TRPM8/transient receptor potential M8

Icilin-evoked TRPM8 currents demonstrate very varied latencies with substantial desensitization. They also have variable activation kinetics and Ca2+ dependence[1].

Rats and mice are behaviorally activated by iscilin, a transient receptor potential cation channel subfamily M (TRPM8) agonist. Mice are given the "Wet dog shake" when given icin (3 mg/kg; sc)[2].

TRPM8, a member of the transient receptor potential family of ion channels, depolarizes somatosensory neurons in response to cold. TRPM8 is also activated by the cooling agents menthol and icilin. When exposed to menthol or cold, TRPM8 behaves like many ligand-gated channels, exhibiting rapid activation followed by moderate Ca(2+)-dependent adaptation. In contrast, icilin activates TRPM8 with extremely variable latency followed by extensive desensitization, provided that calcium is present. Here, we show that, to achieve full efficacy, icilin requires simultaneous elevation of cytosolic Ca2+, either via permeation through TRPM8 channels or by release from intracellular stores. Thus, two stimuli must be paired to elicit full channel activation, illustrating the potential for coincidence detection by TRP channels. Determinants of icilin sensitivity map to a region of TRPM8 that corresponds to the capsaicin binding site on the noxious heat receptor TRPV1, suggesting a conserved molecular logic for gating of these thermosensitive channels by chemical agonists[1].

Cell Viability Assay[1]
Cell Types: TRPM8-expressing oocytes or HEK293 cells
Tested Concentrations: 10 μM
Incubation Duration: 3 minutes for TRPM8-expressing oocytes; 1 minute for HEK293 cells
Experimental Results: Activated membrane currents with variable delay of onset in voltage-clamped TRPM8-expressing oocytes or HEK293 cells.

Animal/Disease Models: The C57BL/ 6 mice 9–10 weeks (adult, 26-30 g) or 24 months (aged, 35-42g)[2].
Doses: 3 mg/kg
Route of Administration: Injected sc
Experimental Results: Produced vivid and quantifiable shaking behaviors (“wet- dog shakes”), which were TRPM8-dependent.

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To determine if “compound 5” effectively inhibits TRPM8 activity in live mice, a WDS assay was performed. In this assay, a potent TRPM8 agonist (icilin 3 mg/kg s.c.) was injected in mice treated with “compound 5” or vehicle. Icilin injections in mice produce vivid and quantifiable shaking behaviors (“wet-dog shakes”), which are TRPM8-dependent (18–22)(2, 3). Accordingly, effective inhibition of TRPM8 is predicted to result in decreased number of icilin-induced behavioral events. “Compound 5” or vehicle was administered 60 minutes prior to icilin. The number of “wet-dog shakes” was counted over a 5-minute period starting 10 minutes after icilin injection.Experimental protocols (summarized in Table 1): In protocol 1, used to establish a method for long-lasting and sustained hypothermia through TRPV1 activation, DHC (2–4 mg/kg) or vehicle (20% DMSO in saline) was infused subcutaneously through the PE-10 line in a conscious mouse. The full dose was delivered in two bolus injections 30 minutes apart (Figure 1B) or as continuous infusion with or without an initial bolus injection (Figure 2). Core temperature was recorded by the thermocouple implanted in the abdomen; a second thermocouple recorded cage ambient temperature (22–24 °C). Temperature was measured at a frequency of 1 sample/min for up to 8 hours after the first infusion.[2]

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Protocol 3 was used to determine the effectiveness of TRPV1 agonists in aged subjects. Young (9–10 weeks) or aged (24 months) mice were injected with a single bolus of DHC (1.25 mg/kg s.c.). Temperature was measured by telemeter for 2 hours after injection (Figure 4). In protocol 4, used to determine the hypothermic effect of TRPM8 inhibition in mildly subneutral and cold environments, “compound 5” (20 mg/kg s.c.) or vehicle was administered to telemeter implanted mice (Figure 5). “Compound 5” is a selective TRPM8 inhibitor (see Drugs section). Core temperature was recorded for 30 minutes starting 30 minutes after the injection, at which point the mice were transferred to a cold room for 2 hours and measured at 20 minute intervals. The individual cages were fitted with a plastic mesh top to allow heat exchange with the atmosphere. Temperature in the cage averaged 8 °C.[2]

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Protocol 5 was used to determine if TRPM8 inhibition potentiates the hypothermic effect of TRPV1 agonists. TRPM8 antagonist “compound 5” (30 mg/kg i.p.) or vehicle was injected in mice with implanted telemeter (Figure 6). After 60 minutes, DHC (0.6, 1.25, or 2.5 mg/kg i.p.) was injected in both groups. Temperature was recorded for 5.5 hours (330 minutes) from the time of “compound 5” or vehicle injection, with a measurement taken every 10 minutes. The depth of hypothermia was determined by calculating the minimum value (nadir) of Tcore with each treatment. The duration of hypothermia was determined by measuring the time from DHC injection until recovery of Tcore to ≥34 °C to the nearest 10 minutes. The 0.6 mg/kg DHC dose failed to reliably drop Tcore below 34 °C during the experiment and was therefore excluded from Tcore recovery analysis. In the two groups treated with higher doses of DHC and pretreated with “compound 5”, the Tcore occasionally failed to return to 34 °C within the 330 minute measurement period. In these cases (6 out of 16 mice), the maximum time value (330 min) was assigned for the recovery time. All mice, however, fully recovered Tcore by the next morning. In addition, in a single mouse in the vehicle group treated with 2.5 mg/kg DHC, Tcore did not drop below 34 °C. This mouse was assigned the minimum time value (0 minutes) for the recovery time.[2]

[1]. The super-cooling agent icilin reveals a mechanism of coincidence detection by a temperature-sensitive TRP channel. Neuron. 2004 Sep 16;43(6):859-69.

[2]. Transient receptor potential melastatin 8 channel inhibition potentiates the hypothermic response to transient receptor potential vanilloid 1 activation in the conscious mouse. Crit Care Med. 2014 May;42(5):e355-63.

[3]. The cooling compound icilin attenuates autoimmune neuroinflammation through modulation of the T-cell response. FASEB J. 2018 Mar;32(3):1236-1249.

3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-1,4-dihydropyrimidin-2-one is a C-nitro compound.

C16H13N3O4

311.29

311.09

C, 61.73; H, 4.21; N, 13.50; O, 20.56

36945-98-9

161930

Typically exists as Light yellow to yellow solids at room temperature

1.4±0.1 g/cm3

594.8±50.0 °C at 760 mmHg

200-208ºC

313.5±30.1 °C

0.0±1.7 mmHg at 25°C

1.672

2.73

2

4

2

23

502

0

O=C1N([H])C(C2C([H])=C([H])C([H])=C(C=2[H])[N+](=O)[O-])=C([H])C([H])([H])N1C1=C([H])C([H])=C([H])C([H])=C1O[H]

RCEFMOGVOYEGJN-UHFFFAOYSA-N

InChI=1S/C16H13N3O4/c20-15-7-2-1-6-14(15)18-9-8-13(17-16(18)21)11-4-3-5-12(10-11)19(22)23/h1-8,10,20H,9H2,(H,17,21)

3-(2-hydroxyphenyl)-6-(3-nitrophenyl)-1,4-dihydropyrimidin-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.03 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)