腺苷A1受体激活在钙调磷酸酶信号通路上对异丙肾上腺素诱导的心肌细胞肥大的抑制作用

目的观察腺苷A1受体激活在钙调磷酸酶(CaN)通路上对异丙肾上腺素(Iso)诱导的心肌细胞肥大的抑制作用及机制。方法体外培养大鼠乳鼠心肌细胞,以Iso 10μmol.L-1诱导心肌细胞肥大,观察腺苷A1受体激动剂R(-)-N6-(2-phenylIsopropyl)adenosine(R-PIA)1μmol.L-1对其作用,进一步探讨钙调神经磷酸酶特异性抑制剂环孢菌素A(CSA)1μmol.L-1、PKA抑制剂cAMP三乙胺盐(RP-cAMPS)1μmol.L-1、百日咳毒素(PTX)5 mg.L-1存在时,腺苷A1受体的激活对心肌细胞肥大的影响。通过Lowry法测心肌细胞蛋白含量;RT-PCR法检测心肌细胞心钠素(ANP)的mRNA表达;Western blot法测心肌细胞CaN的相对表达水平;以Fluo-3/AM为荧光探针,共聚焦显微镜下测量心肌细胞[Ca2+]i瞬变。结果 10μmol.L-1 Iso可以诱导心肌细胞肥大,腺苷A1受体激动剂R-PIA可以使其蛋白含量降低、ANP的mRNA表达减少、CaN相对表达降低、[Ca2+]i荧光强度减小,CSA、RP-cAMPS有类似抑制作用,PTX预处理的情况下,R-PIA对Iso诱导的心肌肥大的抑制作用消失。结论腺苷A1受体可以通过钙调磷酸酶通路抑制Iso诱导的心肌肥大,其机制与降低细胞内[Ca2+]i浓度及CaN表达有关。     

高胆固醇血症致大鼠心室肌细胞L-型钙电流失衡的机制

目的研究高胆固醇血症(HC)对大鼠心室肌细胞L-型钙电流(ICa-L)以及细胞内钙浓度[Ca2+]i的影响。方法Wistar大鼠随机分为高胆固醇血症组和对照组各6只,分别给予高胆固醇饲料和普通饲料饲养4wk后,检测血脂;酶解法急性分离大鼠单个心肌细胞,采用全细胞膜片钳技术和激光扫描共聚焦显微镜观察高胆固醇血症对大鼠心室肌细胞ICa-L以及[Ca2+]i的变化。结果喂高胆固醇饲料4wk后,血清总胆固醇较正常对照组明显增高(P<0·01),甘油三脂无明显变化。膜片钳显示:高胆固醇血症大鼠各实验电压L-型钙电流电流密度均减少。在实验电压为0mV时,大鼠心室肌细胞L-型钙电流密度从正常对照组(-8·56±1·29)pA/pF减少到高胆固醇血症组(-5·24±0·90)pA/pF。激光共聚焦显示:在静息状态下,[Ca2+]i升高,峰值荧光强度由211·88±9·08增加至458·63±23·50(P<0·01)。结论高胆固醇血症可导致细胞内钙超载,使L-型钙通道电流失衡,可能是诱导心律失常的原因之一。     

白藜芦醇对人胃癌SGC-7901细胞形态、线粒体膜电位、活性氧及钙离子浓度的影响

目的研究白藜芦醇对人胃癌SGC-7901细胞的影响。方法 SGC-7901细胞体外培养48h,分为白藜芦醇低、中、高剂量组(44、88、176μmol/L),阳性对照组(5-FU153.8μmol/L);阴性对照组(不含药物同体积培养液),荧光显微镜观察不同浓度的白藜芦醇对SGC-7901细胞的形态学影响;流式细胞仪检测白藜芦醇对肿瘤细胞中线粒体膜电位、活性氧的的影响;激光共聚焦显微镜观察白藜芦醇对肿瘤细胞中钙离子浓度的影响。结果显微镜下可见肿瘤细胞染色程度加深,染色质聚集、断裂,产生大小不等的凋亡小体,且随着白藜芦醇浓度加大,现象越来越明显,表明细胞凋亡的比例不断增加;白藜芦醇能够明显降低肿瘤细胞中线粒体膜电位,随着白藜芦醇浓度不断增加,肿瘤细胞中活性氧也不断增加,说明白藜芦醇能够提高肿瘤细胞中的活性氧水平来诱导其凋亡;白藜芦醇对肿瘤细胞中钙离子的浓度有一定的作用,其中高剂量能够显著提高肿瘤细胞中钙离子的浓度,且呈现一定的剂量依赖关系。结论白藜芦醇通过影响SGC-7901肿瘤细胞线粒体膜电位、活性氧及钙离子浓度导致肿瘤细胞凋亡,且与剂量相关。     

Characterization of [H]LUF5834: A novel non-ribose high-affinity agonist radioligand for the adenosine A1 receptor

The adenosine A₁ receptor is a promising therapeutic target for neurological disorders such as cognition deficits and is involved in cardiovascular preconditioning. Classically adenosine receptor agonists were all derivatives of adenosine, and thought to require a D-ribose moiety. More recently, however, the discovery of non-adenosine agonists for the human adenosine A₁ receptor (hA₁R) has challenged this dogma (Beukers et al., 2004). In this study we characterize the tritiated form of one of these compounds, [³H]LUF5834, as the first non-ribose partial agonist radioligand with nanomolar affinity for the hA₁R. Due to its partial agonist efficacy, [³H]LUF5834 labeled both G protein-coupled and uncoupled receptors with a similar high affinity. Using [³H]LUF5834 we performed competition binding experiments to characterize a range of A₁R ligands varying in efficacy from the full agonist CPA to the inverse agonist DPCPX. Surprisingly, in the control condition both agonists and inverse agonists displayed biphasic isotherms. With the addition of 1 mM GTP the high affinity isotherm of agonists or the low affinity isotherm of inverse agonists was lost revealing the mechanism of action of such inverse agonists at the A₁R. Consequently, [³H]LUF5834 represents a novel high affinity radioligand for the A₁R and may prove a useful tool to provide estimates of inverse agonist efficacy at this receptor.     

Determination of the adenosine A1 agonist N-cyclopentyladenosine in rat blood by solid-phase extraction and HPLC

A solid-phase extraction procedure has been developed for the isolation of the adenosine A1 receptor agonist N⁶-cyclopentyladenosine from rat blood. The biological samples were spiked with N⁶-cyclopentyladenosine and the analogue N⁶-cyclohexladenosine (internal standard), diluted with sodium hydroxide, loaded onto disposable cartridges with subsequent desorption with methanol and analysis by HPLC. The performance of columns pre-packed with different C18-bonded silica phases or with a polymeric reversed-phase sorbent (Oasis HLB) was assessed. The highest extraction efficiencies (recovery rates>83.3%) for the two N6-alkyl substituted adenosines were achieved by the Oasis HLB cartridges. In addition, the polymeric sorbent provided reproducible recoveries (relative standard deviation<4.8%), whereas large variations (relative standard deviation values, 9–16.3%) in the extraction yields were observed using the conventional silica-based C18 cartridges. The described sample preparation method is rapid, simple, selective and it is suitable for pharmacokinetic studies.     

Activation of the A3 adenosine receptor inhibits fMLP-induced Rac activation in mouse bone marrow neutrophils

Adenosine is released from injured or hypoxic tissues where it exerts numerous anti-inflammatory effects including suppression of neutrophil functions. Although most previous work has implicated the A_2AAR, we have recently shown that selective activation of the abundantly expressed A₃AR inhibits neutrophil superoxide production and chemotaxis providing a potential mechanistic explanation for the efficacy of A₃AR agonists in experimental animal models of inflammation. In this study, we hypothesized that the A₃AR suppresses neutrophil functions by inhibiting the monomeric GTPase Rac, a central regulator of chemokine-directed neutrophil migration and superoxide production. We found that pre-treating neutrophils with the highly selective A₃AR agonist CP-532,903 reduced fMLP-induced Rac activation using an ELISA-based assay that detects all three Rac isoforms. CP-532,903 also inhibited fMLP-induced F-actin formation, a downstream effector function of Rac relevant to neutrophil migration, but not activation of ERK1/2 or p38. Pre-treating neutrophils with CP-532,903 did not stimulate cAMP production or alter fMLP-induced calcium transients, implicating that A₃AR stimulation does not inhibit Rac activation or neutrophil activities by suppressing Ca²⁺ signaling, elevating the intracellular concentration of cAMP, or by cross-desensitizing fMLP receptors. Our results suggest that activation of the A₃AR signals to suppress neutrophil functions by interfering with the monomeric GTPase Rac, thus contributing to the ant-inflammatory actions of adenosine.     

Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: Insight into the receptor activation switch

A₃ Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A₃ adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A₃ adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N⁶-position. In contrast, the adenosine-5′-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A₃ agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5′-substituents on ligand–target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation.     

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death in HL-1 mouse cardiomyocytes expressing the A3 receptor

Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A₁ and A_2A adenosine receptors (ARs), but not of A_2B and A₃ARs. Activation of the heterologously expressed human A₃AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4 h exposure to H₂O₂ (750 μM) but not in untransfected cells. The A₃ agonist IB-MECA (EC₅₀ 3.8 μM) and the non-selective agonist NECA (EC₅₀ 3.9 μM) protected A₃ AR-transfected cells against H₂O₂ in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N⁶-chain-functionalized adenosine agonist was synthesized and its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical attachment sites. It non-selectively activated the A₃AR to inhibit forskolin-stimulated cAMP formation (IC₅₀ 66 nM) and, similarly, protected A₃-transfected HL-1 cells from apoptosis-inducing H₂O₂ with greater potency (IC₅₀ 35 nM) than monomeric nucleosides. Thus, a PAMAM conjugate retained AR binding affinity and displayed greatly enhanced cardioprotective potency.     

Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.     

Modulation of metalloproteinase-9 in U87MG glioblastoma cells by A3 adenosine receptors

In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A₃ receptors activation. We revealed that A₃ receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A₃ receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A₃ receptors, by inducing an increase in MMP-9 levels, was responsible for an increase of glioblastoma cells invasion. Collectively, these results suggest that ado, through A₃ receptors activation, modulates MMP-9 protein levels and plays a role in increasing invasion of U87MG cells.     

Cromakalim inhibits contractions of the rat isolated mesenteric bed induced by noradrenaline but not caffeine in Ca-free medium: Evidence for interference with receptor-mediated Ca mobilization

The effects of the K⁺ channel opener cromakalim on phasic contractions induced by noradrenaline and caffeine were studied in the rat isolated mesenteric bed. In the presence of 1.4 mM Ca²⁺, 1-s pulses of noradrenaline increased the perfusion pressure of the preparation concentration dependently (midpoint at 92 ± 10 μM noradrenaline). Cromakalim (0.3 and 1 μM) inhibited these contractions in a non-competitive manner. Contractions elicited by 1-s pulses of noradrenaline (100 μM) were inhibited by the dihydropyridine Ca²⁺ antagonist isradipine by maximally 24 ± 1%, indicating that only a minor component of this contraction depended on Ca²⁺ entry via dihydropyridine-sensitive Ca²⁺ channels. Cromakalim was a much more effective inhibitor of these contractions (maximum inhibition by 80%, midpoint of the inhibition curve at 171 ± 15 nM). The effect of cromakalim was stereoselective, inhibited by the sulphonylurea glibenclamide, and abolished in partially depolarizing media (KC1 = 35 and 50 mM). In Ca²⁺-free medium, cromakalim inhibited the contraction induced by noradrenaline (100 μM) by maximally 69 ± 4%, with a midpoint at 58 ± 14 nM. The effect of cromakalim was again stereoselective, inhibited by glibenclamide, and abolished in the presence of 50 mM KC1. Contractions induced by caffeine (10 and 100 μM) were not affected by cromakalim (1 μM). The results indicate that, in rat mesenteric resistance vessels, cromakalim interferes with the ability of noradrenaline, but not caffeine, to mobilize Ca²⁺ from intracellular stores. The antivasoconstrictor effect of cromakalim against noradrenaline is inhibited by glibenclamide and appears to be linked to the ability of cromakalim to hyperpolarize the cell membrane.     

The modulation of Ca2+ and K+ channels but not changes in cAMP signaling contribute to the inhibition of glutamate release by cannabinoid receptors in cerebrocortical nerve terminals

While cannabinoid receptors activate multiple signaling pathways in the brain, it remains unclear what influence the inhibition of adenylylcyclase has on the inhibition of glutamate release. In cerebrocortical nerve terminals, the cannabinoid receptor agonist WIN55,212-2 reduced KCl-evoked glutamate release through a mechanism that restricted the rise of cytoplasmic free Ca²⁺, but not the changes in plasma membrane depolarization. These effects were consistent with the inhibition of Ca²⁺ channels. Furthermore, WIN55,212-2 reduced 4-aminopyridine (4AP) evoked glutamate release to a larger extent by modulating the behavior of both Ca²⁺ and K⁺-channels. The inhibition of 4AP-evoked release was associated with a decrease in cytoplasmic free Ca²⁺ and in plasma membrane depolarization that was reverted by the potassium channel blocker, tetraethylammonium. Interestingly, the reduction of KCl- and 4AP-evoked release by WIN55,212-2 was independent of adenylylcyclase activity and did not affect cAMP. Forskolin and the β-adrenergic receptor increase intrasynaptosomal cAMP and promote a PKA-dependent tetrodotoxin (TTX)-sensitive increase in the spontaneous release of glutamate. These two responses were reduced by WIN55,212-2. However, the glutamate release induced by Sp-8-Br-cAMPS, which directly activated PKA without affecting cAMP, was also similarly reduced by WIN55,212-2. Hence, we conclude that the inhibition of glutamate release by WIN55,212-2 is unrelated to changes in cAMP and that the inhibition of release that a decrease in cAMP might produce is occluded by the activation of additional pathways such as the inhibition of Ca²⁺ channels and/or the activation of K⁺-channels that strongly depress glutamate release.     

Effect of the selective thromboxane A2 receptor antagonist, S-1452, on antigen-induced sustained bronchial hyperresponsiveness

Long-lasting bronchial hyperresponsiveness to i.v. acetylcholine was observed in actively sensitized guinea-pigs after aerosol ovalbium exposure. The response became significant at 7 h post-challenge and persisted for at least 120 h compared to the response of unsensitized animals. Pretreatment of animals with the specific thromboxane A₂ receptor antagonist, S-1452 (calcium (1R, 2S, 3S, 4S)-(5Z)-7-(((phenylsulfonyl)amino)bicyclo[2.2.1]hept-2-yl) hept-5-enoate dihydrate), almost completely inhibited the onset of bronchial hyperresponsiveness, as assessed at 24 and 120 h post-challenge. However, it was ineffective when administered at 1 h post-challenge or 2 h before assessment of bronchial responsiveness. Lung vascular injury occured transiently immediately after antigen challenge, the kinetics of injury being associated with those for the production of thromboxane B₂ in bronchoalveolar lavage fluid. The vascular injury was dramatically suppressed by pretreatment with S-1452. These findings suggest that acutely generated thromboxane A₂ plays an important role in the pathogenesis of antigen-induced long-lasting bronchial hyperresponsiveness, probably by producing vascular damage in the lungs.     

HPLC法测定复方黄根颗粒中人参皂苷Rg1、人参皂苷Rb1和三七皂苷R1含量

目的:建立复方黄根颗粒(无糖型)中有效成分三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1的含量测定方法。方法:色谱柱为Agilent ZORBAX SB-C18(250 mm×4.6 mm,5μm),以乙腈-水梯度洗脱,流速为1.0 ml·min-1,检测波长为203 nm,柱温25℃,进样量为10μl。结果:三七皂苷R1、人参皂苷Rg1和人参皂苷Rb1的线性范围分别为1.6~10.0μg·ml-1(r=0.999 6)、6.3~39.3μg·ml-1(r=0.999 8)和6.3~39.7μg·ml-1(r=0.999 7),平均加样回收率分别为98.81%(RSD=1.20%)、99.93%(RSD=0.93%)和99.22%(RSD=0.87%)(n=6)。结论:本方法操作简便、重复性好,可以更有效地控制该制剂的质量。     

Cytotoxicity induced in myotubes by a Lys49 phospholipase A2 homologue from the venom of the snake Bothrops asper: Evidence of rapid plasma membrane damage and a dual role for extracellular calcium

Acute muscle tissue damage, myonecrosis, is a typical consequence of envenomations by snakes of the family Viperidae. Catalytically-inactive Lys49 phospholipase A₂ homologues are abundant myotoxic components in viperid venoms, causing plasma membrane damage by a mechanism independent of phospholipid hydrolysis. However, the precise mode of action of these myotoxins remains unsolved. In this work, a cell culture model of C2C12 myotubes was used to assess the action of Bothrops asper myotoxin II (Mt-II), a Lys49 phospholipase A₂ homologue. Mt-II induced a dose- and time-dependent cytotoxic effect associated with plasma membrane disruption, evidenced by the release of the cytosolic enzyme lactate dehydrogenase and the penetration of propidium iodide. A rapid increment in cytosolic Ca²⁺ occurred after addition of Mt-II. Such elevation was associated with hypercontraction of myotubes and blebbing of plasma membrane. An increment in the Ca²⁺ signal was observed in myotube nuclei. Elimination of extracellular Ca²⁺ resulted in increased cytotoxicity upon incubation with Mt-II, suggesting a membrane-protective role for extracellular Ca²⁺. Chelation of cytosolic Ca²⁺ with BAPTA-AM did not modify the cytotoxic effect, probably due to the large increment induced by Mt-II in cytosolic Ca²⁺ which overrides the chelating capacity of BAPTA-AM. It is concluded that Mt-II induces rapid and drastic plasma membrane lesion and a prominent Ca²⁺ influx in myotubes. Extracellular Ca²⁺ plays a dual role in this model: it protects the membrane from the cytolytic action of the toxin; at the same time, the Ca²⁺ influx that occurs after membrane disruption is likely to play a key role in the intracellular degenerative events associated with Mt-II-induced myotube damage.     

Sensitivity to selective adenosine A1 and A2A receptor antagonists of the release of glutamate induced by ischemia in rat cerebrocortical slices

Adenosine released during cerebral ischemia is considered to act as a neuroprotectant, possibly through the inhibition of glutamate release. The involvement of A(1) and A(2A) receptors in the control of the rise of extracellular glutamate during ischemia was investigated by monitoring the effects of selective A(1) and A(2A) receptor antagonists on ischemia-evoked glutamate release in rat cerebrocortical slices.Slices were superfused with oxygen- and glucose-deprived medium and [(3)H]D-aspartate or endogenous glutamate was measured in the superfusate fractions. Withdrawal of Ca(2+) ions or addition of tetrodotoxin more than halved the ischemia-evoked efflux of [(3)H]D-aspartate or glutamate, compatible with a vesicular-like release. The glutamate transporter inhibitor DL-TBOA prevented the ischemia-evoked efflux of [(3)H]D-aspartate by about 40%, indicating a carrier-mediated efflux. The ischemia-evoked efflux of [(3)H]D-aspartate or glutamate was increased by the A(1) receptor antagonist DPCPX. The A(2A) antagonist SCH 58261 decreased [(3)H]D-aspartate or endogenous glutamate efflux (50 and 55% maximal inhibitions; EC(50): 14.9 and 7.6 nM, respectively); the drug was effective also if added during ischemia. No effect of either the A(1) or the A(2A) receptor antagonist was found on the ischemia-evoked efflux of [(3)H]D-aspartate in Ca(2+)-free medium. Our data suggest that adenosine released during cerebral ischemia can activate inhibitory A(1) and stimulatory A(2A) receptors that down- or up-regulate the vesicular-like component of glutamate release.     

Umbelliferone induces changes in the structure and pharmacological activities of Bn IV, a phospholipase A2 isoform isolated from Bothrops neuwiedi

In this paper was demonstrated that umbelliferone induces changes in structure and pharmacological activities of Bn IV, a lysine 49 secretory phospholipase A₂ (sPLA2) from Bothrops neuwiedi. Incubation of Bn IV with umbelliferone virtually abolished platelet aggregation, edema, and myotoxicity induced by native Bn IV. The amino acid sequence of Bn IV showed high sequence similarities with other Lys49 sPLA2s from B. jararacussu (BthTx-I), B. pirajai (PrTx-I), and B. neuwiedi pauloensis (Bn SP6 and Bn SP7). This sPLA2 also has a highly conserved C-terminal amino acid sequence, which has been shown as important for the pharmacological activities of Lys49 sPLA2. Sequencing of Bn IV previously treated with umbelliferone revealed modification of S(1) and S(20). Fluorescent spectral analysis and circular dichroism (CD) studies showed that umbelliferone modified the secondary structure of this protein. Moreover, the pharmacological activity of Bn IV is driven by synergism of the C-terminal region with the α-helix motifs, which are involved in substrate binding of the Asp49 and Lys49 residues of sPLA2 and have a direct effect on the Ca²⁺-independent membrane damage of some secretory snake venom PLA2. For Bn IV, these interactions are potentially important for triggering the pharmacological activity of this sPLA2.     

The inhibition of release by mGlu7 receptors is independent of the Ca2+ channel type but associated to GABAB and adenosine A1 receptors

Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca(2+) channels and adenylyl cyclase. Through Ca(2+) imaging and immunocytochemistry, we have recently shown that adenosine A(1), GABA(B) and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca(2+) channels by analysing this process in nerve terminals from mice lacking the alpha(1B) subunit (Ca(v) 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca(2+) channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABA(B) and adenosine A(1). Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A(1), GABA(B) and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca(2+) channel type but rather, it is associated to functional mGlu7 receptors.     

Analgesic effect of paeoniflorin in rats with neonatal maternal separation-induced visceral hyperalgesia is mediated through adenosine A1 receptor by inhibiting the extracellular signal-regulated protein kinase (ERK) pathway

Paeoniflorin (PF), a chief active ingredient in the root of Paeonia lactiflora Pall (family Ranunculaceae), is effective in relieving colorectal distention (CRD)-induced visceral pain in rats with visceral hyperalgesia induced by neonatal maternal separation (NMS). This study aimed at exploring the underlying mechanisms of PF's analgesic effect on CRD-evoked nociceptive signaling in the central nervous system (CNS) and investigating whether the adenosine A₁ receptor is involved in PF's anti-nociception. Results: CRD-induced visceral pain as well as phosphorylated-extracellular signal-regulated protein kinase (p-ERK) and phospho-cAMP response element-binding protein (p-CREB) expression in the CNS structures of NMS rats were suppressed by NMDA receptor antagonist dizocilpine (MK-801) and ERK phosphorylation inhibitor U0126. PF could similarly inhibit CRD-evoked p-ERK and c-Fos expression in laminae I-II of the lumbosacral dorsal horn and anterior cingulate cortex (ACC). PF could also reverse the CRD-evoked increased glutamate concentration by CRD as shown by dynamic microdialysis monitoring in ACC, whereas, DPCPX, an antagonist of adenosine A₁ receptor, significantly blocked the analgesic effect of PF and PF's inhibition on CRD-induced p-ERK and p-CREB expression. These results suggest that PF's analgesic effect is possibly mediated by adenosine A₁ receptor by inhibiting CRD-evoked glutamate release and the NMDA receptor dependent ERK signaling.     

A comparison of the selectivities of SCH 23390 with BW737C89 for D1, D2 and 5-HT2 binding sites both in vitro and in vivo

The in vitro affinities (K_Is) for SCH 23390 in D₁, D₂ and 5-HT₂ binding assays were 0.4, 631 and 20 nM as compared with 0.3, 79 and 79 nM for BW737C89. The K_B values, derived from their abilities to right-shift dopamine-mediated dose-dependent increases in striatal adenylyl cyclase activity, were 0.8 and 0.5 nM for SCH 23390 and BW737C89, respectively. Thus, BW737C89 was a highly potent dopamine D₁ receptor antagonist and, although it was less D₁/D₂-selective than SCH 23390, it was more D₁/5-HT₂-selective. Both SCH 23390 and BW737C89 (0.1–100 μmol/kg s.c.) exhibited a selective dose-dependent protection of D₁, but not D₂, binding, from inactivation by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ, 8 mg/kg s.c.) as measure by [³H]SCH 23390 (D₁) and [³H]spiperone (D₂) binding. The ED₅₀ values for this selective protection of D₁ binding were similar and were between 1 and 3 μmol/kg s.c. BW737C89 showed no protective effect at all on the inactivation of [³H]ketanserin (5-HT₂) binding by EEDQ whereas SCH 23390 started to show protection at doses of 10 μmol/kg. s.c. and above. A direct comparison of the time course of the effects of pretreatment of a dose of 30 μmol/kg s.c. of both compounds to protect 5-HT₂ binding was carried out. This study confirmed the complete lack of protective effect of BW737C89 from 1 to 4 h of pretreatment whereas SCH 23390 exhibited 62, 29 and 28% protection at 1,2 and 4 h pretreatment respectively. Thus, these data clearly show that BW737C89 is a potent and selective D₁ antagonist which is more selective for D₁ receptors in vivo than is SCH 23390.