Received 2002-03-29 Accepted 2002-09-09

This project was supported by the National Natural Science Foundation of China (No. 30070279).

*Corresponding author. Tel: +86-27-83692622; Fax: +86-27-83692608;

E-mial: Yimeidu@hotmail.com

Acta Physiologica Sinica

Dec. 2002, 54 (6), 479-484

Research Paper

Inhibitory effect of adrenomedullin on L-type calcium currents in guinea-pig ventricular myocytes

DU Yi-Mei*, TANG Ming, LIU Chang-Jin, LUO Hong-Yan, HU Xin-Wu

Department of Physiology, Tongji Medical College of Huazhong University of Science and Technology, Wuhan 430030

Abstract: The effects of adrenomedullin (ADM) on the L-type calcium currents (ICa,L) and the mechanism of the signal transduction process were studied. Enzymatically isolated guinea-pig ventricular myocytes were used to measure ICa,L with whole-cell patch-clamp techniques. ADM at the concentrations of 1-100 nmol/L decreased ICa,L in a dose-dependent manner (P<0.05). ADM22-52 (100 nmol/L), a specific ADM-receptor antagonist, completely abolished the ADM-induced inhibition of ICa,L. Pretreatment of the cells with H-89 (10 μmol/L) , a specific PKA inhibitor, did not attenuate the effects of ADM. Intracellular application of 10 μmol/L PKC19-36 , a specific PKC inhibitor, prevented the ADM-induced inhibition of the ICa,L, while the specific PKC activator PMA could mimic the effects of ADM on the ICa,L. PMA (1 μmol/L) decreased the ICa,L by 32.26±4.20% (P<0.05). These findings indicate that ADM can inhibit the ICa,L in guinea-pig ventricular myocytes, and the inhibition is mediated by the specific ADM-receptor and an activation of protein kinase C.

Key words: adrenomedullin; ventricular myocyte; receptor; protein kinase C; L-type calcium channel; patch clamp technique

肾上腺髓质素对豚鼠心室肌细胞L-型钙通道的调制

杜以梅*, 唐明, 刘长金, 骆红艳, 胡新武

华中科技大学同济医学院生理系, 武汉 430030

摘要: 应用全细胞膜片钳技术研究了肾上腺髓质素(ADM)对豚鼠心室肌细胞L-型钙电流(ICa,L)的影响及其信号传导机制。结果发现: ADM (1-100 nmol/L)浓度依赖性抑制ICa,L(P<0.05), 并可被ADM特异受体阻断剂ADM22-52 (100 nmol/L)完全阻断。用蛋白激酶A特异拮抗剂H-89 (10 μmol/L) 预处理, 对ADM抑制ICa,L的作用无影响。但用蛋白激酶C (PKC)特异性拮抗剂PKC19-36预处理, 可完全阻断ADM的抑制效应; 而PKC特异性激动剂PMA则可以模仿ADM的抑制效应(P<0.05)。上述结果提示: ADM作用于特异性ADM受体可浓度依赖性地抑制豚鼠心室肌细胞ICa,L, 而此作用可能是PKC介导的。

关键词: 肾上腺髓质素; 心室肌细胞; 受体; 蛋白激酶C; L-型钙通道; 膜片钳技术

中图分类号: Q463; R 331.3

Adrenomedullin (ADM) is a vasodilator peptide that was originally isolated from human pheochromocytoma[1]. The peptide consisting of 52 amino acids has an intracellular disulfide bond and shows 20% homology with calcitonin gene-related peptide (CGRP). Subsequent studies have demonstrated that ADM is widely distributed in various tissues and organs, including heart[2-4]. Recently, plasma concentrations of ADM have been shown to be increased in a variety of cardiovascular disorders, including acute myocardial infarction[5] and heart failure[6], suggesting that ADM may be involved in the control of cardiac function.

It has been shown that ADM produces a direct positive inotropic effect in multicellular preparation from human[7] and experimental animals[8, 9], but its exact cellular mechanism remains controversial. The effect of ADM was thought to be the result of an increase in intracellular Ca2+ concentration caused by the stimulation of Ca2+ release from intracellular Ca2+ stores,activation of protein kinase C (PKC) and Ca2+ influx through L-type calcium channel[8]. Other possible actions of ADM that might explain its positive inotropic effect could be the activation of protein kinase A (PKA)[9]. However, Perret et al. showed that ADM had a direct negative inotropic effect in the isolated perfused rat heart[10]. A more recent study also reported a direct negative inotropic effect of ADM in isolated rabbit cardiac ventricular myocytes, which may result from a reduction in [Ca2+]i and ICa,L mediated by NO-3', 5'-cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) system[11].These observations suggest that ADM receptors are coupled to multiple G proteins, and the effects of ADM might be mediated by distinct complex signaling pathways.

L-type calcium currents (ICa,L) play a central role in regulating cardiac inotropic effect. However, very little was known about the role of ADM on ICa,L. In this study we examined the mechanism underlying the ADM-mediated action by focusing on ICa,L in guinea-pig ventricular myocytes.

1 MATERIALS AND METHODS

1.1 Cell isolation

Single ventricular myocytes were isolated enzymatically from guinea-pig (300-400 g), using previously described method[12]. Briefly, the heart was rapidly excised and perfused via the coronary artery with normal Tyrode solution for 2-3 min, followed by Ca2+-free Tyrode solution for 5-10 min, and Ca2+-free Tyrode solution containing 50-100 units/ml collagenase (Yakult, Tokyo, Japan) for 10 min. All solutions were gassed with 100% O2 and warmed to 37℃. Isolated ventricular myocytes were stored in KB medium at 4℃ and studied within 8 h. After 2 h of incubation at 4℃, a few drops of the cell suspension were placed in a 0.3 ml perfusion chamber mounted on an inverted microscope stage. Only rod-shaped noncontracting cells with clear cross striations were used for the whole-cell patch-clamp studies. All experiments were carried out at 37℃.

1.2 Electrophysiological recording

The whole cell configuration of the patch clamp technique was used for recording ICa,L. To record ICa,L, K+ currents were suppressed with Cs+-rich dialysis and K+-free Cs+ containing superfusate. The cell membrane was held at －80 mV and a brief prepulse (50 ms) to －40 mV was applied to inactivate Na+ current and T-type Ca2+ current. Test pulses (300 ms, +10 mV) were applied at 0.1 Hz. Pipette electrodes were made from borosilicate glass capillaries (USA) with a vertical pipette puller (Narishige PP-83, Tokyo, Japan), having a resistance of 1-2 MΩ when filled with the internal solution. Cell capacitance and series resistance cancelled during the capacitive current cancellation ranged from 40-100 pF and 4-10 MΩ respectively. External application of test drugs was given after recording steady state for at least 5 min. But at least 10 min were allowed for dialysis before the effect of ADM on ICa,L was examined. Currents were recorded with an EPC-9 amplifier (HEKA, electronic, Lambrecht, Germany), and directly stored in a Macintosh Quadra 840AV computer (Apple Computer, Inc., Cupertino, CA) at 10 kHz. The capacity and leak-corrected data were digitally filtered at 2 kHz, then analyzed with Pulse/Pulsefit (HEKA electronic), IGOR (Wave Metrics Inc., Lake Oswego, OR) and Kaleida Graph (Synergy Software) on the Macintosh computer.

1.3 Solutions and drugs

Normal Tyrode solution contained (in mmol/L): NaCl 135, KCl 4, NaH2PO4 0.33, MgCl2 1, CaCl2 1.8, D-glucose 10, HEPES 5.5, and HEPES-Na 4.5 (pH 7.4 at 37℃). The KB medium contained (in mmol/L): KOH 90, L-glutamic acid 70, taurine 20, KCl 30, KH2PO4 10, HEPES 10, D-glucose 10, and EGTA 0.5 and the pH was adjusted to 7.3 with KOH. The external solution contained (in mmol/L): NaCl 140, CsCl 4, MgCl2 1, CaCl2 1.8, D-glucose 10, HEPES 10 and the pH was adjusted to 7.4 with NaOH. The internal solution contained (in mmol/L): CsCl 20, CsOH 120, L-aspartic acid 110, HEPES 10, EGTA 10, Na2ATP 5, Na2GTP 0.2, MgCl2 1, and CaCl2 4 and the pH was adjusted to 7.2 with CsOH.

Human ADM and human ADM22-52(Peptide Institute Inc., Osaka, Japan) were dissolved in distilled water. H-89 and PMA were dissolved in dimethyl sulfoxide (DMSO). The final concentration of DMSO (0.05%) did not affect ICa,L. Other drugs were all obtained from Sigma Chemical (St.Louis, MO).

1.4 Data analysis

ICa,L amplitudes were calculated as the difference between peak inward and steady state (at the end of the 300 ms step pulses) currents. The percentage inhibition　(P) at +10 mV test potential was calculated as:

P= 1－[(ICa,L after drug application)/ (ICa,L before application)]

The data from cells in which ICa,L was stable in the control condition and reached a steady state with tested drugs were analyzed by t test. Values were expressed as mean±SE. P<0.05 was considered statistically significant.

2 RESULTS

2.1 Effect of ADM on ICa,L

Figure 1 illustrates a set of three original current traces of whole cell currents at +10 mV test potential. Since these inward currents were completely blocked by cadmium and enhanced by isopreterenal or forskolin (data not shown), it reveals that they represent ICa,L. ICa,L was decreased from 628.21 pA (trace a) to 425.64 pA (trace b) by 100 nmol/L ADM. Upon washing out of ADM (trace c), ICa,L partially reversed back to control values. Figure 2 shows the effect of ADM on the ICa,L at different concentrations. Results are expressed as the percentage inhibition of ICa,L compared with the control condition. ICa,L was inhibited in a

Fig.1. Effects of adrenomedullin (ADM) on L-type calcium current (ICa,L) in guinea-pig ventricular myocytes. The maximal ICa,L was evoked from －40 mV to +10 mV. Time and current calibrations are given in the graph. a: Control; b: 100 nmol/L ADM; c: Wash out.

Fig.2. Effect of PMA and different concentrations of ADM on ICa,L. Bar graph shows degree of inhibition of ICa,L at +10 mV (means±SE). *P<0.05 vs control.dose-dependent manner by ADM. ADM at 1 nmol/L was ineffective and at 10 and 100 nmol/L inhibited ICa,L by 18.33±5.12% (P<0.05) and 35.27±2.64% (P<0.05) respectively.

Figure 3 summarizes the effect of ADM (100 nmol/L) on the current-voltage relationship obtained in 5 myocytes. Current-voltage relationships were determined by voltage steps from -30 to +40 mV. The effects of ADM (100 nmol/L) were not associated with a shift along the voltage axis but were associated with a decrease in ICa,L at all test potential.

Fig.3. Effect of ADM on the ICa,L current-voltage relationships (means±SE). The applied test potentials were between -30 mV and +40 mV by 10 mV step from －40 mV. ○, control; ●, exposed to 100 nmol/L ADM.

2.2 Effect of ADM on ICa,L in the presence of specific ADM-receptor antagonist

The action of ADM was tested in the presence of ADM22-52, a specific ADM-receptor antagonist. As shown in Fig.4, ADM22-52 (100 nmol/L) had no significant effect on ICa,L, but pretreatment of the cell with it for 5 min completely abolished the inhibitory action of ADM. Similar results were obtained from other four cells. It is likely that the inhibitory action of ADM is mainly mediated by specific ADM receptor in guinea-pig ventricular myocytes.

Fig.4. Effect of ADM22-52 on ADM-induced inhibition of ICa,L. Time and current calibrations are given in the graph. a: Control; b: 100 nmol/L ADM22-52; c: 100 nmol/L ADM+100 nmol/L ADM22-52.

2.3 Role of PKA in the inhibitory effect of ADM on ICa,L

Since ADM receptors have been reported to be coupled with adenylate cyclase[3], we examined whether activation of PKA was involved in the ADM-induced inhibition of ICa,L. H-89, a specific PKA inhibitor was used. It inhibits PKA at the ATP site on the catalytic subunit of PKA. H-89 (10 μmol/L) caused a rapid decrease of ICa,L (Fig.5). When a nearly stable level of ICa,L had reached, 100 nmol/L ADM was applied in the presence of H-89, ICa,L was further decreased and recovered after washing out. In four experiments, the percentage inhibition induced by 100 nmol/L ADM in the presence H-89 was 33.42±6.64%, a similar inhibition to those induced by 100 nmol/L ADM alone. These results suggest that ADM-induced inhibition of ICa,L was not mediated by PKA.

Fig.5.

Effect of H-89 on ADM-induced inhibition of ICa,L. Time and current calibrations are given in the graph. a: Control; b: 10 μmol/L H-89; c: 100 nmol/L ADM+10 μmol/L H-89; d: wash out. 2.4 of PKC in the inhibitory effect of ADM on ICa,L

PKC19-36, a specific PKC inhibitor, and PMA, a specific PKC activator, were used to examine the role of PKC involved in the ADM-induced inhibition of ICa,L.2.4.1 Effect of ADM on ICa,L in the presence of a PKC inhibitor 10 μmol/L PKC19-36 was added in the internal solution, this concentration of PKC19-36 is more than 50 times higher than the published IC50 for PKC[15]. ICa,L was not changed during the dialysis procedure. (10 μmol/L PKC19-36). After 10 min of dialysis, subsequent application of 100 nmol/L ADM failed to produced any effect on the ICa,L (Fig. 6). In three myocytes, the percentage inhibition of ICa,L induced by ADM (100 nmol/L) in the presence of PKC19-36 was 4±2.78% (P>0.05).

Fig.6.

Effect of PKC19-36 on ADM-induced inhibition of ICa,L. Time and current calibrations are given in the graph. a: 10 μmol/L PKC19-36; b: 100 nmol/L ADM+10 μmol/L PKC19-36; c: 10 μmol/L PKC19-36.

2.4.2 Effect of PMA on ICa,L

Superfusion of cells with 1 μmol/L PMA decreased ICa,L. In five cells tested, PMA decreased ICa,L amplitude by 32.26±4.20%(P<0.05; Fig.2), the inhibition action was reversed after washing out PMA.

3 DISCUSSION

In this study we demonstrated that (1) ADM inhibits ICa,L in a dose-dependent manner in isolated guinea-pig ventricular myocytes (Fig.2) without a shift along the voltage axis (Fig.3); (2) the inhibitory action of ADM on ICa,L is mainly mediated by specific ADM-receptor in guinea-pig ventricular myocytes; and (3) PKC may be involved in the ADM action on ICa,L.

The inhibitory action of ADM on ICa,L was also found in isolated adult rabbit cardiac ventricular myocytes[11]. However, there were different reports on multicellular preparation. Intravenous administration of ADM in human[5] or mammals resulted in an increase in cardiac output and stroke volume in addition to the decrease in mean arterial pressure. These findings suggest that ADM may have a direct positive inotropic effect on myocardium in vivo. More recently, Ihara et al. reported that ADM produced dose-dependent positive inotropic effects in rat papillary muscles, at least partly through a cAMP-dependent pathway[9]. However, from these findings, it is difficult to conceive that the positive inotropic properties of ADM may be mediated by an activation of L-type calcium channel. Probably,the enhancement of the sarcoplasmic reticulum function may play an important role in the positive inotropic effects of ADM[8]. In our experiment excessive rundown of ICa,L is not an explanation, because (1) low concentration of ADM had minimal effect; (2) high ADM-induced inhibition was reversible; (3) ADM22-52 and PKC19-36 completely blocked the ADM-induced inhibition.

Two subtypes of ADM receptors (CGRP receptor and specific ADM receptor) have been reported[4]. Since it was reported that there was not CGRP receptor expressed in guinea-pig ventricular myocytes[13, 14], we examined whether the specific ADM receptor mediated the ADM-induced inhibition of ICa,L. ADM22-52 is the specific ADM receptor antagonist, an NH2-terminal truncated ADM analogue which lacks the six-membrane ring structure of ADM. We found that ADM22-52 (100 nmol/L) could completely abolish the inhibitory action of ADM (Fig.4). It is likely that the inhibitory action of ADM is mainly mediated by specific ADM-receptor in guinea-pig ventricular myocytes.

ADM elevates intracellular cAMP in various types of cells including platelets, vascular smooth muscle cells and endothelial cells. Similarly, ADM has been reported to stimulate cAMP formation in isolated cardiac myocytes[15]. These observations suggest that the activation of adenylate cyclase-cAMP-PKA system is probably involved in the inotropic effect of ADM. In this experiment the effect of a selective PKA inhibitor, H-89, on ADM induced inhibition of ICa,L was studied. H-89 is extremely selective for PKA: value of inhibition constant (ki) for PKA is 50 nmol/L and more than 500 times lower than for any of five other protein kinases. Application of H-89 (10 μmol/L) caused a rapid decrease in ICa,L, suggesting inhibition of the intrinsic phosphorylation of calcium channels in the cells[16]. When a nearly stable level of ICa,L had reached, isoprenoline at 100 nmol/L showed little enhancement of ICa,L (data not shown), suggesting sufficient inhibition of PKA activity. However, pretreatment of the cells with H-89 (10 μmol/L) did not attenuate the effects of ADM (Fig.5). These results demonstrate that the ADM-induced inhibition of ICa,L was not mediated by PKA.

It was reported that the increase in cAMP level induced by ADM might be due to cross-activation of CGRP receptors, whereas signal transduction via specific ADM receptors may not involve the cAMP pathway[4]. In bovine aortic endothelial cells, ADM have been reported to activate phospholipase C through its specific receptor, leading to inositol triphosphate (IP3) and diacylglycerol (DAG) formation, suggesting that specific ADM-receptor is coupled with PLC[17]. Szokodi's results also suggested the existence of another ADM receptor subtype not coupled to adenylate cyclase in the heart[8]. So PKC may be the second messenger of ADM via specific ADM-receptor.

To determine whether the activation of PKC is responsible for the ADM-induced inhibition of ICa,L, we used PKC19-36, a selective PKC inhibitor, and PMA, a selective PKC activator. Intracellular application of 10 μmol/L PKC19-36 completely abolished the inhibitory effect of ADM on ICa,L, and PMA (1 μmol/L) mimicked the inhibitory effect of ADM. The decrease of ICa,L by PMA in our study confirms Satoh's report[18], which indicates that ICa,L was inhibited by the stimulation of PKC in isolated guinea-pig ventricular cardiomyocytes. These results strongly suggest that ADM-mediated inhibition of ICa,L involves an activation of PKC especially in guinea-pig ventricular myocytes. But in guinea-pig ventricular myocytes ICa,L is increased by PMA[19], which differs somewhat from our results. A possible explanation for the discrepancy is the experimental conditions used since those authors used the conventional microelectrodes technique to record ICa,L. But in fact, they also found the transient decrease in ICa,L by PMA.

We also found that ADM suppressed the ICa,L enhanced by isoprenoline/forskolin (data not shown), which is similar to the report of Ikenouchi[11]. This suggested that ADM could modify the effects of activated PKA systems in guinea-pig ventricular myocytes. It is known that the secretion of ADM is increased under certain pathophysiological conditions, such as acute myocardial infarction[5] or congestive heart failure[6], when the plasma level of catecholamines is elevated. By inhibiting the catecholamine-potentialed ICa,L, ADM may protect against myocardial calcium overload and fatal ventricular arrhythmias. But further investigations are needed to determine the clinical importance of this peptide.

In conclusion, ADM caused an inhibition of L-type calcium currents in isolated guinea-pig ventricular myocytes. Binding to the specific ADM receptors and activation of PKC might contribute to the inhibitory effect of ADM.

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