Abstract
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.
Highlights
Growth of chick atrial cells in medium supplemented terol biosynthetic pathway plays a role in the coordiwith lipoprotein-depletedserum has been shown to re- nate induction mofRNAs coding for receptors, sult in an increase in total cell cholesterol, and an in- G-proteins,and an effector that results in crease in the negative chronotropic response to musca- the induction of a parasympathetic response in the venrinic stimulation in parallel with an increase in levels of tricular cell characteristic of the atrialphenotype
In this study we determined whether growth of chick ventricular cells in medium supplemented withlipoprotein depleted serum could alter levels of muscarinic receptors and G-protein a-subunits and induce a negative chronotropic response to musca
We further determined whether levels The negative chronotropic response to vagal stimulation inof mRNA coding for muscarinic receptors, G-proteins, volves the interaction of the M, subtype of the muscarinic reand the acetylcholine-sensitiveK' channel were coordi- ceptor with a G-protein heterotrimer which activates an innately regulated
Summary
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankrM/EMBL DatBaank with accession number(s)L35555. Theabbreviationsused are: LPDS, lipoprotein-depletedserum; QNB, quinuclidinyl benzilate; HMG-CoA,hydroxymethylglutaryl-CoA, Ini,slowinward current; ACh,acetylcholine; GAF'D, glyceraldehyde phosphatedehydrogenase; FCS, fetal calf serum; MOPS,4-mOrphOlinepropanesulfonic acid; PIA, p-phenylisopropyladenosine. These effects of growth with LPDS were reversedby addition of mevinolin, an inhibitorof HMG-1 CoA reductase activity (37). Mented with LPDS was associated with a 10 fold increase in The ACh (acetylcholine)-sensitive K+ channel, cGIRK1, was cloned the ability of carbamylcholine to mediate a negative chronotropic response.This effect was reversedby growth of cells in LPDS plus mevinolin or in LPDS reconstituted with the LDL fraction of fetal calf serum. From a chick brain cDNAlibrary usinga rat GIRKl cDNA as probe., The clone demonstrated a coding region for a protein witha 96%identity to rat GIRKl(36)A. 307-base pair EcoRI-Sal fragmfernomt the 3' endof cGIRKl was subcloned into pBluescript and linearized with EcoRI
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