Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder

Ahmet Akici, Atila Karaalp, Ece Iskender, Arthur Christopoulos, Esam E. El-Fakahany, Şule Oktay

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Previous studies have suggested the presence of multiple muscarinic receptor subtypes in guinea pig gallbladder smooth muscle, although the relative abundance and functional role of these subtypes remains an area of significant research efforts. The present study utilized both radioligand kinetic and functional experiments to further probe the nature of the muscarinic receptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca2+ sources. Dissociation kinetic studies using [3H]N-methylscopolamine ([3H]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel experiments conducted on the cloned muscarinic M1-M5 subtypes in Chinese hamster ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [3H]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladder, but not the gallbladder, demonstrating biphasic radioligand dissociation kinetics. In functional experiments, carbachol caused a concentration-dependent contraction of guinea pig gallbladder smooth muscle strips in Ca2+-free or 5 mM Sr2+-substituted physiological salt solutions (PSS) with amplitudes of the maximal contractions corresponding to 45.8±8.0% and 33.2±6.6% of control responses in normal PSS, respectively. Furthermore, the stimulus-response characteristics of carbachol-mediated contraction appeared significantly altered in Ca2+-free PSS relative to normal or Sr2+-substituted PSS. The antagonist, methoctramine (1x10-7-3x10-5 M), exerted only a slight inhibition of carbachol (10-5 M)-induced contractions in 5 mM Sr2+-substituted medium, whereas it was significantly more potent in antagonizing gallbladder contractions in response to 10-5 M carbachol in the absence of extracellular Ca2+. Both atropine and tripitramine were equipotent in antagonizing carbachol-induced contractions in Ca2+-free (pIC50: 6.85±0.11 for atropine and 5.75±0.32 for tripitramine) and Sr2+-substituted media (pIC50: 6.88±0.25 for atropine and 5.70±0.16 for tripitramine), and pirenzepine was only slightly more potent in Ca2+-free PSS (pIC50: 5.66±0.23) than in Sr2+-substituted PSS (pIC50: 5.33±0.21). Taken together, our data indicate that carbachol contracts guinea pig gallbladder by stimulating two distinct muscarinic receptor subtypes linked to extracellular Ca2+ influx and intracellular Ca2+ release. These two subtypes may represent the muscarinic M3 and M4 receptors, although the presence of the muscarinic M2 receptor subtype is also suggested from the binding data. Copyright (C) 2000 Elsevier Science B.V.

Original languageEnglish
Pages (from-to)115-123
Number of pages9
JournalEuropean Journal of Pharmacology
Volume388
Issue number1
DOIs
Publication statusPublished - 24 Jan 2000
Externally publishedYes

Keywords

  • Ca
  • Extracellular
  • Gallbladder
  • Guinea pig
  • Intracellular
  • Kinetics
  • Muscarinic receptor
  • Radioligand binding
  • Receptor heterogeneity
  • Sr

Cite this

Akici, Ahmet ; Karaalp, Atila ; Iskender, Ece ; Christopoulos, Arthur ; El-Fakahany, Esam E. ; Oktay, Şule. / Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder. In: European Journal of Pharmacology. 2000 ; Vol. 388, No. 1. pp. 115-123.
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title = "Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder",
abstract = "Previous studies have suggested the presence of multiple muscarinic receptor subtypes in guinea pig gallbladder smooth muscle, although the relative abundance and functional role of these subtypes remains an area of significant research efforts. The present study utilized both radioligand kinetic and functional experiments to further probe the nature of the muscarinic receptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca2+ sources. Dissociation kinetic studies using [3H]N-methylscopolamine ([3H]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel experiments conducted on the cloned muscarinic M1-M5 subtypes in Chinese hamster ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [3H]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladder, but not the gallbladder, demonstrating biphasic radioligand dissociation kinetics. In functional experiments, carbachol caused a concentration-dependent contraction of guinea pig gallbladder smooth muscle strips in Ca2+-free or 5 mM Sr2+-substituted physiological salt solutions (PSS) with amplitudes of the maximal contractions corresponding to 45.8±8.0{\%} and 33.2±6.6{\%} of control responses in normal PSS, respectively. Furthermore, the stimulus-response characteristics of carbachol-mediated contraction appeared significantly altered in Ca2+-free PSS relative to normal or Sr2+-substituted PSS. The antagonist, methoctramine (1x10-7-3x10-5 M), exerted only a slight inhibition of carbachol (10-5 M)-induced contractions in 5 mM Sr2+-substituted medium, whereas it was significantly more potent in antagonizing gallbladder contractions in response to 10-5 M carbachol in the absence of extracellular Ca2+. Both atropine and tripitramine were equipotent in antagonizing carbachol-induced contractions in Ca2+-free (pIC50: 6.85±0.11 for atropine and 5.75±0.32 for tripitramine) and Sr2+-substituted media (pIC50: 6.88±0.25 for atropine and 5.70±0.16 for tripitramine), and pirenzepine was only slightly more potent in Ca2+-free PSS (pIC50: 5.66±0.23) than in Sr2+-substituted PSS (pIC50: 5.33±0.21). Taken together, our data indicate that carbachol contracts guinea pig gallbladder by stimulating two distinct muscarinic receptor subtypes linked to extracellular Ca2+ influx and intracellular Ca2+ release. These two subtypes may represent the muscarinic M3 and M4 receptors, although the presence of the muscarinic M2 receptor subtype is also suggested from the binding data. Copyright (C) 2000 Elsevier Science B.V.",
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Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder. / Akici, Ahmet; Karaalp, Atila; Iskender, Ece; Christopoulos, Arthur; El-Fakahany, Esam E.; Oktay, Şule.

In: European Journal of Pharmacology, Vol. 388, No. 1, 24.01.2000, p. 115-123.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder

AU - Akici, Ahmet

AU - Karaalp, Atila

AU - Iskender, Ece

AU - Christopoulos, Arthur

AU - El-Fakahany, Esam E.

AU - Oktay, Şule

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N2 - Previous studies have suggested the presence of multiple muscarinic receptor subtypes in guinea pig gallbladder smooth muscle, although the relative abundance and functional role of these subtypes remains an area of significant research efforts. The present study utilized both radioligand kinetic and functional experiments to further probe the nature of the muscarinic receptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca2+ sources. Dissociation kinetic studies using [3H]N-methylscopolamine ([3H]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel experiments conducted on the cloned muscarinic M1-M5 subtypes in Chinese hamster ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [3H]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladder, but not the gallbladder, demonstrating biphasic radioligand dissociation kinetics. In functional experiments, carbachol caused a concentration-dependent contraction of guinea pig gallbladder smooth muscle strips in Ca2+-free or 5 mM Sr2+-substituted physiological salt solutions (PSS) with amplitudes of the maximal contractions corresponding to 45.8±8.0% and 33.2±6.6% of control responses in normal PSS, respectively. Furthermore, the stimulus-response characteristics of carbachol-mediated contraction appeared significantly altered in Ca2+-free PSS relative to normal or Sr2+-substituted PSS. The antagonist, methoctramine (1x10-7-3x10-5 M), exerted only a slight inhibition of carbachol (10-5 M)-induced contractions in 5 mM Sr2+-substituted medium, whereas it was significantly more potent in antagonizing gallbladder contractions in response to 10-5 M carbachol in the absence of extracellular Ca2+. Both atropine and tripitramine were equipotent in antagonizing carbachol-induced contractions in Ca2+-free (pIC50: 6.85±0.11 for atropine and 5.75±0.32 for tripitramine) and Sr2+-substituted media (pIC50: 6.88±0.25 for atropine and 5.70±0.16 for tripitramine), and pirenzepine was only slightly more potent in Ca2+-free PSS (pIC50: 5.66±0.23) than in Sr2+-substituted PSS (pIC50: 5.33±0.21). Taken together, our data indicate that carbachol contracts guinea pig gallbladder by stimulating two distinct muscarinic receptor subtypes linked to extracellular Ca2+ influx and intracellular Ca2+ release. These two subtypes may represent the muscarinic M3 and M4 receptors, although the presence of the muscarinic M2 receptor subtype is also suggested from the binding data. Copyright (C) 2000 Elsevier Science B.V.

AB - Previous studies have suggested the presence of multiple muscarinic receptor subtypes in guinea pig gallbladder smooth muscle, although the relative abundance and functional role of these subtypes remains an area of significant research efforts. The present study utilized both radioligand kinetic and functional experiments to further probe the nature of the muscarinic receptors in gallbladder smooth muscle and their mode of coupling to intra- and extra-cellular Ca2+ sources. Dissociation kinetic studies using [3H]N-methylscopolamine ([3H]NMS) indicated that the binding profile in guinea pig gallbladder smooth muscle could not be reconciled with that expected for a single muscarinic receptor subtype, the latter determined in parallel experiments conducted on the cloned muscarinic M1-M5 subtypes in Chinese hamster ovary (CHO) cells. Furthermore, comparison of the gallbladder data with the dissociation characteristics of [3H]NMS in guinea pig urinary bladder revealed a significantly different kinetic profile, with the urinary bladder, but not the gallbladder, demonstrating biphasic radioligand dissociation kinetics. In functional experiments, carbachol caused a concentration-dependent contraction of guinea pig gallbladder smooth muscle strips in Ca2+-free or 5 mM Sr2+-substituted physiological salt solutions (PSS) with amplitudes of the maximal contractions corresponding to 45.8±8.0% and 33.2±6.6% of control responses in normal PSS, respectively. Furthermore, the stimulus-response characteristics of carbachol-mediated contraction appeared significantly altered in Ca2+-free PSS relative to normal or Sr2+-substituted PSS. The antagonist, methoctramine (1x10-7-3x10-5 M), exerted only a slight inhibition of carbachol (10-5 M)-induced contractions in 5 mM Sr2+-substituted medium, whereas it was significantly more potent in antagonizing gallbladder contractions in response to 10-5 M carbachol in the absence of extracellular Ca2+. Both atropine and tripitramine were equipotent in antagonizing carbachol-induced contractions in Ca2+-free (pIC50: 6.85±0.11 for atropine and 5.75±0.32 for tripitramine) and Sr2+-substituted media (pIC50: 6.88±0.25 for atropine and 5.70±0.16 for tripitramine), and pirenzepine was only slightly more potent in Ca2+-free PSS (pIC50: 5.66±0.23) than in Sr2+-substituted PSS (pIC50: 5.33±0.21). Taken together, our data indicate that carbachol contracts guinea pig gallbladder by stimulating two distinct muscarinic receptor subtypes linked to extracellular Ca2+ influx and intracellular Ca2+ release. These two subtypes may represent the muscarinic M3 and M4 receptors, although the presence of the muscarinic M2 receptor subtype is also suggested from the binding data. Copyright (C) 2000 Elsevier Science B.V.

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KW - Gallbladder

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KW - Intracellular

KW - Kinetics

KW - Muscarinic receptor

KW - Radioligand binding

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