小剂量辣椒素对豚鼠心室肌细胞L-型钙电流的影响

1、CHENG Yan-Ping et al: Effects of Low-Dose of CAP on L-type Calcium Current in Guinea Pig Ventricular Myocytes Acta Physiologica Sinica, April 25, 2004, 56(2: 243-247 243Effects of low-dose capsaicin on L-type calcium current in guinea pigventricular myocytesCHENG Yan-Ping, YIN Jing-Xiang, CHENG Li-P

2、ing, HE Rui-Rong*Department of Physiology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang 050017, ChinaAbstract : The purpose of this study was to examine the effects of low-dose capsaicin (CAP on L-type calcium current ( ICa-L in guineapig ventricular myocytes and the underlyin

3、g mechanism. I Ca-L was examined in isolated single guinea pig ventricular myocytes by usingwhole-cell patch clamp technique. CAP (125 nmol/L increased the voltage-dependently activated peak amplitude of I Ca-L and downshiftedthe current-voltage (I -V curve. CAP (1, 10, 25 nmol/L increased the peak

4、amplitude of I Ca-L from 9.670.7 pA/pF to 10.210.8 pA/pF (P 0.05, to 11.370.8 pA/pF and to 12.840.9 pA/pF (P 0.05, respectively. CAP 25 nmol/L shifted the steady-state activation curveof I Ca-L to the left and changed half activation potential (V0.5 from (20.762.0 mV to (26.713.0 mV (P 0.05, indicat

5、ing that low-doseCAP may modify the voltage-dependent activation of calcium channel. Low-dose of CAP did not affect the steady-state inactivationcurve of I Ca-L or half-recovery time of Ca2+ channel from inactivation. Ruthenium red (RR, 10 mol/L, a vanilloid receptor (VR1 blocker, antagonized the ef

6、fects of low-dose CAP. These results suggest that low-dose CAP increases I Ca-L mainly by shifting its steady-state activation curve to the left. Such effects may be mediated by VR1. Key words : capsaicin; patch-clamp technique; myocardium; L-type calcium channel L - , : (125 nmol/LpA/pF 10.21, 25 n

7、mol/L , , , * 050017 (capsaicin, CAP L- CAP 1, 10, 25 nmol/L I Ca-L 0.9 pA/pF ( P 0.05, 1.370.8 pA/pF 12.84 (V 0.5 20.762.0 mV I Ca-L , 26.713.0 mV ( P 0.05, CAP (RR, 10 mol/L VR1 : ; : Q463; ; L-Capsaicin (CAP, the main pungent ingredient in hotchili peppers, exerts positive inotropic and chronotro

8、piceffects on the heart and causes vasodilatation in the pe-ripheral vasculature1. These cardiac actions are mediatedby its interaction with a specific vanilloid receptor (VR1on sensory nerve endings in cardiac muscles2. It is pos-sible that capsaicin acts directly on the heart via a cardiacVR. Our

9、previous studies showed that high-dose capsai- Received 2003-06-20 Accepted 2003-07-14cin exerted a negative chronotropic action in SA nodes ofrabbits and prolonged the duration of action potential inpapillary muscles of guinea pigs. These effects were likelydue to reduction in calcium influx3,4. In

10、 other experiments,we also found that CAP (0.1100 mol/L increasedCa2+i and inhibited L-type calcium current ( I Ca-L in guineapig ventricular myocytes5. The present study was un-dertaken to investigate the effect of low-dose capsaicin *Corresponding author. Tel: +86-311-6062490; Fax: 86-311-6062490;

11、 E-mail: syho244on I Ca-L of ventricular myocytes and its mechanism.1 MATERIALS AND METHODS1.1 Isolation of ventricular myocytes. Single ventricularmyocytes were obtained from the hearts of adult guineapigs (300500 g by using enzymatic dissociation tech-nique similar to that previously described by

12、Wang et al6. The hearts were removed and perfused with oxygen-ated Ca2+ Tyrodes solution for 5 min and then with Ca2+-free Tyrodes solution for 5 min before the addition of0.4 g/L collagenase (type, Sigma to the same solution.After 30 min of digestion with the collagenase-contain-ing solution, a pie

13、ce of ventricle was cut and teased intosmaller pieces in Kraftbrhe (KB solution and then keptin KB solution at room temperature for 1 h. The Ca2+concentration of the solution was gradually increased tonormal (1.5 mmol/L within 30 min. All experiments wereperformed within 20 h after ventricular myocy

14、te isolation.1.2 Measurement of Ca2+ current. Isolated ventricularmyocytes were placed in the experimental chamber (0.4ml mounted on the stage of an inverted microscope (CK2,Olympus. After settling to the bottom of the chamber,cells were superfused with external solution for 10 minat a rate of 23 ml

15、/min at 25C. Transmembrane currentswere recorded with an Axopatch amplifer (200 B, AxonInstruments, Inc. Glass microelectrodes were made us-ing a microelectrode puller (PB-7, Narishige, Japan bytwo-stage pulling and had a resistance of 2.04.0 M, when filled with electrode internal solution. Only rod

16、shaped cells with clear cross-striations were used forexperiments. After gigaseal formation, the membrane wasruptured with a gentle suction to obtain the whole cellvoltage-clamp configuration. Membrane capacitance andseries resistance were compensated after membrane rup-ture to minimize the duration

17、 of capacitive current. Theexternal solution was changed to the Na+-free solutionin which Na+ was replaced by equimolar tetraethylam-monium chloride (TEA-Cl. Na+ current was also inacti-vated at the holding potential 50 mV and blocked bytetrodotoxin (TTX. K+ current was suppressed by sub-stituting K

18、+ by Cs+. Computer-generated voltage pulseswere programmed using the pCLAMP 6.0 software(Axon Instruments, Inc. On-line acquired data werestored on a hard disk of the microcomputer.1.3 Solution and drugs. Capsaicin (CAP, ruthenium red,collagenase type, taurine, TEA-Cl, EGTA, HEPES,CsOH, CsCl, Mg-ATP

19、, and TTX were purchased fromSigma Co.Acta Physiologica Sinica, April 25, 2004, 56(2: 243-247The Ca2+-free Tyrodes solution contained (mmol/LNaCl 135, KCl 5.4, MgCl2 1.0, NaH2PO 4 0.33, glucose 5,and HEPES 10, pH was adjusted to 7.4 with NaOH. KBsolution contained (mmol/L KOH 80, KCl 40, NaH2PO 425,

20、 MgSO4 3, glutamic acid 50, taurine 20, EGTA 1, HEPES10, and glucose 10, pH was adjusted to 7.4 with KOH.The external solution was composed of TEA-Cl 140, MgCl22.0, CaCl2 1.5, glucose 10, HEPES 10, TTX 10 mol/L,gassed with 100 % O2, pH was adjusted with TEA-OH to7.37.4. The electrode internal soluti

21、on for whole-cell re-cording was composed of Mg-ATP 3, CsCl 140, HEPES10, EGTA 2 mmol/L, pH 7.2 adjusted with CsOH. Capsai-cin was dissolved in distilled water containing 10 % etha-nol and 1% Tween-80 and then diluted to final concentra-tion with external solution.1.4 Statistics . The values were ex

22、pressed as meanSD.Statistical analysis was performed using t test. Statisti-cal significance was set at P 0.05, to 11.370.8 pA/pF and to 12.840.9 pA/pF(P 0.05, respectively. Current-voltage (I-V curves ofL-type calcium current was obtained by 350 ms depolariz-ing pulses from the Eh of 50 mV to the t

23、est potentialsranging from 40 mV to +50 mV in 10-mV increments.The pulse frequency was 0.1 Hz. I Ca-L was activated at 30mV and the peak amplitude appeared at the potential of 0mV. CAP 25 nmol/L downshifted the I-V curve (Fig.1.2.2 Effects of CAP on activation of I Ca-LSteady-state activation of L-t

24、ype calcium current wasobtained by depolarizing pulses from the Eh of 50 mV tothe test potentials +50 mV in 10-mV increments for 350ms. The pulse frequency was 0.1 Hz. The activation curveswere fitted according to the Boltzmann equation: G/G max =1/1+ exp (V V 0.5/. V is the membrane potential,V 0.5

25、 is the midpoint voltages of the activation functions,CHENG Yan-Ping et al: Effects of Low-Dose of CAP on L-type Calcium Current in Guinea Pig Ventricular Myocytes245Fig. 1. Effects of CAP on I Ca-L in isolated guinea pig ventricularmyocytes. A : Current traces were recorded during 350 ms depolar-iz

26、ations from a holding potential of 50 mV to 0 mV. B : Effects ofCAP on I-V curves of I Ca-L . V alues are mean SD.and is the Boltzmann slope parameter for activation.CAP 25 nmol/L shifted half activation potential (V 0.5 from(20.762.0 mV to (26.713.0 mV, and slope param-Fig. 2. Effects of 25 nmol/L

27、CAP on steady-state activation kinet-ics of L-type calcium channel in myocytes.eter ( from (4.52.1 mV to (5.42.4 mV (n =12 cellsfrom 6 hearts, P 0.05(Fig. 3.Fig. 3. Effects of CAP (25 nmol/L on steady-state inactivation kinetics of L-type calcium channel in myocytes.2.4 Effects of CAP on recovery of

28、 I Ca-L from inactiva-tionThe recovery of I Ca-L from inactivation was studied us-ing double-pulse protocol consisting of a 300 ms prepulseto +10 mV (P1 followed by a 1000 ms test pulse to +10mV (P2 after a variable P1-P2 coupling interval from 0 to2500 ms at the holding potential of 60 mV. Double p

29、ulsestimulation was repeated every 6 s. CAP (25 nmol/L in-significantly shifted the half-recovery time of Ca2+ channelfrom inactivation from (9157 ms to (12361 ms (n =12cells from 6 hearts, P 0.05 (Fig. 4.2.5 Effects of ruthenium red (RR on the responseof I Ca-L to CAPV anilloid receptor (VR1 blocke

30、r RR (10 mol/L itselfhad no marked effect on I Ca-L . Pretreatment of RR (10246Fig. 4. Effects of CAP on time-dependent recovery of I Ca-L from the steady-state inactivation.mol/L could completely inhibit the effects induced byCAP 25 nmol/L. The peak amplitude of I Ca-L was decreasedfrom 10.410.7 pA

31、/pF (CAP to 8.740.6 pA/pF(CAP+RR (n =5 cells from 3 hearts, P 0.05 (Fig. 5.Fig. 5. Effects of ruthenium red (RR on the response of I Ca-L to low-dose of CAP in guinea pig ventricular myocytes.3 DISCUSSIONIn the present study, the slowly inactivated inward cur-rent recorded was L-type calcium current

32、. The rundownof calcium was minimized by adding Mg-ATP and EGTAin pipette solution. Our previous studies showed thatCAP (0.1100 mol/L inhibited I Ca-L by an acceleration ofcalcium channel inactivation including voltage- and Ca2+-dependent inactivation which was secondary to the eleva-tion of Ca2+i 5

33、. In this study, we found that low-dosecapsaicin (125 nmol/L could dose-dependently increaseI Ca-L and shift the I-V curve downward. CAP 25 nmol/LActa Physiologica Sinica, April 25, 2004, 56(2: 243-247markedly shifted the steady-state activation curve ofI Ca-L to the left, but did not change the ina

34、ctivation curveof I Ca-L and the recovery time from inactivation. Sucheffects indicated that low-dose CAP increased L-typecalcium current in guinea pigs ventricular myocytes,and markedly affected the voltage dependence of acti-vation of I Ca-L.It has been generally accepted that many of the capsai-c

35、in effects are mediated by VR1. VR1, a distant relative ofthe transient release potential (TRP family of stored-op-erated calcium channels, is expressed almost exclusivelyby primary sensory neurons involved in nociceptions andinflammation 9. Many actions of CAP on the heart are me-diated by its inte

36、raction with VR1 on sensory nerve end-ings in cardiac muscles and opening of a non-selectivecation channel, inducing the liberation of neuropeptides,most notably calcitonin gene-related peptide (CGRP, fromthe vanilloid-sensitive innervation of the heart1012. However,the presence of a VR1-like expres

37、sed sequence tags (ESTclone in heart is in accordance with the recognition ofnonneuronal VRs13,14. In this study, RR, a vanilloid recep-tor antagonist, could completely block the stimulatory ef-fects of low-dose CAP on L-type calcium current, indi-cating that the effects of low-dose CAP might be med

38、iatedby VR1 on the membrane of ventricular myocytes. Atpresent, the relationship between VR1 and voltage-depen-dent L-type calcium channel is not clear. The mechanismresponsible for the low-dose CAP increased ICa-L needsfurther investigation.In conclusion, low-dose CAP dose-dependently in-creased I

39、Ca-L mainly by shifting the steady-state activationcurve of L-type calcium channel to the left in guinea pigventricular myocytes. These effects might be mediatedby VR1.REFERENCES1 Ledda F, Amerini S, Fillipi S, Mantelli L, Morbidelli L, RubinoA, Ziche M. Cardiovascular effects of capsaicin-sensitive

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