Journal of Evolutionary Biochemistry and Physiology, Vol. 38, No. 6, 2002, pp. 762—772. Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 38, No. 6, 2002, pp. 599—607. Original Russian Text Copyright © 2002 by Leontieva, Leontiev, Krivchenko.
COMPARATIVE AND ONTOGENIC PHYSIOLOGY
Comparative Study of Mechanisms of Relaxation of Smooth Muscles in the Rat Bronchus and Pulmonary Artery G. R. Leontieva, V. G. Leontiev, and A. I. Krivchenko Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, St. Petersburg, Russia Received June 7, 2001
Abstract—To compare mechanisms of relaxation of the vascular and bronchial smooth muscles that have different structural and functional organization and embryonic origin, some links were studied of the intracellular systems transducing signals of dilation to the smooth-muscular cellular apparatus in the rat main pulmonary artery and main bronchus. The pulmonary artery and bronchus relaxation was measured by change in the value of isometric contraction of isolated ring segments of the artery and bronchus under the effect of isoproterenol, 3-isobutyl-1-methylxanthine, forskolin, sodium nitroprusside, verapamil, adenosine, and papaverine. The results obtained have shown heterogeneity of the responses of the rat arterial and bronchial smooth muscles to action of relaxants. The vascular smooth muscle turned out to be more reactive: all used substances relaxed it, although to a different extent (from 30 to 100%). In the bronchial smooth muscle, the same response, although less pronounced, was found for 4 out of 7 substances. The relaxation produced by stimulation of β-adrenoreceptors turned out to be functionally more important for the pulmonary artery than for the bronchus. Nevertheless, the results obtained have shown that apart from the heterogeneity of the responses to external stimuli and involvement of different signal systems, there are the links of intracellular transduction systems that are equally involved in relaxation of the vascular and bronchial muscles. They proved to be intracellular systems of cyclic nucleotides, cAMP and cGMP.
INTRODUCTION Unlike skeletal musculature that is activated by acetylcholine released from one type of nerves, contraction or relaxation of smooth muscle cells (SMC) can be produced by many hormones and neurotransmitters activating a complicated complex of signal transducing mechanisms, which is seen as diversity of responses of vascular and visceral smooth muscles (respiratory tract, stomach, intestine, urinary bladder, uterus) to physiological and pharmacological stimuli [1]. In spite of an important role played by the smooth muscle practically in all somatic functions, very many aspects of the mechanisms controlling its activity remains unknown and, therefore, study of the processes transducing stimulating (contracting) or
inhibitory (relaxing) signals to the SMC contractile apparatus is paid attention of researchers. It is to be emphasized that the interest to this problem is also due to necessity of elucidation of pathophysiological aspects of many diseases, of which the presumable etiology is disturbance of regulatory mechanisms operating in different types of SMC. For example, constriction of coronary arteries, leading to a decreased heart work, hypertension, difficult breathing in bronchial asthma are results of a sudden, enhanced contraction of vascular or bronchial SMC, which starts due either to an excessive production of excitating agonists or to an increased reactivity of SMC. These disturbances of smooth-muscular function are believed to result, in turn, from disturbances of some intracellular transduction systems. Therefore, the
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study of mechanisms of regulation of the smooth muscle contraction, relaxation, and proliferation is necessary both for understanding of physiology of the smooth muscle work and for therapy and prevention of diseases of blood vessels and visceral organs. All the above predetermined the goal of the present work: to study some links of the intracellular systems transducing signals for relaxation to the SMC contractile apparatus and to compare them in two types of smooth muscles, vascular and bronchial ones, belonging to the same (“pulmonary”) region but having different structural and functional organization and embryonic origin. For this purpose, the rat main pulmonary artery and the main bronchus were used as objects of the study. MATERIALS AND METHODS The work was carried out on male Wistar rats weighing 180–230 g. In decapitated animals, the main pulmonary artery and main bronchus were rapidly dissected out and, after removal of adipose and connective tissues, placed for 40 min into Krebs–Henselite’s physiological solution (mM): 117 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 Mg SO4, 27.2 NaHCO3, 1.0 NaHPO4, 11.1 glucose, 0.11 ascorbic acid, 0.02 EDTA. The reactivity of vascular and bronchial smooth muscles was studied on ring-shaped 1-mm wide segments placed in a thermostated cuvette (10 ml) with the above physiological solution aerated with carbogen at 37°C. Isometric contraction of the smooth-muscular objects was recorded on a highly sensitive installation designed at the laboratory [2]. As a criterion of the reactivity to the agonist action, the maximal response of the ring preparation was used; its value was expressed in mg/ mm3, which allowed comparing the tension developed by smooth muscles. The effect of inhibitors was evaluated from two parameters: their effect on the preparation contraction value after its placing into the cuvette in the process of contraction of the preparation (on the background of its maximal tonic response) or 30 min before the contraction, and their effect on duration of relaxation of the contracted smooth muscle. Used in the experiments were adenosine, 3-isobutyl-1-methylxanthine, verapamil, isoproterenol, sodium nitroprusside (SNP), papaverine, propranolol, and forskolin. Statistical processing of the data with determination of the mean value, standard error of the mean, confidence interval, and evaluation of dif-
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Fig. 1. Histogram of contractile responses of the pulmonary artery (a) to phenylephrine and of the bronchus (b) to 5-methylfurmemide. Vertical axis: value of contraction (mg/mm3) of vascular and bronchial walls (the same for Figs. 2–5). (I ) Phasic contractile component, (II ) tonic component (the same for Fig. 2). Asterisk indicates statistically significant differences as compared with control (p < 0.001; the same for Figs. 2–5), n (the number of animals) = 25.
ferences was performed by Student t-criterion, using the computer program Microsoft Excel. RESULTS The pulmonary artery contraction was achieved by action of the specific agonist of α1-adrenoreceptors phenylephrine (10–5 M) (Fig. 1). It is to be noted that the agonist of muscarinic receptors 5-methylfurmethide (10–5 M) did not produced any significant contractile response of the artery. The bronchial smooth muscle response was opposite: a strong contraction was obtained only by action of the muscarinic agonist (Fig. 1). The contractions of vascular and bronchial muscles consisted of initial (phasic) and tonic components: the phasic component in the pulmonary artery amounted, on average, to 68% (50–80%), while in the bronchus, to 36% (20–50%) of the maximal tonic contraction. At washing out, the time of relaxation was 905.2 ± 13.6 s in the pulmonary artery and 932.4 ± 8.7 s in the bronchus. Effect of isoproterenol and propranolol on vascular and bronchial smooth muscles. Isoproterenol, a nonselective agonist of β1- and β2-adrenoreceptors, when added at different concentrations (2 × 10 –8 –2 × 10–6 M) on the background of the maximal response of the phenylephrine-produced pulmonary artery contraction produced the concentration-dependent relaxation of the vascular smooth muscle. The greatest effect was observed at an isoproterenol concentra-
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Fig. 2. Responses of the vessel and the bronchus to actions of isoproterenol and propranolol. (a) Histogram of contractile responses of the pulmonary artery after the 20-min action of isoproterenol and propranolol. (1 ) Control, (2 ) isoproterenol (10–5 M), (3 ) propranolol (10–5 M); p < 0.05, n = 5. (b) Histogram of contractile responses of the bronchus after the 20-min actions of isoproterenol and propranolol; other designations as those in Fig. 2a; p > 0.05, n = 5. (c) Curves of dependence of contractile responses of the artery and bronchus on concentration of isoproterenol added on the background of the maximal tonic contraction. (1 ) Control; isoproterenol concentration (M): (2 ) 2 × 10–8, (3 ) 10–7, (4 ) 4 × 10–7, (5 ) 2 × 10–6; p < 0.01–0.005, n = 5.
tion of 4 × 10–7 M; a higher concentration (2 × 10–6 M) had a lower inhibitory effect, while at a concentration of 10–5 M, no relaxation was produced. Propranolol, an antagonist of β1- and β2-adrenoreceptors, acted on the artery by an unexpected manner: it did not change the value of relaxation induced by isoproterenol (Figs. 2a, 2c). The time of relaxation of the contracted artery was reduced by 36%. In the bronchus, isoproterenol did not relax the muscle at any concentration both on the background of the bronchus tonic contraction and after a 20-min exposure of the bronchus to isoproterenol before the contraction. No effect was also produced by propranolol on the bronchial smooth muscle. The time of its relaxation also was unchanged (Figs. 2b, 2c; see table). Effect of 3-isobutyl-1-methylxanthine. 3-Isobutyl1-methylxanthine (IBMX) (10–5 M) produced a fast relaxation by 80% of the contracted pulmonary artery. The inhibitory effect of inhibitors was also revealed after a 30-min exposure of the pulmonary artery: the phasic and tonic components of the contrac-
tile response were reduced by 90%. The time of relaxation was shortened almost 3 times. The inhibitor of phosphodiesterase also had an inhibitory effect on the bronchial smooth muscle, although it was considerably less pronounced than on the pulmonary artery: its administration on the background of the maximal tonic contraction produced the bronchus relaxation by 17%, while after a 30-min exposure, by 27%, the time of relaxation being shortened 1.5 times (Figs. 3a, 3b, see table). Forskolin at a concentration of 2 µM, when added on the background of the maximal tonic contraction of the pulmonary artery, produced an instant relaxation (100%) of the vascular smooth muscle. The relaxing effect of forskolin was also recorded after a 30min exposure of the pulmonary artery to forskolin (2 µM); at the end of the exposure there was no contractile response at all to the action of activator. Forskolin also produced relaxation of the bronchial smooth muscle; the maximal effect decreased by 80% at its addition on the background of the tonic con-
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Time of half-relaxation (s) of tonic component of the contractile response of the smooth muscles of bronchus and pulmonary artery after 30-min action of relaxants (x ± Sx) Bronchus
Relaxant
Pulmonary Artery
control
experiment
control
experiment
Isoproterenol
954 ± 8.29 (4)
813 ± 13.62
995 ± 9.24 (5)
639 ± 17.08**
3-Isobutyl-1-methylxanthin
900 ± 1.03 (5)
518 ± 15.07**
917 ± 6.72 (5)
no contractions
Forskolin
751 ± 5.37 (4)
510 ± 13.58
815 ± 17.00 (4)
no contractions
Sodium nitroprusside
1051 ± 6.52 (5)
580 ± 13.52**
1008 ± 15.98 (4)
no contractions
Verapamil
826 ± 7.06 (5)
566 ± 4.47**
814 ± 18.45 (5)
370 ± 19.22*
Adenosine
912 ± 0.97 (6)
591 ± 11.81**
880 ± 4.60 (5)
454 ± 19.02**
Papaverine
1051 ± 6.52 (5)
699 ± 10.36**
1008 ± 15.98 (4)
187 ± 11.75**
Note: Asterisks indicate statistically significant difference as compared with control: *—p < 0.01,**—p < 0.001. Figures in parentheses: the number of animals.
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Fig. 3. Response of the vessel and the bronchus to actions of 3-isobutyl-1-methylxanthine and forskolin. (a) Effect of 3-isobutyl1-methylxanthine on the pulmonary artery contractile responses. (1 ) Control, (2 ) isobutylmethylxanthine (30-min action), (3 ) isobutylmethylxanthine on the background of contraction; p < 0.002, n = 5. (b) Effect of isobutylmethylxanthine on the bronchus contractile responses; designations as those in Fig. 3a; p < 0.008, n = 5. (c) Effect of forskolin on the pulmonary artery contractile responses. (1 ) Control, (2 ) forskolin (30-min action), (3 ) forskolin on the background of contraction; p < 0.002, n = 4. (d) Effect of forskolin on the bronchus contractile responses; designations as those in Fig. 3c; p < 0.01, n = 4. JOURNAL OF EVOLUTIONARY BIOCHEMISTRY AND PHYSIOLOGY Vol. 38 No. 6 2002
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Fig. 4. Response of the vessel and the bronchus to actions of sodium nitroprusside and verapamil. (a) Effect of sodium nitroprusside on the pulmonary artery contractile responses. (I ) Control, (2 ) sodium nitroprusside (30-min action), (3 ) sodium nitroprusside on the background of contraction; p < 0.02, n = 4. (b) Effect of sodium nitroprusside on the bronchus contraction; designations as those in Fig. 4a; p < 0.05, n = 5. (c) Effect of verapamil on the pulmonary artery contraction. (I ) Control, (2 ) verapamil (30-min action), (3 ) verapamil on the background of contraction; p < 0.001, n = 5. (d) Effect of verapamil on the bronchus contraction; designations as those in Fig. 4c; p < 0.05, n = 5.
traction; after the 30-min bronchus exposure the phasic and tonic contractions decreased by 50% and 64%, respectively, while the relaxation time was accelerated by 30% (Figs. 3c, 3e, see table). Effect of sodium nitroprusside. In the pulmonary artery, sodium nitroprusside (SNP) (10–5 M) added on the background of the tonic contraction decreased the maximal contractile response by 80% and accelerated the relaxation time by 32%. After a 30-min action of SNP on the pulmonary artery, the phasic and tonic components of the contraction were decreased by 95% and 104%, respectively. SNP had no effect on the bronchus contraction both at its addition on the background of tonic contraction and after a 30-min exposure, only by accelerating the relaxation time 1.5 times (Figs. 4a, 4b, see table). Effect of verapamil. Verapamil, when added on the background of the pulmonary artery contraction, decreased the maximal response by 40%. A 20-min exposure of the artery in the verapamil solution (10–5 M) before the contraction led to a decrease of the initial phase by 25% and of the tonic one by 54% and also reduced by 55% the relaxation time. Verapamil had
no relaxing effect on the contracted bronchus, it only accelerated the bronchial smooth muscle relaxation (by 31%) (Figs. 4c, 4d; see table.). Effect of adenosine. In the pulmonary artery, adenosine (10–5 M), when added on the background of the tonic contraction, decreased it by 20%. A 30-min action of adenosine on the artery before the contraction also led to the artery relaxation: the phasic component was decreased by 20%, while the tonic one, by 40%; the time of relaxation of the vascular muscle was shortened twice. Response of the bronchial muscle was different: when adenosine (10–5 M) was added on the background of the maximal bronchus contraction, the bronchus was relaxed by 13%, and, after its 30-min action, there occurred a decrease (by 25%) only of the tonic components, whereas the initial component value increased by 44%, the relaxation time decreasing1.5-fold (Figs. 5a, 5b; see table). Effect of papaverine. Figures 5c and 5d present data on the effect of papaverine on the smooth muscles of pulmonary artery and bronchus. In the pulmonary artery, addition of papaverine (10–5 M) on the background of the tonic contraction produced a reduc-
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Fig. 5. Response of the vessel and the bronchus to actions of adenosine and papaverine. (a) Effect of adenosine on the pulmonary artery contraction. (1 ) Control, (2 ) adenosine (30-min action), (3 ) adenosine on the background of contraction; p < 0.02, n = 6. (b) Effect of adenosine on the bronchus contraction; designations as those in Fig. 5a; p < 0.05, n = 5. (c) Effect of papaverine on the pulmonary artery contraction. (1 ) Control, (2 ) papaverine (30-min action), (3 ) papaverine on the background of contraction; p < 0.02, n = 4. (d) Effect of papaverine on the bronchus contraction; designations as those in Fig. 5c; p < 0.05, n = 5.
tion of the maximal contractile response by 60% and accelerated the relaxation 5-fold. A 30-min action of papaverine on the pulmonary artery before its contraction led to an even more pronounced relaxation of the vessel: both the phasic and tonic components of the contraction were decreased by 85%. A different response was recorded for the bronchus: addition of papaverine on the background of the maximal tonic contraction of the bronchus did not change its value. After a 30-min action, papaverine decreased (by 20%) only the tonic contractile phase, accelerating the relaxation 1.5 times (see table). DISCUSSION According to the modern concepts, the SMC relaxation is produced by two mechanisms: by a considerable decrease of intracellular concentration of free calcium and/or by inhibition of actin–myosin interaction due to phosphorylation of MLK protein kinase, which can be induced by various extracellular signals, such as β-adrenergic agonists, endothelium-
dependent factor (EDRF)—NO, calcium channel blockers, nucleosides, etc. [3]. The relaxants, by interacting with receptors and sarcolemmal channels, activate certain intracellular systems of second messengers that deliver the signal from the receptor to the SMC contractile apparatus. In the present study, comparison of the relaxation mechanisms of the pulmonary artery and bronchus smooth muscles was based on the comparison of the effect of relaxants on some links of the transduction systems in smooth-muscular cells, which leads to relaxation of their contractile apparatus. Relaxation of many vascular and visceral smooth muscles is realized through activation of β-adrenoreceptors by catecholamines released from sympathetic adrenergic nerves or circulating in blood [4–7]. We were interested in the question whether identical is the functional role of this mechanism of relaxation in the pulmonary artery and in the bronchus that differ by their performed functions, innervation, embryonic origin, phenotype [8–11]. The data obtained have shown that isoproterenol, a non-selective agonist of
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β1- and β2-adrenoreceptors, produces relaxation of pulmonary artery. These data agree with results of other studies performed on other vessels and in other animals [6,13, 14], i.e., the stimulation of β-adrenoreceptors does lead, indeed, to vasodilatation of most vessels in different animals. It is to be noted that only low concentrations of isoproterenol produce relaxation, whereas concentrations higher than 1 µM have no such effect, while even higher concentrations do not contract the vessel at all, which was also confirmed in our experiments. Meisheri and Breemen [6] believe this occurs due to an activation of α-adrenoreceptor and release of endogenous noradrenaline. The effect of propranolol, an antagonist of β1- and β2-adrenoreceptors, in the pulmonary artery was unusual: this blocker did not change the isoproterenol-produced relaxation. The lack of response to propranolol may be connected with the fact that the relaxation induced by isoproterenol is mediated not only by classic β1and β2-adrenoreceptors, but also by the so-called β3and β4-adrenoreceptors that are not affected by propranolol; these receptors have been revealed in the rat aorta and portal vein [15, 16] and in the dog pulmonary artery [17]. Unlike the pulmonary artery, no relaxation of the bronchus is produced by isoproterenol both at low and high concentrations; propranolol also had no effect. The difference in the type of contraction-mediating receptors (muscarinic receptors in the bronchus and α1-adrenoreceptors in the pulmonary artery) cannot account for the absence of inhibitory response to isoproterenol in the bronchus, as, according to the literature data ([4]), isoproterenol decreases the acetylcholine-produced contraction (activation of muscarinic receptors) of vascular smooth muscle by 95%, while the noradrenaline- or sodium chloride-induced contraction, only by 40% and 30%, respectively. Besides, unlike the rat bronchus, in the dog tracheal smooth muscle, isoproterenol did produce relaxation of the carbachol-induced contraction [18]. Thus, the relaxation produced by activation of βadrenoreceptors plays a certain functional role in the case of the rat pulmonary artery, but not in the case of the bronchus. There are several hypotheses to explain the mechanism of relaxation of smooth muscles through activation of β-adrenoreceptors [5, 6, 19–21]. According to one of them, the agonists interacting with βadrenoreceptors bring about dissociation of GTP-
dependent Gs-proteins into αs- and β-subunits, then αs-subunit is bound to adenylyl cyclase and activates it to catalyze conversion of ATP into cAMP. This water-soluble second messenger is, in turn, bound to and activates protein kinases A and C, which leads to a decrease of intracellular free Ca2+ concentration [5]. Although many details of the intracellular cascade of biochemical processes, their interaction and relay have remained so far unclear, an important role of the system of cyclic nucleotides (cAMP and cGMP) in realization of the smooth muscle relaxation has been shown by the several examples: thus, cAMP inhibits the vascular smooth muscle contraction via a decrease of the cytosolic Ca2+ level and of calcium sensitivity of contractile elements [22–24]. Taking into account all this, we studied participation of the cAMP system in the artery and bronchus dilation; for this purpose, two substances, forskolin and IMBX, increasing intracellular level of cAMP were used. Among these two relaxants, the strongest one turned out to be forskolin, while among the two smooth muscles, the most relaxing effect of forskolin was revealed in the pulmonary artery, in which forskolin induced the maximal (100%) vasodilatation. Even greater difference between the pulmonary artery and the bronchus was observed in the effect of IMBX. The difference in the pulmonary artery and bronchus responses to IMBX might have been due to that their smooth muscles contain heterogeneous, by their sensitivity to IMBX, isozymes of phosphodiesterase, as it is known that mammalian tissues can contain phosphodiesterase isozymes with several different features, including sensitivity to inhibition by pharmacological agents [25]. Since the endothelium plays a significant role in regulation of the smooth muscle tone, and among the dilators released by the endothelium, the major one is NO, the smooth muscle relaxation is produced by NO either through activation of soluble guanylyl cyclase and a subsequent increase of the intracellular cGMP content or through activation of K+ channels and subsequent hyperpolarization of sarcolemma [26], it was this signaling way that we also choose for comparison. As a donor of NO, we used SNP, an activator of soluble guanylyl cyclase. Similar to the effect of forskolin, SNP produced an almost complete relaxation of the pulmonary artery, whereas the bronchus did not respond to the SNP action. We believe that the absence of the rat bronchus response to exogenous NO does not mean the absence in it of the
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mechanism of the bronchial smooth muscle relaxation induced by NO, as it has been shown that in different animal species, including the rat, endogenous NO is a transmitter of the inhibitory effect of non-adrenergic, non-cholinergic nerves on bronchial smooth muscle [27–30], although there is one exception: in the seep, the inhibitory, non-adrenergic, non-cholinergic innervation is absent and NO induces no relaxation of the airway smooth muscle [31]. The absence of the bronchodilatation under the action of exogenous NO observed by us in the rat might be suggested to be due to “competitive interrelations” between exogenous and endogenous NO. For example, it has been shown that the SNP-induced relaxation of the artery contracted by sodium chloride is enhanced after removal of the endothelium that, in the authors’ opinion, releases some factor inhibiting the SNP action [26]. Thus, the intracellular systems of second messengers (cAMP and cGMP) are functionally important links for transduction of the signal for dilation of both the pulmonary artery and the bronchus, although, as shown by the results obtained, there is no complete identity both in the significance of the cyclic nucleotide systems in relaxation of the smooth-muscular cells of a given organ and in the significance of each of the systems in relaxation of the smooth-muscular cells of both organs. Since the change of an intracellular calcium concentration is one of the major mechanisms of realization of the contraction–dilation cycle in smooth muscles, to compare the mechanisms of relaxation in the pulmonary artery and the bronchus, we have chosen one of the links of the transductional, signal, calcium system, such as the entry of extracellular calcium into the cell through L-type potential-dependent calcium channels. Verapamil, a blocker of these channels, relaxed the contracted pulmonary artery, as it was observed by us in other blood vessels [32]. Unlike the blood vessels, the value of the rat bronchus contraction under the effect of verapamil was not changed. By the data of other authors [18, 33], verapamil has no effect on the intracellular concentration of cytosolic calcium and the basal tone of canine tracheal smooth muscle, but it relaxes the contracted trachea, possibly via other mechanisms initiated by verapamil. The absence of the inhibitory effect of verapamil on the 5-methylfurmetid-contracted rat bronchus permits thinking that in the bronchial smooth
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muscle, in contrast to the vascular one, other mechanisms of the contraction exist, which do not depend on an increase of intracellular cytosolic calcium concentration due to the extracellular calcium entry through L-calcium channels. For example, the contraction of canine trachea by carbachol was determined by sensitization of the contractile elements to calcium and/or activation of the calcium-independent mechanism connected with stimulation of hydrolysis of phosphatidylinositols and formation of diacylglycerol, an endogenous activator of protein kinase C, which results in contraction of the smooth muscle even in the absence of the external calcium influx [18]. Thus, there also is a difference between vascular and bronchial muscles in the use of some links of the transduction calcium system for the muscle dilation. To compare the mechanisms of relaxation in the pulmonary artery and in the bronchus, adenosine and papaverine were used as external signals triggering some intracellular signal transduction mechanisms. The nucleoside adenosine is a strong vasodilator that is formed endogenously from adenine nucleotides during metabolic stress (hypoxia, ischemia, increased consumption of oxygen by myocardium) and, by inducing vasodilatation, plays an important role in regulation of blood flow in the heart, brain, skeletal muscle [34, 35]. We were interested in the question, whether adenosine is also a strong bronchodilator. Our results have shown that the pulmonary artery, like the coronary and cerebral vessels, responds to adenosine by relaxation, although less strongly. The response of the bronchus to adenosine differed from that of the artery not only by some lesser extent of dilation in application of the relaxant during the bronchoconstriction, but also by that the contractile tonic phase did not decrease, but, on the contrary, increased. The obtained data on the double effect of adenosine on the bronchus confirmed results of the works showing that, contrary to the common opinion about adenosine as a relaxant, some smooth muscles (vas deferens, smooth muscle of renal vessels, rat bronchial muscle) responded to its action by contraction [34, 36, 37]. At present, the concept of the transducing signaling pathway responsible for the adenosine-induced dilation is contradictory and the mechanisms responsible for the adenosine-induced contraction are quite unclear [38– 40]. Papaverine, a non-selective smooth-muscle relax-
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ant, dilates different smooth-muscle organs. Results of the present study confirmed the commonly accepted opinion about papaverine as vaso- and bronchodilator, although the responses of pulmonary artery and bronchus were not identical. The papaverine-induced relaxation is believed to be connected with three mechanisms: intracellular accumulation of cAMP due to inhibition of phosphodiesterase, inhibition of mitochondrial respiration, and action on Ca2+ translocation; the mechanism of relaxation under the effect of papaverine “demonstrates” an organ heterogeneity, i.e., for the relaxation of the smooth muscle of a given organ, different mechanism may be used [41– 43]. Based on our data obtained, we cannot decide as to which mechanism, or all of them, takes part in the pulmonary artery relaxation. As far the bronchus is concerned, the difference in the responses of initial and tonic contractile phases indicates that papaverine at first stimulates the release of intracellular calcium from the stores (an enhancement of phasic contraction) and only after that the mechanism operates for the relaxation (a decrease of the tonic phase). In this experimental series, the pulmonary artery and bronchus responses to the action of the relaxants adenosine and papaverine also turned out to differ. At present, the heterogeneity of smooth muscle responses has been establish sufficiently well to be considered as an important phenomenon in physiology, pharmacology, and pathophysiology; however, precise molecular mechanisms responsible for this heterogeneity, as well as the mechanisms of its development, are unknown. Some authors believe that a great variety of contractile and pharmacological properties of different smooth muscle is due to heterogeneity of the SMC themselves; their phenotypic heterogeneity, in turn, is due to many molecular determinants [9, 10]. For example, in the pulmonary artery smooth muscle, four subpopulations of SMC were revealed, some of them, only special cells, but not all cells, being responsible for hyperplasia of arterial SMC in hypertension [10]. The heterogeneity of the airway muscles has been established at the tissue and cellular levels: two groups of smooth muscles are identified, the extrapulmonary and intrapulmonary ones, which consist of several types of SMC with non-identical structural and mechanical properties; most of the intrapulmonary smooth muscle cells have specific mechanical properties preventing an excessive constriction of airway pathways under the muscular contraction to
provide optimal conditions for the air passage to the lungs [11]. Thus, the obtained results have demonstrated differences in responses of rat pulmonary artery and bronchus under the effect of relaxants, which confirms once more the heterogeneity of smooth muscles. The vascular smooth muscle turned out to be more reactive: practically all seven substances that we used produced its relaxation, although to a different extent (from 30 to 100%), whereas it was much more difficult to relax bronchial smooth muscle under the effect of these relaxants: this muscle responded to four out of seven substances, the response being less pronounced. The different perception of external signals might also be a reflection of different mechanisms used by smooth-muscle cells for their relaxation. Indeed, the β-adrenoreceptor pathway proved to be more important functionally for relaxation of the pulmonary artery, than for that of the bronchus. At the same time, the obtained results have shown that apart from heterogeneity of responses to external stimuli and involvement of different signal pathways, there are links of intracellular transduction systems, which are equally involved in relaxation of vascular and bronchial muscles; they turned out to be the intracellular systems of cyclic nucleotides (cAMP and cGMP). ACKNOWLEDGMENTS The work was supported by the Program of the Presidium of the Russian Academy of Sciences “Basic Sciences to Medicine.” REFERENCES 1.
2.
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