Dataset Information

Chronic hypoxia increases TRPC6 expression and basal intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle.

ABSTRACT:

Background

Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).

Methods

Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O2 for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracellular Ca2+ concentration ([Ca2+]i) and store-operated Ca2+ entry (SOCE). Quantitative RT-PCR and western blotting were performed to measure the expression of mRNA and levels of canonical transient receptor potential (TRPC) protein respectively.

Results

Hypoxia increased the basal [Ca2+]i and SOCE in both freshly dissociated and serum cultured distal PVSMCs. Moreover, hypoxia increased TRPC6 expression at mRNA and protein levels in both cultured PVSMCs exposed to prolonged hypoxia (4% O2, 60 h) and distal PV isolated from CHPH rats. Hypoxia also enhanced proliferation and migration of rat distal PVSMCs.

Conclusions

Hypoxia induces elevation of SOCE in distal PVSMCs, leading to enhancement of basal [Ca2+]i in PVSMCs. This enhancement is potentially correlated with the increased expression of TRPC6. Hypoxia triggered intracellular calcium contributes to promoted proliferation and migration of PVSMCs.

SUBMITTER: Xu L

PROVIDER: S-EPMC4218830 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Publications

Chronic hypoxia increases TRPC6 expression and basal intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle.

Xu Lei L Chen Yuqin Y Yang Kai K Wang Yingfeng Y Tian Lichun L Zhang Jie J Wang Elizabeth Wenqian EW Sun Dejun D Lu Wenju W Wang Jian J

PloS one 20141103 11

<h4>Background</h4>Hypoxia causes remodeling and contractile responses in both pulmonary artery (PA) and pulmonary vein (PV). Here we explore the effect of hypoxia on PV and pulmonary venous smooth muscle cells (PVSMCs).<h4>Methods</h4>Chronic hypoxic pulmonary hypertension (CHPH) model was established by exposing rats to 10% O2 for 21 days. Rat distal PVSMCs were isolated and cultured for in vitro experiments. The fura-2 based fluorescence calcium imaging was used to measure the basal intracell ...[more]

PMID: 25365342

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Project description:BACKGROUND:Skeletal muscle atrophy is the net loss of muscle mass that results from an imbalance in protein synthesis and protein degradation. It occurs in response to several stimuli including disease, injury, starvation, and normal aging. Currently, there is no truly effective pharmacological therapy for atrophy; therefore, exploration of the mechanisms contributing to atrophy is essential because it will eventually lead to discovery of an effective therapeutic target. The ether-a-go-go related gene (ERG1A) K+ channel has been shown to contribute to atrophy by upregulating ubiquitin proteasome proteolysis in cachectic and unweighted mice and has also been implicated in calcium modulation in cancer cells. METHODS:We transduced C2C12 myotubes with either a human ERG1A encoded adenovirus or an appropriate control virus. We used fura-2 calcium indicator to measure intracellular calcium concentration and Calpain-Glo assay kits (ProMega) to measure calpain activity. Quantitative PCR was used to monitor gene expression and immunoblot evaluated protein abundances in cell lysates. Data were analyzed using either a Student's t test or two-way ANOVAs and SAS software as indicated. RESULTS:Expression of human ERG1A in C2C12 myotubes increased basal intracellular calcium concentration 51.7% (p?<?0.0001; n?=?177). Further, it increased the combined activity of the calcium-activated cysteine proteases, calpain 1 and 2, by 31.9% (p?<?0.08; n?=?24); these are known to contribute to degradation of myofilaments. The increased calcium levels are likely a contributor to the increased calpain activity; however, the change in calpain activity may also be attributable to increased calpain protein abundance and/or a decrease in levels of the native calpain inhibitor, calpastatin. To explore the enhanced calpain activity further, we evaluated expression of calpain and calpastatin genes and observed no significant differences. There was no change in calpain 1 protein abundance; however, calpain 2 protein abundance decreased 40.7% (p?<?0.05; n?=?6). These changes do not contribute to an increase in calpain activity; however, we detected a 31.7% decrease (p?<?0.05; n?=?6) in calpastatin which could contribute to enhanced calpain activity. CONCLUSIONS:Human ERG1A expression increases both intracellular calcium concentration and combined calpain 1 and 2 activity. The increased calpain activity is likely a result of the increased calcium levels and decreased calpastatin abundance.

Project description:Chronic hypoxia causes remodeling and alters contractile responses in both pulmonary arteries and pulmonary veins. Although pulmonary arteries have been studied extensively in these disorders, the mechanisms by which pulmonary veins respond to hypoxia and whether these responses contribute to chronic hypoxic pulmonary hypertension remain poorly understood. In pulmonary arterial smooth muscle, we have previously demonstrated that influx of Ca(2+) through store-operated calcium channels (SOCC) thought to be composed of transient receptor potential (TRP) proteins is likely to play an important role in development of chronic hypoxic pulmonary hypertension. To determine whether this mechanism could also be operative in pulmonary venous smooth muscle, we measured intracellular Ca(2+) concentration ([Ca(2+)](i)) by fura-2 fluorescence microscopy in primary cultures of pulmonary venous smooth muscle cells (PVSMC) isolated from rat distal pulmonary veins. In cells perfused with Ca(2+)-free media containing cyclopiazonic acid (10 ?M) and nifedipine (5 ?M) to deplete sarcoplasmic reticulum Ca(2+) stores and block voltage-dependent Ca(2+) channels, restoration of extracellular Ca(2+) (2.5 mM) caused marked increases in [Ca(2+)](i), whereas MnCl(2) (200 ?M) quenched fura-2 fluorescence, indicating store-operated Ca(2+) entry (SOCE). SKF-96365 and NiCl(2), antagonists of SOCC, blocked SOCE at concentrations that did not alter Ca(2+) responses to 60 mM KCl. Of the seven known canonical TRP (TRPC1-7) and six vanilloid-related TRP channels (TRPV1-6), real-time PCR revealed mRNA expression of TRPC1 > TRPC6 > TRPC4 > TRPC2 ? TRPC5 > TRPC3, TRPV2 > TRPV4 > TRPV1 in distal PVSMC, and TRPC1 > TRPC6 > TRPC3 > TRPC4 ? TRPC5, TRPV2 ? TRPV4 > TRPV1 in rat distal pulmonary vein (PV) smooth muscle. Western blotting confirmed protein expression of TRPC1, TRPC6, TRPV2, and TRPV4 in both PVSMC and PV. Our results suggest that SOCE through Ca(2+) channels composed of TRP proteins may contribute to Ca(2+) signaling in rat distal PV smooth muscle.

Dataset Information

Chronic hypoxia increases TRPC6 expression and basal intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle.

Background

Methods

Results

Conclusions

Publications

Chronic hypoxia increases TRPC6 expression and basal intracellular Ca2+ concentration in rat distal pulmonary venous smooth muscle.

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