Ma SG et al / Acta Pharmacol Sin 2004 May; 25 (5): 587-592

Effect of G_αq/11 protein and ATP-sensitive potassium channels on prostaglandin E₁ preconditioning in rat hearts

Shou-guo MA, Run-fang FU^1,2, Guo-qing¹ FENG, Zhen-ji WANG¹, Xiang-qin MA¹, Shi-ai WENG¹

Department of Cardiology, The 159th hospital of PLA, Zhumadian 463000, China;
¹Department of Pharmacology, Medical College, Zhengzhou University, Zhengzhou, 450052, China

² Correspondence to Prof Run-fang FU. Phn 86-371-695-8936 or 86-371-691-2533. Fax 86-371-697-4754. E-mail frf@zzu.edu.cn or runfangfu@yahoo.com.cn

Received 2003-04-21 Accepted 2003-09-25

KEY WORDS G-proteins; potassium channels; prostaglandin E₁; ischemic preconditioning; signal transduction; ischemia-reperfusion injury

ABSTRACT

AIM: To investigate the effect of G_αq/11 signaling pathway and ATP-sensitive potassium channels (K_ATP channels) on prostaglandin E₁ (PGE₁) induced early and delay-preconditioning protection in rat hearts. METHODS: Two series of experiments were performed in Wistar rat hearts. In the first series of experiment, all rats were pretreated with PGE₁ 40 min or 23 h 20 min before the experiment. Ischemia-reperfusion injury was induced by 30 min coronary artery occlusion followed by 90 min reperfusion. Hemodynamics, infarct size, and scores of ventricular arrhythmias were measured. The expression of G_αq/11 protein in the heart was measured by Western blot analysis in the second series. RESULTS: Preconditioning with PGE₁ (25 µg/kg ) markedly reduced infarct size, left ventricular end-diastolic pressure, and scores of ventricular arrhythmia. The effect of PGE₁ was significantly attenuated by glibenclamide (1 mg/kg, ip), a nonselective K_ATP channel inhibitor. PGE₁ caused a significant increase in the expression of G_αq/11 protein. CONCLUSION: Activations of G_αq/11 signal pathway and K_ATP channel played significant roles in the cardioprotection of PGE₁ preconditioning in rat heart and might be an important mechanism of signal transduction pathway during the PGE₁ preconditioning.

INTRODUCTION

Ischemic preconditioning (IPC), a well-known phenomenon in which brief episodes of ischemia and reperfusion before a prolonged ischemic event limit myocardial cellular damage, has been shown to elicit both an acute and delayed phase of cardioprotection or a second window of protection^[1]. The ATP-sensitive potassium channel has been suggested as an end-effector in the mechanism of ischemic preconditioning^[2]. Recent studies show that the K_ATP channel mediates the myocardial protection induced by pharmacological agents such as adenosine agonist^[3], opioids^[4], flumazenil^[5] and monophosphoryl lipid A (MLA)^[6]. Hide et al^[7] reported that PGE₁ preconditioning reduced myocardial infarct size in the rabbit by activation of K_ATP channels . G_αq/11, a member of G_a protein subunit plays an important role as a signal transduction pathway in protecting mechanism of ET-1 preconditioning and IPC^[8]. The cardioprotective effects of PGE₁ have been attributed to systemic and coronary vasodilation, inhibition of platelet aggregation and in particular, inhibition of neutrophil activation. However, PGE₁ has been suggested a cardioprotection induced by pharmacological preconditioning in rabbit heart and rat heart^[7,9].

In this paper we investigate the effect of G_αq/11 protein and K_ATP channel on PGE₁ preconditioning in rat hearts.

MATERIALS AND METHODS

Animals Male Wistar rats weighing 270-320 g (provided by Henan experimental animal center, Grade II, centificated No 2002LA-193) were anesthetized by intraperitoneal injection of pentobarbital sodium (45 mg/kg). Rats were intubated and ventilated with a respirator using a mixture of 100 % oxygen and room air (total volume of 1.2 mL per 100 g body weight; respiratory rate, 65-70 breaths/min).

Experimental protocol Two series of experiments were performed in the study. In the first series, all rats were subjected to 30 min ischemia and 90 min reperfusion (I/R). Myocardium ischemia/reperfusion (MI/R) group rats, injected with 2 mL saline 40 min before I/R (MI/R). PGE₁ early preconditioning protrection (EPP) group rats were injected 20 min PGE₁ (25 µg/kg) 40 min before I/R. PGE₁ delayed preconditioning protection (DPP) group rats were injected 20 min PGE₁ (25 µg/kg) 23 h 20 min before I/R. Glibenclamide (Gli) group rats were given glibenclamide(1 mg/kg) 30 min before I/R. Gli+EPP and Gli+DPP group rats were treated with glibenclamide 40 min or 23 h 20 min before I/R respectively, and glibenclamide 30 min before I/R. Hemodynamics, infarct size/area at risk (IS/AAR) of myocardium and scores of ventricular arrhythmia were measured. The expression of G_αq/11 protein in sarcolemma was measured in the following groups by Western blot analysis in the second series: sham operated control (Control), MI/R, EPP and DPP.

Hemodynamics and scores of arrhythmia After rats were anesthetized, a catheter tip pressure transducer (PE₅₀) was inserted into the right carotid artery and advanced into the left ventricle for the determination of hemodynamics. Then, a midline thoracotomy was performed , the heart was exposed, and myocardial ischemia was produced by placing a 5-0 silk thread around the left anterior descending coronary artery (LAD), approximately 2-3 mm from its origin. Ischemia was maintained for 30 min. At the end of ischemia, the silk thread was released for 90 min reperfusion. The number of premature ventricular contractions (PVCs), episodes and duration of ventricular tachycardia (VT) and ventricular fibrillation (VF) in ischemia period and reperfusion period^[8], left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular maximum changes in positive pressure over time (+dp/dt) were recorded using ECG monitor and a 4-channel polygraph recorder in 15 min of ischemia period and 30 min of reperfusion period respectively.

Measurements of infarct size At the end of ischemia reperfusion, the LAD was reoccluded and Evans blue dye solution (1 mL of 2 % w/v) was injected into the left ventricle to distinguish perfused and non-perfused (area at risk) sections of the heart. The Evens blue solution stained the perfused myocardium, while the occluded vascular bed remains uncoloured. The rats were killed and the hearts were immediately excised, weighed, frozen, and stored in a freezer. After removal of the atria and right ventricle, the frozen heart was sliced into 1.5 mm thick 5-6 sections, and the slices were incubated in 1 % triphenyltetrazolium chloride (TTC) in pH 7.4 buffer for 20 min at 37 ºC. The slices were immersed in 10 % formalin overnight. Viable myocardium is stained in red color by TTC, whereas infarcted tissue is gray, nonischemic area is blue. The infarcted myocardium was dissected from the AAR under the illumination of a dissecting micro-scope. IS, AAR, and LV were determined by gravi-metric analysis. AAR was expressed as a percentage of the LV (AAR/LV), and IS was expressed as a percentage of the AAR (IS/AAR).

Western blot analysis For G_αq/11 protein assay, heart tissue (100 mg) was homogenized in 2 mL ice-cold lysis buffer (50 mmol/L Tris-HCl, pH 7.2, 0.1 % deoxycholic acid , 0.1 % Triton X-100, 5 mmol/L ethylene diaminotetraacetic acid, 100 µmol/L phenylmethyl-sulfonyl fluoride). The lysates were sonicated on ice and centrifuged at 1000×g at 4 ºC for 10 min. The supernatant was further subjected to centrifugation for 20 000×g for 40 min at 4 ºC. The subsequent crude membrane pellet was resuspended in the homogenizing buffer (20 mmol/L Tris-HCl, pH 7.4, 1 mmol/L ethylene diaminotetraacetic acid, 1 mmol/L dithiothreitol, 100 µmol/L phenylmethylsulfonyl fluoride). Total protein concentration of membrane fractions was measured using the Lowry method. Prestained high molecular mass marker and 150 µg proteins from samples were separated on 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred on to 0.45 µmol/L Nitrocellulose membrane. The membrane was blocked overnight at 4 ºC in 5 % skim milk and probed with primary antibody for G_αq/11. Primary antibody was diluted 1:200 in PBS. Horseradish peroxidase (HRP)-labeled anti-rabbit IgG was diluted in 1:5000 in PBS and used as secondary antibody. G_αq/11 was visualized by enhanced chemiluminescence (ECL). Autoradiographs from Western blot analysis were quantified using Eagle eye II systerm.

Reagents PGE₁ was purchased from Shenyang Biochemical Co (No 010906). Glibenclamide, Evans blue, triphenyltetrazolium chloride (TTC) and BSA were purchased from Sigma Chemical Co. G_αq/11 primary antibody, HRP-labeled IgG, ECL were the product of Santa Cruz Co.

Statistical analysis All values are expressed as mean±SD. One way analysis of variance (ANOVA) followed by Bonferroni's test was used for comparing the differences among multiple groups. Significant differences among groups were defined by P<0.05.

RESULTS

Hemodynamics Myocardical functional parameters, such as heart rate, LVSP, +dp/dt, and LVEDP were not significantly different among the six groups at baseline, the average body and heart weights were also similar among all the groups (data not shown).

In the ischemic-reperfused heart, LVEDP was significantly lower in the PGE₁ pretreated groups as compared with the MI/R (P<0.01 , Tab 1). However, the LVEDP was not significantly different between the EPP and DPP group. The PGE₁-induced improvement in LVEDP was abolished by glibenclamide in Gli+EPP and Gli+DPP (compared with MI/R, P>0.05). Glibenclamide itself had no significant effect on LVEDP. No significant changes in LVSP, +dp/dt, or heart rate were observed among the groups.

Tab 1. Hemodynamics of each group. n=10. ^bP<0.01 vs MI/R. Pre: preischemia; I: ischemia 15 min; R: reperfusion 30 min. HR: heart rate; LVSP: left ventricular systolic pressure; LVEDP: left ventricular end diastolic pressure; +dp/dt: maximum positive change in pressure over time.

	MI/R	EPP	DPP	Gli	Gli+EPP	Gli+DPP
HR /beats·min-1
Pre	416±24	423±31	420±26	419±19	404±19	414±23
I	389±21	406±27	406±26	399±19	388±20	396±22
R	387±21	393±22	383±23	392±27	376±18	382±25
LVSP /mmHg
Pre	128±17	119±18	123±15	132±20	108±19	127±22
I	117±15	124±14	119±20	126±16	98±18	120±18
R	112±17	120±18	120±19	129±17	104±16	123±17
LVEDP /mmHg
Pre	25±5	25±4	26±4	25±5	26±4	24±5
I	31±4	23±3b	22±3b	31±4	34±5	32±5
R	42±4	22±4b	21±4b	39±6	40±5	39±5
+dp/dt /mmHg·s-1
Pre	435±665	4239±743	4407±772	4385±628	4280±692	4291±712
I	428±834	4372±783	4280±685	4328±732	4370±683	4186±677
R	403±657	4166±654	4310±613	4219±650	4173±598	4064±630

Infarct size During the early pretreat phase, preconditioning with PGE₁ resulted in significant decrease in the infarct size (% AAR) from 22.1 %±3.6 % in the MI/R group to 14.7 %±2.0 % in EPP group, a 33.4% reduction compared with the MI/R group. The infarct size increased significantly to 19.6 %±2.8 % (P<0.01) when glibenclamide was given 30 min before I/R in the PGE₁-pretreated rats. Glibenclamide itself had an infarct size of 23.2 %±2.7 %, which was not significantly different compared with the MI/R group (P>0.05). In the delayed preconditioning , the infarct size had a significant reduction (13.4 %±2.9 % in DPP group), compared with MI/R group, P<0.01. PGE₁-induced delayed protection was also abolished by glibenclamide as indicated by increased infarct size (21.4 %±3.1 %, P<0.01). Compared with the early preconditioning, the infarct size was not significantly different in delayed preconditioning. The area at risk (% LV) was not different among the groups (Fig 1).

Fig 1. Effect of PGE₁ on myocardial infarct size. n=10. ^cP<0.01 vs MI/R. IS: infarct size; AAR: area at risk; LV: left ventricular.

Scores of arrhythmia Compared with MI/R group, the scores of ischemia phase (I) and reperfusion phage (R) in EPP and DPP groups significantly decreased (P<0.01). The protective effect was abolished by glibenclamide in Gli+EPP and Gli+DPP, but glibenclamide itself had no significant effect on arrhythmia. Scores of arrhythmia were not significantly different between the early and delayed preconditioning (Fig 2).

Fig 2. Effect of PGE₁ on the score of ventricular arrhythmia during 30-min occlusion and 90-min reperfusion. n=10. ^cP<0.01 vs MI/R. I: ischemia; R: reperfusion.

Expression of cardiac G_αq/11 protein In comparison with Control group, G_αq/11 protein expression was increased by 46.4 % (P<0.01) and 65.8 % (P<0.01) in EPP and DPP group respectively, while there was no significant difference in MI/R group. Interestingly, the expression of G_αq/11 in delayed preconditioning was higher than early preconditioning (P<0.05) (Fig 3).

Fig 3. Immunoblotting analysis of G_αq/11 in left ventricles of rats. Upper panel shows representative Western blots for G_αq/11 and lower panel shows densitometric scores, n=8. ^cP<0.01 vs control.

DISCUSSION

Our results showed that PGE₁ induced an early and delayed cardioprotective effect in the heart as indicated by a significant decrease in the infarct size, scores of ventricular arrhythmias and LVEDP compared with the MI/R animals. The blocker of K_ATP channels glibencla-mide, when administered 30 min before ischemia-reperfusion, abolished the early as well as the delayed cardioprotection induced by PGE₁. No major differences in the heart rate, LVSP, +dp/dt, and AAR/LV were observed among the groups during the infarction protocol, suggesting that the changes in myocardial infarct size and scores of ventricular arrhythmias were independent of the systemic hemodynamics. PGE₁ pretreatment increased the expression of G_αq/11 protein in EPP and DPP rat hearts. Taken together, our data suggested that pretreatment of rats with PGE₁ substantially reduced myocardial infarct size and ventricular arrhythmias, and the cardioprotective effects were mediated by K_ATP channels. Additionally, the G_αq/11 protein signaling was involved in the cardioprotective effect during PGE₁ preconditioning.

In our study, the protective effect in the heart was not significantly different between the early and delayed preconditioning induced by PGE₁. We did not perform time course of protection following PGE₁ treatment. Therefore, it was not clear whether this protection was sustained or was similar to the biphasic effect observed by ischemic preconditioning.

Recent studies have shown that vasodilatation and inhibition of platelet and neutrophil function are not a prerequisite for the cardioprotective effects of prosta-glandins. Hide's study^[7] demonstrated that pretreatment of rabbits with PGE₁ or PGE₀ caused reduction in myocardial infarct size, and the potent cardioprotective effects exerted by opening of K_ATP channels. Yama-moto's results^[10] suggested that the PGE₁ protection of myocardium against ischemia was induced by inhibiting the myocardial L-type Ca²⁺ current. Our previous study^[9] demonstrated that PGE₁ could protect ischemia-reperfusion myocardium from lipid peroxidation and enhence the activity of SOD in experimental rats, then it could modulate the balance of lipid peroxidation and anti-peroxidation effect in vivo.

It is now widely believed that K_ATP channels acts as the "end effector" of preconditioning induced by endogenous stresses^[11-13] as well as pharmacological agents including adenosine agonist^[3], flumazenil^[5], opioid agonist and MLA^[6] etc. Opening of the K_ATP channel has been shown to be protective due to the increase in the outward K⁺ current resulting in the shortening of action potential, which in turn may spare ATP, thereby allowing less entry of Ca²⁺ into the myocyte. Decreased intracellular Ca²⁺ overload then results in less ischemic injury and better myocyte preservation^[14]. Especially, opening of mitochondrial K_ATP channel leads to membrane depolarization, matrix swelling, slowing of ATP synthesis, and accelerated respiration^[2,15,16], which due to myocardical protection by reducing infarct size and ventricular arrhythmias during preconditioning. Our present study also suggested that opening of K_ATP channels was the common mechanism which caused reduction of infarct size and ischemic arrhythmias in pretreatment of rats with PGE₁.

Recent study demonstrated that G_αq/11 signal pathway was related to the protective mechanism of ET-1 pretreatment and ischemic preconditioning^[8]. EP₁, EP₂, EP₃, and EP₄ are the four subtypes of prostaglandin E receptors. The EP₁ and EP₃ are coupled to G_αq/11- phospholipase C (PLC) signal pathway. Interestingly, PGE₁ can act on EP₁ and EP₃ (subgroups A and D) receptors, and then activate PLC to release inositol 1,4,5,-trisphosphate (IP₃) and 1,2-diacylglycerol (DAG)^[7]. The latter compound in combination with intracellular calcium then causes the translocation and activation of protein kinase C (PKC). Activated PKC may phosphorylate secondary effectors. In our experiment, the expression of G_αq/11 protein is significantly increased in PGE₁ pretreated (including EPP and DPP groups) animals. These suggested that the opening of K_ATP channels was based on activation of PKC, while the activation of the G_αq/11 signal pathway (via activation of EP₁ and EP₃ receptors by coupled with PGE₁) is due to activate PLC, which enhences IP₃ /DAG signal pathway for the activation of PKC.

In summary, the present study demonstrated that pretreatment of rats with PGE₁ induced a significant decrease in myocardial infarct size and ventricular arrhythmias during regional ischemia and reperfusion. The cardioprotective effects of PGE₁ were due to activation of K_ATP channels, involved in activation of G_αq/11-PLC signal transduction pathway via activation of EP₁ or more likely EP₃ receptors (coupled with PGE₁).

ACKNOWLEDGEMENT We are indebted to Prof Jie LIU (Department of Physiology and Pathophysiology, Peking University Health Science Center) for supplying primary antibody.

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