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. The results obtained in the present study demonstrate that subthreshold levels of one hormone can create an excitable medium across the liver lobule, which allows global propagation of calcium signals in response to local sympathetic innervation and integration of metabolic regulation by multiple hormones. This enables the liver lobules to respond as functional units to produce full-strength metabolic output at physiological levels of hormone. ABSTRACT: Glucogenic hormones, including catecholamines and vasopressin, induce frequency-modulated cytosolic Ca2+ oscillations in hepatocytes, and these propagate as intercellular Ca2+ waves via gap junctions in the intact liver. We investigated the role of co-ordinated Ca2+ waves as a mechanism for integrating multiple endocrine and neuroendocrine inputs to control hepatic glucose production in perfused rat liver. Sympathetic nerve stimulation elicited localized Ca2+ increases that were restricted to hepatocytes in the periportal zone. During perfusion with subthreshold vasopressin, sympathetic stimulation converted asynchronous Ca2+ signals in a limited number of hepatocytes into co-ordinated intercellular Ca2+ waves that propagated across entire lobules. A similar synergism was observed between physiological concentrations of glucagon and vasopressin, where glucagon also facilitated the recruitment of hepatocytes into a Ca2+ wave. Hepatic glucose production was significantly higher with intralobular Ca2+ waves. We propose that inositol 1,4,5-trisphosphate (IP3 )-dependent Ca2+ signalling gives rise to an excitable medium across the functional syncytium of the hepatic lobule, co-ordinating and amplifying the metabolic responses to multiple hormonal inputs.

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

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.

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 (Ca2+) release from internal stores in hepatocytes. The resultant increase in cytosolic Ca2+ is expected to be accumulated by neighboring mitochondria, which could potentially lead to mitochondrial Ca2+ overload and injury. Our data indicate that total and free mitochondrial matrix Ca2+ levels are, indeed, elevated in hepatocytes isolated from alcohol-fed rats compared with their pair-fed control littermates. In permeabilized hepatocytes, the rates of mitochondrial Ca2+ uptake were substantially increased after chronic alcohol feeding, whereas those of mitochondrial Ca2+ efflux were decreased. The changes in mitochondrial Ca2+ handling could be explained by an up-regulation of the mitochondrial Ca2+ uniporter and loss of a cyclosporin A-sensitive Ca2+ transport pathway. In intact cells, hormone-induced increases in mitochondrial Ca2+ declined at slower rates leading to more prolonged elevations of matrix Ca2+ in the alcohol-fed group compared with controls. Moreover, treatment with submaximal concentrations of Ca2+-mobilizing hormones markedly increased the levels of mitochondrial reactive oxygen species (ROS) in hepatocytes from alcohol-fed rats, but did not affect ROS levels in controls. The changes in mitochondrial Ca2+ handling are expected to buffer and attenuate cytosolic Ca2+ increases induced by acute alcohol exposure or hormone stimulation. However, these alterations in mitochondrial Ca2+ handling may also lead to Ca2+ overload during cytosolic Ca2+ increases, which may stimulate the production of mitochondrial ROS, and thus contribute to alcohol-induced liver 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. 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.

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. We focused on Ca(2+) signaling and the expression of glutamate receptors which play a key role in PC function including synaptic plasticity. We found that the amplitude of depolarization and 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid receptor (AMPAR)-mediated Ca(2+) transients are significantly higher in cultured PMCA2(+/-) PCs than in PMCA2(+/+) PCs. This is due to increased Ca(2+) influx, since P/Q type voltage-gated Ca(2+) channel (VGCC) expression was more pronounced in PCs and cerebella of PMCA2(+/-) mice and VGCC blockade prevented the elevation in amplitude. Neuronal nitric oxide synthase (nNOS) activity was higher in PMCA2(+/-) cerebella and inhibition of nNOS or the soluble guanylate cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway, which mediates nitric oxide (NO) signaling, reduced the amplitude of Ca(2+) transients in PMCA2(+/-) PCs, in vitro. In addition, there was an age-dependent decrease in metabotropic glutamate receptor 1 (mGluR1) and AMPA receptor subunit GluR2/3 transcript and protein levels at 8 weeks of age. These changes were followed by PC loss in the 20-week-old PMCA2(+/-) mice. Our studies highlight the importance of PMCA2 in Ca(2+) signaling, glutamate receptor expression and survival of Purkinje cells.

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. In the present work, we employed the organelle-targeted photoactivatable fluorescent protein technology and electron microscopy to study hepatic mitochondrial structure and dynamics. Hepatocytes in perfused liver as well as in primary cultures showed mostly discrete globular or short tubular mitochondria. The mitochondria showed few fusion events and little movement activity. By contrast, human hepatoma (HepG2)-derived VL-17A cells, expressing the major hepatic ethanol metabolizing enzymes, alcohol dehydrogenase and cytochrome P450 2E1, have elongated and interconnected mitochondria showing matrix continuity and many fusion events. Hepatocytes isolated from chronically ethanol-fed rats showed some increase in mitochondrial volume and exhibited a substantial suppression of mitochondrial dynamics. In VL-17A cells, prolonged ethanol exposure also caused decreased mitochondrial continuity and dynamics. Collectively, these results indicate that mitochondria in normal hepatocytes show relatively slow dynamics, which is very sensitive to suppression by ethanol exposure.

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. OBJECTIVE: The goal of this study was to better understand the role of myocardial UCP2 by examining the effects of UCP2 on bioenergetics, Ca(2+) homeostasis, and excitation-contraction coupling in neonatal cardiomyocytes. METHODS AND RESULTS: Adenoviral-mediated expression of UCP2 caused a mild depression of DeltaPsi(m) and increased the basal rate of oxygen consumption but did not affect total cellular ATP levels. Mitochondrial Ca(2+) uptake was examined in permeabilized cells loaded with the mitochondria-selective Ca(2+) probe, rhod-2. UCP2 overexpression markedly inhibited mitochondrial Ca(2+) uptake. Pretreatment with the UCP2-specific inhibitor genipin largely reversed the effects UCP2 expression on mitochondrial Ca(2+) handling, bioenergetics, and oxygen utilization. Electrically evoked cytosolic Ca(2+) transients and spontaneous cytosolic Ca(2+) sparks were examined using fluo-based probes and confocal microscopy in line scan mode. UCP2 overexpression significantly prolonged the decay phase of [Ca(2+)](c) transients in electrically paced cells, increased [Ca(2+)](c) spark activity and increased the probability that Ca(2+) sparks propagated into Ca(2+) waves. This dysregulation results from a loss of the ability of mitochondria to suppress local Ca(2+)-induced Ca(2+) release activity of the sarcoplasmic reticulum. CONCLUSION: Increases in UCP2 expression that lower DeltaPsi(m) and contribute to protection against oxidative stress, also have deleterious effects on beat-to-beat [Ca(2+)](c) handling and excitation-contraction coupling, which may contribute to the progression of heart disease.

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 (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-ß 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.

Receptor-specific Ca2+ oscillation patterns mediated by differential regulation of P2Y purinergic receptors in rat hepatocytes.

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.

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. Nevertheless, these methods are amendable to a wide variety of primary cell types and cell lines that maintain active mitochondrial metabolism.

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. Typically InsP3-dependent [Ca2+]i spikes manifest as Ca2+ waves that propagate throughout the entire cytoplasm and nucleus, and in the intact liver these [Ca2+]i increases are conveyed through gap junctions to encompass entire lobular units. The translobular movement of Ca2+ provides a means to coordinate the function of metabolic zones of the lobule and thus, liver function. In this article, we describe the characteristics of agonist-evoked [Ca2+]i signals in the liver and discuss possible mechanisms to explain the propagation of intercellular Ca2+ waves in the intact organ.

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. 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.

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. This IP3 buffer slows hormone-induced [IP3] dynamics without changing steady-state [IP3]. GFP-LBD perturbed [Ca(2+)]i oscillations in a dose-dependent manner: it decreased both the rate of [Ca(2+)]i rise and the speed of Ca(2+) wave propagation and, at high levels, abolished [Ca(2+)]i oscillations completely. These data, together with computational modeling, demonstrate that IP3 dynamics play a fundamental role in generating [Ca(2+)]i oscillations and waves.

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. Although this enhances energy supply, it also has the potential for deleterious consequences resulting from increased generation of reactive oxygen species (ROS). The negative consequences of calcium-dependent mitochondrial activation can be ameliorated when the underlying cytosolic calcium signals occur as brief calcium spikes or oscillations, with signal strength encoded through the spike frequency (frequency modulation). Frequency modulation increases signal fidelity, and reduces pathological effects of calcium, including excess mitochondrial ROS production and apoptotic or necrotic outcomes. The present article reviews these issues using data obtained in hepatocytes under physiologic and pathologic conditions.

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. Here we show that AAs induce a rise in intracellular Ca(2+) ([Ca(2+)](i)), which triggers mTOR Complex 1 and hVps34 activation. We demonstrate that the rise in [Ca(2+)](i) increases the direct binding of Ca(2+)/calmodulin (CaM) to an evolutionarily conserved motif in hVps34 that is required for lipid kinase activity and increased mTOR Complex 1 signaling. These findings have important implications regarding the basic signaling mechanisms linking metabolic disorders with cancer progression.

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. The models account for both positive and negative feedbacks of calcium on IP3 metabolism, mediated by calcium activation of phospholipase C and IP3 3-kinase, respectively. The coupled IP3 and calcium oscillations have a greatly expanded frequency range compared to calcium fluctuations obtained with clamped IP3. Therefore the feedbacks can be physiologically important in supporting the efficient frequency encoding of hormone concentration observed in many cell types. This action of the feedbacks depends on the turnover rate of IP3. To shape the oscillations, positive feedback requires fast IP3 turnover, whereas negative feedback requires slow IP3 turnover. The ectopic expression of an IP3 binding protein has been used to decrease the rate of IP3 turnover experimentally, resulting in a dose-dependent slowing and eventual quenching of the Ca2+ oscillations. These results are consistent with a model based on positive feedback of Ca2+ on IP3 production.

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. RT-PCR analysis of isolated hepatocytes showed expression of a truncated type 1 ryanodine receptor, but no type 2 or type 3 message was detected. We also detected binding sites for [3H]ryanodine in the microsomal cellular fraction and in permeabilized hepatocytes. This binding was displaced by caffeine and dantrolene, but not by ruthenium red, heparin or cyclic ADP-Ribose. Ryanodine, by itself, did not trigger Ca2+ oscillations in either primary cultured hepatocytes or hepatocytes within the intact perfused rat liver. In both preparations, however, ryanodine significantly increased the frequency of the cytosolic free [Ca2+] oscillations evoked by an alpha1 adrenergic receptor agonist. Experiments in permeabilized hepatocytes showed that both ryanodine and cyclic ADP-ribose evoked a slow Ca2+ leak from intracellular stores and were able to increase the Ca2+-released response to a subthreshold dose of inositol 1,4,5-trisphosphate. Our findings suggest the presence of a novel truncated form of the type 1 ryanodine receptor in rat hepatocytes. Ryanodine modulates the pattern of cytosolic free [Ca2+] oscillations by increasing oscillation frequency. We propose that the Ca2+ released from ryanodine receptors on the endoplasmic reticulum provides an increased pool of Ca2+ for positive feedback on inositol 1,4,5-trisphosphate receptors.

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. Aldehydes which are substrates of the mitochondrial aldehyde dehydrogenase (mALDH) afford protection, while poor substrates do not. Protection is prevented by the ALDH inhibitor chloral hydrate. We propose that the NADH/NAD(+) ratio is the key to protection. The aldehydes phenylglyoxal (PGO) and 4-hydroxynonenal (4-HNE), which have previously been shown to inhibit the PTP, apparently function by a different mechanism independent of mALDH activity. Both PGO or 4-HNE are themselves potent inhibitors of ALDH, and their protective effect cannot be blocked by an ALDH inhibitor.

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. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.

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. Although studies have established that trauma-hemorrhagic (T/HS) shock but not trauma-sham-shock (T/SS) mesenteric lymph is cytotoxic to endothelial cells, whether T/HS lymph-induced endothelial cell death occurs via an apoptotic or a necrotic pathway is unknown. The mechanisms underlying T/HS lymph-induced cytotoxicity are likewise unknown. METHODS: Human umbilical vein endothelial cell (HUVEC) monolayers were incubated with medium, sham-shock, or post shock mesenteric lymph (5%) for 4 hours, after which the mode of cell death (ie, apoptosis versus necrosis) was determined using morphologic (confocal microscopy), biochemical (nucleosomal release), and DNA-based (gel electrophoresis) assays. To clarify the cellular pathways involved in T/HS lymph-induced HUVEC cell death, caspase-3, caspase-9, caspase-8, and BID activity was measured as was the ability of the pan-caspase inhibitor z-VAD-fmk to prevent T/HS lymph-induced cell death. RESULTS: T/HS, but not T/SS, mesenteric lymph or medium was cytotoxic and caused the appearance of the classic morphologic signs of apoptosis, including membrane blebbing, cell shrinkage, and apoptotic body formation. Nucleosomal release and a DNA laddering pattern was also observed in the HUVECs incubated with T/HS lymph. These signs of apoptosis were associated with increased caspase activity as reflected in activation of the pro-apoptotic caspases, caspase-8, -9, and -3, as well as the pro-apoptotic bcl-2-related protein BID. However, since the broad-spectrum caspase inhibitor z-VAD-fmk delayed T/HS lymph-induced HUVEC cell death, but did not prevent it fully, it appears that other factors besides caspases are involved in the endothelial cell toxicity of T/HS lymph. CONCLUSIONS: Gut-derived factors in T/HS, but not T/SS, mesenteric lymph cause endothelial cell death via an apoptotic mechanism that involves both caspase-dependent and caspase-independent pathways.