P2Y受体介导大鼠胃体环行肌收缩反应及相关受体亚型mRNA和受体蛋白的表达
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摘要
核苷酸类物质对胃肠道动力和分泌功能具有重要的调节作用,其中ATP主要通过P2受体介导胃肠道平滑肌的收缩与舒张反应。有研究发现ATP诱发大鼠近端胃环行肌先舒张后收缩的双相反应。而本室研究曾报道,ATP仅使胃体环行肌产生收缩反应。我们推测,大鼠胃体环行肌的药理学特征可能不同与胃底环行肌,是分析P2受体介导胃平滑肌收缩反应的理想实验标本。P2受体包括P2X1-7和P2Y1-14多种亚型,功能复杂。目前,有关核苷酸类物质诱发大鼠胃体环行肌收缩反应的P2Y受体亚型分析、大鼠胃体平滑肌P2Y受体亚型mRNA表达的绝对定量分析及P2Y1与P2Y2受体在大鼠胃体平滑肌的表达,尚未见文献报道。本实验研究了P2受体介导大鼠胃体环行肌收缩反应的药理学特点,并应用实时荧光定量PCR技术绝对定量分析大鼠胃体平滑肌P2Y受体亚型mRNA的表达和免疫组织化学技术分析大鼠胃体平滑肌P2Y1与P2Y2受体的表达。
第一部分核苷及核苷酸类物质介导大鼠胃体环行肌收缩反应的药理学特点
制备大鼠离体胃体和胃底环行肌标本。利用受体药理学技术观察大鼠胃体和胃底环行肌不同的药理学特征,并进一步分析核苷及核苷酸类物质诱发胃体环行肌收缩反应的作用特点。
1 CCh、5-HT、His和KCl对大鼠胃体环行肌和胃底环行肌的作用
在胃体环行肌KCl所致收缩反应与胃底环行肌无显著性差别;但是,CCh收缩胃体环行肌的EC50值(0.45±0.15μmol?L-1)显著高于胃底环行肌(0.20±0.09μmol?L-1,P<0.01)。5-HT和His收缩两种标本的EC50值无显著差异(P>0.05);但是,在胃体环行肌5-HT和His产生收缩反应的Emax值(0.81±0.26和0.88±0.27 g)显著小于胃底环行肌(2.67±0.61和1.90±0.68 g,P<0.01)。
2 ATP对预收缩大鼠胃体环行肌和胃底环行肌的作用
在预收缩胃体环行肌,ATP(0.1-3000μmol?L-1)诱发浓度依赖性收缩反应,未见舒张反应;在预收缩胃底环行肌标本,同浓度ATP诱发先舒张后收缩的双相反应,并呈浓度依赖性。
3核苷及核苷酸对大鼠胃体环行肌的作用
ATP、UTP、ADP、2-MeSATP和α,β-MeATP浓度依赖性诱发大鼠胃体环行肌收缩反应,2-MeSATP的EC50值为7.2±5.2 nmol?L-1比ACh(3.47±1.20μmol?L-1)低500倍;各药物产生收缩反应的效价序列为:2-MeSATP>>ADP>ATP=UTP>α,β-MeATP>>腺苷。
4酚妥拉明、普萘洛尔、阿托品及河豚毒素对ATP和UTP诱发大鼠胃体环行肌收缩反应的影响
酚妥拉明(1μmol?L-1)、普萘洛尔(1μmol?L-1)、阿托品(1μmol?L-1)及河豚毒素(0.1μmol?L-1)不影响ATP和UTP诱发的胃体环行肌收缩反应。
上述结果表明,大鼠胃体环行肌的药理学特征明显不同于胃底环行肌;核苷及核苷酸类物质诱发大鼠收缩反应的效价序列为2-MeSATP>> ADP> ATP= UTP>α,β-MeATP>>腺苷,提示通过P2Y受体介导胃体环行肌收缩反应;核苷酸类物质是调节胃体环行肌收缩功能的重要介质。
第二部分P2Y受体介导大鼠胃体环行肌收缩反应的受体药理学分析
本研究以2-MeSATP和UTP分别脱敏相应的P2Y受体,观察核苷酸类物质诱发大鼠胃体环行肌收缩反应的变化;同时分析了P2受体阻断剂苏拉明和PPADS对核苷酸类物质诱发大鼠胃体环行肌收缩反应的影响。1 UTP和2-MeSATP脱敏P2Y受体对药物诱发大鼠胃体环行肌收缩反应的影响
ATP(30μmol?L-1)、UTP(3μmol?L-1)、ADP(3μmol?L-1)、2-MeSATP(0.03μmol?L-1)、α,β-MeATP(3μmol?L-1)、ACh(3μmol?L-1)、His(30μmol?L-1)和5-HT(3μmol?L-1)诱发大鼠胃体环行肌的收缩反应在给予溶媒前后均无显著性差异(P>0.05)。UTP(9μmol?L-1)脱敏时,ATP、ADP、2-MeSATP和5-HT诱发的大鼠胃体环行肌收缩反应显著增高(P<0.05),其中ATP组增加了41.1%,ADP组增加了46.7%,2-MeSATP组增加了38.6%,5-HT组增加61.3%;而α,β-MeATP、ACh和His诱发的收缩反应无显著性变化(P>0.05)。2-MeSATP(0.09μmol?L-1)脱敏时,ATP和ADP诱发的大鼠胃体环行肌收缩反应明显降低(P<0.01),其中ATP组降低了86.7%,ADP组的收缩反应完全抑制;UTP、α,β-MeATP、ACh、His和5-HT诱发的收缩反应无显著性变化(P>0.05)。
2苏拉明和PPADS对ATP、UTP和2-MeSATP诱发大鼠胃体环行肌收缩反应的影响
苏拉明(100μmol?L-1)显著抑制ATP(30μmol?L-1)、UTP(3μmol?L-1)和2-MeSATP(0.03μmol?L-1)诱发的大鼠胃体环行肌收缩反应(P<0.01);其中ATP组降低了79.0%,UTP组降低了93.3%,2-MeSATP组降低了87.1%。PPADS(30μmol?L-1)使UTP(3μmol?L-1)诱发大鼠胃体环行肌的收缩反应降低61.6%(P<0.01),而对ATP(30μmol?L-1)和2-MeSATP(0.03μmol?L-1)诱发的收缩反应无明显变化(P>0.05)。
上述结果表明,大鼠胃体环行肌存在多种功能性P2Y受体亚型介导收缩反应;2-MeSATP、ATP和ADP可能激动环行肌细胞的同一P2Y受体亚型产生收缩反应,该收缩反应可被P2Y2和P2Y4受体脱敏所增强。P2Y2和P2Y4受体脱敏对5-HT诱发收缩反应的增强更为明显。
第三部分大鼠胃体平滑肌P2Y受体mRNA表达的定量研究
本研究应用RT-PCR技术分析了大鼠胃体平滑肌P2X受体亚型(P2X1-7)和P2Y受体亚型(P2Y1, 2, 4, 6, 11, 12, 13, 14)mRNA的表达;并进一步应用实时荧光定量PCR技术,绝对定量分析P2Y1, 2, 4, 6, 12, 13, 14受体mRNA在大鼠胃体组织的表达水平。
1 RT-PCR法检测P2X和P2Y受体亚型mRNA在大鼠胃体平滑肌的表达
扩增产物使用2%琼脂糖凝胶电泳分析。结果显示阳性对照β-actin mRNA扩增产物显示与预期相同的特异性条带;P2X1, 2, 4, 5, 7和P2Y1, 2, 4, 6, 12, 13, 14受体mRNA扩增产物在各自相应泳道出现与扩增目的片段大小相同的特异性条带;P2X3, 6和P2Y11受体mRNA扩增产物未出现扩增目的条带。
2实时荧光定量PCR法绝对定量分析大鼠胃体平滑肌P2Y受体mRNA的表达
SYBR-green实时定量PCR检测β-actin mRNA在大鼠胃体平滑肌的表达量为9,809,756.9拷贝数/10ng总RNA,显著高于P2Y受体各亚型mRNA在大鼠胃体平滑肌的拷贝数(P<0.01)。各P2Y受体亚型mRNA在大鼠胃体环行肌表达强度顺序为P2Y2> P2Y1>> P2Y12> P2Y6= P2Y14> P2Y13> P2Y4> P2Y11=0。P2Y2受体和P2Y1受体mRNA在大鼠胃体平滑肌的表达量分别为75,997.5±39,709.3和21,611.5±8,294.7(拷贝数/10 ng总RNA),显著高于其他P2Y受体(P<0.01)。
研究结果表明,除P2X3、P2X6和P2Y11受体亚型外,其他P2X和P2Y受体亚型的mRNA均表达于大鼠胃体平滑肌。大鼠胃体平滑肌P2Y受体亚型mRNA的绝对定量PCR分析结果表明,P2Y2受体和P2Y1受体mRNA是优势表达的P2Y受体,各受体亚型的表达强度顺序为P2Y2> P2Y1 >> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4。
第四部分P2Y1受体和P2Y2受体在大鼠胃体平滑肌细胞的表达
本研究应用免疫组织化学技术分析大鼠胃体平滑肌细胞(SMCs)P2Y1和P2Y2受体的表达,并对P2Y1和P2Y2受体在大鼠胃底与胃体的分布进行了比较。
阴性对照组中,大鼠胃体和胃底全层标本无特异性着色。在大鼠胃体和胃底,粘膜固有层、粘膜肌、纵行肌、环行肌和肌间神经丛可检测到P2Y1和P2Y2受体免疫反应阳性产物。腺体细胞和神经元的胞浆和平滑肌细胞的细胞核出现强阳性着色。
大鼠胃体环行肌SMCs的P2Y1受体和P2Y2受体的阳性细胞率分别为33.08%±9.93%和45.17%±6.11%,显著高于纵行肌和粘膜肌SMCs的阳性细胞率(P<0.01);大鼠胃底纵行肌、环行肌和粘膜肌SMCs P2Y1受体和P2Y2受体的表达与胃体相似。在大鼠胃体和胃底的纵行肌和环行肌层,P2Y2受体的阳性细胞率显著高于P2Y1受体(P<0.05);大鼠胃体粘膜肌SMCs的表达与此类似。大鼠胃体纵行肌、环行肌和粘膜肌SMCs P2Y2受体的阳性细胞率显著低于胃底同类SMCs的阳性细胞率(P<0.05);胃体纵行肌和粘膜肌SMCs的P2Y1受体阳性细胞率显著低于胃底同类SMCs的阳性细胞率(P<0.05),而在环行肌SMCs间无显著性差别(P>0.05)。
研究结果表明,在大鼠胃体和胃底组织,P2Y1受体和P2Y2受体表达于环行肌、纵行肌和粘膜肌SMCs的细胞核;两种受体在环行肌的表达显著高于纵行肌和粘膜肌。P2Y1受体和P2Y2受体在胃体纵行肌和粘膜肌的表达显著低于胃底。此外,在大鼠胃体、胃底环行肌和纵行肌SMCs,P2Y2受体的表达显著高于P2Y1受体;胃体粘膜肌SMCs的P2Y2表达亦显著高于P2Y1。
结论
核苷酸类物质使大鼠胃体环行肌产生浓度依赖性收缩反应,是调节胃体环行肌收缩活动的重要介质;大鼠胃体环行肌的药理学特征明显不同于胃底环行肌。
大鼠胃体环行肌存在多种功能性P2Y受体亚型介导收缩反应。2-MeSATP、ATP和ADP可能作用于环行肌细胞的同一P2Y受体亚型产生收缩反应,该收缩反应可被P2Y2和P2Y4受体脱敏所增强。
P2Y受体家族中的P2Y1受体和P2Y2受体mRNA在大鼠胃体平滑肌组织呈优势表达,各受体亚型mRNA的表达强度顺序为P2Y2 > P2Y1>> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4。
大鼠胃体和胃底组织中P2Y1和P2Y2受体在环行肌层SMCs的表达显著高于纵行肌层,两种受体在胃体纵行肌层SMCs的表达显著低于胃底。在大鼠胃体、胃底的环行肌层和纵行肌层SMCs,P2Y2受体的表达显著高于P2Y1受体。
Nucleotides play an important role in the modulation of both secretory and motor functions in the gastrointestinal tract. Among them, ATP acts mainly via P2 receptors to produce relaxation and contraction of smooth muscle in the gastrointestinal tract. It was reported that ATP induced a biphasic response (relaxation followed by a contraction) in the circular muscle of the rat proximal stomach. In our study, however, ATP produced only contractile response in the circular muscle of the rat gastric body. We therefore propose that there is an obvious difference in pharmacological characteristics of the circular smooth muscle between rat gastric body and gastric fundus, and this circular muscle strips might be a useful preparation to analyze P2 receptor-mediated contraction of the stomach. P2 receptors include P2X1-7 and P2Y1-14 receptor subtypes mediating complicated multiple functions. Up to the present time there is no evidence to show the involvement of P2Y receptor subtypes in circular muscle contraction, the absolute quantitative analysis of mRNA expression of P2Y receptor subtypes, and P2Y1 and P2Y2 receptor expression in the rat gastric body smooth muscle layer. In the present study, we investigated the pharmacological characteristics of P2 receptor-mediated contraction of the circular smooth muscle between rat gastric body and gastric fundus, mRNA expression of P2Y receptor subtypes by absolute quantitative real-time PCR analysis, and the immunohistochemical staining of P2Y1 and P2Y2 receptor in the rat gastric body.
PartⅠPharmacological characteristics of contractile responses to nucleosides and nucleotides in circular smooth muscle of the rat gastric body
Isolated circular muscle strips of the rat gastric body and gastric fundus were prepared and their contractile responses to nucleosides and nucleotides were investigated. The difference in pharmacological characteristics of the isolated circular muscle strips between rat gastric body and gastric fundus was analyzed.
1 Responses to CCh, 5-HT, His and KCl in circular muscle strips of the rat gastric body and gastric fundus
There was no significant difference in the responses to KCl of the circular muscle strips between gastric body and gastric fundus, and no difference in EC50 values of 5-HT and His to produce contractile responses between the two kinds of preparations (P>0.05). However, Emax values of contractile responses to 5-HT and His (0.81±0.26 and 0.88±0.27 g) in gastric body were significantly smaller than those in gastric fundus (2.67±0.61 and 1.90±0.68 g, P<0.01). EC50 value of CCh to produce contractile response (0.45±0.15μmol?L-1) in gastric body was significantly higher than that in gastric fundus (0.20±0.09μmol?L-1, P<0.01).
2 Responses to ATP in circular muscle strips of the rat gastric body and gastric fundus precontracted with ACh
In precontracted circular muscle strips of the gastric body, ATP (0.1-3000μmol?L-1) produced only a contractile response in a concentration dependent manner, but the same concentration of ATP induced a biphasic response (relaxation followed by a contraction) in precontracted circular muscle strips of the gastric fundus.
3 Responses to nucleosides and nucleotides in circular muscle strips of the rat gastric body
ATP, UTP, ADP, 2-MeSATP andα,β-MeATP produced contractile responses in circular muscle strips of the rat gastric body in a concentration dependent manner, respectively. The EC50 value of 2-MeSATP (7.2±5.2 nmol?L-1) was about 500 times lower than that of ACh (3.47±1.20μmol?L-1). The rank order of potency was 2-MeSATP>> ADP> ATP= UTP>α,β-MeATP>>adenosine.
4 Effects of phentolamine, propranolol, atropine and tetrodotoxin on the contractile responses to ATP and UTP
The contractile responses to ATP and UTP were not significantly affected by phentolamine (1μmol?L-1), propranolol (1μmol?L-1), atropine (1μmol?L-1) and tetrodotoxin (0.1μmol?L-1).
These results indicate that there is a significant difference in pharmacological characteristics of circular muscle strips between the rat gastric body and gastric fundus. The rank order of potency for the contraction was 2-MeSATP>> ADP> ATP= UTP>α,β-MeATP>> adenosine, indicating that nucleotides produce contractile responses mainly via P2Y receptors, and nucleotides are important mediators responsible for the contraction in circular smooth muscle of the rat gastric body.
PartⅡPharmacological analysis of P2Y receptor subtypes involved in the contraction in circular smooth muscle of the rat gastric body
Isolated circular muscle strips of the rat gastric body were prepared and their contractile responses to nucleotides were investigated. The contractile responses induced by nucleotides were analyzed in the preparations desensitized with 2-MeSATP or UTP, and analyzed with antagonists of suramin and PPADS.
1 Changes in contractile response to P2 receptor agonists in circular muscle strips of the rat gastric body desensitized with 2-MeSATP or UTP
Solvent did not affect the contractile responses to ATP (30μmol?L-1), UTP (3μmol?L-1), ADP (3μmol?L-1), 2-MeSATP (0.03μmol?L-1),α,β-MeATP (3μmol?L-1), ACh (3μmol?L-1), His (30μmol?L-1) and 5-HT (3μmol?L-1) in circular muscle strips of the rat gastric body (P>0.05). The contractile responses to ATP, ADP, 2-MeSATP and 5-HT were significantly increased by a desensitization with UTP (9μmol?L-1, P<0.05), but those toα,β-MeATP, ACh and His were not affected significantly (P>0.05). ATP, ADP, 2-MeSATP and 5-HT related contractions were increased by 41.1%, 46.7%, 38.6% and 61.3% in the preparations desensitized with UTP, respectively. The contractile responses to ATP and ADP were significantly decreased by a desensitization with 2-MeSATP (0.09μmol?L-1, P<0.01), but those to UTP,α,β-MeATP, ACh, His and 5-HT were not affected significantly (P>0.05). ATP and ADP related contractions were decreased by 86.7% and 100% in the preparations desensitized with 2-MeSATP, respectively.
2 Effects of suramin and PPADS on the contractile response to ATP, 2-MeSATP and UTP in circular muscle strips of the rat gastric body
Suramin (100μmol?L-1) significantly inhibited the contractile responses to ATP (30μmol?L-1), UTP (3μmol?L-1) and 2-MeSATP (0.03μmol?L-1) by 79.0%, 93.3% and 87.1%, respectively (P<0.01). PPADS (30μmol?L-1) significantly inhibited the contractile response to UTP (3μmol?L-1) by 61.6% (P<0.01), but did not affect the contractile responses to ATP (30μmol?L-1) and 2-MeSATP (0.03μmol?L-1) significantly (P>0.05).
These results indicate that there are several P2Y receptor subtypes involved in the contractile responses induced by nucleotides in circular muscle of rat gastric body. 2-MeSATP, ATP and ADP might act on a same P2Y receptor subtype regulating the contraction, and which is potentiated by a desensitization of P2Y2 and P2Y4 receptors. Desensitization of P2Y2 and P2Y4 receptors also potentiates the contraction response to 5-HT.
PartⅢQuantitative analysis of mRNA expression of P2Y receptor subtypes in the rat gastric body muscle layer
The mRNA expression of P2X1-7 and P2Y1, 2, 4, 6, 11, 12, 13, 14 receptors was detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the mRNA expression of P2Y1, 2, 4, 6, 12, 13, 14 receptors was further analyzed by absolute quantitative RT-PCR utilizing SYBR-green fluorescence in the rat gastric body muscle layer.
1 The mRNA expression of P2X and P2Y receptor subtypes detected by RT-PCR analysis in the rat gastric body muscle layer
The PCR products were analyzed by electrophoresis on a 2% agarose gel. We detected a single prominent band of the expected size ofβ-actin mRNA after PCR amplification of RNA preparation. Specific bands of the expected sizes of P2X1, 2, 4, 5, 7 and P2Y1, 2, 4, 6, 12, 13, 14 receptor transcripts were detected by agarose gel electrophoresis, but we were not able to confirm the specific bands of the expected sizes of P2X3, 6 and P2Y11 receptor transcripts.
2 The mRNA expressions of P2Y receptor subtypes detected by absolute quantitative real-time PCR analysis in the rat gastric body muscle layer
Theβ-actin mRNA copy number detected by absolute quantitative real-time PCR utilizing SYBR-green fluorescence were 9,809,756.9 copies per 10 ng of total RNA in the rat gastric body muscle layer, which was significantly higher than the mRNA copy number of each P2Y receptor subtype (P<0.01). The relative rank order of P2Y receptor subtype mRNA expression was P2Y2 > P2Y1 >> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4 > P2Y11 = 0. Expression level of P2Y1 or P2Y2 receptor mRNA was significant higher than that of the other P2Y receptor subtype mRNA in the rat gastric body muscle layer (P<0.01), and P2Y1 and P2Y2 receptor mRNA copy number detected by absolute quantitative real-time PCR were 21,611.5±8,294.7 and 75,997.5±39,709.3 (copies per 10 ng of total RNA), respectively.
The results indicate that P2X and P2Y receptor subtype mRNAs, except for P2X3, P2X6 and P2Y11 receptors, express in the rat gastric body muscle layer. Absolute quantitative real-time PCR analysis shows that P2Y1 and P2Y2 receptor mRNA expressions are significant higher than other P2Y receptor subtype mRNAs in the rat gastric body muscle layer, and the relative rank order of P2Y receptor subtype mRNA expression is P2Y2 > P2Y1 >> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4 .
PartⅣP2Y1 and P2Y2 receptors detected using immunohisto- chemistry in smooth muscle cells of the rat gastric body
P2Y1 and P2Y2 receptor expressions in smooth muscle cells (SMCs) of the rat gastric body were detected using immunohistochemistry techniques, and we compared the distribution profile of P2Y1 and P2Y2 receptors between the gastric body and gastric fundus of the rat.
There was no specific immunoreactivity in the different layers of the gastric body and gastric fundus in control experimental preparations. In the rat gastric body and gastric fundus, P2Y1 receptor-immunoreactive (-ir) cells and P2Y2 receptor-ir cells were observed in the lamina propria, muscularis mucosae, longitudinal muscle (LM) layer, circular muscle (CM) layer and myenteric plexus. Strong immunostaining of P2Y1 and P2Y2 receptors was observed in the cytoplasm of gland cells and neurons, and in the nucleus of SMCs.
Percentages of P2Y1 receptor-ir and P2Y2 receptor-ir SMCs in the CM layer of the gastric body were 33.08%±9.93% and 45.17%±6.11% respectively, which were significantly higher than that in the LM layer and muscularis mucosae of the gastric body (P<0.01). The expression level of P2Y1 and P2Y2 receptor in SMCs of the CM layer, LM layer and muscularis mucosae of the gastric fundus was similar to the gastric body.
In CM and LM layers of the gastric body and fundus, percentages of P2Y2 receptor-ir SMCs were significantly higher than those of P2Y1 receptor-ir SMCs (P<0.05). The same phenomenon was detected in the muscularis mucosae SMCs of the gastric body.
Percentages of P2Y2 receptor-ir SMCs of the CM layer, LM layer and muscularis mucosae in the gastric body were significantly lower than that in the gastric fundus (P<0.05). Percentages of P2Y1 receptor-ir SMCs of the LM layer and muscularis mucosae in the gastric body were significantly lower than that in the gastric fundus (P<0.05). The expression of P2Y1 receptor in SMCs of the CM layer was the same in both gastric body and gastric fundus (P>0.05).
The results indicate that P2Y1 and P2Y2 receptors are highly expressed in nucleus of SMCs of the LM layer, CM layer and muscularis mucosae in the rat gastric body and gastric fundus. Both the receptors expressed in CM layer SMCs of the gastric body and gastric fundus are significantly higher than that in the SMCs of LM layer and muscularis mucosae. Both the receptors expressed in the SMCs of LM layer and muscularis mucosae in the gastric body are significantly lower than that in the gastric fundus. Moreover, in the rat gastric body and gastric fundus P2Y2 receptor expressions in SMCs of the LM layer and CM layer are significantly higher than P2Y1 receptor expressions.
Nucleotides producing muscle contraction in a concentration dependent manner are important mediators in circular muscle strips of the rat gastric body, and the pharmacological characteristics of circular muscle strips in the rat gastric body are different from that in gastric fundus.
There are several P2Y receptor subtypes involved in the contraction induced by nucleotides in circular smooth muscle of the rat gastric body. 2-MeSATP, ATP and ADP might act on a same P2Y receptor subtype regulating the contraction, and which is potentiated by a desensitization of P2Y2 and P2Y4 receptors.
P2Y1 and P2Y2 receptor mRNA expressions are significant higher than other P2Y receptor subtype mRNAs in smooth muscles of the rat gastric body, and the relative rank order of P2Y receptor subtype mRNA expression is P2Y2 > P2Y1 >> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4 .
P2Y1 and P2Y2 receptors expressed in SMCs of the circular muscle layer are significantly higher than that of the longitudinal muscle layer in the rat gastric body and gastric fundus. Both the receptors expressed in SMCs of the longitudinal muscle layer in the rat gastric body are significantly lower than that in the rat gastric fundus. Moreover, in the rat gastric body and gastric fundus, P2Y2 receptor expression in SMCs of the circular and longitudinal muscle layers is higher than P2Y1 receptor expression.
第一部分核苷及核苷酸类物质介导大鼠胃体环行肌收缩反应的药理学特点
制备大鼠离体胃体和胃底环行肌标本。利用受体药理学技术观察大鼠胃体和胃底环行肌不同的药理学特征,并进一步分析核苷及核苷酸类物质诱发胃体环行肌收缩反应的作用特点。
1 CCh、5-HT、His和KCl对大鼠胃体环行肌和胃底环行肌的作用
在胃体环行肌KCl所致收缩反应与胃底环行肌无显著性差别;但是,CCh收缩胃体环行肌的EC50值(0.45±0.15μmol?L-1)显著高于胃底环行肌(0.20±0.09μmol?L-1,P<0.01)。5-HT和His收缩两种标本的EC50值无显著差异(P>0.05);但是,在胃体环行肌5-HT和His产生收缩反应的Emax值(0.81±0.26和0.88±0.27 g)显著小于胃底环行肌(2.67±0.61和1.90±0.68 g,P<0.01)。
2 ATP对预收缩大鼠胃体环行肌和胃底环行肌的作用
在预收缩胃体环行肌,ATP(0.1-3000μmol?L-1)诱发浓度依赖性收缩反应,未见舒张反应;在预收缩胃底环行肌标本,同浓度ATP诱发先舒张后收缩的双相反应,并呈浓度依赖性。
3核苷及核苷酸对大鼠胃体环行肌的作用
ATP、UTP、ADP、2-MeSATP和α,β-MeATP浓度依赖性诱发大鼠胃体环行肌收缩反应,2-MeSATP的EC50值为7.2±5.2 nmol?L-1比ACh(3.47±1.20μmol?L-1)低500倍;各药物产生收缩反应的效价序列为:2-MeSATP>>ADP>ATP=UTP>α,β-MeATP>>腺苷。
4酚妥拉明、普萘洛尔、阿托品及河豚毒素对ATP和UTP诱发大鼠胃体环行肌收缩反应的影响
酚妥拉明(1μmol?L-1)、普萘洛尔(1μmol?L-1)、阿托品(1μmol?L-1)及河豚毒素(0.1μmol?L-1)不影响ATP和UTP诱发的胃体环行肌收缩反应。
上述结果表明,大鼠胃体环行肌的药理学特征明显不同于胃底环行肌;核苷及核苷酸类物质诱发大鼠收缩反应的效价序列为2-MeSATP>> ADP> ATP= UTP>α,β-MeATP>>腺苷,提示通过P2Y受体介导胃体环行肌收缩反应;核苷酸类物质是调节胃体环行肌收缩功能的重要介质。
第二部分P2Y受体介导大鼠胃体环行肌收缩反应的受体药理学分析
本研究以2-MeSATP和UTP分别脱敏相应的P2Y受体,观察核苷酸类物质诱发大鼠胃体环行肌收缩反应的变化;同时分析了P2受体阻断剂苏拉明和PPADS对核苷酸类物质诱发大鼠胃体环行肌收缩反应的影响。1 UTP和2-MeSATP脱敏P2Y受体对药物诱发大鼠胃体环行肌收缩反应的影响
ATP(30μmol?L-1)、UTP(3μmol?L-1)、ADP(3μmol?L-1)、2-MeSATP(0.03μmol?L-1)、α,β-MeATP(3μmol?L-1)、ACh(3μmol?L-1)、His(30μmol?L-1)和5-HT(3μmol?L-1)诱发大鼠胃体环行肌的收缩反应在给予溶媒前后均无显著性差异(P>0.05)。UTP(9μmol?L-1)脱敏时,ATP、ADP、2-MeSATP和5-HT诱发的大鼠胃体环行肌收缩反应显著增高(P<0.05),其中ATP组增加了41.1%,ADP组增加了46.7%,2-MeSATP组增加了38.6%,5-HT组增加61.3%;而α,β-MeATP、ACh和His诱发的收缩反应无显著性变化(P>0.05)。2-MeSATP(0.09μmol?L-1)脱敏时,ATP和ADP诱发的大鼠胃体环行肌收缩反应明显降低(P<0.01),其中ATP组降低了86.7%,ADP组的收缩反应完全抑制;UTP、α,β-MeATP、ACh、His和5-HT诱发的收缩反应无显著性变化(P>0.05)。
2苏拉明和PPADS对ATP、UTP和2-MeSATP诱发大鼠胃体环行肌收缩反应的影响
苏拉明(100μmol?L-1)显著抑制ATP(30μmol?L-1)、UTP(3μmol?L-1)和2-MeSATP(0.03μmol?L-1)诱发的大鼠胃体环行肌收缩反应(P<0.01);其中ATP组降低了79.0%,UTP组降低了93.3%,2-MeSATP组降低了87.1%。PPADS(30μmol?L-1)使UTP(3μmol?L-1)诱发大鼠胃体环行肌的收缩反应降低61.6%(P<0.01),而对ATP(30μmol?L-1)和2-MeSATP(0.03μmol?L-1)诱发的收缩反应无明显变化(P>0.05)。
上述结果表明,大鼠胃体环行肌存在多种功能性P2Y受体亚型介导收缩反应;2-MeSATP、ATP和ADP可能激动环行肌细胞的同一P2Y受体亚型产生收缩反应,该收缩反应可被P2Y2和P2Y4受体脱敏所增强。P2Y2和P2Y4受体脱敏对5-HT诱发收缩反应的增强更为明显。
第三部分大鼠胃体平滑肌P2Y受体mRNA表达的定量研究
本研究应用RT-PCR技术分析了大鼠胃体平滑肌P2X受体亚型(P2X1-7)和P2Y受体亚型(P2Y1, 2, 4, 6, 11, 12, 13, 14)mRNA的表达;并进一步应用实时荧光定量PCR技术,绝对定量分析P2Y1, 2, 4, 6, 12, 13, 14受体mRNA在大鼠胃体组织的表达水平。
1 RT-PCR法检测P2X和P2Y受体亚型mRNA在大鼠胃体平滑肌的表达
扩增产物使用2%琼脂糖凝胶电泳分析。结果显示阳性对照β-actin mRNA扩增产物显示与预期相同的特异性条带;P2X1, 2, 4, 5, 7和P2Y1, 2, 4, 6, 12, 13, 14受体mRNA扩增产物在各自相应泳道出现与扩增目的片段大小相同的特异性条带;P2X3, 6和P2Y11受体mRNA扩增产物未出现扩增目的条带。
2实时荧光定量PCR法绝对定量分析大鼠胃体平滑肌P2Y受体mRNA的表达
SYBR-green实时定量PCR检测β-actin mRNA在大鼠胃体平滑肌的表达量为9,809,756.9拷贝数/10ng总RNA,显著高于P2Y受体各亚型mRNA在大鼠胃体平滑肌的拷贝数(P<0.01)。各P2Y受体亚型mRNA在大鼠胃体环行肌表达强度顺序为P2Y2> P2Y1>> P2Y12> P2Y6= P2Y14> P2Y13> P2Y4> P2Y11=0。P2Y2受体和P2Y1受体mRNA在大鼠胃体平滑肌的表达量分别为75,997.5±39,709.3和21,611.5±8,294.7(拷贝数/10 ng总RNA),显著高于其他P2Y受体(P<0.01)。
研究结果表明,除P2X3、P2X6和P2Y11受体亚型外,其他P2X和P2Y受体亚型的mRNA均表达于大鼠胃体平滑肌。大鼠胃体平滑肌P2Y受体亚型mRNA的绝对定量PCR分析结果表明,P2Y2受体和P2Y1受体mRNA是优势表达的P2Y受体,各受体亚型的表达强度顺序为P2Y2> P2Y1 >> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4。
第四部分P2Y1受体和P2Y2受体在大鼠胃体平滑肌细胞的表达
本研究应用免疫组织化学技术分析大鼠胃体平滑肌细胞(SMCs)P2Y1和P2Y2受体的表达,并对P2Y1和P2Y2受体在大鼠胃底与胃体的分布进行了比较。
阴性对照组中,大鼠胃体和胃底全层标本无特异性着色。在大鼠胃体和胃底,粘膜固有层、粘膜肌、纵行肌、环行肌和肌间神经丛可检测到P2Y1和P2Y2受体免疫反应阳性产物。腺体细胞和神经元的胞浆和平滑肌细胞的细胞核出现强阳性着色。
大鼠胃体环行肌SMCs的P2Y1受体和P2Y2受体的阳性细胞率分别为33.08%±9.93%和45.17%±6.11%,显著高于纵行肌和粘膜肌SMCs的阳性细胞率(P<0.01);大鼠胃底纵行肌、环行肌和粘膜肌SMCs P2Y1受体和P2Y2受体的表达与胃体相似。在大鼠胃体和胃底的纵行肌和环行肌层,P2Y2受体的阳性细胞率显著高于P2Y1受体(P<0.05);大鼠胃体粘膜肌SMCs的表达与此类似。大鼠胃体纵行肌、环行肌和粘膜肌SMCs P2Y2受体的阳性细胞率显著低于胃底同类SMCs的阳性细胞率(P<0.05);胃体纵行肌和粘膜肌SMCs的P2Y1受体阳性细胞率显著低于胃底同类SMCs的阳性细胞率(P<0.05),而在环行肌SMCs间无显著性差别(P>0.05)。
研究结果表明,在大鼠胃体和胃底组织,P2Y1受体和P2Y2受体表达于环行肌、纵行肌和粘膜肌SMCs的细胞核;两种受体在环行肌的表达显著高于纵行肌和粘膜肌。P2Y1受体和P2Y2受体在胃体纵行肌和粘膜肌的表达显著低于胃底。此外,在大鼠胃体、胃底环行肌和纵行肌SMCs,P2Y2受体的表达显著高于P2Y1受体;胃体粘膜肌SMCs的P2Y2表达亦显著高于P2Y1。
结论
核苷酸类物质使大鼠胃体环行肌产生浓度依赖性收缩反应,是调节胃体环行肌收缩活动的重要介质;大鼠胃体环行肌的药理学特征明显不同于胃底环行肌。
大鼠胃体环行肌存在多种功能性P2Y受体亚型介导收缩反应。2-MeSATP、ATP和ADP可能作用于环行肌细胞的同一P2Y受体亚型产生收缩反应,该收缩反应可被P2Y2和P2Y4受体脱敏所增强。
P2Y受体家族中的P2Y1受体和P2Y2受体mRNA在大鼠胃体平滑肌组织呈优势表达,各受体亚型mRNA的表达强度顺序为P2Y2 > P2Y1>> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4。
大鼠胃体和胃底组织中P2Y1和P2Y2受体在环行肌层SMCs的表达显著高于纵行肌层,两种受体在胃体纵行肌层SMCs的表达显著低于胃底。在大鼠胃体、胃底的环行肌层和纵行肌层SMCs,P2Y2受体的表达显著高于P2Y1受体。
Nucleotides play an important role in the modulation of both secretory and motor functions in the gastrointestinal tract. Among them, ATP acts mainly via P2 receptors to produce relaxation and contraction of smooth muscle in the gastrointestinal tract. It was reported that ATP induced a biphasic response (relaxation followed by a contraction) in the circular muscle of the rat proximal stomach. In our study, however, ATP produced only contractile response in the circular muscle of the rat gastric body. We therefore propose that there is an obvious difference in pharmacological characteristics of the circular smooth muscle between rat gastric body and gastric fundus, and this circular muscle strips might be a useful preparation to analyze P2 receptor-mediated contraction of the stomach. P2 receptors include P2X1-7 and P2Y1-14 receptor subtypes mediating complicated multiple functions. Up to the present time there is no evidence to show the involvement of P2Y receptor subtypes in circular muscle contraction, the absolute quantitative analysis of mRNA expression of P2Y receptor subtypes, and P2Y1 and P2Y2 receptor expression in the rat gastric body smooth muscle layer. In the present study, we investigated the pharmacological characteristics of P2 receptor-mediated contraction of the circular smooth muscle between rat gastric body and gastric fundus, mRNA expression of P2Y receptor subtypes by absolute quantitative real-time PCR analysis, and the immunohistochemical staining of P2Y1 and P2Y2 receptor in the rat gastric body.
PartⅠPharmacological characteristics of contractile responses to nucleosides and nucleotides in circular smooth muscle of the rat gastric body
Isolated circular muscle strips of the rat gastric body and gastric fundus were prepared and their contractile responses to nucleosides and nucleotides were investigated. The difference in pharmacological characteristics of the isolated circular muscle strips between rat gastric body and gastric fundus was analyzed.
1 Responses to CCh, 5-HT, His and KCl in circular muscle strips of the rat gastric body and gastric fundus
There was no significant difference in the responses to KCl of the circular muscle strips between gastric body and gastric fundus, and no difference in EC50 values of 5-HT and His to produce contractile responses between the two kinds of preparations (P>0.05). However, Emax values of contractile responses to 5-HT and His (0.81±0.26 and 0.88±0.27 g) in gastric body were significantly smaller than those in gastric fundus (2.67±0.61 and 1.90±0.68 g, P<0.01). EC50 value of CCh to produce contractile response (0.45±0.15μmol?L-1) in gastric body was significantly higher than that in gastric fundus (0.20±0.09μmol?L-1, P<0.01).
2 Responses to ATP in circular muscle strips of the rat gastric body and gastric fundus precontracted with ACh
In precontracted circular muscle strips of the gastric body, ATP (0.1-3000μmol?L-1) produced only a contractile response in a concentration dependent manner, but the same concentration of ATP induced a biphasic response (relaxation followed by a contraction) in precontracted circular muscle strips of the gastric fundus.
3 Responses to nucleosides and nucleotides in circular muscle strips of the rat gastric body
ATP, UTP, ADP, 2-MeSATP andα,β-MeATP produced contractile responses in circular muscle strips of the rat gastric body in a concentration dependent manner, respectively. The EC50 value of 2-MeSATP (7.2±5.2 nmol?L-1) was about 500 times lower than that of ACh (3.47±1.20μmol?L-1). The rank order of potency was 2-MeSATP>> ADP> ATP= UTP>α,β-MeATP>>adenosine.
4 Effects of phentolamine, propranolol, atropine and tetrodotoxin on the contractile responses to ATP and UTP
The contractile responses to ATP and UTP were not significantly affected by phentolamine (1μmol?L-1), propranolol (1μmol?L-1), atropine (1μmol?L-1) and tetrodotoxin (0.1μmol?L-1).
These results indicate that there is a significant difference in pharmacological characteristics of circular muscle strips between the rat gastric body and gastric fundus. The rank order of potency for the contraction was 2-MeSATP>> ADP> ATP= UTP>α,β-MeATP>> adenosine, indicating that nucleotides produce contractile responses mainly via P2Y receptors, and nucleotides are important mediators responsible for the contraction in circular smooth muscle of the rat gastric body.
PartⅡPharmacological analysis of P2Y receptor subtypes involved in the contraction in circular smooth muscle of the rat gastric body
Isolated circular muscle strips of the rat gastric body were prepared and their contractile responses to nucleotides were investigated. The contractile responses induced by nucleotides were analyzed in the preparations desensitized with 2-MeSATP or UTP, and analyzed with antagonists of suramin and PPADS.
1 Changes in contractile response to P2 receptor agonists in circular muscle strips of the rat gastric body desensitized with 2-MeSATP or UTP
Solvent did not affect the contractile responses to ATP (30μmol?L-1), UTP (3μmol?L-1), ADP (3μmol?L-1), 2-MeSATP (0.03μmol?L-1),α,β-MeATP (3μmol?L-1), ACh (3μmol?L-1), His (30μmol?L-1) and 5-HT (3μmol?L-1) in circular muscle strips of the rat gastric body (P>0.05). The contractile responses to ATP, ADP, 2-MeSATP and 5-HT were significantly increased by a desensitization with UTP (9μmol?L-1, P<0.05), but those toα,β-MeATP, ACh and His were not affected significantly (P>0.05). ATP, ADP, 2-MeSATP and 5-HT related contractions were increased by 41.1%, 46.7%, 38.6% and 61.3% in the preparations desensitized with UTP, respectively. The contractile responses to ATP and ADP were significantly decreased by a desensitization with 2-MeSATP (0.09μmol?L-1, P<0.01), but those to UTP,α,β-MeATP, ACh, His and 5-HT were not affected significantly (P>0.05). ATP and ADP related contractions were decreased by 86.7% and 100% in the preparations desensitized with 2-MeSATP, respectively.
2 Effects of suramin and PPADS on the contractile response to ATP, 2-MeSATP and UTP in circular muscle strips of the rat gastric body
Suramin (100μmol?L-1) significantly inhibited the contractile responses to ATP (30μmol?L-1), UTP (3μmol?L-1) and 2-MeSATP (0.03μmol?L-1) by 79.0%, 93.3% and 87.1%, respectively (P<0.01). PPADS (30μmol?L-1) significantly inhibited the contractile response to UTP (3μmol?L-1) by 61.6% (P<0.01), but did not affect the contractile responses to ATP (30μmol?L-1) and 2-MeSATP (0.03μmol?L-1) significantly (P>0.05).
These results indicate that there are several P2Y receptor subtypes involved in the contractile responses induced by nucleotides in circular muscle of rat gastric body. 2-MeSATP, ATP and ADP might act on a same P2Y receptor subtype regulating the contraction, and which is potentiated by a desensitization of P2Y2 and P2Y4 receptors. Desensitization of P2Y2 and P2Y4 receptors also potentiates the contraction response to 5-HT.
PartⅢQuantitative analysis of mRNA expression of P2Y receptor subtypes in the rat gastric body muscle layer
The mRNA expression of P2X1-7 and P2Y1, 2, 4, 6, 11, 12, 13, 14 receptors was detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the mRNA expression of P2Y1, 2, 4, 6, 12, 13, 14 receptors was further analyzed by absolute quantitative RT-PCR utilizing SYBR-green fluorescence in the rat gastric body muscle layer.
1 The mRNA expression of P2X and P2Y receptor subtypes detected by RT-PCR analysis in the rat gastric body muscle layer
The PCR products were analyzed by electrophoresis on a 2% agarose gel. We detected a single prominent band of the expected size ofβ-actin mRNA after PCR amplification of RNA preparation. Specific bands of the expected sizes of P2X1, 2, 4, 5, 7 and P2Y1, 2, 4, 6, 12, 13, 14 receptor transcripts were detected by agarose gel electrophoresis, but we were not able to confirm the specific bands of the expected sizes of P2X3, 6 and P2Y11 receptor transcripts.
2 The mRNA expressions of P2Y receptor subtypes detected by absolute quantitative real-time PCR analysis in the rat gastric body muscle layer
Theβ-actin mRNA copy number detected by absolute quantitative real-time PCR utilizing SYBR-green fluorescence were 9,809,756.9 copies per 10 ng of total RNA in the rat gastric body muscle layer, which was significantly higher than the mRNA copy number of each P2Y receptor subtype (P<0.01). The relative rank order of P2Y receptor subtype mRNA expression was P2Y2 > P2Y1 >> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4 > P2Y11 = 0. Expression level of P2Y1 or P2Y2 receptor mRNA was significant higher than that of the other P2Y receptor subtype mRNA in the rat gastric body muscle layer (P<0.01), and P2Y1 and P2Y2 receptor mRNA copy number detected by absolute quantitative real-time PCR were 21,611.5±8,294.7 and 75,997.5±39,709.3 (copies per 10 ng of total RNA), respectively.
The results indicate that P2X and P2Y receptor subtype mRNAs, except for P2X3, P2X6 and P2Y11 receptors, express in the rat gastric body muscle layer. Absolute quantitative real-time PCR analysis shows that P2Y1 and P2Y2 receptor mRNA expressions are significant higher than other P2Y receptor subtype mRNAs in the rat gastric body muscle layer, and the relative rank order of P2Y receptor subtype mRNA expression is P2Y2 > P2Y1 >> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4 .
PartⅣP2Y1 and P2Y2 receptors detected using immunohisto- chemistry in smooth muscle cells of the rat gastric body
P2Y1 and P2Y2 receptor expressions in smooth muscle cells (SMCs) of the rat gastric body were detected using immunohistochemistry techniques, and we compared the distribution profile of P2Y1 and P2Y2 receptors between the gastric body and gastric fundus of the rat.
There was no specific immunoreactivity in the different layers of the gastric body and gastric fundus in control experimental preparations. In the rat gastric body and gastric fundus, P2Y1 receptor-immunoreactive (-ir) cells and P2Y2 receptor-ir cells were observed in the lamina propria, muscularis mucosae, longitudinal muscle (LM) layer, circular muscle (CM) layer and myenteric plexus. Strong immunostaining of P2Y1 and P2Y2 receptors was observed in the cytoplasm of gland cells and neurons, and in the nucleus of SMCs.
Percentages of P2Y1 receptor-ir and P2Y2 receptor-ir SMCs in the CM layer of the gastric body were 33.08%±9.93% and 45.17%±6.11% respectively, which were significantly higher than that in the LM layer and muscularis mucosae of the gastric body (P<0.01). The expression level of P2Y1 and P2Y2 receptor in SMCs of the CM layer, LM layer and muscularis mucosae of the gastric fundus was similar to the gastric body.
In CM and LM layers of the gastric body and fundus, percentages of P2Y2 receptor-ir SMCs were significantly higher than those of P2Y1 receptor-ir SMCs (P<0.05). The same phenomenon was detected in the muscularis mucosae SMCs of the gastric body.
Percentages of P2Y2 receptor-ir SMCs of the CM layer, LM layer and muscularis mucosae in the gastric body were significantly lower than that in the gastric fundus (P<0.05). Percentages of P2Y1 receptor-ir SMCs of the LM layer and muscularis mucosae in the gastric body were significantly lower than that in the gastric fundus (P<0.05). The expression of P2Y1 receptor in SMCs of the CM layer was the same in both gastric body and gastric fundus (P>0.05).
The results indicate that P2Y1 and P2Y2 receptors are highly expressed in nucleus of SMCs of the LM layer, CM layer and muscularis mucosae in the rat gastric body and gastric fundus. Both the receptors expressed in CM layer SMCs of the gastric body and gastric fundus are significantly higher than that in the SMCs of LM layer and muscularis mucosae. Both the receptors expressed in the SMCs of LM layer and muscularis mucosae in the gastric body are significantly lower than that in the gastric fundus. Moreover, in the rat gastric body and gastric fundus P2Y2 receptor expressions in SMCs of the LM layer and CM layer are significantly higher than P2Y1 receptor expressions.
Nucleotides producing muscle contraction in a concentration dependent manner are important mediators in circular muscle strips of the rat gastric body, and the pharmacological characteristics of circular muscle strips in the rat gastric body are different from that in gastric fundus.
There are several P2Y receptor subtypes involved in the contraction induced by nucleotides in circular smooth muscle of the rat gastric body. 2-MeSATP, ATP and ADP might act on a same P2Y receptor subtype regulating the contraction, and which is potentiated by a desensitization of P2Y2 and P2Y4 receptors.
P2Y1 and P2Y2 receptor mRNA expressions are significant higher than other P2Y receptor subtype mRNAs in smooth muscles of the rat gastric body, and the relative rank order of P2Y receptor subtype mRNA expression is P2Y2 > P2Y1 >> P2Y12 > P2Y6 = P2Y14 > P2Y13 > P2Y4 .
P2Y1 and P2Y2 receptors expressed in SMCs of the circular muscle layer are significantly higher than that of the longitudinal muscle layer in the rat gastric body and gastric fundus. Both the receptors expressed in SMCs of the longitudinal muscle layer in the rat gastric body are significantly lower than that in the rat gastric fundus. Moreover, in the rat gastric body and gastric fundus, P2Y2 receptor expression in SMCs of the circular and longitudinal muscle layers is higher than P2Y1 receptor expression.
引文
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