Functional properties of muscarinic receptor subtypes Hm1, Hm3 and Hm5 expressed in SF9 cells using the baculovirus expression system

Jyrki Kukkonen, Johnny Näsman, Pekka Ojala, Christian Oker-Blom, Karl Åkerman

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Abstract

The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using the baculovirus expression system. Stimulation of all three subtypes with CCh caused an increase in inositol-1,4,5-trisphosphate and intracellular Ca++. The increase in cytosolic free Ca++ was to a large extent due to influx. The levels of receptors (< 0.1-1 pmol/mg protein) increased with infection time in a narrow time span (24-36 h). The changes in the receptor densities did not significantly affect the EC50 values of CCh-mediated Ca++ mobilization with the m3 or the m5 subtype. The EC50 value was higher with the m1 receptor at low expression levels (approximately 100 fmol/mg protein), and it decreased with an increase in receptor density. The receptor subtypes displayed no gross differences in their response to oxotremorine-M, which behaved as a full agonist, or to oxotremorine and pilocarpine, which were less active. With the m3 subtype, there was an increase in the maximal response to oxotremorine with longer infection times. The results demonstrate that the recombinant muscarinic receptors, expressed in Sf9 cells, show many of the characteristics of endogenously expressed receptors when studied at low expression levels and that the receptor density may significantly affect the receptor pharmacology.
Original languageEnglish
Pages (from-to)593-601
Number of pages9
JournalThe Journal of Pharmacology and Experimental Therapeutics
Volume279
Issue number2
Publication statusPublished - 1996
MoE publication typeA1 Journal article-refereed

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Baculoviridae
Muscarinic Receptors
Oxotremorine
Sf9 Cells
Pilocarpine
Inositol 1,4,5-Trisphosphate
Cholinergic Receptors
Infection
Proteins
Pharmacology

Cite this

Kukkonen, Jyrki ; Näsman, Johnny ; Ojala, Pekka ; Oker-Blom, Christian ; Åkerman, Karl. / Functional properties of muscarinic receptor subtypes Hm1, Hm3 and Hm5 expressed in SF9 cells using the baculovirus expression system. In: The Journal of Pharmacology and Experimental Therapeutics. 1996 ; Vol. 279, No. 2. pp. 593-601.
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abstract = "The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using the baculovirus expression system. Stimulation of all three subtypes with CCh caused an increase in inositol-1,4,5-trisphosphate and intracellular Ca++. The increase in cytosolic free Ca++ was to a large extent due to influx. The levels of receptors (< 0.1-1 pmol/mg protein) increased with infection time in a narrow time span (24-36 h). The changes in the receptor densities did not significantly affect the EC50 values of CCh-mediated Ca++ mobilization with the m3 or the m5 subtype. The EC50 value was higher with the m1 receptor at low expression levels (approximately 100 fmol/mg protein), and it decreased with an increase in receptor density. The receptor subtypes displayed no gross differences in their response to oxotremorine-M, which behaved as a full agonist, or to oxotremorine and pilocarpine, which were less active. With the m3 subtype, there was an increase in the maximal response to oxotremorine with longer infection times. The results demonstrate that the recombinant muscarinic receptors, expressed in Sf9 cells, show many of the characteristics of endogenously expressed receptors when studied at low expression levels and that the receptor density may significantly affect the receptor pharmacology.",
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Functional properties of muscarinic receptor subtypes Hm1, Hm3 and Hm5 expressed in SF9 cells using the baculovirus expression system. / Kukkonen, Jyrki; Näsman, Johnny; Ojala, Pekka; Oker-Blom, Christian; Åkerman, Karl.

In: The Journal of Pharmacology and Experimental Therapeutics, Vol. 279, No. 2, 1996, p. 593-601.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Functional properties of muscarinic receptor subtypes Hm1, Hm3 and Hm5 expressed in SF9 cells using the baculovirus expression system

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AU - Oker-Blom, Christian

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AB - The human muscarinic ACh receptor subtypes m1, m3 and m5 have been expressed in Sf9 cells using the baculovirus expression system. Stimulation of all three subtypes with CCh caused an increase in inositol-1,4,5-trisphosphate and intracellular Ca++. The increase in cytosolic free Ca++ was to a large extent due to influx. The levels of receptors (< 0.1-1 pmol/mg protein) increased with infection time in a narrow time span (24-36 h). The changes in the receptor densities did not significantly affect the EC50 values of CCh-mediated Ca++ mobilization with the m3 or the m5 subtype. The EC50 value was higher with the m1 receptor at low expression levels (approximately 100 fmol/mg protein), and it decreased with an increase in receptor density. The receptor subtypes displayed no gross differences in their response to oxotremorine-M, which behaved as a full agonist, or to oxotremorine and pilocarpine, which were less active. With the m3 subtype, there was an increase in the maximal response to oxotremorine with longer infection times. The results demonstrate that the recombinant muscarinic receptors, expressed in Sf9 cells, show many of the characteristics of endogenously expressed receptors when studied at low expression levels and that the receptor density may significantly affect the receptor pharmacology.

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