Postepy Hig Med Dosw. (online), 2011; 65: 584-587
Original Article
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Role of M1 receptor in regulation of gastric fundus smooth muscle contraction
Rola receptora M1 w regulacji mięśniówki gładkiej dna żołądka
Marta GajdusBCDE, Katarzyna Szadujkis-SzadurskaF, Leszek Szadujkis-SzadurskiAC, Izabela GlazaBD, Rafał Szadujkis-SzadurskiC, Joanna OlkowskaF
Department of Pharmacology and Therapy, Collegium Medicum in Bydgoszcz
Corresponding author
MSc Marta Gajdus, ul. Harcerska 14c/9, 87-100 Toruń; e-mail: gajdi@o2.pl

Authors' Contribution:
A - Study Design, B - Data Collection, C - Statistical Analysis, D - Data Interpretation, E - Manuscript Preparation, F - Literature Search, G - Funds Collection

Received:  2011.06.27
Accepted:  2011.08.09
Published:  2011.09.14

Streszczenie
Wstęp: Przedmiotem pracy jest określenie wpływu leków na skurcz mięśniówki gładkiej dna żołądka wyzwalany aktywacją receptorów muskarynowych M1. W przeprowadzonych doświadczeniach badane były interakcje między agonistą receptorów muskarynowych - karbacholem a antagoni­stami receptorów muskarynowych: atropiną oraz pirenzepiną.
Wyniki: Z przeprowadzonych badań wynika, że karbachol, w stężeniu 10-8-10-4 M, w sposób zależny od dawki wyzwala skurcz mięśniówki gładkiej dna żołądka. Przedstawione wyniki wskazują, że kar­bachol spełnia warunki stawiane pełnym agonistom. Atropina natomiast, nieselektywny antagonista receptorów muskarynowych, powoduje przesu­nięcie krzywej stężenie-efekt (dla karbacholu) w sposób zależny od stężenia w prawo, z zacho­waniem maksymalnej reakcji. Na podstawie wyznaczonej krzywej można stwierdzić, że atropi­na spełnia wszystkie warunki stawiane antagonistom kompetycyjnym. Zastosowanie pirenzepiny, agonisty kompetycyjnego receptorów M1, powoduje przesunięcie krzy­wej stężenie-efekt (dla karbacholu) w prawo, z zachowaniem maksymalnej reakcji.
Wnioski: Z przeprowadzonych badań na preparacie dna żołądka wynika, że atropina powoduje przesunię­cie krzywych stężenie-efekt w prawo (dla karbacholu). Podobny efekt wyzwala pirenzepina se­lektywnie blokująca receptory muskarynowe typu M1. Uzyskane wyniki wskazują, iż w prepa­racie mięśniówki gładkiej dna żołądka, receptory typu M1 występują także postsynaptycznie.
Słowa kluczowe: skurcz mięśniówki gładkiej • dno żołądka • karbachol • pirenzepina


Summary
Background: The subject of this study is determination of the influence of drugs on gastric fundus smooth mu­scle contraction induced by activation of muscarinic receptors M1. Experiments tested interac­tions between a receptor agonist, carbachol and muscarinic receptor antagonists, atropine and pirenzepine.
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). br>Results: According to tests, carbachol, in concentrations ranging between 10-8 M to 10-4 M, in a dosage­-dependent way induces gastric fundus smooth muscle contraction. Presented results indicate that carbachol meets the conditions posed to full agonists. On the other hand, atropine, a non-selec­tive muscarinic receptor antagonist, causes a concentration-dependent shift of concentration-ef­fect curve (for carbachol) to the right, maintaining maximum reaction. According to analysis of the curve determined, we can deduce that atropine meets the conditions posed to competitive an­tagonists. The use of pirenzepine, a competitive receptor agonist M1, causes shift of concentra­tion-effect curve (for carbachol) to the right, maintaining maximum reaction.
Conclusions: From the testing conducted on the preparation of the gastric fundus we can deduce that atropi­ne causes shift of concentration-effect curves for carbachol to the right. A similar effect is re­leased by pirenzepine, selectively blocking muscarinic receptors of M1 type. The results indi­cate that in the preparation of the gastric fundus smooth muscle, M1 type receptors occur also postsynaptically.
Key words: smooth muscle contraction • gastric fundus • carbachol • pirenzepine




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 [2,3,8,10]. 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) [1,5,9].
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 [4,6,7].
Material and Methods
Testing was conducted on male Wistar rats with we­ight between 220 g and 360 g. Animals were anestheti­zed with urethane. 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.
Regardless of traditional Krebs fluid, tests were also using Krebs fluid deprived of calcium ions. Tested preparations were added to the dish in the amount between 0.1 and 0.3 ml. The purpose of testing conducted in fluid without cal­cium ions was determination of intracellular role of cal­cium in released contraction. This process can take place through two mechanisms. The first one consists in activa­tion of IP3 receptors, whereas the second one involves ac­tivation of ryanodine receptors with the use of ryanodine or caffeine. In testing conducted in calcium fluid, intracel­lular calcium pool was eliminated by using Ca-ATPase in­hibitor, cyclopiazonic acid or Thapsigargin.
An inflow of calcium to the cell takes place by passive dif­fusion, in accordance with concentration gradient, through two types of channels, VOC - Voltage Operated Calcium Channel (controlled by potential on the cell membrane) and ROC - Receptor Operating Calcium Channel (control­led by receptors). Opening of VOC channels and closing for a flow of calcium ions depend on the volume of elec­tric potential flowing to them, namely on the level of their polarization. A phenomenon of opening of calcium chan­nels as a result of their depolarization is called electrome­chanical coupling. Opening of ROC channels depends on specific receptors located on these channels, meaning me­chanism called pharmacomechanical coupling.
Based on concentration-effect curves determined for te­sted agonists, constants were marked, specifying activity of the preparations used, including:
• EC50 (concentration releasing 50% of maximum reaction),
• dissociation constant of a given drug acting with Ka receptor.
Concentration-effect curves for tested agonists and antago­nists were determined with the use of the van Rossum me­thod (increasing concentrations, every half of logarithm).
Results
Carbachol, in the range of concentrations between 10-8 M and 10-4 M, in a dosage-dependent way induces gastric fundus smooth muscle contraction (Fig. 1). The average value of EC50 for carbachol amounts to 2.17×10-6 M/for n=25. The chart also shows Ka constant for carbachol, de­termined based on 25 concentration-effect curves after ir­reversible inactivation of reserve receptors by phenoxy­benzamine 10-7 M/l. The average value of Ka constant for carbachol amounts to 1.26×10-5.M/l. Fig. 1 presents depen­dence curve between% RA/Rt and carbachol concentration. Comparing concentration-effect curve with RA/Rt/carba­chol concentration dependence curve, we can confirm that the curve presenting% of occupied receptors is shifted to the right. Presented results indicate that carbachol meets the conditions posed to full agonists. In addition, presen­ted data confirms that there is a reserve receptor pool in the tested preparation of the gastric fundus.
Figure 1. Concentration-effect curve determined for contractile activity of carbachol on the gastric fundus smooth muscle and%RA/Rt/carbachol concentration dependence curve

On the other hand, atropine, a non-selective muscarinic re­ceptor antagonist, causes a concentration-dependent shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction. According to analysis of the curve determined, we can deduce that atropine meets the conditions posed to competitive antagonists. Fig. 2 pre­sents concentration-effect dependence curve for antagoni­stic activity of atropine on gastric smooth muscle. Based on curves determined, the average IC50 value for atropine amounts to 1.76×10-8 M/l.
Figure 2. Presentation of concentration-effect curve determined for antagonistic activity of atropine on gastric smooth muscle. The average IC50 value for atropine for n=9 amounts to 1.76 (±0.08)×10-8 M/l

The use of pirenzepine, a competitive receptor agonist M1, causes shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction. According to analysis of the curve determined, we can deduce that piren­zepine meets the conditions posed to competitive antago­nists, and the average IC50 value for pirenzepine amounts to 4.89×10-9 M/l. Fig. 3 presents concentration-effect cu­rve for antagonistic activity of pirenzepine in relation of a non-selective muscarinic receptor agonist and carbachol.
Figure 3. Presentation of concentration-effect curve for activity of pirenzepine on gastric fundus smooth muscle. The average IC50 value for pirenzepine for n=9 amounts to 4.89 (±0.08)×10-8 M/l

Discussion
According to the analysis conducted, in compliance with the principles of the receptor theory, we can deduce that atropine, a non-selective muscarinic receptor antagonist, stops effectively contractions induced by carbachol. It should be remembered that carbachol is also a non-selec­tive antagonist.
Analyzing the curves, in compliance with the principles of the receptor theory, we can deduce that atropine meets the conditions posed to competitive antagonist, and IC50 for this agonist amounts to 1.91×10-8 M/l.
Results obtained from testing with the use of atropine were compared with the activity of pirenzepine, a selec­tive antagonist blocking M1 type muscarinic receptors. It is known that receptors occur primarily on synaptic ends. In addition, they induce contractions. The results indicate that M1 receptors occur not only presynaptically, but also postsynaptically.
Conclusions
1. In testing conducted on preparation of the gastric fun­dus, atropine causes shift of concentration-effect curves (for carbachol) to the right.
2. A similar effect is induced by pirenzepine, selectively blocking M1 type muscarinic receptors.
3. The results indicate that in the preparation of the ga­stric fundus smooth muscle, M1 type receptors occur also postsynaptically.
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The authors have no potential conflicts of interest to declare.