Background and PurposeThe role of extracellular Ca²⁺in the maintenance of chronic vasospasm after subarachnoid hemorrhage (SAH) is largely unknown. Indeed, studies thus far have been limited to demonstrations that L-type Ca²⁺−channel antagonists were unable to reverse the spasm. This study tested whether SAH-induced vasospasm is maintained, at least in part, through the influx of extracellular Ca²⁺and whether the influx of extracellular Ca²⁺occurs through L-type Ca²⁺channels and possibly, in addition, through store operated channels (SOCs). Furthermore, as there is considerable evidence in the literature to suggest that the spasm is mediated through endothelin-1 (ET-1) release, we tested whether the Ca²⁺dependency of the spasm was consistent with the mediation of the spasm by ET-1.MethodsChronic spasm of the basilar artery was induced in a double SAH rabbit model. Relaxation of SAH-, ET-1−, serotonin-, and KCl-constricted basilar artery in response to Ca²⁺-free solution, verapamil, and Ni²⁺was measured in situ with the use of a cranial window.ResultsSAH induced 23% constriction of the basilar artery. Ca²⁺-free solution and 1 μmol/L verapamil reversed the constriction of SAH vessels by 60% and 17%, respectively. In contrast, control vessels challenged with 40 to 50 mmol/L KCl, which induced 34% constriction, relaxed in response to Ca²⁺-free solution and verapamil by 98% and 89%, respectively. In SAH vessels, verapamil followed by 0.1 mmol/L Ni²⁺, which is known to block SOCs, induced a combined relaxation of 67%. Control vessels challenged with 3 nmol/L ET-1, which induced a magnitude of constriction similar to that of SAH (29%), relaxed in response to Ca²⁺-free solution, verapamil, and verapamil plus Ni²⁺by 69%, 20%, and 50%, respectively (P>.05 versus respective values in SAH vessels). In contrast, control vessels challenged with 2 to 8 μmol/L serotonin, which induced a magnitude of constriction similar to those of SAH and ET-1 (22%), completely relaxed in response to Ca²⁺-free solution and verapamil.ConclusionsThese results demonstrate that the maintenance of chronic spasm in the two-hemorrhage rabbit model after SAH is due to smooth muscle cell contractile mechanisms partly dependent on the influx of extracellular Ca²⁺. The influx of extracellular Ca²⁺results from the opening of L-type Ca²⁺channels and an additional channel or channels. We speculate that the L-type Ca²⁺channel−independent influx of extracellular Ca²⁺results from the opening of SOCs. The Ca²⁺-dependent characteristics of the spasm likely reflect the mediation of the spasm by ET-1.

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). 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. Enhanced STIM1 expression significantly increased ER Ca2+ release and proliferation rate of EpCAM(+)CD133(+) cells. 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.