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1212 ТепловUDC 612.741.001.6 © A.Yu.Teplov, S.N.Grishin, A.M.Farkhutdinov, 2010 Possible mechanisms of change contraction of function of isolated striated muscles of the mouse at allergic reorganization of the organism A.Y. Teplov, S.N. Grishin, A.M. Farkhutdinov Kazan State Medical University, Kazan, Russia Teplov A.Yu., Grishin S.N., Farkhutdinov A.M. Possible mechanisms of contractile function change in isolated murine striated muscles at allergic body change // Preventive and clinical medicine. – 2010. - № 1 (34). – Р.105-110. State Educational Establishment for Higher Professional Training «Kazan State Medical University of the Federal Agency for Public Health and Social Development of the Russian Federation». Russia, 420012, Kazan, Butlerova str., 49, tel.: 8(843)236-05-63, e-mail: pathphiz.kgmu@gmail.com or AlikTeplov@mail.ru Summary: The study observes contraction function of striated mouse muscles at protein sensibility – m. diaphragm, m. extensor digitorum longus, m. soleus. It is shown that protein sensibility changes contractile force of all researched muscles. Thece changes differ for muscles with different fenotipe. Key words: skeletal muscle, contraction characteristics, m. extensor digitorum longus, m. soleus, m. diaphragma, non-quantal secretion, ovalbumin sensabilization. Well-known, that airway muscles functional state and, primarily, diaphragm, is essentially changed in asthma, the disease of allergic origin. We have previously studied the influence of ovalbumin sensabilization on contraction function of guinea-pig isolated diaphragm strip. To be more concrete, its ability to change its contractile functions by increase of force and shortening velocity on carbacholine (CCh) in terms of ovalbumin sensabilization was shown. To explain this fact, changes of electrogenic features as well as histochemical profile of muscle fibers were proposed. Furthermore, as the contraction of isolated muscle was initiated by agonist we can suggest the participation of surface membrane cholinergic receptors activation mechanisms in the change of muscle fibers functional features at ovalbumin sensabilization. Diaphragm is a mixed muscle; it consists of “fast” and “slow” muscle fibers (MF). Recently we showed that ovalbumin sensabilization changes contraction functions of isolated – “fast” and “slow” – shank mouse skeletal muscles. Dynamics of contraction function of studied skeletal muscles shows remarkable differences. For clarity, cholinemediated processes of excitement of postsynaptic membrane play an important role in the mechanisms of contraction force change. The ability of mouse diaphragm to change its functions in terms of allergic restructure and its possible mechanisms remain to be determined. The purpose of the present study is to reveal cholinemediated mechanisms in pathogenesis influence of ovalbumin sensabilization on contraction functions of mouse isolated muscles: “mixed” – diaphragm strip (m. Diaphragma); “fast” - m. extensor digitorum longus (m. EDL) and "slow" – m. Soleus. The complex researches were performed to investigate this problem. Two experimental models which are characteristic for cholinemediated processes of excitement of isolated mouse striated muscles were used to study the effects of ovalbumin sensabilization on: 1) the rates of contraction muscles response, caused by agonist CCh and 2) the level of non-quantal secretion of acetylcholine (ACh) in a zone of a trailer plate. Materials and methods. Experiments were conducted on both sexes mice weighing 17-22 g. Animals were twice ovalbumin sensabilitized (OS) with gel hydrate aluminum (2 mkg of dry gel substance + 150 mkg ОА in 0,5 ml of a physiological solution) parenteral. The second injection was made in 14 days after the first one. Animals were got into experiment on a pique of sensabilization – 7-10 days after the second sensabilization injections. Mechanomiographic researches were conducted on a preparation of an isolated muscle in terms of an isometry. Skeletal muscle was stretched during 20 minutes with force of 500 mg at a constant perfusion by a solution Krebs type to maintain isometry and temperature at 20-21 C. Contraction was recorded by the photoelectric converter. Agonist CCh was investigated at submaximal concentration: for m. EDL – 7х10-4М, a m. Diaphragm strip - 2х10-4М, m. Soleus – 5х10-4М. Contraction function was analyzed according muscles contraction parameters on CCh. Muscle contraction force (Poc) and speed (Vос) were estimated. Contraction force developed by isolated muscle was related to its mass (m) (Poc* - numerically equal volume of a muscular preparation) to get objective information at the force characteristics analysis. To study a condition of muscle fiber postsynaptic membrane in the zone of a trailer plate non quantum secretion of Ach was studied. It was measured by glass microelectrodes (with the resistance of 8-12 MΩ, filled with 2,5 M KCl). To determine its size armin action acetylcholinesteraze, then on a muscle was eliminated during 8-12 minutes application m-cholinergic receptors blockader d-tubocurarine (d-TC) (10-5М). The rates difference of membrane potential before and after application d-TC corresponds to the rate of not quantum secretion of ACh (Н-effect). The results were analyzed by comparing data from experiments on intact muscles (control) and sensibility animals (experiment). Statistical analyzes was performed by Student's t test. Results. Biometric parameters of the studied skeletal muscles are represented in Table 1. Contraction parameters of isolated m. EDL, a m. Diaphragm strips and mouse m. Soleus on CCh at submaximal concentration in the control and at ovalbumin sensabilization are represented in Tables 2, 3 and 4. Table 1 Biometric parameters of “fast”, “slow” and “mixed” mouse muscle preparation in control and at ovalbumin sensabilization (experiment)
For "fast" muscle it is shown that CCh at submaximal concentration caused contraction of with force of 76,59±6,51 mg and speed of 14,3±1,6 mg/s. m. EDL contraction force decreased, speed practically did not change at ovalbumin sensabilization (Table 2). Table 2 Parameters of isolated m. extensor digitorum longus contraction (X±Sx) on CCh (7х10-4 М) in control and at ovalbumin sensabilization (experiment)
Note: * - р<0,05; ** - р<0,01 ; *** - р<0,001 Study of non quantum secretion of ACh in "fast" mouse muscle fiber has shown the following data. Initial rate of membrane potential in the terms of rest was 72,3±0,6 мВ (n=150). But at presence of d-TC it increased up to 77,4±1,6 мВ (n=150). Thus, the Н-effect in the terms of control makes 5,1±0,4 мВ (n=150). In the terms of ovalbumin sensabilization initial rate of membrane potential of the rest was 73,9±0,5 мВ (n=150). At presence of d-TC it increased up to 79,7±1,7 мВ (n=150). It means that the Н-effect value has increased, making in the described terms of experiment 5,8±0,5 мВ (n=150, р <0,05). In the "mixed" on intact muscle mouse CCh at submaximal concentration caused contraction with force of 342,8+-18,54 mg and speed 31,0+-1,7 mg/s. Ovalbumin sensabilization resulted in force increase and speed increase - 32,08+-0,89 mg/s of slow muscle contraction (Table 3). Table 3 Parameters of isolated strip of diaphragmic muscle contraction (X±Sx) on CCh (2х10-4 М) in control and at ovalbumin sensabilization (experiment)
Note: * - р<0,05; ** - р<0,01 ; *** - р<0,001 Study of non quantum secretion of ACh of "mixed" muscle has shown: initial rate of membrane potential of the rest was 70,7±1,9 мВ (n=150). At presence of d-TC it increased up to 75,9±0,7 мВ (n=150). Thus, Н-effect in the control makes 5,2±0,4 мВ (n=150). Initial rate of membrane potential of the rest was 70,0±1,5 мВ (n=150). At ovalbumin sensabilization at the presence of d-TC it increased up to 74,4±0,6 мВ (n=150). Tit means that the Н-effect value decreased, making in the described terms of experiment 4,4±0,5 мВ (n=150, р <0,05). At "slow" intact mouse muscle CCh at submaximal concentration caused contraction with the force of 237,77±20,61 mg and speed of 13,1±1,0 mg/s. Ovalbumin sensabilization resulted in force and speed increase (Table 4). Table 4 Parameters of isolated mouse m. soleus contraction (X±Sx) on CCh (5х10-4 М) in control and at ovalbumin sensabilization (experiment)
Note: * - р<0,05; ** - р<0,01 ; *** - р<0,001 Study of non quantum secretion of ACh has shown: initial rate of membrane potential of rest was 70,9±1,7 мВ (n=160). But at presence of d-TC it increased up to 75,9±1,3 мВ (n=160). Thus, Н-effect in the control makes 5,0±0,7 мВ (n=160). Initial rate of membrane potential of rest making 69,4±0,9 мВ (n=150) at ovalbumin sensabilization at presence of d-TC increased up to 72,5±1,0 мВ (n=150). It means that the Н-effect value decreased making in the described conditions of experiment 3,1±0,6 мВ (n=150, р <0,05). Discussion. Results of studies indicate that ovalbumin sensabilization changes contraction function of diaphragm strip as well as isolated shank mouse skeletal muscles (Table 2-4). To clarify, nature of these changes is essentially different for “fast” and “slow” muscles. Basic differences of morph-functional status of studied objects and its change mechanisms in the process of allergic restructure of an organism can explain this fact. Presumably, the fiber structure defines the differences in contractile force of observed muscles of nonsensibility animals on CCh. Soleus mouse muscle contains 50-60% of “slow” filaments, m.EDL – 97-100% of “fast” filaments. Mouse diaphragm, that takes media position, contains 88.6% of fast myosin. Presumably, force differences result from different level of muscle fibers sensibility to CCh that suggests direct dependence on the area of synapse. It is known that the size of a trailer plate of “slow” muscle fiber of soleus mouse muscle is 3 lengths than that of “fast” muscle fiber (m.EDL). Considering similarity of biometric parameters (length and mass, Table 1) of observed muscles, more sensibility to cholinomediate, caused by greater number of cholinergic receptors in the area of synapse, must result in more contractile force of m. soleus and diaphragm on CCh. Changes of diaphragmatic muscle functional features in terms of ovalbumin sensabilization suggest that changes in muscle fiber during allergic restructure of an organism are complex. Changes occurring in muscle fiber during sensabilization may affect surface membrane, electromechanical connected mechanisms or contractile protein system. The absence of corresponding changes of shortening velocity on sub maximal concentration of agonist of all the muscles doesn’t suggest electromechanical connected system changes. However, various of force change vector of “fast” muscle from the one side and “mixed” and “slow” muscles from the other side indicate principle difference of change mechanisms of muscles` functional features at ovalbumin sensabilization. While contractile force (Poc*) of “fast” muscle decreases (Table 2), contractile force of “slow” and “mixed” muscles increases (Table 3, 4). This dynamics confirms the fact that differences of “fast” muscle from the one side and “mixed” and “slow” muscles form the other side at ovalbumin sensabilization affect, primarily, cholinemediate processes of excitement of muscle fibers manifold effect. Evidence that ovalbumin sensabilization is able to affect the mechanisms of excitement of postsynaptic membrane of different muscles in different way comes from comparing muscle contractile force dynamics on CCh with level change of non-quantal secretion in the zone of a trailer plate. Force change vector of observed muscles at ovalbumin sensabilization correlates with level change of non-quantal secretion of Ach in the zone of a trailer plate (H-effect) (Figure 1A, 1B) F  igure 1. Ovalbumin sensabilization effect on: A) contraction force of isolated mouse m.EDL caused by CCh (7х10-4 М). Б) H-effect value We may conclude that the decrease of contractile force of “fast” muscle on CCh results from the decrease of postsynapse sensibility to cholinomediate. The evidence of this fact comes from the increasing H-effect (Fig. 1A, Б). Increase of non-quantal secretion of Ach in the zone of synapse contributes intensification of desensabilization mechanisms of cholinergic receptors of postsynaptic membrane. Correspondingly, we observe reverse picture about “mixed” and “slow” muscles. Increase of contractile force on CCh results from increase of postsynapse sensibility to CCh. Decreasing H-e  ffect reflects this process (Fig. 2A, Б, 3А, Б). Fig. 2 Ovalbumin sensabilization effect on: A) contraction force of isolated mouse m.soleus caused by CCh (5х10-4 М). Б) H-effect value  Fig. 3 Ovalbumin sensabilization effect on: A) contraction force of isolated mouse diaphragm strip caused by CCh (2х10-4 М). Б) H-effect value Conclusion. Thus, allergic restructure of an organism causes changes of contraction function of isolated mouse skeletal muscles. Contractile force (Poc*) on CCh of “fast” muscle decreases and, correspondingly, of “mixed” and “slow” muscles increases. This rate change occurs from the following. Decrease of contractile force of “fast” muscle (m.EDL) results from decrease of postsynaptic membrane sensibility to CCh that is caused by increasing of non-quantal secretion of Ach in the zone of a trailer plate. “Mixed” and “slow” muscles show reverse dynamics of contractile force as well as of non-quantal secretion of ACh. Increase of contractile force of these muscles on CCh at ovalbumin sensabilization results from increasing postsynaptic membrane sensibility to cholinomediate that is caused by decrease of non-quantal secretion of Ach in the zone of a trailer plate. Different changes of contraction function of skeletal muscles at ovalbumin sensabilization are caused, primarily, by dynamics of cholinomediated processes of membrane excitement of muscle fibers. References 1. Белковые спектры и фосфолипидный состав мембран, обогащенных холинорецепторами из скелетных мышц крыс в условиях сенсибилизации / А.Д. Адо [и др.] // Бюлл. эксперим. биол. Медицины. – 1984. – Т.99 (7). – С. 84-86. 2. Ахметзянов Р.Х. Измерение силовых характеристик мышечных волокон с помощью фотоэлектрического преобразователя / Р.Х. Ахметзянов, Е.Б. Филиппов // Физиол. ж. СССР. – 1986. - Т.72 (3). – С. 387-390. 3. Гущин И.С. Анафилаксия гладкой и сердечной мускулатуры / И.С. Гущин. – М.: Медицина, 1973. – 175 с. 4. Экспериментальная модель для разработки и оценки способов контроля немедленной аллергии / И.С. Гущин [и др.] // Патол. физиол. и эксперимент. Терапия. – 1986. – № 4. – С. 18-23. 5. Девятаев А.М. Избирательное влияние сенсибилизации на фенотип быстрой и медленной мышцы морской свинки / А.М. Девятаев, В.В. Валиуллин // Бюлл. эксперим. биол. медицины. – 1994. – № 2. – С. 191-193. 6. Возможные механизмы аллергических реакций скелетных мышц / А.М. Девятаев [и др.] // Бюлл. эксперим. биол. медицины. – 1996. – № 11. – С. 547-550. 7. Теплов А.Ю. Влияние белковой сенсибилизации на сократительные свойства «быстрых» и «медленных» мышц мыши in vitro / А.Ю.Теплов // Нижегородский медицинский журнал. – 2006. – № 3. – С. 21-24. 8. Blank S. Biochemical characteristics of mammalian diaphragms / S. Blank, V. Chen, CD. Ianuzzo // Respir Physiol. – 1988. – № 74 (1). – P. 115-25. 9. Fahim M.A. Topographic comparison of neuromuscular junctions in mouse “slow” and “fast” twitch muscles / M.A. Fahim, J.A. Holley, N. Robbins // Neuroscience. – 1984. – № 13 (1). – P. 227-235. 10. Florendo J.A. Electrophysiologic differences between mouse extensor digitorum longus and soleus / J.A. Florendo, J.F. Reger, P.K. Law // Exp Neurol. – 1983. – № 82 (2). – P. 404-412. 11. Galkin A.V. ATP but not adenosine inhibits nonquantal acetylcholine release at the mouse neuromuscular junction / A.V. Galkin [etc] // Eur. J. Neurosci. – 2001. – № 13 (11). – P. 2047-2053. Information about the authors: Teplov Alexander Yurievich – candidate of biology sciences, assistant professor of the Department of Pathophysiology of Kazan State Medical University, work tel.: 8 (843) 236-05-63, home tel.: 8 (843) 234-59-50, mobile phone 8-904-665-78-30, e-mail: alikteplov@mail.ru Grishin Sergey Nikolaevich - candidate of biology sciences, assistant professor of the Department of Normal Physiology of Kazan State Medical University. E -mail: sngrishin@mail.ru Farkhutdinov Albert Mansurovich – post-graduate student of the Department of Pathophysiology of Kazan State Medical University, work tel.: 8 (843) 236-05-63, e-mail: a.fakhutdinov@gmail.ru Accepted 12.10.2009 г. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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