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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.

A biphasic time course of the agonist-mediated loss of muscarinic cholinergic receptors has been demonstrated in cultured chick embryo heart cells by radioligand binding studies using the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB). This agonist-mediated receptor loss was associated with decreased affinity of the receptor for agonist as judged by competitive binding of the agonist carbamylcholine with [3H]QNB to cell homogenates (Galper, J. B., and Smith, T. W. (1980) J. Biol. Chem. 255, 9571-9579). In the current studies the concentration dependence of agonist-mediated receptor loss was also found to be biphasic. The apparent shift of affinity following brief (15 min) agonist exposure coincided with the agonist-mediated loss of a subclass of high affinity receptors with an IC50 for carbamylcholine inhibition of [3H]QNB binding of 3.9 x 10(-7) M. Those receptors remaining constituted a subclass of low affinity receptors with IC50 = 8.2 x 10(-5) M. The data further suggest that an apparent decrease in agonist affinity after guanine nucleotide exposure represents conversion of high affinity receptors to a similar low affinity state, IC50 = 8.6 x 10(-5) M. The rapid loss of [3H]QNB binding sites in the presence of agonist did not require interaction of agonist with intact cells, but also occurred if cells were homogenized and then subjected to a brief (15 min) exposure to agonist. The slow loss over 3 h of [3H]QNB binding sites could only be demonstrated in intact cells incubated with agonist prior to homogenization. To probe further the later phase of agonist-mediated receptor loss, we developed new assay methods for determining muscarinic antagonist binding to intact cells. In control cells, binding of the hydrophobic antagonist [3H]QNB was quite similar in extent to binding of the more hydrophilic antagonist [3H]methylscopolamine ([3H]MS), with Kd values of 0.11 and 0.47 nM, respectively. Kinetic analysis of the binding of these two ligands was performed to determine whether they might distinguish between two states of the receptor. Both ligands bound to the receptor by a two step mechanism consistent with the formation of a low affinity complex followed by conversion to a high affinity complex. However, the ratio of reverse to forward rate constants of the second step of [3H]MS binding was roughly 100-fold greater than that for the more hydrophobic ligand [3H]QNB. Comparison of the time course of agonist-induced receptor loss as measured by binding of [3H]MS or [3H]QNB was consistent with muscarinic agonist mediation of a stepwise alteration in receptor configuration from a form that bound both [3H]MS and [3H]QNB to a form that bound only [3H]QNB and thence to a form that bound neither [3H]MS nor [3H]QNB. The relationship of such a sequential mechanism to agonist-induced changes in the relationship of the receptor to the cell membrane and agonist-induced endocytosis of the receptor is discussed.

Parasympathetic and sympathetic innervation of the embryonic chick heart proceed non-coordinately. beta-Adrenergic agonists mediate an increase in beating rate in embryonic chick heart prior to ingrowth of the vagus nerve (Culver, N. G., and Fishman, D. A. (1977) Am. J. Physiol. 232, R116-R123) while muscarinic agonists exert relatively little effect on beating rate in hearts 2-4 days in ovo (Papanno, A. J. (1979) Pharmacol. Rev. 29, 3-33). Studies of developmental changes in the ability of muscarinic agonists to inhibit adenylate cyclase activity and their relationship to the development of a physiologic response of the embryonic chick heart to muscarinic stimulation have been inconclusive. In the current studies the ability of isoproterenol to stimulate adenylate cyclase activity did not change during development. Maximum stimulation above basal was 760 pmol of cAMP/10 min/mg of proterin with an IC50 of 1.5 X 10(-6) M for isoproterenol in homogenates of hearts 2 1/2, 3 1/2, and 10 days in ovo and 3 days posthatching. However, inhibition of isoproterenol-stimulated adenylate cyclase activity by carbamylcholine increased from 7.6% with a IC50 for carbamylcholine of 16 +/- 5.0 microM at day 2 1/2 in ovo to 29% with an IC50 of 0.4 +/- 0.1 microM at day 10 in ovo and to 43% with a IC50 of 0.6 +/- 0.1 microM at 3 days posthatching. Since previous data had demonstrated the presence of muscarinic receptors as early as 2 1/2 days in ovo (Galper, J. B., Klein, W., and Catterall, W. A. (1977) J. Biol. Chem. 252, 8692-8699), studies of developmental changes in guanine nucleotide-coupling proteins were carried out to determine whether early in development muscarinic receptors were uncoupled from a physiologic response. Studies of pertussis toxin-catalyzed ADP-ribosylation of homogenates of embryonic chick heart with [32P]NAD demonstrated the presence of two ADP-ribosylated proteins at 39,000 and 41,000 kDa, respectively. Both ADP-ribosylation and immunoblotting of homogenates with an antibody to the 39-kDa guanine nucleotide-binding protein in bovine brain demonstrated that the 39-kDa alpha protein increased 1.8-fold between days 2 1/2 and 3 1/2 in ovo and another 1.8-fold from day 3 1/2 to 10 in ovo in parallel with the increase in the extent of muscarinic inhibition of adenylate cyclase activity.(ABSTRACT TRUNCATED AT 400 WORDS)