Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis <em>In Vitro</em>

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

Anhui medical university

Store-operated Ca(2+) entry (SOCE) is a common and ubiquitous mechanism regulating Ca(2+) influx into cells and participates in numerous biological processes including cell proliferation. Glomerular mesangial cells (GMCs) play a role in the regulation of the glomerular filtration rate. From a clinical point of view, many physiological functions alter with age. In the present study, we used angiotensin II, glucagon, and the sarco/endoplasmic reticulum membrane Ca(2+) pump inhibitor thapsigargin to deplete the internal Ca(2+) stores for the activation of SOCE. We found that SOCE was significantly attenuated in GMCs from aged (22-month-old) rats. The expression of SOCE-related components, stromal interaction molecule 1 (STIM 1) and Orai 1, in freshly isolated glomeruli notably decreased, and STIM 1 and Orai 1 puncta formation significantly reduced in primary-cultured GMCs in aged rats. Moreover, specific knockdown of STIM 1 and Orai 1 by small interfering RNA markedly suppressed SOCE and cell proliferation of GMCs isolated from young (3-month-old) rats. We conclude that the attenuation of GMCs proliferation can be attributed to the decreased SOCE partially caused by reduced expression of STIM 1 and Orai 1.

Attenuated mesangial cell proliferation related to store-operated Ca2+ entry in aged rat: the role of STIM 1 and Orai 1.

High-salt intake is a major risk factor in the development of hypertension. The underlying mechanism of high sodium on the cardiovascular system has received extensive attention. TRPP2 (Polycystin-2) is a Ca(2+) permeable nonselective cation channel that mediates Ca(2+) mobilization to control vascular smooth muscle cells (VSMCs) contraction. Here, we investigated TRPP2 expression change in VSMCs from high-salt intake hypertensive rats and role of TRPP2 in the development of high-salt diet-induced hypertension.

Increased TRPP2 expression in vascular smooth muscle cells from high-salt intake hypertensive rats: The crucial role in vascular dysfunction.

Ca is a crucial factor in the regulation of smooth muscle contraction. Store-operated Ca entry (SOCE) is one pathway that mediates Ca influx and smooth muscle contraction. Vessel contraction function usually alters with aging to cause severe vascular-related diseases. However, the underlying mechanism is still not fully understood. Here, we assessed intracellular Ca and vessel tension and found that SOCE and SOCE-mediated contraction of vascular smooth muscle cells (VSMCs) was reduced in aorta but increased in mesenteric arteries from aged rats. The results of Western blot and immunofluorescence staining show that the expression levels of Orai1, a store-operated Ca channel, were increased in VSMCs of mesenteric arteries but were reduced in VSMCs of aorta with aging. In conclusion, we demonstrated that the changing pattern of SOCE and SOCE-mediated contraction of VSMCs is completely reversed in mesenteric arteries and aorta with aging, providing a potential therapeutic target for clinical treatment in age-related vascular diseases. 

Contrasting Patterns of Agonist-induced Store-operated Ca2+ Entry and Vasoconstriction in Mesenteric Arteries and Aorta With Aging.

The flow-stimulated intracellular Ca(2+) concentration ([Ca(2+)]i) rise in endothelial cells is an important early event leading to flow-induced blood vessel dilation. Transient receptor potential vanilloid subtype 4 (TRPV4), a Ca(2+)-permeable cation channel, facilitates the flow-stimulated [Ca(2+)]i rise. To determine whether TRPV4 is involved in age-related flow-induced blood vessel dilation impairment, we measured blood vessel diameter and nitric oxide (NO) levels and performed Ca(2+) imaging, immunoblotting, and immunostaining assays in rats. We found that the flow-induced and TRPV4 activator 4α-PDD-induced dilation of mesenteric arteries from aged rats were significantly decreased compared with those from young rats. The flow- or 4α-PDD-induced [Ca(2+)]i rise was also markedly reduced in primary cultured mesenteric artery endothelial cells (MAECs) from aged rats. Immunoblotting and immunostaining results showed an age-related decrease of TRPV4 expression levels in MAECs. Additionally, the 4α-PDD-induced NO production was significantly reduced in aged MAECs. Compared with lentiviral GFP-treated aged rats, lentiviral vector delivery of TRPV4 increased TRPV4 expression level in aged MAECs and restored the flow- and 4α-PDD-induced vessel dilation in aged mesenteric arteries. We concluded that impaired TRPV4-mediated Ca(2+) signaling causes endothelial dysfunction and that TRPV4 is a potential target for clinical treatment of age-related vascular system diseases.

Increasing TRPV4 expression restores flow-induced dilation impaired in mesenteric arteries with aging.

Artemisia argyi Lev. et Vant. (A. argyi) is widely utilized for moxibustion in Chinese medicine, and the mechanism underlying terpenoid biosynthesis in its leaves is suggested to play an important role in its medicinal use. However, the A. argyi transcriptome has not been sequenced. Herein, we performed RNA sequencing for A. argyi leaf, root and stem tissues to identify as many as possible of the transcribed genes. In total, 99,807 unigenes were assembled by analysing the expression profiles generated from the three tissue types, and 67,446 of those unigenes were annotated in public databases. We further performed differential gene expression analysis to compare leaf tissue with the other two tissue types and identified numerous genes that were specifically expressed or up-regulated in leaf tissue. Specifically, we identified multiple genes encoding significant enzymes or transcription factors related to terpenoid synthesis. This study serves as a valuable resource for transcriptome information, as many transcribed genes related to terpenoid biosynthesis were identified in the A. argyi transcriptome, providing a functional genomic basis for additional studies on molecular mechanisms underlying the medicinal use of A. argyi.

Jinhang Zhu

Department of Orthopedics,
Anhui Provincial Hospital,
Department of Orthopedics, Anhui Provincial Hospital

Anhui medical university

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Jinhang Zhu

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Orthopedics,Anhui Provincial Hospital,Department of Orthopedics, Anhui Provincial Hospital

Anhui medical university

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Department of Orthopedics,
Anhui Provincial Hospital,
Department of Orthopedics, Anhui Provincial Hospital