AMPK and ACC phosphorylation: effect of leptin, muscle fibre type and obesity.

Leptin stimulates fatty acid oxidation via the phosphorylation of AMPK (AMP-activated protein kinase) and ACC (acetyl-CoA carboxylase). Obesity is associated with resistance to the effects of leptin. We determined the action of leptin on AMPKalpha and ACCbeta phosphorylation and lipid metabolism in soleus (SOL) and extensor digitorum longus (EDL) muscles from lean and obese Wistar rats after 1 and 100 nM leptin. Both leptin doses stimulated phosphorylation of AMPKalpha and ACCbeta (P

Localization of P2X2 and P2X3 receptors in rat trigeminal ganglion neurons.

Purine receptors have been implicated in central neurotransmission from nociceptive primary afferent neurons, and ATP-mediated currents in sensory neurons have been shown to be mediated by both P2X3 and P2X2/3 receptors. The aim of the present study was to quantitatively examine the distribution of P2X2 and P2X3 receptors in primary afferent cell bodies in the rat trigeminal ganglion, including those innervating the dura. In order to determine the classes of neurons that express these receptor subtypes, purine receptor immunoreactivity was examined for colocalization with markers of myelinated (neurofilament 200; NF200) or mostly unmyelinated, non-peptidergic fibers (Bandeiraea simplicifolia isolectin B4; IB4). Forty percent of P2X2 and 64% of P2X3 receptor-expressing cells were IB4 positive, and 33% of P2X2 and 31% of P2X3 receptor-expressing cells were NF200 positive. Approximately 40% of cells expressing P2X2 receptors also expressed P2X3 receptors and vice versa. Trigeminal ganglion neurons innervating the dura mater were retrogradely labeled and 52% of these neurons expressed either P2X2 or P2X3 or both receptors. These results are consistent with electrophysiological findings that P2X receptors exist on the central terminals of trigeminal afferent neurons, and provide evidence that afferents supplying the dura express both receptors. In addition, the data suggest specific differences exist in P2X receptor expression between the spinal and trigeminal nociceptive systems.

Amitriptyline pharmacologically preconditions rat hearts against cardiac ischemic-reperfusion injury.

BACKGROUND/OBJECTIVES: Amitriptyline (AMY) is a tricyclic anti-depressant that has recently been shown to have anti-inflammatory properties. We investigated whether AMY is cardioprotective against reperfusion injury in ex-vivo rat hearts. METHODS: Thirty adult Sprague-Dawley rat hearts were perfused ex-vivo in a Langendorff apparatus. All hearts except SHAM (n = 6, perfused for 110 min.) received 30 min no-flow ischemia followed by 40 min reperfusion (I-R). One group (n = 6) was untreated before I-R (non-preconditioned; NPC), another non-preconditioned group was perfused with 10 µM amitriptyline for 30 min before I-R (NPC-AMY, n = 6). One group was preconditioned with 3 × 5-minute periods of ischemia before I-R (PC, n = 6) and a fifth group was preconditioned in the presence of 10 µM amitriptyline (PC-AMY, n = 6). p38 phosphorylation and HMGB1 levels were quantified using Western blots. Data was analysed using multiway ANOVAs with Tukey HSD and linear regression models with Sobel mediator tests. RESULTS: NPC hearts recovered poorly (LVDP recovered to 26.5 ± 10.5% of pre-ischemic values, compared to PC hearts (82.8 ± 14.9%: P < 0.05)). PC-AMY (69.9 ± 6.16%) and NPC-AMY (90.3 ± 10.0%) groups both recovered well (P < 0.05). The Sobel mediator test suggested that p38 activity may be indirectly involved in the amitriptyline induced cardioprotection (P < 0.05). HMGB1 was lower in amitriptyline treated hearts compared to the non-preconditioned hearts (P < 0.05) but the multiway ANOVA test suggests that HMGB1 was not involved in amitriptyline induced protection. CONCLUSIONS: Amitriptyline at 10 µM protects hearts against ischemic-reperfusion injury which may be partially mediated through p38 phosphorylation.

By what mechanism does ondansetron inhibit colonic migrating motor complexes: does it require endogenous serotonin in the gut wall?

BACKGROUND: 5-HT3 antagonists, such as ondansetron (Zofran), retard colonic transit and provide effective relief of symptoms of chronic diarrhea and diarrhea-predominant irritable bowel syndrome (IBS), but the mechanism by which ondansetron retards transit is unclear. What is clear is that the frequency of colonic migrating motor complexes (CMMCs) is reduced by ondansetron, which could account for reduced transit. Our aim was to determine whether an acute depletion of 5-HT from enteric neurons would inhibit spontaneous CMMCs; and determine whether the sensitivity of ondansetron to reduce CMMC frequency would change in a 5-HT-depleted preparation. METHODS: Mice were injected with reserpine, 24 h prior to euthanasia to deplete neuronally synthesized 5-HT. Mechanical recordings were made from proximal and mid-distal regions of isolated whole mouse colon. Immunohistochemical staining for 5-HT was used to detect neuronal 5-HT. KEY RESULTS: Reserpine depleted all detectable 5-HT from enteric nerves. In whole colons, with mucosa and submucosal plexus removed, the frequency and amplitude of spontaneous CMMCs was not different between groups treated with or without reserpine. Surprisingly, in mucosa and submucosal plexus-free preparations, ondansetron was equally or significantly more effective at inhibiting CMMC frequency compared with control preparations (containing 5-HT). Reserpine pretreatment had no effect on the sensitivity of ondansetron to inhibit CMMCs. CONCLUSIONS & INFERENCES: Endogenous 5-HT in enteric neurons (or the mucosa) is not required for the spontaneous generation or propagation of CMMCs. Furthermore, the primary mechanism by which ondansetron inhibits CMMC frequency is not mediated via the mucosa, submucosal plexus or 5-HT in myenteric neurons.

Peripheral N-methyl-d-aspartate receptors contribute to mechanical hypersensitivity in a rat model of inflammatory temporomandibular joint pain.

The aim of this study was to determine whether peripheral N-methyl-d-aspartate (NMDA) receptors are involved in inflammation-induced mechanical hypersensitivity of the temporomandibular joint (TMJ) region. We developed a rat model of mechanical sensitivity to Complete Freund's Adjuvant (CFA; 2µl containing 1µg Mycobacterium tuberculosis)-induced inflammation of the TMJ and examined changes in sensitivity following injection of NMDA receptor antagonists (dl-2-amino-5-phosphonovaleric acid (AP5) or Ifenprodil) with CFA. CFA injected into the TMJ resulted in an increase in mechanical sensitivity relative to pre-injection that peaked at day 1 and lasted for up to 3days (n=8, P<0.05). There was no change in mechanical sensitivity in vehicle-injected rats at any time-point (n=9). At day 1, there was a significant increase in mechanical sensitivity in animals injected with CFA+vehicle (n=7) relative to those injected with vehicle alone (n=7, P<0.05), and co-injection of AP5 (n=6) or Ifenprodil (n=7) with CFA blocked this hypersensitivity. Subcutaneous injection of AP5 (n=7) and Ifenprodil (n=5) instead of into the TMJ had no significant effect on CFA-induced hypersensitivity of the TMJ region. Western blot analysis revealed constitutive expression of the NR1 and NR2B subunits in trigeminal ganglion lysates. Immunohistochemical studies showed that 99% and 28% of trigeminal ganglion neurons that innervated the TMJ contained the NR1 and NR2B subunits respectively. Our findings suggest a role for peripheral NMDA receptors in inflammation-induced pain of the TMJ region. Targeting peripheral NMDA receptors with peripheral application of NMDA receptor antagonists could provide therapeutic benefit and avoid side effects associated with blockade of NMDA receptors in the central nervous system.

Inflammation-induced increase in hyperpolarization-activated, cyclic nucleotide-gated channel protein in trigeminal ganglion neurons and the effect of buprenorphine.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are active at resting membrane potential and thus contribute to neuronal excitability. Their increased activity has recently been demonstrated in models of nerve injury-induced pain. The major aim of the current study was to investigate altered HCN channel protein expression in trigeminal sensory neurons following inflammation of the dura. HCN1 and HCN2 channel immunoreactivity was observed on the membranes of medium- to large-sized trigeminal ganglion neurons with 76% and 85% of HCN1 and HCN2 expressing neurons also containing the 200 kDa neurofilament protein (associated with myelinated fibers). Western immunoblots of lysates from rat trigeminal ganglia also showed bands with appropriate molecular weights for HCN1 and HCN2. Three days after application of complete Freund's adjuvant (CFA) to the dura mater, Western blot band densities were significantly increased; compared to control, to 166% for HCN1 and 284% for HCN2 channel protein. The band densities were normalized against alpha-actin. In addition, the number of retrogradely labeled neurons from the dura expressing HCN1 and HCN2 was significantly increased to 247% (HCN1) and 171% (HCN2), three days after inflammation. When the opioid receptor partial agonist, buprenorphine, was given systemically, immediately after CFA, the inflammation-induced increase in HCN protein expression in both Western blot and immunohistochemical experiments was not observed. These results suggest that HCN1 and HCN2 are involved in inflammation-induced sensory neuron hyperexcitability, and indicate that an opioid receptor agonist can reverse the protein upregulation.