Alcohol use disorders are associated with altered stress responses, but the impact of stress or stress hormones on alcohol-associated tissue injury remain unknown. We evaluated the effects of chronic restraint stress on alcohol-induced gut barrier dysfunction and liver damage in mice. To determine whether corticosterone is the stress hormone associated with the stress-induced effects, we evaluated the effect of chronic corticosterone treatment on alcoholic tissue injury at the Gut-Liver-Brain (GLB) axis. Chronic restraint stress synergized alcohol-induced epithelial tight junction disruption and mucosal barrier dysfunction in the mouse intestine. These effects of stress on the gut were reproduced by corticosterone treatment. Corticosterone synergized alcohol-induced expression of inflammatory cytokines and chemokines in the colonic mucosa, and it potentiated the alcohol-induced endotoxemia and systemic inflammation. Corticosterone also potentiated alcohol-induced liver damage and neuroinflammation. Metagenomic analyses of 16S RNA from fecal samples indicated that corticosterone modulates alcohol-induced changes in the diversity and abundance of gut microbiota. In Caco-2 cell monolayers, corticosterone dose-dependently potentiated ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. These data indicate that chronic stress and corticosterone exacerbate alcohol-induced mucosal barrier dysfunction, endotoxemia, and systemic alcohol responses. Corticosterone-mediated promotion of alcohol-induced intestinal epithelial barrier dysfunction and modulation of gut microbiota may play a crucial role in the mechanism of stress-induced promotion of alcohol-associated tissue injury at the GLB axis.

The apical junctional complex (AJC), which includes tight junctions (TJs) and adherens junctions (AJs), determines the epithelial polarity, cell-cell adhesion and permeability barrier. An intriguing characteristic of a TJ is the dynamic nature of its multiprotein complex. Occludin is the most mobile TJ protein, but its significance in TJ dynamics is poorly understood. On the basis of phosphorylation sites, we distinguished a sequence in the C-terminal domain of occludin as a regulatory motif (ORM). Deletion of ORM and expression of a deletion mutant of occludin in renal and intestinal epithelia reduced the mobility of occludin at the TJs. ORM deletion attenuated Ca depletion, osmotic stress and hydrogen peroxide-induced disruption of TJs, AJs and the cytoskeleton. The double point mutations T403A/T404A, but not T403D/T404D, in occludin mimicked the effects of ORM deletion on occludin mobility and AJC disruption by Ca depletion. Both Y398A/Y402A and Y398D/Y402D double point mutations partially blocked AJC disruption. Expression of a deletion mutant of occludin attenuated collective cell migration in the renal and intestinal epithelia. Overall, this study reveals the role of ORM and its phosphorylation in occludin mobility, AJC dynamics and epithelial cell migration.

Angiotensin II (Ang II) is a potent vasoconstrictor of vascular smooth muscle cells and implicated in hypertension, but it’s role in the regulation of endothelial function is not well known. We and others have previously shown that mechanosensitive ion channel, TRPV4 mediates flow- and/or receptor-dependent vasodilation via nitric oxide (NO) production in endothelial cells. Ang II was demonstrated to crosstalk with TRPV4 via AT1R and beta-arresting signaling in epithelial and immortalized cells, however, the role of this crosstalk in endothelial cell function is unexplored. Here, we demonstrate that pre-treatment with Ang II (200 nM for 48 h) significantly abolished TRPV4 mediated calcium influx in human microvascular endothelial cells, HMEC-1 (human EC). Further, Ang II treatment significantly downregulated TRPV4 protein expression in human EC without altering TRPV4 mRNA levels. Furthermore, Ang II-treatment significantly inhibited TRPV4-induced eNOS phosphorylation in human EC. Taken together, these results suggest that Ang II, in addition to eliciting smooth muscle contraction, contributes to hypertension by reducing TRPV4 protein expression and dependent NO production in human EC. This work is supported by NIH (1RO1HL119705, 1R15CA202847-01 and R01 HL148585), AHA Grant in Aid (14GRNT20380935, AHA- TPA-971237) and start-up funds from University of Toledo (CKT). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transient receptor potential vanilloid type 4 (TRPV4) is a mechanosensor that regulates endothelial cell (EC) proliferation, migration, and angiogenesis. However, the molecular mechanisms by which TRPV4 regulates EC functions are not well understood. In this study, we investigated if TRPV4 regulates EC function via modulation of mitochondria by employing three types of EC expressing different levels of TRPV4, normal (NEC), TRPV4-deficienct (TEC), and TRPV4 knockout (KOEC). First, we confirmed the functional expression of TRPV4 in these EC using qPCR and calcium imaging. Confocal images upon MitoTracker staining revealed spherical and/or round shaped mitochondria with a perinuclear localization in NEC while in TEC and KOEC the mitochondrial network was elongated and rod shaped with a whole cell distribution. Furthermore, Transmission Electron Microscopy confirmed the presence of clear round mitochondria in NEC compared to elongated mitochondria with distinct cristae in TEC and KOEC. These results indicate increased fusion in TRPV4 deficiency or deletion. When cultured on ECM gels of varying stiffness that mimic stiffness of matrix in pathophysiological conditions such as tumor or heart failure (0.2, 8, and 50 kPa), we found increased distribution of mitochondria in EC with increasing stiffness. Moreover, western blot analysis showed increased expression of a fusion/fission protein ratio (Optic Atrophy 1 (OPA1)/mitochondrial fission factor (MFF)), in TEC and KOEC. Further, uncoupling mitochondrial function by mitochondrial stressors (Oligomycin, FCCP, and Rot/AA) significantly increased basal oxygen consumption rate (OCR), maximal OCR, ATP-linked OCR, and spare capacity in TEC and KOEC than NEC. Finally, a small molecule inhibitor of OPA1, MYLS22, attenuated/normalized the mitochondrial energy metabolism to that of NEC but did not affect NEC. These findings suggest that mechanosensitive TRPV4 channels regulate EC mitochondrial morphology and function through increased OPA1. National Institutes of Health (R15CA202847, R01HL119705, and R01HL148585) to CKT This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.