Inhibition of GH release and synthesis by fatty acids in primary cultured caprine anterior pituitary cells

Abstract

s REGPEP 2000 35 INHIBITION OF GH RELEASE AND SYNTHESIS BY FATTY ACIDS IN PRIMARY CULTURED CAPRINE ANTERIOR PITUITARY CELLS Katoh, Kazuo; Ishiwata, Hiroko; Chen, Chen; Obara, Yoshiyuki Dept. Anim. Physiol., Facul. Agri., Tohoku Univ., Japan Influence of growth hormone (GH) secretion from somatotrophs by nutrients has been demonstrated in a variety of animal species in vivo and in vitro. For example, amino acids increased intracellular free calcium concentration ([Ca2+]i) and raised GH release from caprine somatotrophs (Ohata et al., 1997). In contrast, short-chain fatty acids (propionate and butyrate) suppressed GH release from and total GH content in somatotrophs (Katoh et al., 1999; lshiwata et al., 2000). Present experiment aimed to clarify the mechanism of fatty acids action on GH release, [Ca2+]i, Ca current and GH mRNA levels in primary cultured caprine anterior pituitary cells. Caprine anterior pituitary cells were cultured in DMEM containinglO% FCS for 3 days. 1) Cells were stimulated with GHRH (1 nM, 30 min) in the presence or absence of a fatty acid (butyrate, octanoate or oleate, 0.01-10 mM). Fatty acids significantly suppressed GHRH-induced increase in GH release and cellular GH content in a dose-dependent manner. 2) Octanoate and oleate significantly reduced [Ca2+]i observed in flue-3 loaded cells with a confocal laser microscope. 3) Recorded by patch-clamp in whole-cell configuration, nifedipine-sensitive (I-TX-resistant) L-type Ca current was markedly reduced by octanoate. 4) Northern blot analysis showed that butyrate treatment for 24h reduced the level of GHmRNA in pituitary cells. Based on these results, we conclude that fatty acids suppress Ca2+ influx via L-type voltage-gated Ca channels leading to the decrease in [Ca2+]i and GH release. Fatty acids also reduce the level of GHmRNA resulting in the decrease in GH synthesis in caprine somatotrophs. REFERENCES: Ohata Y., Maruyama Y., Katoh K. and Sasaki Y. (1997) Domestic Animal Endocrinology 14: 99-107; Katoh K., Ohata Y. and Ishiwata H. (1999) Domestic Animal Endocrinology 17: 85-93; lshiwata H., Nagano M., Sasaki Y., Chen C. and Katoh K. (2000) Genera/ and Comparative Ecfocrinology 118:400-406. INHIBITION OF iNOS EXPRESSION AND NITRIC OXIDE PRODUCTION IN MONOCYTES BY THE TREFOIL PEPTIDE TFF2 Giraud, Andy S; Parker, Lorraine M University of Melbourne, Australia Trefoil peptides are secreted products of many epithelia particularly the gut. They are induced following mucosal injury and inflammation and participate in wound healing. We demonstrated that application of recombinant TFF2 in a model of ulcerative colitis both accelerates mucosal reconstitution and reduces inflammatory indices, suggesting multiple modes of action. Since prolonged inflammation results in high level production of nitric oxide (NO) and ultimately formation of damaging peroxynitrite, we aimed to test whether trefoil peptides might limit ongoing mucosal injury in part by inhibiting NO synthesis by the inducible synthase INOS. METHODS: RAW 264.7 monocyte cell line was pre-incubated with 0.3535 uM glycosylated human TFF2 (ghTFF2) or vehicle then challenged with 1 nM LPS for 18 hr. Secreted NO was measured using the Griess reagent and cellular iNOS by Western blotting. Peroxynitrite induced protein nitration was evaluated by immunohistochemistry in control colon or after TFF2 or vehicle treatment for 5 days, 1 week after colitis induction (DNBS/ethnol). Results: ghTFF2 treatment of LPS challenged cells showed a dosedependent reduction (3535pM) in NO production. Similarly, 1.4-14 uM ghTFF2 inhibited iNOS expression to control levels in RAW cell extracts. Mucosal nitrated protein was markedly reduced in rats with colitis treated with ghTFF2 compared to controls. CONCLUSIONS: TFF2 reduces NO production by monocytes by inhibiting iNOS expression after LPS challenge. The TFF2-induced reduction in protein nitration after colitis induction, indicates that peroxynitrite production is reduced due to NO inhibition. These data suggest that NO inhibition may be one mechanism by which trefoil peptides protect the gut against ongoing damage in chronic inflammation.