Characterization of G-protein Signaling in Ventricular Myocytes From the Adult Mouse Heart: Differences From the Rat

We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3–6×106cells/heart. The cells are rod-shaped, roughly 20μM ×100 μM and Ca++tolerant, with viability of 65–80%. Binding studies with [125I]ICYP demonstrate th...

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Veröffentlicht in:Journal of molecular and cellular cardiology 2000-07, Vol.32 (7), p.1211-1221
Hauptverfasser: Hilal-Dandan, Randa, Kanter, Joan R, Brunton, Laurence L
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creator Hilal-Dandan, Randa
Kanter, Joan R
Brunton, Laurence L
description We have developed a high yield technique for isolating ventricular myocytes from adult mouse hearts. This collagenase-trypsin procedure yields 3–6×106cells/heart. The cells are rod-shaped, roughly 20μM ×100 μM and Ca++tolerant, with viability of 65–80%. Binding studies with [125I]ICYP demonstrate the presence of β -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the β1-specific antagonist CGP 20712A on [125I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are β1and 33% are β2, compared to 16–20%β2in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC50≈110±20 n M. A functional Gipathway is demonstrated by inhibition of ISO-stimulated cyclic AMP accumulation by endothelin, carbachol and ATP and by sensitivity of this inhibition to pertussis toxin. As assessed by inositol phosphate production, endothelin and ATP stimulate the activity of the Gq-phospholipase C pathway, whereas carbachol, PGF2 αand α1-adrenergic receptor agonists show no significant effect. The inability of α1-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold α1-adrenergic activation that occurs in rat. Biochemical and pharmacological profiles, as well as the need for modifications in experimental design, indicate that mouse myocytes differ substantially from rat cardiac myocytes.
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subjects Adenylate Cyclase Toxin
Adenylyl Cyclases - metabolism
Adrenergic beta-Agonists - pharmacology
Animals
Cell Culture Techniques - methods
Cells, Cultured
Cholinergic Agonists - pharmacology
Cyclic AMP - metabolism
Dose-Response Relationship, Drug
GTP-Binding Proteins - metabolism
Heart - physiology
Inositol Phosphates - biosynthesis
Isoproterenol - pharmacology
Male
Mice
Myocardium - cytology
Myocardium - metabolism
Pertussis Toxin
Phosphoinositide metabolism
Protein Binding
Rats
Receptors, Adrenergic, beta - metabolism
Signal Transduction
Transgenic mice β -adrenergic receptors α1-adrenergic receptors
Virulence Factors, Bordetella - pharmacology
title Characterization of G-protein Signaling in Ventricular Myocytes From the Adult Mouse Heart: Differences From the Rat
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