Aluminum chloride effect on ca2+,Mg2+-AtPase Activity And dynAmic PArAmeters of skeletAl muscle contrAction

We studied enzymatic activity and measured strain-gauge contraction properties of the frog rana temporaria m. tibialis anterior muscle fascicles during the action of aluminum chloride solution. It was shown that Alcl3 solutions did not affect the dynamic properties of skeletal muscle preparation in concentrations less than 10-4 M. Increasing the concentration of Alcl3 to 10-2 M induce complete inhibition of muscle contraction. A linear correlation between decrease in ca2+,Mg2+-ATPase activity of sarcoplasmic reticulum and the investigated concentrations range of aluminum chloride was observed. The reduction in the dynamic contraction performance and the decrease ca2+,Mg2+-ATPase activity of the sarcoplasmic reticulum under the effect of the investigated Alcl3 solution were minimal in pre-tetanus period of contraction.

We studied enzymatic activity and measured strain-gauge contraction properties of the frog rana temporaria m. tibialis anterior muscle fascicles during the action of aluminum chloride solution. It was shown that Alcl 3  A luminum enters the organism with drinking water and has much higher bioavailability than that from other sources; it is also consumed with vegetable food [1]. There is a plethora of data on toxicity of aluminum and other elements for a living organism [2][3][4]. Pathologies associated with increased aluminum levels in human organism include heart rhythm disorders resulted from its accumulation in the heart muscle. It has been established that aluminum causes specific physiological and biochemical changes in organism of humans and animals, namely disorders of the central nervous system, changes in functional state and development of bone tissue, membrane permeabilization and channel conductance [5].
Aluminum toxicity is attributed to its ability to change concentrations and balance of other ions, e.g. by supplanting other metals, mostly bivalent, from certain enzymes and metalloproteins. It has been established that aluminum may replace magnesium in active sites of enzymes such as phosphodiesterases, acid and alkaline phosphatases. Aluminum was demonstrated to enter myocytes and inhibit Ca 2+ release from sarcoplasmic reticulum (SR) [1,3].
The impact of aluminum on muscular system remains poorly understood. There is limited available data, which is primarily of descriptive nature. It has been established that aluminum may inhibit contractile function. The published data gives ground to the assumption that aluminum may affect both neuro-muscular transmission and contractile apparatus itself [6]. Consequently, understanding the effect of aluminum on muscular contraction mechanics may allow better understanding of the mechanisms of action of this metal and the possibilities of its clinical application.
The effects of biologically active substances on terminal changes in power output of the muscle are currently under active investigations. Nevertheless, the dynamics of this process remains largely unstudied . The onset of the equilibrium stable state of contraction under the effect of biologically active substances may vary within wide margins depending not only on the concentrations of the reagents used [7][8][9], but also on duration of the experiment [10,11]. This fact complicates the interpretation of the obtained experimental data and may result in severe errors in research planning. Accordingly, an im-doi: http://dx.doi.org/10.15407/ubj87.05.038 portant emphasis in our work was made on temporal changes in achievement of equilibrium stable state of contraction under the effect of the investigated compounds. The aim of the study was to investigate the effect of aluminum chloride solutions on Ca 2+ ,Mg 2+ -ATPase activity of sarcoplasmic reticulum and muscular contraction dynamics of isolated fascicles induced by electrostimulation.

materials and methods
The experiments were conducted on m. tibialis anterior fascicles of rana temporaria frog. We determined contractile force, change in length and stimulating signal parameters. The experiments were conducted in closed circuit Ringer solution with relaxation period of 3 min. A strain-gauge device was used to determine contractile forces of skeletal muscle fiber bundles [12].
Incubation medium (1.9 ml) was prepared with the following concentrations in final volume: imidazole -50 mM, KCl -100 mM, MgCl 2 -3.5 mM, NaN 3 -5 mM, EDTA -3 mM, sodium oxalate -2 mM, ATP -3 mM. To this end, we took 0. Test tubes were bathed to 37 °C, and the reaction was started by addition of 0.1 ml of protein (1 mg/ml). The tubes were incubated for 20 min.; the reaction was then stopped by addition of 1.5 ml of cold 10% trichloroacetic acid.
We used the following reagents: reagent 1 -10% ascorbic acid, freshly prepared; reagent 2 -0.42% ammonium molybdate in 1 N solution of H 2 SO 4 ; 3 -1 ml of reagent 1 and 6 ml of reagent 2; 40 µg/ml solution of KH 2 PO4. To measure inorganic phosphate produced as a result of enzyme activity, we placed 0.9 ml of supernatant (as a source of P i ) in a test tube and added 2.1 ml of reagent 3. The mix was incubated for 30 min at 37 °C, and optical density determined at λ = 820 nm.
To facilitate the description and adequate analysis of the results, we attributed various stages of the dynamic response of the muscle to different temporal regions, which correspond to various stages of contractile process. The force response and changes in length were attributed to stages ( Fig. 1): F 1 -initiation of the force response of the muscle; F 2 -the muscle force productivity enters a steady level of contraction without a noticeable trend towards either end; F 3 -terminal muscle activity; L 1 -initiation of changes in muscle length; L 2 -the length of the muscle enters a steady level of contraction; L 3 -terminal changes in muscle length, was not analyzed due to noticeable fluctuations even after stimulation ceased. This may be attributed to transitions in rigid composition of muscle fibers caused by abrupt changes in fiber elasticity, which in turn depends on momentary discontinuance in stimulating signal. Registration and adequate analysis of these processes were very complicated. Thus we used the first two, L 1 and L 2 , to analyze length change curves in these test series.
In order to establish the margins of concentrations within which the experimental substances display physiological effects influencing dynamic properties of muscle contractions, we investigated concentrations from 10 -8 to 10 -4 M. As a result, we demonstrated, that AlCl 3 solutions in concentrations of less than 10 -4 M did not affect performance of skeletal-muscle preparations. As concentrations increased to 10 -2 M the muscle contractile processes were totally suppressed. Consequently, we used AlCl 3 solutions with concentrations of 10 -4 to 10 -2 M.
The experiments were done in accordance with guidelines for keeping and work with laboratory animals laid down in the 'European convention for the protection of vertebrate animals used for experimental and other scientific purposes' (Strasbourg, 1986).
The statistical analysis of data was done with variation statistics methods in Origin 7.0 software, using Student's t-test. The differences between test and control samples were considered significant at P ≤ 0.05.

results and discussion
The experiments using 10 -4 M AlCl 3 demonstrated that muscle contractile force reached steady levels at 4 th min of observations during F 1 and was at 99% of control values. It was in F 2 and F 3 on the 12 th min at 96.6 and 97.7%, accordingly (Fig. 2, a).
The inhibition of changes in length of muscle fibers entered steady level on the 10 th min during L 1 in these experimental conditions reaching 93.7% of control, and at the 8 th min during L 2 reaching 95% of control. A decrease of dynamic characteristics of muscle contraction under the effect of 10 -4 M aluminum chloride solution was of linear nature. The results of exposing muscle fiber bundles to 10 -3 M aluminum chloride solution (Fig. 2, b) demons trate a statistically significant reduction in muscle contraction parameters during F 2 , F 3 , L 1 stages.
The maximum decrease in muscle's contractile force was observed after the 10 th min during F 1 and was at 92.6% of control values. The maximum decrease in muscle's contractile force during F 2 was at the 12 th min and constituted 71.2% from that of control values . The steady level of contraction during F 3 was at the 14 th min and was at 71.2% from that of the corresponding control values.
The maximum reduction in contraction of muscle fibers was at the 12 th min of the experiment during L 1 and L 2 , and constituted 69.1 and 73% of the corresponding control values. The value of changes in muscle fiber length during L 1 was in all instances smaller than that during L 2 .
We observed drastic decrease in dynamic properties of contractions in the experiments where 10 -2 M solutions of AlCl 3 were used (Fig. 2, c).
The maximum reduction in muscle contractile force was after the 6 th min of stimulation dur-ing F 1 , F 2 , and F 3 , and was at 53, 35.6 and 33.9% of correspo ding control values.
The maximum inhibition muscle fiber contraction was found after the 6 th min of the experiment during L 1 and L 2 , and reached zero value. The results of these experiment show the significant linear decrease in Ca 2+ ,Mg 2+ -ATPase activity of sarcoplasmic reticulum (SR) as a result of the effects of all the mentioned concentrations of AlCl 3 (Table 1).
The AlCl 3 solution with concentration of 1.4·10 -4 M caused maximum decrease in muscle contraction force at the 8 th min of the experiment during F 1 and F 2 and constituted 98.4 ± 0.8% and 94.2 ± 1.7%, accordingly. The steady level during F 3 was observed at the 12 th min and was at 93% of control value. The curves dependence of muscular contraction force on duration of exposition to the aluminum chloride was of linear nature, both pa-експериментальні роботи Experiments with 2·10 -4 M solution of aluminum chloride (Fig. 3, b) demonstrated that the maximum reduction in muscle contractile force was at the 10 th min during F 1 and constituted 97.1% of control value. The most profound decrease in muscle contractile force during F 2 and F 3 was correspondingly at the 8 th and 6 th min, and reached 94.1 and 92.3% of control, yet these changes were not statistically significant. The changes in dynamic properties of muscle contraction during these periods were of irregular nature.
We found no significant changes in muscle fibers length under the effect of 2·10 -4 M solution of aluminum chloride in comparison to the effect of 1.4·10 -4 M AlCl 3 solution (Fig. 3, a and b). We detected statistically insignificant decrease in muscle contraction properties in all investigated stages in experiments with 3.3·10 -4 M AlCl 3 solutions (Fig. 3, c). The maximal reduction of muscle contractile force was found at the 8 th min during F 1 and was of 96.2% of control. The most notable decrease in muscle contractile force during F 2 and F 3 was on 12 th and 10 th min, accordingly, and constituted 92.5 and 85.3% of that of control. The most profound decrease in muscle contraction was during the 10 th min in L 1 and L 2 , and was correspondingly of 86.1 and 87.1% of control values.
We found decreased force and changes in muscle fiber length in all studied cases in experiments with 5·10 -4 M aluminum chloride solution (Fig. 3, d).
The maximum in reduction of contractile force was observed at the 10 th min during F 1 and was of 96.1% of that of control. The most noticeable decrease in muscle contractile force during F 2 and F 3 was at the 14 th and 12 th min, accordingly, and was of 90.9% and 85.8% of that of control. The maximum decrease in the length of muscle contraction was at the 14 th min in L 1 and L 2 and constituted correspondingly 88.6% and 89% from that of control values.
Aluminum chloride in concentration of 6.6·10 -4 M caused a decrease in dynamic properties of contraction (Fig. 3, e). The maximum decrease in muscle contractile force was at the 14 th min of the experiment during F 1 and constituted 94.2% of that of control, and at the 12 th min during F 2 and F 3 , and was accordingly 87 and 82.6%. The dependence of muscle contraction force on the duration of exposition to the effector during F 1 , F 2 and F 3 was of linear nature. The maximal statistically significant reduction in muscle fibers contraction was detected at the 14 th min during stages L 1 and L 2 and constituted 80.2 and 83.7% of the corresponding control values.
There was a linear decrease in Ca 2+ ,Mg 2+ -ATPase activity of SR as a result of the effects of AlCl 3 ( Table 2).
Washing of muscle samples with Ringer solution caused restoration of the dynamic properties of contraction to their starting levels in all experimental concentrations of AlCl 3 solutions. The duration of the restorative process up to control values depended linearly on the duration of the effector exposition. The time period of washing increased linearly with the concentration of aluminum chloride.
The experimental data show inhibition of Ca 2+ ,Mg 2+ -ATPase activity of SR, with linear dependence on the concentration of AlCl 3 . The demons trated inhibition of Ca 2+ ,Mg 2+ -ATPase activity of SR corroborates imbalance in intracellular Ca 2+ concentrations under the effect of aluminum that has been found by others [3]. The reduction of Ca 2+ ,Mg 2+ -ATPase activity probably results from compromised SR membrane integrity due to activation of lipid peroxidation [1,3,18].
We found, in accordance with the obtained results, the irregularities in the effect of aluminum chloride in the investigated concentrations (10 -4 to 10 -2 M) on changes in force response and muscle fiber length and inhibiting properties of this compound in concentrations of more than 10 -4 M. These processes may be attributed to the ability of aluminum ions to permeate sarcolemma [3]. It may be supposed that aluminum ions may affect muscle performance at a level of actin-myosin interaction. The ions of this metal can supposedly supplant magnesium ions in ATP [3,19]. It is possible that aluminum ions modulate actin-myosin interaction and change the functional properties of actin-myosin complexes of the muscle. There is data of dose-dependent reduction in myosin ATPase activity under the effect of aluminum ions [14]. It has been demonstrated that aluminum ions in concentration of 5 mM inhibited myosin ATPase from heart muscle to half of its maximum level. The inhibition was observed also at concentrations over 5 mM for myosin ATPase from smooth muscle cells [15]. Aluminum ions were  shown to exert a notable influence on structural changes in actin-myosin complex during ATP hydrolysis. A decrease in rate of superprecipitation of actin-myosin complex has been found in the presence of aluminum in concentrations of 10 -4 to 10 -3 M [16]. This process was totally suppressed in case of aluminum concentration of 10 -2 M.