S1682 Role of the Gasotransmitter Hydrogen Sulfide in Modulation of Contractile Function in Longitudinal Muscle of Rat Jejunum

Abstract

Background: HDC catalyzes the conversion of histidine to histamine, a bioamine which plays an important role in allergy, inflammation, neurotransmission, gastric acid secretion and etc. Regulation of HDC expression occurs at both the transcriptional and posttranslational level. While extensive studies have been performed to study the posttranslational regulation of HDC, an adequate In Vivo model to study HDC gene expression at the transcriptional level has not been reported. Aim: To establish an In Vivomodel to study HDC gene expression at the transcriptional level. Methods: A mouse bacterial artificial chromosome (BAC) clone that contains the entire HDC gene locus was used to generate an HDC/EGFP construct where EGFP gene expression is controlled by ~113 kb of HDC 5' flanking and 75 kb of 3' flanking DNA. An EGFP cassette was inserted into translational start codon (ATG) of mouse HDC gene. The transgene was pronuclearly injected into fertilized mouse oocytes (B6/CBA mixed background), and potential founders were screened by tail DNA PCR. Positive founders were backcrossed to B6 mice. HDC/EGFP mice were infected with H. felis for 3 months or intraperitoneally injected of omeprazole for 2 weeks (350 μmol/kg). Results: Three potential founder mice were obtained, and specific EGFP fluorescence was observed in epithelial cells residing in the lower third of the gastric corpus glands in all F1 mice from three lines. QRT-PCR confirmed that F1 mice from three founders expressed EGFP mRNA in the stomach at a level similar to that of endogenous HDC. Specificity of EGFP fluorescence was also confirmed by immunostaining for EGFP. Expression of EGFP in ECL cells was confirmed by co-immunostaining with HDC and chromogranin A antibodies. EGFP positive cells were also found in small intestine, colon, spleen, lung, and olfactory neurons in brain, while not found in other tissues including liver, pancreas, heart, kidney, skin, testis and thymus. FACS analysis detected EGFP expression in a subpopulation of bone marrow cells. Q-RT-PCR showed that EGFP mRNA expression in the stomach and small intestine was significantly increased after 3 months H. felis infection. EGFP positive cells in the stomach were also increased after omeprazole treatment. Conclusions: Expression of the HDC/EGFP transgene accurately reflects expression of the endogenous HDC gene. With H. felis infection, HDC/ EGFP expression is enhanced in both stomach and small intestine. The HDC/EGFP transgene model offers a robust way to track HDC expression under diverse physiological and pathological conditions and to potentially isolate HDC expression cells for further studies.