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The graphical abstract summarises the effect of hydrogen peroxide on regulatory components of the Ca 2+ signaling pathway in salivary acinar cells. We show that H₂O₂ induced increased amount of reactive oxygen species (ROS) in parotid gland (PG) and submandibular gland (SMG) acinar cells and hypothesize that this disrupts cellular signaling that induce fluid secretion. Protein and mRNA expression analysis indicated that H₂O₂ exposure will interfere with neurotransmitter signaling pathways involving noradrenaline and acetylcholine, since the expression of both receptors was reduced in both PG and SMG cells. Additionally, H₂O₂ impacted Store-Operated Calcium Entry (SOCE) components, specifically Orai1 and <t>STIM1,</t> leading to differential expression changes in the two glands. This dysregulation can affect Ca 2 ⁺ homeostasis and ion transport, and subsequently fluid secretion (Abbreviations: hydrogen peroxide (H 2 O 2 ), reactive oxygen species (ROS), parotid gland (PG), submandibular gland (SMG), α1 adrenergic receptor (α1-AR) and muscarinic receptor 3 (M3R), Store-Operated Calcium Entry (SOCE), stromal interaction molecule 1 (STIM1), Phosphatidylinositol 4,5-bisphosphate (PIP2) and Inositol 3-phosphate (IP3)).
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The graphical abstract summarises the effect of hydrogen peroxide on regulatory components of the Ca 2+ signaling pathway in salivary acinar cells. We show that H₂O₂ induced increased amount of reactive oxygen species (ROS) in parotid gland (PG) and submandibular gland (SMG) acinar cells and hypothesize that this disrupts cellular signaling that induce fluid secretion. Protein and mRNA expression analysis indicated that H₂O₂ exposure will interfere with neurotransmitter signaling pathways involving noradrenaline and acetylcholine, since the expression of both receptors was reduced in both PG and SMG cells. Additionally, H₂O₂ impacted Store-Operated Calcium Entry (SOCE) components, specifically Orai1 and STIM1, leading to differential expression changes in the two glands. This dysregulation can affect Ca 2 ⁺ homeostasis and ion transport, and subsequently fluid secretion (Abbreviations: hydrogen peroxide (H 2 O 2 ), reactive oxygen species (ROS), parotid gland (PG), submandibular gland (SMG), α1 adrenergic receptor (α1-AR) and muscarinic receptor 3 (M3R), Store-Operated Calcium Entry (SOCE), stromal interaction molecule 1 (STIM1), Phosphatidylinositol 4,5-bisphosphate (PIP2) and Inositol 3-phosphate (IP3)).

Journal: Frontiers in Molecular Biosciences

Article Title: Hydrogen peroxide disrupts the regulatory pathway of saliva secretion in two salivary acinar rat cell lines

doi: 10.3389/fmolb.2024.1480721

Figure Lengend Snippet: The graphical abstract summarises the effect of hydrogen peroxide on regulatory components of the Ca 2+ signaling pathway in salivary acinar cells. We show that H₂O₂ induced increased amount of reactive oxygen species (ROS) in parotid gland (PG) and submandibular gland (SMG) acinar cells and hypothesize that this disrupts cellular signaling that induce fluid secretion. Protein and mRNA expression analysis indicated that H₂O₂ exposure will interfere with neurotransmitter signaling pathways involving noradrenaline and acetylcholine, since the expression of both receptors was reduced in both PG and SMG cells. Additionally, H₂O₂ impacted Store-Operated Calcium Entry (SOCE) components, specifically Orai1 and STIM1, leading to differential expression changes in the two glands. This dysregulation can affect Ca 2 ⁺ homeostasis and ion transport, and subsequently fluid secretion (Abbreviations: hydrogen peroxide (H 2 O 2 ), reactive oxygen species (ROS), parotid gland (PG), submandibular gland (SMG), α1 adrenergic receptor (α1-AR) and muscarinic receptor 3 (M3R), Store-Operated Calcium Entry (SOCE), stromal interaction molecule 1 (STIM1), Phosphatidylinositol 4,5-bisphosphate (PIP2) and Inositol 3-phosphate (IP3)).

Article Snippet: Next, membranes were incubated overnight with unlabeled rabbit anti-Orai1 (Alomone Labs, Israel), mouse anti-Stim1 (Alomone Labs), rabbit anti-Chrm3 (Bioss, Massachusetts, United States), rabbit anti-alpha1a adrenergic receptor (Invitrogen, Thermo Fisher Scientific, Massachusetts, United States), mouse anti-beta actin (Proteintech, United States) at 4°C.

Techniques: Expressing

Chrm3 and Adra1a, Orai1, and Stim1 gene expression in PG and SMG acinar cells. Chrm3 (A) , Adra1a (B) , Orai1 (C) and Stim1 (D) mRNA levels were analyzed by RT-qPCR in cells exposed to varying H 2 O 2 concentrations. Gene expressions were normalized to the housekeeping gene Rpl27 . Relative expression from five independent experiments. *p-value<0.05 for each treated group compared to control.

Journal: Frontiers in Molecular Biosciences

Article Title: Hydrogen peroxide disrupts the regulatory pathway of saliva secretion in two salivary acinar rat cell lines

doi: 10.3389/fmolb.2024.1480721

Figure Lengend Snippet: Chrm3 and Adra1a, Orai1, and Stim1 gene expression in PG and SMG acinar cells. Chrm3 (A) , Adra1a (B) , Orai1 (C) and Stim1 (D) mRNA levels were analyzed by RT-qPCR in cells exposed to varying H 2 O 2 concentrations. Gene expressions were normalized to the housekeeping gene Rpl27 . Relative expression from five independent experiments. *p-value<0.05 for each treated group compared to control.

Article Snippet: Next, membranes were incubated overnight with unlabeled rabbit anti-Orai1 (Alomone Labs, Israel), mouse anti-Stim1 (Alomone Labs), rabbit anti-Chrm3 (Bioss, Massachusetts, United States), rabbit anti-alpha1a adrenergic receptor (Invitrogen, Thermo Fisher Scientific, Massachusetts, United States), mouse anti-beta actin (Proteintech, United States) at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Control

Chrm3, Adra1a, Orai1, STIM1 protein expression in SMG and PG acinar cells. Western blot analysis was conducted to measure protein expression of Chrm3, Adra1a, Orai1 and STIM1 (A) in SMG and PG cells exposed to 0 (control), 100 and 150 μM H₂O₂. B-actin used as a reference protein. Orai1 and STIM1 protein expression in SMG and PG acinar cells were assessed by immunofluorescence microscopy, and their fluorescence intensities were measured in SMG (B, C) and PG (D, E) cells exposed to 0 (control), 100 and 150 μM H₂O₂. DAPI was used for nuclear staining. All photos were taken with the same exposure time (500 ms), analog gain (9.3x), and 40x magnification. Representative pictures from three independent experiments.

Journal: Frontiers in Molecular Biosciences

Article Title: Hydrogen peroxide disrupts the regulatory pathway of saliva secretion in two salivary acinar rat cell lines

doi: 10.3389/fmolb.2024.1480721

Figure Lengend Snippet: Chrm3, Adra1a, Orai1, STIM1 protein expression in SMG and PG acinar cells. Western blot analysis was conducted to measure protein expression of Chrm3, Adra1a, Orai1 and STIM1 (A) in SMG and PG cells exposed to 0 (control), 100 and 150 μM H₂O₂. B-actin used as a reference protein. Orai1 and STIM1 protein expression in SMG and PG acinar cells were assessed by immunofluorescence microscopy, and their fluorescence intensities were measured in SMG (B, C) and PG (D, E) cells exposed to 0 (control), 100 and 150 μM H₂O₂. DAPI was used for nuclear staining. All photos were taken with the same exposure time (500 ms), analog gain (9.3x), and 40x magnification. Representative pictures from three independent experiments.

Article Snippet: Next, membranes were incubated overnight with unlabeled rabbit anti-Orai1 (Alomone Labs, Israel), mouse anti-Stim1 (Alomone Labs), rabbit anti-Chrm3 (Bioss, Massachusetts, United States), rabbit anti-alpha1a adrenergic receptor (Invitrogen, Thermo Fisher Scientific, Massachusetts, United States), mouse anti-beta actin (Proteintech, United States) at 4°C.

Techniques: Expressing, Western Blot, Control, Immunofluorescence, Microscopy, Fluorescence, Staining