Stimulation of Nitric Oxide Synthesis in Porcine Granulosa Cells by Phorbol Ester.

Abstract

Abstract. This study was designed to assess the release of nitric oxide (NO) from porcine granulosa cells. The cells obtained from medium-sized ovarian follicles (3-6 mm diameter) were cultured for 48 h with ovine FSH to be developed. A significant accumulation of nitrite was observed during development, showing an increase in NO synthesis. The mRNA of the endothelial NO synthase was detected in the granulosa cells, as revealed by RT-PCR. Furthermore, nitrite was accumulated in the culture medium after 1-h stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA), but not with 4α-phorbol, 4β-phorbol 13-monoacetate (4β-PMA) and forskolin. TPA led to an increase in the nitrite content in a dose-dependent manner. NO was measured directly in the cell suspension with an amperometric NO sensor. The NO sensor signal gradually increased to reach a plateau (5-15 pA) after exposure to TPA in the presence of L-arginine, but not D-arginine. The concentration of NO was approximately 5.5-11 nM, but no detectable change was observed after the addition of 4α-phorbol or 4β-PMA. These results suggest that the synthesis of NO is promoted at least in part through the activation of protein kinase C in granulosa cells. Key words: Nitric oxide, Granulosa cells, eNOS mRNA, Phorbol ester (J. Reprod. Dev. 47: [1][2][3][4][5][6] 2001) itric oxide (NO) is a short-lived messenger molecule that is involved in various cellular processes and/or functions. NO is generated by three isoforms of NO synthase (NOS) that are cl a ss i f ie d i n to t w o co n s ti tu ti ve is o f o r m s , endothelial and neural NO synthases (eNOS and nNOS, respectively) [1, 2] and an inducible isoform (iNOS) [3]. The expression of mRNAs and proteins for eNOS and iNOS in the rat ovary was seen during follicular development, ovulation and pseudopregnancy [4,5]. NO was also reported to be generated in immature ovaries [6,7] and at various stages of follicular development [8]. In a recent study by us [9], NO was proposed to function as a modulator in the differentiation of rat granulosa cells; removal of NO during the differentiation suppressed expression of the epidermal growth factor receptor. Nevertheless, the NO assay employed in these studies was based on indirect measurement of NO formation, i.e., nitrite and nitrate. NO may be naturally released from the ovary, but there is no report concerning