Neonatal Ethanol and Choline Treatments Alter the Morphology of Developing Rat Hippocampal Pyramidal Neurons in Opposite Directions.

Some of the neurobehavioral deficits identified in children with Fetal Alcohol Spectrum Disorders (FASDs) have been recapitulated in a binge model of gestational third trimester-equivalent ethanol (EtOH) exposure, in which Sprague-Dawley rats are intragastrically intubated between post-natal day (PD) 4 and PD9 with high doses of EtOH. In this model, the ameliorating effects of choline (Chol) administration on hippocampus-dependent behaviors altered by EtOH have also been extensively documented. In the present study, we investigated the effects of EtOH (5 g/kg/day) and/or Chol (100 mg/kg/day) on morphometric parameters of CA1 pyramidal neurons by Golgi-Cox staining followed by Neurolucida tracing and analysis. We found that EtOH increased apical dendrite complexity in male and female pups neonatally exposed to EtOH. EtOH did not significantly affect basal dendrite parameters in female and male rats. Interestingly, Chol treatments decreased basal dendrites' length, number, and maximal terminal distance in male pups. When pups were co-treated with EtOH and Chol, Chol did not rescue the effect of EtOH. In conclusion, EtOH increases while Chol decreases dendritic length and arborization of hippocampal CA1 neurons in PD9 rats. We hypothesize that developmental EtOH exposure induces a premature maturation of neurons, leading to early restriction of neuronal plasticity while Chol treatments delay the normal program of neuronal maturation and therefore prolong the window of maximal plasticity. Chol does not prevent the effects of developmental alcohol exposure on hippocampal pyramidal neurons' morphology characterized in the present study, although whether prolonged Chol administration after developmental EtOH exposure rectifies EtOH damage remains to be assessed.

Understanding the addiction cycle: a complex biology with distinct contributions of genotype vs. sex at each stage.

Ethanol abuse can lead to addiction, brain damage and premature death. The cycle of alcohol addiction has been described as a composite consisting of three stages: intoxication, withdrawal and craving/abstinence. There is evidence for contributions of both genotype and sex to alcoholism, but an understanding of the biological underpinnings is limited. Utilizing both sexes of genetic animal models with highly divergent alcohol withdrawal severity, Withdrawal Seizure-Resistant (WSR) and Withdrawal Seizure-Prone (WSP) mice, the distinct contributions of genotype/phenotype and of sex during addiction stages on neuroadaptation were characterized. Transcriptional profiling was performed to identify expression changes as a consequence of chronic intoxication in the medial prefrontal cortex. Significant expression differences were identified on a single platform and tracked over a behaviorally relevant time course that covered each stage of alcohol addiction; i.e., after chronic intoxication, during peak withdrawal, and after a defined period of abstinence. Females were more sensitive to ethanol with higher fold expression differences. Bioinformatics showed a strong effect of sex on the data structure of expression profiles during chronic intoxication and at peak withdrawal irrespective of genetic background. However, during abstinence, differences were observed instead between the lines/phenotypes irrespective of sex. Confirmation of identified pathways showed distinct inflammatory signaling following intoxication at peak withdrawal, with a pro-inflammatory phenotype in females but overall suppression of immune signaling in males. Combined, these results suggest that each stage of the addiction cycle is influenced differentially by sex vs. genetic background and support the development of stage- and sex-specific therapies for alcohol withdrawal and the maintenance of sobriety.

Optimizing a microwave gas ion source for continuous-flow accelerator mass spectrometry.

A 2.45 GHz microwave ion source coupled with a magnesium charge exchange canal (C × C) has been successfully adapted to a large acceptance radiocarbon accelerator mass spectrometry system at the National Ocean Sciences Accelerator Mass Spectrometry (AMS) Facility, Woods Hole Oceanographic Institution. CO(2) samples from various preparation sources are injected into the source through a glass capillary at 370 µl∕min. Routine system parameters are about 120-140 µA of negative (12)C current after the C × C, leading to about 400 (14)C counts per second for a modern sample and implying a system efficiency of 0.2%. While these parameters already allow us to perform high-quality AMS analyses on large samples, we are working on ways to improve the output of the ion source regarding emittance and efficiency. Modeling calculations suggest modifications in the extraction triode geometry, shape, and size of the plasma chamber could improve emittance and, hence, ion transport efficiency. Results of experimental tests of these modifications are presented.

No evidence for general condition-dependence of structural plumage colour in blue tits: an experiment.

Condition-dependence is a central but contentious tenet of evolutionary theories on the maintenance of ornamental traits, and this is particularly true for structural plumage colour. By providing diets of different nutritional quality to moulting male and female blue tits, we experimentally manipulated general condition within the natural range, avoiding deprivation or stressful treatments. We measured reflectance of the structural-coloured UV/blue crown, a sexually selected trait in males, and the white cheek, a nonpigmented structural colour, directly after moult and again during the following spring mating season. We employed a variety of colour indices, based on spectral shape and avian visual models but, despite significant variation in condition and coloration, found no evidence for condition-dependence of UV/blue or white plumage colour during either season. These and previously published results suggest that structural colour might be sensitive to stress, rather than reduced body condition, during moult.

Testosterone manipulation postcastration does not alter cloacal gland growth differences in male quail selected for divergent plasma corticosterone stress response.

Japanese quail selected for reduced (low-stress, LS) rather than exaggerated (high-stress, HS) plasma corticosterone response to brief restraint have consistently shown greater cloacal gland (CG) development, an androgen-dependent trait. In this study, the effects of testosterone implants on levels of plasma testosterone and CG development in castrated LS and HS quail were determined. Stress-line males were castrated and randomly allocated to 1 of 3 testosterone treatments: the empty testosterone (ET), low testosterone (LT), or high testosterone (HT) implant group. Cloacal gland volume was determined at 4 weekly intervals that represented ranges of 1 to 9 d, 8 to 17 d, 15 to 24 d, and 22 to 31 d after castration and testosterone implantation. Levels of plasma testosterone were also assessed at the end of the study. Development of the CG was affected by quail line (LS > HS), testosterone treatment (HT > LT > ET), and time of measurement (1 to 9 d < 8 to 17 d < 15 to 24 d = 22 to 31 d after castration and testosterone implantation). A significant interaction between testosterone treatment and time of measurement on CG volume was also detected (with CG volume generally increasing with time in LT- and HT-treated quail, but not in ET-treated quail). However, even though HT implant treatments induced higher CG development than did LT treatments beyond the first interval of CG volume measurement, and despite the finding of greater CG volumes in LS than HS quail during the last 2 measurement intervals within each of the LT and HT groups, no interaction was observed between testosterone implant dosages and quail stress line on CG volume. Thus, by the end of the study, regardless of testosterone dose, CG volume was consistently greater in LS quail than in their HS counterparts. In addition, although, as expected, the testosterone implant treatment significantly altered levels of plasma testosterone (HT > LT > ET), neither quail line nor its interaction with testosterone treatment affected plasma testosterone. The present findings suggest that the often-observed depressed CG development in the HS line may be independent of testosterone effects.

Physiological, morphological and behavioural effects of selecting zebra finches for divergent levels of corticosterone.

The effects of environmental stress on the physiology and behaviour of higher vertebrates has become an important avenue of research in recent years. Evidence from recent studies has suggested that the avian stress-related hormone corticosterone (CORT) may play a role in immunocompetence and sexual selection. We tested whether CORT is immunosuppressive by studying humoral and cell-mediated immune responses in populations of captive zebra finches selected for divergent peak levels of CORT. We also investigated whether selection for peak CORT has an effect on the quality of several sexually selected regions of the male zebra finch; in addition we compared morphometric parameters and the dominance ranking in males from the different selection lines. We also tested whether different components of the immune system compete for limited resources. We found that selection for divergent levels of peak CORT had little effect on humoral immunity, male sexual signal quality or dominance ranking. However, contrary to expectations, we did find a positive relationship between CORT titre and cell-mediated immunity, as well as a greater cell-mediated response in the birds selected for high CORT titre than those selected for low CORT titre. Consistent with predictions, significant negative relationships were found between both testosterone and CORT titre on humoral immunity. Birds from the low CORT lines were significantly larger in terms of skeletal size than those from the high CORT lines. Overall, our results suggest that the cell-mediated immune response is associated with a reduction in the humoral response, but only in males, and that there is no simple relationship between peak CORT levels and immune function.

Interaction between the blood fluke, Sanguinicola inermis and humoral components of the immune response of carp, Cyprinus carpio.

The effect of Sanguinicola inermis on serum antibody and complement activity in Cyprinus carpio was assessed using an ELISA and haemolytic assays. Possible immune evasion strategies were assessed using immunodetection of host proteins on the surface of the parasite. Carp acclimatized to 20 or 25 degrees C were infected by exposure to 500 cercariae or injected intraperitoneally with 150 cercariae, and serum monitored over a 63-day period. In cercariae-injected carp, irrespective of time and temperature, a significant increase occurred in complement activity being greatest at 25 degrees C. In addition, fish exposed to the cercariae of S. inermis and maintained at 20 degrees C the level of complement activity was significantly higher after 5 weeks compared to controls. At 20 degrees C intraperitoneal injections of parasites increased serum antibody levels which peaked after 7 days. In contrast, at 25 degrees C, antibody levels were maintained over 63 days. Exposure of fish to infection did not appear to stimulate antibody production. Immunofluorescence studies revealed 'host-like' molecules on the surface of the cercarial body exposed to carp serum and adult flukes obtained directly from the fish or cultured for 24 h in L15 medium. The possible role of 'host-like' molecules in immune evasion is discussed and the response at different temperatures is related to infection dynamics.

Recombinant micro-genes and dystrophin viral vectors.

An effective gene therapy for Duchenne muscular dystrophy ideally relies on the ability to provide long-term expression to muscle tissue of the missing protein, dystrophin. Early work in the mdx mouse using a 6.3 kb mini-dystrophin cDNA, carried out in either adenoviral or retroviral vectors was generally successful, however, expression was only transient. In an attempt to remedy this problem, two approaches are being investigated. The first of these is a hybrid vector system that combines the efficacy of gene transfer into skeletal muscle of adenoviral vectors with the long-term stability of retroviral vectors. The second utilises the inherently efficient transducing properties and stability of the adeno-associated viral delivery system. Using highly truncated micro-dystrophin cDNAs we have shown that both vector systems were able to restore dystrophin and dystrophin-associated protein expression at the plasma membrane of mdx mice for prolonged periods of time. Additionally, evaluation of central nucleation indicated a significant inhibition of degenerative dystrophic muscle pathology. These studies suggest that hybrid adenoviral-retroviral and adeno-associated viral vectors are capable of ameliorating dystrophic pathology at the cellular level and as such are useful tools in the development of a gene therapy for Duchenne muscular dystrophy.

Post-mitotic, differentiated myotubes efficiently produce retroviral vector from hybrid adeno-retrovirus templates.

We have examined the ability of proliferating myoblasts and post-mitotic, differentiated myotubes to produce retroviral vector using hybrid adeno-retroviral vectors as templates. We show that production of retroviral vector from myoblasts peaks 48 h after adenoviral infection at 4.8 x 10(4) cfu/ml and is scarcely detectable by 96 h. Both fully and partially differentiated myotubes were able to generate a sustained increase in the levels of retroviral vector compared with myoblasts peaking 48 h at 1.4 x 10(5) cfu/ml and 1.8 x 10(5) cfu/ml, respectively. Addition of the cell cycle inhibitor aphidicolin (5 microg/ml) had no effect on the production of retroviral vector from fully differentiated myotubes, but resulted in an 80% increase in vector production from partially differentiated myotubes. Thus indicating that retroviral vector production is more efficient in post-mitotic myotubes and is independent of muscle cell cycle progression.

Oxygen sensitivity in the sheep adrenal medulla: role of SK channels.

The hypoxia-evoked secretion of catecholamines from the noninnervated fetal adrenal gland is essential for surviving intrauterine hypoxemia. The ion channels responsible for the initial depolarization that leads to catecholamine secretion have not been identified. Patch-clamp studies of adrenal chromaffin cells isolated from fetal and adult sheep revealed the presence of a Ca(2+)-dependent K(+) current that was reduced by hypoxia. Apamin, a blocker of small-conductance K(+) (SK) channels, reduced the Ca(2+)-dependent K(+) current, and the sensitivity of the channels to apamin indicated that the channels involved were of the SK2 subtype. In the presence of apamin, the hypoxia-evoked change in K(+) currents was largely eliminated. Both hypoxia and apamin blocked a K(+) current responsible for maintaining the resting potential of the cell, and the depolarization resulting from both led to an influx of Ca(2+). Simultaneous application of hypoxia and apamin did not potentiate the increase in cytosolic Ca(2+) concentration beyond that seen with either agent alone. Similar results were seen with curare, another blocker of SK channels. These results indicate that closure of SK2 channels would be the initiating event in the hypoxia-evoked catecholamine secretion in the adrenal medulla.

Temperature dependence of human muscle ClC-1 chloride channel.

1. In the present work we investigated the dependence on temperature of the ionic conductance and gating of human muscle ClC-1 chloride channels, transiently expressed in human embryonic kidney (HEK 293) cells. 2. At normal pH, ClC-1 currents deactivated at negative potentials with a double-exponential time course. The time constants of the exponential components, corresponding to the relaxations of the fast and slow gates, were temperature dependent with Q(10) values of approximately 3 and approximately 4, respectively. Current amplitude increased with increasing temperature with a Q(10) of approximately 1.6. 3. The voltage dependence of the two gating processes was shifted towards more positive potentials with increasing temperature. The half-saturation voltage (V(1/2)) of the steady-state open probability (P(o)) was shifted by approximately 23 and approximately 34 mV per 10 degrees C increase in temperature, for the fast and slow gate, respectively. 4. At low pH, the voltage dependence of ClC-1 was reversed and currents were activated by hyperpolarisation with a single-exponential time course. This type of gating in ClC-1 resembled the slow gating of the Torpedo ClC-0 homologue, but differed with respect to its kinetics and temperature dependence, with a Q(10) of gating relaxations at negative potentials of approximately 5. The Arrhenius plot of ClC-1 conductance at low pH had a clear break point at approximately 25 degrees C, with higher Q(10) values at lower temperatures. 5. The temperature sensitivity of relaxation and open probability of the slow gate, which in both ClC-0 and ClC-1 controls two pores simultaneously, implies that the slow gating of ClC-1 is mechanistically different from that of ClC-0.

Fast and slow gating of CLC-1: differential effects of 2-(4-chlorophenoxy) propionic acid and dominant negative mutations.

Our knowledge about ClC-1 muscle chloride channel gating, previously gained from single-channel recording and noise analysis, provides a theoretical basis for further analysis of macroscopic currents. In the present study, we propose a simple method of calculation of open probabilities (P(o)) of fast and slow gates from the relative amplitudes of ClC-1 inward current components. With this method, we investigated the effects of 2-(4-chlorophenoxy) propionic acid (CPP), a drug known to produce myotonia in animals, and dominant negative myotonic mutations, F307S and A313T, on fast and slow gating of ClC-1. We have shown that these mutations affected the P(o) of the slow gate, as expected from their mode of inheritance, and that CPP predominantly affected the fast gating process. CPP's action on the fast gating of mutant channels was similar to its effect in wild-type channels. Comparison of the effects of CPP and the mutations on fast and slow gating with the effects produced by reduction of external Cl(-) concentration suggested that CPP and mutations exert their action by affecting the transition of the channel from its closed to open state after Cl(-) binding to the gating site.

Interaction of hydrophobic anions with the rat skeletal muscle chloride channel ClC-1: effects on permeation and gating.

Permeation of a range of hydrophobic anions through the rat skeletal muscle chloride channel, rClC-1, expressed in Sf-9 (a Spodoptera frugiperda insect cell line) cells has been studied using the whole-cell patch-clamp technique. Bi-ionic reversal potentials measured with external application of foreign anions gave the following permeability sequence: Cl- (1) > benzoate (0.15) > hexanoate (0.12) > butyrate (0.09) > propionate (0.047) approximately formate (0.046). Anions with larger hydrophobic moieties were more permeant, which suggested that ClC-1 selectivity for hydrophobic anions is dominated by their interaction with a hydrophobic region in the external mouth of the pore. All anions studied when applied from outside show an apparently paradoxical voltage-dependent block of inward currents; this voltage-dependent block could be qualitatively described by a discrete-state permeation model with two binding sites and three barriers. Effects of the external anions with aliphatic side-chains on the apparent open probability (Po) suggested that they are unable to gate the channel, but can modulate ClC-1 gating, probably, by changing Cl- affinity to the gating site. Effects of internal application of benzoate, hexanoate or propionate mimicked those of increasing internal pH, and similarly depended on the channel protonation from the external side. Results for internal benzoate support the concept of a negatively charged cytoplasmic particle being involved in the ClC-1 gating mechanism sensitive to the internal pH.

The combination of bcl-2 expression and NGF-deprivation facilitates the selective destruction of BAD protein in living sympathetic neurons.

Bcl-2 overexpression prevents neuronal death after injury or neurotrophic factor-deprivation but the biochemical consequences of survival maintenance by Bcl-2 have hardly been explored. We show that unlike NGF, adenovirally delivered hBcl-2 supports the survival of over 80% of the neurons without activating ERK and Akt phosphorylation, or suppressing JNK phosphorylation, or enhancing cell growth. However, the proapoptotic protein BAD, whose phosphorylation is induced by NGF, is degraded in NGF-deprived neurons expressing hBcl-2, while the level of Bcl-xL remains unaffected. Interestingly, degradation of BAD protein is prevented by the pan-caspase inhibitor Boc.Asp(OMe)fmk. We propose that NGF-deprivation promotes dephosphorylation of BAD while hBcl-2 facilitates its release into the cytoplasm where it is degraded by noncaspase, Boc.Asp(O-Me)fmk-inhibitable proteases. The potential importance of BAD degradation is suggested by our finding that overexpressed BAD kills NGF-maintained sympathetic neurons by apoptosis, while hBcl-2 prevents BAD-induced death.

Calcium bioavailability and parathyroid hormone acute changes after oral intake of dairy and nondairy products in healthy volunteers.

This study was designed to compare calcium bioavailability and serum parathyroid hormone acute changes after oral intake of 500 mg of elemental calcium from liquid milk, yogurt, calcium-citrate-enriched powdered milk or a calcium carbonate pill; or after intake of soybean imitation-milk. After a 12-h fast, blood samples were drawn both at baseline and 1, 2, 3 and 4 h after an oral intake of the above-mentioned products, which were ingested together with a light neutral breakfast. The administration order of the study products was randomly assigned to each of 19 healthy young volunteers (11 females, 8 males). The baseline serum concentrations of ionized calcium, phosphorus and intact parathyroid hormone (iPTH) were normal. Calcium-citrate-enriched powdered milk induced a significant increase in serum ionized calcium (p<0.001) and a significant and continuous decrease in serum iPTH concentration (p<0. 001). Yogurt and the calcium carbonate pill induced a similar but less significant effect, increasing serum ionized calcium (p<0.05) and decreasing serum iPTH (p<0.01). Liquid milk only induced a significant change in serum ionized calcium and iPTH concentration during the first 2 h; this effect was lost during the following 2 h. In conclusion, our study suggests the possibility that the addition of calcium citrate to powered milk may improve calcium bioavailability and enhance the inhibitory effect on serum iPTH in the assayed conditions.

Acid-evoked quantal catecholamine secretion from rat phaeochromocytoma cells and its interaction with hypoxia-evoked secretion.

1. Amperometric recordings using polarized carbon fibre microelectrodes were used to detect exocytosis of catecholamines from rat phaeochromocytoma (PC12) cells in response to a reduction in pHo. 2. Exocytosis was detected at pHo levels of between 7.2 and 6.8. This was probably due to intracellular acidification, since acid-evoked secretion was enhanced by the Na+-H+ exchange blocker ethylisopropylamiloride (30 microM), and was mimicked by sodium propionate (10 mM), which causes selective intracellular acidosis. 3. Acid-evoked exocytosis was abolished by removal of Ca2+o or application of 200 microM Cd2+. It was unaffected by nifedipine, but significantly reduced by either omega-conotoxin GVIA (1 microM) or omega-agatoxin GIVA (200 nM). The two toxins applied together almost completely abolished (> 97 %) acid-evoked secretion. 4. Hypoxia-evoked catecholamine release was potentiated under acidic conditions and suppressed under alkaline conditions in a manner which indicated a greater than additive interaction of these two stimuli. 5. Our results indicate that, like carotid body arterial chemoreceptors, PC12 cells represent model chemoreceptor cells for both hypoxia and acidity and that the release of catecholamines in response to these physiological stimuli is dependent on Ca2+ influx through voltage-gated N- and P/Q-type Ca2+ channels.

Potentiation of quantal catecholamine secretion by glibenclamide: evidence for a novel role of sulphonylurea receptors in regulating the Ca(2+) sensitivity of exocytosis.

Electrochemical detection of quantal catecholamine release from PC-12 cells revealed that glibenclamide, an inhibitor of ATP-sensitive K(+) channels, potentiated Ca(2+)-dependent exocytosis evoked by raised extracellular [K(+)] and by exposure of cells to caffeine. Glibenclamide was without effect on voltage-gated Ca(2+) currents, membrane potential, or rises of [Ca(2+)](i) evoked by either raised extracellular [K(+)] or caffeine. The dependence of K(+)-evoked secretion on extracellular Ca(2+) was shifted leftward in the presence of glibenclamide, with a small increase in the plateau level of release, suggesting that glibenclamide primarily increased the Ca(2+) sensitivity of the exocytotic apparatus. Enhancement of secretion by glibenclamide was reversed by pinacidil and cromakalim, indicating that the effects of glibenclamide were mediated via an action on a sulfonylurea receptor. These results demonstrate that sulfonylurea receptors can modulate Ca(2+)-dependent exocytosis via a mechanism downstream of Ca(2+) influx or mobilization.

Modulation of the gating of CIC-1 by S-(-) 2-(4-chlorophenoxy) propionic acid.

1. Using whole-cell patch-clamping and Sf-9 cells expressing the rat skeletal muscle chloride channel, rCIC-1, the cellular mechanism responsible for the myotonic side effects of clofibrate derivatives was examined. 2. RS-(+/-) 2-(4-chlorophenoxy)propionic acid (RS-(+/-) CPP) and its S-(-) enantiomer produced pronounced effects on CIC-1 gating. Both compounds caused the channels to deactivate more rapidly at hyperpolarizing potentials, which showed as a decrease in the time constants of both the fast and slow deactivating components of the whole cell currents. Both compounds also produced a concentration-dependent shift in the voltage dependence of channel apparent open probability to more depolarizing potentials, with an EC50 of 0.79 and 0.21 mM for the racemate and S-(-) enantiomer respectively. R-(+) CPP at similar concentrations had no effect on gating. RS-(+/-) CPP did not block the passage of Cl- through the pore of rCIC-1. 3. CIC-1 is gated by Cl- binding to a site within an access channel and S-(-) CPP alters gating of the channel by decreasing the affinity of this binding site for Cl-. Comparison of the EC50 for RS-(+/-) CPP and S-(-) CPP indicates that R-(+) CPP can compete with the S-(-) enantiomer for the site but that it is without biological activity. 4. RS-(+/-) CPP produced the same effect on rCIC-1 gating when added to the interior of the cell and in the extracellular solution. 5. S-(-) CPP modulates the gating of CIC-1 to decrease the membrane Cl- conductance (GCl), which would account for the myotonic side effects of clofibrate and its derivatives.

Etiology and treatment of intrinsic circadian rhythm sleep disorders.

Some individuals experience an acute or chronic sleep disturbance, associated with a misalignment between the timing of their sleep and the sleep-wake cycle that is desired, or considered normal by society. It is estimated that 5-10% of insomniacs seeking treatment have this type of disorder, collectively called circadian rhythm sleep disorders. This paper reviews circadian rhythm sleep disorders of the intrinsic type, which include delayed sleep phase syndrome, advanced sleep phase syndrome, non-24-hour sleep-wake syndrome, and irregular sleep-wake pattern. For each disorder, we present data addressing its pathophysiology and potential treatments, including the use of behavioral measures and chronotherapy, bright light treatment and pharmacological treatments such as melatonin. We conclude by addressing some of the limitations and drawbacks of the various treatments.

Interleukin-1 beta and reactive oxygen species mediate activation of c-Jun NH2-terminal kinases, in human epithelial cells, by two independent pathways.

The c-Jun N terminal kinases (JNKs) are members of the mitogen activated protein kinases family, which have been shown to be preferentially activated either by cytokines or stress stimuli. In this study we identify a selective and potent antisense oligonucleotide to RhoA (ISIS 17131) and investigate its effect on JNK activation induced by IL-1beta and H2O2 in A549 cells. The RhoA antisense oligonucleotide was able to inhibit JNK activation when A549 cells were stimulated by H2O2, but did not have any effect on IL-1beta induced JNK activation. Consistent with the idea that the phosphatidylinositol 3-kinase (PI 3-kinase) activates the small G protein exchange factors, H2O2 activated the PI 3-kinase. Additionally, Wortmannin, a potent inhibitor of the PI 3-kinase and phospholipase A2 (PLA2), and AACOCF3, also a PLA2 inhibitor, were able to inhibit JNK activation induced by H2O2, but they had no effect on JNK activation when stimulated by IL-1beta. These results suggest that, in A549, IL-1beta and H2O2 induce JNK activation by two independent pathways.