Cholestasis shuts down calcium signaling in cholangiocytes

BACKGROUND & AIMS: Cholestasis is one of the principal manifestations of liver disease and often results from disorders involving bile duct epithelia rather than hepatocytes. A range of disorders affects biliary epithelia, and no unifying pathophysiologic event in these cells has been identified as the cause of cholestasis. Here we examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R)/Ca(2+) release channel in Ca(2+) signaling and ductular secretion in animal models of cholestasis and in patients with cholestatic disorders. METHODS: The expression and distribution of the InsP3R and related proteins were examined in rat cholangiocytes before and after bile duct ligation or treatment with endotoxin. Ca(2+) signaling was examined in isolated bile ducts from these animals, whereas ductular bicarbonate secretion was examined in isolated perfused livers. Confocal immunofluorescence was used to examine cholangiocyte InsP3R expression in human liver biopsy specimens. RESULTS: Expression of the InsP3R was selectively lost from biliary epithelia after bile duct ligation or endotoxin treatment. As a result, Ca(2+) signaling and Ca(2+)-mediated bicarbonate secretion were lost as well, although other components of the Ca(2+) signaling pathway and adenosine 3',5'-cyclic monophosphate (cAMP)-mediated bicarbonate secretion both were preserved. Examination of human liver biopsy specimens showed that InsP3Rs also were lost from bile duct epithelia in a range of human cholestatic disorders, although InsP3R expression was intact in noncholestatic liver disease. CONCLUSIONS: InsP3R-mediated Ca(2+) signaling in bile duct epithelia appears to be important for normal bile secretion in the liver, and loss of InsP3Rs may be a final common pathway for cholestasis.     

Lipid rafts establish calcium waves in hepatocytes

BACKGROUND & AIMS: Polarity is critical for hepatocyte function. Ca(2+) waves are polarized in hepatocytes because the inositol 1,4,5-trisphosphate receptor (InsP3R) is concentrated in the pericanalicular region, but the basis for this localization is unknown. We examined whether pericanalicular localization of the InsP3R and its action to trigger Ca(2+) waves depends on lipid rafts. METHODS: Experiments were performed using isolated rat hepatocyte couplets and pancreatic acini, plus SkHep1 cells as nonpolarized controls. The cholesterol depleting agent methyl-beta-cyclodextrin (mbetaCD) was used to disrupt lipid rafts. InsP3R isoforms were examined by immunoblot and immunofluorescence. Ca(2+) waves were examined by confocal microscopy. RESULTS: Type II InsP3Rs initially were localized to only some endoplasmic reticulum fractions in hepatocytes, but redistributed into all fractions in mbetaCD-treated cells. This InsP3R isoform was concentrated in the pericanalicular region, but redistributed throughout the cell after mbetaCD treatment. Vasopressin-induced Ca(2+) signals began as apical-to-basal Ca(2+) waves, and mbetaCD slowed the wave speed and prolonged the rise time. MbetaCD had a similar effect on Ca(2+) waves in acinar cells but did not affect Ca(2+) signals in SkHep1 cells, suggesting that cholesterol depletion has similar effects among polarized epithelia, but this is not a nonspecific effect of mbetaCD. CONCLUSIONS: Lipid rafts are responsible for the pericanalicular accumulation of InsP3R in hepatocytes, and for the polarized Ca(2+) waves that result. Signaling microdomains exist not only in the plasma membrane, but also in the nearby endoplasmic reticulum, which in turn, helps establish and maintain structural and functional polarity.     

Ca2+ waves require sequential activation of inositol trisphosphate receptors and ryanodine receptors in pancreatic acini

BACKGROUND & AIMS: The inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) and the ryanodine receptor (RyR) are the principal Ca2+-release channels in cells and are believed to serve distinct roles in cytosolic Ca2+ (Ca(i)2+) signaling. This study investigated whether these receptors instead can release Ca2+ in a coordinated fashion. METHODS: Apical and basolateral Ca(i)2+ signals were monitored in rat pancreatic acinar cells by time-lapse confocal microscopy. Caged forms of second messengers were microinjected into individual cells and then photoreleased in a controlled fashion by either UV or 2-photon flash photolysis. RESULTS: InsP3 increased Ca(i)2+ primarily in the apical region of pancreatic acinar cells, whereas the RyR agonist cyclic adenosine diphosphate ribose (cADPR) increased Ca(i)2+ primarily in the basolateral region. Apical-to-basal Ca(i)2+ waves were induced by acetylcholine and initiation of these waves was blocked by the InsP3R inhibitor heparin, whereas propagation into the basolateral region was inhibited by the cADPR inhibitor 8-amino-cADPR. To examine integration of apical and basolateral Ca(i)2+ signals, Ca2+ was selectively released either apically or basolaterally using 2-photon flash photolysis. Ca(i)2+ increases were transient and localized in unstimulated cells. More complex Ca(i)2+ signaling patterns, including polarized Ca(i)2+ waves, were observed when Ca2+ was photoreleased in cells stimulated with subthreshold concentrations of acetylcholine. CONCLUSIONS: Polarized Ca(i)2+ waves are induced in acinar cells by serial activation of apical InsP3Rs and then basolateral RyRs, and subcellular release of Ca2+ coordinates the actions of these 2 types of Ca2+ channels. This subcellular integration of Ca2+-release channels shows a new level of complexity in the formation of Ca(i)2+ waves.     

Regulation of Ca(2+) signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms

Cytosolic Ca(2+) (Ca(i)(2+)) regulates secretion of bicarbonate and other ions in the cholangiocyte. In other cell types, this second messenger acts through Ca(2+) waves, Ca(2+) oscillations, and other subcellular Ca(2+) signaling patterns, but little is known about the subcellular organization of Ca(2+) signaling in cholangiocytes. Therefore, we examined Ca(2+) signaling and the subcellular distribution of Ca(2+) release channels in cholangiocytes and in a model cholangiocyte cell line. The expression and subcellular distribution of inositol 1,4,5-trisphosphate (InsP(3)) receptor (InsP(3)R) isoforms and the ryanodine receptor (RyR) were determined in cholangiocytes from normal rat liver and in the normal rat cholangiocyte (NRC) polarized bile duct cell line. Subcellular Ca(2+) signaling in cholangiocytes was examined by confocal microscopy. All 3 InsP(3)R isoforms were expressed in cholangiocytes, whereas RyR was not expressed. The type III InsP(3)R was the most heavily expressed isoform at the protein level and was concentrated apically, whereas the type I and type II isoforms were expressed more uniformly. The type III InsP(3)R was expressed even more heavily in NRC cells but was concentrated apically in these cells as well. Adenosine triphosphate (ATP), which increases Ca(2+) via InsP(3) in cholangiocytes, induced Ca(2+) oscillations in both cholangiocytes and NRC cells. Acetylcholine (ACh) induced apical-to-basal Ca(2+) waves. In conclusion, Ca(2+) signaling in cholangiocytes occurs as polarized Ca(2+) waves that begin in the region of the type III InsP(3)R. Differential subcellular localization of InsP(3)R isoforms may be an important molecular mechanism for the formation of Ca(2+) waves and oscillations in cholangiocytes. Because Ca(i)(2+) is in part responsible for regulating ductular secretion, these findings also may have implications for the molecular basis of cholestatic disorders.     

Extracellular polyamines regulate fluid secretion in rat colonic crypts via the extracellular calcium-sensing receptor

BACKGROUND AND AIMS: Polyamines are essential for the normal postnatal development, maintenance, and function of gastrointestinal epithelia. The extracellular Ca(2+) (Ca(2+)(o)/nutrient)-sensing receptor is expressed on both luminal and basolateral membranes of colonocytes, and, in other cell systems, this receptor has been shown to respond to polyamines. Thus, the Ca(2+)-sensing receptor could provide a mechanism for modulation of colonocyte function by dietary and systemic extracellular polyamines. In the present study, we investigated the interaction of polyamines, particularly spermine, and extracellular Ca(2+) on second messenger generation by, and on function of, rat distal colonic crypts. METHODS: Calcium-sensing receptor activation was assessed in colonic epithelial cells and intact crypts freshly isolated from distal colon by monitoring intracellular IP(3) and Ca(2+) accumulation using radioimmunoassay and Fluo-3 fluorometry, respectively. Interactions of extracellular Ca(2+) and spermine on regulation of both basal and forskolin-stimulated fluid transport were measured in crypts microperfused in vitro. RESULTS: Polyamine (spermine > spermidine > putrescine)-mediated enhancement of intracellular D-myo-inositol 1,4,5-trisphosphate (IP(3)) and Ca(2+) accumulation required extracellular Ca(2+), and the EC(50) for extracellular Ca(2+)-mediated activation of the calcium-sensing receptor was reduced by polyamines. Extracellular spermine modulated both basal and forskolin-stimulated fluid secretion in perfused colonic crypts, and the EC(50) for spermine-induced reduction in forskolin-stimulated fluid secretion was inversely dependent on extracellular Ca(2+) (Ca(2+)(o)). CONCLUSIONS: The interactions of extracellular Ca(2+) and polyamines on second messenger accumulation and fluid secretion support a role for the luminal and basolateral calcium-sensing receptors in mediating some of the effects of polyamines on distal colonic epithelial cells.     

Cholangiocyte cilia detect changes in luminal fluid flow and transmit them into intracellular Ca2+ and cAMP signaling

BACKGROUND & AIMS: Cholangiocytes have primary cilia extending from the apical plasma membrane into the ductal lumen. While the physiologic significance of cholangiocyte cilia is unknown, studies in renal epithelia suggest that primary cilia possess sensory functions. Here, we tested the hypothesis that cholangiocyte cilia are sensory organelles that detect and transmit luminal bile flow stimuli into intracellular Ca2+ ([Ca2+]i) and adenosine 3',5'-cyclic monophosphate (cAMP) signaling. METHODS: Scanning electron microscopy, transmission electron microscopy, and immunofluorescent confocal microscopy of rat isolated intrahepatic bile duct units (IBDUs) were used to detect and characterize cholangiocyte cilia. The fluid flow-induced changes in Ca2+ and cAMP levels in cholangiocytes of microperfused IBDUs were detected by epifluorescence microscopy and a fluorescence assay, respectively. RESULTS: In microperfused IBDUs, luminal fluid flow induced an increase in [Ca2+]i and caused suppression of the forskolin-stimulated cAMP increase. The fluid flow-induced changes in [Ca2+]i and cAMP levels were significantly reduced or abolished when cilia were removed by chloral hydrate or when ciliary-associated proteins polycystin-1 (a mechanoreceptor), polycystin-2 (a Ca2+ channel), and the Ca2+-inhibitable adenylyl cyclase isoform 6 were individually down-regulated by small interfering RNAs. CONCLUSIONS: Cholangiocyte cilia are sensory organelles containing polycystin-1, polycystin-2, and adenylyl cyclase isoform 6 through which luminal fluid flow affects both [Ca2+]i and cAMP signaling in the cell. The data suggest a new model for regulation of ductal bile secretion involving cholangiocyte cilia.     

Type 2 inositol 1,4,5-trisphosphate receptor modulates bile salt export pump activity in rat hepatocytes

Bile salt secretion is mediated primarily by the bile salt export pump (Bsep), a transporter on the canalicular membrane of the hepatocyte. However, little is known about the short-term regulation of Bsep activity. Ca(2+) regulates targeting and insertion of transporters in many cell systems, and Ca(2+) release near the canalicular membrane is mediated by the type II inositol 1,4,5-trisphosphate receptor (InsP3R2), so we investigated the possible role of InsP3R2 in modulating Bsep activity. The kinetics of Bsep activity were monitored by following secretion of the fluorescent Bsep substrate cholylglycylamido-fluorescein (CGamF) in rat hepatocytes in collagen sandwich culture, an isolated cell system in which structural and functional polarity is preserved. CGamF secretion was nearly eliminated in cells treated with Bsep small interfering RNA (siRNA), demonstrating specificity of this substrate for Bsep. Secretion was also reduced after chelating intracellular calcium, inducing redistribution of InsP3R2 by depleting the cell membrane of cholesterol, or reducing InsP3R function by either knocking down InsP3R2 expression using siRNA or pharmacologic inhibition using xestospongin C. Confocal immunofluorescence showed that InsP3R2 and Bsep are in close proximity in the canalicular region, both in rat liver and in hepatocytes in sandwich culture. However, after knocking down InsP3R2 or inducing its dysfunction with cholesterol depletion, Bsep redistributed intracellularly. Finally, InsP3R2 was lost from the pericanalicular region in animal models of estrogen- and endotoxin-induced cholestasis. CONCLUSION: These data provide evidence that pericanalicular calcium signaling mediated by InsP3R2 plays an important role in maintaining bile salt secretion through posttranslational regulation of Bsep, and suggest that loss or redistribution of InsP3R2 may contribute to the pathophysiology of intrahepatic cholestasis.     

Functional architecture of inositol 1,4,5-trisphosphate signaling in restricted spaces of myoendothelial projections

Calcium (Ca(2+)) release through inositol 1,4,5-trisphosphate receptors (IP(3)Rs) regulates the function of virtually every mammalian cell. Unlike ryanodine receptors, which generate local Ca(2+) events ("sparks") that transmit signals to the juxtaposed cell membrane, a similar functional architecture has not been reported for IP(3)Rs. Here, we have identified spatially fixed, local Ca(2+) release events ("pulsars") in vascular endothelial membrane domains that project through the internal elastic lamina to adjacent smooth muscle membranes. Ca(2+) pulsars are mediated by IP(3)Rs in the endothelial endoplasmic reticulum of these membrane projections. Elevation of IP(3) by the endothelium-dependent vasodilator, acetylcholine, increased the frequency of Ca(2+) pulsars, whereas blunting IP(3) production, blocking IP(3)Rs, or depleting endoplasmic reticulum Ca(2+) inhibited these events. The elementary properties of Ca(2+) pulsars were distinct from ryanodine-receptor-mediated Ca(2+) sparks in smooth muscle and from IP(3)-mediated Ca(2+) puffs in Xenopus oocytes. The intermediate conductance, Ca(2+)-sensitive potassium (K(Ca)3.1) channel also colocalized to the endothelial projections, and blockage of this channel caused an 8-mV depolarization. Inhibition of Ca(2+) pulsars also depolarized to a similar extent, and blocking K(Ca)3.1 channels was without effect in the absence of pulsars. Our results support a mechanism of IP(3) signaling in which Ca(2+) release is spatially restricted to transmit intercellular signals.     

The type II inositol 1,4,5-trisphosphate receptor can trigger Ca2+ waves in rat hepatocytes

BACKGROUND & AIMS: Ca2+ regulates cell functions through signaling patterns such as Ca2+ oscillations and Ca2+ waves. The type I inositol 1,4,5-trisphosphate receptor is thought to support Ca2+ oscillations, whereas the type III inositol 1,4,5-trisphosphate receptor is thought to initiate Ca2+ waves. The role of the type II inositol 1,4,5-trisphosphate receptor is less clear, because it behaves like the type III inositol 1,4,5-trisphosphate receptor at the single-channel level but can support Ca2+ oscillations in intact cells. Because the type II inositol 1,4,5-trisphosphate receptor is the predominant isoform in liver, we examined whether this isoform can trigger Ca2+ waves in hepatocytes. METHODS: The expression and distribution of inositol 1,4,5-trisphosphate receptor isoforms was examined in rat liver by immunoblot and confocal immunofluorescence. The effects of inositol 1,4,5-trisphosphate on Ca2+ signaling were examined in isolated rat hepatocyte couplets by using flash photolysis and time-lapse confocal microscopy. RESULTS: The type II inositol 1,4,5-trisphosphate receptor was concentrated near the canalicular pole in hepatocytes, whereas the type I inositol 1,4,5-trisphosphate receptor was found elsewhere. Stimulation of hepatocytes with vasopressin or directly with inositol 1,4,5-trisphosphate induced Ca2+ waves that began in the canalicular region and then spread to the rest of the cell. Inositol 1,4,5-Trisphosphate-induced Ca2+ signals also increased more rapidly in the canalicular region. Hepatocytes did not express the ryanodine receptor, and cyclic adenosine diphosphate-ribose had no effect on Ca2+ signaling in these cells. CONCLUSIONS: The type II inositol 1,4,5-trisphosphate receptor establishes a pericanalicular trigger zone from which Ca2+ waves originate in hepatocytes.     

Targeted expression of the inositol 1,4,5-triphosphate receptor (IP3R) ligand-binding domain releases Ca2+ via endogenous IP3R channels

Virtually all functions of a cell are influenced by cytoplasmic [Ca(2+)] increases. Inositol 1,4,5-trisphosphate receptor (IP(3)R) channels, located in the endoplasmic reticulum (ER), release Ca(2+) in response to binding of the second messenger, IP(3).IP(3)Rs thus are part of the information chain interpreting external signals and transforming them into cytoplasmic Ca(2+) transients. IP(3)Rs function as tetramers, each unit comprising an N-terminal ligand-binding domain (LBD) and a C-terminal channel domain linked by a long regulatory region. It is not yet understood how the binding of IP(3) to the LBD regulates the gating properties of the channel. Here, we use the expression of IP(3) binding protein domains tethered to the surface of the endoplasmic reticulum (ER) to show that the all-helical domain of the IP(3)R LBD is capable of depleting the ER Ca(2+) pools by opening the endogenous IP(3)Rs, even without IP(3) binding. This effect requires the domain to be within 50 A of the ER membrane and is impaired by the presence of the N-terminal inhibitory segment on the LBD. These findings raise the possibility that the helical domain of the LBD functions as an effector module possibly interacting with the channel domain, thereby being part of the gating mechanisms by which the IP(3)-induced conformational change within the LBD regulates Ca(2+) release.     

Inositol hexakisphosphate suppresses excitatory neurotransmission via synaptotagmin-1 C2B domain in the hippocampal neuron

Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons. Intracellular exposure to InsP(6) did not affect spontaneous EPSCs or excitatory amino acid-activated currents in neurons lacking autapses. The InsP(6)-induced inhibition of autaptic EPSCs was effectively abolished by coapplication of an antibody to synaptotagmin-1 C2B domain. Importantly, preabsorption of the antibody with a GST-WT synaptotagmin-1 C2B domain fragment but not with a GST-mutant synaptotagmin-1 C2B domain fragment that poorly reacted with the antibody impaired the activity of the antibody on the InsP(6)-induced inhibition of autaptic EPSCs. Furthermore, K(+) depolarization significantly elevated endogenous levels of InsP(6) and occluded the inhibition of autaptic EPSCs by exogenous InsP(6). These data reveal that InsP(6) suppresses excitatory neurotransmission via inhibition of the presynaptic synaptotagmin-1 C2B domain-mediated fusion via an interaction with the synaptotagmin Ca(2+)-binding sites rather than via interference with presynaptic Ca(2+) levels, synaptic vesicle trafficking, or inactivation of postsynaptic ionotropic glutamate receptors. Therefore, elevated InsP(6) in activated neurons serves as a unique negative feedback signal to control hippocampal excitatory neurotransmission.     

Adaptative bile duct proliferative response in experimental bile duct ischemia

BACKGROUND/AIMS: A rat model of bile duct ischemia was established and used to examine the potential of bile duct proliferation to provide an adaptative response in cholestatic disorders. METHODS: Rats underwent partial or complete arterial deprivation of the liver. Serum biochemical tests, histological analyses and bile secretion measurements were performed at different time points up to 6 weeks after surgery. RESULTS: Rats developed biochemical signs of cholestasis exclusively after complete arterial deprivation. Within 4h, cholangiocytes in these rats showed morphological signs of cell damage. After 48h, they displayed VEGF expression and became proliferative. The proportion of Ki67-labeled cholangiocytes ( approximately 30%) was similar in interlobular bile ducts and periportal ductules. A ductular reaction made of well-formed bile ducts confined to portal tracts developed within 1 week. Bile flow which was initially decreased, was restored at 3 weeks, while the biochemical signs of cholestasis completely resolved at 6 weeks. At this time, the number of bile duct sections was maximal. Fibrosis intensity was also maximal, although moderate (相似文献   

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca²⁺ signals can affect excitability and neural circuit formation. Ca²⁺ signals are modified by Ca²⁺ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca²⁺ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA depletion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca²⁺ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP₃R) can partially compensate for their loss of flight. Ca²⁺ measurements show that Orai gain-of-function contributes to the quanta of Ca²⁺-release through mutant InsP₃Rs and elevates store-operated Ca²⁺ entry in Drosophila neurons. Our data show that replenishment of intracellular store Ca²⁺ in neurons is required for Drosophila flight.     

Attenuation of store-operated Ca2+ current impairs salivary gland fluid secretion in TRPC1(-/-) mice

Agonist-induced Ca(2+) entry via store-operated Ca(2+) (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(-/-) mice was severely decreased (by 70%). Further, agonist- and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(-/-) mice. Deletion of TRPC1 also eliminated sustained Ca(2+)-dependent potassium channel activity, which depends on Ca(2+) entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca(2+) signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(-/-) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion.     

Histamine-induced Ca(2+) signaling in human valvular myofibroblasts

In this study, we examined histamine-induced calcium signaling in cultured human valvular myofibroblasts (hVMFs), which are the most prominent interstitial cells in cardiac valves mediating valvular contraction, extracellular matrix secretion, and wound repair. Despite the functional importance of VMFs in cardiac valves, the cellular-signaling pathways, especially those mediated by Ca(2+), are still poorly understood. Using fluorescence imaging microscopy, we measured intracellular Ca(2+) ([Ca(2+)](i)) levels in fura-2-loaded hVMFs. Activation of H(1) receptors released Ca(2+) from one compartment of the endoplasmic reticulum (ER) of hVMFs, but did not induce Ca(2+) entry. This histamine-induced Ca(2+) release was oscillatory and dependent on Ca(2+) re-uptake into the ER by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Application of the reversible SERCA blocker, cyclopiazonic acid (CPA), after depletion of the histamine-sensitive Ca(2+) store revealed the presence of a second, smaller histamine-insensitive Ca(2+) store in the ER. The Ca(2+) content ratio of the histamine-sensitive and histamine-insensitive Ca(2+) stores in the ER was found to be approximately 1.15:1. Another effect of CPA in hVMFs was the activation of store-operated Ca(2+) channels, as demonstrated by maintained [Ca(2+)](i) elevation as well as accelerated Mn(2+) entry. In conclusion, this study establishes for the first time an agonist-induced Ca(2+)-signaling pathway in hVMFs.     

Osteoblasts induce Ca2+ oscillation-independent NFATc1 activation during osteoclastogenesis

Intercellular cross-talk between osteoblasts and osteoclasts is important for controlling bone remolding and maintenance. However, the precise molecular mechanism by which osteoblasts regulate osteoclastogenesis is still largely unknown. Here, we show that osteoblasts can induce Ca(2+) oscillation-independent osteoclastogenesis. We found that bone marrow-derived monocyte/macrophage precursor cells (BMMs) lacking inositol 1,4,5-trisphosphate receptor type2 (IP(3)R2) did not exhibit Ca(2+) oscillation or differentiation into multinuclear osteoclasts in response to recombinant receptor activator of NF-kappaB ligand/macrophage colony-stimulating factor stimulation. IP(3)R2 knockout BMMs, however, underwent osteoclastogenesis when they were cocultured with osteoblasts or in vivo in the absence of Ca(2+) oscillation. Furthermore, we found that Ca(2+) oscillation-independent osteoclastogenesis was insensitive to FK506, a calcineurin inhibitor. Taken together, we conclude that both Ca(2+) oscillation/calcineurin-dependent and -independent signaling pathways contribute to NFATc1 activation, leading to efficient osteoclastogenesis in vivo.     

Cholestasis: human disease and experimental animal models

Cholestasis may result from a failure in bile secretion in hepatocytes or ductular cells, or from a blockade to the free bile flow. Human cholestasis may be induced by many drugs, being antibiotics the more common. Other types of cholestasis seen in humans are a group of familial cholestatic disorders, obstructive cholestasis, primary biliary cirrhosis, extrahepatic biliary atresia, primary sclerosing cholangitis, cholestasis of pregnancy, oral contraceptive-induced cholestasis, and sepsis-induced cholestasis. Experimental animal models allow the understanding of pathophysiological mechanisms involved and their clinical correlates. The most common experimental models of intrahepatic cholestasis are estrogen-induced, endotoxin-induced and drug-induced cholestasis. A well known model of extrahepatic biliary obstruction is common bile duct ligation. Drug-induced cholestasis were described using different drugs. On this regard, alpha naphthylisothiocyanate treatment has been extensively used, permitting to describe not only cholestatic alterations but also compensatory mechanisms. Congenital defficiency of transport proteins also were studied in natural rat models of cholestasis. The experimental animal models allow to define down-regulated alterations of hepatocyte transport proteins, and up-regulated ones acting as compensatory mechanisms. In conclusion, animal model and transport protein studies are necessary for the progressive understanding of congenital and acquired human cholestasis, and regulatory mechanisms that operate on liver cells.     

Ion sensing in the RCK1 domain of BK channels

BK-type K(+) channels are activated by voltage and intracellular Ca(2+), which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different Ca(2+) binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this Ca(2+) binding site. This site is specific for binding to Ca(2+) but not Cd(2+). Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of Ca(2+) by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how Ca(2+) binding at this site opens the activation gate of the channel that is remotely located in the membrane.