Functional consequences of enhanced expression of STIM1 and Orai1 in Huh-7 hepatocellular carcinoma tumor-initiating cells.

BACKGROUND: The endoplasmic reticulum (ER) Ca2+ sensor, stromal interaction molecule1 (STIM1) activates the plasma membrane (PM) channel Orai1 in order to mediate store-operated Ca2+ entry (SOCE) in response to ER store depletion. Enhanced expression of STIM1 in cancer tissue has been associated with poor patient prognosis. Therefore, this study investigated the functional consequences of enhanced expression of STIM1 and Orai1 in a tumor-initiating subpopulation of Huh-7 hepatocellular carcinoma (HCC) cells that express epithelial cell adhesion molecule (EpCAM) and Prominin 1 (CD133). METHODS: We performed qRT-PCR, intracellular Ca2+ monitoring, protein analyses, and real-time cell proliferation assays on EpCAM(+)CD133(+) subpopulation of tumor-initiating Huh-7 HCC cells expressing high levels of STIM1 and/or Orai1. Statistical significance between the means of two groups was evaluated using unpaired Student's t-test. RESULTS: Enhanced STIM1 expression significantly increased ER Ca2+ release and proliferation rate of EpCAM(+)CD133(+) cells. CONCLUSION: STIM1 overexpression may facilitate cancer cell survival by increasing ER Ca2+-buffering capacity, which makes more Ca2+ available for the cytosolic events, on the other hand, possibly preventing Ca2+-dependent enzymatic activity in mitochondria whose Ca2+ uniporter requires much higher cytosolic Ca2+ levels.

Alterations of store-operated calcium entry and cyclopiazonic acid-induced endothelium-derived relaxations in aging rat thoracic aorta.

The purpose of our study was to investigate whether endothelium-derived relaxations induced by store depletion are altered in aging rat thoracic aorta. Vascular responses were measured in aortic segments isolated from young (2-4 month) and old (20-24 month) male Sprague-Dawley rats. In phenylephrine-contracted intact tissues, receptor-mediated and receptor-independent endothelium-derived relaxations were induced by acetylcholine (ACh) and sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) blocker cyclopiazonic acid (CPA), respectively. In addition, CPA-induced changes in intracellular calcium levels were monitored in fura-2-loaded endothelium-denuded tissues. Real-time quantitative reverse transcription polymerase chain reaction and western blot analysis were performed to determine the transient receptor potential canonical (TRPC) 4 mRNA and protein levels. Endothelial TRPC4 mRNA levels were apparently decreased in aging rats. Immunoblot analysis showed that TRPC4 protein levels significantly decreased in intact aorta from 20- to 24-month-old rats compared to that from 2- to 4-month-old rats. ACh- and CPA-induced endothelium-dependent relaxations decreased in old rat aorta without any change in direct vasodilation induced by sodium nitroprusside. Store-operated Ca2+ entry (SOCE) induced by CPA was significantly decreased, whereas sarcoplasmic reticulum Ca2+ release was unaffected in endothelium-denuded aging rat aorta. In conclusion, TRPC4 downregulation could be associated with decreased endothelium-dependent vasorelaxations. As endothelial nitric oxide synthase is activated by SOCE-induced caveolar internalization, tracking the expression levels of SERCA, ion channels, and/or associated proteins involved in SOCE would lead to the development of novel therapeutics for age-related vasospastic disorders with dysfunctional endothelium.

Cyclopiazonic acid alters serotonin-induced responses in rat thoracic aorta.

We previously showed that endothelin A (ETA) receptor antagonist BQ-123 partially inhibited cyclopiazonic acid (CPA)-enhanced endothelin-1 (ET-1)-induced contractions suggesting enhancement of ETA receptor internalization in caveolar structures by sarco/endoplasmic reticulum Ca+2 ATPase (SERCA) blockade. Since serotonin (5-Hydroxytryptamine, 5-HT) receptors are reported to be localized on caveolar membranes, we investigated whether SERCA inhibition affects 5-HT-induced responses and 5-HT receptor antagonism. For this purpose, vascular responses were measured in thoracic aorta segments from male Wistar albino rats using isolated tissue experiments. Data showed that CPA inhibits 5-HT- and PE-induced contractions in intact vessels while potentiating those in endothelium-denuded. Furthermore, non-selective 5-HT receptor blocker methysergide partially inhibited CPA-induced 5-HT contractions. However, α1-adrenergic receptor antagonist prazosin totally inhibited CPA-potentiated PE contractions. We suggest that SERCA inhibition results in 5-HT receptor internalization similar to ETA receptors possibly through protein kinase C activation by increased subsarcolemmal Ca2+ levels, eventually preventing 5-HT receptor antagonism.

Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition prevents endothelin A receptor antagonism in rat aorta.

This study tested whether sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase regulates the ability of endothelin receptor antagonist to inhibit the endothelin-1 constriction. The endothelin A receptor antagonist BQ-123 (1 microM) completely relaxed constriction to 10 nM endothelin-1 in endothelium-denuded rat aorta. Challenge with cyclopiazonic acid (10 microM), a sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor, during the plateau of endothelin-1 constriction enhanced the constriction by approximately 30%. BQ-123 relaxed the endothelin-1 plus cyclopiazonic acid constriction by only approximately 10%. In contrast, prazosin (1 microM), an alpha-adrenergic receptor antagonist, still completely relaxed the 0.3 muM phenylephrine constriction in the presence of cyclopiazonic acid. Verapamil relaxed the endothelin-1 plus cyclopiazonic acid constriction by approximately 30%, whereas Ni(2+) and 2-aminoethoxydiphenyl borate, nonselective cation channel and store-operated channel blockers, respectively, completely relaxed the constriction. These results suggest that lowered sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase activity selectively decreases the ability of endothelin receptor antagonist to inhibit the endothelin A receptor. The decreased antagonism may be related to the opening of store-operated channels and subsequent greater internalization of endothelin A receptor.