Donation programme of returned medicines: role of donors and point of view of beneficiaries.

Background: Donation of returned medicines is a debated health policy issue as it is discouraged by WHO, but accepted in some countries. Methods: Lessons learned from a donation programme of returned medicines carried out in Europe were documented. Results: The donation programme we reviewed followed a strict protocol for collection, sorting and distribution of returned drugs, in order to avoid the major limitations associated with unused medicine donations. Over a period of 3 years, 23 145 boxes of medicines were donated to 14 organizations operating in Europe, Africa and Latin America. Conclusions: The donations covered about one-third of the volume of medicines used by beneficiary organizations. The programme helped to decrease expenditure by both patients and health facilities.

In vitro mitochondrial failure and oxidative stress mimic biochemical features of Alzheimer disease.

Primary cortical neurons exposed to the mitochondrial toxin NaN3 (0.1-3 mM) were submitted to oxidative stress with H2O2 (30-150 µM), to mimic conditions observed in neurodegenerative disorders. The effects of such treatment on a series of parameters useful in characterizing neuronal damage were investigated: (i) the basal release of glutamate, evaluated as (3)H-d-Aspartate efflux, was sharply, concentration-dependently, increased; (ii) the phosphorylation status of intracellular markers known to be involved in the neurodegenerative processes, in particular in Alzheimer disease: tau and GSK3ß were increased, as well as the protein level of ß-secretase (BACE1) and p35/25 evaluated by Western blotting, while (iii) the cell metabolic activity, measured with the MTT method, was reduced, in a concentration- and time-dependent manner. The latter effect, as well as tau hyperphosphorylation, was prevented both by a mixture of antioxidant drugs (100 µM ascorbic acid, 10 µM trolox, 100 µM glutathione) and by the anti-Alzheimer drug, memantine, 20 µM. Since it is well known that hippocampal cholinergic neurons are particularly affected in Alzheimer disease, the effects of NaN3 and H2O2 were also studied in electrically stimulated rat hippocampal slices, evaluating the (3)H-Choline efflux, as an index of acetylcholine release. The neurotoxic treatment depressed the neurosecretory function and the mixture of antioxidant drugs, as well as memantine, were able to restore it. The neuronal damage induced by the in vitro protocol adopted in the present work displays peculiarities of neurodegenerative disorders, e.g. Alzheimer disease, underlining the role of mitochondrial failure and oxidative stress, which appear to occur upstream the neurodegenerative process; such protocol could be utilized to test the efficacy of neuroprotective treatments.

Effects of synthetic peptides on the inflammatory response and their therapeutic potential.

Recently, interest in small peptide molecules as potential drug candidates has revived. In this review, two series of synthetic peptides and their selective effects on the inflammatory response have been described, focusing on the intracellular pathways involved and on their therapeutic potential. A series of F(D)LF(D)LF analogs has been synthesized, including either N- t-Boc or different N-ureido substituents. The free acid derivatives as they are good candidates as antiinflammatory drugs are able to antagonize the multiple neutrophil functions evoked by N-formyl-L-methionyl-L-leucyl-Lphenylalanine (fMLF), i.e. chemotaxis, superoxide anion production and lysozyme release. Their methyl-ester derivatives are ineffective. The second series of peptides derives from the endogenous protein kinase C (PKC) inhibitor PKI55, a 55-amino acid protein, whose synthesis is induced by PKC activation, so that a feedback loop of inhibition is established. In vitro experiments showed that PKI55 inhibits recombinant PKC isoforms α, ß1, ß2, γ, δ, ζ, ; to identify the minimal amino acid sequence of PKI55 protein maintaining the inhibitory effects on PKC, peptides derived from both C- and N-terminal sequences have been synthesized. The N-terminal peptides 5 (MLYKLHDVCRQLWFSC), 8 (CRQLWFSC) and 9 (CRQLW), that in human neutrophils retain the inhibitory activity on PKC, decrease the chemotaxis, and, in mice, display anti-inflammatory and analgesic action, after both central and peripheral administration of very low doses. Furthermore, the peptide 5 shows neuroprotective activity in a model of cerebral ischemia in vitro, favouring the recovery of synaptic function. These findings suggest interesting possible therapeutic applications for these peptides.

Anti-inflammatory and analgesic effects displayed by peptides derived from PKI55 protein, an endogenous protein kinase C inhibitor.

We recently characterized the PKI55 protein as an endogenous protein kinase C (PKC) inhibitor and investigated, in vitro, the potential anti-inflammatory actions of its N-terminal peptides 1-16 (peptide 5), 1-8 (peptide 8) and 1-5 (peptide 9). We showed their ability to inhibit chemotaxis in human polymorphonuclear leukocytes activated by the N-formyl tripeptide for-Met-Leu-Phe-OMe. In this work, we evaluated the anti-inflammatory and the analgesic effects of the selected peptides by in vivo experiments carried out in the mouse. The peptides 5, 8 and 9 (0.1 and 10 nmol i.c.v.) were effective in both the parameters chosen to test the anti-inflammatory activity, i.e., the xylene-induced ear edema and the acetic acid-induced infiltration of neutrophils in the peritoneal cavity. In addition, they displayed analgesic effect, evaluated by the acetic acid-induced writhing test. All the peptides' effects were shared by the reference compounds, dexamethasone and indomethacin (10 mg kg(-1) i.p.), but not by the 9-scramble peptide (10 nmol i.c.v.). The peptide 9, which represents the shortest active sequence of the PKI55 protein, was tested in the ear edema model even following intraperitoneal (i.p.) administration and proved to be effective in the range doses 3-30 mg kg(-1). Moreover, an increase in plasma corticosterone levels was detected in mice treated with the peptide 9, but not with the 9-scramble peptide (both at 10 nmol i.c.v.). The anti-inflammatory and analgesic effects of the PKI55-derived synthetic peptides, possibly related both to PKC inhibition and hypothalamic-pituitary-adrenal axis activation, deserve further investigation in view of potential therapeutic exploitation.

Swim stress enhances nociceptin/orphanin FQ-induced inhibition of rat dorsal raphe nucleus activity in vivo and in vitro: role of corticotropin releasing factor.

The effects of nociceptin/orphanin FQ on putative serotonin (5HT) neurons of the dorsal raphe nucleus (DRN), known to modulate the behavioral responses to stress, were investigated in vivo and in vitro. In DRN slices from unstressed rats, nociceptin/orphanin FQ concentration-dependently inhibited the firing rate of putative 5HT neurons (EC(50) = 21.6 +/- 1.21 nM) and the selective NOP receptor antagonist UFP-101 shifted the concentration-response curve to the right (estimated pA(2) 6.86). Nociceptin/orphanin FQ potency was enhanced in slices prepared from rats previously subjected to a 15 min swim stress (EC(50) = 1.98 +/- 0.11 nM). Swim stress did not change the number or affinity of NOP receptors in DRN. Stress-elicited potentiation involved corticotropin-releasing factor (CRF)(1) receptors, GABA signaling and protein synthesis, being attenuated by pre-treatment with antalarmin (20 mg/kg, i.p.), diazepam (2.4 mg/kg, i.p.) and cycloheximide (2.5 mg/kg, i.p.), respectively. In anesthetized unstressed rats, locally applied nociceptin/orphanin FQ (0.03 and 0.1 ng/30 nl) inhibited the firing rate of DRN neurons (to 80 +/- 7 and 54 +/- 10% of baseline, respectively). Nociceptin/orphanin FQ inhibition was potentiated both 24 h after swim stress and 1 h after CRF (30 ng/30 nl intra-DRN). Stress-induced potentiation was prevented by the selective CRF(1) receptor antagonist, NBI 30755 (20 mg/kg, i.p.). In contrast, the inhibitory response of DRN neurons to the 5HT(1A) agonist, 8OH-DPAT (1 microg/1 microl, intra-DRN) was not potentiated by swim stress, ruling out a non-specific enhanced permeability of GIRK channel. Together, these findings suggest that CRF and the nociceptin/orphanin FQ/NOP system interact in the DRN during stress to control 5HT transmission; this may play a role in stress-related neuropsychopathologies.

Inhibition of serotonin outflow by nociceptin/orphaninFQ in dorsal raphe nucleus slices from normal and stressed rats: Role of corticotropin releasing factor.

In the dorsal raphe nucleus (DRN) many inputs converge and interact to modulate serotonergic neuronal activity and the behavioral responses to stress. The effects exerted by two stress-related neuropeptides, corticotropin releasing factor (CRF) and nociceptin/orphaninFQ (N/OFQ), on the outflow of [(3)H]5- hydroxytryptamine were investigated in superfused rat dorsal raphe nucleus slices. Electrical stimulation (100 mA, 1 ms for 2 min) evoked a frequency-dependent peak of [(3)H]5- hydroxytryptamine outflow, which was sodium and calcium-dependent. Corticotropin releasing factor (1-100 nM), concentration-dependently inhibited the stimulation (3 Hz)-evoked [(3)H]5-hydroxytryptamine outflow; the inhibition by 30 nM corticotropin releasing factor (to 68 +/- 5.7%) was prevented both by the non selective CRF receptor antagonist alpha-helicalCRF(9-41) (alpha-HEL) (300 nM) and by the CRF(1) receptor antagonist antalarmin (ANT) (100 nM). The CRF(2) agonist urocortin II (10 nM) did not modify [(3)H]5- hydroxytryptamine outflow, ruling out the involvement of CRF(2) receptors. Bicuculline (BIC), a GABAA antagonist (10 microM), prevented the inhibitory effect of corticotropin releasing factor (30 nM), supporting the hypothesis that the inhibition was mediated by increased gamma-aminobutyric acid (GABA) release. Nociceptin/ orphaninFQ (1 nM-1 microM) exerted an antalarmin- and bicuculline-insensitive inhibition on [(3)H]5- hydroxytryptamine outflow, with the maximum at 100 nM (to 63+/- 4.2%), antagonized by the NOP receptor antagonist UFP-101 (1 microM). Dorsal raphe nucleus slices prepared from rats exposed to 15 min of forced swim stress displayed a reduced [(3)H]5-hydroxytryptamine outflow, in part reversed by antalarmin and further inhibited by nociceptin/orphaninFQ. These findings indicate that (i) both corticotropin releasing factor and nociceptin/orphaninFQ exert an inhibitory control on dorsal raphe nucleus serotonergic neurons; (ii) the inhibition by corticotropin releasing factor involves gamma-aminobutyric acid neurons; (iii) nociceptin/ orphaninFQ inhibits dorsal raphe nucleus serotonin system in a corticotropin releasing factor- and gamma-aminobutyric acid-independent manner; (iv) nociceptin/orphaninFQ modulation is still operant in slices prepared from stressed rats. The nociceptin/orphaninFQ-NOP receptor system could represent a new target for drugs effective in stress-related disorders.

Sodium azide induced neuronal damage in vitro: evidence for non-apoptotic cell death.

The features of neuronal damage induced by the mitochondrial toxin NaN(3) were investigated in rat primary cortical neuron cultures. Cell viability (MTT colorimetric determination) and transmembrane mitochondrial potential (J-C1 fluorescence) were concentration-dependently reduced 24 h after NaN(3); neither nuclear fragmentation by DAPI, nor Annexin V positivity by flow cytometry were detected, ruling out the occurrence of apoptosis. The loss in cell viability (to 54 +/- 2%) observed 24 h after a 10-min treatment with 3 mM NaN(3) was prevented by the NMDA glutamate receptor antagonist MK801 (1 microM), by the antioxidants trolox (100 microM) and acetyl-L-carnitine (1 mM) and by the nitric oxide synthase inhibitor, L-NAME (100 microM), but not by the guanylylcyclase inhibitor ODQ, 10 microM. The mitochondrial dysfunction induced by NaN(3) provides a common platform for investigating the mechanisms of both ischemic and degenerative neuronal injury, useful for screening potential protective agents against neuronal death.

Study of synthetic peptides derived from the PKI55 protein, a protein kinase C modulator, in human neutrophils stimulated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH.

Elucidation of the involvement of protein kinase C subtypes in several diseases is an important challenge for the future development of new drug targets. We previously identified the PKI55 protein, which acts as a protein kinase C modulator, establishing a feedback loop of inhibition. The PKI55 protein is able to penetrate the cell membrane of activated human T-lymphocytes and to inhibit the activity of alpha, beta(1) and beta(2) protein kinase C isoforms. The present study aimed to identify the minimal amino acid sequence of PKI55 that is able to inhibit the enzyme activity of protein kinase C. Peptides derived from both C- and N-terminal sequences were synthesized and initially assayed in rat brain protein kinase C to identify which part of the entire protein maintained the in vitro effects described for PKI55, and then the active peptides were tested on the isoforms alpha, beta(1), beta(2), gamma, delta, epsilon and zeta to identify their specific inhibition properties. Specific protein kinase C isoforms have been associated with the activation of specific signal transduction pathways involved in inflammatory responses. Thus, the potential therapeutic role of the selected peptides has been studied in polymorphonuclear leukocytes activated by the methyl ester derivative of the hydrophobic N-formyl tripeptide for-Met-Leu-Phe-OH to evaluate their ability to modulate chemotaxis, superoxide anion production and lysozyme release. These studies have shown that only chemotactic function is significantly inhibited by these peptides, whereas superoxide anion production and lysozyme release remain unaffected. Western blotting experiments also demonstrated a selective reduction in the levels of the protein kinase C beta(1) isoform, which was previously demonstrated to be associated with the polymorphonuclear leukocyte chemotactic response.

Effects of PKI55 protein, an endogenous protein kinase C modulator, on specific PKC isoforms activity and on human T cells proliferation.

PKI55 protein, coded by the recently identified KI55 gene [R. Selvatici, E. Melloni, M. Ferrati, C. Piubello, F.C. Marincola, E. Gandini, J. Mol. Evol. 57 (2003) 131-139] is synthesized following protein kinase C (PKC) activation and acts as a PKC modulator, establishing a feedback loop of inhibition. In this work, PKI55 was found to inhibit recombinant alpha, beta(1), beta(2), gamma, delta, zeta and eta PKC isoforms; the effect on conventional PKC was lost in the absence of calcium. Confocal immunofluorescence experiments showed that PKI55 can penetrate into peripheral blood mononuclear cells (PBMC), following a coordinated movement of calcium ions. The addition of PKI55 protein down-regulated the PKC enzyme activity in phytohaemagglutinin-activated PBMC, decreasing the activity of alpha, beta(1) and beta(2) PKC isoforms. Moreover, inhibition in PBMC proliferation was observed. Similar effects were detected in Jurkat T cells transfected with a plasmid containing the coding sequence of PKI55. The PKI55 protein functional role could be to control the pathological over-expression of specific PKC isoforms and to regulate proliferation.

Mechanisms of sodium azide-induced changes in intracellular calcium concentration in rat primary cortical neurons.

An intracellular calcium ([Ca(2+)](i)) increase is involved in sodium azide (NaN(3))-induced neurotoxicity, an in vitro model of brain ischemia. In this study the questions of possible additional sources of calcium influx, besides glutamate receptor activation, and of the time-course of NaN(3) effects have been addressed by measuring [Ca(2+)](i) in rat primary cortical cultures with the FURA-2 method. Basal [Ca(2+)](i) of neuronal populations was concentration-dependently increased 30 min, but not 24h, after a 10-min NaN(3) (3-30 mM) treatment; conversely, the net increase induced by electrical stimulation (10Hz, 10s) was consistently reduced. All the above effects depended on glutamate release and consequent NMDA receptor activation, since the NMDA antagonist MK-801 (1 microM) prevented them, and the spontaneous efflux of [(3)H]-d-aspartate from superfused neurons was concentration-dependently increased by NaN(3). In single neuronal cells, NaN(3) application progressively and concentration-dependently increased [Ca(2+)](i) (to 177+/-5% and 249+/-7% of the controls, 4 and 12 min after a 10mM-treatment, respectively). EGTA (5mM) pretreatment reduced the effect of 10mM NaN(3) (to 118+/-5% at 4 min, and to 148+/-10% at 12 min, respectively), while 1 microM cyclosporin A did not. Both MK-801 and CNQX (a non-NMDA glutamate antagonist, 10 microM) prevented NaN(3) effect at 4 min (to 147+/-8% and 153+/-5%, respectively), but not at 12 min after NaN(3) treatment. Conversely, 10 microM verapamil and 0.1 microM omega-conotoxin (L- and N-type calcium channel blockers, respectively) significantly attenuated NaN(3) effects at 12 min (to 198+/-8% and 164+/-5%, respectively), but not at 4 min; the P/Q-type calcium channel blocker, agatoxin, 0.3 microM, was ineffective. These findings show that the predominant source of calcium increase induced by NaN(3) is extracellular, involving glutamate receptor activation in a first step and calcium channel (mainly of the N-type) opening in a second step.

Differential activation of protein kinase C isoforms following chemical ischemia in rat cerebral cortex slices.

The aim of the current study was to characterize the effects of chemical ischemia and reperfusion at the transductional level in the brain. Protein kinase C isoforms (alpha, beta(1), beta(2), gamma, delta and epsilon) total levels and their distribution in the particulate and cytosolic compartments were investigated in superfused rat cerebral cortex slices: (i) under control conditions; (ii) immediately after a 5-min treatment with 10mM NaN(3), combined with 2mM 2-deoxyglucose (chemical ischemia); (iii) 1h after chemical ischemia (reperfusion). In control samples, all the PKC isoforms were detected; immediately after chemical ischemia, PKC beta(1), delta and epsilon isoforms total levels (cytosol+particulate) were increased by 2.9, 2.7 and 9.9 times, respectively, while alpha isoform was slightly reduced and gamma isoform was no longer detectable. After reperfusion, the changes displayed by alpha, beta(1), gamma, delta and epsilon were maintained and even potentiated, moreover, an increase in beta(2) (by 41+/-12%) total levels became significant. Chemical ischemia-induced a significant translocation to the particulate compartment of PKC alpha isoform, which following reperfusion was found only in the cytosol. PKC beta(1) and delta isoforms particulate levels were significantly higher both in ischemic and in reperfused samples than in the controls. Conversely, following reperfusion, PKC beta(2) and epsilon isoforms displayed a reduction in their particulate to total level ratios. The intracellular calcium chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1mM, but not the N-methyl-d-asparate receptor antagonist, MK-801, 1muM, prevented the translocation of beta(1) isoform observed during ischemia. Both drugs were effective in counteracting reperfusion-induced changes in beta(2) and epsilon isoforms, suggesting the involvement of glutamate-induced calcium overload. These findings demonstrate that: (i) PKC isoforms participate differently in neurotoxicity/neuroprotection events; (ii) the changes observed following chemical ischemia are pharmacologically modulable; (iii) the protocol of in vitro chemical ischemia is suitable for drug screening.

Effects of chemical ischemia on cerebral cortex slices: focus on mitogen-activated protein kinase cascade.

A variety of harmful stimuli, among them energy depletion occurring during transient brain ischemia, are thought to unbalance protein kinase cascades, ultimately leading to neuronal damage. In superfused, electrically stimulated rat cerebral cortex slices, chemical ischemia (CI) was induced by a 5-min treatment with the mitochondrial toxin, sodium azide (10 mM), combined with the glycolysis blocker, 2-deoxyglucose (2 mM). Thereafter, 1 h reperfusion (REP) with normal medium followed. Western blot analysis of p21Ras, extracellular signal-regulated protein kinases (ERK)1/2 (p44/42), phospho-ERK1/2, mitogen-activated protein kinase (MAPK)-p38, phospho-p38, stress-activated protein kinases/c-Jun NH2-terminal protein kinases (SAPK/JNK), phospho-SAPK/JNK was carried out. The level of p21Ras was increased by 40% immediately after CI, and did not return to control values following REP. Both ERK1 and ERK2 levels were reduced by CI and recovered to control values following REP; no significant change in their phosphorylation degree (phosphorylated to total level ratio, about 50% in the controls) was observed. Neither p38 levels, nor phosphorylation degree were changed following CI/REP. The activation of SAPK/JNK was significantly reduced under CI, and did not recover following REP. All CI/REP-induced effects were prevented by the NMDA receptor antagonist MK-801, 10 microM, suggesting the involvement of glutamate. The present findings show that although CI stimulates the p21Ras protein, MAPK levels and/or phosphorylation are reduced, possibly because of acute energy depletion. Because the activation of SAPK/JNK has been related to both apoptosis and neuroprotection, the decrease observed under CI/REP conditions may instead be related to nonapoptotic neuronal death. These results could be of interest in developing preventive treatments for ischemia/REP-induced brain damage.

Studies on the apoptotic activity of natural and synthetic retinoids: discovery of a new class of synthetic terphenyls that potently support cell growth and inhibit apoptosis in neuronal and HL-60 cells.

New terphenyl derivatives have been synthesized and tested for their effect on cell survival in serum-free cultures. These compounds protected HL60 cells from death and supported their growth with an activity higher than that of the natural 14-hydroxy-retro-retinol. Terphenyls 26 and 28 also possess antiapoptotic activity on neuronal cells, proving them as possible candidates for the treatment of neurodegenerative and ischemic diseases.

Synthesis and pharmacology of 6-substituted benztropines: discovery of novel dopamine uptake inhibitors possessing low binding affinity to the dopamine transporter.

A series of 6alpha- and 6beta-substituted benztropines were synthesized. A marked enantioselectivity was observed for the 6beta-methoxylated benztropines, the (1R)-isomers being more potent than the corresponding (1S) compounds. The racemic 6alpha-methoxy-3-(4',4' '-difluorodiphenylmethoxy)tropane (5 g) was the most potent compound. It has been found that modifications at the 6-position of benztropine might reduce the DAT binding affinity, maintaining otherwise a significant dopamine uptake inhibitory activity. A reinvestigation of the absolute configuration of 6beta-methoxytropinone proved the 6R configuration for the (+)-enantiomer.

Early and delayed glutamate effects in rat primary cortical neurons. Changes in the subcellular distribution of protein kinase C isoforms and in intracellular calcium concentration.

Glutamate-induced changes in the subcellular distribution of protein kinase C isoforms and in the intracellular calcium concentration were investigated in rat primary cortical neurons. Western blot analysis of protein kinase C isoforms (alpha, beta1, beta2, gamma, delta, epsilon, zeta and theta), performed 30 min after a 10 min treatment with 30 microM glutamate, revealed a decrease in the total beta1 (-24%) and beta2 (-40%) isoform levels, without any significant change in any of the other isozymes. All conventional isoforms translocated to the membrane compartment, while delta, epsilon, zeta and theta; maintained their initial subcellular distribution. Twenty-four hours after glutamate treatment, the total protein kinase C labelling had increased, particularly the epsilon isoform, which accounted for 34% of the total densitometric signal. At this time, protein kinase C beta1, delta, epsilon and zeta isoforms were mainly detected in the membrane compartment, while gamma and theta; signals were displayed almost solely in the cytosol. Basal intracellular calcium concentration (FURA 2 assay) was concentration-dependently increased (maximum effect +77%) 30 min, but not 24h after a 10 min glutamate (10-100 microM) treatment, while the net increase induced by electrical stimulation (10 Hz, 10s) was consistently reduced (maximum effect -64%). The N-methyl-d-aspartate receptor antagonist, MK-801, 1 microM, prevented glutamate action both 30 min and 24 h after treatment, while non-selective protein kinase C inhibitors, ineffective at 30 min, potentiated it at 24 h. These findings show that protein kinase C isoforms are differently activated and involved in the early and delayed glutamate actions, and that the prevailing effect of their activation is neuroprotective.

Nociceptin inhibition of acetylcholine efflux from different brain areas.

The effects of nociceptin on [3H]choline [3H](Ch) efflux from electrically-stimulated rat cortical, hippocampal and caudatal slices as well as from KCl-depolarized synaptosomes and tetrodotoxin-pretreated slices have been studied. The inhibition of electrically evoked [3H]Ch efflux by nociceptin (0.03-3 microM) was moderate (max -33%), more evident in the neocortex than in the hippocampus and was prevented by [Nphe1]NC(1-13)NH(2) 10 microM. This effect was absent in the caudate nucleus, in cortical synaptosomes and in tetrodotoxin-pretreated cortical slices. These data point to a distinct localization of NOP receptors in the different brain areas and to a prevailing inhibitory control by nociceptin on the cortical cholinergic input at pre-terminal level. However, the reported impairment of neocortical and hippocampal function by nociceptin may be referred to the inhibition not only of the cholinergic signal but also of other transmitters such as glutamate.

Protein kinase C activity, translocation, and selective isoform subcellular redistribution in the rat cerebral cortex after in vitro ischemia.

Protein kinase C (PKC) involvement in ischemia-induced neuronal damage has been investigated in superfused rat cerebral cortex slices submitted to 15 min of oxygen-glucose deprivation (OGD) and in primary cultures of rat cortical neurons exposed to 100 microM glutamate (GLU) for 10 min. OGD significantly increased the total PKC activity in the slices, mostly translocated in the particulate fraction. After 1 hr of reperfusion, the total PKC activity was reduced and the translocated fraction dropped by 84% with respect to the control. Western blot analysis of OGD samples showed an increase in total beta(2) and epsilon PKC isoform levels. After reperfusion, the total levels of alpha, beta(1), beta(2) and gamma isoforms were significantly reduced, whereas the epsilon isoform remained at an increased level. Endogenous GLU release from OGD slices increased to about 15 times the basal values after 15 min of oxygen-glucose deprivation, and to 25 and 35 times the basal level in the presence of the PKC inhibitors staurosporine (0.1 microM) and bisindolylmaleimide (1 microM), respectively. Western blot analysis of GLU-treated cortical neurons showed a significant decrease only in the total level of beta(2) isoforms. Cell survival was reduced to 31% in GLU-treated neuronal cultures; PKC inhibitors were not able to modify this effect. These findings demonstrate that the cell response to OGD and GLU involves PKC in a complex way. The net role played by PKC during OGD may be to reduce GLU release and, consequently, neurotoxicity. The isoforms beta(2) and epsilon are affected the most and may play a significant role in the mechanisms underlying neurotoxicity/neuroprotection.

Dual effects of 5-HT4 receptor activation on GABA release from guinea pig hippocampal slices.

The effects of BIMU-8, a 5-HT4 receptor agonist, were studied on GABA release in guinea pig hippocampal slices. BIMU-8 did not modify GABA outflow at rest but did display a complex action in electrically stimulated slices: at low concentrations it increased, and at higher concentrations inhibited, GABA release. These responses were competitively counteracted by GR 125487, a selective 5-HT4 receptor antagonist. The dual effects of BIMU-8 are consistent with its indirect cholinergic action since the M1 and M3 antagonist, 4-DAMP, prevented BIMU-8-elicited GABA facilitation, whereas the M2 antagonist AFDX-116 cancelled GABA inhibition. These results provide evidence that serotonin exerts a complex modulation on the GABAergic system, via 5-HT4 receptors, and suggest that the amine releases acetylcholine which, in turn, bidirectionally modulates GABA release.

Direct and indirect inhibition by nociceptin/orphanin FQ on noradrenaline release from rodent cerebral cortex in vitro.

1 The modulation exerted by nociceptin/orphanin FQ (NC) on noradrenaline (NE) release in rodent cerebral cortex slices and synaptosomes was studied. 2 Rat, mouse and guinea-pig cortical slices and synaptosomes were preincubated with 0.1 micro M [(3)H]-NE and superfused. NE release was evoked by 2 min of electrical (3 Hz) stimulation in slices and by 1 min pulse of 10 mM KCl in synaptosomes. 3 In rat cortical slices, 0.01-3 micro M NC reduced the evoked [(3)H]-NE efflux (E(max)-54%), with a bell-shaped concentration-response curve, which regained its monotonic nature in the presence of either 0.1 micro M naloxone (NX) or 30 micro M bicuculline. In synaptosomes, the NC effect curve was sygmoidal in shape and reached a plateau at 1 micro M concentration. 4 In the rat, both 1 micro M [Phe(1)psi(CH(2)-NH)Gly(2)]NC(1-13)NH(2) and 10 micro M [Nphe(1)]NC(1-13)NH(2) (NPhe) antagonised NC-induced inhibition, without per se modifying [(3)H]-NE efflux. The effects of 0.3-1 micro M NC concentrations were partially prevented by 1 micro M NX; 1 micro M D-Phe-Cys-Thr-D-Trp-Orn-Thr-Pen-Thr-NH(2) (CTOP) was also an effective antagonist, but 0.1 micro M norbinaltorphimine was not. 5 In the mouse cerebral cortex, NC-induced inhibition of NE release (pEC(50) 6.87, E(max)-61%, in the slices) was prevented by Nphe but was NX-insensitive. In guinea-pig cortical slices, NC effect (pEC(50) 6.22, E(max)-38%) was prevented by Nphe, but was NX-insensitive. 6 These findings demonstrate that NC inhibits NE release from rodent cerebral cortex via presynaptically located ORL(1) receptors. In the rat, micro opioid and GABA(A) receptors are involved as well.