Effects Of Exhaustive Exercise On P2X₁ Receptor Mediated Vasoconstrictions And MRNA Expression Of P2X Receptor Subtypes In Rat Arteries

Regular moderate exercise has many well-established health benefits; however the benefits are lost with exhaustive exercise which even induces significant impairments. At present, the impairment of heart was primally focused during the exhaustive exercise, and the pathological changes of heart tissue were observed at both molecular and cell levels. However, little research revealed the characteristics induced by exhaustive exercise in different types of arteies. It is well known that the sympathetic and purinergic co-transmission involving NA and adenosine 5'-triphosphate (ATP) normally exists in a variety of blood vessels. The neurogenic vasoconstriction consists of purinergic component and adrenergic component in different animals, even in different blood vessels from the same animal. The P2X₁ receptor is the principal P2X receptor subtype expressed on most vascular smooth muscles and responsible for the purine nucleotides-induced arterial contraction. However, an appropriate administration regime ofα,β-methylene ATP (α,β-MeATP) in vitro using isolated arteries has not been yet evaluated carefully, because the agent causes profound desensitization of P2X₁ receptors. In this study, therefore, we evaluated the effects of different ways ofα,β-MeATP administration on the P2X₁ purinoceptor-mediated vasoconstriction in the rat mesenteric artery, differential inhibition by exhaustive swimming exercise of P2X purinoceptor- andα₁-adrenoceptor-mediated vasoconstrictions, and expression levels of P2X receptor subtype mRNAs in the rat arteries.Partâ… Effects of different ways of administration on the P2X₁ purinoceptor-mediated vasoconstriction in the rat mesenteric arteryIsometric vasoconstrictive responses toα,β-MeATP, administered by non-cumulative manner or single concentration manner, were recorded in the rat isolated mesenteric arterial rings. Pharmacological characteristics of the two administrations were analyzed.1 Vasoconstrictive responses and wet weight of the mesenteric arteryThe first cumulative administration of NA (0.0001-100μmol·L^-1) produced vasoconstrictive responses in a concentration-dependent manner, it was not significantly different among the group of non-cumulative administration ofα,β-MeATP, the group of single concentration ofα,β-MeATP, and NA control group (P > 0.05). Wet weight of the mesenteric arterial ring segments in the three groups was 0.82±0.16, 0.82±0.11, 0.86±0.05 (mg), respectively. There was no significant difference in the wet weight among the three groups (P > 0.05).2 Vasoconstrictive responses toα,β-MeATP administered in different manners in the mesenteric arteryα,β-MeATP (0.1-100μmol·L^-1) produced concentration-dependent vasoconstrictive responses in different manners in the mesenteric artery. When the responses were normalized to tissue wet weight or the maximal contraction to 120 mmol·L^-1 KCl, the concentration-response curve forα,β-MeATP in the preparations exposed to a single concentration of the agent was significantly different from that exposed to a non-cumulative administration of the agent (P < 0.01). Vasoconstrictive responses to 100μmol·L^-1α,β-MeATP administered in single concentration manner or non-cumulative manner were 0.71±0.10 or 0.44±0.12 (g·mg^-1 tissue) when the responses were normalized to tissue wet weight (P < 0.05), and that were 48.57±11.52% and 38.00±2.52 % when the responses were normalized to the maximal contraction to 120 mmol·L^-1 KCl.3 Vasoconstrictive responses to NA in the mesenteric arteryThe first and second cumulative concentration-response curves for NA (0.0001-100μmol·L^-1) were all produced vasoconstrictive responses in a concentration-dependent manner in the mesenteric arteries. When the vasoconstrictive responses to NA were normalized to the tissue wet weight (g·mg^-1 tissue) or to the maximal contraction to 120 mmol·L^-1 KCl, two concentration-response curves for NA were not significantly different (P > 0.05).4 Vasoconstrictive responses to KCl in the mesenteric arteryThe vasoconstrictive responses to 120 mmol·L^-1 KCl in preparations used in the group of non-cumulative concentration ofα,β-MeATP were significantly smaller than that of single administration ofα,β-MeATP and NA control (P < 0.01). The vasoconstrictive responses to 120 mmol·L^-1 KCl were 1.05±0.12, 1.48±0.24, 1.46±0.17 (g·mg^-1 tissue), respectively.Partâ…¡Physiological significance of P2X₁ receptor-mediated vasoconstriction in five different types of arteries in ratsIsolated arteries were prepared and the contractile responses toα,β-MeATP and NA were investigated in the rat pulmonary, mesenteric, internal carotid, caudal arteries and aorta. The pharmacological characteristics of the vasoconstriction induced byα,β-MeATP were analyzed. Pressor responses toα,β-MeATP and NA were also observed in the anesthetized mice.1 Vasoconstrictive responses and wet weight of arteriesIn the pulmonary, mesenteric, internal carotid, caudal arteries and aorta, each type of artery was randomly devided into two groups: NA group and the single administration ofα,β-MeATP group. The first concentration-response curve of each artery for NA in preparations used in the group where single concentration ofα,β-MeATP was subsequently applied was not significantly different from that in the NA group (P > 0.05). In addition, the wet weight of each type of the preparations used in the NA experiments was not significantly different from that used in theα,β-MeATP experiments in the five types of arteries, respectively (P > 0.05).2 Vasoconstrictive responses to KCl in the pulmonary, mesenteric, internal carotid, caudal arteries and aortaThe maximal vasoconstrictive response to KCl of each type of the preparations used in the NA experiments was not significantly different from that used in theα,β-MeATP experiments in the pulmonary, mesenteric, internal carotid, caudal arteries and aorta, respectively (P > 0.05). The range of EC₅₀ (mmol·L^-1) values for KCl in the 5 types of arteries was 16.77 29.89. The EC₅₀ values of KCl in the 5 types of arteries used in NA group were significantly smaller than those inα,β-MeATP group (P < 0.05,P < 0.01).3 Vasoconstrictive responses to NA in the pulmonary, mesenteric, internal carotid, caudal arteries and aortaThe second cumulative administration of NA (0.0001-100μmol·L^-1) produced vasoconstrictive responses in a concentration-dependent manner in in the pulmonary, mesenteric, internal carotid, caudal arteries and aorta. When the vasoconstrictive responses to NA were normalized to the tissue wet weight (g/mg tissue), the rank order of the maximal vasoconstrictive responses (Emax) was caudal > mesenteric > internal carotid > pulmonary = aorta. In addition, when the vasoconstrictive responses to NA were normalized to the maximal vasoconstriction induced by KCl (% KCl Emax), the rank order of the Emax was internal carotid > aorta > pulmonary = mesenteric > caudal. The range of -log EC₅₀ (mol/L) values for NA in the 5 types of arteries was 6.90 7.83, and the rank order was caudal = mesenteric < internal carotid < aorta = pulmonary.4 Vasoconstrictive responses to a single concentration ofα,β-MeATP in the pulmonary, mesenteric, internal carotid, caudal arteries and aortaα,β-MeATP (0.1-100μmol·L^-1) produced a concentration-dependent vasoconstrictive responses in the pulmonary, mesenteric, internal carotid, caudal arteries and aorta (each arterial preparation was only exposed toα,β-MeATP once). The rank order of the Emax was caudal > mesenteric > internal carotid > pulmonary = aorta when the vasoconstrictive responses toα,β-MeATP were normalized to tissue wet weight (g·mg^-1 tissue), whereas it was internal carotid = caudal > aorta > pulmonary = mesenteric when normalized to maximal vasoconstriction induced by KCl (% KCl Emax). The range of -log EC₅₀ (mol·L^-1) values forα,β-MeATP in the 5 types of arteries was 4.86 5.93, and their rank order was pulmonary = aorta = internal carotid -1 injected into the tail vein of the mice produced a rapid increase in BP and reached its peak value within 10 s, then the increased BP returned to the base level 2 min after the administration. Repeated administration ofα,β-MeATP at the same dose at 20 min interval induced a similar pressor response, and the increased SBP, DBP and MBP were not significantly different from those by the first administration ofα,β-MeATP (P > 0.05). In other two groups of anesthetized mice, NA andα,β-MeATP (1³0 nmol·kg^-1) increased the SBP, DBP and MBP in a dose-dependent manner, respectively. Before intravenous administration of the drugs, the values of SBP, DBP and MBP in the NA group were not significantly different from those in theα,β-MeATP group (P > 0.05). However, the increases in SBP, DBP and MBP induced byα,β-MeATP were much bigger than those induced by NA (P < 0.01).α,β-MeATP at 30 nmol·kg^-1 increased the SBP, DBP and MBP by 65.83±7.00, 65.67±5.00 and 65.72±5.51 (mm Hg), while the same dose of NA increased the SBP, DBP and MBP just by 36.83±4.59, 33.33±4.90 and 34.50±4.71 (mm Hg, P < 0.01).Partâ…¢Differential inhibition by exhaustive swimming exercise of P2X receptor- andα₁-adrenoceptor-mediated vasoconstrictions in the different types of arteriesMale Wistar rats were divided into two groups of sedentary control group (SCG) and exhaustive swimming exercise group (ESEG). The rats of ESEG with a weight equivalent to 3% of body weight tied to the tail were subjected to swim to exhaustion once daily for two weeks. And then isometric vasoconstrictive responses toα,β-MeATP or NA were recorded in the rat pulmonary, caudal, mesenteric and internal carotid arteries. The difference in pharmacological characteristics was analyzed.1 Effects of exhaustive swimming exercise on the tissue wet weight and the vasoconstrictive responses to KClWet weight of the mesenteric arterial ring segments in SCG was 0.73±0.02 mg, which was significantly lower than that (0.85±0.02 mg) in ESEG (P < 0.01). The values of wet weight of the pulmonary, caudal and internal carotid arterial segments in SCG were not significantly different from those in ESEG (P > 0.05). Maximal vasoconstrictive responses to KCl in the mesenteric and caudal arteries in SCG were 1.72±0.05 g·mg^-1 tissue and 3.27±0.10 g·mg^-1 tissue, which were much greater than the values of 1.46±0.06 g·mg^-1 tissue and 2.85±0.10 g·mg^-1 tissue in ESEG (P < 0.01). There were no significant differences in the maximal vasoconstrictive responses to KCl of the pulmonary and internal carotid arteries between SCG and ESEG rats (P > 0.05). However, the range of EC₅₀ (mmol·L^-1) values for KCl in the 4 types of arteries subsequently exposing to the second administration of NA was 17.23 33.44, and the EC₅₀ values of KCl in SCG were not significance with those in ESEG.2 Vasoconstrictive responsiveness of the pulmonary, caudal, mesenteric and internal carotid arteries to NAThe first cumulative administration of NA (0.0001-100μmol·L^-1) produced vasoconstrictive responses in a concentration-dependent manner in the pulmonary, caudal, mesenteric and internal carotid arteries. In the rats of SCG, there were no significant differences in the vasoconstrictive responses to the first exposure to NA in the pulmonary, caudal, mesenteric and internal carotid arteries between the preparations subsequently exposing to the second administration of NA and those subsequently exposing toα,β-MeATP (P > 0.05). Same results were obtained in the rats of ESEG (P > 0.05). 3 Effects of exhaustive swimming exercise on vasoconstrictive responses to NA in the pulmonary, caudal, mesenteric and internal carotid arteriesThe second exposure of the pulmonary, caudal, mesenteric or internal carotid artery to NA (0.0001-100μmol·L^-1) produced vasoconstrictive responses in a concentration-dependent manner in the rats of SCG and ESEG. The exhaustive swimming exercise decreased the vasoconstrictive responses to NA in the pulmonary and caudal arterial preparations significantly but slightly (P < 0.01), reaching a maximal inhibition of 13.68% in the pulmonary artery and 9.48% in the caudal artery. In the mesenteric arterial preparation, however, the inhibition by exhaustive swimming exercise of vasoconstrictive response to NA was more potent, reaching a maximal inhibition of 21.02% (P < 0.01). The vasoconstrictive response to NA in the internal carotid arterial preparation was not significantly affected by exhaustive swimming exercise (P > 0.05). The range of -log EC₅₀ (mol·L^-1) values for NA in the 4 types of arteries was 6.70 7.58, and the EC₅₀ values of NA in SCG were not significantly different from those in ESEG.4 Effects of exhaustive swimming exercise on vasoconstrictive responses toα,β-MeATP in the pulmonary, caudal, mesenteric and internal carotid arteriesα,β-MeATP (0.1-100μmol·L^-1) produced the vasoconstrictions of the pulmonary, caudal, mesenteric and internal carotid arteries concentration-dependently in the rats of SCG and ESEG. The vasoconstrictive responses toα,β-MeATP in the pulmonary, caudal and mesenteric arteries were not significantly affected by exhaustive swimming exercise (P > 0.05). On the contrary, exhaustive swimming exercise significantly decreased the vasoconstrictive responses toα,β-MeATP in the internal carotid arterial preparations, reaching a maximal inhibition of 50.38% (P < 0.01). The range of -log EC₅₀ (mol·L^-1) values forα,β-MeATP in the 4 types of arteries was 4.71 6.19, and the EC₅₀ values ofα,β-MeATP in SCG were not significantly different from those in ESEG.Partâ…£mRNA expression of P2X receptor subtypes in different types of the rat arteriesThe mRNA expression of P2X receptor subtypes was detected by reverse transcription-polymerase chain reaction (RT-PCR) analysis, and the relative expression of P2X receptor subtype mRNAs was further analyzed by real time RT-PCR utilizing SYBR-green fluorescence in the rat internal carotid, pulmonary, mesenteric, caudal arteries and aorta.1 The mRNA expression of P2X receptor subtypes detected by RT-PCR analysis in the rat internal carotid, pulmonary, mesenteric, caudal arteries and aortaThe PCR products were analyzed by agarose gel electrophoresis. The amplified PCR products of the predicted size were detected for HPRT, P2X₁, P2X₄ and P2X₇ receptors in the rat internal carotid, pulmonary, mesenteric, caudal arteries and aorta. However, those were barely detected for the P2X₂, P2X₃, P2X₅ and P2X₆ receptors in the five types of arteries.2 The relative expression of P2X₁, P2X₄ and P2X₇ receptor subtype mRNAs detected by real time fluorescence quantitative RT-PCR in the rat internal carotid, pulmonary, mesenteric, caudal arteries and aortaThe expression level of P2X₁ receptor mRNA in rat caudal artery was significantly higher than that in the internal carotid, pulmonary, mesenteric arteries or aorta (P < 0.01),but there was not significantly different among the latter four arteries. The rank order of P2X₇ receptor mRNA expression levels was pulmonary < caudal = aorta < internal carotid < mesenteric. However, there were no significant differences in P2X₄ receptor mRNA expression level among the five types of arteries (P > 0.05).ConclusionP2X₁ receptor-mediated vasoconstriction and KCl-induced vasoconstriction become significantly smaller in the rat isolated mesenteric artery exposed to non-cumulative administration ofα,β-MeATP, and this administration regime may lead to a wrong conclusion.P2X₁ receptor-mediated vasoconstrictions are equally important in rat internal carotid, pulmonary, mesenteric arteries and aorta, but much greater in the tail artery suggesting its special role in physiological function.The exhaustive swimming exercise has a selective inhibition between theα₁-adrenoceptor-medicated and P2X₁ receptor-medicated vasoconstrictions in the rat different type of arteries, and the inhibition characteristics in the internal carotid artery is quiet different from the peripheral arteries. The expression level of P2X₁ receptor mRNA in rat caudal artery is significantly higher than that in the internal carotid, pulmonary, mesenteric arteries or aorta. The rank order of P2X₇ receptor mRNA expression levels is pulmonary < caudal = aorta < internal carotid < mesenteric. However, there are no significant differences in P2X₄ receptor mRNA expression level among the five types of arteries.