Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)‐sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N‐terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re‐accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady‐state Ca2+ concentration was obtained using Sr2+, a well‐known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady‐state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3‐fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10‐fold drop in a few minutes.

Pancreatic cancer remains as one of the most deadly cancers, and responds poorly to current therapies. The prognosis is extremely poor, with a 5-year survival of less than 5%. Therefore, search for new effective therapeutic drugs is of pivotal need and urgency to improve treatment of this incurable malignancy. Synthetic alkyl-lysophospholipid analogs (ALPs) constitute a heterogeneous group of unnatural lipids that promote apoptosis in a wide variety of tumor cells. In this study, we found that the anticancer drug edelfosine was the most potent ALP in killing human pancreatic cancer cells, targeting endoplasmic reticulum (ER). Edelfosine was taken up in significant amounts by pancreatic cancer cells and induced caspaseand mitochondrial-mediated apoptosis. Pancreatic cancer cells show a prominent ER and edelfosine accumulated in this subcellular structure, inducing a potent ER stress response, with caspase-4, BAP31 and c-Jun NH 2 -terminal kinase (JNK) activation, CHOP/GADD153 upregulation and phosphorylation of eukaryotic translation initiation factor 2 a-subunit that eventually led to cell death. Oral administration of edelfosine in xenograft mouse models of pancreatic cancer induced a significant regression in tumor growth and an increase in apoptotic index, as assessed by TUNEL assay and caspase-3 activation in the tumor sections. The ER stress-associated marker CHOP/ GADD153 was visualized in the pancreatic tumor isolated from edelfosine-treated mice, indicating a strong in vivo ER stress response. These results suggest that edelfosine exerts its pro-apoptotic action in pancreatic cancer cells, both in vitro and in vivo, through its accumulation in the ER, which leads to ER stress and apoptosis. Thus, we propose that the ER could be a key target in pancreatic cancer, and edelfosine may constitute a prototype for the development of a new class of antitumor drugs targeting the ER.

We have monitored specifically the [Ca2+] in the lumen of the endoplasmic reticulum (ER) of intact HeLa cells using an ER-targeted low-Ca2+-affinity aequorin. The steady-state [Ca2+] in the ER was around 600 microM. Histamine induced a concentration-dependent decrease in lumenal [Ca2+], whose rate increased near one order of magnitude and became "quantal" when cytosolic [Ca2+] ([Ca2+]c) was clamped with the Ca2+ chelator BAPTA. This effect was not due to decreased Ca2+ pumping because simultaneous addition of a SERCA inhibitor produced only additive effects. Given that inhibition by [Ca2+]c of the inositol 1,4,5-trisphosphate-gated channels requires a [Ca2+]c much higher than that observed in the bulk cytosol after histamine addition, we conclude that local [Ca2+]c microdomains at the site of release strongly inhibit agonist-induced Ca2+ mobilization in intact cells. This effect should play a key role in the mechanism controlling cytosolic [Ca2+] oscillations and waves, and therefore in the generation of spatio-temporal Ca2+ patterns.

The filling state of the intracellular Ca2+ stores of rat thymocytes regulates plasma membrane permeability to Mn2+, used here as a Ca2+ surrogate for plasma membrane Ca2+ channels. Emptying of the Ca2+ stores accelerated Mn2+ entry about 10-fold, and refilling with Ca2+ restored low Mn2+ permeability. The acceleration of Mn2+ entry observed in cells with empty intracellular Ca2+ stores was prevented by cytochrome P450 inhibitors. Imidazole antimycotics, especially econazole and miconazole, were the most potent inhibitors (IC50 approximately equal to 10(-6) M). The inhibitor sensitivity profile was similar to IA-type cytochrome P450. Calmodulin antagonists increased the plasma membrane permeability to Mn2+ in cells with filled Ca2+ stores, and this effect was also blocked by imidazole antimycotics. On this basis, we propose a model in which activation of a cytochrome P450, situated at the Ca2+ stores, opens a plasma membrane Ca2+ pathway. This activity would be inhibited by Ca2+ inside the stores by a calmodulin-dependent mechanism.