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Excess consumption of carbohydrates, fat, and calories leads to non-alcoholic fatty liver disease (NAFLD) and hepatic insulin resistance; major factors in the pathogenesis of type II diabetes. Hormones and catecholamines acting through G-protein coupled receptors (GPCRs) linked to phospholipase C (PLC) and increases in cytosolic Ca2+ ([Ca2+]c) regulate many metabolic functions of the liver. In the intact liver, catabolic hormones such as glucagon, catecholamines and vasopressin integrate and synergize to regulate the frequency and extent to which [Ca2+]c waves propagate across hepatic lobules to control metabolism. Dysregulation of hepatic Ca2+ homeostasis has been implicated in the development of metabolic disease, but changes in hepatic GPCR-dependent Ca2+ signalling have been largely unexplored in this context. We show that short-term, 1-week, high fat diet (HFD) feeding of mice attenuates norepinephrine-stimulated Ca2+ signalling, reducing the number of cells responding and suppressing the frequency of [Ca2+]c oscillations in both isolated hepatocytes and intact liver. The 1-week HFD feeding paradigm did not change basal Ca2+ homeostasis; endoplasmic reticulum Ca2+ load, store-operated Ca2+ entry and plasma membrane Ca2+ pump activity were unchanged compared to low fat diet (LFD) fed controls. However, norepinephrine-induced IP3 production was significantly reduced after HFD feeding, demonstrating an effect of HFD on receptor-stimulated PLC activity. Thus, we have identified a lesion in the PLC signalling pathway induced by short-term HFD feeding, which interferes with hormonal Ca2+ signalling in isolated hepatocytes and the intact liver. These early events may drive adaptive changes in signalling, which lead to pathological consequences in fatty liver disease.Key points summaryNon-alcoholic fatty liver disease (NAFLD) is a growing epidemic.In healthy liver, the counteracting effects of catabolic and anabolic hormones regulate metabolism and energy storage as fat. Hormones and catecholamines promote catabolic metabolism via increases in cytosolic Ca2+ ([Ca2+]c).We show that 1 week high fat diet (HFD) feeding of mice attenuated the Ca2+ signals induced by physiological concentrations of norepinephrine. Specifically, HFD suppressed the normal pattern of periodic [Ca2+]c oscillations in isolated hepatocytes and disrupted the propagation of intralobular [Ca2+]c waves in the intact perfused liver.Short-term HFD inhibited norepinephrine-induced inositol 1,4,5-trisphosphate (IP3) generation, but did not change basal endoplasmic reticulum Ca2+ load or plasma membrane Ca2+ fluxes.We propose that impaired Ca2+ signalling plays a key role in the earliest phases of the etiology of NAFLD, and is responsible for many of the ensuing metabolic and related dysfunctional outcomes at the cellular and whole tissue level.

Other grants supported: Programa de Apoio ao Desenvolvimento Científico da Faculdade de Ciências Farmacêuticas da UNESP (PADC); and funding from the Thomas P. Infusino Endowment at Rutgers University. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-finance code 001 (I.A.P.L., T.G.P., K.B.I., and J.M.V.). M.A.R.B., A.V.G.N., and M.A.S.G. are recipients of a Research Productivity Scholarship from the National Council for Research and Development (CNPq). Submitted by Angela Maria Arenas Velasquez (a.velasquez@unesp.br) on 2022-05-04T17:54:05Z No. of bitstreams: 1 Velasquez_2022_AAC.00767-21.pdf: 2923911 bytes, checksum: 21833fd4c858b3b80135c83c2c090390 (MD5) Approved for entry into archive by Maria Irani Coito (irani@fcfar.unesp.br) on 2022-05-17T22:52:55Z (GMT) No. of bitstreams: 1 Supplemental Material_Velasquez_2022_AAC.00767-21.pdf: 2923911 bytes, checksum: 21833fd4c858b3b80135c83c2c090390 (MD5) Made available in DSpace on 2022-05-17T22:52:55Z (GMT). No. of bitstreams: 1 Supplemental Material_Velasquez_2022_AAC.00767-21.pdf: 2923911 bytes, checksum: 21833fd4c858b3b80135c83c2c090390 (MD5) Previous issue date: 2022-01-18 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The current treatment of leishmaniasis is based on a few drugs that present several drawbacks, such as high toxicity, difficult administration route, and low efficacy. These disadvantages raise the necessity to develop novel antileishmanial compounds allied with a comprehensive understanding of their mechanisms of action. Here, we elucidate the probable mechanism of action of the antileishmanial binuclear cyclopalladated complex [Pd(dmba)(m-N3)]2 (CP2) in Leishmania amazonensis. CP2 causes oxidative stress in the parasite, resulting in disruption of mitochondrial Ca2+ homeostasis, cell cycle arrest at the S-phase, increasing the reactive oxygen species (ROS) production and overexpression of stress-related and cell detoxification proteins, and collapsing the Leishmania mitochondrial membrane potential, and promotes apoptotic-like features in promastigotes, leading to necrosis, or directs programmed cell death (PCD)-committed cells toward necrotic-like destruction. Moreover, CP2 reduces the parasite load in both liver and spleen in Leishmania infantum-infected hamsters when treated for 15 days with 1.5 mg/kg body weight/day CP2, expanding its potential application in addition to the already known effectiveness on cutaneous leishmaniasis for the treatment of visceral leishmaniasis, showing the broad spectrum of action of this cyclopalladated complex. The data presented here bring new insights into the CP2 molecular mechanisms of action, assisting the promotion of its rational modification to improve both safety and efficacy. São Paulo State University (Unesp), School of Pharmaceutical Sciences, Araraquara, São Paulo, Brazil Department of Pharmacology, Physiology, and Neuroscience, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, New Jersey, USA Department of Chemistry, São Carlos Institute of Chemistry–IQSC, University of São Paulo (USP), São Carlos, São Paulo, Brazil Department of Cellular and Molecular Biology and Pathogenic Bioagents, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil Laboratory of Biochemistry, Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo (USP), Bauru, São Paulo, Brazil São Paulo State University (Unesp), Institute of Chemistry, Araraquara, São Paulo, Brazil 2016/05345-4 2016/177115 2017/03552-5 2018/23015-7 2016/19289-9 2016/18191-5 2019/21661-1 2020/04415-4

Summary Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists., Graphical abstract, Highlights • Distinct stereotypic Ca2+ oscillations are elicited by P2Y1 and P2Y2 receptors • P2X receptors do not contribute to the generation of Ca2+ oscillations • Agonist-specific Ca2+ spike shapes reflect discrete modes of PKC negative feedback • Bifurcation of IP3/PKC signaling yields unique Ca2+ oscillation signatures, Biological sciences; Cell biology; Cellular physiology; Functional aspects of cell biology

Background: Leishmaniasis is a parasitic disease that affects millions of people and lacks new molecules for treatment. The current treatment is associated with high toxicity, a difficult administration route, and the emergence of resistant strains. These disadvantages raise the necessity to develop novel compounds with effective leishmanicidal activity and elucidate their action mechanism. Methods: To provide new information about of mechanism of action of the binuclear cyclopalladated complex [Pd(dmba)(µ-N3)]2 (CP2) in Leishmania, we have utilized two-dimensional SDS-PAGE for proteomic analyses, analyses of the progression of the cell cycle by flow cytometry. The measurement of reactive oxygen species (ROS) and mitochondrial membrane potential by fluorescence, live-cell Fura-2 Ca2+ imaging, and a dual acridine orange/ethidium bromide (AO/EB) staining method for the cell death mechanism. In vivo leishmanicidal activity has been performed in a golden hamster model. Findings: Dysregulation of parasite Ca2+ levels coming mainly from mitochondria, increasing ROS production and collapsing the Leishmania mitochondrial membrane potential. The presence of CP2 promotes apoptotic-like features in promastigotes six hours post-CP2-treatment. Exposure of parasites for 24 h or 48 h to CP2 leads to necrosis or directs programmed cell death (PCD)-committed cells toward necrotic-like destruction of cells. Besides, CP2 leads to arrests of cell cycle progression at S-phase, even in the absence of ROS production, and increases the expression of stress-related proteins, including calreticulin, Heat shock 10 kDa protein 1 (Hsp10), Hsp70, GRP78/BiP, and protein disulfide-isomerase (PDI) and cell detoxification proteins (trypanothione reductase, peroxiredoxin, tryparedoxin peroxidase). These studies provide new insight into the mechanism of action of CP2. When Leishmania infantum-infected Golden hamsters were treated for 15 days with 1·5 mg/Kg/day, CP2 caused a reduction in the parasite load of both liver and spleen by ~50%, which is similar to the leishmanicidal activity of amphotericin B. Interpretation: Our finding established the ability of CP2 to cause oxidative stress with disruption of mitochondrial Ca 2+ homeostasis of Leishmania, leading to other effects observed downstream to CP2-treatment. This new insight into the cyclopalladated complex's mechanism of action may have wider therapeutic applications targeting the parasite's mitochondria since structural modification to improve the leishmanicidal effect of CP2 may offer an alternative therapeutic agent for this neglected disease. Funding Information: This work was supported by the Sao Paulo Research Foundation (FAPESP) grants: #2016/05345-4, #2016/177115, #2017/03552-5 and #2018/23015-7; Programa de Apoio ao Desenvolvimento Cientifico da Faculdade de Ciencias Farmaceuticas da UNESP (PADC); and Funding from the Thomas P. Infusino Endowment at Rutgers University. AMAV (#2016/19289-9 and #2019/21661-1) and SV (#2016/18191-5) were supported by FAPESP. This study was financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior - Brasil (CAPES) - Finance Code 001, IAPL, TGP, KBI, JMV. MARB, AVGN and MASG are recipienu of a Research Productivity Scholarship from the National Council for Research and Development (CNPq). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The Ethics Committee approved this study for Animal Experimentation of Sao Paulo State University (UNESP), the School of Pharmaceutical Sciences (CEUA/FCF/CAr, 18/2015 and 44/2015) in agreement with the guidelines of Sociedade Brasileira de Ciencia de Animais de Laboratorio (SBCAL) and Conselho Nacional de Controle da Experimentacao Animal (CONCEA).

Receptor-coupled phospholipase C (PLC) is an important target for the actions of ethanol. In the ex vivo perfused rat liver, concentrations of ethanol >100 mM were required to induce a rise in cytosolic calcium (Ca2+) suggesting that these responses may only occur after binge ethanol consumption. Conversely, pharmacologically achievable concentrations of ethanol (≤30 mM) decreased the frequency and magnitude of hormone-stimulated cytosolic and nuclear Ca2+ oscillations and the parallel translocation of protein kinase C-β to the membrane. Ethanol also inhibited gap junction communication resulting in the loss of coordinated and spatially organized intercellular Ca2+ waves in hepatic lobules. Increasing the hormone concentration overcame the effects of ethanol on the frequency of Ca2+ oscillations and amplitude of the individual Ca2+ transients; however, the Ca2+ responses in the intact liver remained disorganized at the intercellular level, suggesting that gap junctions were still inhibited. Pretreating hepatocytes with an alcohol dehydrogenase inhibitor suppressed the effects of ethanol on hormone-induced Ca2+ increases, whereas inhibiting aldehyde dehydrogenase potentiated the inhibitory actions of ethanol, suggesting that acetaldehyde is the underlying mediator. Acute ethanol intoxication inhibited the rate of rise and the magnitude of hormone-stimulated production of inositol 1,4,5-trisphosphate (IP3), but had no effect on the size of Ca2+ spikes induced by photolysis of caged IP3. These findings suggest that ethanol inhibits PLC activity, but does not affect IP3 receptor function. We propose that by suppressing hormone-stimulated PLC activity, ethanol interferes with the dynamic modulation of [IP3] that is required to generate large, amplitude Ca2+ oscillations.

Calcium (Ca2+) oscillations in hepatocytes have a wide dynamic range. In particular, recent experimental evidence shows that agonist stimulation of the P2Y family of receptors leads to qualitatively diverse Ca2+ oscillations. We present a new model of Ca2+ oscillations in hepatocytes based on these experiments to investigate the mechanisms controlling P2Y-activated Ca2+ oscillations. The model accounts for Ca2+ regulation of the IP3 receptor (IP3R), the positive feedback from Ca2+ on phospholipase C (PLC) and the P2Y receptor phosphorylation by protein kinase C (PKC). Furthermore, PKC is shown to control multiple cellular substrates. Utilising the model, we suggest the activity and intensity of PLC and PKC necessary to explain the qualitatively diverse Ca2+ oscillations in response to P2Y receptor activation.

Extracellular nucleotides are key regulators of liver physiology. In primary rat hepatocytes, P2Y1 receptor (P2Y1R) activation by ADP generates cytosolic calcium ([Ca2+]c) oscillations with narrow spikes, whereas P2Y2/4R activation by UTP led to more complex broad [Ca2+]coscillations. Both [Ca2+]coscillation signatures were observed with the common agonist ATP. Inhibition of Gαqsignaling with YM-254890 abolished ATP-induced [Ca2+]coscillations, indicating that they depend on inositol 1,4,5-trisphosphate (IP3), and are not mediated by P2X receptors. The narrow P2Y1-linked [Ca2+]cspikes and the broad P2Y2/4-linked [Ca2+]cspikes are shaped by differential and complex PKC-mediated feedback mechanisms. Downregulation of PKC broadened both ADP- and UTP-induced [Ca2+]coscillations, with a more pronounced effect on the former. PKC downregulation also selectively elicited a more robust response to ADP stimulation, enhancing oscillatory and sustained [Ca2+]cresponses. Acute PKC modulation confirmed the importance of the negative PKC feedback regulation of P2Y1R-linked [Ca2+]csignals; such that PKC activation decreased [Ca2+]coscillation frequency and PKC inhibition increased [Ca2+]cspike width. However, both PKC activation and inhibition decreased the spike width of P2Y2/4R-induced [Ca2+]coscillations, suggesting that multiple opposing PKC feedback mechanisms shape P2Y2/4R responses. Significantly, plasma membrane Ca2+entry was required for negative PKC feedback on P2Y1R-linked [Ca2+]coscillations, whereas P2Y2/4R-linked [Ca2+]coscillations were less sensitive to negative regulation by PKC and independent of Ca2+influx. Thus, differential feedback regulation by PKC gives rise to receptor-specific [Ca2+]coscillation profiles, which can encode the diverse physiological and pathophysiological responses to distinct agonists that all act through the IP3signaling cascade.

Calcium (Ca

Cytosolic Ca(2+) regulates multiple steps in the host-cell invasion, growth, proliferation, and egress of blood-stage Plasmodium falciparum, yet our understanding of Ca(2+) signaling in this endemic malaria parasite is incomplete. By using a newly generated transgenic line of P. falciparum (PfGCaMP3) that expresses constitutively the genetically encoded Ca(2+) indicator GCaMP3, we have investigated the dynamics of Ca(2+) release and influx elicited by inhibitors of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps, cyclopiazonic acid (CPA), and thapsigargin (Thg). Here we show that in isolated trophozoite phase parasites: (i) both CPA and Thg release Ca(2+) from intracellular stores in P. falciparum parasites; (ii) Thg is able to induce Ca(2+) release from an intracellular compartment insensitive to CPA; (iii) only Thg is able to activate Ca(2+) influx from extracellular media, through a mechanism resembling store-operated Ca(2+) entry, typical of mammalian cells; and (iv) the Thg-sensitive Ca(2+) pool is unaffected by collapsing the mitochondria membrane potential with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone or the release of acidic Ca(2+) stores with nigericin. These data suggest the presence of two Ca(2+) pools in P. falciparum with differential sensitivity to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pump inhibitors, and only the release of the Thg-sensitive Ca(2+) store induces Ca(2+) influx. Activation of the store-operated Ca(2+) entry–like Ca(2+) influx may be relevant for controlling processes such as parasite invasion, egress, and development mediated by kinases, phosphatases, and proteases that rely on Ca(2+) levels for their activation.

Summary Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists.

Key points Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca2+-mobilizing hormones resulting in a leftward shift in the concentration–response relationship and the transition from oscillatory to more sustained and prolonged Ca2+ increases. Our data demonstrate that alcohol-dependent adaptation in the Ca2+ signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP3) production and does not involve changes in the sensitivity of the IP3 receptor or size of internal Ca2+ stores. We suggest that prolonged and aberrant hormone-evoked Ca2+ increases may stimulate the production of mitochondrial reactive oxygen species and contribute to alcohol-induced hepatocyte injury. Abstract ‘Adaptive’ responses of the liver to chronic alcohol consumption may underlie the development of cell and tissue injury. Alcohol administration can perturb multiple signalling pathways including phosphoinositide-dependent cytosolic calcium ([Ca2+]i) increases, which can adversely affect mitochondrial Ca2+ levels, reactive oxygen species production and energy metabolism. Our data indicate that chronic alcohol feeding induces a leftward shift in the dose–response for Ca2+-mobilizing hormones resulting in more sustained and prolonged [Ca2+]i increases in both cultured hepatocytes and hepatocytes within the intact perfused liver. Ca2+ increases were initiated at lower hormone concentrations, and intercellular calcium wave propagation rates were faster in alcoholics compared to controls. Acute alcohol treatment (25 mm) completely inhibited hormone-induced calcium increases in control livers, but not after chronic alcohol-feeding, suggesting desensitization to the inhibitory actions of ethanol. Hormone-induced inositol 1,4,5 trisphosphate (IP3) accumulation and phospholipase C (PLC) activity were significantly potentiated in hepatocytes from alcohol-fed rats compared to controls. Removal of extracellular calcium, or chelation of intracellular calcium did not normalize the differences in hormone-stimulated PLC activity, indicating calcium-dependent PLCs are not upregulated by alcohol. We propose that the liver ‘adapts’ to chronic alcohol exposure by increasing hormone-dependent IP3 formation, leading to aberrant calcium increases, which may contribute to hepatocyte injury.

IP3-dependent Ca2+ oscillations have been described in different cell types and ascribed to either biphasic Ca2+ regulation of the IP3 receptor (IP3R), or requiring feedback mechanisms controlling IP3 levels. IP3 uncaging in hepatocytes elicits Ca2+ transients that are often localized at the subcellular level, and increase in magnitude with stimulus-strength. However, this does not reproduce the broad baseline-separated global Ca2+ oscillations elicited by vasopressin. Addition of hormone to cells activated by IP3 uncaging initiates a qualitative transition from high frequency spatially disorganized Ca2+ transients, to low frequency, oscillatory Ca2+ waves that propagate throughout the cell. A mathematical model with dual coupled oscillators that integrate Ca2+-induced Ca2+ release at the IP(sub>3R, and feedback mechanisms of cross-coupling between Ca2+ and IP3 reproduces this behavior. Thus, multiple Ca2+ oscillation models can functionally coexist in the same cell, and hormonal stimulation can switch from the simpler to the more complex to yield robust signaling.

Calcium (Ca2+) oscillations in hepatocytes control many critical cellular functions, including glucose metabolism and bile secretion. The mechanisms underlying repetitive Ca2+ oscillations and how these mechanisms regulate these oscillations is not fully understood. Recent experimental evidence has shown that both Ca2+ regulation of the inositol 1,4,5-trisphosphate (IP3) receptor and IP3 metabolism generate Ca2+ oscillations and co-exist in hepatocytes. To investigate the effects of these feedback mechanisms on the Ca2+ response, we construct a mathematical model of the Ca2+ signalling network in hepatocytes. The model accounts for the biphasic regulation of Ca2+ on the IP3 receptor (IP3R) and the positive feedback from Ca2+ on IP3 metabolism, via activation of phospholipase C (PLC) by agonist and Ca2+. Model simulations show that Ca2+ oscillations exist for both constant [IP3] and for [IP3] changing dynamically. We show, both experimentally and in the model, that as agonist concentration increases, Ca2+ oscillations transition between simple narrow-spike oscillations and complex broad-spike oscillations. The model predicts that narrow-spike oscillations persist when Ca2+ transport across the plasma membrane is blocked. This prediction has been experimentally validated. In contrast, broad-spike oscillations are terminated when plasma membrane transport is blocked. We conclude that multiple feedback mechanisms participate in regulating Ca2+ oscillations in hepatocytes.

Background Phosphoinositides (PIs) and their derivatives are essential cellular components that form the building blocks for cell membranes and regulate numerous cell functions. Specifically, the ability to generate myo-inositol 1,4,5-trisphosphate (InsP3) via phospholipase C (PLC) dependent hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to InsP3 and diacylglycerol (DAG) initiates intracellular calcium signaling events representing a fundamental signaling mechanism dependent on PIs. InsP3 produced by PI turnover as a second messenger causes intracellular calcium release, especially from endoplasmic reticulum, by binding to the InsP3 receptor (InsP3R). Various PIs and the enzymes, such as phosphatidylinositol synthase and phosphatidylinositol 4-kinase, necessary for their turnover have been characterized in Apicomplexa, a large phylum of mostly commensal organisms that also includes several clinically relevant parasites. However, InsP3Rs have not been identified in genomes of apicomplexans, despite evidence that these parasites produce InsP3 that mediates intracellular Ca2+ signaling. Conclusion Evidence to supporting IP3-dependent signaling cascades in apicomplexans suggests that they may harbor a primitive or non-canonical InsP3R. Understanding these pathways may be informative about early branching eukaryotes, where such signaling pathways also diverge from animal systems, thus identifying potential novel and essential targets for therapeutic intervention.

Acidocalcisomes are acidic calcium stores rich in polyphosphate and found in a diverse range of organisms. The mechanism of Ca 2+ release from these organelles was unknown. Here we present evidence that Trypanosoma brucei acidocalcisomes possess an inositol 1,4,5-trisphosphate receptor (TbIP 3 R) for Ca 2+ release. Localization studies in cell lines expressing TbIP 3 R in its endogenous locus fused to an epitope tag revealed its partial colocalization with the vacuolar proton pyrophosphatase, a marker of acidocalcisomes. IP 3 was able to stimulate Ca 2+ release from a chicken B-lymphocyte cell line in which the genes for all three vertebrate IP 3 Rs have been stably ablated (DT40-3KO) and that were stably expressing TbIP 3 R , providing evidence of its function. IP 3 was also able to release Ca 2+ from permeabilized trypanosomes or isolated acidocalcisomes and photolytic release of IP 3 in intact trypanosomes loaded with Fluo-4 elicited a transient Ca 2+ increase in their cytosol. Ablation of TbIP 3 R by RNA interference caused a significant reduction of IP 3 -mediated Ca 2+ release in trypanosomes and resulted in defects in growth in culture and infectivity in mice. Taken together, the data provide evidence of the presence of a functional IP 3 R as a Ca 2+ release channel in acidocalcisomes of trypanosomes and suggest that a Ca 2+ signaling pathway that involves acidocalcisomes is required for growth and establishment of infection.

Chronic alcohol consumption causes a spectrum of liver diseases, but the pathogenic mechanisms driving the onset and progression of disease are not clearly defined. We show that chronic alcohol feeding sensitizes rat hepatocytes to Ca

How Ca(2+) oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca(2+) oscillations report signal strength via frequency, whereas Ca(2+) spike amplitude and wave velocity remain constant. IP3 uncaging also triggers oscillatory Ca(2+) release, but, in contrast to hormones, Ca(2+) spike amplitude, width, and wave velocity were dependent on [IP3] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP3 uncaging are driven by the biphasic regulation of the IP3 receptor by Ca(2+), and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca(2+) did not perturb Ca(2+) oscillations elicited by IP3 uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca(2+) influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca(2+) oscillations but had no effect on Ca(2+) increases induced by uncaging IP3. Importantly, PKC activation and inhibition differentially affected Ca(2+) spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP3 metabolism to attenuate IP3 levels and suppress the generation of Ca(2+) oscillations. Inhibition of PKC relieves negative feedback regulation of IP3 accumulation and, thereby, shifts Ca(2+) oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca(2+) wave velocity, whereas responses to IP3 uncaging are enhanced. The ability to assess Ca(2+) responses in the absence of PLC activity indicates that IP3 receptor modulation by PKC regulates Ca(2+) release and wave velocity.

The magnitude and temporal nature of intracellular signaling cascades can now be visualized directly in single cells by the use of protein domains tagged with enhanced green fluorescent protein (eGFP). In this study, signaling downstream of G protein-coupled receptor-mediated phospholipase C (PLC) activation has been investigated in a cell line coexpressing recombinant M(3) muscarinic acetylcholine and alpha(1B) -adrenergic receptors. Confocal measurements of changes in inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)), using the pleckstrin homology domain of PLCdelta1 tagged to eGFP (eGFP-PH(PLCdelta)), and 1,2-diacylglycerol (DAG), using the C1 domain of protein kinase Cgamma (PKCgamma) (eGFP-C1(2)-PKCgamma), demonstrated clear translocation responses to methacholine and noradrenaline. Single cell EC(50) values calculated for each agonist indicated that responses to downstream signaling targets (Ca(2+) mobilization and PKC activation) were approximately 10-fold lower compared with respective Ins(1,4,5)P(3) and DAG EC(50) values. Examining the temporal profile of second messenger responses to sub-EC(50) concentrations of noradrenaline revealed oscillatory Ins(1,4,5)P(3), DAG, and Ca(2+) responses. Oscillatory recruitments of conventional (PKCbetaII) and novel (PKCepsilon) PKC isoenzymes were also observed which were synchronous with the Ca(2+) response measured simultaneously in the same cell. However, oscillatory PKC activity (as determined by translocation of eGFP-tagged myristoylated alanine-rich C kinase substrate protein) required oscillatory DAG production. We suggest a model that uses regenerative Ca(2+) release via Ins(1,4,5)P(3) receptors to initiate oscillatory second messenger production through a positive feedback effect on PLC. By acting on various components of the PLC signaling pathway the frequency-encoded Ca(2+) response is able to maintain signal specificity at a level downstream of PKC activation.

A major role of the liver is to integrate multiple signals to maintain normal blood glucose levels. The balance between glucose storage and mobilization is primarily regulated by the counteracting effects of insulin and glucagon. However, numerous signals converge in the liver to ensure energy demand matches the physiological status of the organism. Many circulating hormones regulate glycogenolysis, gluconeogenesis and mitochondrial metabolism by calcium-dependent signaling mechanisms that manifest as cytosolic Ca(2+) oscillations. Stimulus-strength is encoded in the Ca(2+) oscillation frequency, and also by the range of intercellular Ca(2+) wave propagation in the intact liver. In this article, we describe how Ca(2+) oscillations and waves can regulate glucose output and oxidative metabolism in the intact liver; how multiple stimuli are decoded though Ca(2+) signaling at the organ level, and the implications of Ca(2+) signal dysregulation in diseases such as metabolic syndrome and non-alcoholic fatty liver disease.

Circulating hormones stimulate the phospholipase Cβ (PLC)/Ca(2+) influx pathway to regulate numerous cell functions, including vascular tone. It was proposed previously that Ca(2+)-independent phospholipase A(2) (iPLA(2))-dependent store-operated Ca(2+) influx channels mediate hormone-induced contractions in isolated arteries, because bromoenol lactone (BEL), a potent irreversible inhibitor of iPLA(2), inhibited such contractions. However, the effects of BEL on other channels implicated in mediating hormone-induced vessel contractions, specifically voltage-gated Ca(2+) (Ca(V)1.2) and transient receptor potential canonical (TRPC) channels, have not been defined clearly. Using isometric tension measurements, we found that thapsigargin-induced contractions were ∼34% of those evoked by phenylephrine or KCl. BEL completely inhibited not only thapsigargin- but also phenylephrine- and KCl-induced ring contractions, suggesting that Ca(V)1.2 and receptor-operated TRPC channels also may be sensitive to BEL. Therefore, we investigated the effects of BEL on heterologously expressed Ca(V)1.2 and TRPC channels in human embryonic kidney cells, a model system that allows probing of individual protein function without interference from other signaling elements of native cells. We found that low micromolar concentrations of BEL inhibited Ca(V)1.2, TRPC5, TRPC6, and heteromeric TRPC1-TRPC5 channels in an iPLA(2)-independent manner. BEL also attenuated PLC activity, suggesting that the compound may inhibit TRPC channel activity in part by interfering with an initial PLC-dependent step required for TRPC channel activation. Conversely, BEL did not affect endogenous voltage-gated K(+) channels in human embryonic kidney cells. Our findings support the hypothesis that iPLA(2)-dependent store-operated Ca(2+) influx channels and iPLA(2)-independent hormone-operated TRPC channels can serve as smooth muscle depolarization triggers to activate Ca(V)1.2 channels and to regulate vascular tone.

Activation of sphingosine kinase (SPHK), thereby increasing cellular levels of sphingosine 1-phosphate (S1P), may be involved in a variety of intracellular responses including Ca 2+ signaling. This study uses mammalian SPHK1a, tagged with enhanced green fluorescent protein (eGFP), to examine whether translocation of this enzyme is linked with Ca 2+ -mobilizing responses. Real-time confocal imaging of SPHK1a-eGFP in human SH-SY5Y neuroblastoma cells visualized a relocation of the enzyme from the cytosol to the plasma membrane in response to Ca 2+ -mobilizing stimuli (muscarinic M 3 - or lysophosphatidic acid receptor activation, and thapsigargin-mediated store release). This redistribution was preceded by a transient increase in cytosolic SPHK1a-eGFP levels due to liberation of SPHK from localized higher intensity regions. Translocation was dependent on Ca 2+ mobilization from intracellular stores, and was prevented by pretreatment with the Ca 2+ /calmodulin inhibitor W-7, but not W-5 or KN-62. In functional studies, pretreatment with W-7 lowered basal and M 3 -receptor-mediated cellular S1P production. However, this pretreatment did not alter agonist-mediated Ca 2+ responses, and SPHK1a-eGFP activity itself appeared insensitive to Ca 2+ /calmodulin and W-7. These data suggest a role for Ca 2+ /calmodulin in controlling the subcellular distribution but not the activity of SPHK1a.

SUMMARY Receptor-mediated oscillations in cytosolic Ca2+ concentration ([Ca2+]i) could originate either directly from an autonomous Ca2+ feedback oscillator at the inositol 1,4,5-trisphosphate (IP3) receptor or as a secondary consequence of IP3 oscillations driven by Ca2+ feedback on IP3 metabolism. It is challenging to discriminate these alternatives, because IP3 fluctuations could drive Ca2+ oscillations or could just be a secondary response to the [Ca2+]i spikes. To investigate this problem, we constructed a recombinant IP3 buffer using type-I IP3 receptor ligand-binding domain fused to GFP (GFP-LBD), which buffers IP3 in the physiological range. This IP3 buffer slows hormone-induced [IP3] dynamics without changing steady-state [IP3]. GFP-LBD perturbed [Ca2+]i oscillations in a dose-dependent manner: it decreased both the rate of [Ca2+]i rise and the speed of Ca2+ wave propagation and, at high levels, abolished [Ca2+]i oscillations completely. These data, together with computational modeling, demonstrate that IP3 dynamics play a fundamental role in generating [Ca2+]i oscillations and waves., Graphical Abstract, In Brief Gaspers et al. use a genetically encoded IP3 buffer to suppress IP3 dynamics during hormonal stimulation. Using this approach, they find that positive feedback of Ca2+ on IP3 formation is an essential component, generating long-period, baseline-separated Ca2+ oscillations and intracellular Ca2+ waves.