An increase in the intracellular (cytoplasmic) con- centration of calcium ions is the key event in triggering the contraction of vascular smooth muscles. This.
Doklady Biological Sciences, Vol. 398, 2004, pp. 357–359. Translated from Doklady Akademii Nauk, Vol. 398, No. 2, 2004, pp. 272–274. Original Russian Text Copyright © 2004 by Sobol’, Nesterov.
PHYSIOLOGY
Dual Effect of Inorganic Calcium-Channel Blockers on Contraction of Smooth Muscles of the Frog K. V. Sobol’ and V. P. Nesterov Presented by Academician V.L. Sviderskii March 19, 2004 Received March 19, 2003
An increase in the intracellular (cytoplasmic) concentration of calcium ions is the key event in triggering the contraction of vascular smooth muscles. This increase may be due to the mobilization of these ions from intracellular stores and/or calcium influx into the cell from the environment (predominantly through the calcium-dependent plasma membrane channels). When contraction is induced by an increase in the concentration of potassium ions in the environment, mobilization of calcium ions from the intracellular stores is not observed, and calcium enters into the cell through the plasma membrane voltage-gated calcium channels, which open in response to membrane depolarization [1–3]. Calcium channels may be effectively blocked both by organic and inorganic blockers [4]. Organic blockers of calcium channels are widely used in clinical practice as antiarrhythmic and vasodilating agents. In contrast to organic blockers, inorganic agents can effectively block both voltage-gated and ligandoperated calcium channels [4, 5]. Heavy metal ions (La3+, Cd2+, Co2+) are known as effective inorganic blockers of calcium channels [5, 6]. However, some studies showed that heavy metal ions themselves may induce contraction of vessels [7−9]. Furthermore, heavy metal ions may substitute for calcium and stimulate proliferation of smooth muscle cells, thereby being involved in the pathogenesis of some diseases, such as atherosclerosis and hypertension [10]. The goal of this work was to study the effects of inorganic calcium channel blockers, La3+ and Co2+, on frog vessel contraction induced by an increase in potassium concentration in the environment. It was found that addition of La3+ or Co2+ blocks the contraction of frog subclavian vein segment. However, a combination of these ions at certain concentrations may induce a substantial vessel contraction.
Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, pr. Morisa Toreza 44, St. Petersburg, 194223 Russia
MATERIALS AND METHODS Experiments were performed with isolated ring segments of subclavian vein of the frog Rana temporaria (diameter, 0.5–0.8 mm; excised segment width, approximately 1 mm). Steel rings made of mandrin (total weight, 1 mg) were inserted in isolated ring segments and attached to a strain indicator (sensitivity, 5.7 mV/mH) [11] at one side and a fixed lever on the other side. Samples were stretched to the optimal length and left under a passive load of 2 mH for 60 min, which gradually dropped to 0.1–0.3 mH. After passive stretching, three control isometric contractions induced by high concentration of KCl (110 mM) were performed. Contractions were recorded on an Endim 622.01 plotter. Physiological solution contained 110 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 0.75 mM NaH2PO4, and 0.5 mM NaHCO3 (pH 7.4). Solution with high concentration of KCl (110 mM) was prepared by equimolar substitution of 107.5 mM NaCl with KCl. Calcium-free physiological solution contained 1 mM K2EGTA instead of CaCl2. All experiments were performed at 21°C in a temperature-controlled cuvette. RESULTS AND DISCUSSION An increase in potassium concentration in the environment induced depolarization of smooth muscle membranes and Ca2+ influx through voltage-gated calcium channels. This led to an increase in intracellular Ca2+ concentration, which eventually resulted in blood vessel contraction (Fig. 1). Contraction induced by potassium ions in calcium-free solution was completely suppressed (Fig. 1), which was indicative of its dependence on extracellular calcium. The blockade of calcium influx through calcium channels by inorganic calcium antagonists also suppressed potassium-induced contraction. Figure 2 shows partial blockade of potassium-induced contraction by 2 mM La3+. A similar effect was observed when potassium-induced contraction was blocked by 1 and 2 mM Co2+ (Fig. 3). These findings are consistent with the common notion on the blocking effect of
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SOBOL’, NESTEROV 0.5 mH 2 min 20 min
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0 Ca2+ 110 KCl
110 KCl
Fig. 1. Contraction developed by frog vein in a hyperpotassium solution (110 mM) in the presence and absence of Ca2+. Before recording contraction in a calcium-free hyperpotassium solution, samples were incubated for 35 min in calcium-free physiological solution supplemented by 1 mM EGTA. Arrows and time show corresponding interruptions in recording response of samples to save place on the plot.
0.5 mH 2 min
calcium antagonists on calcium channels in smooth muscle cells. However, a combination of these ions at certain concentrations may have an opposite effect—initiate vessel contraction. When potassium-induced contraction was partly blocked by Co2+ (Fig. 3), subsequent blockade of contraction with increasing La3+ concentrations, starting with a certain concentration, caused a strong contraction. In our experiments, the concentration of La3+ that induced contraction against the background of partial channel blockade with 2mM Co2+, was also 2 mM. This effect was observed in four independent experiments. Note that, in the absence of Co2+, 2 mM La3+ partly blocked potassium-induced contraction (Fig. 2). Another characteristic feature of contraction that was induced by the combination of inorganic blockers was that, after washing vessel, the amplitude of subsequent contractions induced both by high potassium concentration (110 mM) and noradrenalin (0.01 mM) decreased by 60–90%. However, after 2 h of incubation in normal physiological solution, the ability of vessel segment to normally respond both to an increase in external potassium concentration and noradrenalin was restored. It should be also noted that the nature of contraction induced by a combination of inorganic blockers in our case is not quite clear.
110 KCl 1
2 La3+
Fig. 2. Contraction developed by frog vein in a hyperpotassium solution (110 mM) in the presence of La3+. The blocker was added during potassium-induced contraction at concentrations 1 and 2 mM.
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110 KCl 1
2 1
Co2+ 2 La3+
Fig. 3. Contraction developed by frog vein in a hyperpotassium solution (110 mM) in the presence of Co2+ and La3+. Additions were made in the following order: first, 1 and 2 mM Co2+; then, 1 and 2 mM La3+.
For example, Gallagher et al. accounted for the nature of rabbit aorta segment contraction induced by addition of Co2+ to the solution by the fact that Co2+ activates voltage-gated and ligand-operated calcium channels in the plasma membrane, thereby increasing the concentration of Ca2+ in smooth muscle cells [8]. However, in our case, subsequent reversible decrease in the contractile response of the vessel to an increase in external potassium concentration possibly indicates that Co2+ and/or La3+ themselves may penetrate across the plasma membrane, thereby suppressing vasoactive response of the vessel. This assumption is corroborated by the fact that heavy metal ions may substitute for Ca2+ and stimulate proliferation of smooth muscle cells [10]. Thus, we found that La3+ or Co2+ block the contraction of frog subclavian vein segment induced by an increase in external potassium concentration. However, a combination of these ions at certain concentrations may induce substantial vessel contraction, the nature of which is poorly understood. The results of this study indicate that a combination of inorganic potassium channel blockers at concentrations greater than threshold ones may have an opposite effect (expressed in potentiation rather than suppression of contraction), thus being involved in pathogenesis of some diseases (e.g., hypertension). DOKLADY BIOLOGICAL SCIENCES
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DUAL EFFECT OF INORGANIC CALCIUM-CHANNEL BLOCKERS
ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research (project no. 03-04-49495). REFERENCES 1. Loutzenhiser, R. and Epstein, M., Eur. J. Pharmacol., 1984, vol. 106, pp. 47–52. 2. Raeburn, D., Roberts, J.A., Rodger, I.W., and Thomson, N.C., Eur. J. Pharmacol., 1986, vol. 121, pp. 251–255. 3. Wali, F.A., Greenidge, E., and Tugwell, A.C., Acta Physiol. Acad. Sci. Hung., 1988, vol. 72, pp. 115–121. 4. Gouw, M.A., Wilffert, B., Wermelskirchen, D., and van Zwieten, P.A., Pharmacology, 1990, vol. 40, pp. 277–287.
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5. Ruegg, U.T., Wallnofer, A., Weir, S., and Cauvin, C., J. Cardiovasc. Pharmacol., 1989, pp. S49–S58. 6. Van Renterghem, C. and Lazdunski, M., Pflügers Arch., 1994, vol. 429, pp. 1–6. 7. Bakos, M. and Rubanyi, G., Acta Physiol. Acad. Sci. Hung., 1982, vol. 59, pp. 175–180. 8. Gallagher, M.J., Alade, P.I., Dominiczak, A.F., and Bohr, D.F., J. Cardiovasc. Pharmacol., 1994, vol. 24, pp. 293–297. 9. Evans, D.H. and Weingarten, K., Toxicology, 1990, vol. 61, pp. 275–281. 10. Lu, K.P., Zhao, S.H., and Wang, D.S., Sci. China, B, 1990, vol. 33, pp. 303–310. 11. Sobol’, K.V., Fiziol. Zh., 1991, vol. 77, no. 7, pp. 108–111.
