УДК 577.352.4


© 2013 A. V. Maltsev, M. N. Nenov, O. Y. Pimenov, Yu. M. Kokoz*

Institute of Theoretical and Experimental Biophysics, Russian Academy of Science, Pushchino, Moscow obl., 142290 Russia; *e-mail: goicr@rambler.ru Received 02.07.2012

It is shown that agmatine inhibits L-type Ca2+ currents in isolated cardiomyocytes of rats in a dose-dependent manner. The inhibitory analysis indicates that imidazoline receptors of type I (IiRs) rather than ^-adrenoceptors (a2-ARs) are implicated in mediating the effects of agmatine. Agmatine affects the dynamics of intracellular Ca2+ concentration changes in spontaneously active cardiomyocytes. The averaged intracellular Ca2+ concentration ([Ca2+]in) varied biphasically, depending on the agmatine dose: at 1—500 цМ, agmatine decreased [Ca2+]in; at 500 цМ—2 mM, [Ca2+]in remained unchanged, and at concentrations above 2 mM agmatine caused an increase of [Ca2+]in. The effects of low agmatine concentrations were inhibited by 7NI, an inhibitor of NO synthases (NOS), as well as by the inhibitors of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) thapsigargin and cyclopiazonic acid. In contrast, ODQ, a blocker of NO-sensitive guanylate cyclase, and the antagonist of IiRs efaroxan were ineffective. At low concentrations agmatine did not affect the increase in [Ca2+]in induced by stimulating doses of ryanodine (40 nM). In addition, agmatine at low doses was found to markedly stimulate NO production. When efaroxan (10 цМ) or ryanodine (200 цМ) were added to the bath to inhibit IiRs and ryanodine receptors (RyRs), respectively, [Ca2+]in became much less sensitive to millimolar agmatine. In contrast to low concentrations (100 цМ), high agmatine doses (10—15 mM) did not stimulate the NO synthesis but were effective as NOS inducer in cells pretreated with efaroxan. The selective I1R agonist ril-menidine increased [Ca2+]in in a dose-dependent manner. The effect of rilmenidine was similar to that of agma-tine at high doses and was abolished by RyRs inhibition. Our findings indicate that in spontaneously active car-diomyocytes agmatine at low concentrations decreases [Ca2+]in, does not stimulate ^Rs but most likely enhances NO synthase followed by an increase in SERCA activity due to the direct nitrosylation of SERCA and/or phos-pholamban. The effects of high agmatine doses are apparently mediated by ^Rs and involve RyRs.

Keywords: L-type Ca2+ currents, spontaneous Ca2+ waves, agmatine, rilmenidine, imidazoline receptors.

DOI: 10.7868/S0233475513020059

Agmatine is an amine formed by decarboxylation of Z-arginine by arginine decarboxylase. It was first identified as an intermediate during the synthesis of polyamines in bacteria, plants and many invertebrates and then was found in the aorta, large and small intestines, spleen, lungs, kidney, heart, liver, muscle tissue, testis, and blood plasma of mammals [1]. Agmatine produces a variety of physiological effects on the nervous, endocrine, and cardiovascular systems, and it might therefore be used as a potential drug. It was shown that agmatine is a ligand of a2-ARs and imidazoline receptors (IRs). The intravenous administration of agmatine decreases the arterial pressure in a dose-dependent manner [2, 3] and induces a cardioprotective effect in an ischemia—reperfusion model [4]. At the tissue and cellular levels agmatine inhibits the proliferation of vascular smooth muscle cells [5], exhibits a negative chronotropic activity by diminishing the heart rate and the amplitude of the action potential in pacemaker cells of the rabbit sinoatrial node

[6], and partakes in the regulation of voltage-dependent Ca2+ currents and intracellular Ca2+ concentration ([Ca2+]in) in cardiomyocytes [7, 8]. However, the mechanisms of its action on L-type Ca2+ currents and on [Ca2+]in remain undefined. The available data on the effects of agmatine on the activity of NO synthases (NOS) are contradictory: agmatine has been reported to inhibit NOS in homogenates of the brain and lung artery [9, 10] as well as to increase NOS activity in endothelial cells [11] and in isolated kidney [12]. In the present study we focused on evaluating the role of agmatine in the regulation of L-type Ca2+ currents and intracellular Ca2+ in isolated rat cardiomyocytes and on revealing the underlying mechanisms.


Isolation of cells. Cardiomyocytes were isolated from hearts of Sprague—Dawley and Wistar male rats (200—250 g) as described earlier [13]. Basic extracellu-

lar solution contained (mM, pH 7.25): NaCl, 80; KCl, 10; KH2PO4, 1.2; MgSO4, 5; glucose, 20; taurine, 50; HEPES, 10, and Z-arginine, 1. The isolated heart was perfused according to Langensdorf. Solutions were continuously delivered through a buffer container (10 mL). Preparations were washed from blood for 3— 5 min using the medium DMEM + 10 mM HEPES, pH 7.25. After stabilization of the heart rate, the perfusion was carried out using the basic solution with the Ca2+—EGTA buffer (pCa 7) until the cardiac arrest. Then the perfusion was continued using the solution containing the basic solution (100 mL) to which pronase E (20 mg), bovine serum albumin (100 mg, fraction 5), and CaCl2 (140 ^M) were added. After 3—4 min the solution draining down the preparation acquired the form of viscous drops, after the disappearance of which the perfusion was stopped (the perfusion with the pronase-containing solution did not last longer than 10 min). The heart was removed from the cannula, placed in the basic solution supplemented with CaCl2 (200 ^M), and cut into small fragments of 6—10 mm3. The dispersion of the tissue into individual cells and the entire cell isolation procedure were carried out at 37°C in the same solution supplemented with pronase (2 mg/ml) and stirred at 1—2 revolution/s. Immediately prior to the tissue dispersion, 2—3 mg of collagenase (type IV) per 10 ml of the pronase-containing solution was added. After 20-min incubation, the first discharge was taken. Cell suspension was passed through a nylon net to separate from undispersed tissue aggregates. The rest of the tissue was dispersed again on a stirrer in a solution containing pronase and collagenase. The disintegration was usually completed after 2—4 cycles. Cells were stored at room temperature in the basic solution containing 200 ^M Ca2+.

Registration of L-type Ca2+ currents. The extracellular solution contained (mM, pH 7.25): NaCl, 80; CaCl2, 2; MgSO4, 5; KH2PO4, 1.2; CsCl, 10; TEA-Cl, 20; glucose, 20; HEPES, 10, and Z-arginine, 1. The intracellular solution contained (mM, pH 7.25): CsCl, 130; MgSO4, 5; HEPES, 10. K+ currents were blocked by the addition of TEA-Cl to the extracellular solution and replacement of potassium ions by cesium ions (Cs+). Voltage-gated (VG) Ca2+ currents were recorded with the perforated patch clamp approach at 20—22°C. Membranes were perforated using amphotericin B (160—170 ^g/mL). Electrodes were manufactured of soft molybdenum glass with resistance of 1— 5 MD. Typically, cells were held at a holding potential of —40 mV and depolarized to 40 mV for 300 ms to evoke VG Ca2+ currents.

Measurements of [Ca2+]in and the level of NO in cardiomyocytes. Individual cardiomyocytes were put into in a camera on a round cover glass with Hanks balanced salt solution (HBSS) containing (mM, pH 7.4): NaCl, 138; CaCl2, 1.3; MgSO4, 0.4; MgCl2, 0.5; KCl, 5.3; KH2PO4, 0.45; NaHCO3, 4; Na2HPO4, 0.3; glucose, 10; Z-arginine, 1; HEPES, 10. After cell sedi-

mentation and attachment to the glass for 15 min, the bath solution was replaced with HBSS supplemented with 5 ^M Fluo4-AM or DAF-FM (4-amino-5-me-thylamino-2',7'-difluorofluorescein diacetate) for monitoring [Ca2+]in or NO, respectively, followed by a 40-min incubation of cells at 37°C. Subsequently the cells were washed three times with HBSS and incubated for 10 min. Dye-loaded cardiomyocytes could be used in the experiments within 3 h.

Cell fluorescence was recorded using an inverted microscope Eclipse TS100F equipped with a fluorescent module T1-FM (Nikon, Japan) and Leica TCS SP5 laser scanning confocal microscope. The intracellular levels of Ca2+ and NO was assessed by Fluo4 and DAF-FM fluorescence, respectively . The time course of the changes in [Ca2+]in and NO was present-F' — F

ed as F = -0, where F' is the fluorescence inten-


^ aver

sity in the current moment of time, F0 is the background value, Faver is the average fluorescence intensity in the control (before the application of compounds used). Measurements were carried out on spontaneously active cardiomyocytes. The results were processed using ImageJ and SigmaPlot 8.0 software. Quantitatively, variations of [Ca2+]in and NO were characterized by a difference of current fluorescence of Fluo4-AM and DAF-FM,respectively, averaged in time (50—60 s) before and 8—10 min after the application of substances.

Statistics. The data obtained (except the original records of single experiments) were presented as a mean ± SD. The statistical significance of the effect of a particular compound was evaluated using the Student i-test atp < 0.05.

Materials. Most reagents were from Sigma—Ald-rich (USA). Fluo4-AM and DAF-FM were from In-vitrogen (USA); collagenase type IV and protease were from Worthington (USA) and Fluka (Germany), respectively.


Effects of agmatine on L-type Ca2+ currents in isolated cardiomyocytes. Agmatine decreased the amplitude of L-type Ca2+ currents (Fig. 1a) in isolated rat cardiomyocytes in a dose-dependent manner (Fig. 1b). Particularly, L-type Ca2+ current were virtually unchanged at the agmatine concentrations of20 ^M (n = 4) and 100 ^M (n = 4), while 200 ^M agmatine caused a detectable reduction of current amplitude by 3.2 ± ± 0.6% on average (n = 3). In further experiments agmatine was used at the concentration of 2 mM, at which the inhibitory effect reached 23.8 ± 1.9% (n = 6) (Fig

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