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HORMONES AND SIGNALING

Short food deprivation inhibits orexin receptor 1 expression and orexin-A induced intracellular calcium signaling in acutely isolated duodenal enterocytes

¹Department of Neuroscience, Division of Physiology, Uppsala University, Uppsala, Sweden; ²Institute of Biomedicine, Department of Physiology, Oulu, Finland; and ³A. I. Virtanen Institute for Molecular Sciences and Dept of Internal Medicine, Kuopio University Hospital, Kuopio, Finland

Submitted 18 June 2008 ; accepted in final form 27 December 2008

Close intra-arterial infusion of the appetite regulating peptide orexin-A stimulates bicarbonate secretion from the duodenal mucosa. The aim of the present study was to elucidate the ability of orexin-A to induce intracellular calcium signaling in acutely isolated duodenal enterocytes. Freshly isolated clusters of enterocytes, obtained from rat duodenal mucosa or human duodenal biopsies, were loaded with fura 2-AM and mounted in a perfusion chamber. Cryptlike enterocytes were selected (caged), and changes in intracellular calcium concentration ([Ca²⁺]_i) were evaluated by fluorescence imaging. Total RNA was extracted from pellets of enterocytes and reverse transcribed to cDNA, and expression of orexin receptors 1 and 2 (OX₁R and OX₂R) was measured by quantitative real-time PCR. Orexin-A at all concentrations tested (1–100 nM) increased [Ca²⁺]_i in enterocytes isolated from continuously fed rats, and the OX₁R-antagonist SB-334867 (10 nM) attenuated the response. The primary [Ca²⁺]_i response was a slow increase to a sustained plateau persisting after orexin-A removal, and a similar response was observed in enterocytes from human biopsies. In contrast to orexin-A, the OX₂R agonist (Ala¹¹,D-Leu¹⁵)-orexin-B (1–10 nM) did not induce calcium signaling. There were no significant [Ca²⁺]_i responses in enterocytes from animals food deprived overnight, and overnight fasting decreased (P < 0.01) enterocyte OX₁R as well as OX₂R mRNA. Induction of intracellular calcium signaling in isolated duodenal enterocytes is thus mediated primarily by OX₁R receptors. Short (overnight) food deprivation markedly depresses receptor expression and inhibits orexin-A induced increases in [Ca²⁺]_i. Studies of enterocyte signaling and intestinal secretion requires particular evaluation regarding feeding status.

(Ala¹¹,D-Leu¹⁵)-orexin-B; bicarbonate secretion; fasting; feeding; SB- 334867