Gordan Kilic

Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events.

Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules. Stained cores are not released, but remain attached to the cell and are eventually endocytosed. However, in cells exposed to dopamine (or an analog, bromocriptine), the cores dissolve and are secreted after several seconds. Solubilization of dense cores is mediated through a reduction in cytoplasmic cyclic AMP. Thus, the composition of secretions from individual secretory granules is regulated.

Characterization of ionotrophic purinergic receptors in hepatocytes

Ionotrophic purinergic (P2X) receptors function as receptor‐gated cation channels, where agonist binding leads to opening of a nonselective cation pore permeable to both Na+ and Ca2+. Based on evidence that extracellular adenosine 5′‐triphosphate (ATP) stimulates glucose release from liver, these studies evaluate whether P2X receptors are expressed by hepatocytes and contribute to ATP‐dependent calcium signaling and glucose release. Studies were performed in isolated hepatocytes from rats and mice and hepatoma cells from humans and rats. Transcripts and protein for both P2X4 and P2X7 were detectable, and immunohistochemistry of intact liver revealed P2X4 in the basolateral and canalicular domains. In whole cell patch clamp studies, exposure to the P2X4/P2X7 receptor agonist 2′3′‐O‐(4‐benzoyl‐benzoyl)‐adenosine 5′‐triphosphate (BzATP; 10 μM) caused a rapid increase in membrane Na+ conductance. Similarly, with Fluo‐3 fluorescence, BzATP induced an increase in intracellular [Ca2+]. P2X4 receptors are likely involved because the calcium response to BzATP was inhibited by Cu2+, and the P2X4 modulators Zn2+ and ivermectin (0.3‐3 μM) each increased intracellular [Ca2+]. Exposure to BzATP decreased cellular glycogen content; and P2X4 receptor messenger RNA increased in glycogen‐rich liver samples. Conclusion: These studies provide evidence that P2X4 receptors are functionally important in hepatocyte Na+ and Ca2+ transport, are regulated by extracellular ATP and divalent cation concentrations, and may constitute a mechanism for autocrine regulation of hepatic glycogen metabolism. (HEPATOLOGY 2007.)

Pannexin1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated free fatty acids in liver cells

Hepatocyte lipoapoptosis induced by saturated free fatty acids (FFA) contributes to hepatic inflammation in lipotoxic liver injury, and the cellular mechanisms involved have not been defined. Recent studies have shown that apoptosis in nonhepatic cells stimulates ATP release via activation of pannexin1 (panx1), and extracellular ATP functions as a proinflammatory signal for recruitment and activation of the inflammatory cells. However, it is not known whether lipoapoptosis stimulates ATP release in liver cells. We found that lipoapoptosis induced by saturated FFA stimulated ATP release in liver cells that increased extracellular ATP concentration by more than fivefold above the values observed in healthy cells. This sustained pathophysiological ATP release was not dependent on caspase-3/7 activation. Inhibition of c-Jun NH(2)-terminal kinase (JNK), a key mediator of lipoapoptosis, with SP600125 blocked pathophysiological ATP release in a dose-dependent manner. RT-PCR analysis indicated that panx1 is expressed in hepatocytes and multiple liver cell lines. Notably, inhibition of panx1 expression with short hairpin (sh)RNA inhibited in part pathophysiological ATP release. Moreover, lipoapoptosis stimulated uptake of a membrane impermeable dye YoPro-1 (indicative of panx1 activation), which was inhibited by panx1 shRNA, probenecid, and mefloquine. These results suggest that panx1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated FFA. Thus panx1 may play an important role in hepatic inflammation by mediating an increase in extracellular ATP concentration in lipotoxic liver injury.

Sustained stimulation of exocytosis triggers continuous membrane retrieval in rat pituitary somatotrophs.

1 We studied the relationship between exocytosis and endocytosis in rat pituitary somatotrophs using patch‐clamp capacitance, FM1‐43 fluorescence imaging and amperometry. 2 Stimulation of exocytosis through voltage‐dependent Ca2+ channels by depolarizations (1‐5 s) increased the capacitance by 4.3 ± 0.9 % and the fluorescence by 6.6 ± 1.1 % (10 cells). The correlation between the capacitance and fluorescence changes indicated that the cell membrane and granule membrane added via exocytosis were stained with the membrane‐bound fluorescent dye FM1‐43 in a quantitatively similar manner. 3 Intracellular dialysis (0.5‐4.5 min) with elevated Ca2+ (1.5‐100 μm) evoked continuous exocytosis that was detected with a carbon fibre electrode from dopamine‐loaded cells (10 cells) or as an increase in FM1‐43 fluorescence (56 ± 10 %; 21 cells). Interestingly during Ca2+ dialysis the capacitance did not significantly change (2 ± 1 %; 31 cells), indicating that endocytosis efficiently retrieved increased cell membrane. 4 Sustained endocytosis was not blocked when the intracellular GTP (300 μm) was replaced with GTPγS. Replacing intracellular Ca2+ (100 μm) with Ba2+ (300 μm) or Sr2+ (200 μm), or reducing the pH of the intracellular solution from 7.2 to 6.2 did not block sustained endocytosis. 5 Our results suggest that pituitary somatotrophs have the ability to undergo continuous exocytosis and membrane retrieval that persist in whole‐cell recordings.

Volume-sensitive tyrosine kinases regulate liver cell volume through effects on vesicular trafficking and membrane Na+ permeability

In liver cells, the influx of Na+ mediated by nonselective cation (NSC) channels in the plasma membrane contributes importantly to regulation of cell volume. Under basal conditions, channels are closed; but both physiologic (e.g. insulin) and pathologic (e.g. oxidative stress) stimuli that are known to stimulate tyrosine kinases are associated with large increases in membrane Na+ permeability to ∼80 pA/pF or more. Consequently, the purpose of these studies was to evaluate whether volume-sensitive tyrosine kinases mediate cell volume increases through effects on the activity or distribution of NSC channel proteins. In HTC hepatoma cells, decreases in cell volume evoked by hypertonic exposure increased total cellular tyrosine kinase activity ∼20-fold. Moreover, hypertonic exposure (320–400 mosm) was followed after a delay by NSC channel activation and partial recovery of cell volume toward basal values (regulatory volume increase (RVI)). The tyrosine kinase inhibitors genistein and erbstatin prevented both NSC channel activation and RVI. Similarly, hypertonic exposure resulted in an increase in p60c-src activity, and intracellular dialysis with recombinant p60c-src led to activation of NSC currents in the absence of an osmolar gradient. Utilizing FM1-43 fluorescence, exposure to hypertonic media caused a rapid increase in the rate of exocytosis of ∼40% (p < 0.01), and genistein inhibited both exocytosis and channel activation. These findings indicate that volume-sensitive increases in p60c-src and/or related tyrosine kinases play a key role in the regulation of membrane Na+ permeability, suggesting that increases in the NSC conductance may be mediated in part through rapid recruitment of a distinct pool of channel-containing vesicles.

Exocytosis in bovine chromaffin cells: Studies with patch-clamp capacitance and FM1-43 fluorescence

In response to physiological stimuli, neuroendocrine cells secrete neurotransmitters through a Ca(2+)-dependent fusion of secretory granules with the plasma membrane. We studied insertion of granules in bovine chromaffin cells using capacitance as a measure of plasma membrane area and fluorescence of a membrane marker FM1-43 as a measure of exocytosis. Intracellular dialysis with [Ca(2+)] (1.5-100 microM) evoked massive exocytosis that was sufficient to double plasma membrane area but did not swell cells. In principle, in the absence of endocytosis, the addition of granule membrane would be anticipated to produce similar increases in the capacitance and FM1-43 fluorescence responses. However, when endocytosis was minimal, the changes in capacitance were markedly larger than the corresponding changes in FM1-43 fluorescence. Moreover, the apparent differences between capacitance and FM1-43 fluorescence changes increased with larger exocytic responses, as more granules fused with the plasma membrane. In experiments in which exocytosis was suppressed, increasing membrane tension by osmotically induced cell swelling increased FM1-43 fluorescence, suggesting that FM1-43 fluorescence is sensitive to changes in the membrane tension. Thus, increasing membrane area through exocytosis does not swell chromaffin cells but may decrease membrane tension.

Influence of conductance changes on patch clamp capacitance measurements using a lock-in amplifier and limitations of the phase tracking technique

We characterized the influence of conductance changes on whole-cell patch clamp capacitance measurements with a lock-in amplifier and the limitations of the phase-tracking method by numerical computer simulations, error formulas, and experimental tests. At correct phase setting, the artifacts in the capacitance measurement due to activation of linear conductances are small. The cross talk into the capacitance trace is well approximately by the second-order term in the Taylor expansion of the admittance. In the case of nonlinear current-voltage relationships, the measured conductance corresponds to the slope conductance in the range of the sine wave amplitude, and the cross talk into the capacitance trace corresponds to the second-order effect of the slope conductance. The finite gating kinetics of voltage-dependent channels generate phase-shifted currents. These lead to major artifacts in the capacitance measurements when the angular frequency of the sine wave is close to the kinetic rate constant of the channel. However, when the channel kinetics are sufficiently slow, or sufficiently fast, the cross talk is still close to the second-order effect of the measured conductance. The effects of activation of voltage-dependent currents on the capacitance measurements may be estimated, provided a detailed characterization of the kinetics and voltage dependence is available. A phase error of the lock-in amplifier of a few degrees leads to significant projections. The phase-tracking method can be used to keep the phase aligned only during periods of low membrane conductance. However, nonideal properties of the equivalent circuit, in particular the fast capacitance between the pipette and the bath solutions, may lead to large phase errors when the phase-tracking method is used, depending on the electrical properties of the cell. In this article we provide practical values, setting the range where possible artifacts are below defined limits. For proper evaluation of capacitance measurements, the capacitance and conductance traces should always be displayed together.

5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles

Cells release ATP in response to physiologic stimuli. Extracellular ATP regulates a broad range of important cellular functions by activation of the purinergic receptors in the plasma membrane. The purpose of these studies was to assess the role of vesicular exocytosis in cellular ATP release. FM1-43 fluorescence was used to measure exocytosis and bioluminescence to measure ATP release in HTC rat hepatoma and Mz-Cha-1 human cholangiocarcinoma cells. Exposure to a Cl− channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (50–300 μm) stimulated a 5–100-fold increase in extracellular ATP levels within minutes of the exposure. This rapid response was not a result of changes in cell viability or Cl− channel activity. NPPB also potently stimulated ATP release in HEK293 cells and HEK293 cells expressing a rat P2X7 receptor indicating that P2X7 receptors are not involved in stimulation of ATP release by NPPB. In all cells studied, NPPB rapidly stimulated vesicular exocytosis that persisted many minutes after the exposure. The kinetics of NPPB-evoked exocytosis and ATP release were similar. Furthermore, the magnitudes of NPPB-evoked exocytosis and ATP release were correlated (correlation coefficient 0.77), indicating that NPPB may stimulate exocytosis of a pool of ATP-enriched vesicles. These findings provide further support for the concept that vesicular exocytosis plays an important role in cellular ATP release and suggest that NPPB can be used as a biochemical tool to specifically stimulate ATP release through exocytic mechanisms.

Increased Phosphoenolpyruvate Carboxykinase Gene Expression and Steatosis during Hepatitis C Virus Subgenome Replication ROLE OF NONSTRUCTURAL COMPONENT 5A AND CCAAT/ENHANCER-BINDING PROTEIN β

Background: Chronic hepatitis C virus (HCV) infection increases the risk of type 2 diabetes and hepatic steatosis. Results: Phosphoenolpyruvate carboxykinase (PEPCK) and associated transcription factors are up-regulated in HCV-infected Huh.8 cells. Conclusion: Increased CCAAT/enhancer-binding protein β (C/EBPβ) and nonstructural component 5A (NS5A) are essential components for increased gluconeogenesis. Significance: NS5A and C/EBPβ may possibly be considered as a new pharmacological target during HCV infection. Chronic hepatitis C virus (HCV) infection greatly increases the risk for type 2 diabetes and nonalcoholic steatohepatitis; however, the pathogenic mechanisms remain incompletely understood. Here we report gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK) transcription and associated transcription factors are dramatically up-regulated in Huh.8 cells, which stably express an HCV subgenome replicon. HCV increased activation of cAMP response element-binding protein (CREB), CCAAT/enhancer-binding protein (C/EBPβ), forkhead box protein O1 (FOXO1), and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) and involved activation of the cAMP response element in the PEPCK promoter. Infection with dominant-negative CREB or C/EBPβ-shRNA significantly reduced or normalized PEPCK expression, with no change in PGC-1α or FOXO1 levels. Notably, expression of HCV nonstructural component NS5A in Huh7 or primary hepatocytes stimulated PEPCK gene expression and glucose output in HepG2 cells, whereas a deletion in NS5A reduced PEPCK expression and lowered cellular lipids but was without effect on insulin resistance, as demonstrated by the inability of insulin to stimulate mobilization of a pool of insulin-responsive vesicles to the plasma membrane. HCV-replicating cells demonstrated increases in cellular lipids with insulin resistance at the level of the insulin receptor, increased insulin receptor substrate 1 (Ser-312), and decreased Akt (Ser-473) activation in response to insulin. C/EBPβ-RNAi normalized lipogenic genes sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and liver X receptor α but was unable to reduce accumulation of triglycerides in Huh.8 cells or reverse the increase in ApoB expression, suggesting a role for increased lipid retention in steatotic hepatocytes. Collectively, these data reveal an important role of NS5A, C/EBPβ, and pCREB in promoting HCV-induced gluconeogenic gene expression and suggest that increased C/EBPβ and NS5A may be essential components leading to increased gluconeogenesis associated with HCV infection.

Heterotrimeric G-proteins Activate Cl− Channels through Stimulation of a Cyclooxygenase-dependent Pathway in a Model Liver Cell Line

Circulating hormones produce rapid changes in the Cl− permeability of liver cells through activation of plasma membrane receptors coupled to heterotrimeric G-proteins. The resulting effects on intracellular pH, membrane potential, and Cl− content are important contributors to the overall metabolic response. Consequently, the purpose of these studies was to evaluate the mechanisms responsible for G-protein-mediated changes in membrane Cl− permeability using HTC hepatoma cells as a model. Using patch clamp techniques, intracellular dialysis with 0.3 mm guanosine 5′-O-(3-thiotriphosphate) (GTPγS) increased membrane conductance from 10 to 260 picosiemens/picofarads due to activation of Ca2+-dependent Cl− currents that were outwardly rectifying and exhibited slow activation at depolarizing potentials. These effects were mimicked by intracellular AlF 4 − (0.03 mm) and inhibited by pertussis toxin (PTX), consistent with current activation through Gαi. Studies using defined agonists and inhibitors indicate that Cl− channel activation by GTPγS occurs through an indomethacin-sensitive pathway involving sequential activation of phospholipase C, mobilization of Ca2+ from inositol 1,4,5-trisphosphate-sensitive stores, and stimulation of phospholipase A2 and cyclooxygenase (COX). Accordingly, the conductance responses to GTPγS or to intracellular Ca2+were inhibited by COX inhibitors. These results indicate that PTX-sensitive G-proteins regulate the Cl− permeability of HTC cells through Ca2+-dependent stimulation of COX activity. Thus, receptor-mediated activation of Gαi may be essential for hormonal regulation of liver transport and metabolism through COX-dependent opening of a distinct population of plasma membrane Cl− channels.