Effect of acetylethylcholine mustard on muscarinic receptor-coupled attenuation of cAMP formation in intact GH3 cells.
The effects of acetylethylcholine mustard (Aech-M) on the muscarinic receptor coupled adenylate cyclase system of intact GH3 cells were investigated. The concentration of Aech-M and Ach that inhibited specific [3H]N-methylscopolamine binding by 50% was similar for both compounds (9.3 microM for Ach and 10.7 microM for Aech-M). Pretreatment of intact GH3 cells or isolated membranes with 10 to 50 microM Aech-M, followed by washing, reduced the [3H]N-methylscopolamine binding capacity by 60% and 75-77%, respectively, without changing the KD value for the radioligand to the remaining receptors. Both Aech-M and Ach attenuated forskolin (1 microM) stimulated cAMP formation with half-maximal effects (EC50) occurring at 0.84 microM for Ach and 0.24 microM for Aech-M. The maximal inhibition was 70-80% for Ach and 30-40% for Aech-M. However, the dose-response for Aech-M was biphasic such that at high concentrations (greater than 50 microM) there was a reduction in its ability to attenuate cAMP formation. After 3 min incubation with either Ach or Aech-M, the addition of atropine completely reversed their inhibitory effect even though with Aech-M there was a greater than 50% reduction in receptor capacity. Furthermore, over a 12-min incubation, Ach produced a relatively stable 67-76% reduction in cAMP accumulation, whereas with Aech-M the initial attenuation was gradually reduced such that by 10 min of incubation, no effect was observed. Finally, pretreatment with Aech-M resulted in a reduced sensitivity to the action of Ach as the EC50 values for inhibition of cAMP accumulation were increased 7.6- and 13.5-fold, respectively, with little or no change in the maximal response. The data indicate that Aech-M produces a transient agonist effect to attenuate cAMP formation in intact GH3 cells followed by an antagonist action probably after irreversible binding to the receptor.
Bolden
C P
CP
Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville.
Baker
S P
SP
D000109:Acetylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D000242:Cyclic AMP / Q000096:biosynthesis*; D004305:Dose-Response Relationship, Drug; D019832:N-Methylscopolamine; D010277:Parasympathomimetics / Q000494:pharmacology*; D051381:Rats; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000378:metabolism; D012602:Scopolamine Derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured
D010277:Parasympathomimetics; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; C002561:acetylcholine mustard; D000242:Cyclic AMP; D000109:Acetylcholine; D019832:N-Methylscopolamine