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 3-6x10(6)cells/heart. The cells are rod-shaped, roughly 20 microM x 100 microM and Ca(++)tolerant, with viability of 65-80%. Binding studies with [(125)I]ICYP demonstrate the presence of beta -adrenergic receptors at a density of 83 fmol/mg membrane protein. Assessment of the effects of the beta(1)-specific antagonist CGP 20712A on [(125)I]ICYP binding and on isoproterenol (ISO)-sensitive adenylyl cyclase activity indicates that 67% of the receptors are beta(1)and 33% are beta(2), compared to 16-20%beta(2)in rat myocytes. Mouse myocytes respond to isoproterenol to produce cyclic AMP with an EC(50) approximately 110+/-20 n M. A functional G(i)pathway 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 G(q)-phospholipase C pathway, whereas carbachol, PGF(2 alpha)and alpha(1)-adrenergic receptor agonists show no significant effect. The inability of alpha(1)-adrenergic receptor agonists to induce phosphoinositide hydrolysis in mouse myocytes differs from a several fold alpha(1)-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.
Hilal-Dandan
R
R
Departments of Pharmacology and Medicine, UCSD School of Medicine, La Jolla, CA 92093, USA.
Kanter
J R
JR
Brunton
L L
LL
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D018679:Cholinergic Agonists / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006321:Heart / Q000502:physiology*; D007295:Inositol Phosphates / Q000096:biosynthesis; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D051379:Mice; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D037342:Pertussis Toxin; D011485:Protein Binding; D051381:Rats; D011943:Receptors, Adrenergic, beta / Q000378:metabolism; D015398:Signal Transduction; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D000318:Adrenergic beta-Agonists; D018679:Cholinergic Agonists; D007295:Inositol Phosphates; D011943:Receptors, Adrenergic, beta; D010566:Virulence Factors, Bordetella; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol