P2Y Receptor-mediated Contraction And MRNA And Protein Expressions Of P2Y Receptor Subtype In Circular Smooth Muscle Of The Rat Gastric Body

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 bodyIsolated 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 fundusThere 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 AChIn 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 bodyATP, 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 UTPThe 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 bodyIsolated 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 UTPSolvent 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 bodySuramin (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 layerThe 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 layerThe 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 layerTheβ-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 bodyP2Y1 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.