Streszczenie

Wstęp: Przedmiotem pracy jest określenie wpływu leków na skurcz mięśniówki gładkiej jelita krętego wyzwalany aktywacją receptorów muskarynowych typu M₁. Leki wpływające na receptory mu­skarynowe dzielą się na agonistów, leki o dużym powinowactwie do receptora i dużej aktywno­ści wewnętrznej oraz antagonistów, niemających aktywności wewnętrznej. W przeprowadzonych doświadczeniach badano interakcje między szerokospektralnym agonistą receptorów muskaryno­wych – karbacholem a selektywnym antagonistą receptorów muskarynowych M₁ – pirenzepiną.
Materiał/Metody: Badania przeprowadzono na tkankach wyizolowanych z jelita szczura. Samce szczurów szcze­pu Wistar o masie 220-360 g usypiano uretanem (120 mg/kgm.c.) wstrzykiwanym dootrzewno­wo. Krzywe stężenie – efekt wyznaczano metodą stężeń kumulowanych, zgodnie z metodą van Rossuma (1963) w modyfikacji Kenakin (2006).
Wyniki: Celem pracy było wyznaczenie krzywych stężenie – efekt dla karbacholu. Krzywa ta została po­równana z krzywą zajęcia receptorów w zależności od stężenia tego leku. Na podstawie uzyska­nych krzywych stężenie – efekt obliczono średnią wartość EC₅₀ dla karbacholu, która wyniosła 2,44×10^-6 [M/l].
Wnioski: Uzyskane wyniki potwierdziły, że atropina skutecznie hamuje skurcze wywołane karbacholem, spełnia więc warunki stawiane antagonistom kompetycyjnym. Atropina powodowała przesunię­cie krzywych dla karbacholu w prawo. Pirenzepina, selektywnie blokująca receptory muskary­nowe typu M₁ dała podobne rezultaty. Dowiedziono, że w preparacie mięśniówki gładkiej dna żołądka, receptory typu M₁ występują nie tylko presynaptycznie, lecz także postsynaptycznie.

Słowa kluczowe:mięśniówka gładka • jelito kręte • receptory muskarynowe • atropina • pirenzepina

Summary

Background: The subject of the study was determination of the effect of drugs on ileal smooth muscle con­traction induced by activation of M₁ type muscarinic receptors. Drugs that have an effect on mu­scarinic receptors are divided to agonists, with close ties to the receptor and high internal activi­ty and antagonists, with no internal activity. Conducted experiments tested interactions between a broad-spectrum agonist of muscarinic receptors, carbachol and a selective muscarinic receptor antagonist of M₁ type, pirenzepine.
Material/Methods: Testing was conducted on tissues isolated from rat’s intestine. Male Wistar rats with weight be­tween 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963) in Kenakin modification (2006).
Results: The purpose of the study was determination of concentration-effect curves for carbachol. This curve was compared with the curve of receptor occupation depending on concentration of this drug. Based on concentration-effect curves, the average value of EC₅₀ was calculated for carba­chol, amounting to 2.44×10^-6 [M/l].
Conclusions: The results confirmed that atropine is effective in stopping contractions caused by carbachol, meeting the conditions of competitive antagonists. Atropine caused the shift of curves for carba­chol to the right. Pirenzepine, selectively blocking muscarinic receptors of M₁ type gave similar results. It was proved that in the preparation of gastric fundus smooth muscle, M₁ type receptors occur not only presynaptically, but also postsynaptically.

Key words:smooth muscle • ileum • muscarinic receptors • atropine • pirenzepine

Introduction

The subject of the study was determination of the ef­fect of drugs on ileal smooth muscle contraction indu­ced by activation of M₁ type muscarinic receptors. Drugs that have an effect on muscarinic receptors are divided to agonists, with close ties to the receptor and high in­ternal activity and antagonists, with no internal activi­ty [1,4]. Conducted experiments tested interactions be­tween a broad-spectrum agonist of muscarinic receptors, carbachol and a selective muscarinic receptor antagonist of M₁ type, pirenzepine.

In accordance with the receptor theory, the condition for activity of drug is its reaction with the cell protein (recep­tor), resulting in changes in its activity. Receptor means unique places of bonding of drug with the cell, which in­termediate in activity of the drug. They can be found on the surface of the cell or inside cytoplasm [9]. Muscarinic receptors M₁ are frequently called neuronal receptors. They occur on parietal cells, in the central and peripheral nervo­us system. M₁ receptors release stimulation effects; one of the examples is slow muscarinic stimulation depending on acetylcholine in sympathetic ganglions and central neurons [9,11,12]. This simulation is caused by drop in conduction for potassium ions, resulting in depolarization of the neu­ronal membrane. Muscarinic receptors M₁ also stimulate secretion of hydrochloric acid in the stomach after vagus nerve stimulation [2,9,15].

Material and Methods

Testing was conducted on tissues isolated from rat’s in­testine. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg). The intestine was dissected under anesthetic; after dissection it was cut out and placed in a dish for insulated organs with 20 ml in capacity, filled with oxidized Krebs fluid. Content of Krebs fluid: 71.8 mM NaCl, 4.7 mM KCl, 1.7 mM CaCl₂, 28.4 mM NaHCO₃, 11.7 mM glucose, 2.4 mM MgSO₄, 1.2 mM KH ₂PO₄. A series of testing with inadequate blood supply was con­ducted under controlled conditions. The intestine was dis­sected and arteries were pressed with Klem clamps for a period of 30 minutes, in order to induce ischemia; after that, Klem clamps were removed and the intestine was dissected after 90 minutes.

In addition to that testing, another one was performed; after 30 minutes from closing blood vessels vascularizing the intestine, Klem tools were used to dissect the organ. After that, it was placed in oxidized Krebs fluid, followed by analysis of reaction. Testing was carried out 30 minu­tes after closing dishes and 90 minutes after reperfusion of the dish. Preparations were added to the dish in the amo­unt between 0.1 ml and 0.3 ml.

Concentration-effect curves for tested agonists and anta­gonists were determined with the use of the Van Rosum method – concentrations increasing every 0.5 log. Based on these concentration-effect curves, constants were de­termined for tested agonists, specifying activity of a given preparation, i.e. EC₅₀. The control curve of EC₅₀ value was determined based on 25 curves and under controlled con­ditions it amounts to 2.44(±0.11) ×10^-7; it can serve as ba­sis for recreation of the theoretical curve.

Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963) in Kenakin modification (2006).

Results

Carbachol, in the range of concentrations between 10^-8 and 10^-3, causes ileal smooth muscle contraction that depends on concentration. Concentration-effect curves for carba­chol were used for determination of the average value of EC₅₀, which amounts to 2.44(±0.11)×10^-7 for n=9. Results are presented on Fig. 1 and in Table 1.

Figure 1. Concentration-effect curve for carbachol and RA/RT dependence curve for carbachol. Constant K_A 1.26(±0.09)×10^-5. Points marked on the curve present average values and SE for n=9

Table 1. Influence of carbachol on the reaction of ileal smooth muscle contraction before and after the use of increasing concentrations of pirenzepine

A series of experiments made with dibenamine, an irre­versible antagonist, was used for determination of Ka, a dissociation constant, which amounts to 1.26(±0.09)×10^-5 for n=9. This constant was used for determination of a cu­rve, presenting dependence between%RA/RT and concen­tration of carbachol (Fig. 1).

Pirenzepine, a relatively selective receptor antagonist of M₁ type, in the range of concentrations between 10^-7 and 10^-6 [M/l], causes concentration-dependent shift of concentra­tion-effect curve for carbachol to the right, maintaining ma­ximum reaction. Based on these curves, EC₅₀ values were determined in the presence of increasing concentrations of pirenzepine. Results are shown in Table 1. The average for n=9 is shown by concentration-effect curve for carba­chol in the presence of pirenzepine in concentrations be­tween 10^-7 and 10^-6, presented on Fig. 2.

Figure 2. Influence of increasing concentrations (from 10^-7 to 10^-6 [M/l]) of pirenzepine on concentration-effect curve for carbachol. Points marked on the curve present average values and SE ± for n =9

The use of pirenzepine, a competitive receptor agonist M₁, causes shift of concentration-effect curve (for carbachol) to the right, maintaining maximum reaction (Fig. 3).

Figure 3. Presentation of concentration-effect curve determined for antagonistic activity of pirenzepine on ileal smooth muscle. The average value of IC₅₀ pirenzepine for n=9 amounts to 1.89(±0.16)×10^-8 [M/l]

According to analysis of curves, we can deduce that pirenze­pine meets the conditions posed to competitive antagonists, whereas the average value of IC₅₀ determined for pirenze­pine amounts to 1.89×10^-9 [M/l]. Fig. 3 presents concen­tration-effect curve for antagonistic activity of pirenzepine in relation to a non-selective muscarinic receptor agonist and carbachol.

Atropine, as a non-selective muscarinic receptor antago­nist, causes concentration-dependent shift of concentra­tion-effect curve (for carbachol) to the right, maintaining maximum reaction. According to the determined curve, we can deduce that atropine meets the conditions posed to competitive antagonists (Fig. 4).

Figure 4. Presentation of concentration-effect curve determined for antagonistic activity of atropine on ileal smooth muscle. The average value of IC₅₀ atropine for n=9 amounts to 1.76(±0.08)×10^-8 [M/l]

Discussion

According to testing conducted to date, we can confirm that rat’s ileum contains muscarinic receptors. Existence of the­se receptors acknowledges antagonistic activity of atropine, a non-selective muscarinic receptor antagonist [13,14,15]. According to presented data, pirenzepine, a relatively se­lective receptor antagonist shows activity similar to atro­pine. According to analysis of curves and EC₅₀ values for carbachol determined during experiments, we can deduce that pirenzepine meets the conditions of competitive an­tagonism in relation to carbachol, a non-selective musca­rinic receptor agonist. The IC₅₀ value for pirenzepine, de­termined during experiments, amounts to 1.89(±0.16)×10^-8 [M/l]. During testing conducted in the laboratory on an in­sulated gastric fundus with darifenacin, it was determined that M3 type receptor occurs in smooth muscle and inter­mediates in contraction release. IC₅₀ value for darifena­cin amounts to 3.89(±0.12)×10^-8 [M/l]. In addition, accor­ding to testing conducted to date, it was determined that M₁ type muscarinic receptors occur mainly on ends of the parasympathetic nervous system and fulfill a function mo­dulating secretion of acetylcholine [3,10,16]. Activation of these receptors causes release of acetylcholine from ends of the parasympathetic nervous system. During testing, it was determined that M₁ type receptor occurs in smooth muscle and intermediates in contraction release [8,11,12].

Conclusions

• Carbachol in concentrations between 10^-7 and 10^-6 [M/l] causes concentration-dependent isometric ileal smooth muscle contraction.
• Atropine, a non-selective muscarinic receptor antago­nist, meets the conditions of competitive antagonists.
• Pirenzepine, a relatively selective muscarinic receptor antagonist of M₁ type, breaks contraction induced by carbachol and meets the conditions posed to competiti­ve antagonists.
• The results suggest occurrence of M₁ type receptors in rat’s ileum.

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.

REFERENCES

[1] Aihara T., Nakamura Y., Taketo M.M., Matsui M., Okabe S.: Cholinergically stimulated gastric acid secretion is mediated by M₃ and M₅ but not M₁ muscarinic acetylcholine receptors in mice. Am. J. Physiol. Gastrointest. Liver Physiol., 2005; 288: G1199-G1207
[PubMed]  [Full Text PDF]  

[2] Aleksander S.P., Mathie A., Peters J.A.: Guide to receptors and channels (GRAC), 2^nd edition (2007 revision). Br. J. Pharmacol., 2007; 150 (Suppl. 1): S6-S7
[PubMed]  [Full Text PDF]  

[3] Ashkenazi A., Peralta E.G., Winslow J.W., Ramachandran J., Capon D.J.: Functional diversity of muscarinic receptor subtypes in cellular signal transduction and growth. Trends Pharmacol. Sci., 1989; Suppl.: 16-22
[PubMed]  

[4] Billington C.K., Penn R.B.: m3 muscarinic acetylcholine receptor regulation in the airway. Am. J. Respir. Cell Mol. Biol., 2002; 26: 269-272
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[5] Braverman A.S., Tibb A.S., Ruggieri M.R.Sr.: M₂ and M₃ muscarinic receptor activation of urinary bladder contractile signal transduction. I. Normal rat bladder. J. Pharmacol. Exp. Ther., 2006; 316: 869-874
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[6] Cabrera-Vera T.M., Vanhauwe J., Thomas T.O., Medkova M., Preininger A., Mazzoni M.R., Hamm H.E.: Insights into G protein structure, function, and regulation. Endocr. Rev., 2003; 24: 765-781
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[7] Caulfield M.P., Birdsall N.J.: International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol. Rev., 1998; 50: 279-290
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[8] Felder C.C.: Muscarinic acetylcholine receptors: signal transduction through multiple effectors. FASEB J., 1995; 9: 619-625
[PubMed]  [Full Text PDF]  

[9] Jacob L.S.: Pharmacology. Wydanie I polskie pod red. M. Wilimowskiego, Urban & Partner Wyd. Med. Wrocław 1994; 1: 1-17

[10] Jaffrey S.R., Snyder S.H.: Nitric oxide: a neural messenger. Annu. Rev. Cell Dev. Biol., 1995; 11: 417-440
[PubMed]  

[11] Karlin A.: Structure of nicotinic acetylcholine receptors. Curr. Opin. Neurobiol., 1993; 3: 299-309
[PubMed]  

[12] Kenakin T.: Inverse, protean, and ligand-selective agonism: matters of receptor conformation. FASEB J., 2001; 15: 598-611
[PubMed]  [Full Text HTML]  [Full Text PDF]  

[13] Waelbroeck M., Tastenoy M., Camus J., Christophe J.: Binding of selective antagonists to four muscarinic receptors (M₁ to M₄) in rat forebrain. Mol. Pharmacol., 1990; 38: 267-273
[PubMed]  

[14] Wess J.: Molecular biology of muscarinic acetylcholine receptors. Crit. Rev. Neurobiol., 1996; 10: 69-99
[PubMed]  

[15] Wielosz M.: Przekaźnictwo noradrenergiczne. In: Farmakologia kliniczna, 1995: 136

[16] Xie G., Drachenberg C., Yamada M., Wess J., Raufman J.P.: Cholinergic agonist-induced pepsinogen secretion from murine gastric chief cells is mediated by M1 and M3 muscarinic receptors. Am. J. Physiol. Gastrointest. Liver Physiol., 2005; 289: G521-G529
[PubMed]  [Full Text HTML]  [Full Text PDF]  

The authors have no potential conflicts of interest to declare.

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