to cooling (30%). This suggests that these channels have also other functions than cold perception in these cells. Furthermore, our results suggest that IMR-32 cells have sensory characteristics and can be used to study native TRPA1 and TRPM8 channel function as well as developmental expression.
Louhivuori
Lauri M
LM
Biomedicum Helsinki, Institute of Biomedicine/Physiology, University of Helsinki, Helsinki, Finland.
Bart
Genevieve
G
Larsson
Kim P
KP
Louhivuori
Verna
V
Näsman
Johnny
J
Nordström
Tommy
T
Koivisto
Ari-Pekka
AP
Akerman
Karl E O
KE
D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D017879:Isothiocyanates / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000074025:TRPA1 Cation Channel; D050053:TRPM Cation Channels / Q000235:genetics / Q000378:metabolism*; D050051:Transient Receptor Potential Channels / Q000235:genetics / Q000378:metabolism*
D015220:Calcium Channels; D017879:Isothiocyanates; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050053:TRPM Cation Channels; C425966:TRPM8 protein, human; D050051:Transient Receptor Potential Channels; C041942:2,3,4-tri-O-acetylarabinopyranosyl isothiocyanate; D002118:Calcium