Canavanine induces insulin release via activation of imidazoline I3 receptors

The aim of the present study was to identify the effect of canavanine on the imidazoline receptor because canavanine is a guanidinium derivative that has a similar structure to imidazoline receptor ligands. Transfected Chinese hamster ovary‐K1 cells expressing imidazoline receptors (nischarin (NISCH)‐CHO‐K1 cells) were used to elucidate the direct effects of canavanine on imidazoline receptors. In addition, the imidazoline I₃ receptor has been implicated in stimulation of insulin secretion from pancreatic β‐cells. Wistar rats were used to investigate the effects of canavanine (0.1, 1 and 2.5 mg/kg, i.v.) on insulin secretion. In addition the a specific I₃ receptor antagonist KU14R (4 or 8 mg/kg, i.v.) was used to block I₃ receptors. Canavanine decreased blood glucose by increasing plasma insulin in rats. In addition, canavanine increased calcium influx into NISCH‐CHO‐K1 cells in a manner similar to agmatine, the endogenous ligand of imidazoline receptors. Moreover, KU12R dose‐dependently attenuated canavanine‐induced insulin secretion in HIT‐T15 pancreatic β‐cells and in the plasma of rats. The data suggest that canavanine is an agonist of I₃ receptors both in vivo and in vitro. Thus, canavanine would be a useful tool in imidazoline receptor research.     

Dexmedetomidine protects against oxygen-glucose deprivation-induced injury through the I2 imidazoline receptor-PI3K/AKT pathway in rat C6 glioma cells

Objectives To explore the protection and the mechanism of dexmedetomidine on the oxygen–glucose deprivation (OGD) insults in rat C6 glioma cells. Methods Cells were subjected to OGD then assessed by viability studies. After dexmedetomidine treatment, p‐AKT, hypoxia‐inducible factor (HIF)‐1α, vascular endothelial growth factor (VEGF) and RTP801 expression were measured. Key findings Three hours of OGD decreased cell viability to 48.8%, which was reversed to 67.4% by 1 µm dexmedetomidine. Hoechst 33342 and propidium iodide double stains showed that the protection of dexmedetomidine was mainly by an anti‐apoptosis effect, which was also strengthened by decreasing caspase‐3 expression. Dexmedetomidine protection was mainly blocked by the I2 imidazoline receptor antagonist idazoxan and BU 224, but not by the α₁‐adrenoceptor antagonist prazosin, the α₂‐adrenoceptor antagonist yohimbine and RX 821002, or the I1 imidazoline receptor antagonist efaroxan. On the other hand, dexmedetomidine enhanced AKT phosphorylation. Furthermore, the protection of dexmedetomidine was blocked by the PI3K/AKT inhibitor wortmannin. The proteins of HIF‐1α, VEGF and RTP801 were significantly increased by dexmedetomidine treatment. Conclusions Dexmedetomidine activated the I2 imidazoline receptor‐PI3K/AKT pathway, and up‐regulated HIF‐1α, VEGF and RTP801 expression to protect against OGD‐induced injury in rat C6 cells.     

Carnosic acid as a component of rosemary extract stimulates skeletal muscle cell glucose uptake via AMPK activation

Compounds that increase the activity of the energy sensor AMP‐activated kinase (AMPK) have the potential to regulate blood glucose levels. Although rosemary extract (RE) has been reported to activate AMPK and reduce blood glucose levels in vivo, the chemical components responsible for these effects are not known. In the present study, we measured the levels of the polyphenol carnosic acid (CA) in RE and examined the effects and the mechanism of action of CA on glucose transport system in muscle cells. High performance liquid chromatography (HPLC) was used to measure the levels of CA in RE. Parental and GLUT4myc or GLUT1myc overexpressing L6 rat myotubes were used. Glucose uptake was assessed using [³H]‐2‐deoxy‐d ‐glucose. Total and phosphorylated levels of Akt and AMPK were measured by immunoblotting. Plasma membrane GLUT4myc and GLUT1myc levels were examined using a GLUT translocation assay. Statistics included analysis of variance (ANOVA) followed by Tukey's post‐hoc test. At concentrations found in rosemary extract, CA stimulated glucose uptake in L6 myotubes. At 2.0 μmol/L CA a response (226 ± 9.62% of control, P=.001), similar to maximum insulin (201 ± 7.86% of control, P=.001) and metformin (213 ± 10.74% of control, P=.001) was seen. Akt phosphorylation was not affected by CA while AMPK and ACC phosphorylation was increased and the CA‐stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C. Plasma membrane GLUT4 or GLUT1 glucose transporter levels were not affected by CA. Our study shows increased muscle cell glucose uptake and AMPK activation by low CA concentrations, found in rosemary extract, indicating that CA may be responsible for the antihyperglycemic properties of rosemary extract seen in vivo.     

Radiosynthesis of a 2‐substituted 4,5‐dihydro‐1H‐[2‐11C] imidazole: the I2 imidazoline receptor ligand [11C] benazoline

Benazoline (2‐naphthalen‐2‐yl‐4,5‐dihydro‐1H‐imidazole) is a selective high‐affinity ligand for the imidazoline I₂ receptor. This compound was labelled with carbon‐11 (T_1/2=20.4 min) at the number two carbon atom of its 2‐imidazoline ring. Cyclotron‐produced [¹¹C]carbon dioxide reacted with 2‐naphthylmagnesium bromide to give 2‐[carboxyl‐¹¹C]naphthoic acid in 60% radiochemical yield. The latter was heated with a mixture of ethylenediamine and its dihydrochloride at 300°C to give [¹¹C]benazoline in 16% overall yield, relative to [¹¹C]carbon dioxide and with a specific radioactivity of 54 GBq/μmol, decay corrected for end of irradiation. The procedure requires about 45 min from end of cyclotron irradiation. This method should be extendable to other imidazolines. Copyright © 2003 John Wiley & Sons, Ltd.     

A newly synthetic chromium complex—Chromium (d-phenylalanine)3 activates AMP-activated protein kinase and stimulates glucose transport

We synthesized the chromium (phenylalanine)₃ [Cr(d-phe)₃] by chelating chromium(III) with d-phenylalanine ligand in aqueous solution to improve the bioavailability of chromium, and reported that Cr(d-phe)₃ improved insulin sensitivity. AMP-activated protein kinase (AMPK) is a key mediator for glucose uptake and insulin sensitivity. To address the molecular mechanisms by which Cr(d-phe)₃ increases insulin sensitivity, we investigated whether Cr(d-phe)₃ stimulates glucose uptake via activation of AMPK signaling pathway. H9c2 myoblasts and isolated cardiomyocytes were treated with Cr(d-phe)₃ (25 μM). Western blotting was used for signaling determination. The glucose uptake was determined by 2-deoxy-d-glucose-³H accumulation. HPLC measured concentrations of AMP. The mitochondrial membrane potential (Δψ) was detected by JC-1 fluorescence assay. Cr(d-phe)₃ stimulated the phosphorylation of α catalytic subunit of AMPK at Thr¹⁷², as well the downstream targets of AMPK, acetyl-CoA carboxylase (ACC, Ser²¹²) and eNOS (Ser¹¹⁷⁷). Moreover, Cr(d-phe)₃ significantly stimulated glucose uptake in both H9c2 cells and cardiomyocytes. AMPK inhibitor compound C (10 μM) dramatically inhibited the glucose uptake stimulated by Cr(d-phe)₃, while it did not affect insulin stimulation of glucose uptake. Furthermore, in vivo studies showed that Cr(d-phe)₃ also activated cardiac AMPK signaling pathway. The increase of cardiac AMP concentration and the decrease of mitochondrial membrane potential (Δψ) may contribute to the activation of AMPK induced by Cr(d-phe)₃. Cr(d-phe)₃ is a novel compound that activates AMPK signaling pathway, which contributes to the regulation of glucose transport during stress conditions that may be associated the role of AMPK in increasing insulin sensitivity.     

Discovery of Novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole Derivatives as Potential Anti‐Inflammatory Agents

A novel 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 5 with good anti‐inflammatory activity was identified from our in‐house library. Based on hit compound 5 , two series of 2‐(piperidin‐4‐yl)‐1H‐benzo[d]imidazole derivative 6a – g and 7a – h were designed and synthesized as novel anti‐inflammatory agents. Most of synthesized compounds exhibited good inhibitory activity on NO and TNF‐α production in LPS‐stimulated RAW 264.7 macrophages, in which the compound 6e showed most potent inhibitory activity on NO (IC₅₀ = 0.86 μm ) and TNF‐α (IC₅₀ = 1.87 μm ) production. Further evaluation revealed that compound 6e displayed more potent in vivo anti‐inflammatory activity than ibuprofen did on xylene‐induced ear oedema in mice. Additionally, Western blot analysis revealed that compound 6e could restore phosphorylation level of IκBα and protein expression of p65 NF‐κB in LPS‐stimulated RAW 264.7 macrophages.     

Investigation of morin‐induced insulin secretion in cultured pancreatic cells

Morin is a flavonoid contained in guava that is known to reduce hyperglycemia in diabetes. Insulin secretion has been demonstrated to increase following the administration of morin. The present study is designed to investigate the potential mechanism(s) of morin‐induced insulin secretion in the MIN6 cell line. First, we identified that morin induced a dose‐dependent increase in insulin secretion and intracellular calcium content in MIN6 cells. Morin potentiated glucose‐stimulated insulin secretion (GSIS). Additionally, we used siRNA for the ablation of imidazoline receptor protein (NISCH) expression in MIN6 cells. Interestingly, the effects of increased insulin secretion by morin and canavanine were markedly reduced in Si‐NISCH cells. Moreover, we used KU14R to block imidazoline I₃ receptor (I‐3R) that is known to enhance insulin release from the pancreatic β‐cells. Without influence on the basal insulin secretion, KU14R dose‐dependently inhibited the increased insulin secretion induced by morin or efaroxan in MIN6 cells. Additionally, effects of increased insulin secretion by morin or efaroxan were reduced by diazoxide at the dose sufficient to open K_ATP channels and attenuated by nifedipine at the dose used to inhibit L‐type calcium channels. Otherwise, phospholipase C (PLC) is introduced to couple with imidazoline receptor (I‐R). The PLC inhibitor dose‐dependently inhibited the effects of morin in MIN6 cells. Similar blockade was also observed in protein kinase C (PKC) inhibitor‐treated cells. Taken together, we found that morin increases insulin secretion via the activation of I‐R in pancreatic cells. Therefore, morin would be useful to develop in the research and treatment of diabetic disorders.     

Stimulation of 5‐HT4 receptor enhances differentiation of mouse induced pluripotent stem cells into neural progenitor cells

Activation of serotonin (5‐hydroxytryptamine; 5‐HT) receptors plays a role in adult neurogenesis and differentiation of neural progenitor cells (NPC). Herein, we examined the involvement of 5‐HT receptors in the differentiation of mouse induced pluripotent stem (iPS) cells into NPC. To induce embryoid body (EB) formation, mouse iPS cells were cultured on ultralow‐attachment dishes. All‐trans retinoic acid (ATRA; 1 μmol/L) and/or 5‐HT (0.03 or 0.1 μmol/L) was added to the EB cultures for 4 days and then EB plated on gelatin‐coated plates were cultured for 7 or 14 days. Immunofluorescence staining revealed that mouse iPS cells expressed both 5‐HT_2A and 5‐HT₄ receptors and, to a lesser extent, 5‐HT_1A receptors. Treatment with 5‐HT significantly enhanced the ATRA‐induced expression of nestin, a specific marker for NPC, and phosphorylation of cAMP response element‐binding protein (CREB). Pretreatment of EB cultures with either 1 μmol/L GR113808 (a selective 5‐HT₄ receptor antagonist) or 1 μmol/L H89 (a protein kinase (PKA) inhibitor) significantly inhibited these effects of 5‐HT. These findings suggest that stimulation of 5‐HT₄ receptors may enhance ATRA‐induced neural differentiation of mouse iPS cells through activation of PKA and CREB.     

Enhancing effects of anagliptin on myoblast differentiation and the expression of mitochondrial biogenetic factors in C2C12 mouse skeletal muscle cells

To investigate the regulatory effects of anagliptin, a DPP-IV inhibitor used to treat type 2 diabetes mellitus (T2DM), on myoblast differentiation and mitochondrial biogenesis in C2C12 mouse skeletal muscle cells. C2C12 myoblasts were differentiated into myotubes and then treated with anagliptin (10, 25, and 50 μmol/L) for 24 hours. In C2C12 myotubes, anagliptin treatment was significantly increased the expression of MHC, PGC1α, Sirt-1, NRF-1, and TFAM and the phosphorylation of AMPK and ACC in a concentration-dependent manner. Anagliptin also significantly increased the total ATP levels in the myotubes. These results suggest that anagliptin can help prevent skeletal muscle dysfunction in T2DM by promotion of myoblast differentiation and enhancement of energy production via upregulation of mitochondrial biogenetic factors and activation of the AMPK/ACC signalling pathway.     

Specific Role of α2A‐ and α2B‐, but not α2C‐, Adrenoceptor Subtypes in the Inhibition of the Vasopressor Sympathetic Out‐flow in Diabetic Pithed Rats

Several lines of evidence have shown an association of diabetes with a catecholamines' aberrant homeostasis involving a drastic change in the expression of adrenoceptors. This homeostatic alteration includes, among other things, atypical actions of α₂‐adrenoceptor agonists within central and peripheral α₂‐adrenoceptors (e.g. profound antinociceptive effects in diabetic subjects). Hence, this study investigated the pharmacological profile of the α₂‐adrenoceptor subtypes that inhibit the vasopressor sympathetic out‐flow in streptozotocin‐pre‐treated (diabetic) pithed rats. For this purpose, B‐HT 933 (up to 30 μg/kg min) was used as a selective α₂‐adrenoceptor agonist and rauwolscine as a non‐selective α_2A/2B/2C‐adrenoceptor antagonist; in addition, BRL 44408, imiloxan and JP‐1302 were used as subtype‐selective α_2A‐, α_2B‐ and α_2C‐adrenoceptor antagonists, respectively (all given i.v.). I.v. continuous infusions of B‐HT 933 inhibited the vasopressor responses induced by electrical sympathetic stimulation without affecting those by i.v. bolus injections of noradrenaline in both normoglycaemic and diabetic rats. Interestingly, the ED₅₀ for B‐HT 933 in diabetic rats (25 μg/kg min) was almost 1‐log unit greater than that in normoglycaemic rats (3 μg/kg.min). Moreover, the sympatho‐inhibition induced by 10 μg/kg min B‐HT 933 in diabetic rats was (i) abolished by 300 μg/kg rauwolscine or 100 and 300 μg/kg BRL 44408; (ii) partially blocked by 1000 μg/kg imiloxan; and (iii) unchanged by 1000 μg/kg JP‐1302. Our findings, taken together, suggest that B‐HT 933 has a less potent inhibitory effect on the sympathetic vasopressor responses in diabetic (compared to normoglycaemic) rats and that can probably be ascribed to a down‐regulation of α_2C‐adrenoceptors.     

Vasorelaxation induced by methyl cinnamate,the major constituent of the essential oil of Ocimum micranthum,in rat isolated aorta

The aim of the present study was to investigate the vascular effects of the E‐isomer of methyl cinnamate (E‐MC) in rat isolated aortic rings and the putative mechanisms underlying these effects. At 1–3000 μmol/L, E‐MC concentration‐dependently relaxed endothelium‐intact aortic preparations that had been precontracted with phenylephrine (PHE; 1 μmol/L), with an IC₅₀ value (geometric mean) of 877.6 μmol/L (95% confidence interval (CI) 784.1–982.2 μmol/L). These vasorelaxant effects of E‐MC remained unchanged after removal of the vascular endothelium (IC₅₀ 725.5 μmol/L; 95% CI 546.4–963.6 μmol/L) and pretreatment with 100 μmol/L N^G‐nitro‐l ‐arginine methyl ester (IC₅₀ 749.0 μmol/L; 95% CI 557.8–1005.7 μmol/L) or 10 μmol/L 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (IC₅₀ 837.2 μmol/L; 95% CI 511.4–1370.5 μmol/L). Over the concentration range 1–3000 μmol/L, E‐MC relaxed K⁺‐induced contractions in mesenteric artery preparations (IC₅₀ 314.5 μmol/L; 95% CI 141.9–697.0 μmol/L) with greater potency than in aortic preparations (IC₅₀ 1144.7 μmol/L; 95% CI 823.2–1591.9 μmol/L). In the presence of a saturating contractile concentration of K⁺ (150 mmol/L) in Ca²⁺‐containing medium combined with 3 μmol/L PHE, 1000 μmol/L E‐MC only partially reversed the contractile response. In contrast, under similar conditions, E‐MC nearly fully relaxed PHE‐induced contractions in aortic rings in a Ba²⁺‐containing medium. In preparations that were maintained under Ca²⁺‐free conditions, 600 and 1000 μmol/L E‐MC significantly reduced the contractions induced by exogenous Ca²⁺ or Ba²⁺ in KCl‐precontracted preparations, but not in PHE‐precontracted preparations (in the presence of 1 μmol/L verapamil). In addition, E‐MC (1–3000 μmol/L) concentration‐dependently relaxed the contractions induced by 2 mmol/L sodium orthovanadate. Based on these observations, E‐MC‐induced endothelium‐independent vasorelaxant effects appear to be preferentially mediated by inhibition of plasmalemmal Ca²⁺ influx through voltage‐dependent Ca²⁺ channels. However, the involvement of a myogenic mechanism in the effects of E‐MC is also possible.     

Chemical modification of canavanine with p-nitrophenylglyoxal. Factors influencing the chemistry and reactivity of α-dicarbonyl-guanidino reactions

The role of structural features and deprotonation of guanidino derivatives on chemical reactions with p-nitrophenylglyoxal has been investigated. Canavanine, an arginine analog, reacts to form a yellow product, which absorbs maximally at 350 nm (? = 6500) and at 278 nm (?= 14 500). Elemental analysis, fast atom bombardment mass spectral analysis, n.m.r. and i.r. studies suggest that the product is a 5-(p-nitrophenyl)4-oxo-2 imidazoline derivative of canalaline. Kinetic studies show that the second order rate constant for the reaction increases with increasing pH in the range of pH 7–11.0. It is concluded that the pH dependence of the reaction can be explained by general base catalysis and not simply by a deprotonation of the guanidinoxy side chain. The reaction of arginine, polyarginine, and other derivatives differs markedly from that of canavanine. The results suggest that change in the tautomeric equilibria between the imino and amino forms of the guanidino group may partly account for differences in reaction of canavanine and arginine and the reactions of specific arginyl residues in proteins.     

5‐Aminoimidazole‐4‐carboxamide‐1‐β‐d‐ribofuranoside Increases Myocardial Glucose Uptake during Reperfusion and Induces Late Pre‐conditioning: Potential Role of AMP‐Activated Protein Kinase

Abstract: Late pre‐conditioning protects against myocardial ischaemic‐reperfusion injury. AMP‐activated protein kinase (AMPK) is activated by exercise and 5‐aminoimidazole‐4‐carboxamide‐1‐β‐d ‐ribofuranoside (AICAR). Early pre‐conditioning involves AMPK activation and increased myocardial glucose uptake. The aim of the present study was to determine whether AICAR activates myocardial AMPK and induces late pre‐conditioning and whether myocardial glucose uptake during reperfusion was modulated. Twenty‐four hours after AICAR treatment or exercise, Wistar rats were subjected to ischaemia and reperfusion in a Langendorff model and compared to control rats. AMPK activity increased immediately 2.5‐fold in AICAR‐treated animals (P < 0.01) and twofold in exercised animals (P < 0.05). AICAR and exercise reduced infarct size by 60% and 50% (both P < 0.01), respectively, and increased myocardial glucose uptake during reperfusion (AICAR; 45%, P < 0.05, exercise; 40%, P < 0.05). In conclusion, AICAR induces late pre‐conditioning and increases myocardial glucose uptake during reperfusion in rat hearts. AICAR and exercise activate AMPK, suggesting a role of AMPK in the signalling mechanisms behind late pre‐conditioning.     

1‐[(Imidazolidin‐2‐yl)imino]‐1H‐indoles as new hypotensive agents: synthesis and in vitro and in vivo biological studies

A series of 1‐[(imidazolidin‐2‐yl)imino]‐1H‐indole analogues of hypotensive α₂‐AR agonists, 1‐[(imidazolidin‐2‐yl)imino]‐1H‐indazoles, was synthesized and tested in vitro for their activities at α₁‐ and α₂‐adrenoceptors as well as imidazoline I₁ and I₂ receptors. The most active 1‐[(imidazolidin‐2‐yl)imino]‐1H‐indoles displayed high or moderate affinities for α₁‐ and α₂‐adrenoceptors and substantial selectivity for α₂‐adrenoceptors over imidazoline‐I₁ binding sites. The in vivo cardiovascular properties of indole derivatives 3 revealed that substitution at C‐7 position of the indole ring may result in compounds with high cardiovascular activity. Among them, 7‐fluoro congener 3g showed the most pronounced hypotensive and bradycardic activities in this experiment at a dose as low as 10 μg/kg i.v. Metabolic stability of the selected compounds of type 3 was determined using both in vitro and in silico approaches. The results indicated that these compounds are not vulnerable to rapid first‐phase oxidative metabolism.     

Remote per‐conditioning reduces oxidative stress,downregulates cyclo‐oxygenase‐2 expression and attenuates ischaemia–reperfusion‐induced acute kidney injury

相似文献   

Preparation of 14C2‐cis‐1,2‐dichloroethylene from 14C2‐trichloroethylene using a cobalt porphyrin catalyst

A protocol for producing ¹⁴C₂‐cis‐1,2‐dichloroethylene (¹⁴C₂‐cis‐DCE) from ¹⁴C₂‐trichloroethylene (¹⁴C₂‐TCE) was developed as an economical alternative to commercial, custom synthesis of ¹⁴C₂‐cis‐DCE. The method uses 5,10,15,20‐tetrakis‐(4‐carboxyphenyl) porphyrin cobalt(II) as a catalyst in the presence of titanium(III) citrate to convert ¹⁴C₂‐TCE to ¹⁴C₂‐cis‐DCE as shown in Figure 1. A purge‐and‐trap technique was used to isolate ¹⁴C₂‐cis‐DCE to provide a methanolic stock solution of high purity (97% cis‐DCE). The overall ¹⁴C₂‐cis‐DCE yield of the process was approximately 53%. Copyright © 2005 John Wiley & Sons, Ltd.     

Acetaminophen‐induced hepatocyte injury: C2‐ceramide and oltipraz intervention,hepatocyte nuclear factor 1 and glutathione S‐transferase A1 changes

Acetaminophen (APAP) is an antipyretic and analgesic, which is commonly associated with drug‐induced hepatic injury. C2‐ceramide plays a key role in mediating cell life activities, and oltipraz was extensively studied as a cancer chemopreventive agent. Glutathione S‐transferase A1 (GSTA1) acts as a vital liver detoxification enzyme. Hepatocyte nuclear factor 1 (HNF‐1) regulates various cellular signaling pathways. In this study, we investigated the effects of C2‐ceramide and oltipraz on APAP‐induced hepatocyte injury and the changes of HNF‐1 and GSTA1. Results showed that C2‐ceramide (6 μmol/L) exacerbated APAP‐induced hepatocyte injury and caused a significant decrease (P < .01) in HNF‐1 and GSTA1 expressions. Meanwhile, GSTA1 content in supernatant was significantly increased (P < .01). In contrast, oltipraz (8 μmol/L) reduced the injury and significantly elevated (P < .01) HNF‐1 and GSTA1 expressions while GSTA1 content in supernatant was significantly decreased (P < .01). In conclusion, these findings revealed that C2‐ceramide inhibited HNF‐1 and GSTA1 expression and exacerbated hepatocyte injury, while oltipraz treatment results in the reduction of hepatocyte injury, and promoted HNF‐1 and GSTA1 expression. Additionally, the changes in HNF‐1 and GSTA1 were related to APAP‐induced hepatocyte injury. These results were useful to investigate the mechanism of an antipyretic and analgesic drug combination.     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.     

Mechanism of the vasorelaxant effect induced by trans‐4‐methyl‐β‐nitrostyrene,a synthetic nitroderivative,in rat thoracic aorta

Mechanisms underlying the vasorelaxant effects of trans‐4‐methyl‐β‐nitrostyrene (T4MeN) were studied in rat aortic rings. In endothelium‐intact preparations, T4MeN fully and similarly relaxed contractions induced by phenylephrine (PHE) (IC₅₀ = 61.41 [35.40‐87.42] μmol/L) and KCl (IC₅₀ = 83.50 [56.63‐110.50] μmol/L). The vasorelaxant effect of T4MeN was unchanged by endothelium removal, pretreatment with L‐NAME, indomethacin, tetraethylammonium, ODQ or MDL‐12,330A. Under Ca²⁺‐free conditions, T4MeN significantly reduced with a similar potency: (i) phasic contractions induced by PHE, but not by caffeine; (ii) contractions due to CaCl₂ in aortic preparations stimulated with PHE (in the presence of verapamil) or high KCl; (iii) contractions evoked by the restoration of external Ca²⁺ levels after depletion of intracellular Ca²⁺ stores in the presence of thapsigargin. In contrast, T4MeN was more potent at inhibiting contractions evoked by the tyrosine phosphatase inhibitor, sodium orthovanadate, than those induced by the activator of PKC, phorbol‐12,13‐dibutyrate. These results suggest that T4MeN induces an endothelium‐ independent vasorelaxation that appears to occur intracellularly through the inhibition of contractions that are independent of Ca²⁺ influx from the extracellular milieu but involve phosphorylation of tyrosine residues.     

Mechanisms underlying the renoprotective effect of GABA against ischaemia/reperfusion‐induced renal injury in rats

Excitation of the renal sympathetic nervous system is important for the development of ischaemic acute kidney injury (AKI) in rats. We reported that intravenous treatment with GABA has preventive effects against ischaemia/reperfusion (I/R)‐induced renal dysfunction with histological damage in rats; however, the mechanisms underlying these effects on renal injury remain unknown. Thus, the aim of the present study was to clarify how GABA mechanistically affects ischaemic AKI in rats. Ischaemic AKI was induced in rats by clamping the left renal artery and vein for 45 min and then reperfusing the kidney to produce I/R‐induced injury. Treatment with the GABA_B receptor antagonist CGP52432 (100 nmol/kg, i.v., or 1 nmol/kg, i.c.v.) abolished the suppressive effects of 50 μmol/kg, i.v., GABA on enhanced renal sympathetic nerve activity (RSNA) during ischaemia, leading to elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with 0.5 μmol/kg GABA or i.v. treatment with 1 μmol/kg baclofen, a selective GABA_B receptor agonist, prevented the I/R‐induced renal injury equivalent to i.v. treatment with GABA. Conversely, i.v. treatment with 10 μmol/kg bicuculline, a GABA_A receptor antagonist, failed to affect the preventive effects of GABA against ischaemic AKI. We therefore concluded that GABA_B receptor stimulation in the central nervous system, rather than peripheral GABA_B receptor stimulation, mediates the preventive effect of GABA against ischaemic AKI by suppressing the enhanced RSNA induced by renal ischaemia.