Substance P (SP) is colocalized with ACh in splanchnic nerves that innervate into adrenal medulla and the peptide has been shown to inhibit nicotinic agonists-induced catecholamine secretion. To elucidate the effects of SP on cytosolic Ca(2+) dynamics, the present study was conducted using fura-2-loaded isolated bovine adrenal chromaffin cells. Stimulation of the cells with nicotine (10-100mu-M) produced a rapid rise of cytosolic Ca(2+) concentration ([Ca(2+)]i), the peak level of which increased in a dose-dependent manner, followed by a gradual decay. In the presence of 10mu-M SP, the dose-response relationship of the peak levels shifted downward. Quantitative analyses implied that SP inhibits the nicotine-induced Ca(2+) influx in a noncompetitive manner. Nicotinic acetylcholine receptor is composed of two major functional domains: an agonist-binding site and an ionophore or channel domain. Agonist binding activates ionophore / channel domain and causes mainly Na(+) influx. This Na(+) influx depolarizes the cell and activates voltage-dependent Ca(2+) channels. Based on this fact, the present results indicate that SP dose not block nicotine binding sites but interferes with other sites of nicotinic receptor / channel molecule, most probably a channel domain. It was suggested that SP colocalized with ACh in splanchnic nerves functions as a physiological modulator of catecholamine secretion by non-competitively suppressing ACh-induced cytosolic Ca(2+) dynamics in bovine a