Reconstitution of muscarinic cholinergic inhibition of adenylate cyclase activity in homogenates of embryonic chick hearts by membranes of adult chick hearts.

B.T Liang,J.B Galper

doi:10.1016/s0021-9258(18)61531-5

Abstract

We have previously demonstrated that during embryonic development of the chick heart between days 2 1/2 and 10 days in ovo, muscarinic cholinergic inhibition of isoproterenol-stimulated adenylate cyclase activity increased 4-fold, and the sensitivity of isoproterenol-stimulated adenylate cyclase activity to inhibition by carbamylcholine increased 26-fold. Although the number of muscarinic receptors remained constant between days 2 1/2 and 10 in ovo, the levels of a 39- and 41-kDa pertussis toxin substrate increased in parallel with the ability of muscarinic agonist to inhibit adenylate cyclase activity (Liang. B.T., Hellmich, M. R., Neer, E. J., and Galper, J. B. (1986) J. Biol. Chem. 261, 9011-9021). These data are consistent with the hypothesis that between days 2 1/2 and 10 in ovo muscarinic receptors were uncoupled from inhibition of adenylate cyclase activity because of limiting levels of pertussis toxin substrates. In the current studies, in order to test this hypothesis homogenates of embryonic chick hearts 3 1/2 days in ovo were reconstituted with membranes from hearts of hatched chicks. In order to rule out reconstitution by factors from hatched chick hearts other than pertussis toxin substrates, muscarinic receptors from hatched chick hearts were inactivated by covalent binding of benzilycholine mustard and adenylate cyclase inactivated by N-ethylmaleimide prior to reconstitution. Reconstitution of benzilylcholine mustard/N-ethylmaleimide treated hatched chick heart membranes with homogenates of embryonic chick hearts 3 1/2 days in ovo resulted in a 2 1/2-fold increase in the ability of carbamylcholine to inhibit adenylate cyclase activity and reconstitution of hatched chick heart membranes with homogenates of hearts 2 1/2 days in ovo resulted in an approximately 10-fold increase in the sensitivity of isoproterenol-stimulated adenylate cyclase activity to inhibition by carbamylcholine. Membranes from hearts of hatched chicks which had been injected with pertussis toxin were incapable of reconstituting muscarinic inhibition of adenylate cyclase activity in homogenates of hearts 3 1/2 days in ovo. These data support the conclusion that early in embryonic development coupling of muscarinic receptors to inhibition of adenylate cyclase activity is limited by the availability of a pertussis toxin substrate.

Highlights

From the Cardiovascular Division, Brighum and Women’s Hospital, andthe Departments of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
2‘/2 and 10days in ouo, muscarinic cholinergic inhibition of isoproterenol-stimulatedadenylate cyclase activity increased 4-fold, anthde sensitivityof isoproterenol-stimulated adenylate cyclase activity to inhibition Parasympathetic stimulationof the heart causes a decrease by carbamylcholine increased 26-fold
We previously demonstrated that duringembryonic development the ability of carbamylcholine to inhibit GTP-stimulated adenylatecyclase activity increased in parallwelith the ability of carbamylcholine to inhibit isoproterenol-stimulated adenylate cyclase activity from 32 pmol/mg/lO min at 3%

Summary

RESULTS

The level of muscarinicinhibition of adenylate cyclase activity in embryonic chick heart3s%days i n ouo is markedly depressed comparedtoinhibitioninhatched chick hearts [11]. 2, we determined theeffect muscarinicreceptors, and NEM inactivates both hormoneof incubating hatched chick heart membranes with various sensitive as well as the Mn2+ and Mn2+ pfloursskolin stimuconcentrations of NEM on isoproterenol-stimulated adenyl- latable adenylate cyclase activity. BCM/NEM-treated membrane from 3-day hatched chick hearts in the presence of the indicated concentration of GTP, or GTP plus carbamylcholine. GTP-stimulated adenylatecyclase activity by 64 pmol/mg/lO min from 293 k 15 (S.E., n = 5) pmol/mg/lO min t o 229 & 10 pmol/mg/lOmin These data suggest that mixing of homogenates of hearts 3% days i n ouo with membranes from hatched chick hearts increases the level of muscarinic inhibition of GTP-stimulated adenylate cyclase to that seen in hearts 10 days i n ouo.

GTP plus isoproterenol

Reconstitution of HatchedChickHeartMembraneswith

TABLEI V

Heated Trypsinized

DISCUSSION