The Ca 2؉ -permeable channel TRPM8 is thought to play an important role in the pathophysiology of prostate cancer. We have investigated the intracellular location of TRPM8 and its role as a Ca 2؉ -permeable channel in an androgen-responsive and an androgen-insensitive prostate cancer cell line. We report evidence from immunofluorescence experiments that in the androgen-responsive LNCaP cell line, the TRPM8 protein is expressed in the endoplasmic reticulum and plasma membrane, acts as a Ca 2؉ -permeable channel (assessed using Fura-2 to measure increases in the cytoplasmic Ca 2؉ concentration) in each of these membranes, and is regulated by androgen. Although TRPM8 was detected in the androgeninsensitive PC-3 cell line, no evidence was obtained for regulation of its expression by androgen. The results of experiments using LNCaP cells, the TRPM8 antagonist capsazepine, and small interference RNA targeted to TRPM8 indicate that TRPM8 is required for cell survival. These results indicate that TRPM8 is an important determinator of Ca 2؉ homeostasis in prostate epithelial cells and may be a potential target for the action of drugs in the management of prostate cancer.

The progression of cells from a normal differentiated state in which rates of proliferation and apoptosis are balanced to a tumorigenic and metastatic state involves the accumulation of mutations in multiple key signalling proteins and the evolution and clonal selection of more aggressive cell phenotypes. These events are associated with changes in the expression of numerous other proteins. This process of tumorigenesis involves the altered expression of one or more TRP proteins, depending on the nature of the cancer. The most clearly described changes are those involving TRPM8, TRPV6 and TRPM1. Expression of TRPM8 is substantially increased in androgen-dependent prostate cancer cells, but is decreased in androgen independent and metastatic prostate cancer. TRPM8 expression is regulated, in part, by androgens, most likely through androgen response elements in the TRPM8 promoter region. TRPM8 channels are involved in the regulation of cell proliferation and apoptosis. Expression of TRPV6 is also increased in prostate cancer and in a number of other cancers. In contrast to TRPM8, expression of TRPV6 is not directly regulated by androgens. TRPM1 is highly expressed in early stage melanomas but its expression declines with increases in the degree of aggressiveness of the melanoma. The expression of TRPV1, TRPC1, TRPC6, TRPM4, and TRPM5 is also increased in some cancers. The level of expression of TRPM8 and TRPV6 in prostate cancer, and of TRPM1 in melanomas, potentially provides a good prognostic marker for predicting the course of the cancer in individuals. The Drosophila melanogaster, TRPL, and the TRPV1 and TRPM8 proteins, have been used to try to develop strategies to selectively kill cancer cells by activating Ca(2+) and Na(+) entry, producing a sustained increase in the cytoplasmic concentration of these ions, and subsequent cell death by apoptosis and necrosis. TRPV1 is expressed in neurones involved in sensing cancer pain, and is a potential target for pharmacological inhibition of cancer pain in bone metastases, pancreatic cancer and most likely in other cancers. Further studies are required to assess which other TRP proteins are associated with the development and progression of cancer, what roles TRP proteins play in this process, and to develop further knowledge of TRP proteins as targets for pharmaceutical intervention and targeting in cancer.

Two cellular proteins, stromal interaction molecule 1 (STIM1) and Orai1, are recently discovered essential components of the Ca 2+ release activated Ca 2+ (CRAC) channel. Orai1 polypeptides form the pore of the CRAC channel, while STIM1 plays the role of the endoplasmic reticulum Ca 2+ sensor required for activation of CRAC current (I CRAC ) by store depletion. It is not known, however, if the role of STIM1 is limited exclusively to Ca 2+ sensing, or whether interaction between Orai1 and STIM1, either direct or indirect, also defines the properties of I CRAC . In this study we investigated how the relative expression levels of ectopic Orai1 and STIM1 affect the properties of I CRAC . The results show that cells expressing low Orai1 : STIM1 ratios produce I CRAC with strong fast Ca 2+ -dependent inactivation, while cells expressing high Orai1 : STIM1 ratios produce I CRAC with strong activation at negative potentials. Moreover, the expression ratio of Orai1 and STIM1 affects Ca 2+ , Ba 2+ and Sr 2+ conductance, but has no effect on the current in the absence of divalent cations. The results suggest that several key properties of Ca 2+ channels formed by Orai1 depend on its interaction with STIM1, and that the stoichiometry of this interaction may vary depending on the relative expression levels of these proteins.

Receptor-activated Ca2+ channels (RACCs) play a central role in regulation of the functions of animal cells. Together with voltage-operated Ca2+ channels (VOCCs) and ligand-gated non-selective cation channels, RACCs provide a variety of pathways by which Ca2+ can be delivered to the cytoplasmic space and the endoplasmic reticulum (ER) in order to initiate or maintain specific types of intracellular Ca2+ signal. Store-operated Ca2+ channels (SOCs), which are activated by a decrease in Ca2+ in the ER, are a major subfamily of RACCs. A careful analysis of the available data is required in order to discern the different types of RACCs (differentiated chiefly on the basis of ion selectivity and mechanism of activation) and to properly develop hypotheses for structures and mechanisms of activation. Despite much intensive research, the structures and mechanisms of activation of RACCs are only now beginning to be understood. In considering the physiological functions of the different RACCs, it is useful to consider the specificity for Ca2+ of each type of cation channel and the rate at which Ca2+ flows through a single open channel; the locations of the channels on the plasma membrane (in relation to the ER, cytoskeleton and other intracellular units of structure and function); the Ca2+-responsive enzymes and proteins; and the intracellular buffers and proteins that control the distribution of Ca2+ in the cytoplasmic space. RACCs which are non-selective cation channels can deliver Ca2+ directly to specific regions of the cytoplasmic space, and can also admit Na+, which induces depolarization of the plasma membrane, the opening of VOCCs and the subsequent inflow of Ca2+. SOCs appear to deliver Ca2+ specifically to the ER, thereby maintaining oscillating Ca2+ signals.

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The compound 2-aminoethyl diphenylborate (2-APB), an inhibitor of Ins(1,4,5)P3 receptor action in some cell types, has been used to assess the role of Ins(1,4,5)P3 receptors in the activation of store-operated Ca2+ channels (SOCs) [Ma, Patterson, van Rossum, Birnbaumer, Mikoshiba and Gill (2000) Science 287, 1647–1651]. In freshly-isolated rat hepatocytes, 2-APB inhibited thapsigargin- and vasopressin-stimulated Ca2+ inflow (measured using fura-2) with no detectable effect on the release of Ca2+ from intracellular stores. The concentration of 2-APB which gave half-maximal inhibition of Ca2+ inflow was approx. 10µM. 2-APB also inhibited Ca2+ inflow initiated by a low concentration of adenophostin A but had no effect on maitotoxin-stimulated Ca2+ inflow through non-selective cation channels. The onset of the inhibitory effect of 2-APB on thapsigargin-stimulated Ca2+ inflow was rapid. When 2-APB was added to rat hepatocytes in the presence of extracellular Ca2+ after a vasopressin-induced plateau in the cytoplasmic free Ca2+ concentration ([Ca2+]cyt) had been established, the kinetics of the decrease in [Ca2+]cyt were identical with those induced by the addition of 50µM Gd3+ (gadolinium). 2-APB did not inhibit the release of Ca2+ from intracellular stores induced by the addition of Ins(1,4,5)P3 to permeabilized hepatocytes. In the H4-IIE rat hepatoma cell line, 2-APB inhibited thapsigargin-stimulated Ca2+ inflow (measured using fura-2) and, in whole-cell patch-clamp experiments, the Ins(1,4,5)P3-induced inward current carried by Ca2+. It was concluded that, in liver cells, 2-APB inhibited SOCs through a mechanism which involved the binding of 2-APB to either the channel protein or an associated regulatory protein. 2-APB appeared to be a novel inhibitor of SOCs in liver cells with a mechanism of action which, in this cell type, is unlikely to involve an interaction of 2-APB with Ins(1,4,5)P3 receptors. The need for caution in the use of 2-APB as a probe for the involvement of Ins(1,4,5)P3 receptors in the activation of SOCs in other cell types is briefly discussed.

During the past 5 years it has emerged that the transient receptor potential (TRP) family of Ca 2+ -and Na + -permeable channels plays a diverse and important role in cell biology and in pathology. One member of this family, TRPM8, is highly expressed in prostate cancer cells but the physiological and pathological functions of TRPM8 in these cells are not known. Here we address these questions, and the issue of whether or not TRPM8 is an effective diagnostic and prognostic marker in prostate cancer. TRPM8 is known to be activated by cool stimuli (17-25 C) and cooling compounds such as menthol. The activation mechanism(s) involves voltage sensing of membrane potential, phosphatidylinositol 4,5-bisphosphate and Ca 2+. In addition to prostate cancer cells, TRPM8 is expressed in sensory neurons where it acts as a sensor of cold. In prostate epithelial cells, expression of TRPM8 is regulated by androgen and is elevated in androgen-sensitive cancerous cells compared with normal cells. While there is some evidence that in prostate cancer cells Ca 2+ and Na + inflow through TRPM8 is necessary for survival and function, including secretion at the apical membrane, the function of TRPM8 in these cells is not really known. It may well differ from the role of TRPM8 as a cool sensor in sensory nerve cells. Androgen unresponsive prostate cancer is difficult to treat effectively and there are limited diagnostic and prognostic markers available. TRPM8 is a potential tissue marker in differential diagnosis and a potential prognostic marker for androgen-unresponsive and metastatic prostate cancer. As a consequence of its ability to convey Ca 2+ and Na + and its expression in only a limited number of cell types, TRPM8 is considered to be a promising target for pharmaceutical, immunological and genetic interventions for the treatment of prostate cancer.

Acetaminophen (paracetamol) is the most frequently used analgesic and antipyretic drug available over the counter. At the same time, acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. Acetaminophen overdose causes a multitude of interrelated biochemical reactions in hepatocytes including the formation of reactive oxygen species, deregulation of Ca 2+ homeostasis, covalent modification and oxidation of proteins, lipid peroxidation, and DNA fragmentation. Although an increase in intracellular Ca 2+ concentration in hepatocytes is a known consequence of acetaminophen overdose, its importance in acetaminophen-induced liver toxicity is not well understood, primarily due to lack of knowledge about the source of the Ca 2+ rise. Here we report that the channel responsible for Ca 2+ entry in hepatocytes in acetaminophen overdose is the Transient Receptor Potential Melanostatine 2 (TRPM2) cation channel. We show by whole-cell patch clamping that treatment of hepatocytes with acetaminophen results in activation of a cation current similar to that activated by H 2 O 2 or the intracellular application of ADP ribose. siRNA-mediated knockdown of TRPM2 in hepatocytes inhibits activation of the current by either acetaminophen or H 2 O 2 . In TRPM2 knockout mice, acetaminophen-induced liver damage, assessed by the blood concentration of liver enzymes and liver histology, is significantly diminished compared with wildtype mice. The presented data strongly suggest that TRPM2 channels are essential in the mechanism of acetaminophen-induced hepatocellular death.