Although human pannexins (PanX) are homologous to gap junction molecules, their physiological function in vertebrates remains poorly understood. Our results demonstrate that overexpression of PanX1 results in the formation of Ca2+-permeable gap junction channels between adjacent cells, thus, allowing direct intercellular Ca2+ diffusion and facilitating intercellular Ca2+ wave propagation. More intriguingly, our results strongly suggest that PanX1 may also form Ca2+-permeable channels in the endoplasmic reticulum (ER). These channels contribute to the ER Ca2+ leak and thereby affect the ER Ca2+ load. Because leakage remains the most enigmatic of those processes involved in intracellular calcium homeostasis, and the molecular nature of the leak channels is as yet unknown, the results of this work provide new insight into calcium signaling mechanisms. These results imply that for vertebrates, a new protein family, referred to as pannexins, may not simply duplicate the connexin function but may also provide additional pathways for intra- and intercellular calcium signaling and homeostasis.

Several lines of evidence indicate that insulin-like growth factor-I (IGF-I) is a potent mediator of vasodilation. To elucidate the mechanism and site of action of IGF-I, we performed continuous monitoring of nitric oxide (NO) release from endothelial cells using a highly-sensitive amperometric NO-sensor. Two types of cultured cells were used: human umbilical vein endothelial cells and immortalized rat renal interlobar artery endothelial cells. In separate experiments, [Ca2+]i changes in response to IGF-I were measured spectrofluorometrically in fura-2-loaded cells. Stimulation with IGF-I resulted in a rapid, dose-dependent increase in [NO] as detected by the NO-probe positioned 1 mm above the monolayers, followed by a sustained elevation lasting for at least five minutes. The effect of IGF-I was significantly suppressed by pretreatment with anti-IGF-I antibody, suggesting that it was specific for IGF-I. NG-nitro-L-arginine methyl ester, an inhibitor of NO synthesis, significantly blunted responses to IGF-I, but dexamethasone preincubation did not reduce the IGF-I-induced release of NO. These results indicate that the observed IGF-I-induced release of NO is a result of activation of the constitutive, rather than the inducible type of NO synthase in endothelial cells. Genistein, a tyrosine kinase inhibitor, resulted in a profound suppression of the IGF-I-induced release of NO. IGF-I did not affect [Ca2+]i in either type of cells. Therefore, IGF-I-induced NO production by both types of endothelial cells is mediated via a tyrosine kinase-dependent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I cold/menthol ) that shows inward rectification and high Ca 2؉ selectivity, which are dramatically different properties from "classical" TRPM8-mediated I cold/menthol . Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I cold/menthol and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca 2؉ release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca 2؉ release channel, potentially involved in a number of Ca 2؉ -and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis. Mammalian homologues of the Drosophila transient receptor potential (TRP)7 channel, which initially emerged as a channel specifically linked to phospholipase C-catalyzed inositol phospholipid breakdown signaling pathways, have now grown into a broad family of channelforming proteins displaying extraordinarily diverse activation mechanisms (for reviews, see Refs. 1-5). At present, these channels are grouped into six subfamilies based on structural homology and have been given a standard nomenclature (5).A number of mammalian TRPs show a unique mode of gating, in response to thermal stimuli as well as to the chemical imitators of burning and cooling sensations, capsaicin and menthol, respectively. As such, they represent a group of thermal receptors covering a wide range of physiological temperatures. Most thermal receptors belong to the vanilloid TRP subfamily (TRPV, Ref. 6) including warm-sensitive (Ͻ40°C) TRPV3 (7-9) and heat-and capsaicin-sensitive TRPV1 (Ͼ43°C) (10) and TRPV2 (Ͼ52°C) (11). In contrast, sensitivity to cooling temperatures (Ͻ22°C) and menthol is mediated by a structurally distant thermal receptor, TRPM8, belonging to the melastatine (TRPM) subfamily of TRP channels (12, 13); the ankyrin transmembrane protein 1 (ANKTM1 or TRPA1) is involved in the detection of noxious cold (14).Consistent with their role in the sensation of distinct physiological temperatures, thermal receptors are mostly expressed in subsets of...

The main contributors to increases in [Ca2+]i and tension are the entry of Ca2+ through voltage-dependent channels opened by depolarization or during action potential (AP) or slow-wave discharge, and Ca2+ release from store sites in the cell by the action of IP3 or by Ca(2+)-induced Ca(2+)-release (CICR). The entry of Ca2+ during an AP triggers CICR from up to 20 or more subplasmalemmal store sites (seen as hot spots, using fluorescent indicators); Ca2+ waves then spread from these hot spots, which results in a rise in [Ca2+]i throughout the cell. Spontaneous transient releases of store Ca2+, previously detected as spontaneous transient outward currents (STOCs), are seen as sparks when fluorescent indicators are used. Sparks occur at certain preferred locations--frequent discharge sites (FDSs)--and these and hot spots may represent aggregations of sarcoplasmic reticulum scattered throughout the cytoplasm. Activation of receptors for excitatory signal molecules generally depolarizes the cell while it increases the production of IP3 (causing calcium store release) and diacylglycerols (which activate protein kinases). Activation of receptors for inhibitory signal molecules increases the activity of protein kinases through increases in cAMP or cGMP and often hyperpolarizes the cell. Other receptors link to tyrosine kinases, which trigger signal cascades interacting with trimeric G-protein systems.