Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors

Abstract

Nonsteroid anti-inflammatory drugs (NSAIDs) are major drugs against inflammation and pain. They are well known inhibitors of cyclooxygenases (COXs). However, many studies indicate that they may also act on other targets. Acidosis is observed in inflammatory conditions such as chronic joint inflammation, in tumors and after ischemia, and greatly contributes to pain and hyperalgesia. Administration of NSAIDs reduces low-pH-induced pain. The acid sensitivity of nociceptors is associated with activation of H(+)-gated ion channels. Several of these, cloned recently, correspond to the acid-sensing ion channels (ASICs) and others to the vanilloid receptor family. This paper shows (1) that ASIC mRNAs are present in many small sensory neurons along with substance P and isolectin B4 and that, in case of inflammation, ASIC1a appears in some larger Abeta fibers, (2) that NSAIDs prevent the large increase of ASIC expression in sensory neurons induced by inflammation, and (3) that NSAIDs such as aspirin, diclofenac, and flurbiprofen directly inhibit ASIC currents on sensory neurons and when cloned ASICs are heterologously expressed. These results suggest that the combined capacity to block COXs and inhibit both inflammation-induced expression and activity of ASICs present in nociceptors is an important factor in the action of NSAIDs against pain.

Fig. 1.

Expression of ASIC mRNA in DRG nociceptors in normal and inflamed conditions. A, Colocalization (yellow) on DRG of ASIC1a or ASIC3 (green) with SP or IB4 (red).Arrows indicate characteristic cells:arrowhead indicates a double-labeled cell;pointing-up arrow indicates an ASIC-labeled cell; andpointing-down arrow indicates an SP- or IB4-labeled cell. B, Histograms represent the ASIC1a- and ASIC3-labeled cell proportion according to cell cross-sectional areas in normal (green) and inflamed (red) conditions. n = 3 animals for ASIC1a (control, n = 221 cells; inflamed,n = 210), and n = 4 animals for ASIC3 (control, n = 185 cells; inflamed,n = 214).

Fig. 2.

In vivo studies of the expression of ASIC mRNA levels in inflamed conditions and after action of NSAIDs.A, In situ hybridization experiments on DRG with ASIC1a and ASIC3 probes. It presents L4 DRG on the inflamed side of the animal (middle), contralateral non-inflamed DRG (left), and DRG from inflamed animal treated with aspirin (right). Arrowheads show examples of unlabeled (white) and labeled (black) cells (on 5 animals at least). B shows semiquantitative RT-PCR results. Top, Representative experiments showing RT-PCR in normal (−) and inflamed (+) conditions (−NSAID) and after treatment with NSAID (+NSAID, here aspirin). Bottom, Induction factors (mean ± SEM) of mRNA levels by inflammation (Freund) and after inflammation plus treatment with dexamethasone or NSAIDs (n = 3–10 animals per experiment). Band densities are measured and normalized to that of actin (act) in the same preparation, and the induction factor corresponds to the ratio of the normalized densities in treated over untreated conditions.

Fig. 3.

Action of NSAIDs on DRG cells in primary culture. Salicylic acid (A) and aspirin (B) inhibit H⁺-induced currents; inset in A shows a 2.5× magnification of the inhibition. C, Flurbiprofen (500 μm) reversibly inhibits the psalmotoxin-1-sensitive current (n = 9). D, Capsaicin-activated currents are insensitive to salicylic acid.E, Current-clamp experiments show that aspirin reduces acid-induced spiking on DRG neurons.

Fig. 4.

Action of NSAIDs on ASIC1a transfected in COS cells. Flurbiprofen (A) or ibuprofen (B) inhibit the current in a dose-dependent manner (C) (5–10 cells per data point).

Fig. 5.

Action of NSAIDs on ASIC3 expressed in COS cells. Salicylic acid, aspirin, and diclofenac inhibit the current in whole-cell (A–C) and outside-out (D) patches. Dose dependence of the inhibition of the sustained component by salicylic acid (E) or diclofenac (F). Five to 10 cells per data point.

Fig. 6.

Chemical formulas of the NSAIDs tested on ASIC currents showing NSAIDs inhibiting ASIC activity (A) and those with no action on ASICs (B).