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Effect of 15-hydroxyazomethine atisine, a diterpenoid alkaloid atisine derivative, on Na+/Ca 2+ exchanger mediated...
Section 2. Biology
Zaynabiddinov Anvar Erkinjonovich, post-graduate, Sadykov Institute of Bioorganic Chemistry, Uzbek Academy of Sciences. E-mail: azaynobiddinov@bk.ru Usmanov Pulat B., Doctor of science, professor; Sadykov Institute of Bioorganic Chemistry, Uzbek Academy of Sciences. Salimov Bahodir T. Doctor of science; Yunusov Institute of Chemistry of Plant Substances, Uzbek Academy of Sciences. E-mail: miconypharms@yahoo.com
Effect of 15-hydroxyazomethine atisine, a diterpenoid alkaloid atisine derivative, on Na+/Ca2+ exchanger mediated contraction of rat papillary muscle
Abstract: We investigated the effects of 15-hydroxyazomethine atisine (15-HAA), a derivative of atisine, a diterpenoid alkaloid, on the force of contraction of rat papillary muscle induced by low Na+ (35 mmol/l) solution and ouabaine (20 pmol/l). The force of contraction induced by these two different procedures in the presence of nifedipine (10 pmol/l) and ryanodine (4 pmol/l) dose-dependently inhibited by 15-HAA. At 25 pmol/l 15-HAA almost complete inhibited the force of contraction induced by low Na+ solution and ouabaine, with IC50 values of 16.9±3.9 and 15.8±4.7 pmol/l, respectively. Since under used experimental conditions Ca 2+ influx through slow Ca 2+-channels and Ca 2+ release from sarcoplasmic reticulum were blocked by nifedipine and ryanodine, the results suggest that 15-HAA inhibits the force of contraction mediated by Ca 2+ influx via the Na+/ Ca 2+. This suggestion was further confirmed when we used KB-R7943, a specific blocker of the reverse mode of Na+/Ca 2+ exchanger, which significantly attenuated the inhibitory effects of alkaloid on the force of contraction increased by two different procedures. In conclusion, we found that 15-HAA possess a potent inhibitory action on Ca 2+ influx via Na+/Ca 2+ exchanger suggesting that this action may also be involved in its negative inotropic effect. The significant potency of 15-HAA to block reverse mode of Na+/Ca 2+ exchanger suggests that it can be useful in development of novel approaches to protect heart against severe cardiac pathologies associated with Ca 2+ overload induced by exchanger.
Keywords: Alkaloids, negative inotropic effect, Na+/ Ca 2+ exchanger.
Introduction
The 15-hydroxyazomethine atisine (15-HAA), a derivative of atisine, a diterpenoid alkaloid, isolated from Aconitum plant species, has a pronounced antiarrhythmic effect [1]. The mechanism of antiarrhythmic action of 15-HAA is similar to that of class 1 antiarrhythmic drugs which mainly is due to blockage of voltage-gated Na+-channels in cardiac cells [2]. The blockage of Na+-channels with these antiarrhythmic drugs causes a decrease in the depolarization rate of the action potential, slows conduction velocity and reduces the excitability of the heart tissue [3, 4]. At the same time, all class 1 antiarrhythmic drugs exert significant negative inotropic effect related to Na+-channels blockage and the subsequent decreases in the intracellular Са 2+ concentration [5; 6]. It is assumed that the blockade of the Na+-channels by
these drugs may alter Ca 2+ transport through Na+/Ca 2+ exchanger so that a decrease in intracellular Na+ promotes Ca 2+ extrusion at a polarized state and inhibits Ca 2+ influx through the exchanger at a depolarized state [7]. These changes in Ca 2+ transports would secondarily reduce Ca 2+ content in the sarcoplasmic reticulum (SR), limit SR Ca 2+ release and, thus, decrease contractile force [8]. Recently we found that alkaloid 15-HAA also produced a strong negative inotropic effect which was attenuated by lidocaine and after inactivation of Na+-channels [9]. In addition, we observed that this alkaloid significantly decreased the post-rest potentiation of contraction, which mainly reflects the amount of Ca 2+ in SR. Since Ca 2+ content in SR is directly associated with functional state of Na+/Ca 2+ exchanger [10], these data suggest that the inhibition of the exchanger may also be involved in
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Section 2. Biology
the negative inotropic effect of 15-HAA. Therefore, to test this hypothesis we examined the effects of 15-HAA on the contractions of rat papillary muscle induced by low Na+ solution and ouabaine, which are mainly due to the Ca 2+ entry via Na+/Ca 2+-exchanger.
Material and methods
Adult male Wistar rats (200-250 g) were used according to a protocol of the Institute of Bioorganic Chemistry Animal care and Use Committee. After brief anesthesia with sodium pentobarbital hearts were rapidly removed, and papillary muscles (1-3 mm in length and 0,5-0,8 mm in diameter) were dissected from the left ventricles. Preparation was mounted in a tissue bath (STEIRT, HSE, Germany) by one end to a force-displacement transducer (Type F30, HSE), and the other end to the bottom of the tissue bath. The tissue bath (3 ml volume) was perfused at a rate of 20 ml min-1 with Krebs solution containing (in mmol/l): NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4,1.2; KH2PO4, 1.2; NaHCO3, 24; glucose 10 (pH 7.4). The solution was bubbled with 95% O2 and 5% CO2 and maintained at 37 °C. The preparation was stretched to a length at which maximum developed force was evoked and allowed to equilibrate for at least 1 h with changes in Krebs solution every 15 min. The preparation was field-stimulated at a rate of 0.15 Hz by two platinum electrodes with rectangular wave pulses of10 ms duration at twice the threshold voltage delivered from the electronic stimulator (ESL-2, Russia). The amplitudes of elicited maximal isometric contraction were used as the control (taken as 100%), and changes in the contraction after drugs action were expressed as a percentage of the maximal response. Contractions recorded on the chart recorder (TZ 4620, Czech Rep.) and simultaneously stored on computer for analysis. To examine the effect of alkaloid on contraction mediated by Ca 2+ influx through Na+/Ca 2+-exchanger, both low Na+ Krebs solution and ouabain, a cardiac glycoside, (20 pmol/l) were used. In a low Na+ Krebs solution, the Na+ concentration was reduced to 35 mmol/l by replacing the NaCl with equimolar choline chloride and atropine (10 pmol/l) was added to avoid any parasympathetic effects. To block Ca 2+ influx through L-type slow Ca 2+ channels and Ca 2+ release from SR in these experiments nifedipine (10 pmol/l) and ryanodine (4 pmol/l) were used. Chemicals such as atropine, nifedipine, ouabaine, ryanodine and KB-R-7943 were purchased from Sigma-Aldrich (Sigma, St. Louis, MO, U. S.A.). Data are expressed as mean ± SEM with n for number of experiments. Student’s f-test or one way ANOVA used for analysis, P<0.05 was considered statistically significant.
Results
To examine the role of Na+/Ca 2+ exchanger in the negative inotropic effect of 15-HAA its action on the contractions of rat papillary muscle induced by low Na+ solution and oua-baine were evaluated. In control experiments the basal force of contraction of rat papillary muscle perfused with normal Krebs solution and driven at 0.1 Hz, was about 3.6±0.2 mN, taken as 100%. When to this perfusion solution nifedipine (10 pmol/l) and ryanodine (4 pmol/l) were added simulta-
neously the force of contraction decreased to 16.1±5.3% of control (n=6). After replacement of the normal Krebs solution with modified low Na+ solution, containing nifedipine and ryanodine, the force of contraction increased to 72.6±4.8% of control (n=6). Similarly, addition of ouabaine (20 pmol/l) to the normal Krebs solution containing nifedipine and ryanodine increased the force of contraction to 68.6±3.6% of control (n=6). Since in these experiments Ca 2+ influx through slow Ca 2+-channels and Ca 2+ release from SR were blocked by nifedipine and ryanodine, the results indicate that increases in the force of contraction induced by low Na+ solution and ouabaine were mediated by the same mechanism involving Ca 2+ influx via the Na+/Ca 2+ exchanger. The experiments with KB-R7943, a specific blocker of Ca 2+ influx via Na+/Ca 2+ exchanger [11], confirmed the involvement of Ca 2+ influx via Na+/Ca 2+ exchanger in contractions induced by these two different procedures. As shown in (Fig.1), KB-R7943 almost completely inhibited the force of contraction induced by these two different procedures. These results provide clear evidence for the involvement of Ca 2+ influx via Na+/Ca 2+ exchanger in contractions induced by these two different procedures.
Figure 1: Effects of KB-R7943 on the force of contractions of rat papillary muscle induced by low Na+ solution (solid circles) and ouabaine (open circles)
The experiments were performed in preparations preincubated with nifedipine (10 pmol/l) and ryanodine (4 pmol/l) for 30 min before low Na+ solution and ouabaine were added. Data are shown as mean (±SD) and expressed as a percentage of the force of control contraction, obtained in normal Krebs solution at 0.1 Hz before the addition of drugs, which was taken as 100%. (n=6). *p<0.05, when compared with control value.
Therefore, to test the effect of 15-HAA on contraction mediated by Na+/Ca 2 exchanger we first studied its action on the force of contraction induced by low Na+ solution. In these experiments was observed that 15-HAA dose-dependently inhibited the force of contraction increased by low Na+ solu-
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Effect of 15-hydroxyazomethine atisine, a diterpenoid alkaloid atisine derivative, on Na+/Ca 2+ exchanger mediated...
tion and at 25 pmol/l maximally reduced it from 72.6±4.8% to 11.6±3.1% (Fig.2). The IC50 value of 15-HAA, a concentration producing 50% of maximal inhibition, determined graphically, was 16.9±3.9 pmol/l. Similar inhibitory effect of 15-HAA was observed in papillary muscles contracted by ouabaine in the presence of nifedipine and ryanodine. Under these conditions 15-HAA in similar dose-dependent manner inhibited the force of contraction increased by ouabaine and at 25 pmol/l maximally reduced it from 68.6±3.6% to 6.9±3.1% (Fig.2).
Figure 2: Effects of 15-HAA on the force of contraction of rat papillary muscle induced by low Na+ solution (solid circles) and ouabaine (open circles)
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I I low Na+ (35 mmol/l)
I I KB-R7943 (20,6 pmol/l)
H KB-R7943 + 15-HAA (25 pmol/l)
The effects of 15-HAA were examined in preparations precontracted with low Na+ solution and ouabaine in the presence of nifedipine (10 pmol/l) and ryanodine (4 pmol/l). Data are shown as mean (±SD) and expressed as a percentage of the force of control contraction, obtained in normal Krebs solution at 0.1 Hz before the addition ofdrugs, which was taken as 100%. (n=6). *p<0.05, when compared with control value.
The IC50 value of 15-HAA obtained in these experiments was 15.8±4.7 pmol/l, which is nearly equal to those obtained from experiments with low Na+ solution. The data indicate that 15-HAA with the almost identical potency inhibits the force of contraction induced by low Na+ solution and ouabaine, suggesting that this inhibitory effect of alkaloid is due to the blockage of Ca 2+ influx via Na+/Ca 2+ exchanger. This suggestion was further confirmed in the experiments with KB-R7943, which significantly attenuated the inhibitory effect of 15-HAA on the force of contraction increased by these two different procedures. In these experiments the preparations were pretreated with KB-R7943 at concentration of 20.6 pmol/l, at which the force of contraction increased by low Na+ solution or ouabaine was reduced by about a half. In these experimental conditions administration of 15-HAA (25 pmol/l) on top of KB-R7943 further reduced the force of contraction induced by low Na+ solution, but to a much lesser extent, than that obtained in the absence of KB-R7943 (Fig.3A).
The experiments were performed in preparations preincubated with nifedipine (10 pmol/l) and ryanodine (4 pmol/l). KB-R7943 was used at 20.6 pmol/l, concentration corresponding to its IC50 value. Data are shown as mean (±SD). *p<0.05, when compared with control value.
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I I Ouabaine (20 pmol/l)
I I KB-R7943 (20,6 pmol/l)
H KB-R7943 + 15-HAA (25 pmol/l)
Figure 3: KB-R7943 attenuates the inhibitory effects of 15-HAA on the force of contraction induced by low Na+ solution (A) and ouabaine (B) in rat papillary muscle
Similarly, KB-R7943 attenuates the inhibitory effect of 15-HAA on the force of contraction induced by ouabaine. As shown in Fig.3B, after administration of 15-HAA (25 pmol/l) on top of KB-R-7943 the force of contraction induced by oua-
baine further reduced, but also to a much lesser extent, than that observed in the absence of KB-R7943. These data again support the suggestion that the inhibitory effect of 15-HAA is due to the blockade of Ca 2+ influx via Na+/Ca 2+ exchanger.
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Section 2. Biology
Discussion
In this study we have examined the effects of 15-HAA on the force of contraction of rat papillary muscle induced by low Na+ solution and ouabaine. The present results demonstrated that 15-HAA markedly and dose-dependently inhibited the force of contraction induced by these two different procedures. The increase in the force of contraction induced by low Na+ solution is mainly due to changes in the thermodynamic driving force of the Na+/Ca 2+ exchanger which promotes Ca 2+ influx via the reverse mode of the exchanger [12; 13]. Since under the present experimental conditions Ca 2+ influx through slow Ca 2+-channels and Ca 2+ release from SR were blocked by nifedipine and ryanodine, inhibition by 15-HAA of the force of contraction induced by low Na+ solution suggests that this effect of alkaloid is due to the blockage of Ca 2+ influx via the Na+/Ca 2+. This suggestion was confirmed by observation that 15-HAA also potently inhibited the force of contraction induced by ouabaine, which also is mediated by Ca 2+ influx via the Na+/Ca 2+ exchanger. Ouabaine inhibits Na+, K+-ATPase, and increases intracellular Na+ concentration which in turn reverses the operating mode of Na+/Ca 2+ exchanger, thus promoting Ca 2+ influx [14, 15]. Since in the experiments with ouabaine, nifedipine and ryanodine also were present, the results indicate that inhibitory effect of 15-HAA on the force of contraction induced by ouabaine is a result of blockage of Ca 2+ influx via the Na+/Ca 2+. Furthermore, from these results it is evident that 15-HAA inhibited the force of contraction induced by ouabaine almost to the same extent and with nearly equal IC50 value as those observed in the experiments with low Na+ solution. Thus, a similar inhibitory action of 15-HAA on the force of contractions induced by two
different procedures which activates Ca 2+ influx via Na+/Ca 2+ exchanger provides the clear evidence that these effects of alkaloid are a result of direct blockage of Ca 2+ influx through this exchange mechanism. The potency of 15-HAA to block of Ca 2+ influx via Na+/Ca 2+ exchanger was further confirmed in the experiments with KB-R7943, which significantly attenuated the inhibitory effects of 15-HAA on the force of contraction increased by two different procedures.
Conclusion
The present results demonstrate that 15-HAA markedly inhibited the force of contraction induced by low Na+ solution and ouabaine indicating that this inhibitory effect of alkaloid is due to the blockage of Ca 2+ influx via Na+/Ca 2+ exchanger. These results suggest that the blockage of Ca 2+ influx via Na+/Ca 2+ exchanger may also be involved in the negative inotropic effect of 15-HAA. It seems likely that the negative inotropic effect of 15-HAA is provided with a complex mechanism and, apparently, represents the sum of their Na+ channel and Na+/Ca 2+ exchanger blocking activities. The inhibition of Na+/Ca 2+ exchanger in combination with a blockage of Na+ channel by this alkaloid may significantly reduce Ca 2+ content in SR, and thus producing strong negative inotropic effect. There is no doubt that due to the significant potency to block reverse mode of Na+/Ca 2+ exchanger 15-HAA represent a valuable structure which will be useful in development of novel approaches to protect heart against severe cardiac pathologies associated with Ca 2+ overload induced by exchanger.
Acknowledgments. This work was supported by a grant FA-F6-T083 from the Coordinating Committee for Development of Science and Technology under the Cabinet of Ministers of the Republic of Uzbekistan.
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Effect of 15-hydroxyazomethine atisine, a diterpenoid alkaloid atisine derivative, on Na+/Ca 2+ exchanger mediated...
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