Xylazine is an α₂-adrenergic agonist extensively used in veterinary medicine and animal experimentation for producing antinociception, sedation, and muscle relaxation. The nitric oxide (NO) / cGMP / ATP-sensitive K⁺ (K_ATP) channel pathway has been proposed as the action mechanism of peripheral antinociception of several groups of drugs, including opioids and nonsteroidal analgesics. Considering the lack of knowledge regarding the mechanisms involved in xylazine effects, the present study investigated the contribution of K⁺ channels on peripheral antinociception induced by xylazine using the rat paw pressure test, in which hyperalgesia was induced by intraplantar injection of prostaglandin E₂. Xylazine administered into the right hind paw elicited a local antinociceptive effect, since only much higher doses produced a systemic effect in the contralateral paw. The peripheral antinociceptive effect induced by xylazine was antagonized by glibenclamide, a specific blocker of K_ATP channels. In another experiment, tetraethylammonium, a voltage-dependent K⁺-channel blocker, and paxilline and dequalinium, which are selective blockers for the large- and small-conductance Ca²⁺-activated K⁺ channels, respectively, were ineffective at blocking xylazine antinociception. These results provide evidence that the peripheral antinociceptive effect of xylazine probably results from K_ATP-channel activation, while the voltage-dependent K⁺ channels, small- and large-conductance Ca²⁺-activated K⁺ channels, appear not to be involved in this mechanism.

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Some of the pharmacological properties of phytosteryl ferulates may be linked to their antioxidant potential. In this study, 2,2-diphenyl-1-picrylhydrazyl (DPPH), electron spin resonance (ESR), and thiobarbituric acid–reactive substances (TBARS) assays demonstrated that phytosteryl ferulates such as cycloartenyl ferulate (CAF), 24-methylenecycloartanyl ferulate (24-mCAF), and β-sitosteryl ferulate (β-SF) and ferulic acid (FA) each exerted strong free radical scavenging and antioxidation of lipid membrane, which were comparable to α-tocopherol. However, the sterol moiety alone, such as cycloartenol (CA), had neither activity. Since, the reactive oxygen species (ROS) production in the cell complex defense mechanism cannot be ruled out with the cell free system, we measured ROS production in NIH 3T3 fibroblast cells induced by H₂O₂. CAF and ethyl ferulate (eFA) greatly decreased the ROS level in this system. CA also significantly inhibited the ROS level, suggesting that CA could inhibit ROS production in living cells. Besides these, CAF, 24-mCAF, β-SF, as well as eFA and CA, all these chemicals significantly inhibited the NF-κB activity as analyzed by measuring translocation of NF-κB p65 in LPS-stimulated RAW 264.7 macrophages. These observations revealed that phytosteryl ferulates are responsible for the antioxidant and anti-inflammatory activity via ROS scavenging and inhibition of ROS production.

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This study examines the influence of receptor expression level on signaling pathways activated via endothelin type A receptor (ET_AR) expressed in Chinese hamster ovary cells at 32,100 (ET_AR-high-CHO) and 893 (ET_AR-low-CHO) fmolmg protein⁻¹. Endothelin-1 (ET-1) elicited a sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), which was dependent on G_q/11 protein, phospholipase C (PLC), Na⁺/H⁺ exchanger (NHE), and p38 mitogen–activated protein kinase (p38MAPK) in ET_AR-high-CHO, whereas the sustained [Ca²⁺]_i increase was negligible in ET_AR-low-CHO. Functional study with Cytosensor^TM microphysiometer showed that ET-1 evoked an NHE1-mediated increase in extracellular acidification rate (ECAR) in ET_AR-high-CHO and ET_AR-low-CHO. In ET_AR-high-CHO, the ECAR response at 30 min after ET-1 stimulation was insensitive to G_q/11 and PLC inhibitors, but sensitive to the p38MAPK inhibitor. In ET_AR-low-CHO, the ECAR response at 30 min was sensitive to these inhibitors. Western blot analysis demonstrated that ET-1–induced p38MAPK phosphorylation in ET_AR-low-CHO but not in ET_AR-high-CHO was mediated via G_q/11 and PLC. The G_q/11/PLC-independent p38MAPK phosphorylation in ET_AR-high-CHO was suppressed by expression of the C terminus of G_α12 protein to disrupt receptor-G₁₂ protein coupling. These results provide evidence for multiple signaling pathways of ET_AR that were activated via at least the G_q/11/PLC/NHE, G₁₂/p38MAPK/NHE, and G_q/11/PLC/p38MAPK/NHE cascades in an expression level–dependent manner.

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NADPH oxidase is an enzyme that converts molecular oxygen into reactive oxygen species, which cause severe damage in several organs. Cyclooxygenase (COX)-2 is an inducible enzyme that is important in gastric mucosal defense and repair processes. It is unclear whether NADPH oxidase is related to COX expression in the gastric mucosa, so we investigated the correlation. Under urethane anesthesia, a male Sprague Dawley rat stomach was mounted in an ex-vivo chamber, and ischemia/reperfusion (I/R) was performed through a cannula in the femoral vein. I/R significantly increased NADPH oxidase activity, H₂O₂ production, and myeloperoxidase (MPO) activity. In contrast, ischemia alone clearly enhanced both NADPH oxidase activity and H₂O₂ production but not MPO activity. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI) suppressed I/R-induced mucosal damage. On the other hand, the selective COX-2 inhibitor rofecoxib exhibited a tendency to enhance the severity of gastric damage induced by I/R, although the selective COX-1 inhibitor SC-560 and the nonselective COX inhibitor indomethacin had no effect. I/R also increased the expression of COX-2, and this increase was suppressed by pretreatment with DPI. These findings suggest that the increase in NADPH oxidase activity is involved in the occurrence of gastric mucosal damage induced by I/R and that this enzyme activity may be causally related to the upregulation of COX-2 during I/R.

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Exendin-4, a long-acting glucagon-like peptide-1–receptor agonist, is known to enhance β-cell function, but the active mechanism by which it modulates β-cell mass still remains unclear. We investigated what the long-term effects of exendin-4 (300 pmol/kg body weight per day) on β-cell function and mass would be in 90% pancreatectomized (Px) Sprague Dawley rats; half of whom were intraperitoneally injected with streptozotocin (STZ, 20 mg/kg body weight) and half of whom were not. Exendin-4 improved glucose tolerance by elevating serum insulin levels in both STZ-treated and untreated Px rats. At hyperglycemic clamp, STZ attenuated both first and second phase insulin secretion in STZ- and saline-treated Px rats, but exendin-4 incompletely reversed the attenuation. Since STZ mostly removed the remaining β-cells by increasing apoptosis after Px, their regeneration was initiated through neogenesis, which was determined by the number of β-cells budding from pancreatic duct layers and small clusters. Exendin-4 enhanced β-cell proliferation and neogenesis in STZ-treated and -untreated Px rats and reduced β-cell apoptosis partly by attenuating the expression of endoplasmic reticulum stress-response genes such as X-box-binding protein-1, activating transcription factor (ATF)-4, ATF6, and C/EBP-homologous protein. In conclusion, exendin-4 improved glycemic control by potentiating β-cell function and increasing β-cell mass by increasing β-cell neogenesis and proliferation and by decreasing apoptosis in diabetic rats.

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We previously reported that endothelium-derived hyperpolarizing factor (EDHF)–mediated response time-dependently suppressed methoxamine-induced vasoconstriction in mesenteric vascular beds isolated from 8-week-old rats. We investigated age-related changes in endothelial regulation of methoxamine-induced vasoconstriction. Mesenteric vascular beds isolated from young (8-week-old) to adult (16-week-old) rats were perfused, and changes in perfusion pressure induced by continuous perfusion of methoxamine or high KCl (60 mM) were measured over 180 min. In young preparations with intact endothelium, methoxamine-induced vasoconstriction time-dependently decreased to 20% of the initial levels, while time-dependent reduction was not observed in adult preparations. High KCl–induced vasoconstriction in young and adult preparations did not show time-dependent reduction. Endothelium removal abolished time-dependent reduction of methoxamine-induced vasoconstriction in young preparations and significantly attenuated vasoconstriction in adult preparations. Indomethacin, seratrodast, or tempol but not catalase significantly reduced methoxamine-induced vasoconstriction in adult preparations with endothelium. A23187 (Ca²⁺-ionophore)–, but not acetylcholine-, induced endothelium-dependent vasodilation in the presence of N^G-L-nitro arginine methyl ether in adult preparations was significantly smaller than that in young preparations. These findings suggest that the inhibitory effect of mesenteric vascular endothelium on methoxamine-induced vasoconstriction disappears with aging by reducing EDHF and increasing endothelium-derived contracting factors and reactive oxygen species.

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The skin lesions of inflammatory skin diseases (e.g., atopic dermatitis or psoriasis) accompany infiltration of inflammatory cells like macrophages, where abnormal sensory innervations and elevation of nerve growth factor (NGF) level are observed. It is thought that increased NGF mediates the abnormal innervations and this may cause the hypersensitivity of the skin. However, the mechanism of this increased NGF production in the skin is still unknown. Here, we show that tumor necrosis factor (TNF)-α, but not interferon-γ or interleukin-6, enhanced the NGF production in human keratinocytes. The enhanced NGF production was abolished by both Raf-1 kinase and MEK inhibitors, whereas specific inhibitors of p38 mitogen–activated protein kinase and c-Jun N-terminal kinase did not. The extracellular signal–regulated kinase (ERK) phosphorylation and expression of NGF mRNA were accelerated by TNF-α treatment. Furthermore, serum was necessary for the NGF production and epidermal growth factor could substitute for serum in the effect on NGF secretion. These results indicate that TNF-α enhances NGF production via the Raf-1 / MEK / ERK pathway in human keratinocytes, suggesting that regulating TNF-α is a therapeutic target to control NGF production and subsequent sensory innervations.

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We investigated the effects of brefeldin A and ilimaquinone, inhibitors of membrane trafficking, using serotonin transporter (SERT)–expressing COS-7 cells. Both drugs significantly inhibited the serotonin uptake activity of SERT and caused SERT to be retained in the endoplasmic reticulum (ER), indicating that membrane trafficking is an important factor for SERT functional regulation. In agreement with previous reports, a C-terminal–deletion mutant of SERT (SERTΔCT) mostly localized to the ER and completely lacked serotonin uptake activity. To further elucidate the role of the C-terminus of SERT, we investigated whether overexpression of FLAG-tagged SERT C-terminus (FLAG-SERT-CT) affected the serotonin uptake activity and glycosylation of SERT. Interestingly, when concomitantly expressed with full-length FLAG-SERT in COS-7 cells, FLAG-SERT-CT increased the serotonin uptake activity and mature glycosylation of FLAG-SERT. These results indicate that the C-terminal region of SERT plays a crucial role in the functional regulation of SERT via membrane trafficking and glycosylation. In addition, proteasome inhibitors induced apparent ER stress, significantly decreased the serotonin uptake activity and mature glycosylation of SERT and caused SERT to be localized to the ER, suggesting that SERT function would be attenuated via membrane trafficking in pathological states that trigger ER stress.

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Genetic depletion of the dystrophin-related glycoprotein (DRGP) complex causes cardiomyopathy in animals and humans. The present study was undertaken to explore the possible involvement of alterations in DRGP in the development of the right ventricular failure in monocrotaline-administered rats (MCT rats). At the 6th and 8th weeks after subcutaneous administration of 60 mg/kg monocrotaline, echocardiographic examination showed that cardiac output indices were decreased and that the right ventricular Tei indices were increased, suggesting that right ventricular failure occurs, at the latest, by 6 weeks after monocrotaline-administration. The levels of α- and β-sarcoglycan and β-dystroglycan in the right ventricle of the MCT rats at the 6th and 8th weeks were markedly decreased, and these decreases were inversely related to the increase in the right ventricular Tei index of the MCT-administered animals. The content and activity of the Ca²⁺-activated neutral protease m-calpain in the right ventricle of the MCT rats were increased at the 4th to 8th weeks and those of matrix metalloproteinase-2, at the 6th and 8th weeks. These results suggest that m-calpain– and/or matrix metalloproteinase-2–mediated alterations in the contents of α-sarcoglycan, β-sarcoglycan, and β-dystroglycan may be involved in the development of right ventricular failure in MCT rats.

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In this study, we investigated the roles of calmodulin kinase II (CaMKII) and calmodulin (CaM) in the reversal of run-down of L-type Ca²⁺ channels. Single Ca²⁺-channel activities in guinea-pig ventricular myocytes were recorded using the patch-clamp technique, and run-down of the channel activities was induced by inside-out patch formation in the basic internal solution. At 1 min after patch excision, 1 – 30 μM CaMKII mutant T286D (CaMKIIT286D), a constitutively active type of CaMKII, induced the Ca²⁺-channel activities to only 2% – 10% of that recorded in the cell-attached mode. However, in the presence of CaMKIIT286D, the time-dependent attenuation of CaM’s effects in the reversal of run-down was abolished. A GST-fusion protein containing amino acids 1509 – 1789 of the C-terminal region of guinea-pig Cav1.2 (CT1) was prepared. In pull-down assays, CT1 treated with CaMKIIT286D showed a higher affinity for CaM compared with CT1 treated with phosphatase. We propose a model in which CaMKII-mediated phosphorylation of the channels regulates the binding of CaM to the channels in the reversal of run-down of L-type Ca²⁺ channels.

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Methylglyoxal (MG), a reactive dicarbonyl produced during glucose metabolism, induces oxidative stress and apoptosis. Under hyperglycemic conditions, the abnormal accumulation of MG is related to the development of diabetic complications. We examined the effects of MG on thioredoxin (Trx) and glutaredoxin (Grx) systems, two thiol-disulfide oxidoreductase systems that protect against oxidative damage of proteins, in bovine aortic endothelial cells (BAECs). The levels of protein carbonyls as markers of protein oxidation increased in BAECs exposed to MG at 5 mM, resulting in the loss of cell viability. Western blot analysis demonstrated that Trx protein level decreased when BAECs were exposed to 5 mM MG. MG also inactivated Trx reductase, which maintains Trx in the reduced/active state. Moreover, peroxiredoxin, which is dependent on Trx and Trx reductase to maintain its reduced state, was oxidized by 5 mM MG. No significant difference in the levels of Trx, Trx reductase, or peroxiredoxin was observed in BAECs exposed to MG at 1 mM; this concentration had little effect on protein carbonyl formation and cell viability. MG failed to decrease Grx activity, indicating that Trx is more susceptible to MG than Grx. Taken together, these findings suggest that MG causes dysfunction of the Trx system, including Trx and Trx reductase, in BAECs.

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Cisplatin causes chronic interstitial disease with fibrosis, but the development mechanism of interstitial fibrosis is not yet understood. We examined the effect of an antioxidant, N,N'-diphenyl-1,4-phenylenediamine (DPPD), on development of interstitial fibrosis induced by cisplatin. Cisplatin increased blood urea nitrogen (BUN), plasma creatinine, and elicited glucosuria and enzymuria at 3 days after administration, but these changes were restored to the normal level after 14 days. Type III collagen increased from 7 days after administration of cisplatin and the expansion of the interstitial fibrosis area became evident at 14 days. Sustained renal fibrosis worsened renal function again at 56 days. Administration of DPPD, which was started at 3 days after cisplatin treatment, significantly inhibited the increase in renal type III collagen contents and the expansion of the interstitial fibrosis area without affecting enzymuria and increased BUN. These results indicate that anti-fibrotic action of DPPD is not secondary due to the inhibition of acute renal injury but is rather a direct effect on renal fibrogenesis. DPPD did not prevent the infiltration of macrophages by cisplatin, suggesting that anti-fibrotic action of DPPD was not mediated by the inhibition of inflammatory cellular influx. It is suggested that reactive oxygen species are involved in cisplatin-induced renal interstitial fibrosis.

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Developmental inadequacy in hepatic antioxidant defenses may contribute to chemical toxicity and pediatric liver diseases. We measured a comprehensive panel of antioxidants in liver tissue from 27 normal pediatric donors. Glutathione reductase declined with age (P = 0.008, r = −0.54, Spearman) while microsomal glutathione-S-transferase increased (GST, P<0.001, r = 0.81). Males had significantly lower superoxide dismutase and vitamin E (P<0.05) and may have lower glutathione reductase (P = 0.06), while females show less cytosolic GST (P = 0.07). Hepatic antioxidants are high in neonates, decline throughout childhood, and then increase in adolescence to adult levels.

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Diabetic cardiomyopathy may be accompanied by myocardial fibrosis. We have previously reported that cardiac fibrosis and protein O-glycosylation are elevated in diabetes. In this study, we examined if the hexosamine biosynthesis pathway (HBP) was involved with collagen expression in rat cardiac fibroblasts (RCFs). Long-term glucose load significantly increased type III collagen expression in RCFs, but did not affect the protein O-glycosylation. In addition, glucosamine treatment not only induced expressions of collagen types I and III, but also increased the O-glycosylated protein. These results suggest that O-glycosylation of protein induced by HBP activation modifies collagen expression and contributes to diabetic cardiomyopathy.

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