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The contractile effect of substance P on the longitudinal muscle of the isolated guinea-pig small intestine and the desensitization of the muscle which occurs on prolonged exposure to the peptide have been investigated. All experiments were performed in the presence of atropine. The response to a substance P concentration which produced a nearly maximal effect was not sustained but faded rapidly. It was found that not elimination of substance P from the bath, but desensitization of the muscle to substance P was the main cause for the fading of contraction. Desensitization of the muscle to substance P only developed if the muscle was exposed to the peptide for a certain time. The degree of and the time needed for recovery from desensitization were directly related to concentration of substance P and contact time. Tetraethylammonium (3 mM), which reduces the membrane conductance for K+, enhanced the potency of substance P in contracting the muscle and reduced the fading of contraction. Noradrenaline (295 nM), which increases the K+ conductance, produced opposite effects. Lowering the extracellular Ca2+ concentration to one-tenth decreased the potency of substance P in contracting the muscle, accelerated the fading of contraction, and reduced the ability of the muscle to respond to a second addition of substance P after the response to the first addition had faded away. Concentrations of substance P (22 nM) and tetraethylammonium (30 mM), which produced nearly maximal contractions, slightly enhanced the efflux of 86Rb from pre-loaded muscle strips. Both substances, however, caused a sustained reduction of 86Rb efflux from strips depolarized by high [K+], the effect of substance P being smaller than that of tetraethylammonium. The effect of substance P and tetraethylammonium on 86Rb efflux appeared independent of the extracellular [Ca2+]. On exposure of the muscle to substance P (22 nM) for 8 min the intracellular uptake of 45Ca was first decreased and then increased while the 45Ca influx was instantly enhanced by tetraethylammonium (30 mM) or K+ (108 mM). The delayed increase in 45Ca influx caused by substance P was also observed in muscle strips depolarized with high [K+].(ABSTRACT TRUNCATED AT 400 WORDS)