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Recently, we identified, for the first time, two-pore channels (TPCs, TPCN for gene name) as a novel family of nicotinic acid adenine dinucleotide phosphate (NAADP)-gated, endolysosome-targeted calcium release channels. Significantly, three subtypes of TPCs have been characterized, TPCI-3, with each being targeted to discrete acidic calcium stores, namely lysosomes (TPC2) and endosomes (TPC1 and TPC3). That TPCs act as NAADP-gated calcium release channels is clear, given that NAADP binds to high--and low-affinity sites associated with TPC2 and thereby induces calcium release and homologous desensitization, as observed in the case of endogenous NAADP receptors. Moreover, NAADP-evoked calcium signals via TPC2 are ablated by short hairpin RNA knockdown of TPC2 and by depletion of acidic calcium stores with bafilomycin. Importantly, however, NAADP-evoked calcium signals were biphasic in nature, with an initial phase of calcium release from lysosomes via TPC2, being subsequently amplified by calcium-induced calcium release (CICR) from the endoplasmic reticulum (ER). In marked contrast, calcium release via endosome-targeted TPC1 induced only spatially restricted calcium signals that were not amplified by CICR from the ER. These findings provide new insights into the mechanisms that cells may utilize to 'filter' calcium signals via junctional complexes to determine whether a given signal remains local or is converted into a propagating global signal. Essentially, endosomes and lysosomes represent vesicular calcium stores, quite unlike the ER network, and TPCs do not themselves support CICR or, therefore, propagating regenerative calcium waves. Thus 'quantal' vesicular calcium release via TPCs must subsequently recruit inositol 1,4,5-trisphoshpate receptors and/or ryanodine receptors on the ER by CICR to evoke a propagating calcium wave. This may call for a revision of current views on the mechanisms of intracellular calcium signaling. The purpose of this review is, therefore, to provide an appropriate framework for future studies in this area. two-pore segment channel; nicotinic acid adenine dinucleotide phosphate; sarco (endo)plasmic reticulum; lysosome doi:10.1152/ajpcell.00475.2009

The Caenorhabditis elegans intestinal epithelium generates rhythmic inositol 1,4,5-trisphosphate ([IP.sub.3])-dependent [Ca.sup.2+] oscillations that control muscle contractions required for defecation. Two highly [Ca.sup.2+]-selective transient receptor potential (TRP) melastatin (TRPM) channels, GON-2 and GTL-1, function with PLC[gamma] in a common signaling pathway that regulates [IP.sub.3]-dependent intracellular [Ca.sup.2+] release. A second PLC, PLC[beta], is also required for [IP.sub.3]-dependent [Ca.sup.2+] oscillations, but functions in an independent signaling mechanism. PLC[gamma] generates [IP.sub.3] that regulates [IP.sub.3] receptor activity. We demonstrate here that PLC[gamma], via hydrolysis of phosphatidylinositol 4,5-bisphosphate ([PIP.sub.2]) also regulates GON-2/GTL-1 function. Knockdown of PLC[gamma] but not PLC[beta] activity by RNA interference (RNAi) inhibits channel activity ~80%. Inhibition is fully reversed by agents that deplete [PIP.sub.2] levels. [PIP.sub.2] added to the patch pipette has no effect on channel activity in PLC[gamma] RNAi cells. However, in control cells, 10 [micro]M [PIP.sub.2] inhibits whole cell current ~80%. Channel inhibition by phospholipids is selective for [PIP.sub.2] with an [IC.sub.50] value of 2.6 [micro]M. Elevated [PIP.sub.2] levels have no effect on channel voltage and [Ca.sup.2+] sensitivity and likely inhibit by reducing channel open probability, single-channel conductance, and/or trafficking. We conclude that hydrolysis of [PIP.sub.2] by PLC[gamma] functions in the activation of both the [IP.sub.3] receptor and GON-2/GTL-1 channels. GON-2/GTL-1 functions as the major intestinal cell [Ca.sup.2+] influx pathway. Calcium influx through the channel feedback regulates its activity and likely functions to modulate [IP.sub.3] receptor function. [PIP.sub.2]-dependent regulation of GON-2/ GTL-1 may provide a mechanism to coordinate plasma membrane [Ca.sup.2+] influx with PLC[gamma] and [IP.sub.3] receptor activity as well as intracellular [Ca.sup.2+] store depletion. Caenorhabditis elegans; inositol 1,4,5-trisphosphate; transient receptor potential channels; patch-clamp electrophysiology; phospholipase C; phosphatidylinositol 4,5-bisphosphate doi: 10.1152/ajpcell.00394.2009

Evidence for a modulatory effect of cyclosporin A (CsA) on calcium signaling and cell survival in dystrophin-deficient cells is presented. Our previous works strongly supported the hypothesis of an overactivation of [Ca.sup.2+] release via inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in dystrophin-deficient cells, both during membrane depolarization and at rest, through spontaneous [Ca.sup.2+] release events. Forced expression of mini-dystrophin in these cells contributed, during stimulation and in resting condition, to the recovery of a controlled calcium homeostasis. In the present work, we demonstrate that CsA exposure displayed a dual-modulator effect on calcium signaling in dystrophin-deficient cells. Short-time incubation induced a decrease of IP3-dependent calcium release, leading to patterns of release similar to those observed in myotubes expressing mini-dystrophin, whereas long-time incubation reduced the expression of the type I of IP3 receptors (IP3R-1) RNA levels. Moreover, both IP3R-1 knockdown and blockade through 2-aminoethoxydiphenyle borate or CsA induced improved survival of dystrophin-deficient myotubes, demonstrating the cell death dependence on the IP3-dependent calcium signaling as well as the protective effect of CsA. Inhibition of the IP3 pathway could be a very interesting approach for reducing the natural cell death of dystrophin-deficient cells in development. calcium regulation; cyclosporin A; muscular dystrophy doi: 10.1152/ajpcell.00048.2009.

The coupling mechanism between depletion of [Ca.sup.2+] stores in the endoplasmic reticulum and plasma membrane store-operated ion channels is fundamental to [Ca.sup.2+] signaling in many cell types and has yet to be completely elucidated. Using [Ca.sup.2+] release-activated [Ca.sup.2+] (CRAC) channels in RBL2H3 cells as a model system, we have shown that CRAC channels are maintained in the closed state by an inhibitory factor rather than being opened by the inositol 1,4,5-trisphosphate receptor. This inhibitory role can be fulfilled by the Drosophila protein INAD (inactivation-no after potential D). The action of INAD requires [Ca.sup.2+] and can be reversed by a diffusible [Ca.sup.2+] influx factor. Thus the coupling between the depletion of [Ca.sup.2+] stores and the activation of CRAC channels may involve a mammalian homologue of INAD and a low-molecular-weight, diffusible store-depletion signal. store-operated ion channels; inositol 1,4,5-trisphosphate receptor

Kwan, H. Y., Y. Huang, S. K. Kong, and X. Yao. cGMP abolishes agonist-induced [[Ca.sup.2+].sub.i] oscillations in human bladder epithelial cells. Am J Physiol Renal Physiol 281: F1067-F1074, 2001. First published August 9, 2001; 10.1152/ajprenal.00031.2001.--Cytosolic calcium oscillations may permit cells to respond to information provided by increases in intracellular [Ca.sup.2+] concentration ([[Ca.sup.2+]sub.i]) while avoiding prolonged exposure to constantly elevated [[Ca.sup.2+]sub.i]. In this study, we demonstrated that agonists could induce [Ca.sup.2+] oscillations in human bladder epithelial cells. Application of 10 [micro]M acetylcholine or 200 nM bradykinin triggered an initial [Ca.sup.2+] transient that was followed by periodic [[Ca.sup.2+]sub.i] oscillations. The oscillations did not depend on extracellular [Ca.sup.2+]. 8-Bromoguanosine 3',5'-cyclic monophosphate abolished acetylcholine- or bradykinin-induced oscillations. Elevation of cellular cGMP by dipyridamole, an inhibitor of cGMP-specific phosphodiesterase, also terminated the [[Ca.sup.2+]sub.i] oscillations. The inhibitory effect of cGMP could be reversed by KT-5823, a highly specific inhibitor of protein kinase G (PKG), suggesting that the action of cGMP was mediated by PKG. Comparison of the effect of cGMP with that of xestospongin C, an inhibitor of the inositol 1,4,5-trisphosphate (I[P.sub.3]) receptor, revealed similarities between the action of cGMP and xestospongin C. Therefore, it is likely that cGMP and PKG may target a signal transduction step(s) linked to I[P.sub.3] receptor-mediated [Ca.sup.2+] release. protein kinase G; inositol 1,4,5-triphosphate; calcium release; nitric oxide; intracellular calcium concentration; guanosine 3',5'-cyclic monophosphate Received 5 February 2001; accepted in final form 23 July 2001

A study was conducted to determine whether reduced type I IP3 receptor (IP3R) is essential in diabetic kidney disease. In evaluating type I IP3R expression in the kidney of streptozotocin-induced diabetic rats and mice, it was found that there is reduced type I IP3R in glomerular and vascular smooth muscle cells in the diabetic kidney, which may have a part in the modified renal vasoregulation and renal hypertrophy of diabetes.

A study was conducted to examine the effects of Mg2+ and Ba2+ on the single-channel properties of inositol 1,4,5-triphosphate receptors. The identity of the Xenopus IP3R channel was determined in outer nuclear membrane patches while stage VI oocytes were isolated from the ovary. Results indicated that divalent cations minimize currents using a mechanism supporting the permeation block of the IP3R.

The effects of long-term, hypoxemia-induced reductions in cerebrovascular contractility on the density and affinity of inositol 1,4,5-triphosphate [Ins(1,4,5)P3] were analyzed in the cerebral arteries of sheep. Vascular Ins(1,4,5)P3-R density increased as a function of developmental age in the common carotid artery and aorta of sheep. High-altitude, long-term hypoxia was also associated with a significant decrease in Ins(1,4,5)P3-R density in the common carotid artery and main bran cerebral blood vessels of fetal and adult sheep.

The effects of 2-omicron, C-methylene-myo-inositol (MMI) on myo-inositol transport were analyzed in Madin-Darby canine kidney (MDCK) cells to determine the role of myo-inositol under hypertonic conditions. Analysis of myo-inositol transport in MDCK cells after the administration of MMI indicated the presence of inhibited myo-inositol transport due to the inactivation of the Na+-dependent component. Furthermore, MMI coupled with betaine increased the colony-forming efficiency of MDCK cells.

The role of alpha1-adrenergic receptor (alpha1-AR) density and norepinephrine (NE)-induced inositol 1,4,5-triphosphate (IP3) in NE-mediated contraction changes were analyzed in sheep cerebral arteries. Development differences in the contractility of cerebral artery to NE is mediated by changes in aplha1-AR density which affects vascular agonist sensitivity. Furthermore, the common carotid arteries of fetus and newborn infants exhibited little IP3 response to NE compared to the anterior and middle cerebral arteries.

Perfused rat hearts were analyzed to determine the effects of an 8-week dietary supplementation of n-3 long chain polyunsaturated fatty acids (n-3 PUFA) on cardiac D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Dietary supplementation with n-3 PUFA inhibited the release of reperfusion-induced Ins(1,4,5)P3 in rat ischemic models. Reperfusion arrythmia was also inhibited by dietary Ins(1,4,5)P3 in rat regional ischemia models.

The stimulation of acinar cells by inositol 1,4,5-triphosphate produced asynchronous increases in Ca2+ levels that are characterized as a coordinated oscillatory cycle that passes between the cells. The focal application of additional antagonists to the acinar cells did not alter the generation and propagation of Ca2+ signals indicating an activation-mechanism that is not dependent on the generation of secondary messengers. Enhanced acinar secretions after stimulation also indicate the possible role of gap-junctional communication.

Receptors for 1,4,5-trisphosphate signal the need for changes in the calcium ion concentration within the cell. The two receptor types for inositol interact in regulating calcium ion influx. Changing the ratio of both types of receptors seems to change the signal induced by inositol. Dominance of type 1 receptors raises the speed of propagation of ion release in the membrane. Meanwhile, more type 3 receptors increase the amount and length of calcium influx.

Neural and endothelial cells are able to osmotically regulate their intake of myoinositol in a manner similar to renal cells. These cells raised their production of myoinositol transporter which then directly increased the amount of transported myoinositol when the cells were exposed to hypertonic conditions. Hypotonic conditions led to lower myoinositol transporter production and, consequently, lower myoinositol levels in the cells.

Betaine and inositol decrease the glycerophosphocholine (GPC) content of cultured Madin-Darby canine kidney cells by increasing GPC breakdown by GPC:choline phosphodiesterase. The effect of betaine and inositol is higher in isosmotic or high-NaCl medium than that in high-urea medium. This is due to inhibition of betaine and inositol accumulation by urea. Maintenance of total osmolyte level involves the reciprocal control of GPC, with respect to other related osmolytes, in response to high tonicity.

Development of rat mesangial cell cultures with high galactose or glucose levels to characterize factors that help upregulate myo-inositol (MI) transport reveals that high level glucose or galactose initiates protein synthesis with intact polymerized actin to help upregulate MI transport in the cells. Tolrestat, an aldose reductase inhibitor, suppresses the upregulation of MI transport in the isolated glomeruli and cultured mesangial cells, suggesting that activation of the polyol pathway is essential for the upregulation process.

CK1.4 Chinese hamster ovary cells produced by transfection with the gene for cattle heart Na+-Ca2+ exhanger were studied. The study showed that exchanging external Na+ with K+ resulted in a significant increase in Ca2+ uptake by Na+-loaded cells. Nontransfected cells showed a decrease in the 45Ca2+ uptake when the external Na+ was replaced with K+. CK1.4 cells provide excellent study models because of the stable expression of sufficient exchanger to biochemical and innunological analysis.

The expression of the osmosensitive sodium/myo-inositol cotransporter (SMIT) is regulated by multiple tonicity-responsive enhancers (TonEs) in the 5'-flanking region of the gene. In response to hypertonicity, the nuclear abundance of the transcription factor TonE-binding protein (TonEBP) is increased, and the transcription of the SMIT gene is induced. Transport system A for neutral amino acids, another osmosensitive mechanism, is progressively stimulated if amino acid substrates are not present in the extracellular compartment. Under this condition, as in hypertonicity, cells shrink and mitogen-activated protein kinases are activated. We demonstrate here that a clear-cut nuclear redistribution of TonEBP, followed by SMIT expression increase and inositol transport activation, is observed after incubation of cultured human flbroblasts in Earle's balanced salts (EBSS), an isotonic, amino acid-free saline. EBSS-induced SMIT stimulation is prevented by substrates of system A, although these compounds do not compete with inositol for transport through SMIT. We conclude that the incubation in isotonic, amino acid-free saline triggers an osmotic stimulus and elicits TonEBP-dependent responses like hypertonic treatment. hypertonic stress; system A; glutamine; stress proteins; cell volume; tonicity-responsive enhancer-binding protein

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells. Am J Physiol Cell Physiol 250: C22-C33, 2001.-- To examine the nature of inositol 1,4,5-trisphosphate ([IP.sub.3])-sensitive and ryanodine (Ryn)-sensitive [Ca.sup.2+] stores in isolated canine pulmonary arterial smooth cells (PASMC), agonist-induced changes in global intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) were measured using fura 2-AM fluorescence. Properties of elementary local [Ca.sup.2+] release events were characterized using fluo 3-AM or fluo 4-AM, in combination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum [Ca.sup.2+] stores with Ryn (300 [micro]M) and caffeine (Caf; 10 mM) eliminated subsequent Caf-induced intracellular [Ca.sup.2+] transients but had little or no effect on the initial [IP.sub.3]-mediated intracellular [Ca.sup.2+] transient induced by ANG II (1 [micro]M). Cyclopiazonic acid (CPA; 10 [micro]M) abolished [IP.sup.3]-induced intracellular [Ca.sup.2+] transients but failed to attenuate the initial Caf-induced intracellular [Ca.sup.2+] transient. These results suggest that in canine PASMC, [IP.sub.3]-, and Ryn-sensitive [Ca.sup.2+] stores are organized into spatially distinct compartments while similar experiments in canine renal arterial smooth muscle cells (RASMC) reveal that these [Ca.sup.2+] stores are spatially conjoined. In PASMC, spontaneous local intracellular [Ca.sup.2+] transients sensitive to modulation by Caf and Ryn were detected, exhibiting spatial-temporal characteristics similar to those previously described for '[Ca.sup.2+] sparks' in cardiac and other types of smooth muscle cells. After depletion of Rynsensitive [Ca.sup.2+] stores, ANG II (8 nM) induced slow, sustained [[[Ca.sup.2+]].sub.i] increases originating at sites near the cell surface, which were abolished by depleting [IP.sub.3] stores. Discrete quantal-like events expected due to the coordinated opening of [IP.sub.3] receptor clusters ('[Ca.sup.2+] puffs') were not observed. These data provide new information regarding the functional properties and organization of intracellular [Ca.sup.2+] stores and elementary [Ca.sup.2+] release events in isolated PASMC. pulmonary artery; smooth muscle cells; sarcoplasmic reticulum; intracellular calcium; inositol 1,4,5-trisphosphate; calcium ion sparks

Normalization of hyperosmotic-induced inositol uptake by renal and endothelial cells is regulated by NF-[Kappa]B. Am J Physiol Cell Physiol 278: C1011-C1018, 2000.--Hyperosmolarity is a stress factor that has been shown to cause an increase in the transcription of the [Na.sup.+]-dependent myo-inositol cotransporter (SMIT). However, regulation of the reversion of SMIT mRNA levels and transporter activity following removal of hyperosmotic stress is less understood. Previously we have shown that postinduction normalization of SMIT mRNA levels and myo-inositol accumulation following removal of hyperosmotic stress is inhibited by actinomycin D and cycloheximide, suggesting that normalization requires RNA transcription and protein synthesis. We now demonstrate that removal of hyperosmotic stress causes an activation of the transcription factor NF-[Kappa]B in renal and endothelial cells. Inhibiting NF-[Kappa]B activation with pyrrolidine dithiocarbamate (PD) blocks the normalization of SMIT mRNA levels and myo-inositol accumulation on removal of the cells from hyperosmotic medium. These studies demonstrate that the downregulation of the myo-inositol transporter following reversal of hyperosmotic induction is regulated via the activation of NF-[Kappa]B. myo-inositol; hyperosmolarity; nuclear factor [Kappa]B; sodiumdependent myo-inositol cotransporter

[IP.sub.3] receptors, [IP.sub.3] transients, and nucleus-associated [Ca.sup.2+] signals in cultured skeletal muscle. Am J Physiol Cell Physiol 278: C998-C1010, 2000.--Inositol 1,4,5-trisphosphate ([IP.sub.3]) receptors ([IP.sub.3R]) and ryanodine receptors (RyR) were localized in cultured rodent muscle fractions by binding of radiolabeled ligands ([IP.sub.3] and ryanodine), and [IP.sub.3]R were visualized in situ by fluorescence immunocytological techniques. Also explored was the effect of [K.sup.+] depolarization on [IP.sub.3] mass and [Ca.sup.2+] transients studied using a radio-receptor displacement assay and fluorescence imaging of intracellular fluo 3. RyR were located in a microsomal fraction; [IP.sub.3]R were preferentially found in the nuclear fraction. Fluorescence associated with anti-[IP.sub.3]R antibody was found in the region of the nuclear envelope and in a striated pattern in the sarcoplasmic areas. An increase in external [K.sup.+] affected membrane potential and produced an [IP.sup.3] transient. Rat myotubes displayed a fast-propagating [Ca.sup.2+] signal, corresponding to the excitation-contraction coupling transient and a much slower [Ca.sup.2+] wave. Both signals were triggered by high external [K.sup.+] and were independent of external [Ca.sup.2+]. Slow waves were associated with cell nuclei and were propagated leaving 'glowing' nuclei behind. Different roles are proposed for at least two types of [Ca.sup.2+] re]ease channels, each mediating an intracellular signal in cultured skeletal muscle. myotubes; inositol 1,4,5-trisphosphate; excitation-contraction coupling; signal transduction

Inositol is a widespread intracellular organic osmolyte and phosphoinositide precursor maintained at millimolar intracellular concentrations through the action of membrane-associated Na+-myo-inositol cotransporters (SMIT). Functional cloning and expression of a canine SMIT cDNA, which conferred SMIT activity in Xenopus oocytes, predicted a 718-amino acid peptide homologous to the Na+-glucose cotransporter with a potential protein kinase A phosphorylation site and multiple protein kinase C phosphorylation sites. An open reading frame on human chromosome 21 homologous to that of the canine cDNA is composed of an intronless human SMIT gene. An alternate splice donor site is proposed within the coding region that extends the open reading frame into the otherwise untranslated 3' exons, possibly generating novel SMIT isoforms.