Acetylcholine (ACh), synthesized in the pituitary, can act locally to modulate pituitary function. We used rat primary anterior pituitary (AP) cells to investigate how ACh affects pituitary prolactin (PRL) secretion in the presence or absence of known PRL regulators: thyrotropin-releasing hormone (TRH), 17β-estradiol (E₂) and triiodothyronine (T₃). Cultured AP cells were prepared from ovariectomized rats and pretreated with diluent, 0.6 nM E₂, 10 nM T₃, or E₂ plus T₃ for 5 days, then challenged with various doses of ACh or muscarinic receptor agonists (oxotremorine or carbachol) and TRH (100 nM) for 20 min. Significant ACh (10^–5M) suppression of both basal and TRH-induced PRL secretion was not evident in diluent-, E₂- or T₃-pretreated cells, but observed only in cells pretreated with both E₂ and T₃. Moreover, in E₂ plus T₃-pretreated cells, oxotremorine and carbachol, like ACh (10^–7–10^–5M), suppressed both responses in a dose- related manner. Pertussis toxin (PTX; 100 ng/ml) as well as atropine (a muscarinic receptor antagonist; 1 mM) blocked these effects of cholinomimetics. ACh also inhibited both PRL responses elicited by drugs elevating intracellular cAMP (10 µM forskolin) or Ca²⁺ (1 µM Bay K-8644) in a PTX-sensitive manner. ACh inhibition of basal PRL secretion was unaltered by intracellular Ca²⁺ mobilization blockers, TMB-8 (100 µM) and thapsigargin (1 µM), but abrogated by the nitric oxide synthase inhibitor (300 µM L-NAME). ACh inhibition of TRH-induced PRL secretion was accentuated by TMB-8 and alleviated by thapsigargin or L-NAME. In summary, muscarinic inhibition of either basal or TRH-induced PRL secretion was augmented by E₂ and T₃, and involved the PTX-sensitive cAMP/Ca²⁺ pathways. Furthermore, nitric oxide mediated the basal rather than TRH-induced PRL response to ACh, whereas the intracellular Ca²⁺ mobilization concerned the TRH-induced rather than the basal PRL response to ACh. Thus, ACh synthesized in the AP appears to inhibit basal vs. TRH-induced PRL secretion via different mechanisms.

The mechanisms of exogenous nitric oxide (NO)-enhanced growth of the U937 human myeloid leukemic cells were examined using sodium nitroprusside (SNP) as a NO donor. Treatment with 0.1 mM SNP for 72 h caused a 45 ؎ 2% increase in U937 cell growth with significantly increased S/G 2 ؉M-phase and decreased G 0 /G 1 -phase of the cell cycle. The growth-enhancing effect of SNP was blocked by indomethacin, a cyclooxygenase inhibitor, but not by H7, a broad spectrum kinase inhibitor, or PD98059, a mitogenactivated protein kinase inhibitor. SNP treatment resulted in a dose-dependent increase in prostaglandin E 2 (PGE 2 ) production. Furthermore, the addition of exogenous PGE 2 not only enhanced U937 cell growth but restored the indomethacin-inhibited mitogenic effect of SNP. We suggest that NO can enhance cell growth through activating the cyclooxygenase pathway and that PGE 2 may be an effector molecule for NO-regulated cell proliferation. Our data provide a mechanistic insight into the regulatory role of NO in myelopoiesis. J. Leukoc. Biol. 64: 451-458; 1998.