Elevated Serotonin in COVID-19 Stimulates Chloride Secretion in Lung and Intestinal Epithelial Cells
Elevated 5-hydroxytryptamine in COVID-19 Stimulates ANO1 Mediated Cl Secretion in Lung & Intestinal Epithelial Cells
Earlier studies demonstrated that blood 5-hydroxytryptamine (5-HT) is elevated in patients with COVID-19-associated diarrhea with higher severity of symptoms. We hypothesize that disruption of vectorial Cl transport by 5-HT may be critical in determining the alveolar flooding and abnormalities in intestinal Cl secretion through stimulation of anoctamin 1(ANO1) Cl channel. Using western blot, immunostaining, and electrophysiology, we characterized the localization of human ANO1 and its stimulation by 5-HT on Cl secretion in lung and intestinal epithelium. Because calcium-activated chloride current in human intestinal epithelia remains controversial, we examined the localization of ANO1 in the human terminal ileum and colonic tissue using confocal microscopy.
Our results indicated that ANO1 was localized predominantly at the brush-border membrane and co-localized with the brush-border membrane marker villin. ANO1 is not present in goblet cells. The anti-ANO1 antibody recognized a protein of appropriate size in human colonic tissue. The specificity of the ANO1 antibody was tested by immunoblot analysis of lysates from human colorectal cancer tissues, where it displayed amplified ANO1 protein expression. We next confirmed ANO1-currents activated by 5-HT in the Caco-2 cells by patch-clamp measurements of whole-cell current. The application of 100 nM 5-HT produced a typical outward rectification. CaCCinh-A01, a specific ANO1 blocker, inhibited the currents. The half-maximal effective concentration value for the effects of 5-HT was estimated at 21.8 ± 13.7 nM with a Hill coefficient of 0.89 ± 0.16.
These results indicated that 5-HT evoked calcium-activated Cl currents through ANO1 channels. ANO1 is expressed in Calu 3 cells. We next confirmed the presence of ANO1 currents activated by 5-HT in Calu-3 cells by the Ussing chamber experiments. Serosal addition of 5-HT produced an immediate and significant increase in Isc in Calu-3 cells that was inhibited by the ANO1 selective inhibitor T16Ainh-A01. Finally, we demonstrate that SARS-CoV2 infection led to enterochromaffin cell hyperplasia in the intestinal epithelium of Syrian Hamster with a possible elevation of 5-HT, which could explain the severity of symptoms in COVID-19 associated diarrheal patients.
In conclusion, SARS-CoV2 infection resulted in intestinal enterochromaffin cells hyperplasia that could elevate 5-HT. Elevated 5-HT activates luminal ANO1 CaCC in the intestinal and lung epithelium by a mechanism that appears to involve the rise of [Ca2+]i. Our data suggest that 5-HT may be a critical determinant of the COVID-19 associated diarrhea and flooding of alveoli that have considerable implications for COVID-19 therapy.
ANO1 is a transmembrane protein that functions as a calcium-activated chloride channel. Ca2+, Sr2+, and Ba2+ activate the channel.
Its overexpression was reported in esophageal squamous cell carcinoma and breast cancer progression.
Niflumic acid is a drug used for joint and muscular pain. In experimental biology, it has been employed to inhibit ANO1.
Inhibition of ANO1 calcium-activated chloride channels by avermectins is essential for their anticancer effects
Anoctamin 1 (ANO1) encoded Ca2+-activated Cl- channels were found to be involved in tumorigenesis. Previous studies suggest the effect of ANO1 gene amplification on tumorigenic proliferation is exerted through its channel function. ANO1-specific and potent small molecule inhibitors have been proposed to potentially be useful for the treatment of cancer. Thus, we screened six analogues of avermectin for their inhibitory activities on ANO1 mediated currents. A whole-cell patch technique was used to record the currents. The IC50 and Emax values for ANO1 inhibition of five tested avermectins (avermectin B1, ivermectin, doramectin, selamectin, and moxidectin) were 0.15-1.32 μM and 65-87 %, respectively. In addition, these avermectins significantly inhibited endogenous ANO1 mediated currents and thus, the proliferation, migration, inducing apoptosis of LA795 cancer cells. Eprinomectin (4"-(acetylamino)-4"-deoxy-avermectin B1) and two other important macrolides (erythromycin and azithromycin), which have minimal or no ANO1 inhibitory effects, were used as negative control drugs. These drugs were found to have limited effects on the proliferation, migration, and apoptosis of LA795 cells. Finally, avermectin B1 and ivermectin dramatically inhibited the growth of xenograft tumors in mice. These data demonstrate that avermectins are novel ANO1 inhibitors and are potentially useful in specific cancer therapies. These findings also provide a new opportunity to develop ANO1 modulators.