Abstract
Cultures of myocytes from embryonic chick atria grown in medium supplemented with fetal calf serum from which lipoproteins had been removed demonstrated a nearly 10-fold increase in sensitivity of beating to the muscarinic cholinergic agonist carbamylcholine compared to cells grown with control serum. This effect was reversed by growth of cells in medium supplemented with lipoprotein-depleted serum (LPDS) reconstituted with the low density lipoprotein fraction from fetal calf serum. In cells grown in LPDS, total cell cholesterol was increased 32% over control levels and returned to control levels in cells grown with LPDS reconstituted with low density lipoprotein. Growth of cells in LPDS plus mevinolin, an inhibitor of endogenous cholesterol synthesis, also reversed the effects of LPDS on cholesterol content and sensitivity of beating to carbamylcholine. The ability of mevinolin (30 microM) to reverse the effect of LPDS on sensitivity of beating to carbamylcholine was inhibited by mevalonic acid, a metabolic precursor to cholesterol, with an IC50 of 7 x 10(-5) M. These data suggest that mevinolin reverses the effects of LPDS by altering cellular cholesterol levels. Enhanced responsiveness of embryonic chick heart cells to muscarinic stimulation was associated with a 2-fold increase in the number of muscarinic receptors with high affinity for agonist from 82 +/- 10 fmol/mg protein in media containing fetal calf serum to 175 +/- 12 fmol/mg protein in cells grown in the presence of LPDS. The distribution of receptors between high affinity (RH) and low affinity (RL) forms changed from 41% RH and 59% RL in cells grown in control serum to 66.5% RH and 33.5% RL in cells grown in LPDS. Quantitation of the effect of growth in LPDS on the levels of guanine nucleotide regulatory proteins No and Ni which couple the muscarinic receptor to a physiologic response, demonstrated that the relative levels of the 39-kDa alpha subunits of No and 41-kDa alpha subunits of Ni determined by ADP ribosylation with pertussis toxin and immunoblotting increased 2-fold compared to control cells grown with fetal calf serum. Growth of cells with medium supplemented with LPDS plus mevinolin reduced the levels of alpha 39 and alpha 41 to below the levels in control cells. Levels of the beta subunit of No and Ni were unaffected by growth with LPDS.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
Growth of cells in media supplemented with LPDS had a marked effect on the relative distribution of muscarinic receptors between high and low affinity states
Comparison of the computer drawn plots for high and low affinity binding in Fig. 7, a and b, demonstrates that the ratio of low affinity to high affinity receptors is reversed in cells grown with LPDS
We have previously demonstrated that pertussis toxin stimulatedADP ribosylation of embryonic chick heart homogenates in the presence of [32P]NADfollowed by polyacrylamide gel electrophoresis and autoradiography was a reliable method for determining the relative levels of the a subunits of No and Ni [29], a 3 9 and a 4 1 in chicken heart homogenates
Summary
The assay mixture contained 10 p M NAD, 0.3-0.5 pCi of [3ZP]NAD, 2.5 mM ATP, 2 mM GTP, 10 mM isoniazid, 10 mM thymidine, 10 p~ where R1 = molar concentration of receptors with affinity Kzl for. MgC12,25 ng of pertussis toxin, mM phosphocreatine, and 84 units agonist or K11 for antagonist: Rz = molar concentration of receptors of creatine phosphokinase in a total volume of 25 pl. 7.6, 10 mM MgClZ,0.2 M sucrose, 1.0 mM dithiothreitol, 1mM EDTA) tration; FI = free [3H]QNB concentration; and FP= free carbamylwere frozen and thawed once followed by Douncehomogenization choline concentration. In these analyses, Kll and K12are kept conand added to the reaction mixture at 50-100pg of protein/assay. Proteaseinhibitors (soybean trypsininhibitor, lima beantrypsin inhibitor, and leupeptinw)ere present at the concentrationdsescribed
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