Histamine intolerance (HIT) is a common diagnosis with an estimated prevalence of 1% in the Western population. Patients suffering from HIT exposed to histamine present with various symptoms such as rhinal congestion, headache, tachycardia, diarrhea, urticaria, and eventually bronchoconstriction. Food-derived histamine is modulated by different mechanisms in the intestine. Two intestinal enzymes, diamine oxidase (DAO) and histamine-N-methyltransferase (HNMT), are reported to metabolize histamine. Moreover, transport proteins, such as Organic Cation Transporter 3 (OCT3) or Plasma Membrane Monoamine Transporters (PMAT), are believed to contribute to the transmembrane transport and thus the overall handling of histamine.
Using an immortalized cell line originating from colon adenocarcinoma, Caco-2, we aim to elucidate the influence of herbal remedies used in clinics for their influence on the intestinal handling of histamine. In pre-experiments we successfully validated the expression of DAO and HNMT in Caco-2 cells. In a dose-finding experiment, we will first test a dilution series of herbal extracts in cell viability assays in order to exclude a negative effect on Caco-2 cell viability. Next we will investigate the effect of different concentrations of herbal extracts on DAO and HNMT mRNA and protein expression by real-time qPCR and Western blot analysis, respectively. Previously, it has been reported that Caco-2 cells exposed to heparin showed a significant increase in DAO release and DAO enzymatic activity. Accordingly, we will examine the effect of herbal extract on DAO content and DAO activity in the enriched secretome of Caco-2 cells applying Western blot analysis and a fluorometric DAO enzyme activity assay, respectively.
Since herbal extract may potentially interact with the transcellular transport of food-derived histamine in the intestine, we will apply Caco-2 transwell transport studies to assess the influence on the uptake of radioactive [3H]-histamine as a tracer. Adding herbal extracts to the apical side of the transwell (representing the luminal side of the intestine) we will determine the apical-to-basal as well as the basal-to-apical transfer of histamine.
Concluding we will perform transport interaction experiments testing for the impact of histamine handling by OCT3 or PMAT in presence or absence of herbal extracts. To investigate the role of OCT3 we will apply a MDCKII cell line stably expressing OCT3 (MDCKII-OCT3) and readily available in our laboratory. PMAT will be cloned from human brain cDNA. We will then use the vaccinia virus-assisted expression system to transiently overexpress PMAT in HeLa cells.