The binuclear cyclopalladated complex CP2 is targeting ubiquinol-cytochrome c reductase (complex III) of Leishmania amazonensis.

Leishmaniasis is a neglected disease that remains with a limited number of drugs available for chemotherapy and has an increased drug resistance that affects treatment outcomes. Metal-based drugs such as cyclopalladated complex [Pd(dmba)(μ-N)] (CP2), a Leishmania topoisomerase IB inhibitor involved in calcium dysregulation and mitochondrial dysfunction of the parasite, had been an alternative to outline the appearance of chemoresistance. To identify new molecular targets and point out possible resistance mechanisms, a CP2-resistant Leishmania amazonensis (LaR) was selected by stepwise exposure to increasing drug pressure until a line capable of growth in 13.

Receptor-specific Ca oscillation patterns mediated by differential regulation of P2Y purinergic receptors in rat hepatocytes.

Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP) formation elicit cytosolic Ca oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca 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 Ca oscillations, whereas UTP acting through P2Y2R elicits broad Ca oscillations, with composite patterns observed for ATP.

Ethanol Disrupts Hormone-Induced Calcium Signaling in Liver.

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 (Ca) 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 Ca oscillations and the parallel translocation of protein kinase C-β to the membrane.

Intercellular calcium waves integrate hormonal control of glucose output in the intact liver.

Key Points: Sympathetic outflow and circulating glucogenic hormones both regulate liver function by increasing cytosolic calcium, although how these calcium signals are integrated at the tissue level is currently unknown. We show that stimulation of hepatic nerve fibres or perfusing the liver with physiological concentrations of vasopressin only will evoke localized cytosolic calcium oscillations and modest increases in hepatic glucose production. The combination of these stimuli acted synergistically to convert localized and asynchronous calcium responses into co-ordinated intercellular calcium waves that spread throughout the liver lobule and elicited a synergistic increase in hepatic glucose production.

Chronic alcohol feeding potentiates hormone-induced calcium signalling in hepatocytes.

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 -mobilizing hormones resulting in a leftward shift in the concentration-response relationship and the transition from oscillatory to more sustained and prolonged Ca increases. Our data demonstrate that alcohol-dependent adaptation in the Ca signalling pathway occurs at the level of hormone-induced inositol 1,4,5 trisphosphate (IP ) production and does not involve changes in the sensitivity of the IP receptor or size of internal Ca stores.

The effect of chronic alcohol consumption on mitochondrial calcium handling in hepatocytes.

The damage to liver mitochondria is universally observed in both humans and animal models after excessive alcohol consumption. Acute alcohol treatment has been shown to stimulate calcium (Ca) release from internal stores in hepatocytes. The resultant increase in cytosolic Ca is expected to be accumulated by neighboring mitochondria, which could potentially lead to mitochondrial Ca overload and injury.

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations.

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.

Hormone-induced calcium oscillations depend on cross-coupling with inositol 1,4,5-trisphosphate oscillations.

Receptor-mediated oscillations in cytosolic Ca(2+) concentration ([Ca(2+)]i) could originate either directly from an autonomous Ca(2+) feedback oscillator at the inositol 1,4,5-trisphosphate (IP3) receptor or as a secondary consequence of IP3 oscillations driven by Ca(2+) feedback on IP3 metabolism. It is challenging to discriminate these alternatives, because IP3 fluctuations could drive Ca(2+) oscillations or could just be a secondary response to the [Ca(2+)]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.

Calcium-dependent regulation of glucose homeostasis in the liver.

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.

Purkinje cell dysfunction and delayed death in plasma membrane calcium ATPase 2-heterozygous mice.

Purkinje cell (PC) dysfunction or death has been implicated in a number of disorders including ataxia, autism and multiple sclerosis. Plasma membrane calcium ATPase 2 (PMCA2), an important calcium (Ca(2+)) extrusion pump that interacts with synaptic signaling complexes, is most abundantly expressed in PCs compared to other neurons. Using the PMCA2 heterozygous mouse as a model, we investigated whether a reduction in PMCA2 levels affects PC function.

Calcium-dependent physiologic and pathologic stimulus-metabolic response coupling in hepatocytes.

A recurrent paradigm in calcium signaling is the coordination of the target response of the calcium signal with activation of metabolic energy production to support that response. This occurs in many tissues, including cardiac and skeletal muscle where contractile activity and ATP production are coordinately regulated by the frequency and amplitude of calcium transients, endocrine and exocrine cells that use calcium to drive the secretory process, and hepatocytes where the downstream targets of calcium include both catabolic and anabolic processes. The primary mechanism by which calcium enhances the capacity for energy production is through calcium-dependent stimulation of mitochondrial oxidative metabolism, achieved by increasing NADH production and respiratory chain flux.

Mitochondrial morphology and dynamics in hepatocytes from normal and ethanol-fed rats.

Mitochondrial structure and function are central to cell physiology and are mutually interdependent. Mitochondria represent a primary target of the alcohol-induced tissue injury, particularly in the liver, where the metabolic effects of ethanol are predominant. However, the effect of ethanol on hepatic mitochondrial morphology and dynamics remain to be established.

Uncoupling protein-2 modulates myocardial excitation-contraction coupling.

Rationale: Uncoupling protein (UCP)2 is a mitochondrial inner membrane protein that is expressed in mammalian myocardium under normal conditions and upregulated in pathological states such as heart failure. UCP2 is thought to protect cardiomyocytes against oxidative stress by dissipating the mitochondrial proton gradient and mitochondrial membrane potential (DeltaPsi(m)), thereby reducing mitochondrial reactive oxygen species generation. However, in apparent conflict with its uncoupling role, UCP2 has also been proposed to be essential for mitochondrial Ca(2+) uptake, which could have a protective action by stimulating mitochondrial ATP production.

L-lysine uptake in giant vesicles from cardiac ventricular sarcolemma: two components of cationic amino acid transport.

Cationic L-amino acids enter cardiac-muscle cells through carrier-mediated transport. To study this process in detail, L-[(14)C]lysine uptake experiments were conducted within a 10(3)-fold range of L-lysine concentrations in giant sarcolemmal vesicles prepared from rat cardiac ventricles. Vesicles had a surface-to-volume ratio comparable with that of an epithelial cell, thus representing a suitable system for initial uptake rate studies.

Calcium-dependent activation of mitochondrial metabolism in mammalian cells.

Endogenous fluorophores provide a simple, but elegant means to investigate the relationship between agonist-evoked Ca2+ signals and the activation of mitochondrial metabolism. In this article, we discuss the methods and strategies to measure cellular pyridine nucleotide and flavoprotein fluorescence alone or in combination with Ca2+-sensitive indicators. These methods were developed using primary cultured hepatocytes and neurons, which contain relatively high levels of endogenous fluorophores and robust metabolic responses.

Amino acids activate mTOR complex 1 via Ca2+/CaM signaling to hVps34.

Excess levels of circulating amino acids (AAs) play a causal role in specific human pathologies, including obesity and type 2 diabetes. Moreover, obesity and diabetes are contributing factors in the development of cancer, with recent studies suggesting that this link is mediated in part by AA activation of mammalian target of rapamycin (mTOR) Complex 1. AAs appear to mediate this response through class III phosphatidylinositol 3-kinase (PI3K), or human vacuolar protein sorting 34 (hVps34), rather than through the canonical class I PI3K pathway used by growth factors and hormones.

Ryanodine receptors in liver.

The ryanodine receptor has been mainly regarded as the Ca2+ release channel from sarcoplasmic reticulum controlling skeletal and cardiac muscle contraction. However, many studies have shown that it is widely expressed, with functions not restricted to muscular contraction. This study examined whether ryanodine receptor plays a role in calcium signaling in the liver.

Models of IP3 and Ca2+ oscillations: frequency encoding and identification of underlying feedbacks.

Hormones that act through the calcium-releasing messenger, inositol 1,4,5-trisphosphate (IP3), cause intracellular calcium oscillations, which have been ascribed to calcium feedbacks on the IP3 receptor. Recent studies have shown that IP3 levels oscillate together with the cytoplasmic calcium concentration. To investigate the functional significance of this phenomenon, we have developed mathematical models of the interaction of both second messengers.

Calcium signaling in liver.

In hepatocytes, hormones linked to the formation of the second messenger inositol 1,4,5-trisphosphate (InsP3) evoke transient increases or spikes in cytosolic free calcium ([Ca2+]i), that increase in frequency with the agonist concentration. These oscillatory Ca2+ signals are thought to transmit the information encoded in the extracellular stimulus to down-stream Ca2+-sensitive metabolic processes. We have utilized both confocal and wide field fluorescence microscopy techniques to study the InsP3-dependent signaling pathway at the cellular and subcellular levels in the intact perfused liver.

Trauma-hemorrhagic shock mesenteric lymph induces endothelial apoptosis that involves both caspase-dependent and caspase-independent mechanisms.

Objective: To determine the mechanism by which gut-derived factors present in mesenteric lymph from rats subjected to trauma-hemorrhagic shock (T/HS) induce endothelial cell death.

Summary Background Data: Intestinal ischemia after hemorrhagic shock results in gut barrier dysfunction and the subsequent production of biologically active and tissue injurious factors by the ischemic gut. These factors are carried in the mesenteric lymph and reach the systemic circulation via the mesenteric lymph, thereby ultimately resulting in distant organ injury.

Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes.

Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation.

Inhibition of the mitochondrial permeability transition by aldehydes.

Fructose has been shown to protect hepatocyte viability during hypoxia or exposure to mitochondrial electron transport inhibitors. We report here that the fructose metabolite D-glyceraldehyde (D-GA) is a good inhibitor of the mitochondrial permeability transition pore (PTP) in isolated rat liver mitochondria. We propose that a substantial portion of the protective effect of fructose on hepatocytes is due to D-GA inhibition of the permeability transition.