Enterochromaffin Cells of the Human Gut: Sensors for Spices and Odorants  Thomas Braun, Petra Voland, Lars Kunz, Christian Prinz, Manfred Gratzl  Gastroenterology  Volume 132, Issue 5, Pages 1890-1901 (May 2007) DOI: 10.1053/j.gastro.2007.02.036 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Expression of olfactory receptors by a cell line derived from human enterochromaffin cells (BON) and human small intestine biopsy samples. Genomic DNA (gDNA) of the intronless OR genes was removed from the samples by treatment with DNase I (controls). Complementary DNA (cDNA) was generated, and PCR was performed using the primers listed (see Table 1). gDNA from BON cells was used as positive control. Expression of VMAT1 (marker for EC cells) and β-actin was analyzed with primers designed to amplify exon sequences of these genes. (A) Presence of ORs in BON cells was shown with primers established for the detection of consensus sequences of ORs (conOR). Expression of VMAT1 and β-actin was analyzed in parallel. (B) Using specific primers, the nature of 4 ORs (OR73, hOR17-7/11, OR1G1, and hOR17-210) expressed by BON cells is revealed. (C) Nested PCR using the amplicon of the PCR reaction in B was performed showing amplification of the signal and confirming the presence of ORs in BON cells. (D) Presence of olfactory receptors in small intestinal biopsy samples. The receptors OR73, hOR17-7/11, OR1G1, and hOR17-210 were detected by nested PCR using the primers indicated. Expression of VMAT1 and β-actin was analyzed in parallel. Gastroenterology 2007 132, 1890-1901DOI: (10.1053/j.gastro.2007.02.036) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Expression of olfactory receptors in human ileum mucosa samples. The presence of ORs in paraffin-embedded human ileum sections was investigated using primers detecting the receptor OR1G1 with a broad ligand range. Signals were obtained by nested PCR in sections from 3 different tissue samples (1, 2, 3). Expression of VMAT1 (nested PCR) and β-actin was analyzed in the same samples. Gastroenterology 2007 132, 1890-1901DOI: (10.1053/j.gastro.2007.02.036) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Microdissection and expression of olfactory receptors in human enterochromaffin cells. EC cells were identified in paraffin-embedded tissue sections by their immunoreactivity for VMAT1. (A) Immunostaining of 3 enterochromaffin cells (arrows) in the crypt of human small intestine done on a glass slide is shown. (B) Prior to laser microdissection, tissue sections were placed on a polyethylene naphtalene membrane and immunostained. (C and D) Subsequently, the VMAT1-immunostained cells were microdissected and harvested. RNA was isolated from laser microdissected EC cells, and cDNA was generated after DNase I treatment. (E) Nested PCR was performed using the indicated primers (see Table 1), which allowed identifying the ORs OR73, hOR17-7/11, OR1G1, and hOR17-210. Gastroenterology 2007 132, 1890-1901DOI: (10.1053/j.gastro.2007.02.036) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Effect of thymol on intracellular free Ca2+ levels in enterochromaffin BON cells. (A) Stimulation of BON cells with the odorant thymol (100 μmol/L) caused an elevation of intracellular Ca2+ in virtually all cells. (B) Addition of nifedipine (8 μmol/L) inhibited the effect of thymol. (C) The time course of a Ca2+ transient of a single cell upon addition of thymol reveals that the transient effect of thymol is reversibly inhibited by nifedipine. The color bar on the left indicates intracellular free Ca2+ levels (black/dark red: low; yellow/white: high). Gastroenterology 2007 132, 1890-1901DOI: (10.1053/j.gastro.2007.02.036) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Effect of odorants on intracellular free Ca2+ levels and serotonin release by enterochromaffin BON cells. (A) Elevation of intracellular Ca2+ levels in BON cells depends on the concentration of the odorant eugenol. (B) Methyl isoeugenol (MIE, 500 μmol/L) has no effect but completely and reversibly inhibited the effect of eugenol (EG). (C) Eugenol (250 μmol/L) but not methyl isoeugenol (250 μmol/L) elicits serotonin release, and the effect of eugenol is inhibited by preincubation of the cells with equimolar concentrations of methyl isoeugenol (n = 6, P values correspond to Student–Newman–Keuls tests). (D) The effect of the odorant bourgeonal on intracellular Ca2+ levels was reversibly inhibited by addition of nifedipine (8 μmol/L). Gastroenterology 2007 132, 1890-1901DOI: (10.1053/j.gastro.2007.02.036) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Effects of thapsigargin, PLC inhibitor, and IP3 receptor blocker on Ca2+ transients triggered by thymol. (A) BON cells still react to the odorant thymol (100 μmol/L) after intracellular Ca2+ stores have been depleted by thapsigargin (1 μmol/L). (B) PLC inhibitor U73122 (10 μmol/L) strongly reduced the signal caused by thymol (100 μmol/L), whereas the inactive analogue U73343 (10 μmol/L) had no effect. (C) Xestospongin C (1 μmol/L), an antagonist of IP3R, completely blocked the thymol-induced calcium signals. The cells still respond to depolarization (56 mmol/L K+). Gastroenterology 2007 132, 1890-1901DOI: (10.1053/j.gastro.2007.02.036) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Odorants stimulate serotonin release by enterochromaffin BON cells. BON cells were incubated for 15 minutes in serum-free medium with different odorants at concentrations indicated. Serotonin release was measured in the culture supernatant by a serotonin enzyme immunoassay kit (ELISA). Concentration-dependent serotonin release by BON cells was triggered by eugenol (A), thymol (B), bourgeonal (C), and helional (D) but not phenol (shaded bars in B). The solvent ethanol at equivalent final concentrations (0.25% to 1.0%) caused a maximum of 1.58- ± 1.32-nmol/L serotonin release from BON cells. P values correspond to Student–Newman–Keuls tests. Amperometrical recording of exocytotic serotonin release by BON cells caused by the odorants thymol and bourgeonal are shown in (E) and (F). Thymol and bourgeonal (both 100 μmol/L) caused quantal release of serotonin (recorded at 850 mV). Spikes represent single exocytotic events. Gastroenterology 2007 132, 1890-1901DOI: (10.1053/j.gastro.2007.02.036) Copyright © 2007 AGA Institute Terms and Conditions