Key pointsr Vascular oxidative stress increases with advancing age. r We hypothesized that resistance vessels develop resilience to oxidative stress to protect functional integrity and tested this hypothesis by exposing isolated pressurized superior epigastric arteries (SEAs) of old and young mice to H 2 O 2 . r H 2 O 2 -induced death was greater in smooth muscle cells (SMCs) than endothelial cells (ECs) and lower in SEAs from old vs. young mice; the rise in vessel wall [Ca 2+ ] i induced by H 2 O 2 was attenuated with ageing, as was the decline in noradrenergic vasoconstriction; genetic deletion of IL-10 mimicked the effects of advanced age on cell survival. r Inhibiting NO synthase or scavenging peroxynitrite reduced SMC death; endothelial denudation or inhibiting gap junctions increased SMC death; delocalization of cytochrome C activated caspases 9 and 3 to induce apoptosis.r Vascular cells develop resilience to H 2 O 2 during ageing by preventing Ca 2+ overload and endothelial integrity promotes SMC survival.Abstract Advanced age is associated with elevated oxidative stress and can protect the endothelium from cell death induced by H 2 O 2 . Whether such protection occurs for intact vessels or differs between smooth muscle cell (SMC) and endothelial cell (EC) layers is unknown. We tested the hypothesis that ageing protects SMCs and ECs during acute exposure to H 2 O 2 (200 µM, 50 min). Mouse superior epigastric arteries (SEAs; diameter, ß150 µm) were isolated and pressurized to 100 cmH 2 O at 37˚C. For SEAs from young (4 months) mice, H 2 O 2 killed 57% of SMCs and 11% of ECs in males vs. 8% and 2%, respectively, in females. Therefore, SEAs from males were studied to resolve the effect of ageing and experimental interventions. For old (24 months) Charles E. Norton III is currently a postdoctoral fellow in Steven Segal's laboratory at the University of Missouri, Columbia. His interest in vascular physiology developed as an undergraduate and PhD student studying mechanisms of chronic hypoxia-induced pulmonary hypertension at the University of New Mexico. Dr Norton's primary interest is in understanding how chemical and electrical communication between endothelial cells and smooth muscle cells modulate vasomotor tone and cell death. Currently, he is exploring how conditions of chronic oxidative stress alter resilience to apoptosis.C. E. Norton and others J Physiol 597.15 mice, SMC death was reduced to 10% with diminished accumulation of [Ca 2+ ] i in the vessel wall during H 2 O 2 exposure. In young mice, genetic deletion of IL-10 mimicked the protective effect of ageing on cell death and [Ca 2+ ] i accumulation. Whereas endothelial denudation or gap junction inhibition (carbenoxolone; 100 µM) increased SMC death, inhibiting NO synthase (L-NAME, 100 µM) or scavenging peroxynitrite (FeTPPS, 5 µM) reduced SMC death along with [Ca 2+ ] i . Despite NO toxicity via peroxynitrite formation, endothelial integrity protects SMCs. Caspase inhibition (Z-VAD-FMK, 50 µM) attenuated cell death with immunostaining for annex...
Background: Local injection of BaCl 2 is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl 2 causes muscle injury is unresolved. Because Ba 2+ inhibits K + channels, we hypothesized that BaCl 2 induces myofiber depolarization leading to Ca 2+ overload, proteolysis, and membrane disruption. While BaCl 2 spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl 2 treatment. Methods: The intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3-4 months) and irrigated with physiological salt solution (PSS) at 37°C. Myofiber membrane potential (V m) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca 2+ ] i) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl 2 (50-75 μL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant. Results: Addition of 1.2% BaCl 2 to PSS depolarized myofibers from − 79 ± 3 mV to − 17 ± 7 mV with a corresponding rise in [Ca 2+ ] i ; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl 2 exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca 2+ from PSS prevented the rise in [Ca 2+ ] i and ameliorated myonuclear staining with PI during BaCl 2 exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl 2 and morphological integrity deteriorated through 72 h. Conclusions: BaCl 2 injures myofibers through depolarization of the sarcolemma, causing Ca 2+ overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.
Growth suppression mediated by connexin 37 (Cx37; also known as GJA4) requires interaction between its C-terminus and functional pore-forming domain. Using rat insulinoma cells, we show that Cx37 induces cell death and cell cycle arrest, and slowed cell cycling. Whether differential phosphorylation might regulate intramolecular interactions, and consequently the growth-suppressive phenotype, is unknown. Protein kinase C inhibition increased the open state probability of low-conductance gap junction channels (GJChs) and reduced GJCh closed state probability. Substituting alanine at serine residues 275, 302 and 328 eliminated Cx37-induced cell death, supported proliferation and reduced the GJCh closed state probability. With additional alanine for serine substitutions at residues 285, 319, 321 and 325, Cx37-induced cell death was eliminated and the growth arrest period prolonged, and GJCh closed state probability was restored. With aspartate substitution at these seven sites, apoptosis was induced and the open state probability of large conductance GJChs (and hemichannels) was increased. These data suggest that differential phosphorylation of the C-terminus regulates channel conformation and, thereby, cell cycle progression and cell survival.
A Western-style diet (WD; high in fat and carbohydrates) increases vascular oxidative stress. We hypothesized that vascular cells adapt to a WD by developing resilience to oxidative stress. Male and female C57BL/6J mice (4 wk of age) were fed a control diet (CD) or a WD for 16–20 wk. Superior epigastric arteries (SEAs; diameter, ~125 µm) were isolated and pressurized for study. Basal reactive oxygen species production was greatest in SEAs from males fed the WD. During exposure to H2O2 (200 μM, 50 min), propidium iodide staining identified nuclei of disrupted endothelial cells (ECs) and smooth muscle cells (SMCs). For mice fed the CD, death of SMCs (21%) and ECs (6%) was greater ( P < 0.05) in SEAs from males than females (9% and 2%, respectively). WD consumption attenuated cell death most effectively in SEAs from males. With no difference at rest, H2O2 increased intracellular Ca2+ concentration ([Ca2+]i) to the greatest extent in SEAs from males, as shown by fura 2 fluorescence. Selective disruption of the endothelium (luminal air bubble) increased [Ca2+]i and SMC death during H2O2 exposure irrespective of sex; the WD reduced both responses most effectively in males. Nonselective transient receptor potential (TRP) channel inhibition (ruthenium red, 5 μM) attenuated the rise of [Ca2+]i, as did selective inhibition of TRP vanilloid type 4 (TRPV4) channels (HC-067047, 1 μM), which also attenuated cell death. In contrast, inhibition of voltage-gated Ca2+ channels (diltiazem, 50 μM) was without effect. Thus, for resistance arteries during acute oxidative stress: 1) ECs are more resilient than (and can protect) SMCs, 2) vessels from females are inherently more resilient than those from males, and 3) a WD increases vascular resilience by diminishing TRPV4 channel-dependent Ca2+ entry.
Differential phosphorylation of the carboxyl-terminus of connexin 37 (Cx37-CT) regulates phenotypic switching between cell growth phenotypes (cell death, cell cycle arrest, proliferation). The specific phosphorylation events in the Cx37-CT that are necessary for these growth regulatory effects are currently unknown. Through the combined use of deletion and site specific (de)phospho-mimetic Cx37-CT mutants, our data suggest a phosphorylation-dependent interaction between the mid-tail (aa 273–317) and end-tail (aa 318–333) portions of the Cx37-CT that regulates cell survival. As detected by mass spectrometry, Cx37 was phosphorylated at serines 275, 321, and 328; phosphomimetic mutations of these sites resulted in cell death when expressed in rat insulinoma cells. Alanine substitution at S328, but not at S275 or S321, also triggered cell death. Cx37-S275D uniquely induced the death of only low density, non-contact forming cells, but neither hemichannel open probability nor channel conductance distinguished death-inducing mutants. As channel function is necessary for cell death, together the data suggest that the phosphorylation state of the Cx37-CT controls an intra-domain interaction within the CT that modifies channel function and induces cell death.
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