Effect of SP on pLH Secretion by Static Cultures of Porcine Pituitary Cells The next objective of the present study was to determine whether SP affects LH secretion from porcine gonadotrophs. Exposure of cultured pituitary cells to SP concentrations ranging from 0.01 nM to 10 (xM during 4 h induced increases in pLH release from gonadotrophs that were significant at, or above, 100 nM (Fig. 2a). Conversely, no significant changes in hormonal content of cellular homoge-nates were detected at any of the concentrations tested (Fig. 2b). buy flovent inhaler
We then analyzed the possible interaction between SP and GnRH in their stimulatory activity on gonadotrophs by incubating pituitary cells with GnRH (10 nM), SP (100 nM), or a combination of both peptides (Fig. 3). As expected, a 4-h incubation with each of these agents alone induced an increase in LH release, which was more pronounced in the case of GnRH (312% of control) than with SP (186%). Interestingly, the combined administration of both peptides resulted in a synergistic enhancement of LH release (787%, Fig. 3a). With regard to intracellular hormone contents, treatment with GnRH alone significantly decreased (40% below control) the average pLH cell content. However, this decrease was prevented by the combined administration of SP with GnRH. Similarly, SP alonedid not modify the average pLH cell content with respect to control values in spite of its LH-releasing action (Fig. 3b). To examine whether the SP effect on gonadotrophs was mediated through a GnRH receptor, each of these peptides was incubated at the concentrations mentioned above in the presence of 1 (jt,M antide, a potent GnRH antagonist specific for the GnRH receptor. As illustrated in Figure 4, addition of antide completely inhibited GnRH-induced LH release from cultured gonadotrophs, whereas it did not affect the LH-releasing activity of SP, thus indicating that the SP effect was not mediated via a GnRH receptor. FIG. 2. LH secretory response to SP in static cultures of porcine pituitary cells, a) Effect of various SP doses on LH release by porcine gonadotrophs. Cells were incubated with various concentrations of SP for 4 h, and the hormone secreted into the medium was evaluated by EIA. Results are the mean ± SEM of triplicate independent experiments and are expressed as a percentage of the control value (100%; dotted line). Control value of LH secreted into the medium was 21.1 ± 3.2 ng/ml. a, p < 0.05 vs. control, b) Intracellular pLH content measured in porcine pituitary cultures after a 4-h treatment with various SP doses. After treatment, cells were solubilized, and hormonal contents were measured by EIA. Results correspond to the mean ± SEM of 3 separate experiments and are expressed as a percentage of the control value (100%; dotted line). LH content in control gonadotrophs was 3413.0 ± 692.4 ng/100 000 cells, a, p < 0.05 vs. control. FIG. 3. Response of static cultures of porcine pituitary cells to treatment with SP and/or GnRH. a) Effect of 100 nM SP on basal and GnRH (10 nM)-stimulated LH release from porcine gonadotrophs, (n = 3; control 21.6 ± 2.1 ng LH/ml). a, p < 0.05 vs. control; b, p < 0.05 vs, GnRH; c, p < 0.05 vs. SP. b) Intracellular pLH content in porcine gonadotroph cells after a 4-h treatment with 100 nM SP and/or 10 nM GnRH. (n = 3; control LH content 2660 ± 406 ng LH/100 000 cells), a, p < 0.05 vs. control. See Figure 2 for further details. FIG. 4. Effect of the GnRH antagonist antide on SP- or GnRH-induced LH release by porcine pituitary cells in static culture. Cells were incubated (4 h) with 100 nM SP or 10 nM GnRH in the presence or absence of 1 m-M antide; (n = 3; control MEM alone 18.5 ± 2.3 ng LH/ml; control MEM + antide 21.4 ± 3.0 ng LH/ml). a, p < 0.05 vs. control MEM; b, p < 0.05 vs. GnRH + antide; с, p < 0.05 vs. control MEM + antide. See Figure 2 for further details.