CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 µM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.

cAMP-PKA signaling is involved in regulation of spinal HCN channels function in diabetic neuropathic pain.

The cyclic adenosine monophosphate-protein kinase A (cAMP-PKA) signaling acts a pivotal part in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels-mediated neuropathic and inflammatory pain. However, there has been no evidence of cAMP-PKA signaling is involved in regulation of spinal HCN channels function in the occurrence of diabetic neuropathic pain (DNP). The study aimed to elucidate the impact of HCN channels on neuropathic pain in a rat model of diabetes induced by streptozotocin, and whether cAMP-PKA signaling is involved in regulation of HCN channels function. In this report, we evaluated the effect of intrathecal administration of HCN channel blockers ZD7288, cAMP inhibitor SQ22536 and PKA inhibitor H-89 on nociceptive behavior in DNP rats. The mechanical withdrawal threshold (MWT) was measured to evaluate pain behavior in rats. Protein expression levels of HCN2, HCN4 channels and PKA in the spinal dorsal horn of rats were assessed. Furthermore, the levels of cAMP in rat spinal dorsal horn was analyzed. We discovered that DNP rats showed significant mechanical allodynia and are related to the increased HCN2 and HCN4 channels expression, enhanced cAMP production and elevated the expression of PKA protein in the spinal dorsal horn, which were attenuated by intrathecal ZD7288. Furthermore, intrathecal injection of SQ22536 and H-89 significantly reduced the HCN2 and HCN4 channels expression in the spinal dorsal horn of DNP rats. Our findings indicate that HCN channels of the spinal dorsal horn participate in the pathogenesis of allodynia in rats with DNP, which could be regulated by cAMP-PKA signaling. Therefore, HCN channels and cAMP-PKA signaling are potential targets for hyperalgesia treatment in DNP patients.

Dietary arginine supplementation enhances antioxidative capacity and improves meat quality of finishing pigs.

The present study was conducted to test the hypothesis that dietary arginine supplementation may improve meat quality of finishing pigs. Beginning at approximately 60 kg body weight, pigs were fed a corn- and soybean meal-based diet supplemented with 0, 0.5 or 1% L-arginine until they reached a body weight of approximately 110 kg. On the last day of the experiment, pigs were food-deprived for 16 h before blood samples were obtained for analysis of amino acids, insulin, and other metabolites. Immediately thereafter, pigs were slaughtered for determination of carcass composition, muscle biochemical parameters, and meat quality. The result showed that arginine did not affect pig growth performance or carcass traits. However, 1% arginine decreased drip loss of pork muscle at 48 h postmortem, while increasing intramuscular fat content (P < 0.05). Supplementing 0.5 or 1% arginine to the diet increased arginine concentration and decreased cortisol level in serum, while enhancing antioxidative capacity and glutathione peroxidase activity in serum (P < 0.05). Additionally, 1% arginine increased antioxidative capacity in skeletal muscle (P < 0.05). Furthermore, 0.5 or 1% arginine decreased the cortisol receptor mRNA level in muscle (P < 0.05). Collectively, these results indicate that supplemental arginine improved meat quality and attenuated oxidative stress of finishing pigs.

[Effects of Rhein on the hypertrophy of renal proximal tubular epithelial cells induced by high glucose and angiotensin II in rats].

OBJECTIVE: To explore the effect of Rhein on the hypertrophy of renal proximal tubular epithelial cells induced by high glucose and angiotensin II in rats. METHODS: Studies were performed on anesthetized SD rats. Renal proximal tubular were gained by microdissection and cultured in RPMI-1640 medium. The cell types were identified by immunocytochemistry. The renal proximal tubular epithelial cells were incubated with high glucose (30 mmol/L) and angiotensin II (10(-7) mol/L) to induce the hypertrophy of cells. To observe the effect of Rhein on hypertrophy induced by high glucose and angiotensin II, renal proximal tubular epithelial cells were cultured with different concentrations of Rhein (30, 15, 5 mg/L) for 72 h, then cell size, 3H-leucine incorporation, and cellular protein content were detected to observe the changes. RESULTS: High glucose (30 mmol/L) and Ang II (10(-7) mol/L) induced hypertrophy of renal proximal tubular epithelial cells result in, cell size, 3H-leucine incorporation and cellular protein content increased significantly. On the contrary, Rhein inhibited the hypertrophy of renal proximal tubular epithelial cells induced by high glucose and Angiotensin II. Rhein 30 mg/L significantly decreased cell size, 3H-leucine incorporation and cellular protein content. Rhein 15 mg/L decreased 3H-leucine incorporation and cellular protein content. Rhein 5 mg/L decreased cellular protein content. CONCLUSIONS: Rhein can inhibit the hypertrophy of renal proximal tubular epithelial cells induced by high glucose and Angiotensin II in rats.

Effects of constant or intermittent high temperature on egg production, feed intake, and hypothalamic expression of antioxidant and pro-oxidant enzymes genes in laying ducks.

Heat stress is a major environmental factor contributing to lower production of poultry. The objective of present study was to evaluate the influence of constant or intermittent high temperature on the production performance and redox status of plasma and hypothalamus in laying ducks. A total of 288 weight- and laying-matched laying ducks were randomly assigned to 1 of 4 treatments (each with 6 replicates of 12 birds): control, pair-fed, constant high temperature (24 h, 34 ± 1°C), and intermittent high temperature (10 h, 34 ± 1°C). Blood and hypothalamic tissue samples were collected on days 1, 21, and 55 to determine redox status. Average daily feed intake and egg weight was reduced (P < 0.001) during imposition of both high-temperature treatments but was not different (P > 0.05) among the treatments during the recovery period. Lower (P < 0.05) egg mass was observed in pair-fed and intermittent high-temperature treatment during high-temperature period and in constant high temperature during the recovery period. Haugh units from high temperature-treated ducks were significantly lower than those from control or pair-fed ducks (P < 0.05) during the high-temperature period. Both models of heat exposure decreased plasma concentrations of glutathione (GSH) at day 1, and constant high temperature decreased plasma activity of GSH peroxidase (GSH-PX) at day 21 (P < 0.05). Hypothalamic expression of antioxidant genes GSH reductase (GR) and mitochondrial NADH dehydrogenase subunit (Complex Ι) were decreased by both high-temperature treatments at day 1. Hypothalamic expression of genes for pro-oxidant enzymes cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX), and cytochrome P450 7A1 (CYP7A1) were decreased (P < 0.05) by both models of high temperature but transcripts of cyclooxygenase-1 (COX-1) of ducks that were pair-fed or were exposed to constant high temperature were increased at day 21. The transcripts of NADPH oxidase 1 (NOX-1) were decreased at day 1 by both high-temperature treatments (P < 0.05) but increased during the recovery period. These results indicate that, for laying ducks, intermittent high temperature caused much greater negative production performance effects than constant high temperature during high-temperature period, but laying ducks exposed to constant high temperature tend to take longer to recover their production performance. High-temperature stress, either constant or intermittent, altered hypothalamic expression of antioxidation and pro-oxidation genes.

Dietary supplementation with a high dose of daidzein enhances the antioxidant capacity in swine muscle but experts pro-oxidant function in liver and fat tissues.

BACKGROUND: Although isoflavones are natural dietary antioxidants, they may have toxicological effects. This study aimed to evaluate the redox system in tissues of finishing pigs by supplementation with high dose of daidzein (640 mg/kg). RESULTS: The supplementation of high dose of daidzein for 64 d increased the activity of superoxide dismutase and total antioxidant capacity in longissimus muscle but down-regulated the expression of reactive oxygen species (ROS)-producing enzyme NADPH oxidase-2 and cyclooxygenase-2. In contrast, high-level supplementation with daidzein exerted pro-oxidant changes in back fat, abdominal fat, liver, and plasma, as reflected by increased contents of malondialdehyde, a lipid peroxidation product, in these tissues. Furthermore, daidzein supplementation resulted in higher expression of ROS-producing enzymes, including NADPH oxidase-1 and cyclooxygenase-1 in liver, 5-lipoxygenase (5-LOX) in backfat and NADPH oxidase-2 both in abdominal fat and backfat. The supplementation of daidzein did not affect meat quality parameters in longissimus muscle, including marbling score, eye muscle areas, intramuscular fat, shear force, drip loss, pH and meat color. CONCLUSIONS: This experiment suggests that dietary supplementation of finishing pigs with daidzein at a high dose level improves redox status in muscle but exerts pro-oxidant effect in liver and fat tissues.