Streszczenie

Wstęp: Przedmiotem pracy jest określenie wpływu kalmoduliny oraz wapnia na skurcz mięśniówki gład­kiej dna żołądka. W przeprowadzonych doświadczeniach badano wpływ agonisty receptorów se­rotoninowych – serotoniny (5-HT), wywołującej skurcz mięśni gładkich.
Materiał/Metody: Badania przeprowadzono na tkankach wyizolowanych z żołądka szczura. Samce szczurów szcze­pu Wistar o masie 220-360 g usypiano uretanem (120 mg/kg m.c.) wstrzykiwanym dootrzewno­wo. Preparowano żołądek, po czym izolowano dno żołądka. Krzywe stężenie- efekt wyznacza­no metodą stężeń kumulowanych, zgodnie z metodą van Rossuma (1963) w modyfikacji Kenakin (2006).
Wyniki: Z przeprowadzonych badań wynika, że 8 Br cGMP hamuje reakcję skurczu mięśniówki gładkiej żołądka wyzwalaną serotoniną. Zastosowanie 8Br-cGMP w zakresie stężeń 10-300 µM powo­duje obniżenie maksymalnego efektu 100-46%. Podobne zmiany uzyskano po zastosowaniu ak­tywatora cyklazy guanylanowej (CG) – YC-1. Krzywe dla kurczącego działania serotoniny wraz ze wzrostem stężenia YC-1 ulegają przesunięciu w prawo, a efekt maksymalny reakcji obniża się. Wzrastające stężenia flunaryzyny – antagonisty kalmoduliny, w sposób zależny od stężenia hamuje wiązanie wapnia z kalmoduliną i jednocześnie powoduje przemieszczenie krzywych stę­żenie – efekt dla serotoniny w prawo oraz obniżenie maksymalnej reakcji. Wzrastające stężenia ODQ – inhibitora cyklazy guanylanowej w sposób istotny statystycznie po­wodują przesunięcie krzywych w lewo, obniżenie wartości EC₅₀ i jednocześnie podwyższenie maksymalnej reakcji na serotoninę.
Wnioski: Z przeprowadzonych badań wynika, że serotonina powoduje skurcz mięśniówki gładkiej żołąd­ka w sposób zależny od stężenia. Reakcja skurczu wyzwolonego serotoniną hamowana jest przez antagonistę kalmoduliny – flunaryzynę. Doświadczenia potwierdziły ponadto udział cyklicznych nukleotydów w hamowaniu reakcji skurczu dna żołądka.

Słowa kluczowe:skurcz mięśniówki gładkiej • dno żołądka • kalmodulina • wapń

Summary

Background: The subject of this study is determination of the influence of calmodulin and calcium on gastric fundus smooth muscle contraction. During experiments, the author tested the influence of a se­rotonin receptor agonist, serotonin (5-HT), causing smooth muscle contraction.
Material/Methods: Testing was conducted on tissues isolated from rat’s stomach. Male Wistar rats with weight be­tween 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). The stomach was dissected, and later the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid. Results contained in the stu­dy are average values ± SE. In order to determine statistical significance, the principles of recep­tor theory were used (Kenakin modification).
Results: According to conducted tests, we can deduce that 8 Br cGMP stops the reaction of gastric fun­dus smooth muscle contraction induced by serotonin. The use of 8Br-cGMP in the range of con­centrations between 10 and 300 µM leads to reduction of maximum effect from 100% to 46%. Similar changes were obtained after the use of guanylate cyclase activator (CG) – YC-1. Curves for the contractile activity of serotonin along with an increase of concentration YC-1 are shifted to the right, and the maximum effect of reaction decreases. Increasing concentrations of fluna­rizine, a calmodulin antagonist, in a concentration-dependent way blocks binding between cal­cium and calmodulin, and at the same time leads to the shift of concentration-effect curves for serotonin to the right and a decrease of maximum reaction. Increasing concentrations of ODQ, a guanylate cyclase inhibitor lead to statistically significant shift of the curves to the left, decrease of EC₅₀ value and simultaneous increase of maximum re­action to serotonin.
Conclusions: According to conducted testing, serotonin causes gastric fundus smooth muscle contraction de­pendent on concentration. Reaction of contraction induced by serotonin is stopped by a calmo­dulin antagonist, flunarizine. In addition, experiments confirmed participation of cyclical nucle­otides in blocking reaction of gastric fundus contraction.

Key words:smooth muscle contraction • gastric fundus • calmodulin • calcium

Introduction

The subject of this study is determination of the influence of calmodulin and calcium on gastric fundus smooth mu­scle contraction. Regulation of smooth muscle contraction is important for the course of many essential physiologi­cal mechanisms such as motor functions of the alimenta­ry canal, including the gastric fundus. The main mecha­nism causing smooth muscle contraction is an increase of the intracellular concentration of calcium ions, although sensitivity of the contractile apparatus can be subject to many modifications as a result of activity of specific ago­nists [7,8]. Reaction of contraction induced by serotonin can be modified by cGMP, cAMP, as well as factors bloc­king the release of calcium from the endoplasmic reticu­lum. In addition, contraction can be caused by an inflow of calcium ions from extracellular fluids to cytoplasm by canals located in the cell membrane. Diastole, however, is related to, among others, activation of guanylate cyclase receptors (CG) [1,2,6,7,12,13,14].

The study analyzed interaction between serotonin agonists, serotonin inducing smooth muscle contraction and cycli­cal nucleotide – 8Br cGMP, YC-1, ODQ (guanylate cycla­se inhibitor) and flunarizine [3,4,5,8,9,10,11,15].

Material and Methods

Testing was conducted on tissues isolated from rat’s sto­mach. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). The stomach was dissected, and la­ter the gastric fundus was isolated. Tissue was placed in a dish for insulated organs with 20 ml in capacity, filled with Krebs fluid [9,17].

Regardless of traditional Krebs fluid, tests were also using Krebs fluid deprived of calcium ions. The purpose of testing conducted in fluid without calcium ions was determination of intracellular role of calcium in released contraction [8,16].

Based on determined concentration-effect curves, con­stants were marked, specifying activity of the used prepa­rations: EC₅₀ and a dissociation constant of a given drug acting with receptor – Ka. Concentration-effect curves for tested agonists and antagonists were determined with the use of the Van Rosum method. The control curve of EC₅₀ value was determined based on 25 curves and under con­trolled conditions it amounts to 2.17×10^-6; it can serve as basis for recreation of the theoretical curve. Results con­tained in the study are average values ± SE. In order to de­termine statistical significance, the principles of receptor theory were used (Kenakin modification) [13].

The following reagents were used in testing: Serotonin (3-[2-Aminoethyl]-5-hydroxyindol), 8 Br cGMP (Beringher), YC-1 (Sigma) and 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

Serotonin (5HT) with concentration amounting to 3.2×10^-7 (±0.06) M/l releases concentration-dependent gastric fun­dus smooth muscle contraction. The use of 8Br-cGMP in the range of concentrations of 10, 30, 100 µM, respectively, causes decrease of maximum effect from 100% to 46%, with simultaneous shift of concentration-effect curves to the right. As a result of this activity, the value of EC50 for 5HT incre­ases from 3.2×10^-7 to 3.72×10^-6 (±0.23) uM/l, which means that the value of EC₅₀ increases approximately twelvefold (Table 1, Fig. 1).

Table 1. Determined values EC₅₀ ± SE and N for substances used during experiments conducted under controlled conditions and conditions of gastric fundus smooth muscle contraction

Figure 1. Concentration-effect curve for 5HT before and after the use of 8Br cGMP. Concentration-effect curve for 5HT after the use of various concentrations 8Br cGMP shifts to the right, as a result of which EC₅₀ increases to 6.7 µM/l ±0.17, 1.2 µM/l ±0.17, 3.72 µM/l ±0.17, for n=9

Just like in the case of using 8Br cGMP, the use of YC-1 in concentrations: 1 uM/l, 3 uM/l and 10 uM/l, causes de­crease of maximum effect from 100% to 38%, shift of con­centration-effect curves to the right, an increase of EC₅₀ va­lue, and decrease of maximum effect of reaction. Therefore, EC₅₀ value increased approximately elevenfold (Fig. 2).

Figure 2. Concentration-effect curve determined for contractile activity of 5-HT on the gastric fundus, determined before and after the use of YC-1; (EC₅₀ values for consecutive concentrations YC-1 – Tab. 1)

The author also analyzed activity of ODQ (10 uM, 30 uM, 100 uM) on contracted gastric fundus 5-HT (Fig.). EC₅₀ for serotonin after the use of increasing ODQ concentrations decreases respectively to the value EC50: 1.17×10^-6 [M/l], (±0.23), 6.61×10^-8 [M/l], (±0.16) and 3.79×10^-8 [M/l] (±0.14). In addition, after the use of ODQ, maximum re­action of Em value was increasing, respectively (Fig. 3).

Figure 3. Concentration-effect curve determined for contractile activity of 5-HT on the gastric fundus, determined before and after the activity of ODQ (EC₅₀ values for consecutive concentrations ODQ – Tab. 1)

The use of flunarizine with concentrations: 1 µM, 3 µM and 10 µM, causes decrease of maximum effect from 100% to 30%, shift of concentration-effect curves to the right, and an increase of EC₅₀ value to: 4.22×10^-8 [M/l] (±0.06), 7.11×10^-7 [M/l] (±0.22) and 2.97×10^-6 [M/l] (±0.17) (Fig. 4).

Figure 4. Concentration-effect curve determined for contractile activity of 5-HT on the gastric fundus, determined before and after the activity of flunarizine (EC₅₀ values for consecutive concentrations of flunarizine – Tab. 1)

Discussion

Calcium ions are essential for smooth muscle contractions. These contractions can be regulated through many various substances stimulating the calcium ions’ release from the cellular storages. Cyclic nucleotide analogs, such as cAMP and cGMP, which through the protein kinase regulate the function of the ion channels allowing smooth muscle con­tractions. Experiments conducted in earlier years showed the blocking influence of cGMP analogs on the reaction of smooth muscle contractions, including gastric fundus smo­oth muscle contractions. The author also tested the influ­ence of a serotonin receptor agonist, serotonin. According to tests, 8 Br cGMP stops the reaction of gastric fundus smooth muscle contraction induced by serotonin. Under the influence of this nucleotide, concentration-effect cu­rves for serotonin shift to the right with simultaneous de­crease of the effect of maximum reaction.

Similar changes in the shape of concentration-effect curves were obtained after the use of guanylate cyclase activator (CG)- YC-1. Curves for the contractile activity of seroto­nin along with an increase of YC-1 concentration shift to the right with simultaneous decrease of the effect of ma­ximum reaction [16,17].

Increasing concentrations of ODQ, a guanylate cyclase in­hibitor, have a statistically significant effect on the shift of curves to the left, decrease of EC₅₀ value and an increased maximum reaction to serotonin.

On the other hand, experiments conducted with flunari­zine showed that this calmodulin antagonist in a concen­tration-dependent way blocks binding of calcium with calmodulin, causing simultaneous shift of concentration­-effect curves for serotonin to the right and decrease of maximum reaction.

Conclusions

1. In testing conducted on preparation of the gastric fun­dus, serotonin causes concentration-dependent smooth muscle contraction.
2. Reaction of contraction induced by serotonin is blocked by flunarizine, a calmodulin antagonist.
3. YC-1 – CG activator and 8 BrcGMP cause similar chan­ges in reaction of the gastric fundus to serotonin.
4. Confirmation of participation of CG and cGMP in re­gulation of smooth muscle contraction induced by se­rotonin was obtained by using ODQ inhibitor.
5. Confirmation of participation of CG and cGMP in re­gulation of smooth muscle contraction induced by car­bachol was obtained by using ODQ inhibitor

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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