Development of muscarinic cholinergic inhibition of adenylate cyclase in embryonic chick heart. Its relationship to changes in the inhibitory guanine nucleotide regulatory protein

Parasympathetic and sympathetic innervation of the embryonic chick heart proceed non-coordinately. β-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 μM at day 2 1/2 in ovo to 29% with an IC₅₀ of 0.4 ± 0.2 μM at day 10 in ovo and to 43% with a IC₅₀ of 0.6 ± 0.1 μM 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 α 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. Between day 10 in ovo and 3 days posthatching the 39-kDa protein remained constant, but muscarinic receptor number measured by [³H]quinuclidinyl benzilate binding increased 2.6-fold from 200 fmol/mg of protein to 520 fmol/mg of protein in parallel with the increase in inhibition of adenylate cyclase activity by carbamylcholine. The data suggest that early in embryonic development coupling of muscarinic receptors to inhibition of adenylate cyclase activity may be limited by the availability of the 39-kDa guanine nucleotide regulatory protein but that later in development physiologic response to muscarinic stimulation might be limited by muscarinic receptor number.