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Non-specific TRPM8 agonist menthol was shown to inhibit voltage- and agonist-evoked contractions of the smooth muscle (SM) of rat vas deferens. Here we compared the action of menthol with the action of more specific TRPM8 agonist icilin on depolarization- (60 mM KCl), carbachol-(CCh) and noradrenalin-(Nor)-evoked contractions of the SM strips from the prostatic and epididymal portions of the vas deferens of normal and castrated (60-137 days) rats. Inhibitory action of menthol (100 microM) and icilin (10 microM) on the amplitude of KCl-, CCh- and Nor-induced contractions of normal as well as castrated rats was similar consisting about 50%, despite castration per se strongly potentiated CCh- and Nor-evoked contractions compared to the control animals. In the epididymal portion of the control animals menthol suppressed KC 1- and CCh-evoked contractions by 46 +/- 5% and 32 +/- 3% and icilin by only 14 +/- 4% and 6 +/- 7%, respectively, whilst after castration both compounds became virtually ineffective. Considering that TRPM8 may localize in the sarcolemma and sarcoplasmic reticulum (SR) membrane and that menthol can also block voltage-gated calcium channels (VGCCs), our data indicate that in the prostatic portion TRPM8 modulates contractility by primarily decreasing the SR Ca2+ stores content, whilst in the epididymal one by both decreasing the SR filling and supporting Ca2+ entry. Drop in the circulation androgens as a result of castration changes the menthol- and icilin-mediated modulation of the rat vas deferens SM contractility via the decrease of the expression of L-type VGCCs and increase of the expression of TRPM8.

By means of real-time RT-PCR and immunofluorscent staining combined with confocal microscopy we show for the first time the expression ofmRNA and protein ofthe cold/menthol-sensitive cationic channel, TRPM8, in the smooth muscle cells (SMC) from the epididimal and prostatic portions of the rat vas deferens. Expression of TRPM8 mRNA correlated with the expression ofmRNA for androgen receptor (AR): orchidectomy of the animals resulted in the enhancement of the expression of both mRNAs, which likely reflects specific for the vas deferens compensatory response to the decreasing levels of circulating androgens. TRPM8 protein in the SMC from both parts of the vas deferens primarily localized outside the plasma membrane (PM), however, in the SMC from prostatic portion we observed higher TRPM8 protein targeting specifically the endoplasmic reticulum and PM, where it can form functional cold/menthol-sensitive cationic channel capable of modulating contractile activity of the smooth muscle.

For the first time by means of electrophysiology we demonstrate functional expression of cold/menthol sensitive TRPM8 channel in secretory epithelial cells plasma membrane from rat prostate. Under whole-cell patch-clamp technique rapid cooling from 33 to 21 degrees C of freshly isolated rat prostate epithelial cells elicited the significant outward rectifying membrane current with close to 0 mV reversal potential characteristic of TRPM8 activation. Extracellular application of chemical analog of cooling, menthol (100 microM) at 33 degrees C evoked similar current responses. Cold- and menthol-evoked currents could be detected only in elongated epithelial cells, which in prostate slices were labeled with anti-cytokeratin 8, suggesting that they belong to the apical secretory phenotype. No such current could be detected in round apparently basal epithelial cells. We conclude that in normal rat prostate functional expression of TRPM8 is restricted to plasma membrane of luminal apical epithelial cells.