The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on muscarinic receptor-induced Ca2+ mobilization in a human salivary epithelial cell line

Abstract

We have investigated the effect of W-7, a calmodulin (CaM) antagonist, on Ca2+ mobilization in a human salivary epithelial cell line, HSG-PA, after muscarinic receptor stimulation. In a medium containing 1.5 mmol/l Ca2+, W-7 reduced both the maximum peak increase in cytosolic Ca2+ [( Ca2+]i) which follows stimulation by carbachol (Cch, 100 mumol/l) and the sustained nature of the response. Using an experimental approach which allows separate visualization of the intracellular Ca2+ release and extracellular Ca2+ entry phases, W-7 was shown preferentially to inhibit Ca2+ release. At 100 mumol/l W-7, Cch-induced Ca2+ release was completely inhibited, but Cch-induced Ca2+ entry was partially (approximately 40%) maintained. This W-7 residual Ca2+ entry response was abolished when cells were depolarized with high K+ or gramicidin D. W-7 also substantially inhibited Cch-induced inositol trisphosphate (IP3) production (approximately 5%). W-5, a less potent CaM antagonist than W-7, had markedly smaller effects on Cch-induced Ca2+ mobilization and IP3 formation. W-7 (100 mumol/l) completely blocked (comparable to 10 mumol/l atropine) the binding of the muscarinic antagonist [3H] quinuclidinyl benzilate (QNB) to muscarinic receptors on cell membranes, whereas Cch (at 100 mumol/l) had minimal effects on ligand binding. W-7 and W-5 were equipotent in their ability to inhibit [3H] QNB binding. These results suggest that W-7 reduces Ca2+ mobilization in HSG-PA cells by a mechanism which likely involves the antagonism of a CaM regulatory step(s) but may also involve at least a partial blockade of the muscarinic receptor.