心肌肥厚对κ阿片受体介导的信号通路受损

目的：分离正常及慢性缺氧性右心肥厚的心室肌细胞,观察细胞内[Ca^2 ]i及细胞内pHi对心肌κ－阿片受体激动后的反应。方法：以fura-2和BCECF分别为[Ca^2 ]i和pHi的指示剂,用光谱荧光法测定电刺激引起的细胞内[Ca^2 ]i瞬变及pHi。结果：κ阿片受体选择性激动剂U50,488H可降低电刺激引起的[Ca^2 ]i瞬变和增加pHi,该作用是由蛋白激酶C（PKC）所介导。在肥厚的心室肌细胞,U50,488H的上述作用显著减弱。与此相对应,PKC的激动剂PMA引起的[Ca^2 ]i瞬变降低和pHi的增加作用在肥厚的心室肌细胞亦消失。用NH4C1法观察Na^ -H^ 交我器的功能显示其在肥厚的心室肌细胞无明显改变。结论：心肌肥厚时κ－阿片受体介导的信号通路受损,受损部位发生在PKC与效应器之间。     

前胡丙素对分离的成年鼠正常及肥厚心室肌细胞内游离钙的影响(英文)

目的:研究分离的成年大鼠正常及肥厚左室肌细胞[Ca~(2 )]_i及前胡丙素的作用.方法:用Fura 2-AM测定单细胞[Ca~(2 )]_i.结果:外钙为1.0mmol·L~(-1)时,正常左室肌细胞静息钙87±4 nmol·L~(-1),肥厚细胞123±7 nmol·L~(-1).肥厚心肌细胞中,加入KCl 20,40,60 mmol·L~(-1),[Ca~(2 )]_i增加29％,78％和185％,幅度高于正常细胞.前胡丙素1,10,100 μmol·L~(-1)浓度依赖地抑制KCl及去甲肾上腺素诱导[Ca~(2 )]_i增加.作用与硝苯啶相似.结论:肥厚心肌细胞静息钙高于正常细胞;前胡丙素抑制激动剂引起的[Ca~(2 )]_i升高源于其钙通道阻断作用.     

槲皮素降低大鼠心率和心肌钙振荡频率和抑制小鼠心肌肥厚(英文)

目的:研究槲皮素对心肌兴奋收缩耦联及构型重建的影响。方法:经动脉插管记录大鼠血流动力学;缩窄小鼠腹主动脉致心肌肥厚;检测Fura 2-AM负载的培养大鼠心肌细胞内游离钙([Ca~(2 )]_i)及钙振荡,结果:槲皮素剂量相关地降低大鼠窦性心率,而动脉血压、左室压及其微分改变轻微;10-250 μmol·L~(-1)时浓度依赖性降低培养心肌自发钙振荡频率,100μmol·L~(-1)时预防异丙肾上腺素及哇巴因加速钙振荡频率,但不抑制静息[Ca~(2 )]_i.和自发钙振荡及二药增高的振荡幅度,槲皮素还抑制血管紧张素Ⅱ而非高钾的升[Ca~(2 )]_i作用,槲皮素120 mg·kg~(-1)·d~(-1)5 d灌胃不改变假手术鼠的心室与体重之比,但显著抑制腹主动脉结扎小鼠的心室肥大,结论:槲皮素降低心肌钙振荡频率和心肌肥厚但不直接影响心脏兴奋收缩耦联。     

阿片受体激动剂U50,488H通过Cx43介导的抗心律失常作用与抑制钙通道有关

目的研究U50,488H(选择性κ阿片受体激动剂)是否通过抑制钙通道减少外钙内流参与了κ阿片受体的抗缺血性心律失常作用。方法将30只SD大鼠完全随机设计分为5组(每组6只):对照组(Sham)、缺血组(Isc)、缺血+U50,488H组(Isc+U50)、缺血+U50,488H+钙离子通道激动剂Bay k8644组(Isc+U50+Bay)、缺血+Bay k8644组(Isc+Bay)。建立大鼠急性心肌缺血模型,施行30 min心肌缺血前应用U50,488H(1.5 mg.kg-1)及Bay k8644(66.7μg.kg-1)进行干预,观察各组心律失常发生情况及连接蛋白43(Cx43)在蛋白和基因水平表达的变化。结果①在体动物模型中,Bay k8644可以翻转U50,488H激活κ阿片受体后产生的抗心律失常作用(P<0.05)。②在蛋白表达水平,Bay k8644可以阻断U50,488H对缺血心肌Cx43的保护性上调作用(P<0.01)。③在mRNA表达水平,Bay k8644亦可翻转U50,488H激活κ阿片受体后对缺血心肌Cx43mRNA的调控作用(P<0.01)。结论 U50,488H可通过抑制钙通道减少外钙内流,参与κ阿片受体对缺血心肌Cx43的稳定性调控及抗缺血性心律失常作用。     

尼卡地平升高小鼠胸腺细胞胞浆钙浓度(英文)

研究尼卡地平(nicardipine,Nic)对小鼠胸腺细胞胞浆钙浓度([Ca~(2 )]_i)及增殖的影响.方法:用Fura-2掺入细胞的荧光测定法测定[ca~(2 )]_i;用[~3H]thymidine掺入法测定胸腺淋巴细胞的增殖.结果:无论在含Ca~(2 )或无Ca~(2 )介质中,Nic 1—30 μmol·L~(-1),以浓度依赖的方式升高静息胸腺细胞的[Ca~(2 )]_i.丝裂原Con A 5 mg·L~(-1)也从细胞内库释放Ca~(2 ),而Nic抑制Con A引起的[Ca~(2 )]_i升高.在上述升高[Ca~(2 )]_i的浓度中,Nic不刺激静息胸腺淋巴细胞增殖,但显著抑制Con A的增殖反应.结论:Nic升高[Ca~(2 )]_i,干扰了细胞Ca~(2 )稳态,因而抑制淋巴细胞对丝裂原的反应.     

多非替利衍生物CPU 228(V03)对大鼠心室肌细胞钙瞬变的调节

目的在β-受体激动情况下,研究多非替利衍生物CPU 228对电刺激触发心肌细胞的胞浆Ca2+浓度([Ca2+]i)变化及钙瞬变动力学参数的影响.方法游离单个大鼠心室肌细胞,Fluo-3/AM负载,电刺激诱发心室肌细胞钙瞬变,定量测定心室肌细胞内Ca2+浓度变化,异丙肾上腺素(isoproterenol, ISO)100nmol·L-1激动β-受体,观察CPU 228 1 μmol·L-1对钙瞬变动力学参数、[Ca2+]i及钙负荷水平的影响.结果CPU 228 1 μmol·L-1对大鼠心室肌细胞[Ca2+]i的影响不明显(P>0.05);ISO 100 nmol·L-1显著升高大鼠心室肌细胞[Ca2+]i和细胞内钙负荷水平,缩短钙瞬变时程,并且增加钙瞬变的消除速率.CPU 228 1 μmol·L-1显著抑制ISO的上述作用.结论在β-受体激动情况下,CPU 228可以降低心肌细胞[Ca2+]i,使ISO改变的钙瞬变动力学参数恢复到正常水平.     

细胞内游离钙与柯萨奇病毒B_3诱导的大鼠培养心肌细胞凋亡(英文)

目的:探讨[Ca~(2 )]_i在柯萨奇病毒(CV)B_3诱导心肌培养细胞凋亡中的作用。方法:DNA裂点检测法(3'-末端标记)及扫描电镜检测细胞凋亡,Fluo3-AM负载心肌细胞,共聚焦显微镜观察[Ca~(2 )]_i荧光强度变化,结果:感染24 h,心肌细胞内CVB_3的浓度达峰值,感染10 h未见凋亡的心肌细胞,17,24和36 h凋亡细胞分别为5％,60％和90％,感染17 h心肌[Ca~(2 )]_i浓度达峰值,电镜结果提示凋亡的特征,结论:CVB_3可诱导培养心肌细胞的凋亡过程,细胞内Ca~(2 )参与凋亡细胞的信息转导过程,并在凋亡早期发挥重要作用。     

U50,488H抗大鼠缺血/再灌注损伤心律失常作用与抑制钠通道电流有关

目的观察κ阿片受体选择性激动剂(U50,488H)对大鼠心脏缺血/再灌注(ischemia and reperfusion,I/R)室性心律失常的影响并探讨其可能的机制。方法观察U50,488H对大鼠I/R整体动物模型血浆肌酸磷酸激酶(CK)、乳酸脱氢酶(LDH)水平的影响;分离正常大鼠心脏,采用Langen-dorff离体心脏灌流方法,预先给予U50,488H(5mmol·L-1)和NE(100μmol·L-1),测定其对血流动力学指标和对心律失常的影响;常规酶解法分离成年大鼠心室肌细胞,采用全细胞膜片钳技术观察U50,488H对钠电流的作用。结果①U50+I/R组血浆中CK和LDH的含量较I/R组明显降低(P<0.01)。②与I/R组相比,给予U50,488H后,心率明显下降(P<0.05)。③对照组偶发早搏,I/R组心律失常发生频率明显增加,与I/R组相比较,I/R+NE组心律失常评分明显增高(P<0.01);如果提前给予U50,488H,发现不仅可以明显降低I/R组缺血和再灌注期间室速和室颤的发生率(P<0.01)及降低I/R组心律失常的评分,而且明显降低I/R+NE组心律失常的评分(P<0.01)。④U50,488H(100μmol·L-1)可以明显降低心室肌细胞的钠电流(P<0.01)。结论κ阿片受体激动剂U50,488H对I/R时的心律失常有拮抗作用,其作用可能是通过抑制心肌细胞的钠电流而实现的。     

小檗胺对高钾、去甲肾上腺素及咖啡因引起大鼠心肌细胞内钙动员的拮抗作用(英文)

目的:研究小檗胺(Ber)对氯化钾、NE及咖啡因引起大鼠培养心肌细胞[Ca~(2 )]_i动员的影响,方法:Fluo 3-AM负载后,共聚焦法测定心肌细胞[Ca~(2 )]_i荧光强度的变化。结果:Ber对心肌细胞静息[Ca~(2 )]_i水平无影响,但可剂量依赖性地抑制KCl60mmol·L~(-1)及NE 30 μmol·L~(-1)引起的内钙动员(P<0.01),此作用与维拉帕米相似.Egtazic acid3 mmol·L~(-1)并不能增强Ber对NE引起的[Ca~(2 )]_i升高的抑制作用,无外钙时,咖啡因80-160μmol·L~(-1)的[Ca~(2 )]_i动员不受Ber的影响(P>0.05),结论:Ber与维拉帕米相似,对大鼠心肌细胞靠电压依赖性和受体操纵性钙通道而升高的胞[Ca~(2 )]_i有拮抗作用,并不影响[Ca~(2 )]_i释放。     

地尔硫唑对慢性充血性心力衰竭患者淋巴细胞膜β肾上腺素受体下调的影响(英文)

目的:研究钙拮抗剂地尔硫唑是否逆转慢性充德性心力衰竭(CHF)患者淋巴细胞膜β肾上腺素受体(β-AR)的下调.方法:同位素放射配基法测定地尔硫唑治疗前后CHF患者淋巴细胞β-AR密度,Fura 2-AM荧光指示法测定血小板胞内[Ca~(2 )]_i,放射免疫法测定血浆去甲肾上腺素(NE)浓度的变化.结果:CHF患者淋巴细胞β-AR密度低于对照组.血小板[Ca~(2 )]_i及血浆NE水平均高于对照组.地尔硫唑降低血小板[Ca~(2 )]_i,升高淋巴细胞β-AR密度.治疗前后血浆NE水平无显著变化.无论治疗前后,血小板[Ca~(2 )]_i与淋巴细胞β-AR密度均呈负相关.结论:地尔硫唑能够部分逆转心衰患者β-AR的下调,这种作用与血浆NE水平变化无关,而可能与降低细胞内[Ca~(2 )]_i有关.     

Role of protein kinase C-epsilon in the development of kappa-opioid receptor tolerance to U50,488H in rat ventricular myocytes

The role of protein kinase C-epsilon (PKC-epsilon) in the development of kappa-opioid receptor (kappa-OR) tolerance to the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl) (U50,488H), the selective agonist of kappa-OR, was determined in rat ventricular myocytes. Incubation of ventricular myocytes with 1 microM U50,488H for 24 h significantly attenuated the inhibitory effects of 30 microM U50,488H on the electrically-induced [Ca(2+)](i) transient and forskolin-stimulated cyclic AMP accumulation, indicating the development of tolerance to the kappa-OR agonist. Chronic treatment of ventricular myocytes with U50,488H also induced translocation of PKC-epsilon to the particulate fraction. On the other hand, administration of 30 microM U50,488H for 10 min induced translocation of PKC-alpha to the particulate fraction in na?ve ventricular myocytes, but not in cells pretreated with 1 microM U50,488H for 24 h. In ventricular myocytes incubated for 24 h with 1 microM U50,488H together with 1 microM chelerythrine or 1 microM GF109203X, PKC inhibitors, or 0.1 microM epsilonV1-2 peptide, a selective inhibitor of PKC-epsilon, 30 microM U50,488H still produced the inhibitory effect on the electrically-induced [Ca(2+)](i) transient as it did in na?ve ventricular myocytes. Chronic treatment of ventricular myocytes with U50,488H and chelerythrine also attenuated the development of tolerance to acute U50,488H on cyclic AMP accumulation. Cells exposed to chelerythrine, GF109203X, or epsilonV1-2 peptide alone did not show an altered [Ca(2+)](i) response to U50,488H. These results indicate that activation of PKC-epsilon is a critical step in the development of tolerance in the kappa-OR.     

kappa-opioid receptor stimulation inhibits cardiac hypertrophy induced by beta1-adrenoceptor stimulation in the rat

To test the hypothesis that kappa-opioid receptor stimulation inhibits cardiac hypertrophy induced by beta1-adrenoceptor stimulation, we determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on cardiac hypertrophy induced by isoprenaline, a selective beta-adrenoceptor agonist, in neonatal ventricular myocytes upon blockade of beta2-adrenoceptor. Hypertrophy of cardiomyocytes was determined by increases in (i) total protein content; (ii) [3H]leucine incorporation; and iii) cell size. 10 micromol/l isoprenaline increased all three parameters. The effects were abolished by 2 micromol/l propranolol, a beta-adrenergic receptor antagonist, or 300 nmol/l CGP20712A, a beta1-adrenoceptor antagonist, but not by 100 nmol/l ICI118,551, a beta2-adrenoceptor antagonist. The effects were also abolished by Rp-cAMPs 100 micromol/l, a protein kinase A inhibitor and not by pertussis toxin 5 mg/l. The effects of isoprenaline in the presence or absence of ICI118,551 were also abolished by 1 micromol/l U50,488H. The inhibitory effects of U50,488H were abolished by 1 micromol/l nor-binaltorphimine, a selective kappa-opioid receptor antagonist. U50,488H also abolished the increases in the amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by 10 micromol/l isoprenaline in the presence or absence of ICI118,551, an effect also abolished by nor-binaltorphimine. In conclusion the results show that kappa-opioid receptor stimulation abolished both the cardiac hypertrophy and enhanced amplitude and frequency of the spontaneous intracellular Ca2+ transient induced by beta1-adrenoceptor stimulation.     

kappa-Opioid receptor stimulation inhibits augmentation of Ca(2+) transient and hypertrophy induced by isoprenaline in neonatal rat ventricular myocytes - Role of CaMKIIdelta(B)

We aimed to further define the pathway mediating the inhibitory effects of kappa-opioid receptor stimulation on Ca(2+) transients and hypertrophic responses to beta(1)-adrenoceptor stimulation. We determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on the enhancement of spontaneous Ca(2+) transients and the induction of hypertrophy by isoprenaline, a beta-adrenoceptor agonist, in cultured neonatal ventricular myocytes. The results were compared with those found with KN93, a selective Ca(2+)/calmodulin-dependent kinase (CaMKII) inhibitor, propranolol, a beta-adrenoceptor antagonist, and verapamil, a L-type Ca(2+) channel antagonist. Hypertrophy of cardiomyocytes was characterized by increases in (i) total protein content; (ii) cell size; and (iii) [(3)H]leucine incorporation. 10 micromol/l isoprenaline increased all three parameters. We also determined the expression of nuclear CaMKIIdelta in response to U50,488H in the presence or absence of isoprenaline. To determine whether the effects of U50,488H were receptor-mediated, its effects were also measured following blockade of the kappa-opioid receptor with nor-binaltorphimine. kappa-Opioid receptor stimulation suppressed the stimulatory effect of isoprenaline on Ca(2+) transients and cardiac hypertrophy, as did KN93, propranalol and verapamil. U50,488H also suppressed the expression of nuclear CaMKIIdelta(B) in the presence, but not in the absence of isoprenaline. These results suggest that the inhibitory effect of kappa-opioid receptor stimulation on beta(1)-adrenoceptor stimulation may also involve CaMKIIdelta.     

The function of calcineurin and ERK1/2 signal in the antihypertrophic effects of kappa-opioid receptor stimulation on myocardial hypertrophy induced by isoprenaline

The aim of the present study, performed in an in vitro model of cardiac hypertrophy, was to examine the possible function of calcineurin and ERK1/2 in the inhibitory effects of kappa-opioid receptor stimulation on Ca2+ transients and myocardial hypertrophy induced by beta1-adrenoceptor stimulation. We determined the effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on the enhancement of spontaneous Ca2+ transients and the induction of hypertrophy by isoprenaline, a beta-adrenoceptor agonist, in cultured neonatal ventricular myocytes. Total protein content, [3H]leucine incorporation and cell size were used as indices of hypertrophy; calcineurin activity and phospho-ERK1/2 level were determined by immunoblotting. Isoprenaline (10 micromol x L(-1)) increased all the three indices of hypertrophy, Ca2+ transients, calcineurin activity and the level of phospho-ERK1/2. The effects of isoprenaline were abolished by 1 micromol x L(-1) U50,488H in the absence but not in the presence of nor-binaltorphimine, a kappa-opioid receptor antagonist. The inhibitory effects of U50,488H were reproduced by cyclosporine-A, an inhibitor of calcineurin, U0126, the inhibitor of ERK1/2 and verapamil, a L-type Ca2+ channel antagonist. In addition, suppression of calcineurin activity by cyclosporine-A was associated with modest suppression of ERK1/2 phosphorylation. Meanwhile, suppression of ERK1/2 phosphorylation by U0126 was associated with modest suppression of calcineurin activity. In conclusion, the inhibitory effects of kappa-opioid receptor stimulation involved calcineurin and ERK1/2, and the two signaling pathways showed interaction in the mechanism of antihypertrophic effects afforded by kappa-opioid receptor stimulation.     

Effects of κ-opioid receptor stimulation in the heart and the involvement of protein kinase C

1. The role of protein kinase C (PKC) in mediating the action of κ-receptor stimulation on intracellular Ca²⁺ and cyclic AMP production was determined by studying the effects of trans-(±)-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl] cyclohexyl) benzeneacetamide methanesulphonate (U50,488H), a selective κ-receptor agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC agonist, on the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation in the presence and absence of a PKC antagonist, staurosporine or chelerythrine, in the single rat ventricular myocyte.
2. U50,488H at 2.5–40 μM decreased both the electrically-induced [Ca²⁺]_i transient and forskolin-stimulated cyclic AMP accumulation dose-dependently, effects which PMA mimicked. The effects of the κ-agonist, that were blocked by a selective κ-antagonist, nor-binaltorphimine, were significantly antagonized by the PKC antagonists, staurosporine and/or chelerythrine. The results indicate that PKC mediates the actions of κ-receptor stimulation.
3. To determine whether the action of PKC was at the sarcoplasmic reticulum (SR) or not, the [Ca²⁺]_i transient induced by caffeine, that depletes the SR of Ca²⁺, was used as an indicator of Ca²⁺ content in the SR. The caffeine-induced [Ca²⁺]_i transient was significantly reduced by U50,488H at 20 μM. This effect of U50,488H on caffeine-induced [Ca²⁺]_i transient was significantly attenuated by 1 μM chelerythrine, indicating that the action of PKC involves mobilization of Ca²⁺ from the SR. When the increase in IP₃ production in response to κ-receptor stimulation with U50,488H in the ventricular myocyte was determined, the effect of U50,488H was the same in the presence and absence of staurosporine, suggesting that the effect of PKC activation subsequent to κ-receptor stimulation does not involve IP₃. The observations suggest that PKC may act directly at the SR.
4. In conclusion, the present study has provided evidence for the first time that PKC may be involved in the action of κ-receptor stimulation on Ca²⁺ in the SR and cyclic AMP production, both of which play an essential role in Ca²⁺ homeostasis in the heart.

     

Effects of pharmacological preconditioning with U50488H on calcium homeostasis in rat ventricular myocytes subjected to metabolic inhibition and anoxia

1. The effects of pharmacological preconditioning with U50488H (U(50)), a selective kappa-opioid receptor agonist, on Ca(2+) homeostasis in rat ventricular myocytes subjected for 9 min to metabolic inhibition (MI) and anoxia (A), consequences of ischaemia, were studied and compared with those of preconditioning with brief periods of MI/A. 2. Precondition with 30 micro M of U(50) for three cycles of 1 min each cycle separated by 3 min of recovery (UP) significantly increased the percentage of non-blue cells following MI/A. The effect of UP is the same as that of preconditioning with an inhibitor of glycolysis and an oxygen scavenger for three 1-min cycles separated by three-minute recovery (MI/AP). The results indicate that like MI/AP, UP also confers cardioprotection. 3. MI/A increased intracellular Ca(2+) ([Ca(2+)](i)) and reduced the amplitude of caffeine-induced [Ca(2+)](i) transients, an indication of Ca(2+) content in the sarcoplasmic reticulum (SR). MI/A also reduced the electrically-induced [Ca(2+)](i) transient, that indicates Ca(2+)-release during excitation-contraction coupling, and Ca(2+) sparks in unstimulated myocytes, that indicates spontaneous Ca(2+)-release from SR. It also prolonged the decline of the electrically-induced [Ca(2+)](i) transient and slowed down the recovery of the electrically-induced [Ca(2+)](i) transient after administration of caffeine. In addition, MI/A prolonged the decline of caffeine induced [Ca(2+)](i) transient, an indication of Na(+)-Ca(2+) exchange activity, and UP prevented it. So UP, that confers cardioprotection, prevented the changes induced by MI/A. With the exception of Ca(2+)-spark, which was not studied, the effects of MI/AP are the same as those of UP. 4. It is concluded that pharmacological preconditioning with U(50), that confers immediate cardioprotection, prevents changes of Ca(2+) homeostasis altered by MI/A in the rat heart. This may be responsible, at least partly, for the cardioprotective action. 5. The study also provided evidence that MI/A causes mobilization of Ca(2+) from SR to cytoplasm causing Ca(2+)-overload which may be due to reduced Ca(2+)-uptake by SR. MI/A also reduces spontaneous and electrically induced Ca(2+) release from SR.     

Kappa-opioid receptor agonist inhibits the cholera toxin-sensitive G protein in the heart

To explore the signaling mechanisms of the negative modulation of beta-adrenoceptors by kappa-Opioid receptors (kappa-OR) in the heart, the possibility of the interaction at the level of G protein and receptor was determined. Cholera toxin, an activator of the stimulatory G protein (Gs), elevated electrically induced intracellular Ca2+ ([Ca2+]i) transients and induced ribosylation of the alpha-subunit of Gs (Gsalpha) in rat ventricular myocytes. The effects were significantly attenuated by U50,488H, a specific agonist of kappa-OR, and were abolished by nor-binaltorphimine, a selective kappa-OR antagonist. The content of Gsalpha, however, was not affected by U50,488H. Receptor binding experiments showed that neither Bmax nor Kd of the binding of [3H]CGP-12177, a beta-adrenoceptor antagonist, was affected by U50,488H. The current study provides the first evidence that kappa-OR stimulation inhibits the ribosylation of the alpha-subunit of the Gs protein, thus inhibiting the action of cholera toxin on the protein.     

Dopamine D2 receptor stimulation inhibits angiotensin II-induced hypertrophy in cultured neonatal rat ventricular myocytes

1. Myocardial hypertrophy is a common pathological change that accompanies cardiovascular disease. Dopamine D2 receptors have been demonstrated in cardiovascular tissues. However, the pathophysiological involvement of D2 receptors in myocardial hypertrophy is unclear. Therefore, the effects of the D2 receptor agonist bromocriptine and the D2 receptor antagonist haloperidol on angiotensin (Ang) II- or endothelin (ET)-1-induced hypertrophy of cultured neonatal rat ventricular myocytes were investigated in the present study. 2. Protein content and protein synthesis, determined by examining [(3)H]-leucine uptake, were used as estimates of cardiomyocyte hypertrophy. The expression of D2 receptor protein in neonatal rat ventricular myocytes was determined using western blotting. Changes in [Ca(2+)](i) in cardiomyocytes were observed by laser scanning confocal microscopy. 3. Angiotensin II and ET-1, both at 10 nmol/L, induced myocyte hypertrophy, as demonstrated by increased protein content and synthesis, [Ca(2+)](i) levels, protein kinase C (PKC) activity and phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase and mitogen-activated protein kinase (MAPK) p38 (p38). Concomitant treatment of cells with 10 nmol/L AngII plus 10 micromol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy, MAPK phosphorylation and PKC activity in the membrane, as well as [Ca(2+)](i) signalling pathways, compared with the effects of AngII alone. In addition, 10 micromol/L bromocriptine significantly inhibited cardiomyocyte hypertrophy induced by 10 nmol/L ET-1. However, pretreatment with haloperidol (10 micromol/L) had no significant effects on cardiomyocyte hypertrophy induced by either AngII or ET-1. 4. In conclusion, D2 receptor stimulation inhibits AngII-induced hypertrophy of cultured neonatal rat ventricular myocytes via inhibition of MAPK, PKC and [Ca(2+)](i) signalling pathways.     

Effects of U50,488H on transient outward and ultra-rapid delayed rectifier K+ currents in young human atrial myocytes

The effects of trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide methanesulfonate salt (U50,488H), a selective kappa-opioid receptor agonist, on transient outward K+ current (Ito1) and ultra-rapid delayed rectifier K+ current (IKur) in young human atrial myocytes were evaluated with a whole-cell patch-clamp technique. At +10 mV, U50,488H decreased Ito1 in a concentration-dependent manner (IC50=12.4+/-3.5 microM), while at +50 mV, U50,488H produced biphasic effects on Ito1-increasing and decreasing the current at 1-3 and 10-30 microM, respectively. U50,488H at 10 microM shifted the midpoint (V0.5) of Ito1 activation in a depolarizing direction by approximately 5 mV, accelerated the inactivation, and slowed the recovery from inactivation of Ito1. In addition, U50,488H inhibited IKur in a concentration-dependent manner (IC50=3.3+/-0.6 microM). The effects of U50,488H on the two types of K+ currents were not antagonized by either 5 microM nor-binaltorphimine or 300 nM naloxone. These results indicate that U50,488H affects both Ito1 and IKur in young human atrial myocytes in an opioid receptor-independent manner.