Biol. Stud. 2019: 13(2); 3–10 • DOI: https://doi.org/10.30970/sbi.1302.595

TWO ISOFORMS OF Trpm8 IN RAT VAS DEFERENS

K. Gulak, A. Kondratskyi

Abstract


Trpm8 is a nonselective Ca2+-permeable ion channel activated by temperatures below 28 °C and cooling chemical compounds such as menthol and icilin. Trpm8 is expressed in sensory neurons where it functions for temperature detection. However, Trpm8 is also expressed in various internal organs where temperature is stably higher than 36 °C, that is much higher than cool temperature needed for Trpm8 activation. This opens possible makes roles for Trpm8, such as nociception. Trpm8 mRNA is expressed in vas deferens (VD), a smooth muscle organ of male reproductive system. However, no Trpm8-mediated currents were previously registered in the myocytes. VD is located outside of testes, and it consists of smooth muscle tube covered by the epithelium. It actively contracts and transfers sperm from testes to ejaculatory ducts prior to ejaculation. Temperature in VD is stably high, thus, Trpm8 role there could be outside of cold detection. The objective of this study was to analyze Trpm8 mRNA and protein expression in rat VD, as well as splice variant analysis. Trpm8 mRNA expression in VD was confirmed with RT-PCR, and Trpm8 protein was detected by the Western-blot analysis. Additionally, we found that both mRNA and protein of shorter non-classical isoform, as well as canonical isoform of Trpm8 in VD. We isolated smooth muscle cells from VD and performed a multi-cell PCR. This technique makes possible non-myocytic mRNA detection that could be isolated from e.g. sensory neurons termini where Trpm8 is expressed at much higher levels. Interestingly, in the isolated smooth muscle cells, no canonical Trpm8 transcript was found, though the non-classical isoform was present. We propose the shorter isoform could be formed as a result of alternative splicing. This would account for a difference in Trpm8 function in VD, i.e. no Trpm8-mediated currents registered in the myocytes. A shorter isoform could have a truncated N-terminal domain, that is consistent with known human Trpm8 isoforms sM8-6 and sM8-18.

Keywords: Trpm8, cold receptor, splice variant, vas deferens, myocytes, gene expression

 


Full Text:

PDF

References


1. Artimo P., Jonnalagedda M., Arnold K., Baratin D., Csardi G., de Castro E., Duvaud S., Flegel V., Fortier A., Gasteiger E., Grosdidier A., Hernandez C., Ioannidis V., Kuznetsov D., Liechti R., Moretti S., Mostaguir K., Redaschi N., Rossier G., Xenarios I., Stockinger H. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Research, 2012: 40(W1): W597-W603.
https://doi.org/10.1093/nar/gks400
PMid:22661580 PMCid:PMC3394269
2. Behrendt H-J., Germann T., Gillen C., Hatt H., Jostock R. Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay. Br J Pharmacol, 2004; 141(4): 737-745.
https://doi.org/10.1038/sj.bjp.0705652
PMid:14757700 PMCid:PMC1574235
3. Belevych A.E., Zima A.V., Vladimirova I.A., Hirata H., Jurkiewicz A., Jurkiewicz N.H., Shuba M.F. TTX-sensitive Na+ and nifedipine-sensitive Ca2+ channels in rat vas deferens smooth muscle cells. Biochimica et Biophysica Acta, 1999; 1419(2): 343-352.
https://doi.org/10.1016/S0005-2736(99)00075-9
4. Bidaux G., Beck B., Zholos A., Gordienko D., Lemonnier L., Flourakis M., Roudbaraki M., Borowiec A-S., Fernбndez J., Delcourt P., Lepage G., Shuba Y., Skryma R., Prevarskaya N. Regulation of activity of transient receptor potential melastatin 8 (TRPM8) channel by its short isoforms. J Biol Chem, 2012; 287(5): 2948-2962.
https://doi.org/10.1074/jbc.M111.270256
PMid:22128173 PMCid:PMC3270952
5. Boldyrev O.I., Sotkis H.V., Kuliieva I.M., Vladymyrova I.A., Filippov I.B., Skryma R., Pre­vars'ka N., Shuba I.M. Expression of the cold receptor TRPM8 in the smooth muscles of the seminal ejaculatory ducts in rats. Fiziolohichnyi Zhurnal, 2009; 55(5): 17-27.
6. Filippov I.B., Vladymyrova I.A., Kuliieva I.M., Skryma R., Prevarskaia N., Shuba I.M. Modulation of the smooth muscle contractions of the rat vas deferens by TRPM8 channel agonist menthol. Fiziolohichnyi Zhurnal, 2009; 55(6): 30-40.
7. McKemy D.D., Neuhausser W.M., Julius D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature, 2002; 416(6876): 52-58.
https://doi.org/10.1038/nature719
PMid:11882888
8. Noyer L., Grolez G.P., Prevarskaya N., Gkika D., Lemonnier L. TRPM8 and prostate: a cold case? Pflugers Archiv: European Journal of Physiology, 2018; 470(10): 1419-1429
https://doi.org/10.1007/s00424-018-2169-1
PMid:29926226
9. Pan Y., Thapa D., Baldissera L., Argunhan F., Aubdool A.A., Brain S.D. Relevance of TRPA1 and TRPM8 channels as vascular sensors of cold in the cutaneous microvasculature. Pflugers Archiv: European Journal of Physiology, 2017; 470(5): 779-786.
https://doi.org/10.1007/s00424-017-2085-9
PMid:29164310 PMCid:PMC5942358
10. Peier A.M., Moqrich A., Hergarden A.C., Reeve A.J., Andersson D.A., Story G.M., Earley T.J., Dragoni I., McIntyre P., Bevan S., Patapoutian A. A TRP channel that senses cold stimuli and menthol. Cell, 2002;108(5): 705-715.
https://doi.org/10.1016/S0092-8674(02)00652-9
11. Stothard P. The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques, 2000; 28(6): 1102, 1104.
https://doi.org/10.2144/00286ir01
PMid:10868275
12. Tsavaler L., Shapero M.H., Morkowski S., Laus R. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer Res, 2001; 61(9): 3760-9.
13. Weyer A.D., Lehto S.G. Development of TRPM8 Antagonists to Treat Chronic Pain and Migraine. Pharmaceuticals (Basel), 2017;10(2). pii: E37.
https://doi.org/10.3390/ph10020037
PMid:28358322 PMCid:PMC5490394




DOI: http://dx.doi.org/10.30970/sbi.1302.595

Refbacks

  • There are currently no refbacks.


Copyright (c) 2019 Studia biologica