pyrimidinones and 3-iodothyronamine

3-Iodothyronamine (3T1AM) is an endogenous thyroid hormone metabolite that interacts with the human trace amine-associated receptor 1 (hTAAR1), a G-protein-coupled receptor, to induce numerous physiological responses including dose-dependent body temperature lowering in rodents. 3T1AM also directly activates cold-sensitive transient receptor potential melastatin 8 (TRPM8) channels in human conjunctival epithelial cells (HCjEC) at constant temperature as well as reducing rises in IL-6 release induced by transient receptor potential vanilloid 1 (TRPV1) activation by capsaicin (CAP). Here, we describe that 3T1AM-induced TRPM8 activation suppresses through crosstalk TRPV1 activation in immortalized human corneal epithelial cells (HCEC). RT-PCR and immunofluorescent staining identified TRPM8 gene and protein expression. Increases in Ca(2+) influx induced by the TRPM8 agonists either 3T1AM (0.1-10 μM), menthol (500 μM), icilin (15-60 μM) or temperature lowering (either <17°C or >17°C) were all blocked by 10-20 μM BCTC, a mixed TRPV1/TRPM8 antagonist. BCTC blocked 3T1AM-induced recombinant TRPM8 activation of Ca(2+) transients in an osteosarcoma heterologous expression system. The effects of BCTC in HCEC were attributable to selective TRPM8 inhibition since whole-cell patch-clamp currents underlying Ca(2+) rises induced by 20 μM CAP were BCTC insensitive. On the other hand, Ca(2+) transients induced by activating TRPV1 with either CAP or a hyperosmolar medium were suppressed during exposure to either 1 μM 3T1AM or 15 μM icilin. All of these modulatory effects on intracellular Ca(2+) regulation induced by the aforementioned agents were attributable to changes in underlying inward and outward current. Taken together, TRPM8 activation by 3T1AM markedly attenuates and even eliminates hyperosmolar and CAP induced TRPV1 activation through crosstalk.

Topics: Calcium; Capsaicin; Cell Line; Epithelial Cells; Humans; Interleukin-6; Membrane Potentials; Patch-Clamp Techniques; Pyrazines; Pyridines; Pyrimidinones; Thyronines; TRPM Cation Channels; TRPV Cation Channels

3-Iodothyronamine (T1AM), an endogenous thyroid hormone (TH) metabolite, induces numerous responses including a spontaneously reversible body temperature decline. As such an effect is associated in the eye with increases in basal tear flow and thermosensitive transient receptor potential melastatin 8 (TRPM8) channel activation, we determined in human conjunctival epithelial cells (IOBA-NHC) if T1AM also acts as a cooling agent to directly affect TRPM8 activation at a constant temperature. RT-PCR and quantitative real-time PCR (qPCR) along with immunocytochemistry probed for TRPM8 gene and protein expression whereas functional activity was evaluated by comparing the effects of T1AM with those of TRPM8 mediators on intracellular Ca(2+) ([Ca(2+)]i) and whole-cell currents. TRPM8 gene and protein expression was evident and icilin (20μM), a TRPM8 agonist, increased Ca(2+) influx as well as whole-cell currents whereas BCTC (10μM), a TRPM8 antagonist, suppressed these effects. Similarly, either temperature lowering below 23°C or T1AM (1μM) induced Ca(2+) transients that were blocked by this antagonist. TRPM8 activation by both 1µM T1AM and 20μM icilin prevented capsaicin (CAP) (20μM) from inducing increases in Ca(2+) influx through TRP vanilloid 1 (TRPV1) activation, whereas BCTC did not block this response. CAP (20μM) induced a 2.5-fold increase in IL-6 release whereas during exposure to 20μM capsazepine this rise was completely blocked. Similarly, T1AM (1μM) prevented this response. Taken together, T1AM like icilin is a cooling agent since they both directly elicit TRPM8 activation at a constant temperature. Moreover, there is an inverse association between changes in TRPM8 and TRPV1 activity since these cooling agents blocked both CAP-induced TRPV1 activation and downstream rises in IL-6 release.

Topics: Calcium; Capsaicin; Cell Line; Conjunctiva; Epithelial Cells; Humans; Interleukin-6; Membrane Potentials; Patch-Clamp Techniques; Pyrimidinones; RNA, Messenger; Thyronines; TRPM Cation Channels; TRPV Cation Channels