The membranes were stripped and reprobed with anti-Akt and Anti-GSK3 antisera (1:1,000) again
The membranes were stripped and reprobed with anti-Akt and Anti-GSK3 antisera (1:1,000) again. acts as a pain-inducing peptide in the dorsal horn. By activating specific receptors (likely AM2) and the PI3K/Akt/GSK3 signaling pathway, AM could play a significant role in long-lasting heat hypersensitivity and inflammatory heat hyperalgesia. and and and and and and and and < 0.01) at 2 h after injection. The dose of 20 g exerted the maximum effect, whereas 1 and 5 g were ineffective. (< 0.001). By 48 h after injection, AM-reduced TFL returned to control level. i.t. CGRP (20 g) significantly reduced TFL at 30 and 60 min after injection (?, < 0.01). (< 0.01). This effect was blocked or reversed by i.t.AM22-52, CGRP8-37, or BIBN4096BS 1 h before and after i.t. rAM1-50. i.t. vehicle, AM22-52, CGRP8-37, and BIBN4096BS failed to significantly affect TFL on their own. Mean SEM, = 5C10 per group. Preemptive i.t. injection of the AM receptor antagonist, AM22-52 (20 g), significantly blocked AM-reduced TFL from 2 (Fig. 3and 10and Fig. 10< 0.001), but this effect was blocked or reversed by pre- or posttreatment with AM22C52 (20 g), LY294002 (10 g) and wortmannin (0.1 g). Veh, vehicle. Moreover, pretreatment with both LY294002 and Wortmannin also significantly increased TFL compared with control (?, < 0.01). Mean SEM, = 8 in each group. (= 10) of AM-IR neurons coexpressed TRPV1 (Fig. 5and and 11 and < 0.001). i.t. AM22-52 (20 g) and Wortmannin (0.1 g) 1 h before (= 7 or 8 per group. (hybridization using highly selective riboprobes to establish the expression of AM mRNA in these two type of nociceptors. Interestingly, it has recently been reported that AM mRNA is expressed in DRG tissues (12). In accordance with findings that capsaicin treatment eliminated AM-IR in perivascular nerves (12), we observed that the majority of AM-IR neurons coexpressed TRPV1. AM has been recognized as an inflammatory mediator (3, 19). Circulating AM is dramatically increased by multiple cellular sources during local and systemic inflammation such as sepsis (3). AM is believed to stimulate the production of cytokines from immune cells and to increase blood flow and vascular permeability in inflamed tissues. The presence of AM in DRG neurons suggests these neurons as an important source of AM released in target tissues. We have shown here that CLR and RAMP 1-, 2- and 3-IR neurons are enriched in the superficial dorsal horn. RCP-IR neurons are also present in this area (20). Thus, the various components required to generate functional CGRP1, (-)-Blebbistcitin AM1, and AM2 (1) receptors are expressed in dorsal horn neurons. Rabbit polyclonal to Amyloid beta A4 Moreover, specific [125I]AM13-52-binding (-)-Blebbistcitin sites are predominantly localized in the area, with AM1-52 and AM22-52 potently competing for these sites, whereas CGRP8-37 and BIBN4096BS were less effective. This ligand-selectivity profile reveals that specific [125I]AM13-52-binding sites mostly represent AM receptors (1, 21). AM could activate both pre- and postsynaptic receptors in the spinal cord because CLR and all RAMPs are expressed in DRG neurons (22). As shown for CGRP (23), AM may act on presynaptic autoreceptors to regulate DRG functions. Functional Evidence That AM Is a Pain Neuropeptide. We observed that i.t. rAM1-50 induced a long-lasting heat hyperalgesia in rats. Both pre- and posttreatments with AM22-52, CGRP8-37, or BIBN4096BS significantly blocked or reversed AM-induced heat hyperalgesia. This pharmacological profile is similar to that of the AM2 receptor subtype, although a (-)-Blebbistcitin role for CGRP1 and/or AM1 receptors cannot be excluded at this time. (-)-Blebbistcitin i.t. CGRP was shown earlier to induce only a transient heat hyperalgesia (14) in contrast to the long-lasting hyperalgesia shown here by i.t. AM1-50. These distinct response profiles could be related to the following observations. First, AM is extensively distributed in both CGRP-containing and IB4-binding nociceptors and highly colocalized with TRPV1. Second, AM acts on AM1, AM2, and CGRP1 receptors, whereas CGRP binds mostly to CGRP1 receptors (1, 2). Finally, AM was also shown to be a more potent vasorelaxing peptide than CGRP (24). However, it has yet to be fully established whether AM-induced pain response is mediated by a direct activation of AM receptors located on nociceptive neurons in the dorsal horn or via an indirect mechanism (as suggested for CGRP), AM potentially inducing the release of other pain-stimulating substances such as substance P.