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Research ArticleArticle

Selectivity Profile of Muscarinic Toxin 3 in Functional Assays of Cloned and Native Receptors

Maria C. Olianas, Angela Ingianni, Carlo Maullu, Abdu Adem, Evert Karlsson and Pierluigi Onali
Journal of Pharmacology and Experimental Therapeutics January 1999, 288 (1) 164-170;
Maria C. Olianas
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Angela Ingianni
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Carlo Maullu
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Abdu Adem
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Evert Karlsson
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Pierluigi Onali
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Abstract

By using acetylcholine-induced stimulation of [35S]guanosine-5′-O-(3-thio)triphosphate ([35S]GTPγS) binding to membrane G proteins as a functional assay of the cloned human m1–m4 muscarinic receptor subtypes stably expressed in Chinese hamster ovary cells, muscarinic toxin 3 (MT3) was found to block the m4 receptor with a potency (pA2 = 8.33) much higher than those displayed at the m1 (pA2 = 6.78), m3 (pA2 = 6.3), and m2 (pA2 < 6.3) subtypes. In N1E-115 cells, which have been reported to express m4 receptors coupled to inhibition of cAMP, MT3 potently antagonized the carbachol-induced inhibition of adenylyl cyclase with a pA2 of 8.81 and displayed monophasic inhibitory curves. Unexpectedly, in NG108-15 cells, known to express only m4 receptors, MT3 counteracted the carbachol inhibition of adenylyl cyclase with a lower potency (pA2 = 7.60) and showed a biphasic inhibitory curve, suggesting the participation of both m4 and m2 receptors. This possibility was supported by radioligand binding data showing that MT3 failed to completely displace the binding of [3H]N-methylscopolamine to NG108-15 cell membranes and by reverse transcription-polymerase chain reaction analysis, revealing the presence of mRNAs for both m4 and m2 receptor subtypes. These data demonstrate that MT3 possesses a high functional receptor selectivity for both the cloned and native m4 receptors and that in cell systems containing m4 and m2 receptors coupled to a common response, the toxin constitutes a powerful tool to resolve the relative contribution by each receptor subtype.

Footnotes

  • Send reprint requests to: Pierluigi Onali, M.D., Section on Biochemical Pharmacology, Department of Neurosciences, University of Cagliari, via Porcell 4, 09124 Cagliari, Italy. E-mail:onali{at}unica.it.

  • ↵1 This work was supported by Grant CHRXCT940689 from the European Communities (to P.O.) and by MURST.

  • Abbreviations:
    ACh
    acetylcholine chloride
    CCh
    carbachol chloride
    CHO
    Chinese hamster ovary
    BSA
    bovine serum albumin
    EGTA
    ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
    GTPγS
    guanosine-5′-O-(3-thio)triphosphate
    HEPES
    4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
    MT3
    muscarinic toxin 3
    NMS
    N-methylscopolamine
    PACAP
    pituitary adenylate cyclase activating polypeptide
    RT
    reverse transcription
    PCR
    polymerase chain reaction
    • Received April 15, 1998.
    • Accepted June 25, 1998.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 288 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 288, Issue 1
1 Jan 1999
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Research ArticleArticle

Selectivity Profile of Muscarinic Toxin 3 in Functional Assays of Cloned and Native Receptors

Maria C. Olianas, Angela Ingianni, Carlo Maullu, Abdu Adem, Evert Karlsson and Pierluigi Onali
Journal of Pharmacology and Experimental Therapeutics January 1, 1999, 288 (1) 164-170;

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Research ArticleArticle

Selectivity Profile of Muscarinic Toxin 3 in Functional Assays of Cloned and Native Receptors

Maria C. Olianas, Angela Ingianni, Carlo Maullu, Abdu Adem, Evert Karlsson and Pierluigi Onali
Journal of Pharmacology and Experimental Therapeutics January 1, 1999, 288 (1) 164-170;
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