核因子-κB诱骗剂局部应用对胶原诱导性关节炎大鼠治疗作用的研究

探讨核因子-κB诱骗剂(NF-κB decoy)局部应用对Ⅱ型胶原诱导关节炎(CIA)大鼠的治疗作用及其对滑膜基质金属蛋白酶-3(MMP-3)、IL-17、TGF-β表达的影响。建立Ⅱ型胶原诱导大鼠关节炎模型,用NF-κB诱骗剂注射到大鼠右后踝关节腔内,并设对照组、CIA模型组和实验组。42d后观察各组关节炎指数、病理指标,采用ELISA法检测各组大鼠关节液中IL-17、IL-12和TGF-β的水平,免疫组织化学方法检测关节组织MMP-3、TGF-β和IL-17表达。与对照组比较,CIA模型组大鼠关节炎指数、关节液中IL-17、IL-12水平均明显升高,滑膜组织MMP-3、IL-17阳性表达的积分光密度(IOD)值明显增加;实验组经NF-κB诱骗剂注射后,与CIA模型组比较,关节炎指数、关节液中IL-17、IL-12水平均明显降低,而TGF-β的含量显著增高,滑膜组织MMP-3、IL-17阳性表达的IOD值明显降低,TGF-β阳性表达的IOD值显著增高。NF-κB诱骗明显减轻CIA大鼠的关节炎症状,延缓关节破坏的进展,对CIA有较好的治疗作用。     

环氧合酶-2抑制剂对骨关节炎大鼠关节软骨中白介素-1β表达的影响

目的研究环氧合酶-2抑制剂对骨关节炎大鼠关节软骨中自介素-1β（IL-1β）表达的影响。方法健康雄性Wistar大鼠30只,随机分为对照组、骨性关节炎组（OA组）和环氧合酶-2抑制剂（吲哚美辛）处理组,每组10只。利用膝关节注射4％的木瓜蛋白酶溶液的方法制作骨关节炎模型。采用关节炎评分法评定各组大鼠平均关节炎指数（MAI）,免疫组化方法与Westernblot方法检测关节软骨中IL-1β蛋白的表达变化。结果OA模型组关节出现重度红肿现象,对照组关节无异常,环氧合酶-2抑制剂组炎症反应比0A模型组减轻。环氧合酶-2抑制剂组平均关节炎指数比0A模型组显著降低（p〈0．01）。IL-1β蛋白在对照组大鼠关节软骨仅有微量表达,而在OA组表达则显著升高（P〈0．01）,环氧合酶-2抑制剂组比0A组显著降低（P〈0．01）。结论吲哚美辛对骨关节炎的治疗作用与其下调IL-1β蛋白表达相关。     

雷公藤多甙对胶原诱导性关节炎大鼠IL-10表达的影响

目的研究雷公藤多甙对类风湿关节炎大鼠的作用,探讨类风湿关节炎的发病机制及雷公藤多甙的作用机制,为临床应用提供理论基础。方法本实验应用鸡Ⅱ型胶原蛋白,建立胶原诱导性关节炎(collagen-induced arthritis,CIA)大鼠模型。应用酶联免疫吸附试验方法(ELISA)检测观察正常对照组、CIA模型组和雷公藤多甙治疗组大鼠血液及关节液中白细胞介素-10(interleukin-10,IL-10)的浓度。结果 ELISA检测结果显示,类风湿关节炎大鼠血清和关节液中IL-10含量明显减低,而雷公藤多甙可以升高IL-10的浓度,使之趋向正常。结论雷公藤多甙可有效减轻佐剂型关节炎大鼠的关节病变,并能使CIA大鼠血清、关节浸液IL-10分子水平增高。     

昆明山海棠对CIA大鼠模型中HIF-1α表达的影响

目的:研究昆明山海棠(THH)对胶原诱导性关节炎(CIA)大鼠模型中低氧诱导因子1α(HIF-1α)表达的影响及其作用机制。方法:建立CIA大鼠模型,随机分组,治疗组分别采用高、中、低剂量THH每天灌胃1次,连续用药30d。动态观察关节炎指数(AI)及关节的病理改变,用RT-PCR及免疫组化染色法分别检测HIF-1αmRNA及其蛋白的表达。结果:THH可明显抑制CIA大鼠的足爪肿胀,明显减轻滑膜增殖及炎症反应,HIF-1α表达明显降低。结论:THH通过降低HIF-1α的表达,减轻CIA模型大鼠关节的炎症反应。     

昆明山海棠对CIA大鼠模型中HIF-1α表达的影响

目的:研究昆明山海棠(THH)对胶原诱导性关节炎(CIA)大鼠模型中低氧诱导因子1α(HIF-1α)表达的影响及其作用机制.方法:建立CIA大鼠模型,随机分组,治疗组分别采用高、中、低剂量THH每天灌胃1次,连续用药30d.动态观察关节炎指数(AI)及关节的病理改变,用RT-PCR及免疫组化染色法分别检测HIF-1 αmRNA及其蛋白的表达.结果:THH可明显抑制CIA大鼠的足爪肿胀,明显减轻滑膜增殖及炎症反应,HIF-1α表达明显降低.结论:THH通过降低HIF-1α的表达,减轻CIA模型大鼠关节的炎症反应.     

雷公藤内酯醇对大鼠胶原性关节炎及足爪组织基质金属蛋白-2表达的影响

目的:研究雷公藤内酯醇(TP)对大鼠胶原性关节炎(CIA)及足爪组织基质金属蛋白酶-2(MMP-2)表达的影响。方法:采用鸡Ⅱ型胶原建立CIA大鼠模型,观察药物对CIA大鼠体重及足肿胀度的影响,免疫组化法观察大鼠足爪组织中MMP-2蛋白的表达。结果:TP可有效缓解CIA大鼠体重减轻及明显抑制CIA大鼠足爪肿胀;治疗后TP组足爪组织MMP-2阳性表达比模型对照组明显降低(P<0.01)。结论:TP对CIA大鼠具有治疗作用,其机制可能与其下调MMP-2表达、降低血清炎性细胞因子水平有关。     

乌梢蛇水解液对大鼠胶原性关节炎的防治作用

本研究观察乌梢蛇水解液对大鼠胶原性关节炎(CIA)的作用。实验分预防性和治疗性疗效观察两部分,均设低剂量组(0.5 mg/kg)、中剂量组(5 mg/kg)、高剂量组(15 mg/kg)和空白对照组,预防性疗效观察于大鼠致炎前14、11、8、5和2d,给药共5次;治疗性疗效观察于大鼠致炎后第18天,将患关节炎的大鼠分组,每天给药一次,共21d。分别于预防给药大鼠致炎后第25天和治疗给药大鼠致炎后第39天记录各组大鼠关节炎的发病率、关节炎症状指数、血清Ⅱ型胶原抗体水平和皮肤对Ⅱ型胶原的迟发型超敏反应(DTH)。结果表明预防给药中剂量和高剂量乌梢蛇水解液能降低大鼠CIA的发病率、改善关节炎症状(P<0.05),治疗给药中剂量和高剂量能减轻关节炎症状(P<0.05);低剂量无预防发病率和治疗作用。中、高剂量药物均能降低预防和治疗给药CIA的血清抗Ⅱ型胶原抗体水平和皮肤对Ⅱ型胶原的DTH。实验提示乌梢蛇水解液对大鼠CIA有预防和治疗作用。     

环氧合酶-2抑制剂对大鼠骨关节炎模型软骨中血管内皮生长因子表达的影响

目的研究环氧合酶-2抑制剂(吲哚美辛)对大鼠骨关节炎模型关节软骨中血管内皮生长因子(VEGF)表达的影响。方法健康雄性wistar大鼠30只,随机均分为对照组、骨性关节炎组(OA组)和吲哚美辛处理组。利用膝关节注射4%的木瓜蛋白酶溶液的方法制作骨关节炎模型。采用关节炎评分法评定各组大鼠平均关节炎指数(MAI),免疫组化方法与Western blot方法检测关节软骨中VEGF蛋白的表达变化。结果 OA模型组随着造模的时间延长,关节出现了重度红肿现象,对照组关节无任何异常变化;与OA模型组比较,吲哚美辛组的关节炎症反应呈现消退现象。VEGF蛋白在对照组大鼠关节软骨仅有微量表达,而在术后8周OA组大鼠关节软骨表达则显著升高(P0.01);与OA组相比,吲哚美辛组大鼠关节软骨中VEGF蛋白表达显著降低(P0.01)。结论吲哚美辛对骨关节炎的抑制作用可能与下调VEGF蛋白的表达相关。     

THH在CIA大鼠模型中对HIF-1α的影响及其作用机制

目的:研究昆明山海棠(THH)在胶原性关节炎(CIA)大鼠模型中对于低氧诱导因子(HIF-1α)、血管内皮生长因子(VEGF)表达的影响,从而探讨其作用机制。方法:建立Wistar大鼠Ⅱ型胶原诱导性关节炎(CIA)模型,动态观察关节炎指数(AI),HE染色观察关节的病理改变,RT-PCR以及免疫组化法检测HIF-1α的表达。结果:给药30天后,治疗组明显抑制胶原性关节炎大鼠的足爪肿胀,降低关节炎指数,HE染色显示明显减轻滑膜增殖以及炎症反应,免疫组化以及PT-PCR显示治疗组HIF-1α明显表达降低。结论:THH治疗大鼠CIA模型具有显著疗效,研究提示THH通过HIF-1α的途径调节其下游基因的表达可能是治疗RA患者的作用机制之一。     

甘草附子汤加减方通过抑制GSDMD介导的焦亡治疗CIA大鼠的作用机制研究

目的：探讨甘草附子汤加减方（GCFZD）抑制gasdermin D (GSDMD)介导细胞焦亡途径对胶原诱导性关节炎（CIA）大鼠的治疗效果。方法：SD大鼠尾跟部注射由牛Ⅱ型胶原与弗氏佐剂混合形成的乳化剂，构建CIA大鼠模型并对其关节炎指数进行评估，将成功诱发关节炎的30只雄性SD大鼠随机分为模型组、甘草附子汤低（4 g/kg）、中（8 g/kg）和高（16 g/kg）剂量组及甲氨蝶呤（1 mg/kg）组，每组6只，连续给药30 d。测定大鼠的体重、关节炎指数和足爪肿胀指数；X线影像学观察骨质破坏和软组织厚度改变；HE染色观察脾脏和关节组织病理学改变；番红O-固绿染色观察关节软骨改变；TUNEL染色观察关节内细胞焦亡发生情况；Western blot测定TLR4、caspase-1、NLRP3和GSDMD蛋白表达；real-time PCR和ELISA测定IL-1β、IL-6和IL-10细胞因子表达。结果：与模型组相比，GCFZD治疗显著改善CIA大鼠软组织肿胀和骨质破坏，降低脾脏和胸腺指数；HE染色结果显示，GCFZD治疗减轻CIA大鼠脾脏和踝关节组织病理学改变；番红O-固绿染色结果...     

The mechanism of action of the new antiinflammatory compound ML3000: inhibition of 5-LOX and COX-1/2

OBJECTIVE: We examined the effects of ML3000 and several non-steroidal antiinflammatory drugs (NSAIDs) on the synthesis of products of 5-LOX (LTB4, LTC4) and COX-1/2 (TXB2, PGE2) in vitro and ex vivo in order to further elucidate the mechanism of action of ML3000. METHODS AND RESULTS: Using a human whole blood assay the effect of ML3000 on the shunt of arachidonic acid to the lipoxygenase pathway when COX is blocked was studied. ML3000 (0.3, 1, 3, 10, 30 microg/ml) and indomethacin (0.3, 1, 3, 10, 30 microg/ml) concentration-dependently inhibited the synthesis of PGE2 (IC50 = 3.9 and 4.5 microM). In contrast to ML3000, indomethacin produced an increase of LTC4 of up to 155.5% of control. 5-lipoxygenase inhibition was further tested in a basophilic leukemia cell assay using RBL-1 cells. ML3000 (1-10 microM) inhibited the synthesis of LTB4 in a concentration related manner (IC50: 3.6 microM). In carrageenan induced rat paw edema, ML3000 and indomethacin completely blocked the formation of PGE2 in the inflamed tissue. The LTB4 production in the inflamed paw was reduced to basal levels by ML3000 (10 +/- 1.4 pg/paw saline control and 7.5 +/- 1.3-5.9 +/- 3.2 pg/paw ML3000), whereas LTB4 levels remained markedly elevated as compared to saline control by indomethacin (30.7 pg/paw). 5-LOX inhibition in the inflamed rat colon was investigated by measuring LTB4 synthesis. MK-886 and ML3000 at 10 mg/kg p.o. reduced LTB4 production to 29.8 +/- 4.9 and 30.1 +/- 2.8 pg/mg tissue as compared to control (54.2 +/- 7.4 mg/kg tissue). LTB4 levels in the rat stomach were comparable to control (2.5 +/- 0.4 pg/mg protein) after oral administration of ML3000 (10, 30, 100 mg/kg), whereas oral treatment with indomethacin (0.3, 1, 3 mg/kg) or diclofenac (1, 3 mg/kg) increased LTB4 up to 9.2 +/- 2.3 or 8.9 +/- 1.6 pg/mg protein. This effect was significant at 1 mg/kg diclofenac and 0.3 mg/kg indomethacin. CONCLUSIONS: These results provide further evidence, that ML3000 inhibits 5-LOX as well as COX-1 and COX-2 in vitro and in animal experiments. The favourable gastrointestinal (GI) tolerability of the compound is believed to be linked to the mechanism of combined 5-LOX and COX-1/2 inhibition of ML3000.     

Aspirin-intolerant asthma: role of cyclo-oxygenase enzymes

Aspirin-induced asthma and rhinitis (AIAR) appear to be precipitated by the inhibition of cyclo-oxygenase (COX). By inhibiting COX pathway aspirin diverts arachidonic acid metabolites to the lipoxygenase pathway. There are two isoforms of COX, namely COX-1 and COX-2. Metabolites derived from COX-1 are involved in cellular housekeeping functions. COX-2 can be induced in cells exposed to proinflammatory substances and growth factors. Recent studies have reported that patients with AIAR have decreased activity of COX-2 and lower production of PGE₂ in the upper airway and peripheral blood cells. Considering the protective effect of exogenous PGE₂ on aspirin-induced bronchoconstriction and the interdependence of PGE₂ and cisteinyl leukotriene production, a reduced PGE₂ synthesis may render aspirin-sensitive patients more susceptible to the inhibitory effect of NSAIDs drugs and also lead to an increase in cysteinyl leukotriene release.     

Induction of endothelin-1 expression by oxidative stress in vascular smooth muscle cells

Atherosclerosis is based on endothelial dysfunction leading to impaired vasomotor function. This is partially due to nitric oxide (NO) depletion caused by oxidative stress. Since the vasoconstrictor endothelin-1 (ET-1) might also be involved in endothelial dysfunction, we investigated whether oxidative stress regulates ET-1 expression in vascular smooth muscle cells (VSMC). Human aortic VSMC were treated with H₂O₂ (200 μM) for up to 8 h. mRNA expression of preproendothelin (prepro-ET) was analyzed by RT-PCR. ET-1 protein and the marker for oxidative stress, 8-isoprostane, were determined by ELISA. Activity of cytosolic phospholipase A2 (cPLA₂) as an indicator of ET-1 autocrine activity was measured photometrically. Stimulation of VSMC with H₂O₂ resulted in increased expression of prepro-ET mRNA after 1 h with a maximum after 6 h (fourfold), similar to treatment with angiotensin II. ET-1 protein was significantly increased by H₂O₂ treatment with a maximum after 8 h (P<.05). This effect was inhibited by the antioxidants resveratrol (100 μM) and quercetin (50 μM). In quiesced VSMC, incubation with H₂O₂-conditioned medium resulted in increased cPLA₂ activity compared to the controls (P<.05). This activity was partially inhibited by the ET_A-receptor antagonist, PD 142893 (10 μM), indicating functional ET-1 in the conditioned medium. The presence of oxidative stress in H₂O₂-treated VSMC was associated by significantly increased formation of 8-isoprostane (P<.05). The data indicate for the first time that oxidative stress increases ET-1 generation and autocrine ET-1 activity in VSMC, a mechanism that might contribute to endothelial dysfunction in atherosclerosis.     

脂氧素A4抑制内毒素诱导的人支气管上皮细胞环氧合酶2及前列腺素E2的表达

目的: 探讨脂氧素A₄对人支气管上皮细胞(HBECs)环氧合酶2(COX-2)表达的影响。方法: 应用不同浓度(0.1、1、10 mg/L)的内毒素(LPS)刺激HBECs 9 h,或者用1 mg/L LPS分别刺激HBECs不同时点(3 h、6 h、9 h)后,测定HBECs的COX-2 mRNA表达和细胞上清液前列腺素E₂(PGE₂)水平。应用不同浓度 (0、100、400 μmol/L) 的脂氧素A₄作用于经过LPS(1 mg/L)刺激培养9 h的HBECs,采用酶联免疫吸附法(ELISA)检测细胞上清液PGE₂的水平, 同时分别应用RT-PCR和Western blotting分别检测HBECs COX-2 mRNA及蛋白的表达。结果: LPS刺激培养条件下HBECs的COX-2 mRNA表达及其上清液PGE₂水平增加,并呈时间、剂量依赖性。脂氧素A₄能抑制LPS刺激培养HBECs COX-2蛋白和mRNA的表达及上清液PGE₂的水平,并呈剂量依赖性。结论: 脂氧素A₄能抑制LPS诱导的HBECs COX-2表达及上清液PGE₂的水平。     

普罗布考通过TGF-β1/Smad 2/3通路减轻大鼠脑缺血再灌注损伤

目的:探讨普罗布考对大鼠脑缺血再灌注(I/R)的影响及其可能机制。方法:线栓法建立大鼠大脑中动脉栓塞(MCAO)模型,用1.5、3 mg/kg普罗布考进行干预,将200只大鼠分为假手术组、I/R组、溶剂组、小剂量组(1.5 mg/kg)及大剂量组(3mg/kg)。术后15 min腹腔注射药物,于1、3、5 d和7 d取材。观察普罗布考对脑缺血再灌注炎性损伤急性期及恢复早期神经功能评分、梗死灶体积、脑组织水含量的影响,并用ELISA检测脑组织匀浆环氧酶(COX-2)和5脂氧酶(5-LOX)活性变化,免疫印迹检测各组TGF-β1和p-Smad2/3蛋白表达量的变化。结果:与假手术组比较,MCAO术后小鼠出现严重的神经功能障碍、脑水肿和脑梗死,COX-2和5-LOX蛋白表达显著升高,而TGF-β1和p-Smad2/3蛋白表达明显降低;与手术组比较,经普罗布考干预后,小鼠神经功能评分、脑水肿和脑梗死情况明显改善,COX-2和5-LOX蛋白表达明显降低,而TGF-β1和p-Smad2/3蛋白表达明显升高,且大剂量组优于小剂量组。结论:普罗布考能缓解大鼠脑缺血再灌注炎性损伤,其机制可能与激活TGF-β1/Smad2/3信号通路,抑制脑内COX-2与5-LOX的活性有关。     

心肌内向整流钾通道激动剂抑制异丙肾上腺素所致大鼠心室重构

目的:观察和探讨内向整流钾通道(Ⅰ_(K1))激动剂盐酸扎考必利(zacopride,Zac)对异丙肾上腺素(isoproterenol,Iso)所致心室重构的影响及作用机制。方法:SD大鼠随机分为正常对照组、Iso模型组、Zac干预组、Zac+氯喹干预组和卡托普利阳性对照组。腹腔注射异丙肾上腺素3 mg/kg,每天1次,连续给药10 d,观测各组全心质量/体质量比和左心室质量/体质量比。用全细胞膜片钳技术检测大鼠心室肌细胞电压门控钙电流(Ⅰ_(Ca-L))、静息膜电位(RMP)及动作电位时程(APD)的变化。选用新生1~3 d的SD乳鼠,用0.08%胰蛋白酶和0.04%Ⅱ型胶原酶消化心脏组织,经差速贴壁法和5-溴脱氧尿嘧啶核苷纯化心肌细胞后随机分成正常对照组、Iso模型组、Zac干预组、Zac+BaCl_2干预组和Zac+氯喹干预组,培养24 h后用激光共聚焦显微镜检测心肌细胞内游离钙离子浓度。结果:Iso模型组与正常对照组比较,全心肥厚指数、左心室肥厚指数明显增加,膜片钳结果提示RMP减小APD明显延长;Zac干预组明显抑制心肌肥大,并增大RMP,缩短APD。同时应用低剂量Ⅰ_(K1)抑制剂氯喹可明显抑制Zac的抗心室重构作用,并逆转Zac对RMP和APD影响。在乳鼠心肌细胞,Iso可使细胞表面积增大,细胞内[Ca~(2+)]_i增高;Zac干预后细胞形态恢复至正常或接近正常水平,并显著减轻钙超载。Ⅰ_(K1)阻断剂BaCl_2和氯喹可阻断Zac的效应。结论:Ⅰ_(K1)选择性激动剂Zac明显抑制异丙肾上腺素所致的心室重构,其机制可能为增强Ⅰ_(K1),进而增大RMP,缩短APD,从而阻断心肌细胞内钙超载依赖的信号通路。     

Combination therapies that inhibit cyclooxygenase-2 and leukotriene synthesis prevent disease in murine collagen induced arthritis

Objective and design:  To determine the effect of combinations of cyclooxygenase (COX) inhibitors and inhibitors of leukotriene (LT) syntheses on collagen induced arthritis (CIA) in mice. Methods:  The CIA model was evaluated for the presence of eicosanoids in the paw tissue. Several selective cyclooxygenase 2 (COX-2) inhibitors or non-selective non-steroidal anti inflammatory drugs (NSAIDs) were evaluated alone or in combination with leukotriene (LT) synthesis inhibitors in the CIA model. Results:  Arthritic paw tissue showed increased levels of prostaglandins and leukotrienes in comparison to normal paws. Analysis of mRNA levels indicated the inducible form of the COX-2 enzyme to be the source of prostaglandins. NSAIDs, COX-2 or leukotriene synthesis inhibitors administered alone in CIA decreased severity but had little effect on disease incidence. However, the combination of selective COX-2 inhibitors with leukotriene synthesis inhibitors produced significant decreases in both incidence and severity, suggesting an additive or synergistic effect. This effect was reversible with removal of drug. Little decrease in incidence was observed with the NSAID/5-LO inhibitor combinations. Conclusions:  These results suggest that the induction of the disease in CIA is mediated by products of the COX-2 enzyme and LTB4 production, and that blockade of both pathways is required to prevent CIA. Received 9 July 2008; returned for revision 20 August 2008; received from final revision 19 September 2008; accepted by J. A. Battista 15 October 2008     

Immunohistochemical localization of anabolic and catabolic enzymes for anandamide and other putative endovanilloids in the hippocampus and cerebellar cortex of the mouse brain

An increasing body of evidence indicates that: 1) the endocannabinoid anandamide (AEA) and other unsaturated N-acylethanolamines (NAEs), 2) 12-(S)-lipoxygenase (12-LOX) products of arachidonic acid, and 3) unsaturated N-acyldopamines (NADAs), act as endogenous ligands of transient receptor potential vanilloid type 1 (TRPV1) channels at intracellular binding sites. AEA is synthesized and released "on demand" in neurons from its membrane precursor, N-arachidonoyl-phosphatidylethanolamine, by an N-acyl-phosphatidylethanolamine-specific phospholipase D (NAPE-PLD), and is inactivated by intracellular hydrolysis by fatty acid amide hydrolase (FAAH), whereas catechol-O-methyl-transferase (COMT) was suggested to inactivate NADAs. However, it is not known whether these enzymes or 12-LOX co-localize to any extent with TRPV1 receptors in the brain. In this study we used immunohistochemical techniques (single peroxidase and double immunofluorescence staining), and analyzed the localization of the TRPV1 channel in mouse hippocampal and cerebellar neurons with respect to NAPE-PLD, FAAH, 12-LOX and COMT. Cycloxygenase-2 (COX-2), another putative AEA-degrading enzyme, was also studied. Co-localization between TRPV1 and either NAPE-PLD or FAAH, COX-2, 12-LOX and COMT was found in Ammon's horn (CA3) hippocampal pyramidal neurons and (with the exception of 12-LOX) in some Purkinje cells. At the cellular level, both anabolic and catabolic enzymes appeared as fine grains with immunoperoxidase labeling and were observed in the somatodendritic compartment of CA3 pyramidal cells as well as (with the exception of 12-LOX) in the cytoplasm of Purkinje neurons, in which FAAH and COX-2 immunoreactivities were, however, preferentially localized in the large extension of the dendritic arbor. Our data agree with the hypothesis that, in potential "endovanillergic" neurons, endogenous TRPV1 agonists, and AEA in particular, act as intracellular mediators by being produced from and/or degraded by the same mouse brain cells that express TRPV1 receptors.