In vitro effects of toxogonin, HI-6 and HLo-7 on the release of [H-3]acetylcholine from peripheral cholinergic nerves in rat airway smooth muscle

The purpose of this work was to evaluate the possible non-reactivating effects of toxogonin (1,1'[oxybis(methylene)]bis[4-[(hydroxyimino)me thyl]pyridinium]-dichloride), HI-6 (1-[[[(4-aminocarbonyl)pyridinio]methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium-dichloride) and HLo-7 (pyridinium, 1-[[[4-(aminocarbonyl)pyridino]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl]diiodide) on the release of acetylcholine from cholinergic nerves. The oximes have been tested in our rat bronchial smooth muscle model, with respect to the effects of oximes on the K+ (51 mM)-evoked release of [H-3]acetylcholine in the presence and absence of soman (1.0 mu M). Toxogonin (100 mu M) had no effect on the K+-evoked release of [H-3]acetylcholine in the presence or absence of soman (1.0 mu M). Similar results were found for HI-6 (100 mu M). In contrast, HLo-7 (100 mu M) enhanced the K+-evoked release of [H-3]acetylcholine in the absence of soman. In the presence of soman HLo-7 did not alter the release of [H-3]acetylcholine induced by K+ stimulation. The potentiating effect of HLo-7 on the release of [H-3]acetylcholine could be blocked by the L-, N- and P-Ca2+ channel blockers verapamil (0.1 and 1.0 mu M), omega-conotoxin GVIA (1.0 mu M) and omega-agatoxin IV-A (0.2 mu M), respectively. Muscarinic receptor antagonists (atropine (10 mu M), pirenzepine (M(1)) (1.0 mu M) and methoctramine (M(2)) (1.0 mu M)) had no effects on the HLo-7 (100 mu M)-enhanced release of [H-3]acetylcholine. Protein kinase inhibitors (H-7 (20 mu M), calphostin C (1.0 mu M) and KN-62 (10 mu M)) inhibited the HLo-7; (100 mu M)-enhanced K+-evoked release of [H-3]acetylcholine. The results showed that only HLo-7 had a direct enhancing effect on the release of acetylcholine through activation or opening of Ca2+ channels and a subsequent protein phosphorylation in the nerve terminal.