Low density lipoproteins induce parasympathetic responsiveness in embryonic chick ventricular myocytes in parallel with a coordinate increase in expression of genes coding for the M2 muscarinic receptor, G alpha i2, and the acetylcholine-sensitive K+ channel.
Growth of chick atrial cells in medium supplemented with lipoprotein-depleted serum has been shown to result in an increase in total cell cholesterol, and an increase in the negative chronotropic response to muscarinic stimulation in parallel with an increase in levels of muscarinic receptors and the G-protein alpha-subunits alpha i and alpha o (Haigh, L. S., Leatherman, G. F., O'Hara, D. S., Smith, T. W., and Galper, J. B. (1988) J. Biol. Chem. 263, 15608-15618). In this study we determined whether growth of chick ventricular cells in medium supplemented with lipoprotein depleted serum could alter levels of muscarinic receptors and G-protein alpha-subunits and induce a negative chronotropic response to muscarinic stimulation. We further determined whether levels of mRNA coding for muscarinic receptors, G-proteins, and the acetylcholine-sensitive K+ channel were coordinately regulated. Growth of embryonic chick ventricular cells from hearts 14 days in ovo in medium supplemented with lipoprotein depleted serum resulted in a 21 +/- 5% (n = 3, +/- S.E.) increase in muscarinic receptor number as demonstrated by [3H]quinuclidinyl benzilate binding and a 4.7 +/- 1.0 (+/- S.E., n = 4)-fold increase in G alpha i2 as demonstrated by Western blot analysis. These changes in receptor and G-protein were associated with a coordinate increase in levels of mRNA coding for the M2 muscarinic receptor, G alpha i2 and the acetylcholine sensitive K+ channel as determined by RNase protection. These increases were reversed by addition of 30 microM mevinolin, an inhibitor of HMG-CoA reductase activity. Carbamylcholine (0.1 mM) had no effect on beat rate in ventricular cells grown in medium supplemented with fetal calf serum. Cells grown in medium supplemented with lipoprotein depleted serum demonstrated a 40 +/- 8% (+/- S.E., n = 10, p < 0.0001) decrease in beat rate in response to 0.1 mM carbamylcholine which was reversed by the addition of 30 microM mevinolin. These data suggest that, during growth in medium supplemented with lipoprotein depleted serum, a component of the cholesterol biosynthetic pathway plays a role in the coordinate induction of mRNAs coding for receptors, G-proteins, and an effector (ion channel) that results in the induction of a parasympathetic response in the ventricular cell characteristic of the atrial phenotype.
Gadbut
A P
AP
Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115.
Toupin
D K
DK
Kilbourne
E J
EJ
Galper
J B
JB
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002217:Carbachol / Q000494:pharmacology*; D002642:Chick Embryo; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D015870:Gene Expression / Q000187:drug effects*; D006321:Heart / Q000187:drug effects / Q000502:physiology*; D006339:Heart Rate / Q000187:drug effects; D006352:Heart Ventricles; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000494:pharmacology*; D008148:Lovastatin / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D015221:Potassium Channels / Q000096:biosynthesis* / Q000187:drug effects; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011976:Receptors, Muscarinic / Q000096:biosynthesis* / Q000378:metabolism
D008077:Lipoproteins, LDL; D046911:Macromolecular Substances; D015221:Potassium Channels; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D011813:Quinuclidinyl Benzilate; D002217:Carbachol; D008148:Lovastatin; D019204:GTP-Binding Proteins; D000109:Acetylcholine