Modulation of the cold-activated channel TRPM8 by lysophospholipids and polyunsaturated fatty acids

Abstract

We investigated the role of phospholipase A2 (PLA2) and the effects of PLA2 products (polyunsaturated fatty acids and lysophospholipids) on the cold-sensitive channel transient receptor potential (melastatin)-8 (TRPM8), heterologously expressed in Chinese hamster ovary cells. TRPM8 responses to cold and the agonist icilin were abolished by inhibitors of the calcium-independent (iPLA2) form of the enzyme, whereas responses to menthol were less sensitive to iPLA2 inhibition. Inhibition of PLA2 similarly abolished the cold responses of the majority of cold-sensitive dorsal root ganglion neurons. The products of PLA2 had opposing effects on TRPM8. Lysophospholipids (LPLs) (lysophosphatidylcholine, lysophosphatidylinositol, and lysophosphatidylserine) altered the thermal sensitivity of TRPM8, raising the temperature threshold toward normal body temperature. Polyunsaturated fatty acids (PUFAs), such as arachidonic acid, inhibited the activation of TRPM8 by cold, icilin, and menthol. The relative potencies of lysophospholipids and PUFAs are such that lysophosphatidylcholine is able to modulate TRPM8 in the presence of an equimolar concentration of arachidonic acid. Positive modulation by LPLs provides a potential physiological mechanism for sensitizing and activating TRPM8 in the absence of temperature variations.

Figure 1.

The PLA₂ inhibitor ACA inhibits TRPM8. A–C, Cold (A), icilin (B), and menthol (C) evoked large [Ca²⁺]_i responses in CHO cells expressing TRPM8. Superfusing the cells with ACA before agonist challenge abolished responses induced by a cold ramp (A) and icilin (B) and significantly reduced responses to menthol (C). Traces are the mean responses of groups of cells monitored individually (n = 28–41). Veh, Vehicle; temp, temperature.

Figure 2.

ACA and BEL concentration–response curves. A, B, The nonselective PLA₂ inhibitor ACA (A) and the selective iPLA₂ inhibitor BEL (B) concentration-dependently reduced [Ca²⁺]_i responses evoked by icilin and menthol. Both inhibitors were significantly more potent against icilin than menthol. Data points are mean ± SEM measured in triplicates, and the data shown are representative of n = 3 separate experiments.

Figure 3.

Arachidonic acid inhibits TRPM8. A, B, Arachidonic acid (AA) (10 μm) almost completely inhibited [Ca²⁺]_i responses induced by a cold ramp from 37°C to 12°C (A), 100 nm icilin (B), and 100 μm menthol (C). Each trace is the average response from a group of cells (n = 25–50) monitored individually. The average peak response amplitude of experiments like those in A–C is shown in D (n = 3–4).

Figure 4.

TRPM8 is sensitive to polyunsaturated fatty acids. A, B, Eicosapentaenoic acid (EPA) (A) and docosahexaenoic acid (DOHA) (B) prevented [Ca²⁺]_i responses to stimulation with a cold ramp (from 37°C to 12°C). The traces in A and B are average responses in groups of cells (n = 25–50 cells). C, Average peak amplitude from experiments like those in A, B, and Figure 3A [the arachidonic acid (AA) data are the same as those shown in Fig. 3D].

Figure 5.

TRPM8 is activated by lysophospholipids. A, Lysophosphatidylcholine elicited [Ca²⁺]_i responses in cells expressing TRPM8 but not in untransfected CHO cells. After a longer initial delay, LPC also activated TRPM8 at 37°C (traces are averages of 25–40 cells monitored individually). B, TRPM8 was activated by lysophospholipids with different head groups. The negatively charged LPI and LPS evoked [Ca²⁺]_i responses of amplitude similar to the zwitterionic LPC, whereas SPC was much less effective (all at a concentration of 3 μm). Experiments were performed at 29°C, and the data in B are mean ± SEM of the number of experiments indicated.

Figure 6.

Menthol and LPC stimulate TRPM8 single-channel activity. A, C, In cell-attached patches, menthol (50 μm) stimulated channel activity within a few seconds of application. B, D, LPC (3 μm) elicited very similar single-channel activity but after a much longer initial delay. E, F, All point histograms from traces recorded in the presence of menthol (E) and LPC (F) show a similar distribution. Both histograms have been fitted with the sum of three Gaussian functions. G, Traces were recorded in cell-attached configuration in the absence and presence of LPC (3 μm). When channel activity had developed, the patch was excised to inside-out configuration. After patch excision, the channel activity stimulated by LPC persisted. Data were recorded at a holding potential of +60 mV. Veh, Vehicle.

Figure 7.

LPC modulates the cold sensitivity of TRPM8. A, LPC potentiates responses to cold by increasing the current amplitude and by shifting the activation threshold to higher temperatures (temp). B, Pseudocolored ratio images from [Ca²⁺]_i imaging experiments illustrating that LPC shifts the temperature activation threshold of TRPM8 closer to physiologically relevant values. No response was seen in the absence of extracellular Ca²⁺.

Figure 8.

LPC is resistant to inhibition of PLA₂ but sensitive to low pH. A, The agonist effect of LPC (3 μm) on TRPM8 is unaffected by the presence of 10 μm ACA. Cells incubated in pH 6 for 1 min before stimulation failed to respond to LPC. B, Application of equimolar concentrations of LPC and arachidonic acid (AA) only produced a small reduction of the LPC induced [Ca²⁺]_i responses. C, However, 10 μm arachidonic acid completely inhibited the response to 3 μm LPC. D, Average of the peak amplitude in three determinations in groups of 20–40 cells.