001 versus “”with external calcium”"). Direct measurement of the ER Ca2+-concentration ([Ca2+]ER) is not reliably feasible. Therefore, we used an indirect

approach. SERCA were inhibited using 1 μM cyclopiazonic acid (CPA) leading to a net Ca2+-efflux out of the ER. The resulting increase in [Ca2+]c was used as an estimate of the [Ca2+]ER [4]. In the lung cancer cell lines in which Ca2+-influx contributed to the Rigosertib mouse ATP-induced Ca2+-increase (EPLC 272 and LCLC) the [Ca2+]ER was equal to the [Ca2+]ER in NHBE (Figure 3A). In those lung cancer cell lines in which the Ca2+-influx did not contribute to the ATP-induced Ca2+-increase (H1339 and HCC) [Ca2+]ER was lower than in NHBE. The SCLC line H1339 showed the lowest

[Ca2+]ER (Figure 3B). Figure 3 SERCA were inhibited using 1 click here μM cyclopiazonic Dactolisib acid leading to a net Ca 2+ -efflux out of the ER. The resulting increase in [Ca2+]c was used as an estimate of the [Ca2+]ER and expressed as percentage of NHBE. (A) In EPLC and LCLC cells in which Ca2+-influx contributed to the ATP-induced Ca2+-increase the [Ca2+]ER was equal to the [Ca2+]ER of NHBE. (B) In H1339 and HCC cells in which the Ca2+-influx did not contribute to the ATP-induced Ca2+-increase the ER Ca2+-content was lower than in NHBE cells (n = 50 – 153 cells, * = P < 0.001 versus all other groups). Next, we investigated the expression of the proteins that regulate the [Ca2+]ER. SERCA pump calcium from the cytoplasm into the ER. Three isoforms of SERCA of have been identified so far and, of these, SERCA 2 has been reported to be the most widely expressed [5]. Analyzing the SERCA expression using Western Blot analysis, we found the isoforms SERCA 1 and 3 to be very weakly expressed (data not shown), while SERCA 2 showed strong expression as confirmed by immuno-fluorescence (Figure 4). Comparing the expression of SERCA 2 in NHBE, H1339, and HCC cells, we found lower levels of expression in the lung cancer cells with expression in H1339 cells being the lowest (Figure 5). Figure 4 Immunohistochemical

staining Anidulafungin (LY303366) of SERCA 2 in a H1339 cell. Note the ER-typical pattern of the staining as SERCA is an ER-trans-membrane protein. Bar = 2 μm. Figure 5 The expression of SERCA 2 was analyzed in NHBE, H1339, and HCC cells using Western Blot analysis and expressed as percentage of the SERCA 2 expression in NHBE cells. In H1339 and HCC cells, the expression of SERCA 2 was found to be reduced with H1339 showing the weakest expression (n = 3, * = P < 0.01 versus all other groups). Ca2+-release channels of the ER are RyR and IP3R. In NHBE, H1339, and HCC cells, we found the expression RyR to be hardly detectable at all (data not shown). In contrast, IP3R showed substantial expression, which was higher in the lung cancer cell lines, and the highest in H1339 cells (Figure 6).