八肽膽囊收縮素激活鈣離子通道和酪氨酸激酶誘導(dǎo)豚鼠心肌細(xì)胞內(nèi)的游離鈣增高

1、31 ZHAO Xiao-Yun:CCK-8 Increases Intracellular Calcium of Cardiomyocytes Received 2003-05-12 Accepted 2003-06-24This work was supported by the grants of Education Committee of Hebei Province (No.200122.*Cholecystokinin octapeptide increases free intracellular calcium of guinea pig cardiomyocytes thr

2、ough activation of Ca 2+ channel and tyrosine kinaseZHAO Xiao-Yun 1, LING Yi-Ling 2,*, SHANG Zhong-Lin 3, LI Qing 4, YIN Jing-Xiang 4 , TAN Guo-Jun 41Hebei Provincial Geriatric Key Laboratory , Shijiazhuang 050051; Departments of 2Pathophysiology and 4Physiology ,Hebei Medical University , Shijiazhu

3、ang 050017; 3College of Biological Science , Hebei Normal University , Shijiazhuang050017, Chinaof Ca2+in isolated guinea pig cardiomyocytes, in which tyrosine kinase may be involved.Key words: cholecystokinin; cardiomyocyte; fluorescent intensity; Ca 2+channel; confocal microscopy; receptor12,*3444

4、1,050051;24,050017;3,050017:(CCK-8(Ca2+iFluo 3-AM,Ca 2+iCa2+iFi(F i/F%: (1Ca 2+1.0 mmol/LTyrodes , CCK-8 (1104 pmol/LCa 2+i(P0.01(2EGTA (3 mmol/Lnisoldipine (0.5mol/L5 min, CCK-8 (102pmol/L Ca2+i (P0.01(3CCK(proglumide 6mol/Lgenistein (1mol/L5 min,CCK-8Ca 2+i (P0.01CCK-8Ca2+, Ca2+,Ca2+,Ca 2+i,:;: Q4

5、63; R363Acta Physiologica Sinica, February 25, 2004, 56(1: 31-3532Cholecystokinin (CCK functions as a neurotransmitter in the central nervous system and gastrointestinal tract as well as a hormone in the gastrointestinal tract, and has nu-merous biologic effects 1. Cholecystokinin octapeptide (CCK-8

6、 is a key biological fragment of endogenous CCK.Previous studies showed that CCK-8 elicited dose-depen-dant increase in arterial blood pressure 2,3, an effect that was inhibited by CCK-A receptor (CCK-AR antagonist 3,4.Our recent study indicated that CCK-8 could enhance cardiac function in a dose-de

7、pendent manner through ac-tivation of CCK- receptor (CCK-R on myocardium 5 . It is well established that the increase in Ca 2+i is the key factor that augments the strength of myocardial contraction.However, the effects of CCK-8 on Ca 2+i level in cardi-omyocytes remain unclear . The present study w

8、as under-taken to observe the effects of CCK-8 on Ca 2+i level and the signal transduction mechanism involved.1 MATERIALS AND METHODS1.1 Preparation of guinea pig cardiomyocytes . Six male guinea pigs (300500 g were provided by the Experi-mental Animal Center of Hebei Province (grade , Cer-tificate

9、No 04064. Animals were first injected intraperi-toneally with 1000 U of heparin and were decapitated 15min later. Hearts were removed quickly by thoracotomyand risen in oxygenated ice-cold Ca 2+-free Tyrodes solution.Single ventricular myocytes were isolated as Rubin de-scribed 6. Briefly guinea pig

10、 hearts were perfused ret-rogradely through the aorta at a rate of 9 ml/min with Ca 2+-free Tyrodes solution for 5 min and then with thesame solution containing collagenase 360 mg/L bovine serum albumin (BSA 160 mg/L, and CaCl 2 34mol/L for 10 min at 37. The ventricles were cut, and then minced, inc

11、ubated for 10 min in Ca 2+-free Tyrodes solution containing 0.1% BSA. Myocytes were harvested after filtration through a nylon mesh, and resuspended in Tyrodes solutions containing different concentrations of Ca 2+. The concentration of Ca 2+was gradually increased to 1 mmol/L.1.2 Fluo 3-AM loading

12、. Isolated ventricular myocytes were incubated with Fluo 3-AM working solution con-taining 0.03 % Pluronic F-127 (the final concentration of Fluo 3-AM is 20 mol/L at 25 for 60 min. Afterincubation, the cells were washed with Ca 2+-free Tyrodes solutions three times to remove the extracellular Fluo3-

13、AM at 25.1.3 Calcium measurement. The cells were placed into asmall pool of Teflon printed slice (from Electromicroscopy Sciences, and covered by cover glass. Only the cells with rod shaped and visible striations were used for experi-ments . The fluorescence signal was detected with confo-cal laser

14、scanning system (BioRad MR/A2, which was equipped with a Nikon E-600 eclipse microscope . An argon laser was used to excite Fluo-3 at 488 nm and emitat 530 nm. Ca 2+i changes were represented with fluorescent intensity (F i or relative fluorescent intensity (F i /F 0 %. F i /F 0 % is the ratio of ch

15、ange in F i to the fluorescence under quiescent condition (F 0. The images were collected with computer and then processed with confocal assistant andphotoshop software. Therefore, the distribution of Ca 2+i was recorded both in the quiescent condition and after application of CCK-8 or other drugs.1

16、.4 Experimental protocols . The experiments were consisted of 3 groups (n =6: (1 effect of CCK-8 on Ca 2+i : F i was measured after adding CCK-8 (1104pmol/L to the normal Tyrodes solution containing 1.0mmol/ L Ca 2+; (2 effect of EGTA or nisoldipine on Ca 2+i elevation induced by CCK-8: the preparat

17、ion was pre-treated with the Ca 2+ chelator EGTA 7 (3 mmol/L or thecalcium channel blocker nisoldipine 8 (0.5mol/L for 5min, and F i was measured after adding CCK-8 (102 pmol/L; (3 effect of proglumide and genistein on Ca 2+i elevation induced by CCK-8: the preparation was pre-treated with the nonse

18、lective CCK-R antagonist proglu-mide (6 mol/L or tyrosine kinase inhibitor genistein (1mol/L for 5 min, and F i was measured after adding CCK-8 (102 pmol/ L.1.5 Chemicals and solution . Sulfated CCK-8, collage-nase type , taurine, MOPS, HEPES, EGTA,nisoldipine,proglumide and genistein were purchased

19、 from Sigma Co.The Ca 2+-free Tyrodes solution contained NaCl 100, KCl 10, MgSO 4 5, NaH 2PO 4 1.2, glucose 20, taurine 10,MOPS 10 mmol/L, pH was adjusted to 7.4 with KOH. The normal Tyrodes solution was used by adding 1.0 mmol/LCaCl 2 to the Ca 2+-free Tyrodes solution.1.6 Statistic analysis. All d

20、ata were expressed as meansSD, and analized by one-way ANOVA followed by the paired t test for within-group comparison and the un-paired t test for between group comparison. P values less than 0.05 were considered statistically significant.2 RESULTS2.1 Effect of CCK-8 on Ca 2+i in cardiomyocytes CCK

21、-8 (1104 pmol/ L elicited a rapid and marked33ZHAO Xiao-Yun:CCK-8 Increases Intracellular Calcium of Cardiomyocytes increase in Ca 2+ i in isolated cardiomyocytes in normal Tyrodes solution containing 1.0 mmol/L Ca 2+ (P 0. 01.The Ca 2+i reached the peak level at 30 s, and thenrestored to baseline a

22、fter 200 s. CCK-8 (102 pmol/Lproduced a marked increase in Ca 2+i (P 0. 01 (Figs.1A and 2. 2.2 Effect of EGTA and nisoldipine on Ca 2+ i elevation induced by CCK-8 in cardiomyocytesCCK-8 (102 pmol/ L induced a small and slow increase in Ca 2+ i in cardiomyocytes after pretreatment with EGTA (3 mmol/

23、L and nisoldipine (0.5 mol/L for 5 min. The increase in Ca 2+ i was inhibited by administration of either EGTA or nisoldipine (P 0. 01. The Ca 2+i reached the peak level at 3060 s and then restored to baseline after 120150 s (Figs. 1B and 3,Table 1.2.3 Effect of proglumide and genistein on Ca 2+i el

24、evation in cardiomyocytes induced by CCK-8Pretreatment with the non-selective CCK-R antagonist proglumide (6 mol/L or the tyrosine kinase inhibitor genistein (1 mol/L for 5 min inhibited the increase in Ca 2+ i induced by CCK-8 (102pmol/L in isolated cardi-Fig. 2. Effect of CCK-8 (1104 pmol/L on Ca

25、2+i changes in single isolated cardiomyocytes. Ca 2+i changes were repre-sented by relative fluorescent intensity (F i /F 0 %. , addi-tion of CCK-8 (102 pmol/L.Fig. 1. Fluorescent images of Ca 2+i changes in isolated cardiomyocytes (dynamic images were captured every 30 s. A : Effect of CCK-8 (102 p

26、mol/L on Ca 2+i in normal Tyrodes solution containing 1.0 mmol/L Ca 2+. B : Effect of CCK-8 (102 pmol/L on Ca 2+ i after pretreatment with EGTA (3 mmol/L. , addition of CCK-8 (102pmol/L.Acta Physiologica Sinica, February 25, 2004, 56(1: 31-3534omyocytes (P 0.01. The Ca 2+i fell to values below quies

27、cent condition after 150 s (Fig. 4, Tab1e 1. 3 DISSCUSIONIntracellular calcium is an important second messenger and mediates a series of biological actions. It is well known that the increase of Ca 2+ i in cardiomyocyte is the key factor for modulating the strength of myocardial contraction. It is r

28、eported that CCK-8 elicited a dose-de-pendant increase in arterial blood pressure in both con-scious and pithed rats 24. Our laboratory has shown that CCK-8 enhances cardiac function and mean arterial pres-sure (MAP in a dose-dependent manner in anesthetized rats 5. However, the roles of Ca 2+ in th

29、e effects of CCK-8on cardiac function were unknown. In the present study,we measured directly the effect of CCK-8 on Ca 2+ i in isolated single cardiomyocyte with confocal microscopy,which is a sensitive method to detect the low level fluo-rescence and has almost no deleterious effect on living cell

30、s. Our results showed that CCK-8 (1104 pmol/ L sig-nificantly increased the Ca 2+i in isolated cardiomyocytes,which could provide a further explanation for the effects of CCK-8 on cardiac function. Ca 2+ i stemmed from the influx of Ca 2+ through the Ca 2+ channels and /or triggered the release of C

31、a 2+ from the sarcoplasmic reticulum (SR via the Ca 2+-induced Ca 2+release (CICR mechanism. Since calcium entry through the Ca 2+ channel plays an important part in excitation-contraction coupling, it was thought that the Ca 2+ current (I Ca might be enhanced by CCK-8. The present study demonstrate

32、d that CCK-8 only caused a slight and slow increase in Ca 2+i after pretreatment with EGTA or nisoldipine in isolated cardiomyocytes,suggesting that CCK-8 is able to activate the Ca 2+ channels and elicit Ca 2+influx in turn leads to intracellular Ca 2+ increase or directly triggers the release of C

33、a 2+ from the SR, while the latter effect was weaker than the former.Our recent study showed that CCK-A/BR mRNA ex-pression was identified by RT-PCR in myocardium of rats 5.Yule reported that stimulation with CCK-8 resulted in an increase in polyphosphoinositides (PPI hydrolysis and Ca 2+i in the CH

34、O cell line when the CCK-AR was trans-fected 9. Furthermore, studies in rat pancreatic acini sug-gested that CCK-8 (103 pmol/L rapidly stimulated tyrosine phosphorylation by activation of both high and low affinity CCK-AR and produced an increase in Ca 2+i 10. Similarly,Lutz et al. reported that CCK

35、 as a IP 3 /Ca 2+-mediated ago-nist could induce tyrosine phosphorylation of regulatoryTable 1. Effect of CCK-8 (102 pmol/L on Ca 2+i changes in single isolated cardiomystotes pretreated with EGTA nisoldipine proglumide or genisteinGroupnF i /F 0 (% Quiescent condition 6 100.021.17 CCK -8 6 245.9310

36、.24* EGTA+CCK-86 136.994.88*+ Nisoldipine+CCK-8 6 121.687.03*+ Proglumide+CCK-8 6 102.866.78+ Genistein+CCK-86109.633.51*+#Ca 2+ i changes are represented by F i /F 0%. Mean SD. *P 0.01 vs quiescent condition; +P 0.01 vs CCK-8 (102 pmol/L in normal Tyrodes solution; #P 0.01 vs pretreatment withprogl

37、umide.Fig. 3. Effect of EGTA and nisoldipine on Ca 2+i elevation induced by CCK-8 (102 pmol/L in single isolated cardi-omyocytes .Ca 2+i changes were represented by relative fluorescent intensity (F/F 0%. , addition of CCK-8 (102pmol/L.Fig. 4. Effect of proglumide and genistein on Ca 2+i elevation i

38、nduced by CCK-8 (102 pmol/L in single isolated cardiomyo-cytes .Ca 2+i changes were represented by F i /F 0%., addition of CCK-8 (102 pmol/L.35 ZHAO Xiao-Yun:CCK-8 Increases Intracellular Calcium of Cardiomyocytesproteins, and stimulate pancreatic enzyme secretion. Thetyrosine kinase inhibitor genis

39、tein inhibited the secretory response to CCK11. In contrast, tyrosine kinases may regu-late not only cellular growth, but also the activity of some ion channels12. Recent studies demonstrated that tyrosine kinases inhibitors decreased the cardiac contractile force and affected the ionic currents in

40、cardiomyocytes13. In our experiments, pretreatment with proglumide or genistein inhibited the Ca2+ielevation induced by CCK-8, suggest-ing that CCK-8 could activate receptor-operated Ca2+channel and increase Ca2+i, which might be mediated by tyrosine kinase. However, whether the effect of CCK-8 on i

41、ntracellular Ca2+ release is mediated by the increase inIP3induced by CCK-8 in cardiomyocytes is to be further studied.In conclusion, CCK-8 can increase Ca2+ivia activat-ing the receptor-operated Ca2+ channel, eliciting the influx of Ca2+, and inducing intracellular Ca2+ release in isolated guinea p

42、ig cardiomyocytes. Such an effect may be medi-ated by tyrosine kinase.REFERENCES1 Crawley JN,Corwin RL.Biological actions of cholecysto-kinin .Peptides 1994;15(4:731-755.2 Janssen PJ, Gardiner SM, Compton AM, Bennett T.Mechanisms contributing to the differential haemody-namic effects of bombesin and

43、 cholecystokinin in consciousLong Evans rats.Br J Pharmacol 1991;102(1: 123-134.3 Gaw AG, Hills DM, Spraggs CF. Characterization of thereceptors and mechanisms involved in the cardiovascularactions of sCCK-8 in the pithed rat. Br J Pharmacol 1995;115(4:660-664.4 Bunting MW, Beart PM, Widdop RE.Effec

44、ts of the CCK(A receptor antagonists SR 27897B and PD140548 onbaroreflex function in conscious rats.Eur J Pharmacol1997;337(1:35-39.5 Zhao XY, Ling YL, Meng AH, Shan BE, Zhang JL.Effects of cholecystokinin octapeptide on rat cardiac func-tion and the receptor mechanism. Acta Physiol Sin (2002;54(3:239-243.6 Rubin LJ, Keller RS, Parker JL, Adams HR. Contractiledysfunction of ventricular myocyt