Dose-dependent down-regulation of ß-adrenoceptors by isoproterenol in rat C6 glioma cells

Nano- and micromolar isoproterenol concentrations were compared by studying cyclic AMP, ß-adrenoceptor density and ß₁-adrenoceptor mRNA in rat C₆ glioma cells. 1 μM isoproterenol significantly changed all parameters at 15–30 min. The ß₁-antagonist metoprolol attenuated the response. No effects of nanomolar isoproterenol on these early changes were observed, although the density of ß-adrenoceptors was significantly reduced beginning at 12 h. The results indicate a different process for ß-adrenoceptor desensitization in C₆ cells following physiologically low agonist concentrations.     

Effect of removing the endothelial cells on the reactivity of rat aortic segments to different α-adrenoceptor agonists

Summary The influence of removing the endothelial cells on the -receptor-mediated contractile response in segments of rat aorta was investigated using agonists with a range of affinity for ₁ and ₂. The preferential ₁ were methoxamine, cirazoline, ST 587, and Sgd 101/75 and the preferential ₂ were B-HT 920, clonidine, and guanfacine. When the endothelium was intact, the intrinsic activity (compared to noradrenaline) varied widely (0.0–0.7) for both groups of agonists. After removal of the endothelium the intrinsic activity was increased in each case to that of noradrenaline, or close to it. Furthermore, an increase in potency was obtained for each agonist, although to different degrees. No correlation, however, was found between the selectivity of the agonists and the degree of enhancement caused by the removal of the endothelium, in terms of either the intrinsic activity or the potency. Moreover, the use of the selective ₂ antagonist rauwolscine on intact tissues did not mimic the effect of removing the endothelium. Therefore, the -receptors of the endothelium could not be classified as either of the ₁ or ₂.This study was supported by a grant of the Deutsche Forschungsgemeinschaft.     

Influence of the A and B subunits of cholera toxin (CT) and Escherichia coli toxin (LT) on TNF-α release from macrophages

In this study an in vitro model was developed with the aim of investigating the modulatory effect of cholera toxin (CT) and its counterpart the heat labile toxin of Escherichia coli (LT) on TNF-α release induced by murine macrophages and primary human monocytes. Previous studies have demonstrated that the enzymatic activity of CT and LT molecules can inhibit TNF-α release by macrophages. The results obtained in this study showed that CT and LT are both, in a dose dependent manner, able either to induce or inhibit TNF-α release by murine macrophages and primary human monocytes. The results also showed that recombinant B subunits of CT and LT in the absence of their A subunit induce high levels of TNF-α release by macrophages and, in addition, increase the level of TNF-α release induced by LPS. The ability of both B subunits (CTB and LTB) in inducing TNF-α release by macrophages is not related to the level of LPS contamination, since direct measurements of LPS made in the samples employed in this study showed only traces of LPS (3.4 × 10⁻⁸ EU/ml) which is in our system does not induce TNF-α release by macrophages. In contrast to the results obtained with the B subunits, incubation of cells with the A subunit of CT (CTA) inhibit TNF-α release induced by native CT, native LT, recombinant LTB and LPS. This inhibitory effect must be related to the activity of the A subunit since viability tests performed in terms of metabolic rate demonstrated that high concentrations of CTA are not toxic to the cells. The data presented herein demonstrate that the A subunits of CT and LT have an inhibitory effect on TNF-α release in macrophages, whereas their B subunits have a stimulatory effect on TNF-α. The results also suggest that the dose dependent bi-modal effect of native CT and native LT on TNF-α release by macrophages is a result of the combined effect of their individual A and B subunits.     

effect of ginsenoside Rd on nitric oxide system induced by lipopolysaccharide plus TNF-α in C6 rat glioma cells

Effects of ginsenosides on nitric oxide (NO) production induced by lipopolysaccharide plus TNF-alpha (LNT) were examined in C6 rat glioma cells. Among several ginsenosides, ginsenoside Rd showed a complete inhibition against LNT-induced NO production. Ginsenoside Rd attenuated LNT-induced increased phosphorylation of ERK. Among several immediate early gene products, only Jun B and Fra-1 protein levels were increased by LNT, and ginsenoside Rd attenuated Jun B and Fra-1 protein levels induced by LNT. Furthermore, LNT increased AP-1 DNA binding activities, which were partially inhibited by ginsenoside Rd. Our results suggest that ginsenoside Rd exerts an inhibitory action against NO production via blocking phosphorylation of ERK, in turn, suppressing immediate early gene products such as Jun B and Fra-1 in C6 glioma cells.     

Effects of anodal polarization on protein kinase Cγ (PKCγ) in the rat brain

An anodal direct current of 0.3 microA or 30.0 microA was unilaterally applied for 30 min or 3 hr to the surface of the sensorimotor cortex of rats, and the effects of anodal polarization on protein kinase C (PKCgamma) activity were examined. The brains were processed by means of immunocytochemistry using the monoclonal antibody 36G9 raised against purified PKCgamma. In sham-operated animals, PKCgamma-like-immunoreactivity (PKCgamma LI) was noted in neuronal cytoplasm, as well as in processes in the cerebral cortex and in the hippocampus. Anodal polarization with 3.0 miroA for 30 min resulted in a pronounced increase in the number of PKCgamma-like-positive neurons in accordance with the intensity of immunostaining in the cerebral cortex, and an increase in the polarized hemispheres was highlighted by repeated applications of the currents. Polarization with 0.3 microA for 3 hr also increased the PKCgamma LI, but 0.3 microA for 30 min or 30.0 microA for any duration had no effects. The effect of polarization on PKCgamma activity, as evaluated by the intensity of immunostaining and the number of neurons, began to increase 1 h after polarization, peaked at 3 hr and thereafter decreased to the control levels by 72 hr. These results indicated the involvement of the gamma-isoform of PKC in the neurochemical mechanism of long-standing central and behavioral changes induced by anodal polarization.     

Increased transport of (G-14C)cytidine into rat liver after administration of α-hexachlorocyclohexane

Summary The transport of cytidine into liver over a wide dose range (0.003–100 mol per animal) proceeds as an nonsaturable process. After the administration of -hexachlorocyclohexane (-HCH)¹ the transport of (G-¹⁴C) cytidine is markedly activated if the concentration of nucleoside administered is low; the differences between the control and experimental group disappear after the administration of higher doses of the nucleoside. The incorporation of (G-¹⁴C) cytidine into RNA cytosine is enhanced after the administration of -HCH. The degree of utilization of labeled cytidine for the RNA synthesis decreases in proportion to the logarithm of its dose, both in the control and in the experimental group. Increased transport of (G-¹⁴C)cytidine is observable even between the 4th and 6th day after the administration of a single dose of -HCH.Abbreviations used -HCH -1,2,3,4,5,6-hexachlorocyclohexane - MFO mixed-function oxygenases     

Involvement of adenylate cyclase and protein kinase C in the α2-adrenoceptor-mediated inhibition of noradrenaline and 5-hydroxytryptamine release in rat hypothalamic slices

Summary In superfused rat hypothalamic slices prelabelled with [³H]-noradrenaline, the ₂-adrenoceptor agonist UK 14304 inhibited in a concentration-dependent manner the electrically-evoked release of tritium. This inhibition was antagonized by the ₂-adrenoceptor blocking agent idazoxan, which by itself increased the electrically-evoked tritium overflow. Exposure to forskolin, an adenylate cyclase activator, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of forskolin (1 mol/l), both the inhibitory effect of UK 14304 and the increasing effect of idazoxan on the electrically-evoked release of [³H]-noradrenaline were less pronounced than in the absence of the adenylate cyclase activator. Exposure to forskolin and to the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine shifted to the right the concentration-effect curve for UK 14304 in a similar manner as that observed in the presence of forskolin alone. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l), a drug which activates protein kinase C, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), the concentration effect curve for UK 14304 on tritium overflow was significantly shifted to the right. The increasing effect of idazoxan on tritium overflow was significantly less pronounced in the presence of 1 mol/l phorbol-12,13-dibutyrate.In superfused rat hypothalamic slices prelabelled with [³H]-5-hydroxytryptamine, the ₂-adrenoceptor agonist UK 14304 significantly inhibited the electrically-evoked release of tritium. Exposure to forskolin increased in a concentration-dependent manner [³H]-5-hydroxytryptamine overflow, but did not modify the UK 14304-mediated inhibition. Exposure to 3-isobutyl-1-methylxanthine enhanced the electrically-evoked release of [³H]-5-hydroxytryptamine. In the presence of both forskolin (1 mol/l) and 3-isobutyl-l-methylxanthine (1 mmol/l), the concentration-response curve for UK 14304 was significantly shifted to the right. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l) enhanced in a concentration-dependent manner the electrically-evoked overflow of [³H]-5-hydroxytryptamine. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), UK 14304 was significantly less potent to inhibit tritium release than in the absence of the protein kinase C activator.It is concluded that both cyclic AMP and phosphoinositide turnover are involved in the modulation of noradrenaline and 5-hydroxytryptamine release by presynaptic ₂-adrenoceptors in rat hypothalamic slices. However, these interactions do not represent definitive proof for a cause-effect relationship for the second messengers mediating the ₂-adrenoceptor induced inhibition of transmitter release either as autoreceptor or as heteroreceptor.Send offprint requests to S. Z. Langer at the above address     

Correlation of rat cortical Fas-associated death domain (FADD) protein phosphorylation with the severity of spontaneous morphine abstinence syndrome: role of α(2)-adrenoceptors and extracellular signal-regulated kinases

Fas-associated death domain (FADD) phosphorylation was recently implicated in opiate-induced neuroplasticity. To further explore the role of FADD in the mechanisms of morphine-induced physical dependence, the regulation of cortical p-FADD (and their interactions with α(2)-adrenoceptors and other signalling pathways) was assessed during spontaneous opiate withdrawal (SW) in morphine-dependent rats (10-100 mg/kg for 6 days). The main results indicated that oligomeric p-FADD in the cerebral cortex mirrored the time course of morphine SW (12-96 h), which resulted in a striking correlation between p-FADD and the intensity (behavioural scores) of morphine abstinence (Spearman correlation coefficient: 0.59, n = 39, p < 0.0001). The inactivation of brain α(2)-adrenoceptors (EEDQ at SW 12 h) further enhanced morphine abstinence intensity and cortical p-FADD content at SW 24 h. The disruption of ERK1/2 signalling (SL 327 at SW 4 h and SW 8 h) did not alter morphine abstinence at SW 12 h, but it attenuated the behavioural syndrome at SW 24 h. This inhibition of ERK1/2, however, did not prevent the up-regulation of oligomeric p-FADD at SW 12 h and 24 h. These data indicate that cortical oligomeric p-FADD, mainly through an interaction with inhibitory α(2)-adrenoceptors, plays a functional role in the behavioural expression of morphine abstinence in rats.     

Mechanisms related to the cardioprotective effects of protein kinase C epsilon (PKC ɛ) peptide activator or inhibitor in rat ischemia/reperfusion injury

The role of protein kinase C epsilon (PKC varepsilon) in polymorphonuclear leukocyte (PMN)-induced myocardial ischemia/reperfusion (MI/R) injury and novel-related mechanisms, such as regulation of vascular endothelium nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) release from blood vessels, have not been previously evaluated. A cell-permeable PKC varepsilon peptide activator (1-10 muM) significantly increased endothelial NO release from non-ischemic rat aortic segments (p < 0.01). By contrast, PKC varepsilon peptide inhibitor (1-10 muM) dose-dependently decreased NO release (p < 0.01). Then, these corresponding doses of PKC varepsilon activator or inhibitor were examined in MI/R. The PKC varepsilon inhibitor (5 muM given during reperfusion, n = 6) significantly attenuated PMN-induced postreperfused cardiac contractile dysfunction and PMN adherence/infiltration (both p < 0.01), and expression of intracellular adhesion molecule-1 (ICAM-1; p < 0.05). By contrast, only PKC varepsilon activator pretreated hearts (5 muM PKC varepsilon activator given before ischemia (PT), n = 6), not PKC varepsilon activator given during reperfusion (5 muM, n = 6) exerted significant cardioprotection (p < 0.01). Moreover, the NO synthase inhibitor, N(G)-nitro-L: -arginine methyl ester, did not block the cardioprotection of PKC varepsilon inhibitor, whereas it completely abolished the cardioprotective effects of PKC varepsilon activator PT. In addition, PKC varepsilon inhibitor (0.4 mg/kg) significantly decreased H(2)O(2) release during reperfusion in a femoral I/R model (p < 0.01). Therefore, the cardioprotection of PKC varepsilon inhibitor maybe related to attenuating ICAM-1 expression and H(2)O(2) release during reperfusion. By contrast, the cardioprotective effects of PKC varepsilon activator PT may be mediated by enhancing vascular endothelial NO release before ischemia.     

Chronic exposure to sub-lethal concentration of a glyphosate-based herbicide alters hormone profiles and affects reproduction of female Jundiá (Rhamdia quelen)

This work was carried out to verify the effect of a glyphosate-based herbicide on Jundiá hormones (cortisol, 17β-estradiol and testosterone), oocyte and swim-up fry production. Earthen ponds containing Jundiá females were contaminated with glyphosate (3.6 mg/L); blood samples were collected from eight females from each treatment immediately before, or at 1, 10, 20, 30 and 40 days following contamination. A typical post-stress rise in cortisol levels was observed at the 20th and 40th days following exposure to glyphosate. At the 40th day, 17β-estradiol was decreased in the exposed females. A similar number of oocytes were stripped out from females from both groups; however, a lower number of viable swim-up fry were obtained from the herbicide exposed females, which also had a higher liver-somatic index (LSI). The results indicate that the presence of glyphosate in water was deleterious to Rhamdia quelen reproduction, altering steroid profiles and egg viability.     

Comparison of the effects of nicotine upon the transcellular electrical resistance and sucrose permeability of human ECV304/rat C6 co-cultures and human CaCo₂ cells

It is now well established that nicotine adversely affects the integrity of the blood-brain barrier (BBB). In contrast, nicotine has been reported to increase the transendothelial electrical resistance (TEER) of CaCo₂ colon cancer cells. In the present study, the effects of nicotine upon the TEER and sucrose permeability of ECV304/C6 co-cultures and, for comparative purposes, CaCo₂ cells has been investigated. Neither ECV304 nor C6 cells were found to express measurable membrane levels of nicotinic acetylcholine receptors, as assessed by [³H]-epibatidine binding. Nicotine treatment (0.01-1 μM) for up to 48 h had little or no effect upon the TEER or sucrose permeability of either ECV304/C6 co-cultures or CaCo₂ cells. It is concluded that in contrast to the situation for the BBB, ECV304 cells lack nicotinic acetylcholine receptors and the barrier properties of ECV304/C6 co-cultures are not affected to any important extent by nicotine. This study underlines the conclusions made by other authors that the ECV304/C6 co-culture system is of limited validity as a model of the BBB.     

AM404 and VDM 11 non-specifically inhibit C6 glioma cell proliferation at concentrations used to block the cellular accumulation of the endocannabinoid anandamide

AM404 [ N-(4-hydroxyphenyl)arachidonylamide] and VDM 11 [(5 Z,8 Z,11 Z,14 Z)- N-(4-hydroxy-2-methylphenyl)-5,8,11,14-eicosatetraenamide] are commonly used to prevent the cellular accumulation of the endocannabinoid anandamide, and thereby to potentiate its actions. However, it has been reported that AM404 can produce an influx of calcium into cells, which might be expected to have deleterious effects on cell proliferation. In the present study, AM404 and VDM 11 were found to reduce C6 glioma cell proliferation with IC(50) values of 4.9 and 2.7 microM, respectively. The inhibition of cell proliferation following a 96-h exposure was not accompanied by dramatic caspase activation, and was not prevented by either a combination of cannabinoid and vanilloid receptor antagonists, or by the antioxidant alpha-tocopherol, suggestive of a non-specific mode of action. Similar results were seen with palmitoylisopropylamide, although this compound only produced significant inhibition of cell proliferation at 30 microM concentrations. AM404 (1 microM), VDM 11 (1 microM) and palmitoylisopropylamide (3-30 microM), i.e. concentrations producing relatively modest effects on cell proliferation per se, reduced the vanilloid receptor-mediated antiproliferative effects of anandamide, as would be expected for compounds preventing the cellular accumulation of anandamide (and thereby access to its binding site on the vanilloid receptor). It is concluded that concentrations of AM404 and VDM 11 that are generally used to reduce the cellular accumulation of anandamide have deleterious effects upon cell proliferation, and that lower concentrations of these compounds may be more appropriate to use in vitro.     

Characterization of α(2B)-adrenoceptor ligand binding in the presence of muscarinic toxin α and delineation of structural features of receptor binding selectivity

Nicotinic acetylcholine receptors mediate fast cholinergic modulation of glutamatergic transmission and synaptic plasticity. Here we investigated the effects of subtype selective activation of the α7 nicotinic acetylcholine receptors on hippocampal transmission and the inhibition of synaptic long-term potentiation by the Alzheimer's disease associated amyloid ?-protein (A?). The α7 nicotinic acetylcholine receptor agonist "compound A" ((R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl))thiophene-2-carboxamide) induced a rapid-onset persistent enhancement of synaptic transmission in the dentate gyrus in vitro. Consistent with a requirement for activation of α7 nicotinic acetylcholine receptors, the type II α7-selective positive allosteric modulator PheTQS ((3aR, 4S, 9bS)-4-(4-methylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) potentiated, and the antagonist methyllycaconitine (MLA) prevented the persistent enhancement. Systemic injection of the agonist also induced a similar MLA-sensitive persistent enhancement of synaptic transmission in the CA1 area in vivo. Remarkably, although compound A did not affect control long-term potentiation (LTP) in vitro, it prevented the inhibition of LTP by A?1-42 and this effect was inhibited by MLA. These findings strongly indicate that activation of α7 nicotinic acetylcholine receptors is sufficient to persistently enhance hippocampal synaptic transmission and to overcome the inhibition of LTP by A?.     

4-Nitrophenol induces Leydig cells hyperplasia,which may contribute to the differential modulation of the androgen receptor and estrogen receptor-α and -β expression in male rat testes

4-Nitrophenol (PNP) is generally regarded as an environmental endocrine disruptor capable of estrogenic and anti-androgenic activities. To investigate PNP-induced reproductive effects, immature male rats were injected subcutaneously with PNP (0.1, 1, 10 mg/kg body weight or vehicle) daily for 4 weeks. We assessed reproductive tract alterations, sex hormone balance in the serum and estrogen receptor (ER)-α, -β and androgen receptor (AR) expression in testes. Although no significant difference was observed in body weight or testes weights of PNP-treated rats compared with the controls, the serum concentrations of testosterone in the 10 mg/kg PNP-treated group were significantly elevated. This effect was accompanied by Leydig cells hyperplasia in the testes. Conversely, there was a significant decrease in estradiol concentration and aromatase expression in the testes of the 10 mg/kg PNP-treated group. Furthermore, we observed a significant increase in ERα expression in the testes of the 10 mg/kg PNP-treated group compared with the control group. Conversely, ERβ expression displayed a significant reduction. Moreover, AR expression was significantly increased in the 10 mg/kg PNP-treated group compared with the control group. The existence of AR, ER-α and -β in the testes suggests that estradiol and testosterone directly affect germ cells and that differential modulation of AR, ER-α and -β in the testis may be involved in the direct effects of PNP or either the indirect effects of PNP-induced disruption of the estradiol-to-testosterone balance or the Leydig cells hyperplasia. Thus, the measurement of many endpoints is necessary for good risk assessment.     

Effect of N-methyl-D-aspartate (NMDA) receptor antagonists on α-synuclein-evoked neuronal nitric oxide synthase activation in the rat brain

α-Synuclein (ASN), a small presynaptic protein that is abundant in the brain, is implicated in the pathogenesis of neurodegenerative disorders including Parkinson’s and Alzheimer’s disease. The central domain of α-synuclein, the non-amyloid β component of the Alzheimer’s disease amyloid (NAC) is probably responsible for its toxicity. However, the molecular mechanism of α-synuclein action remains largely elusive. The present study examined the effect of α-synuclein and the NAC peptide on nitric oxide synthase (NOS) activity in rat brain cortical and hippocampal slices using a radiochemical technique. Moreover, nitrite levels in brain slices incubated in the presence of α-synuclein were measured using the Griess reaction. ASN and the NAC stimulated NOS activity by about 70% and 40%, respectively. β-Synuclein, a homologous protein of ASN that lacks the NAC domain, had no effect on NOS activity. Under the same experimental conditions, α-synuclein increased nitrite levels by 27%. α-Synuclein and the NAC affected the activity of constitutive neuronal isoform of NOS, but had no impact on the endothelial or inducible NOS isoforms. The effect of α-synuclein and the NAC peptide on NOS activity was inhibited by MK-801 and APV, antagonists of the NMDAreceptor. These results indicate that the NMDAreceptor plays an important role in α-synuclein-evoked nitric oxide synthesis. We suggest that nitric oxide liberated by the over-activated neuronal isoform of NOS could react with superoxide to form peroxynitrite, which modulates the function of a variety of biomolecules including proteins, lipids, and DNA.     

Role of protein kinase C epsilon (PKCɛ) in the reduction of ethanol reinforcement due to mGluR5 antagonism in the nucleus accumbens shell

Rationale  The type 5 metabotropic glutamate receptor (mGluR5) and the epsilon isoform of protein kinase C (PKCɛ) regulate ethanol intake, and we have previously demonstrated that mGluR5 receptor antagonism reduces ethanol consumption via a PKCɛ-dependent mechanism. Objectives  We explored the potential neuroanatomical substrates of regulation of ethanol reinforcement by this mGluR5-PKCɛ signaling pathway by infusing selective inhibitors of these proteins into the shell or core region of the nucleus accumbens (NAc). Methods  Male Wistar rats were trained to self-administer ethanol intravenously and received intra-NAc infusions of vehicle or the selective mGluR5 antagonist 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine (MTEP) alone and in combination with a PKCɛ translocation inhibitor (ɛV1–2) or a scrambled control peptide (sɛV1–2). The effects of intra-NAc MTEP on food-reinforced responding and open-field locomotor activity were also determined. Results  MTEP (1 μg/μl) had no effect on ethanol or food reinforcement or locomotor activity when infused into either region. MTEP (3 μg/μl) reduced ethanol reinforcement when infused into the NAc shell but not the core, and this effect was reversed by ɛV1–2 (1 μg/μl) but not sɛV1–2 (1 μg/μl). In both regions, this concentration of MTEP did not alter food-reinforced responding or locomotor activity, and infusion of ɛV1–2 alone did not alter ethanol reinforcement. MTEP (10 μg/μl) reduced locomotor activity when infused into the shell; therefore, this concentration was not further tested on responding for ethanol or food. Conclusions  Blockade of mGluR5 receptors in the NAc shell reduces ethanol reinforcement via a PKCɛ-dependent mechanism.