Physiological neuronal cholinergic antagonism of isoproterenol inhibitory effect on rabbit jejunum spontaneous activity

The regular spontaneous motility (s.m.) of rabbit jejunum makes this isolated preparation suitable to asses the mechanisms underlying the action of various neurotrasmitters and neuromodulators and their interactions. In a previous work (L. Romanelli et al., Autonomic and Autacoid Pharmacol. 23; 105-115, 2003) we have shown that the s.m. of rabbit jejunum is predominantly mediated by neuronal release of acetylcholine (ACh) that inhibits myogenic activity and strongly antagonizes beta-adrenoceptor-induced inhibition of the s.m. amplitude. Our aim in this study was to determine whether M2 or M3-muscarinic receptors were involved in the s.m. and in the functional antagonism of the beta-adrenoceptor isoproterenol (ISO) inhibitory effect. By use of isotonic tension measurement, the s.m. amplitude were measured and ISO (5,5x10-8 M) inhibitionwas investigated in absence or presence of increased concentration of selective muscarinic antagonist methoctramine (MET) (2,8x 10-8 Ð 11 x 10-7 M) (M2 over M3) or 4-diphenyl-acetoxy-N-piperidine (4-DAMP) (0,22 x 10-8 Ð 4,4 x 10-8M)(M3 over M2). ISO at the concentration up 5 x 10-7 M produced small or no inhibitory responses in na•ve tissues. The two muscarinic antagonists inhibited in a concentration-related way the s.m. amplitude and made ISO able to decrease the remained activity. However the MET concentration needful for ISO (5,5 x 10-8 M) to inhibit 50% of the s.m. amplitude was about 20 fold higher than that of 4-DAMP. In na•ve preparation the L-type Ca++ channels bloker nifedipine (5,7 x 10-8 M) decreased the s.m. amplitude but did not increase the inhibitory effect of ISO. In TDX treated tissues the Ca++ channels activator(-) BayK 8644, the K+ channels blocker TEA (3mM) and the inhibitor of sarcoplasmic reticulum (SR) Ca++ - ATPase, cyclopiazonic acid (ACP) (20 microM) all increased s.m. amplitude leaving unaffected the inhibitory ISO effect when administered 5 min after, but blocked, in a dose-related way, its effect when administered 20min after as did ACh. These results seem to indicate that in rabbit jejunum the release of ACh activates M3 receptors eliciting both a contractile response and _-adrenoceptor antagonism. The biochemical mechanism involved in M3/_ adenoreceptor antagonism is less studied and still unclear than the one involved M2 receptors. However both SR and Ca++ play a fundamental role but further studies are necessary to explain the delayed time elapsing for this antagonism.