Nimesulide inhibits protein kinase C epsilon and substance P in sensory neurons – comparison with paracetamol

Abstract

In this paper we describe new actions of nimesulide and paracetamol in cultured peripheral neurons isolated from rat dorsal root ganglia (DRG). Both drugs were able to decrease in a dose-dependent fashion the number of cultured DRG neurons showing translocation of protein kinase C epsilon (PKCɛ) caused by exposure to 1 μM bradykinin or 100 nM thrombin. In addition, the level of substance P (SP) released by DRG neurons and the level of preprotachykinin mRNA expression were measured in basal conditions and after 70 minutes or 36 hours of stimulation with nerve growth factor (NGF) or with an inflammatory soup containing bradykinin, thrombin, endothelin-1, and KCl. Nimesulide (10 μM) significantly decreased the mRNA levels of the SP precursor preprotachykinin in basal and in stimulated conditions, and decreased the amount of SP released in the medium during stimulation of neurons with NGF or with the inflammatory soup. The effects of paracetamol (10 μM) on such response was lower. Nimesulide completely inhibited the release of prostaglandin E2 (PGE2) from DRG neurons, either basal or induced by NGF and by inflammatory soup, while paracetamol decreased PGE2 release only partially. Our data demonstrate, for the first time, a direct effect of two drugs largely used as analgesics on DRG neurons. The present results suggest that PKCɛ might be a target for the effect of nimesulide and paracetamol, while inhibition of SP synthesis and release is clearly more relevant for nimesulide than for paracetamol mechanism of action.

Effects of NSAIDs and paracetamol (acetaminophen) on protein kinase C epsilon translocation and on substance P synthesis and release in cultured sensory neurons

“…In a recent paper, we proposed that nimesulide and paracetamol may exert novel mechanisms of action, likely relevant for their analgesic action, by modulating protein kinase C epsilon (PKCɛ) and substance P (SP) in the peripheral sensory neurons 9. In the present paper, we extend a similar analysis to other NSAIDs in general use.…”

“…The subcellular localization of the epsilon isoform of protein kinase C (ie, PKCɛ) was investigated as previously described 9,13,24. In brief, rat DRG neurons cultured for 2–3 days were rapidly exposed to BK (at 1 μM concentration) or THR (at 100 nM concentration) for 30 seconds, then fixed for 10 minutes at room temperature with paraformaldehyde (4% formaldehyde and 4% sucrose, dissolved in phosphate-buffered saline [PBS]/distilled water 2:1).…”

“…At Day 2/3 in vitro, cultures were stimulated using a cocktail of inflammatory/pro-algesic mediators (termed “inflammatory soup” [IS]) composed of 100 nM THR, 1 μM BK, 100 nM endothelin-1, 25 mM potassium chloride, dissolved in culture medium (DMEM + 10% FBS) as previously described 9. When required, at a concentration of 10 μM, each of the drugs to be tested was pre-applied to cultures before treatment with IS for the time considered appropriate to be fully effective (see “Results”).…”

“…Translocation can be easily visualized with immunocytochemistry 9–13. Inhibition of translocation by nimesulide – and by paracetamol to a lesser extent – has been recently proposed by our group as a novel analgesic mechanism activated by these drugs 9…”

Gabapentin Inhibits Protein Kinase C Epsilon Translocation in Cultured Sensory Neurons with Additive Effects When Coapplied with Paracetamol (Acetaminophen)

“…Finally, the secondary antibody was rinsed off with PBS and cells were analyzed using a confocal microscope (Leica SP2, Leica Microsystems, Milan, Italy). Activation of PKC ε following activation of bradykinin or prokineticin receptors (PKRs) resulted in translocation from the cytoplasm to the neuronal cell membrane, as shown several times before [19, 20]. Translocation was quantified by determining fluorescence intensity along a line positioned across the cell in order to avoid the nucleus (for details, see Cesare et al [21]) using semiautomated proprietary software which significantly improved efficiency of data analysis.…”