CaV2.3 channels are subthreshold voltage-gated calcium channels that play crucial roles in neurotransmitter release and regulation of membrane excitability, yet modulation of these channels with endogenous molecules and their role in pain processing is not well studied. Here, we hypothesized that an endogenous amino acid l-cysteine could be a modulator of these channels and may affect pain processing in mice. To test this hypothesis, we employed conventional patch-clamp technique in the whole-cell configuration using recombinant CaV2.3 subunit stably expressed in human embryonic kidney (HEK-293) cells. We found in our in vitro experiments that l-cysteine facilitated gating and increased the amplitudes of recombinant CaV2.3 currents likely by chelating trace metals that tonically inhibit the channel. In addition, we took advantage of mouse genetics in vivo using the acetic acid visceral pain model that was performed on wildtype and homozygous Cacna1e knockout male littermates. In ensuing in vivo experiments, we found that l-cysteine administered both subcutaneously and intraperitoneally evoked more prominent pain responses in the wildtype mice, while the effect was completely abolished in knockout mice. Conversely, intrathecal administration of l-cysteine lowered visceral pain response in the wildtype mice, and again the effect was completely abolished in the knockout mice. Our study strongly suggests that l-cysteine-mediated modulation of CaV2.3 channels plays an important role in visceral pain processing. Furthermore, our data are consistent with the contrasting roles of CaV2.3 channels in mediating visceral nociception in the peripheral and central pain pathways.
Introduction. β-Caryophyllene (BCP) has been shown to be an effective anti-inflammatory agent in chronic and inflammatory pain models. Since limited data are available for BCP in acute pain, we tested efficacy of BCP in an acute post-surgical pain model. Methods. BCP was tested in an acute postsurgical pain model. Animals were treated with vehicle, 10, 25, 50 and 75 mg/kg BCP that was injected intra-peritoneally (i.p.). Time dependent paw withdrawal response (PWR) were evaluated using von Frey filaments and plasma and tissue samples were taken. BCP levels were determined in tissue (paw and spine) and plasma using an HPLC-MS based approach. Endocannabinoids (2-arachidonoylglycerol) were also evaluated in plasma and tissues using an HPLC-MS based approach. Monoacylglycerol lipase (MAGL) activity was evaluated in-vitro as well as ex vivo. Results. A dose-dependent improvement of hyperalgesia was observed up to 85% of the baseline value 30 minutes after administration of the highest BCP dose of 75 mg/kg. A BCP dose-dependent increase in the 2-arachidonoylglycerol (2-AG) levels was observed with 9.9 ± 6.4 ng/mL in the 75 mg/kg dose group as compared to vehicle controls with 3.0 ± 2.5 ng/mL. In vitro MAGL enzyme activity assessment using 2-AG as the substrate revealed an IC50 of 7.4 µM of BCP for MAGL inhibition. Conclusion. These data showed that BCP inhibits MAGL activity in-vitro and in-vivo causing 2-AG levels to rise. Since the endocannabinoid 2-AG is a CB1 and CB2 receptor agonist, we propose the 2-AG-mediated cannabinoid receptor activation may contribute to BCP’s mechanism of analgesia.
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