Streszczenie

Wstęp: Przedmiotem pracy jest określenie działania aktywatorów cyklazy guanylanowej na reakcję skur­czu mięśniówki gładkiej tchawicy. W pracy oznaczano wpływ wzrastających stężeń aktywato­ra cyklazy guanylanowej YC-1 oraz 8Br cGMP na reakcję skurczu mięśniówki gładkiej wyzwa­laną karbacholem. Badano także wpływ wzrastających stężeń inhibitora cyklazy guanylanowej ODQ na krzywe stężenie-efekt dla karbacholu.
Materiał/Metody: Badania przeprowadzono na izolowanej tchawicy szczurów szczepu Wistar obu płci o masie 350-450 g. Tchawice preparowano zgodnie z metodą (Akcasu) (1959) w modyfikacji Szadujkis-Szadurski (1996). Krzywe stężenie-efekt wyznaczano metodą stężeń kumulowanych, zgodnie z metodą van Rossuma (1963) w modyfikacji Kenakin (2006).
Wyniki: Z przeprowadzonych badań wynika, że aktywacja cyklazy guanylanowej za pomocą YC-1 i 8Br cGMP powoduje obniżenie reakcji mięśniówki gładkiej tchawicy na karbacholu średnio do 80%. Z porównania krzywych stężenie-efekt dla karbacholu przed i po zastosowaniu 8Br cGMP uzy­skano podobne wyniki do wyzwalanych przez YC-1.
Natomiast wzrastające stężenia inhibitora cyklazy guanylanowej – ODQ powodują przesunięcie krzywych w lewo, obniżenie wartości EC₅₀ i podwyższenie maksymalnej reakcji na karbacholu. Można więc stwierdzić, że działanie ODQ jest odwrotne do działania 8Br cGMP i YC-1.
Wnioski: Karbachol w sposób zależny od stężenia powoduje skurcz mięśniówki gładkiej tchawicy. Z prze­prowadzonych badań wynika, że aktywacja cyklazy guanylanowej powoduje obniżenie reakcji mięśniówki gładkiej tchawicy na karbachol średnio do 80%.

Słowa kluczowe:cyklaza guanylanowa • mięśniówka gładka • tchawica • karbachol • 8Br cGMP • ODQ • YC-1

Summary

Introduction: The subject of the study compare the influences of YC-1 guanylyl cyclase activator with ODQ guanylyl cyclase inhibitor on the tracheal smooth muscle contraction induced by carbachol. The study specified the influence of increasing concentrations of soluble guanylyl cyclase activators YC-1 and 8Br cGMP on the reaction of tracheal smooth muscle contraction released by carba­chol. The author also examined the effect of increasing concentrations of soluble guanylyl cyc­lase inhibitor ODQ on the concentration-effect curves for carbachol.
Material/Methods: Testing was conducted on an isolated trachea of both sexes of Wistar rats with weight ranging between 350 g and 450 g. Tracheas were prepared in accordance with the Akcasu (1959) method in Szadujkis-Szadurski (1996) modification. Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963) in Kenakin (2006) modification.
Results: According to conducted testing, activation of soluble guanylyl cyclase with the use of YC-1 and 8Br cGMP caused reduced reaction of the tracheal smooth muscle with carbachol on average to 80%. Comparing concentration-effect curves for carbachol before and after the use of 8Br cGMP, similar results were obtained for those released by YC-1.
On the other hand, increasing concentrations of guanylyl cyclase inhibitor – ODQ cause shift of curves to the left, decrease of EC₅₀ value and an increase of maximum reaction to carbachol.
Conclusions: Carbachol, depending on concentration, causes tracheal smooth muscle contraction. According to testing, we can confirm that activation of guanylyl cyclase leads to reduction of the reaction of tracheal smooth muscle to carbachol on average up to 80%.

Key words:guanylyl cyclase • smooth muscle • trachea • carbachol • 8Br cGMP • ODQ • YC-1

Introduction

Nitric oxide (NO) induces airway smooth muscle cell (SMC) relaxation, but the underlying mechanism is not well under­stood. In the airways and lungs, nitric oxide (NO) is pro­duced by epithelial ciliated cells, type II alveolar cells, and neural fibers that innervate the airway smooth muscle cells (SMCs) [19]. The NO released by these cells decreases air­way resistance and that NO, released by neural fibers, is a major nonadrenergic, noncholinergic neurotransmitter re­sponsible for airway SMC relaxation [5]. Furthermore, the signaling cascade by which NO induces smooth muscle re­laxation has been mainly studied on vascular smooth mu­scle cells. In these blood vessels, NO is synthesized in the endothelial cells and diffuses to the adjacent SMCs, where it activates soluble guanylate cyclase (sGC) to synthesize cGMP. From these studies it is well known that cGMP acts as a second messenger to activate cGMP-dependent PKG and/or other effector proteins, including ion channels, ion pumps, and phosphodiesterases (PDEs) [6,14]. Thus cGMP were involved in the phosphorylation of one or more target molecules by PKG and/or a direct activation/inhibition of ion channels by cGMP are believed to lead to smooth mu­scle relaxation. For these reason, we investigated the ef­fects of ODQ an inhibitor and YC-1 an activator of soluble guanylyl cyclase (CG_s) on airway SMC contraction indu­ced by carbachol and we extended this study to investiga­te and compare the effects of ODQ and YC-1 with the ef­fect of 8Br cGMP on airway relaxation.

The subject of the study is determination of the activi­ty of the nitric oxide (NO)-independent activators of so­luble guanylyl cyclase activators YC-1 and selctive inhi­bitors ODQ in the modulation of the reaction of tracheal smooth muscle contraction. The study specified the influ­ence of increasing concentrations of guanylyl cyclase ac­tivators YC-1 and 8Br cGMP on the reaction of tracheal smooth muscle contraction released by carbachol. The au­thor also examined the effect of increasing concentrations of guanylyl cyclase inhibitor ODQ on the concentration­-responses curves for carbachol.

Natural activators of guanylyl cyclase include nitric oxide and carbon monoxide. Both nitric oxide (NO) and carbon monoxide (CO) activate soluble guanylyl cyclase, binding with the heme group of this enzyme [10,22]. NO is produ­ced through the conversion of l-arginine to citrulline by the enzyme NO synthase (NOS). In the lung, endothelial NOS (eNOS) is found in both vascular endothelium and airway epithelium [15]. CO shows significantly lower activity in this process than NO [24]. NO and cGMP have an impor­tant role in regulating pulmonary vascular tone and deve­lopment. NO produced by nitric oxide synthase (NOS) in pulmonary endothelial cells diffuses into subjacent smo­oth muscle cells (SMC) where it stimulates soluble gu­anylyl cyclase (sGC) to increase cGMP production [3,18].

Although cGMP interacts with several proteins in SMC, cGMP regulates pulmonary vascular tone primarily by sti­mulating cGMP-dependent protein kinase I (PKGI). Cyclic GMP-activated PKGI phosphorylates several cytosolic pro­tein targets that regulate intracellular Ca²⁺ levels, the cal­cium sensitivity of the contraction apparatus, and thin fi­lament proteins and thereby cause vasodilatation [3,18].

Smooth muscle contraction may be regulated by Ca2⁺ thro­ugh two pathways initiated by depolarization and agonist, respectively. Depolarization of the cell membrane activates voltage-gated Ca²⁺ channels resulting in Ca²⁺ influx, where­as agonist stimulation generally activates G protein-coupled receptors (GPCRs) leading to inositol 1,4,5-trisphosphate formation and Ca²⁺ release from the sarcoplasmic reticu­lum. The increase in cytosolic Ca²⁺ leads to smooth musc­le contraction through myosin light chain kinase (MLCK) activation by Ca²⁺/calmodulin and myosin regulatory light chain (RLC) phosphorylation. Additionally, activation of GPCRs leads to inactivation of MLCP by agonist-indu­ced protein kinase C (PKC) and RhoA/ROCK activation. These inhibitory mechanisms thus enhance RLC phospho­rylation and force development [23].

Tests, conducted in this study, indicate that in addition to the modulating component related to guanylyl cyclase in reaction of contraction to carbachol, there is a component independent of this nucleotide participating in this process.

Material and Methods

Testing was conducted on an isolated trachea of both se­xes of Wistar rats with weight ranging between 350 g and 450 g. Tracheas were prepared in accordance with the Akcasu method (1959) [1] in Szadujkis-Szadurski mo­dification (1996) [20]. A chain of combined segments of the trachea with 4 cm in length was placed in a dish for isolated organs. This dish was filled with Krebs fluid and oxidized, after previous addition of 5% CO₂. Reaction of the trachea was registered with the use of isotonic trans­ducer Biograf F-60 and recorded by polyphysiograph Narco Bio-System Narcotrace 40 (USA). Tested compo­unds were added directly to the dish, in which the tra­chea was placed.

Concentration-effect curves for tested agonists were de­termined with the use of traditional van Rossum’s phar­macometric method. EC₅₀ values were determined using the linear regression method for the range between 20% and 80% of reaction. The value of dissociation constant of agonist-receptor complex was determined with the use of the Furchgott and Bursztyn method in Kenakin T mo­dification (1997) [13].

The following reagents were used in testing: Carbachol, 8 Br cGMP (Beringher), 3-(5′-hydroxymethyl-2′-furyl)­-1-benzylindazole (YC-1), (Sigma), 1,2,4-oxodiazolo-[4,3­-a]quinoxalin-1-one (ODQ) (Sigma).

The experiments were carried out using of Krebs’ fluid (nor­mal) – PSS – composition: NaCl (71.8 mM/L), KCl (4.7 mM/L), CaCl₂ (1.7 mM/L), NaHCO₃ (28.4 mM/L), MgSO₄ (2.4 mM/L), KH₂PO₄ (1.2 mM/L), glucose (11.1 mM/L) with the addition of EGTA (30 µM/L).

Results

Carbachol in the range of concentrations between 10^-10 and 10^-6 M/l leads to tracheal smooth muscle contraction, de­pendent on concentration. The average EC₅₀ value was de­termined from concentration-effect curves for carbachol, amounting to 2.76 (±0.11)×10^-8 M/l for n=9.

Results are shown on Fig. 1 and 2.

Figure 1. Concentration-effect curves for carbachol 2.76 (±0.11)×10^-8. Points marked on the curve present average values and SE ± for n=9

Figure 2. Effect of increasing concentrations (from 10^-8 to 10^-7 [M/l]) 8Br cGMP on the concentration-effect curve for carbachol. Points marked on curves present average values and SE ± for n =9

Increasing concentration 8Br cGMP (10-100 µM/l) cau­ses shifting of the concentration -response curve to the right with the decrease of maximum reaction to carba­chol. Under the influence of this activity, EC₅₀ for carba­chol increases along with an increase of concentration 8Br cGMP. Average values EC₅₀ for carbachol are pre­sented in Table 1.

Table 1. Influence of carbachol on the reaction of tracheal smooth muscle contraction before and after the use of increasing concentrations 8Br cGMP

Increasing concentration YC-1(1-10 µM/l) causes shift of the curve into the right, with the decrease of maxi­mum reaction to carbachol. Under the influence of the activity, EC₅₀ for carbachol increases along with concen­tration YC-1 (Fig. 3). Average values EC₅₀ for carbachol are pre­sented in Table 2.

Figure 3. Effect of increasing concentrations (from 10^-8 to 10^-7 [M/l]) YC-1 on the concentration-effect curve for carbachol. Points marked on curves present average values and SE ± for n =9

Table 2. Influence of carbachol on the reaction of tracheal smooth muscle contraction before and after the use of increasing concentrations YC-1

Increasing concentration ODQ (10-100 µM/l) causes shift of the curve into the left with simultaneous increase of ma­ximum reaction to carbachol. Average value Em for carba­chol under the influence of ODQ increases by 31(±6.6)%. Under the presence of ODQ, the average value EC₅₀ for carbachol decreases from 2.76 (±0.11)×10^-8 M/l to 4.11 (±0.14)×10^-9 M/l (Fig. 4). Average results obtained in this series of experiments are summarized in Table 3.

Figure 4. Effect of increasing concentrations (from 10^-9 to 10^-8 [M/l]) ODQ on the concentration-effect curve for carbachol. Points marked on curves present average values and SE ± for n =9

Table 3. Influence of carbachol on the reaction of tracheal smooth muscle contraction before and after the use of increasing concentrations ODQ

Discussion

In the airways and lungs, nitric oxide (NO) is produced by epithelial ciliated cells, type II alveolar cells, and neural fibers that innervate the airway smooth muscle cells [19]. Earlier research on modulating activity of cyclical nucle­otides on the reaction of tracheal smooth muscle contrac­tion indicated that cAMP and analogy of this nucleotide cause both relaxation and reduce the reaction of tracheal smooth muscle to contracting agents. Similar activity mo­dulating smooth muscle contraction induces NO which ac­tivate of the soluble guanylyl cyclase and 8Br cGMP per­meable analog of cGMP [4,8,11,12].

According to presented research, 8 Br cGMP – induced concentration depending reduces the reaction of tracheal smooth muscle contraction induced by carbachol. Under the influence of this cyclic nucleotide, concentration-effect curves for carbachol shift to the right with simultaneous reduction of the effect of maximum reaction. From the analysis of concentration-effect curves for carbachol, de­termined before and after the use of 8Br cGMP, we can deduce that this nucleotide behaves like allosteric antago­nist. Further testing established that YC-1 selective acti­vator of soluble guanylyl cyclase modulates in a similar way reaction of the trachea to carbachol. Antagonistic ac­tion of YC-1 in relation to carbachol in accordance with the principles of the receptor theory also meets the condi­tions of allosteric antagonist [2,7,13,21].

Additional testing analyzed the influence of soluble gu­anylyl cyclase inhibitor – ODQ on the reaction of trache­al smooth muscle contraction induced by carbachol. The analysis of concentration responses curves (CRC) to car­bachol shows that the increasing concentrations of ODQ have a statistically significant effect on the shift of curves to the left with simultaneous reduction of EC₅₀ values and increase of maximum reaction to carbachol by 31(±6.6)% for n=9. The activity of ODQ is opposite to the activity of 8Br cGMP and YC-1 [9,11].

Increase of cGMP results in decrease of the tracheal smo­oth muscle contraction.

These results suggest a possibility of the use of activators of guanylyl cyclase in treatment of spastic bronchitis [8,16].

Conclusions

• Carbachol, depending on concentration, causes trache­al smooth muscle contraction.
• According to testing, we can confirm that activation of guanylate cyclase leads to reduction of the reaction of tracheal smooth muscle to carbachol on average up to 80%.
• Comparing concentration-effect curves for carbachol, before and after the use of 8Br cGMP, similar results were obtained for those released by YC-1.
• It indicates that regardless of modulating component re­lated to guanylate cyclase in reaction of contraction to carbachol, a component independent of this nucleotide participates in the process.
• Confirmation of participation of CG and cGMP in re­gulation of tracheal smooth muscle contraction released by carbachol was obtained by the use of inhibitor ODQ.

Acknowledgment

I would like to express my gratitude to Professor Leszek Szadujkis-Szadurski for his kindness, understanding, va­luable instructions and assistance in analysis of results and writing of my dissertation.

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The authors have no potential conflicts of interest to declare.

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