ISSN 10623590, Biology Bulletin, 2014, Vol. 41, No. 1, pp. 55–59. © Pleiades Publishing, Inc., 2014. Original Russian Text © O.V. Boiko, B.N. Manukhin, 2014, published in Izvestiya Akademii Nauk, Seriya Biologicheskaya, 2014, No. 1, pp. 48–52.
ANIMAL AND HUMAN PHYSIOLOGY
Intracellular Transmission of the Cholinergic Signal in the Chick Amnion O. V. Boiko and B. N. Manukhin Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119334 Russia email:
[email protected] Received June 4, 2013
Abstract—The role of the system of deposited calcium in the mediation of contractile reactions to carbachol in an isolated amnion of 11–13 day old chicken embryo was studied. It was found that thapsigargin (2 µM, 20 min), an inhibitor of the endoplasmic reticulum Ca2+ATPases, decreases the tonic reaction to carbachol by 40 ± 2%. In the presence of U73122 (5–10 µM, 10 min), a phosphoinositidespecific phospholipase C inhibitor, the rhythmic contractile reaction of the amnion to carbachol is blocked, whereas the tonic reaction decreases to 47 ± 9% of the initial one. Ryanodine (10 µM, 5 min) inhibits the spontaneous contractile activ ity of the amnion and decreases the tonic reaction to carbachol to 36 ± 3% relative to control. In the presense of ryanodine, nifedipine (0.05 µM) completely blocks the tonic reaction to carbachol. Thus, calcium mobi lized from intracellular stores via inositol trisphosphate and ryanodine receptors is involved in realization of contractile reactions, mediated by M3 receptors, in the chick amnion. DOI: 10.1134/S1062359014010026
and Bowers, 1996). In terms of affinity to antagonists, muscarinic receptors of the amnion are characterized as М3cholinoreceptors (Boiko and Manukhin, 2007). Thus, the chick amnion allows to study the mecha nisms of the nonneuronal influence of ACh on M3receptors of smooth muscle cells. Activation of М3receptors in innervated smooth muscles leads to an increase in the cytoplasmic concentration of cal cium ions, entering into the cell through voltagegated calcium channels and as a result of release of Ca2+ into the cytoplasm from the sarcoplasmic reticulum (Bai et al., 2009; Wray and Burdyga, 2010). In the chick amnion, contractile responses to cholinomimetics are mediated to a great extent by calcium ions that enter through the Ltype voltagedependent channels (Boiko and Manukhin, 2009). The role of the system of the deposited calcium has not been studied earlier. The aim of this work is to study the contribution and mobilization pathways of sarcoplasmic reticulum deposited Ca2+ in response to CCh action in the amnion.
INTRODUCTION It is known that the substances called neurotrans mitters (mediators) are synthesized and perform regu latory functions in protozoa, during metazoan pre embryonic and prenervous stages of development, and in nonneuronal cells and tissues of vertebrates (Buznikov et al., 2001; Eglen, 2006). Nonneuronal cholinergic systems invariably draw the interest of researchers. Membrane cholinoreceptors were found in vascular endothelial cells, airway epithelial cells, cancer cells, keratinocytes, gametes, macrophages, lymphocytes, etc. (Eglen, 2006; Kawashima and Fujii, 2008; Wessler and Kirkpatrick, 2008). In studying the nature of the nonnervous cholinergic regulatory sys tems and their biological significance, uninnervated extraembryonic tissues can be used as an experimen tal model. Cholinoreceptors in the chick amnion mediate the stimulation of its contractions: in response to the cho linergic agonist action, the tone, amplitude, and fre quency of contractions of the amniotic membrane increase (Cuthbert, 1963; Boiko and Manukhin, 1989a; Bowers, 1989). The spontaneous contractile activity of the amnion correlates with the level of tissue differentiation, acetylcholine content (ACh), and cholinesterase activity (Bunkina, 1963; Polyakova, 1970; Boiko and Manukhin, 1989b). The reaction of the amnion to ACh and carbachol (CCh) is blocked by atropine (Boiko and Manukhin, 1989a; Nechaeva and Turpaev, 1995). The muscarinic nature of cholinore ceptors was confirmed in experiments carried out on dissociated smooth muscle cells of the amnion (Dahm
MATERIALS AND METHODS The study was conducted on an isolated strip of uninnervated amnion of 11–13 day old chicken embryo. Contractile reactions were registered in the isometric regime using 6MX1B transducers (Russia) on H339 and H399 recorders. We simultaneously studied the contractions of two fragments of the amnion placed into 10 mL thermostated (38°C) aer ated chambers, which contained Hanks’ balanced salt 55
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BOIKO, MANUKHIN (a)
CCh
(b)
CCh Thapsigargin 2 µM
CCh
(c)
CCh U73122 10 µM
CCh
CCh Ryanodine 10 µM
Fig. 1. Inhibition of the reaction of the amnion of a chicken embryo (11–13day old) to carbachol (50 µM) by (a) thapsigargin, (b) U73122, and (c) ryanodine. Calibration: 1 min, 25 mg.
solution of the following composition, mM: NaCl (137), KCl (5.4), CaCl2 (1.26), MgSO4 (0.41), MgCl2 (0.49), Na2HPO4 (0.34), KH2PO4 (0.44), NaHCO3 (4.2), and glucose (5.6). The initial load on the prepa ration was 100 mg. The tested substances were intro duced into the chamber in the volume of 100 µL after a halfhour preincubation of the tissue. The contrac tile activity of the amnion was stimulated using a cho linergic agonist CCh (50 µM). The used concentra tion of the agonist induces the maximum response in terms of the frequency of the contractions and a sub maximum tonic reaction (Boiko and Manukhin, 1989a). The following reagents were used: thapsigragin (Tocris Bioscience, United States), ryanodine (Ascent Scientific, United States), carbachol, U73122 (1[6 [[(17beta)3methoxyestra1,3,5(10)trien17yl]ami
Tonic reaction to carbachol
% 120
1 2
80 *
* **
40
0
a
b
c
Fig. 2. Reaction of the amnion of a chicken embryo (11– 13day old) to carbachol. (1) controls; (2) effect of (a) thapsigargin, (b) U73122, and (c) ryanodine. * P < 0.05, ** P < 0.01.
no]hexyl]1Hpyrrole2,5dione), (Sigma, United States).
and
nifedipine
RESULTS Two types of receptors are responsible for the release of Ca2+ from intracellular stores: ryanodine and inositol1,4,5triphosphate (IP3) receptors. The degree of participation of the deposited calcium in the contractile reactions mediated by М3receptors can be estimated using thapsigargin, an inhibitor of the endo plasmic Ca2+ATPases, which leads to a depletion of IP3sensitive and IP3insensitive sources, thus cancel ing the possibility of the additional release of Ca2+ in response to the action of the cholinomimetic. Incuba tion of the tested amnion strip for 20 min with thapsi gargin (2 µM) decreases the tonic reaction to CCh by 40 ± 2% (Figs. 1a, 2a), which means the dependence of CChinduced contraction on the release of the intracellular calcium. Participation of calcium ions mobilized from the intracellular stores through ІР3receptors in realiza tion of muscarinic reaction in the amnion was estab lished by action of U73122. Aminosteroid U73122 is an inhibitor of the phosphoinositidespecific phos pholipase C, which is a key enzyme in the metabolism of phosphatidyl inositol. Hydrolysis of phosphatidyl inositol leads to the formation of diacylglycerol and IP3 that mobilizes Ca2+. In the presense of U73122 (5–10 µM, 10 min), the rhythmic contractile reaction of the amnion to CCh is abolished, whereas the tonic reaction decreases to 47 ± 9% from the initial one (Figs. 1b, 2b). To reveal the contribution of the deposited calcium released into the cytoplasm through ryanodine recep tors, we studied the activity of the modulator of these receptors: plant alkaloid ryanodine. The action of ryanodine (10 µM, 5 min) leads to a blockade of the BIOLOGY BULLETIN
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spontaneous contractile activity of the amnion and decreases the tonic reaction to CCh to 36 ± 3% (Figs. 1c, 2c). In the reactions without a pronounced tonic com ponent, ryanodine at a low concentration (1 µM) induces an increase in the amplitude of the contractile reaction to CCh (Fig. 3a). At higher concentrations (10–75 µM), it dosedependently inhibits the ampli tude of CChinduced contractions till their complete cessation (Fig. 3a). In presense of 20 µM of ryanodine, the cholinergic reaction becomes highly sensitive to nifedipine, a blocker of L type voltagedependent cal cium channels. Nifedipine at a concentration of 0.05 µM completely blocks the reaction to CCh (Fig. 3b), including its tonic component.
(a)
CCh
1
10
25
No. 1
75
µM
(b)
Provisional extraembryonic tissues attract the everincreasing attention of researchers for a number of reasons. It was established that the epithelial cells of the human amnion possess the characteristics of pluri potent stem cells and, under certain conditions, have the potential to differentiate into various types of cells (Miki and Strom, 2006). The unique characteristics of the amnion cells allow to consider them a potential source of material for regenerative medicine (Toda et al., 2007; Miki et al., 2009). It was shown that the amnion cells of embryos of higher vertebrates are capable to synthesize and release catecholamines and acetylcholine (Sakuragawa et al., 2001) and possess the receptors of mediators and hormones (Di Renzo et al., 1984; Collins, 1993). The mechanisms of real ization of the receptor signals in the amniotic tissue remain little researched. The muscarinic receptors of the chick amnion, which mediate the activation of its contractions, were identified by testing the activity of selective antago nists. Pirenzepine, metoctramine, 4DAMP, and tropicamide were used as М1, М2, М3, and М4antagonists, respectively. In terms of the choli nolytic activity (–logIC50) in relation to CCh induced reaction, they constituted the following sequence: 4DAMP (8.29) > tropicamide (6.97) > pirenzepine (5.85) > metoctramine (5.63). According to the obtained data, muscarinic receptors of the smooth muscle cells of the chick amnion belong to the М3 subtype (Manukhin and Boiko, 2008). It was established that in response to the action of 100 µM of CCh, the concentration of calcium in the amnion cells increase more than tenfold compared with the basal Ca2+ concentration (Cross et al., 2000). The latent period between the application of the ago nist and the subsequent increase in the concentration of Ca2+ constituted 0.7 s. In the case of the direct depolarization of the plasmatic membrane by increas ing the concentration of the external К+, the latent period proved to be less by half; however, the increase in the concentration of Ca2+ occurred more slowly and Vol. 41
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Ryanodine
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20
0.01
0.05
µM
Ryanodine Nifedipine Fig. 3. Effect of ryanodine on the contractile response of the amnion of a chicken embryo (11day old) induced by carbachol (50 µM). (a) Cumulative effect of ryanodine, (b) inhibitory action of ryanodine and blockade of the reac tion by nifedipine. Calibration: (a) 1 min, 25 mg; (b) 1 min, 10 mg.
the attained maximum of the peak was significantly lower. As the reaction to high K+ in the amnion is pro vided by the entrance of the extracellular Ca2+ (Boiko and Manukhin, 2009), this indirectly indicates the mobilization of additional sources of Ca2+ in response to CCh action and implies that both extracellular and intracellular sources of Ca2+ can participate in realiza tion of the cholinergic influence. It was shown that nifedipine (0.1–1 µM), a blocker of L type voltagedependent calcium channels, com pletely suppressed the spontaneous rhythmic contrac tions as well as the ones caused by CCh (50 µM), and the tonic reaction to high K+. However, the CChinduced tonic reaction in normal Hanks’ solution and in pres ence of high K+ is not eliminated completely by nife dipine (Boiko and Manukhin, 2009). Hence, the acti vation of the muscarinic receptors of the amnion leads to an exogenic influx of Ca2+ into the cytoplasm of the cell through plasma membrane Ltype channels, which open up in response to depolarization and, apparently, to its release from intracellular sources. The latter is confirmed by experiments with thapsigargin, a specific inhibitor of endoplasmatic Ca2ATPases. As in other smooth muscles (An et al., 2002; Quinna et al.,
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2004), the depletion of the intracellular calcium stores in the amnion by action of thapsigargin for 20 min leads to a significant decrease in the reaction to CCh (Fig. 2a). It is known that in the smooth muscles cholinergic agonists activate the metabolism of the membrane phosphoinositides through M3cholinoreceptors. The binding of ACh with the receptors initiates a signal cascade, which leads to hydrolysis of phosphatidyl inositol by phosphoinositidespecific phospholipase C with the formation of diacylglycerol and IP3, which mobilizes Ca2+ (Eglen, 2005). According to our data, CChinduced contraction of the chick amnion is mediated by the activation of phospholipase C. In the presence of U73122, which suppresses the activity of phospholipase C, the rhythmic component of the response of the amnion to CCh was canceled, whereas the tonic reaction decreased more than two fold (Fig. 2b). Therefore, we can assume that CCh induced contractions of the chick amnion are medi ated by the release of calcium through IP3sensitive receptors of the sarcoplasmic reticulum owing to the dependent on phospholipase C increase in the level of intracellular IP3. These data agree with the results obtained by the activation of M3receptors of other smooth muscle cells and indicate the participation of the IP3dependent mobilization of the intracellular pool of calcium in the smooth muscles (Mimata et al., 1997; Bai et al., 2009). Ryanodine receptors, involved in realization of the contractile responses in the skeletal and cardiac mus cles, are also revealed in various types of smooth mus cle cells and mediate muscarinic reactions (White and McGeown, 2002; Du et al., 2005). To study the role of ryanodine receptors in CChinduced responses of the chick amnion, CCh was applied in the presense of ryanodine. In our experiments, ryanodine at a con centration of 1 µM potentiated the contractile reac tion of the amnion to CCh (Fig. 3a); i.e., apparently, its influence at a low concentration activated ryanod ine receptors and retained them in an open state. This led to an additional release of the deposited Ca2+. Ryanodine at a concentration of 5 µM and more caused a blockade of ryanodine receptors of the amnion and effectively and reliably decreased the reaction to CCh (Fig. 2c). The simultaneous action of nifedipine (0.05 µM) and ryanodine (10 µM) com pletely blocked the cholinergic reaction and increased the sensitivity to nifedipine. Thus, the contractile reactions of the chick amnion caused by the activation of M3cholinoreceptors depend on extracellular Ca2+, which enters through the Ltype voltagedependent channels and mobiliza tion of Ca2+ from the intracellular stores through inos itoltriphosphate receptors and ryanodine receptors. The involvement of various sources of Ca2+ provides a significant Ca2+ signal and a longterm highampli tude reaction.
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Translated by N. Smolina
