Substance P has been previously shown to stimulate luteinizing hormone (LH) secretion and synergistically enhance gonadotropin-releasing hormone (GnRH)-evoked LH release from cultured pig pituitary cells. To investigate the mechanisms involved in these responses, the effects of substance P (100 nM; 4 h) and/or GnRH (10 nM, 4 h) on LH release, LH intracellular content, and betaLH mRNA accumulation were evaluated in the absence or presence of extracellular Ca(2+). Likewise, the effects of substance P on the dynamics of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) were examined in single cells. Extracellular Ca(2+) deprivation abolished both substance P- and GnRH-stimulated LH release, as well as their synergistic interaction. The substance P antagonist D-Arg1,D-Phe5,-Trp7,9,Leu11-substance P (100 nM) blocked the stimulatory effect of substance P on LH release and its interaction with GnRH without affecting GnRH-induced LH secretion. Whereas substance P did not modify betaLH transcript levels, GnRH stimulated betaLH mRNA accumulation through a mechanism dependent upon extracellular Ca(2+). Substance P directly increased [Ca(2+)](i) in a 30% of gonadotropes by causing two distinct types of response kinetics with single-peak (predominant, 83.3%) or sustained-plateau profiles. Reduction of external [Ca(2+)] decreased by half the percent of responsive cells, which only showed single-peak profiles. Taken together, our results demonstrate that the ability of substance P to stimulate basal and GnRH-induced LH release is exerted directly upon gonadotropes, is extracellular Ca(2+)-dependent and does not seem to require net increases in betaLH mRNA levels. Moreover, [Ca(2+)](i) measurements revealed that although substance P action in pig gonadotropes is strongly dependent on extracellular Ca(2+) influx, it would also involve intracellular Ca(2+) mobilization. Finally, extracellular Ca(2+) also plays a requisite role to sustain GnRH-stimulated increases in both betaLH mRNA and LH release.