-opioid receptor stimulation on angiotensin -induced inhibition of cardiac hypertrophy

1、 -opioid receptor stimulation on angiotensin -induced inhibition of cardiac hypertrophy Of: Zhang, Wang and new Guo-Qiang Wu, Jie Abstract Objective To observe the -opioid receptor stimulation by angiotensin II induced cardiac hypertrophy on the role of opioid receptor agonists on cardiac hypertroph

2、y induced by angiotensin II inhibition. Methods in cultured neonatal rat cardiomyocytes in vitro cells as a model, angiotensin II1 M induced cardiac hypertrophy, observed U50488H1 M of its role, and, and losartan (los1 M to do comparison group observed the activation of -opioid receptor on cardiac h

3、ypertrophy. with the Lowry assay protein content of myocardial cells, isolated by digestion method and computer image analysis system for detection of myocardial cell volume, using 3H-leueine incorporation of myocardial cells in protein synthesis. Results AngII1 M total protein content of myocardial

4、 cells and protein expression and cell volume increased significantly, U50488H1 M to reduce the AngII induced myocardial cell protein content, protein expression, and the increase in volume, thereby inhibiting cardiac hypertrophy, and the degree of inhibition similar with los1 M. Conclusions -opioid

5、 receptor agonist can inhibit the AngII induced U50488H myocardial cell hypertrophy. Keywords: cardiac hypertrophy Angiotensin II U50488H opioid receptor Abstract: Objective To demonstrate the inhibitory effect of -Opioid on AngII-induced hypertrophy in cultured myocardial cells of neonatal rats. Me

6、thods Based on the model of cardiac bistiocyte cells in vitro culture, the experiment applies the angiotensin II1 M to induce the myocardium to be hypertrophy. Compared with the group of (los) 1 M, the experiment observes the effects of the activation of -Opioid on myocardial hypertrophy. The protei

7、n content was measured by Lowry s method. The protein synthetic rate was obtained through measuring the incorporation of 3H-leueine into myocyte protein by liquid scintillation method, the cardiomyocytes volumes were measured by computer photograph analysis system; the proteinic compound of the card

8、iac bistiocyte s was measured by the incorporation method of 3H-leueine. Results AngII (1 M) can increase the overall protein content, proteinemia expression, and the cells volumes. U50488H1 M can reduce the protein content of cardiac bistiocyte, protein expression and the cells volumes, which are i

9、nduced by AngII. Therefore, U50488H1 M inhibits the myocardial hypertrophy and it has the similar inhibitive extent with the los1 M. Conclusions -Opioid receptor stimulation U50488H can inhibit hypertrophy induced by AngII in neonatal rat ventricular myocytes. Keywords: myocardial hypertrophy; -Opio

10、id; U50488H; Angiotersin II The existence of myocardial -opioid receptor (-OR 1, in recent years on the -OR on cardiac cell growth and hypertrophy of more research in this laboratory has confirmed that -OR is a negative myocardial cells regulator 2 can inhibit norepinephrine-induced cardiac hypertro

11、phy 3, and that selective -OR agonist U50488H through the activation of -OR interaction with the 1-AR inhibition of Iso-induced cardiac hypertrophy 4 . angiotensin II (angiotensin II, AngII is a peptide hormone, its adrenal gland, liver, kidney and blood vessels have a role in a variety of organs an

12、d tissues. but also has been demonstrated also the existence of myocardial vascular tone receptor 1 (AT1 1 438, AngII myocardial cells is an important target organs, many studies have shown that AngII can induce cardiac hypertrophy. has also been reported in the literature, endogenous opioid peptide

13、s and the renin - angiotensin ( RAS system on 5,6. Therefore, to further explore the -OR agonist induced cardiac hypertrophy and Ang relationship, the experimental induction of myocardial hypertrophy in Ang , based on the key observation of selective -OR agonist and selective U50488H -OR antagonist-

14、nor-BNI (norbinaltorphimine of Ang induced cardiac hypertrophy. 1 Materials and methods 1.1 Animals, drugs and reagents 2 3 days after birth, the SD rats, either male or female, by the Experimental Animal Center of Liaoning Medical College. Calf serum was purchased from Biological Engineering Materi

15、als Co., Ltd. Hangzhou Evergreen, DMEM medium, a selective receptor agonist (U50488H ) and the specific receptor antagonist (nor-BNI, angiotensin , losartan (los), SDS were purchased from Sigma, Coomassie brilliant blue were purchased from Shanghai Health Workers. other reagents were of analytical g

16、rade. 1.2 cardiac myocytes in primary culture Under sterile conditions, take 1 3 d after birth, the SD rat heart, into D-Hanks solution washed three times, cut into approximately 1 mm 3 mm small pieces, with 0.8 g L-1 trypsin digestion and separation of cells, the isolated cells with 15% calf serum,

17、 84% DMEM, 1% of the double anti-solution (containing 1 10 U L-1 penicillin, 100 g streptomycin) medium, and 0.1 mmol / L 5 - bromodeoxyuridine (mainly inhibit the growth of fibroblasts, even after the wind and percussion to 5 109 L-1 of the density of seeded in 24-well culture plates, into the pass

18、 to 0.05 CO2 and 0.95 in air of carbon dioxide incubator culture. 1.3 Grouping and administration methods 2 days after myocardial cells were cultured routinely in order to reduce the composition of the serum effect on the experimental results, the replacement of 0.4% calf serum containing medium and

19、 various concentrations of reagents. As control group, other groups were added to AngII 1 M, AngII 1 M + los 1 M, AngII 1 M + U50488H 1 M, AngII 1 M + U50488H 1 M + nor-BNI 1 M. 48 h after administration of the determination of the index. 1.4 cultured myocardial cell protein content To absorb the cu

20、lture medium in each well plate with D-Hanks solution quickly washed three times, adding 10g L-1SDS 0.5 mL lysed cells, according to count the number of cells per well is about 5 105 , Lowry et al 7 determined protein content per well. 1.5 Determination of cultured myocardial cell volume Cell volume

21、 is obtained by measuring the cell diameter, D-Hanks solution with a quick rinse holes covered with cells in culture 3 times, each well add 0.3 mL / (g L trypsin, placed in 37 incubator 30 min, then add 0.2 mL of serum containing 0.1 volume fraction of medium to terminate digestion, the cells were c

22、ollected into a cell interior (bottom of the cell chamber is the coverslip by silicification, in case of myocardial cell adhesion, in the 400 times magnification under an inverted microscope observed one of the cells showed almost spherical. CIAS Daheng cells by computer image analysis system to mea

23、sure the diameter of individual cells, then calculate the volume of cells. Each randomly selected four view holes, measuring 20 cells per field. 1.6 protein synthesis in cultured myocardial cells measured Replaced after 48 h culture with 37TBq L-1 3H - leucine and various concentrations of reagents

24、in the medium, drained after 72 h culture with medium, with cold D-Hanks solution quickly washed three times, each well adding 1 mL 10 g L-1 SDS lysed cells, the application 49 glass fiber membrane filter, followed by 2 mL trichloroacetic acid (0.2 kg L-1 precipitated protein, 2 mL of ethanol volume

25、 fraction of 0.95 to 0.99 filtration, dry membrane, with the liquid scintillation counter (Paclard, Tricard2200 CA, United States, measuring 3H - leucine binding, the analysis of protein synthesis. According to count the number of cells per well is approximately 5 104 , the experimental results to t

26、he cpm value of each hole. 1.7 Statistical analysis All data are mean + - standard deviation (+-s) that the single factor analysis of variance and LSD method for statistical analysis. 2 Results 2.1 Different Factors on the protein content of myocardial cells Table 1 data show that compared with the

27、control group, AngII myocardial cell protein content increased by 54%, compared with AngII, AngII + los, AngII + U50488H group decreased protein content of myocardial cells, were reduced by 37% , 35%, indicating that U50488H and losartan inhibited AngII-induced increase in protein content of myocard

28、ial cells, and inhibition of U50488H similar role with the los, AngII + U50488H + nor-BNI group, protein content compared with the AngII no significant change, indicating that nor-BNI can be completely inhibited U50488H role. Table 1 Different Factors protein content of myocardial cells (slightly Di

29、fferent Factors acting on the myocardial cells 48 h, * P <0.01, compared with the normal group, # P <0.05, compared with the AngII group, P <0.01 compared with AngII 2.2 Different Factors on the volume of myocardial cells Table 2 results show that compared with the control group, AngII incr

30、eased myocardial cell volume 84%, compared with AngII, AngII + los, AngII + U50488H group were reduced volume of myocardial cells were reduced by 51 %, 48%, indicating that U50488H and losartan inhibited AngII-induced myocardial cell size, and inhibition of U50488H similar role with the los, AngII +

31、 U50488H + nor-BNI group, cell volume compared with the AngII group no significant change than that nor-BNI can be completely inhibited U50488H role. Links to free paper download Table 2 Different Factors of myocardial cell volume (slightly * P <0.01, compared with the normal group, # P <0.01,

32、 compared with the AngII group; P <0.01, compared with the AngII 2.3 Different Factors of protein synthesis Table 3 shows that compared with control group, AngII group of protein synthesis increased by 32% compared with AngII, AngII + los, AngII + U50488H group decreased protein synthesis, decrea

33、sed by 24%, 22 % that U50488H and losartan inhibited AngII-induced increase in protein synthesis, and inhibition of U50488H similar role with the los, AngII + U50488H + nor-BNI group compared protein synthesis and no significant AngII changes that nor-BNI can be completely inhibited U50488H role. Ta

34、ble 3 Different Factors of protein synthesis (slightly * P <0.01, compared with the normal group, # P <0.01, compared with the AngII group, P <0.01 compared with AngII 3 Discussion Cardiac hypertrophy without increasing myocardial cells refers to cell division, is a hemodynamic overload and

35、 / or neurohumoral stimulation of the adaptive response. The initial stage of the disease, cardiac hypertrophy can be improved by balancing the stress of increased cardiac function, However, Zhang Shijian stress-induced cardiac hypertrophy in sustained due to cardiac dysfunction eventually lead to h

36、eart failure. At present, cardiac hypertrophy has been recognized as a sudden death, heart failure, cardiovascular events such as strong independent risk factor 7 Therefore, the development of proven mechanisms of the cardiovascular field has been an important issue 8. Many studies have shown that c

37、ardiac hypertrophy induced by angiotensin II as a stimulus for one of the neurohumoral, the vast majority belonging to 7 times by the cardiovascular effects through membrane-type G protein-coupled receptor family in the AngII receptor type 1 ( AT1-mediated complete 9. has been shown that myocardial

38、cells in vitro after addition of AngII can induce myocardial cell volume increases, RNA transcription and protein synthesis in a dose and time dependent 10. So In this study, the AngII 1 M concentrations, can significantly induced cardiomyocyte hypertrophy. The experimental results show, AngII stimu

39、lation of cardiac cells, protein synthesis, protein content and cell volume increased significantly. antagonized these effects when using Losartan can be completely suppressed, AngII stimulation of myocardial cells demonstrated by AT1-mediated. The experiment also found that, -OR agonist U50488H rol

40、e in AngII induced cardiac hypertrophy can significantly reduce protein expression, content and the increase in cell volume and this effect was selective -OR antagonist nor-BNI blocked by. that U50488H can inhibit AngII-induced cardiac hypertrophy, and also found its role and los inhibited AngII-ind

41、uced cardiac hypertrophy similar effects, indicating that -OR and the interaction between AT1, this interaction may be through G protein-coupled receptor family achieved. has been shown that stimulation of -opioid receptor (-OR can be made through various channels Ca2 + i transient amplitude decreas

42、ed 11-13. Meanwhile, the excitement cardiac opioid receptors, may activate pertussis toxin - sensitive G protein - phospholipase C-inositol triphosphate-Ca2 + channels in the SR Ca2 + depletion. in cardiac cells, U50488H can produce activation of K + outflow 14, leaving the action potential to reduc

43、e the time. repolarization speed up will make through the L-type Ca2 + channel influx Ca2 + decrease and lead to SR of Ca2 + back photo reduction of the Ca2 + i transient amplitude to decline further. All these mechanisms have resulted in intracellular calcium release and Ca2 + i concentration decre

44、ased, thereby inhibiting cardiac hypertrophy and development. However, AngII stimulation of cardiac myocytes hypertrophy also stimulated the release of Ca2 + on Ca2 + also affect the internal flow, so U50488H on AngII induced inhibition of cardiac hypertrophy may be through regulation of intracellul

45、ar Ca2 + homeostasis to achieve. Although the experimental results show that opioid receptor stimulation on AngII-induced inhibition of myocardial hypertrophy, but direct regulation of opioid peptides AngII level is difficult to say. Because sympathetic in the hemodynamic effect of obstacles is very

46、 complex due to , and hemodynamic disorders, the role of opioid peptides on the AngII is also very complex 15, so U50488H specific signal transduction pathways by which AngII inhibition of play it is less clear. to be further explored. References 1 Rogers TB, Gan ST, Allen Is.Identification and char

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49、Pharmacol, 2007,555 (2-3) :100-105. 5 Fukuhara M, Matsumura K, Abe I, et al.Interaction of opioids and vasopressin in central action of angiotensin II in conscious rabbits J. Hypertens Res, 1998,21:89-95. 6 Takai S, Song K, Tanaha T, Okunishi H, Miyazaki M. Antinociceptive effects of angiotensin-converting enzyme inhibitors and an angiotensin II recepto