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Mirzayeva Yulduzkhon T., Master of biology, scientific researcher, Sadykov Institute of Bioorganic Chemistry, Uzbekistan Academy of Sciences E-mail: [email protected] Sultankhodzaev Mukhlis N., Doctor of science, Yunusov Institute of Chemistry of Plant Substances, Uzbekistan Academy of Sciences
Usmanov Pulat B., Doctor of science, professor, Sadykov Institute of Bioorganic Chemistry
The possible role of the Na+/Ca 2+ exchanger in the vasorelaxant effect of 1-O- benzoylkarakoline, a diterpenoid alkaloid, in rat aortic rings
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
Abstract: The aim of this study was to examine the role of the Na+/Ca 2+ exchanger in the vasorelaxation induced by 1-O-benzoylkarakoline (1-O-BK), a diterpenoid alkaloid, in rat aortic rings. We found that 1-O-BK potently and in a concentration-dependent manner inhibited the aortic rings contraction induced by low-Na+ solution and ouabaine. The comparison of our results showed that 1-O-BK inhibited the aortic rings contraction induced by these two procedures almost to the same extent and with nearly equal IC50 values. Since the contractions induced by low Na+ solution and ouabaine were mainly due to increased Ca 2+ influx mediated by the reverse mode of the Na+/Ca 2+ exchanger, these findings indicate that the inhibitory effect of 1-O-BK is a result of direct blockage of Ca 2+ influx via this exchanger. Taken together, the present results provide the clear evidence that 1-O-BK potently inhibited the Ca 2+ influx via Na+/Ca 2+-cexchanger, suggesting that this effect of alkaloid also may be involved in its vasorelaxant activity. The finding that 1-O-BK exhibits significant potency to block Ca 2+- influx via Na+/Ca 2+ exchanger may be important under some pathological conditions where the exchanger, operating in the reverse mode, induces Ca 2+ overload and, hence, may exacerbate overall vasoconstriction.
Keywords: rat aorta; vasorelaxation; diterpenoid alkaloid 1-O-benzoylkarakoline; Na+/Ca 2+ exchanger.
Introduction with a-adrenoreceptor stimulation [6,163-165; 7, 606-718]. These
Diterpenoid alkaloids produced by the plants of the genera data suggested that relaxant effect of 1-O-BK is probably due to
Aconitum and Delphinium comprise a large group of natural com- the inhibition of the Ca 2+ influx through both VDCCs and ROCCs,
pounds, still continue to deliver a great variety of structural templates resulting in decreased intracellular Ca 2+ concentration ([Ca 2+]in),
for drug discovery and development [1, 620-624; 2, 209-221]. and consequently, in reduced contractility. Furthermore, 1-O-BK
1-O-benzoylkarakoline (1-O-BK), a derivative of diterpenoid al- also effectively reduced the contraction induced by PE in a Ca 2+ —
kaloid karakoline, isolated from Aconitum karakolicum, has been free medium, suggesting that the inhibition Ca 2+ release from sar-
reported to possess diverse pharmacological properties, including coplasmic reticulum (SR), in addition to the blockage of VDCCs
spasmolytic, antiarrhythmic and anesthetic activities [3, 390-392; and ROCCs, also may be involved in the relaxant effect of 1-O-BK.
4, 254-269]. Recently, we found that 1-O-BK exhibited marked va- In smooth muscle cells the amount of Ca 2+ available for release, as
sorelaxant activity and significantly inhibited the contraction of rat well as the Ca 2+ content in SR is modulated by Na+/Ca 2+ exchanger,
aortic rings induced by KCl and phenylephrine (PE) [5, 8-11]. that operating in reverse mode provides a source of Ca 2+ for refill-
The relaxant effect of 1-O-BK was not significantly different in ei- ing this store [8, 657-660; 9, 763-854; 10, 519-529]. Considering
ther the presence or absence of functional endothelium and was this, we suggested that the reduction of PE-induced contraction in
not influenced by inhibition of nitric oxide synthase by L-NAME a Ca 2+-free medium by 1-O-BK may probably be due to the inhi-
or cyclooxygenase by indomethacin. At the same time, the relaxant bition of the Ca 2+ influx mediated by reverse mode of Na+/Ca 2+
effect of 1-0-BK on KCl- and PE-induced contractions was signifi- exchanger. Therefore, to address this possibility, we examined the
cantly attenuated in the presence ofverapamil, a voltage-dependent effects of 1-O-BK on the contractions of rat aortic rings induced by
Ca 2+ channels (VDCCs) blocker, and phentolamine which inhibits low-Na+ solution and ouabaine, which mainly are mediated by Ca 2+ receptor-operated Ca 2+ channels (ROCCs) activation associated entry via Na+/Ca 2+- exchanger.
ThepossibleroleoftheNa+/Ca2+exchangerinthevasorelaxanteffectof1-O-benzoylkarakoline,aditerpenoidalkaloid,inrataorticrings
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, rat was decapitated and the thoracic aorta was isolated. The connective tissue was removed and rings 3 mm long were mounted vertically under isometric conditions in a 5 ml organ bath perfused with Krebs solution containing (in mM): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, NaH2PO4 1.2, NaHCO3 25.0, EGTA 1.0 and glucose 11.1 (pH 7.35). Krebs solutio tained at 37 ± 0.5 °C and continuously bubbled with O2/CO2 mixture (95 %/5 %). An initial load of 1 g. was applied and maintained throughout a 60 min. equilibration period. During equilibration the bathing solution was changed every 15 min. with readjustment of baseline tension, when necessary. Tension was recorded on a pen recorder (Endim 621,02, Germany) via force-displacement transducers (FT03, Grass Instrument, Ma, USA). To assess the involvement of Na+/Ca 2+ exchanger in the relaxant action of 1-O-BK, we investigated its effect on contractions of rat aortic rings induced by low-Na+ Krebs solution and ouabaine. The low-Na+ Krebs solution was obtained by replacing 118 mM. of NaCl with iso-osmolar amount of choline chloride. In these experiments after equilibration period in normal Krebs solution the viability of aortic rings was tested by KCl (50 mM.) or phenylephrine (1 ^M.). After this procedure, the aortic rings were repeatedly washed with normal Krebs solution, and when the baseline tension was re-established the aortic rings were exposed to low-Na+ Krebs solution containing 10 ^M. verapamil, and steady contraction was obtained. To test the effect of 1-O-BK on ouabaine-induced contraction, the aortic rings after incubation for 30 min. in normal Krebs solution, containing 10 ^M ofverapamil, were exposed to ouabaine. The concentration-response curves for 1-O-BK were obtained by incubation of aortic rings for 30 min. in Krebs solution with concentrations of the alkaloid increasing from 0.1 to 30 ^M., prior to exposure to low-Na+ Krebs solution or ouabaine. The functional state of the Na+/Ca 2+ exchanger, as well as its contribution to 1-O-BK-induced vasorelaxation, was verified by KB-R7943, a selective blocker of reverse mode of exchanger. KB-R7943 was dissolved in dimethyl Table 1. - Effect of KB-R7943 on low-Na+ and
sulfoxide; the final concentration of vehicle was no more than 0.1 % v/v, which did not affect aortic rings tension. All experiments were performed on endothelium-denuded aortic rings, the endothelium was removed by gently rubbing the lumen with a stainless steel rod and its absence was confirmed by the inability of acetylcholine (10 ^M.) to induce relaxation. We used acetylcholine, phenylephrine, ouabaine, verapamil, and KB-R7943. All reagents were of analytical grade and were obtained from Sigma Chemical Co (St Louis, Mo, USA). All values are expressed as mean ± standard error of mean (S. E.M.). Student's t test was used for unpaired variants. P < 0.05 was considered statistically significant.
Results and discussion
In order to examine the effect of 1-O-BK on the Na+/Ca 2+ exchanger function its action on the contractions of rat aortic rings induced by low-Na+ Krebs solution was studied first. The reduction of extracellular Na+ ([Na+]o ) is a common experimental procedure to test function of Na+/Ca 2+ exchanger, that exchanges Na+ and Ca2+ in either a Ca 2+ efflux or influx mode, depending on the net electrochemical gradients for Na+ and Ca 2+ [11, 421-427; 12, 617-635; 13, 486-493]. In our study the exposure of aortic rings to low-Na+ solution induced contraction, the rate of which corresponds to 53.4 ± 3.6 % of the contraction induced by 1 ^M. of PE, taken as 100 %. Since, during these experiments the VDCCs were blocked by verapamil, the low-Na+-induced contraction is most likely a result of the influx of Ca 2+ through Na+/Ca 2+ exchanger, as reducing the Na+ gradient across the membrane makes the exchanger operate in reverse mode [14, 167-173; 15, 35-42]. Indeed, as shown in Table 1, the contraction induced by low-Na+ was significantly attenuated in the presence of KB-R7943, a selective inhibitor of the reverse mode of the Na+/Ca 2+ exchanger [10, 519-529; 16, 555-563]. This effect of KB-R7943 was concentration-dependent, and maximal inhibition of low-Na+-induced contraction to 20.4 ± 4.2 % of the control, was obtained at 25 ^M. The half-maximal inhibitory concentration (IC50) for KB-R7943 calculated from concentration-response curves was 9.1 ^M. The marked inhibition of low-Na+ induced contraction by KB-R7943 indicated that this contraction is mainly due to elevation in [Ca 2+]in, resulting from Ca 2+ influx mediated by reverse mode of Na+/Ca2+ exchanger. ouabaine-induced contractions in rat aortic rings
Treatment KB-R7943 (yM) IC50 (HM)
5 10 15 20 25
Low-Na+ 55.2 ± 4.2 47.8 ± 4.1 39.2 ± 4.5 24.6 ± 4.3 20.4 ± 4.2 9.1
Ouabaine 65.9 ± 4.3 52.6 ± 4.2 37.6 ± 4.2 24.7 ± 3.8 18.4 ± 4.1 11.3
Note: The aortic rings were preincubated for 30 min. with KB-R7943 prior to exposure to low-Na+ solution or ouabaine (20yM). Values (mean ± S. E.M. n = 5-7) are expressed as a percentage of contraction induced by phenylephrine (1 yM), taken as 100 %. P < 0.05 vs. control.
To examine the effect of 1-O-BK on contraction induced by low-Na+ solution, the aortic rings were preincubated for 30 min. in normal Krebs solution with increasing concentrations of the alkaloid from 0.1 to 30 ^M. prior to their exposure to low-Na+ solution. As shown in Fig. 1a, 1-O-BK effectively inhibits the contraction of aortic rings induced by low-Na+ solution in concentration-dependent manner. The maximal reduction in low-Na+ induced contraction to 23.2 ± 4.1 % by 1-O-BK was obtained at 30 ^M., providing IC50 value of11.1 ^M. These results indicated that 1-O-BK also effectively inhibited the low Na+ induced contraction similarly to KB-R-7943, suggesting that this effect of the alkaloid may be due to blockage of Ca2+ influx through Na+/Ca 2+ exchanger. Indeed, analysis of obtained results showed that 1-O-BK and KB-R7943 at nearly equal concentrations (30 ^M. and 25 ^M., respectively) maximally reduced the low-Na+ induced contractions to almost a similar extent (76.8 % and 79.6 %, respectively).
Furthermore, the inhibitory potency (IC50 values) of 1-O-BK and KB-R7943 (11.1 ^M and 9.1 ^M, respectively) on low-Na+-induced contraction showed no significant difference either, suggesting similar action. To further verify similarities of action between 1-O-BK and KB-R7943, we compared the inhibitory effects produced by 1-O-BK alone and in combination with KB-R7943. The results summarized in Fig. 1b. demonstrate that the simultaneous incubation of aortic rings with KB-R7943 and 1-O-BK resulted in slight additional inhibition of low-Na+ induced contraction to 17.1 ± 4.4 %, which was not significantly different from that produced by KB-R7943 or 1-O-BK separately. These results indicated that the effects of 1-O-BK and KB-R7943 were not additive in their combined presence; the low-Na+ — induced contraction was decreased to a level not significantly greater than that elicited by each of these agents alone. These results again support the suggestion that
the inhibitory effect of 1-O-BK on low-Na+-induced contraction is due to blockage of the Ca 2+ influx via Na+/ Ca 2+ exchanger.
To further investigate the involvement ofNa+/Ca 2+ exchanger in the relaxant effect of 1-O-BK, we studied its action on aortic rings contraction induced by ouabaine. Ouabaine is a potent inhibitor of Na+, K+-ATPase, which induces aortic rings contraction associated with an increase of intracellular Na+ concentration ([Na+]i), which in its turn reverses the operating mode of Na+/Ca 2+ exchanger, thus promoting Ca 2+ influx [17, 87-94; 8, 129-149; 19, 1367-1387]. In our study in aortic rings ouabaine induced the concentration-dependent contraction with peak amplitude of 76.4 ± 3.4 %, obtained at 20 ^M., which corresponds to that induced by PE (1 ^M). The contraction induced by ouabaine was significantly attenuated by KB-R7943 which maximally reduced it to 18.4 ± 4.3 %, at 25 ^M. with IC50 value of11.3 ^M. (Table 1). The significant inhibition ofouabaine-induced contraction by KB-R7943 indicated that this contraction is mainly due to elevation in [Ca 2+] resulting from Ca 2+ influx mediated by reverse mode of Na+/Ca 2+ exchanger. Therefore, to further verify the involvement of Na+/ Ca 2+ exchanger in relaxant effect of 1-O-BK, we studied its action on aortic rings contraction induced by ouabaine. As shown in Fig. 2a, 1-O-BK also effectively and in concentration-dependent manner inhibited the contraction of aortic rings induced by ouabaine. In these experimental conditions 1-O-BK maximally reduced ouabaine-induced contraction to 22.3 ± 4.1 %, at 30 ^M., providing a IC50 value of 10.5 ^M. These results indicated that 1-O-BK also effectively inhibited the ouabaine — induced contractions similarly to that produced by KB-R-7943. A comparison of the effects of 1-O-BK and KB-R7943 showed that these drugs at nearly
equal concentration (30 ^M and 25 ^M, respectively) maximally reduced the ouabaine-induced contraction to almost a similar extent (77.7 ± 4.4 % and 81.6 ± 4.1 %, respectively) and with almost similar potency (IC50 values, 10.5 ^M and 11.3 ^M, respectively). These data suggested that 1-O-BK inhibited the ouabaine-induced contraction by mechanism similar to that of KB-R-7943, involving the blockage of Ca 2+ influx via Na+/ Ca 2+ exchanger. This suggestion was further confirmed when the inhibitory effect of 1-O-BK was compared to those produced by alkaloid alone and in combination with KB-R7943.
As illustrated in Fig. 2b, the combined application of maximally effective concentrations of KB-R7943 and 1-O-BK resulted in small additional inhibition of ouabaine-induced contraction to 16.6 ± 4.6, a reduction not significantly greater than that produced by 1-O-BK alone. These results indicated that the effects of 1-O-BK and KB-R7943 on ouabaine-induced contraction were not additive, suggesting that these agents probably were acting at a common site, i. e. via the Na+/Ca 2+ exchanger. Furthermore, the comparison of results showed that 1-O-BK inhibited the ouabaine- and low-Na+ induced contractions almost to the same extent (77.7 ± 4.4 % and 76.8 ± 4.1 %, respectively) and with nearly equal IC50 values (10.5 ^M. and 11.1 ^M, respectively). Thus, a similar inhibitory action of 1-O-BK on aortic rings contractions induced by two different procedures which induced Ca 2+ influx via Na+/ Ca 2+ exchanger indicates that these effects of the alkaloid are a result of direct blockage of Ca 2+ influx through this exchange mechanism. Taken together, the present results provide the clear evidence that 1-O-BK potently inhibited the Ca 2+ influx via Na+/ Ca 2+- exchanger, suggesting that this effect of the alkaloid also may be involved in its vasorelaxant activity.
Fig. 1. (a) Effect of 1-O-BK on low-Na+ induced contraction in rat aortic rings; (b) Effect of 1-O-BK (30 mM) on low-Na+ induced contraction alone and in combination with KB-R7943 (25 mM). Results (mean ± S. E.M. n = 5) are expressed as a percentage of contraction induced by low-Na+ solution, taken as 100 %. P < 0.05 vs. control
Fig. 2. (a) Effect of 1 -0-BK on ouabaine-induced contraction in rat aortic rigs; (b) Effect of 1-O-BK (30 mM) on ouabaine-induced contraction alone and in combination with KB-R7943 (25 mM). Results (mean ± s. e.m.) are expressed as a percentage of contraction induced by ouabaine (20 mM), taken as 100 %. P < 0.05 vs. control
Particular structure of fruits and seeds ephemers in the arid zone of Central Asia
Conclusion
In this work, the possible role of the Na+/Ca 2+ exchanger in the vasorelaxation produced by 1-O-BK, a diterpenoid alkaloid, in rat aortic rings was elucidated for the first time. The present results support the idea that the 1-O-BK may relax vascular smooth muscle by a mechanism related to a decrease in [Ca 2+]i not only by inhibiting Ca 2+-influx through VDCCs or ROCCs, but also by blocking Ca 2+-entry via Na+/Ca 2+ exchanger. The finding that 1-O-BK
exhibits significant potency to block Ca 2+-influx via Na+/Ca 2+ exchanger may be important under pathological conditions where the exchanger, operating in reverse mode, induced Ca 2+ overload and, hence, may exacerbate overall vasoconstriction. Taken together, these data suggest that 1-O-BK is a promising compound for further development of novel approaches in the treatment of conditions associated with vascular smooth muscle disorders, such as hypertension or ischemia.
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Tursinbaeva Gulbakhor Sultanovna, Pedagogical State University name Nizami, Uzbekistan, Tashkent
Butnik Antonina Anatolievna, Institute of Gene Pool of Plants and Animals, Academy of Sciences
E-mail: [email protected]
Particular structure of fruits and seeds ephemers in the arid zone of Central Asia
Abstract: We describe the morphology and structure of the integuments of fruits and seeds 23 species from 18 genera, 5 families of ephemers from the most common (dominant) in the Kyzylkum desert. The next adaptive features were allocated: sclerification, slimy structure, water-carring tissue which promote the conservation of species in arid conditions. That was a negative correlation between the complexity of the pericarp and seed-coat structure. Keywords: morphology, anatomy, adaptive sign, diaspore, Kyzylkum.
