Received 2002-04-02 Accepted 2002-05-24

*Corresponding author. Tel: +86-311-6062490; Fax: +86-311-6062490;

E-mail: syho@Hebmu.edu.cn

Acta Physiologica Sinica

Dec. 2002, 54 (6), 467-472

Research Paper

Effect of agmatine on intracellular free calcium concentration in

isolated rat ventricular myocytes

LI Qing1, SHANG Zhong-Lin2, YIN Jing-Xiang1, WANG Yi-He3, HE Rui-Rong1, *

1Department of Physiology, Hebei Medical University, Shijiazhuang 050017;

2College of Life Science, Hebei Normal University, Shijiazhuang 050016;

3Department of Physiology, Medical College of Chinese People's Armed Police Forces, Tianjin 300162

Abstract: The present study was to investigate the effects of agmatine (Agm) on free intracellular calcium concentration ([Ca2+]i) of isolated rat ventricular myocytes. [Ca2+]i was measured by confocal microscopy in single rat ventricular myocytes which were dissociated by enzymatic dissociation method and loaded with Fluo 3-AM. The changes in [Ca2+]i were represented by fluorescence intensity (FI) or relative fluorescence intensity (F/F0%). The results showed that the control level of FI value of single rat ventricular myocytes was 128.8±13.8 and 119.6±13.6 in the presence of normal Tyrode's solution containing Ca2+ 1.0 mmol/L and Ca2+-free Tyrode's solution, respectively. There was no difference between these two groups (P>0.05). Agm 0.1, 1, and 10 mmol/L significantly reduced the [Ca2+]i in both extracellular solutions in a concentration-dependent manner. The similar effect of Agm on [Ca2+]i was also observed in the presence of EGTA 3 mmol/L. KCl 60 mmol/L, PE 30 μmol/L, and Bay-K-8644 10 μmol/L, all these substances induced [Ca2+]i elevations in ventricular myocytes. Agm (0.1, 1, and 10 mmol/L) markedly inhibited the increase in [Ca2+]i induced by KCl, phenylephrine (PE), and Bay-K-8644. When Ca2+ waves were produced by increasing extracellular Ca2+ concentration from 1 to 10 mmol/L, 1 mmol/L Agm could block the propagating waves of elevated [Ca2+]i, and reduce the velocity and duration of propagating waves. These results suggest that Agm possesses an inhibitory effects on [Ca2+]i via blocking voltage-dependent Ca2+ channel, and possibly by alleviating calcium release from SR in single isolated rat ventricular myocytes.

Key words: agmatine; fluorescence intensity; myocytes; intracellular calcium; Ca2+ channel; intracellular

Ca2+ release; confocal microscopy

胍丁胺对大鼠心室肌细胞内游离钙浓度的影响

李清1, 尚忠林2, 尹京湘1, 王义和3, 何瑞荣1,

1河北医科大学基础医学研究所生理室, 石家庄 050017;

2河北师范大学生命科学学院, 石家庄 050016；

3中国人民武警医学院生理教研室, 天津 300162

摘要: 本研究旨在观察胍丁胺(agmatine, Agm) 对分离大鼠心室肌细胞内游离钙浓度([Ca2+]i)的影响。用酶解方法分离大鼠心室肌细胞, 用Fluo 3-AM负载, 然后用激光共聚焦法测定单个心室肌细胞[Ca2+]i的荧光强度(fluorescence intensity, FI), 结果以FI 或相对荧光强度(F/F0%)表示。实验结果表明, 在正常台氏液(含钙 1.0 mmol/L)和无钙台氏液中, 单个大鼠心室肌细胞的荧光密度分别为128.8±13.8和119.6±13.6, 两者无差异。Agm 0.1、1和10 mmol/L浓度依赖性地显著降低细胞的钙浓度; 在正常台氏液中加入EGTA 3 mmol/L, Agm同样降低细胞的钙浓度。KCl 60 mmol/L, PE 30 μmol/L, 和Bay-K-8644 10 μmol/L均升高心室肌细胞的[Ca2+]i。 Agm同样降低高浓度KCl、Bay-K-8644和PE诱发的心室肌细胞[Ca2+]i升高。当细胞外液钙浓度由1 mmol/L增加到10 mmol/L时, 诱发心室肌细胞钙超载, 同时部分心室肌细胞产生可传播的钙波（Ca2+ wave）, Agm 1 mmol/L 降低钙波的传播速度和持续时间, 最终阻断钙波。以上结果提示, Agm 对心室肌细胞的胞浆[Ca2+]i 具有抑制作用, 此作用通过阻断电压依赖性钙通道而实现; 并可能与抑制大鼠心室肌细胞内钙释放有关。

关键词: 胍丁胺; 荧光密度; 心室肌细胞; 细胞内钙; 钙通道; 钙释放; 共聚焦显微镜

中图分类号: Q463

Agmatine (Agm) is a polycationic amine synthesized by decarboxylation of L-arginine by the enzyme arginine decarboxylase and has been identified as an endogenous clonidine-displacing substance (CDS) in mammalian brain[1]. Our previous works demonstrated that Agm reduced the amplitude of action potential (APA), maximal rate of depolarization (Vmax), overshoot (OS), velocity of diastolic (phase 4) depolarization (VDD) and rate of pacemaker firing (RPF) in sinoatrial node pacemaker cells of rabbits, and human atrial fibers[2,3]. Moreover, Agm inhibited early afterdepolarization (EAD) and delayed afterdepolarization (DAD) induced by isoproterenol in guinea pig papillary muscles[4]. Elevation of Ca2+ concentration antagonized the decrease of VDD and RPF induced by Agm[5]. It has also been found that Agm exerts antihypertensive[6] and antiarrhythmic[7]effects.Our recent studies indicated that Agm had protective effects against myocardial ischemia-reperfusion injury in rabbits[8] and could inhibit L-type voltage-dependent calcium channel in rat ventricular myocytes[9]. Thus, Agm possesses potential clinical value in preventing and treating myocardial ischemia-reperfusion injury, and its cardioprotective effects may be related to the reduction of free intracellular calcium concentration ([Ca2+]i). The present study was undertaken to observe the effects of Agm on [Ca2+]i level in rat ventricular myocytes.

1 MATERIALS AND METHODS

1．1 Solutions and Drugs

Agmatine, collagenase type Ⅱ, bovine serum albumin (BSA), taurine, HEPES, EGTA, and phenylephrine (PE) were purchased from Sigma Co. 3-(N-morpholino) propanesulfonic acid (MOPS) was purchased from Shanghai Boao Biotech Co.

The Ca2+-free Tyrode's solution contained NaCl 100, KCl 10, MgSO4 5, NaH2PO4 1.2, glucose 20, taurine 10, and MOPS 10 mmol/L, pH was adjusted to 7.4 with KOH. The normal Tyrode's solution was prepared by adding 1.0 mmol/L CaCl2 to Ca2+-free Tyrode's solution. Agm was dissolved in the Ca2+-free Tyrode's or normal Tyrode's solution at the concentrations (0.1, 1, and 10 mmol/L) before experiment.

1．2 Isolation of ventricular myocytes

Single rat ventricular myocytes were obtained by enzymatic dissociation technique. The Sprague-Dawley rats (n=15, either sex, weighing 260±38 g, provided by Experimental Animal Center of Hebei Province, grade Ⅱ, Certificate No.04064), were stunned by heavy blow on the heads and the hearts were rapidly excised, rinsed in oxygenated ice-cold Ca2+-free Tyrode's solution, and a Langendorff retrograde perfusion was performed through the aorta at a rate of 9 ml/min with Ca2+-free Tyrode's solution for 5 min, and then with the same solution containing collagenase Ⅱ 360 mg/L, bovine serum albumin (BSA)160 mg/L, and CaCl2 34 μmol/L for 9 min at 37℃. The ventricles were cut, minced, and then incubated for 10 min in Ca2+-free Tyrode's solution containing 0.1% bovine serum albumin. Myocytes were harvested after filtration through a 200 μm nylon mesh, and resuspended in Tyrode's solutions containing different concentrations of Ca2+. The concentration of Ca2+ was gradually increased to 1 mmol/L.

1.3 Fluo 3-AM loading

Isolated ventricular myocytes were incubated with Flu 3-AM working solution containing 0.03% Pluronic F-127 (the final concentration of Fluo 3-AM is 20 μmol/L ) at 37℃ for 60 min. After incubation, the cells were washed at 25℃ with Tyrode's or Ca2+-free Tyrode's solutions three times to remove the extracellular Fluo 3-AM.

1．4 Measurement of [Ca2+]i

After Fluo 3-AM loading, the cells were mounted in the small pool of Teflon printed slice, and covered by cover glass. Only the cells with rod shape and visible striations were used for experiments. The fluorescence signal was detected with confocal laser scanning system (Biorad lasersharp MRA2, Oxfordshire, UK) equipped with a Nikon E-600 eclipse microscope. An argon laser was used to excite Fluo-3 at 488 nm and emit at 530 nm. [Ca2+]i changes were represented by fluorescence intensity (FI). Total 50-120 images were scanned with each experiment and the data were stored in diskette.

1．5 Experimental protocols

The experiments consisted of 5 groups: (1) Effect of Agm on [Ca2+]i: FI was measured after adding 0.1, 1, or 10 mmol/L Agm to normal Tyrode's solution (n=12 cells from 8 hearts), Ca2+-free Tyrode's solution (n=8 cells from 8 hearts) or normal Tyrode's solution containing EGTA 3 mmol/L (n=8 cells from 8 hearts). (2) Effects of Agm on KCl-induced increase in [Ca2+]i: the preparation was pretreated with 60 mmol/L KCl for 5 min, then FI was measured after adding 0.1, 1, or 10 mmol/L Agm to the normal Tyrode's solution (n=9 cells from 8 hearts). (3) Effects of Agm on Bay-K-8644-induced increase in [Ca2+]i: the preparation was pretreated with 10 μmol/L Bay-K-8644 for 5 min, then FI was measured after adding 0.1, 1, or 10 mmol/L Agm to the normal Tyrode's solution (n=11 cells from 8 hearts). (4) Effects of Agm on PE-evoked increase in [Ca2+]i: FI was measured after treating with 30 μmol/L PE followed by adding 0.1, 1, or 10 mmol/L Agm, respectively (n=10 cells from 8 hearts). (5) Effects of Agm on the overload of [Ca2+]i induced by high concentration Ca2+: after extracellular Ca2+ concentration was increased from 1 to 10 mmol/L for 30 s to produce Ca2+ overload, and Ca2+ waves were observed, FI was measured after adding 0.1, 1, or 10 mmol/L Agm to the normal Tyrode's solution (n=8 cells from 6 hearts).

1．6 Statistics

The values were expressed as means±SD. Statistical analysis was performed using Student's t test. P values less than 0.05 were considered to be significant.

2 RESULTS

2．1 Effect of Agm on [Ca2+]i

Agm (0.1, 1, and 10 mmol/L) reduced the [Ca2+]i in both Ca2+-free Tyrode's solution (Fig.1A) and the normal Tyrode's solution in a concentration-dependent manner (Table 1). The similar effect of Agm on [Ca2+]i was also observed in the normal Tyrode's solution containing EGTA 3 mmol/L.

2．2 Effects of Agm on KCl-induced increase in [Ca2+]i

Normal Tyrode's solution containing KCl 60 mmol/L increased FI by 4.37 folds in rat ventricular myocytes, while 0.1, 1, and 10 mmol/L Agm remarkably inhibited KCl-induced FI elevation in a concentration-dependent manner (Table 1).

Fig.1. Fluorescence images of [Ca2+]i changes in single isolated rat ventricular myocytes loaded with Fluo 3-AM and measured by confocal microscope. A: Effect of 1 mmol/L agmatine on [Ca2+]i in Ca2+-free Tyrode's solution. Fluorescence images recorded at 3 s intervals. B: Ca2+ wave fluorescence image induced by “Ca2+ overload” after extracellular Ca2+ concentration increased from 1 to 10 mmol/L, which was recorded at 500 ms intervals. C: Effect of agmatine on Ca2+ waves induced by Ca2+ overload. Fluorescence images were recorded at 500 ms intervals. arrow, addition of 1 mmol/L Agm.

Table１. Effects of agmatine (Agm) on intracellular fluorescence intensity (FI, an indicator of [Ca2+]i) in isolated rat ventricular myocytes during different conditions

	A (n=12 cells)	B(n=8 clls)	C (n=8 cells)	D (n=9 cells)	E (n=11 cells)
Control	128.8±13.8	119.6±13.6	121.4±14.3	564.1±46.8	483.2±37.5
Agm (mmol/L)
0.1	109.8±11.4**	108.1±11.2*	106.1±11.6*	423.8±44.8 **	394.1±32.8 **
1	70.3±10.8**	89.2±9.8**	92.4±10.3**	294.1±28.8**	321.8±29.4**
10	30.7±8.1**	45.8±9.6**	51.4±9.8**	211.6±40.6**	226.3±25.8**

*P<0.05, **P<0.01 vs control.

2．3 Effects of Agm on Bay-K-8644-induced increase in [Ca2+]i

After pretreatment with 10 μmol/L Bay-K-8644 for 5 min, the FI of rat ventricular myocytes was increased by 3.75 folds. Agm (0.1, 1, and 10 mmol/L) remarkably inhibited Bay-K-8644-induced [Ca2+]i elevation in a concentration-dependent manner (Table 1).

2．4 Effects of Agm on PE-evoked increase in [Ca2+]i

After pretreating with 30 μmol/L PE followed by adding 0.1, 1, or 10 mmol/L Agm, the FI of rat ventricular myocytes was increased by 4 folds in the normal Tyrode's solution. The effect of PE was significantly inhibited in a concentration-dependent manner by adding of 0.1, 1, and 10 mmol/L Agm (Fig.2).

Fig.2 Effects of agmatine on [Ca2+]i elevation induced by PE in single rat ventricular myocytes. [Ca2+]i change was represented by relative fluorescence intensity (F/F0 100%). F/F0 is the ratio of change of fluorescence (F) to the fluorescence under quiescent condition (F0).

Fig.3a. Fig.3b

[Ca2+]i changes in single isolated rat ventricular myocytes under Ca2+ overload condition after extracellular Ca2+ concentration increased from 1 to 10 mmol/L. A: Ca2+ waves fluorescence images induced by “Ca2+ overload”. B: Effects of agmatine on Ca2+ waves induced by Ca2+ overload. The change in [Ca2+]i was represented by fluorescence intensity (FI). ↑, addition of 1 mmol/L Agm.

2．5 Effects of Agm on the overload of [Ca2+]i induced by high Ca2+

As Ca2+ in Tyrode's solution was increased from 1 to 10 mmol/L, a number of cells quickly “rounded-up” into a contracted state (calcium intolerant). Meanwhile, Ca2+ overload was evoked (Figs.1B, 3A). Under these conditions, the propagating waves of elevated [Ca2+]i regularly appeared. Agm (1 mmol/L) could block the propagating waves of elevated [Ca2+]i, and reduce the velocity and duration of propagating waves (Figs.1C, 3B).

3 DISCUSSION

In the present study, the effect of Agm on [Ca2+]i in isolated rat ventricular myocytes loaded with Fluo 3-AM was directly examined with confocal microscopy, which is a sensitive method to detect low level of fluorescence and almost no deleterious effect on living cell. The results demonstrated that Agm reduced [Ca2+]i in both Ca2+-free and normal Tyrode's solution, suggesting the inhibitory effect of Agm on [Ca2+]i in isolated rat ventricular myocytes.

[Ca2+]i are stemmed from the influx of Ca2+ through the Ca2+ channels and/or Ca2+ released from the sarcoplasmic reticulum (SR). The present study demonstrated that Agm could reduce [Ca2+]i in Ca2+-free Tyrode's solution or by pretreatment with EGTA. Such a result indicates that Agm is able to inhibit the Ca2+ release from internal store because the influx of extracellular Ca2+ is suppressed by either the omission of Ca2+ from the external solution or the addition of EGTA.

High K+ which causes depolarization of membrane and opens voltage-dependent calcium channels (VDC)[11], and Bay-K-8644, a Ca2+-channels agonist, could enhance the calcium channel current by accelerating activation of voltage-dependent calcium channel[12]. In our experiment, both KCl 60 (mmol/L) and Bay-K-8644 (10 μmol/L) increased the [Ca2+]i. Agm markedly and concentration-dependently inhibited [Ca2+]i elevation induced by KCl and Bay-K-8644 in ventricular myocytes. This finding corresponded to our previous report that Agm could inhibit L-type voltage-dependent calcium channel in rat ventricular myocytes[9].

The regulation of G proteins on Ca2+ channels has been an attractive area for research. G proteins not only regulate Ca2+ channels indirectly via cytoplasmic second message, but also act directly on Ca2+ channels[13]. Since G proteins couple a variety of receptors to Ca2+ channels. PE can activate the proper G protein-coupled receptors to open Ca2+ channels. In our experiment, Agm inhibited PE-induced increase in [Ca2+]i, suggesting that Agm inhibited Ca2+ influx induced by activation of G protein-coupled receptors (α1-adrenergic receptors).

[Ca2+]i overload would result in an irreversible injury and is a common pathway for cell death. In ventricular muscle, membrane depolarization during the action potential activates the Ca2+ influx through Ca2+ channels (calcium current) that normally triggers the release of Ca2+ from SR. Interventions that cause an increase in the resting level of cytoplasmic [Ca2+]i lead to appearance of propagating SR Ca2+ release (Ca2+ waves), a form of SR Ca2+ release that does not depend on Ca2+ current trigger. Although first detected as propagating waves of contraction within apparently quiescent cardiac muscle preparations[14], propagating Ca2+ waves were observed directly after the development of fluorescence Ca2+ indicators[15,16]. The Ca2+ waves are particularly important because they cause a depolarizing membrane current that can result in contractile dysfunction and diverse arrhythmias[17]. In the present experiment, under Ca2+ overload, the Ca2+ waves were produced, and Agm reduced Ca2+ overload and decreased the velocity and duration of Ca2+ waves. Thus, the cardioprotective effects of Agm were exerted by decrease in [Ca2+]i via inhibiting Ca2+ influx and Ca2+ release.

As the concentrations of Agm used in this study seemed to be high, it was likely that the observed effects of Agm might be nonspecific. However, when the ventricular myocytes were pretreated with idazoxan, an antagonist at imidazoline receptors (IR) and alpha-2 adrenergic receptors (α2-AR), the inhibitory effects of Agm on [Ca2+]i in isolated rat ventricular myocytes were completely blocked in our pilot experiment (data not shown), thus implying that the effects of Agm were not nonspecific but exclusively mediated by IR and/or α2-AR. The results that higher concentrations of agmatine were required for exerting its effects on [Ca2+]i in isolated rat ventricular myocytes might be due to the low affinity of the relevant myocardial receptors to Agm. The results were supported by our standard microelectrode studies[5] showing that Agm at 1 mmol/L showed no effect on maximum diastolic potential (MDP), maximal rate of depolarization in phase 0 (Vmax), and action potential durations at 50% and 90% of repolarization (APD50,90) in human right atrial fibers, while at 5,10 mmol/L induced a marked decrease in Vmax, APD50, APD90, and action potential amplitude in a concentration-dependent manner. Idazoxan (0.1 mmol/L) could completely block the above-mentioned effects induced by Agm (5 mmol/L).

In summary,Agm inhibits calcium influx by blocking voltage-dependent calcium channel, and possibly by alleviating calcium release from SR in single isolated rat ventricular myocytes.

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