17Beta-estradiol elevates cGMP and, via plasma membrane recruitment of protein kinase GIalpha, stimulates Ca2+ efflux from rat hepatocytes.

Rapid non-genomic effects of 17beta-estradiol, the principal circulating estrogen, have been observed in a wide variety of cell types. Here we investigate rapid signaling effects of 17beta-estradiol in rat hepatocytes. We show that, above a threshold concentration of 1 nm, 17beta-estradiol, but not 17alpha-estradiol, stimulates particulate guanylyl cyclase to elevate cGMP, which through activation and plasma membrane recruitment of protein kinase G isoform Ialpha, stimulates plasma membrane Ca(2+)-ATPase-mediated Ca(2+) efflux from rat hepatocytes. These effects are extremely rapid in onset and are mimicked by a membrane-impermeant 17beta-estradiol-BSA conjugate, suggesting that 17beta-estradiol acts at the extracellular face of the plasma membrane. We also show that 17beta-estradiol binds specifically to the intact hepatocyte plasma membrane through an interaction that is competed by an excess of atrial natriuretic peptide but also shows many similarities to the pharmacological characteristics of the putative gamma-adrenergic receptor. We, therefore, propose that the observed rapid signaling effects of 17beta-estradiol are mediated either through the guanylyl cyclase A receptor for atrial natriuretic peptide or through the gamma-adrenergic receptor, which is either itself a transmembrane guanylyl cyclase or activates a transmembrane guanylyl cyclase through cross-talk signaling.

Information transfer in signaling pathways: a study using coupled simulated and experimental data.

BACKGROUND: The topology of signaling cascades has been studied in quite some detail. However, how information is processed exactly is still relatively unknown. Since quite diverse information has to be transported by one and the same signaling cascade (e.g. in case of different agonists), it is clear that the underlying mechanism is more complex than a simple binary switch which relies on the mere presence or absence of a particular species. Therefore, finding means to analyze the information transferred will help in deciphering how information is processed exactly in the cell. Using the information-theoretic measure transfer entropy, we studied the properties of information transfer in an example case, namely calcium signaling under different cellular conditions. Transfer entropy is an asymmetric and dynamic measure of the dependence of two (nonlinear) stochastic processes. We used calcium signaling since it is a well-studied example of complex cellular signaling. It has been suggested that specific information is encoded in the amplitude, frequency and waveform of the oscillatory Ca(2+)-signal. RESULTS: We set up a computational framework to study information transfer, e.g. for calcium signaling at different levels of activation and different particle numbers in the system. We stochastically coupled simulated and experimentally measured calcium signals to simulated target proteins and used kernel density methods to estimate the transfer entropy from these bivariate time series. We found that, most of the time, the transfer entropy increases with increasing particle numbers. In systems with only few particles, faithful information transfer is hampered by random fluctuations. The transfer entropy also seems to be slightly correlated to the complexity (spiking, bursting or irregular oscillations) of the signal. Finally, we discuss a number of peculiarities of our approach in detail. CONCLUSION: This study presents the first application of transfer entropy to biochemical signaling pathways. We could quantify the information transferred from simulated/experimentally measured calcium signals to a target enzyme under different cellular conditions. Our approach, comprising stochastic coupling and using the information-theoretic measure transfer entropy, could also be a valuable tool for the analysis of other signaling pathways.

ANP stimulates hepatocyte Ca2+ efflux via plasma membrane recruitment of PKGIalpha.

In rat hepatocytes, atrial natriuretic peptide (ANP) elevates cGMP through activation of particulate guanylyl cyclase and attenuates Ca(2+) signals by stimulating net plasma membrane Ca(2+) efflux. We show here that ANP-stimulated hepatocyte Ca(2+) efflux is mediated by protein kinase G (PKG) isotype I. Furthermore, we show that ANP recruits endogenous PKGIalpha, but not PKGIbeta, to the plasma membrane. These effects are mimicked by 8-bromo-cGMP, but not by the soluble guanylyl cyclase activators, sodium nitroprusside and YC-1. We propose that ANP, through localized cGMP elevation, promotes plasma membrane recruitment of PKGIalpha, which, in turn, stimulates Ca(2+) efflux.

Spatio-temporal modelling explains the effect of reduced plasma membrane Ca2+ efflux on intracellular Ca2+ oscillations in hepatocytes.

In many non-excitable eukaryotic cells, including hepatocytes, Ca(2+) oscillations play a key role in intra- and intercellular signalling, thus regulating many cellular processes from fertilisation to death. Therefore, understanding the mechanisms underlying these oscillations, and consequently understanding how they may be regulated, is of great interest. In this paper, we study the influence of reduced Ca(2+) plasma membrane efflux on Ca(2+) oscillations in hepatocytes. Our previous experiments with carboxyeosin show that a reduced plasma membrane Ca(2+) efflux increases the frequency of Ca(2+) oscillations, but does not affect the duration of individual transients. This phenomenon can be best explained by taking into account not only the temporal, but also the spatial dynamics underlying the generation of Ca(2+) oscillations in the cell. Here we divide the cell into a grid of elements and treat the Ca(2+) dynamics as a spatio-temporal phenomenon. By converting an existing temporal model into a spatio-temporal one, we obtain theoretical predictions that are in much better agreement with the experimental observations.

Establishing the stochastic nature of intracellular calcium oscillations from experimental data.

Calcium has been established as a key messenger in both intra- and intercellular signaling. Experimentally observed intracellular calcium responses to different agonists show a variety of behaviors from simple spiking to complex oscillatory regimes. Here we study typical experimental traces of calcium oscillations in hepatocytes obtained in response to phenylephrine and ATP. The traces were analyzed with methods of nonlinear time series analysis in order to determine the stochastic/deterministic nature of the intracellular calcium oscillations. Despite the fact that the oscillations appear, visually, to be deterministic yet perturbed by noise, our analyses provide strong evidence that the measured calcium traces in hepatocytes are prevalently of stochastic nature. In particular, bursting calcium oscillations are temporally correlated Gaussian series distorted by a monotonic, instantaneous, time-independent function, whilst the spiking behavior appears to have a dynamical nonlinear component whereby the overall determinism level is still low. The biological importance of this finding is discussed in relation to the mechanisms incorporated in mathematical models as well as the role of stochasticity and determinism at cellular and tissue levels which resemble typical statistical and thermodynamic effects in physics.

Atrial natriuretic peptide attenuates elevations in Ca2+ and protects hepatocytes by stimulating net plasma membrane Ca2+ efflux.

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.

Transition from stochastic to deterministic behavior in calcium oscillations.

Simulation and modeling is becoming more and more important when studying complex biochemical systems. Most often, ordinary differential equations are employed for this purpose. However, these are only applicable when the numbers of participating molecules in the biochemical systems are large enough to be treated as concentrations. For smaller systems, stochastic simulations on discrete particle basis are more accurate. Unfortunately, there are no general rules for determining which method should be employed for exactly which problem to get the most realistic result. Therefore, we study the transition from stochastic to deterministic behavior in a widely studied system, namely the signal transduction via calcium, especially calcium oscillations. We observe that the transition occurs within a range of particle numbers, which roughly corresponds to the number of receptors and channels in the cell, and depends heavily on the attractive properties of the phase space of the respective systems dynamics. We conclude that the attractive properties of a system, expressed, e.g., by the divergence of the system, are a good measure for determining which simulation algorithm is appropriate in terms of speed and realism.

Bile acids induce a cationic current, depolarizing pancreatic acinar cells and increasing the intracellular Na+ concentration.

Biliary disease is a major cause of acute pancreatitis. In this study we investigated the electrophysiological effects of bile acids on pancreatic acinar cells. In perforated patch clamp experiments we found that taurolithocholic acid 3-sulfate depolarized pancreatic acinar cells. At low bile acid concentrations this occurred without rise in the cytosolic calcium concentration. Measurements of the intracellular Na(+) concentration with the fluorescent probe Sodium Green revealed a substantial increase upon application of the bile acid. We found that bile acids induce Ca(2+)-dependent and Ca(2+)-independent components of the Na(+) concentration increase. The Ca(2+)-independent component was resolved in conditions when the cytosolic Ca(2+) level was buffered with a high concentration of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). The Ca(2+)-dependent component of intracellular Na(+) increase was clearly seen during stimulation with the calcium-releasing agonist acetylcholine. During acetylcholine-induced Ca(2+) oscillations the recovery of cytosolic Na(+) was much slower than the recovery of Ca(2+), creating a possibility for the summation of Na(+) transients. The bile-induced Ca(2+)-independent current was found to be carried primarily by Na(+) and K(+), with only small Ca(2+) and Cl(-) contributions. Measurable activation of such a cationic current could be produced by a very low concentration of taurolithocholic acid 3-sulfate (10 microm). This bile acid induced a cationic current even when applied in sodium- and bicarbonate-free solution. Other bile acids, taurochenodeoxycholic acid, taurocholic acid, and bile itself also induced cationic currents. Bile-induced depolarization of acinar cells should have a profound effect on acinar fluid secretion and, consequently, on transport of secreted zymogens.

Atrial natriuretic peptide attenuates Ca2+ oscillations and modulates plasma membrane Ca2+ fluxes in rat hepatocytes.

BACKGROUND & AIMS: Oscillations in cytosolic free Ca2+ concentration are a fundamental mechanism of intracellular signaling in hepatocytes. The aim of this study was to examine the effects of atrial natriuretic peptide (ANP) on cytosolic Ca2+ oscillations in rat hepatocytes. METHODS: Cyclic guanosine monophosphate (cGMP) was measured by enzyme immunoassay. Cytosolic Ca2+ oscillations were recorded from single aequorin-injected hepatocytes. Ca2+ efflux from hepatocyte populations was measured by using extracellular fura-2. Ca2+ influx was estimated by Mn2+ quench of fluorescence of fura-2 dextran injected into single hepatocytes. RESULTS: ANP attenuated cytosolic Ca2+ oscillations through a decrease in their frequency. In addition, ANP dramatically stimulated plasma membrane Ca2+ efflux and modestly inhibited basal Ca2+ influx. All of the observed effects of ANP were mimicked by the cGMP analogue 8-bromo-cGMP (8-Br-cGMP), and were prevented by inhibition of protein kinase G. In contrast, activation of cytosolic guanylyl cyclase by sodium nitroprusside had no effect on Ca2+ efflux, Ca2+ influx, or Ca2+ oscillations. CONCLUSIONS: ANP decreases the frequency of Ca2+ oscillations and modulates plasma membrane Ca2+ fluxes in rat hepatocytes. Attenuation of oscillatory Ca2+ signaling in hepatocytes may represent a key role for ANP in vivo.