An SEM compatible plasma cell for in situ studies of hydrogen-material interaction.

An in situ hydrogen (H) plasma charging and in situ observation method was developed to continuously charge materials, while tensile testing them inside a scanning electron microscope (SEM). The present work will introduce and validate the setup and showcase an application allowing high-resolution observation of H-material interactions in a Ni-based alloy, Alloy 718. The effect of charging time and pre-straining was investigated. Fracture surface observation showed the expected ductile microvoid coalescence behavior in the uncharged samples, while the charged ones displayed brittle intergranular and quasi-cleavage failure. With the in situ images, it was possible to monitor the sample deformation and correlate the different crack propagation rates with the load-elongation curves. H-charging reduced the material ductility, while increasing pre-strain decreased hydrogen embrittlement susceptibility due to the possible suppression of mechanical twinning during the tensile test and, therefore, a reduction in H concentration at grain and twin boundaries. All the presented results demonstrated the validity of the method and the possibility of in situ continuously charging of materials with H without presenting any technical risk for the SEM.

Quantification of A. fumigatus-specific CD154+ T-cells-preanalytic considerations.

Fungal specific CD154+ T-cells have been described as a biomarker in invasive aspergillosis. The influence of sample storage on the detection of these cells was assessed. Six-hour delay prior to PBMC isolation is associated with an 18% decrease of cell viability and alterations of the cellular composition of the sample. This results in 87% reduction of CD154+ A. fumigatus specific cells due to reduced assay sensitivity and increased background values in unstimulated samples. If prompt cell measurement is not feasible, isolated PBMCs can be frozen (at -20°C and -80°C) and processed later with comparable assay reliability (mean value fresh vs. thawing: 0.126, 0.133; Pearson-Coefficient: 0.962).

Direct evidence for grain boundary motion as the dominant restoration mechanism in the steady-state regime of extremely cold-rolled copper.

Ultra-fine-grained high-purity copper (99.99%) deformed by means of high-pressure torsion into the steady-state regime was subjected to additional rolling deformation. The microstructural changes as a function of the applied strain were analysed by means of orientation imaging microscopy. It was found that after a distinctive rolling strain a steady state with respect to microstructural features such as grain size, misorientation distribution and texture evolves again. A special spilt specimen technique was used to perform quasi in situ observations of the microstructure between additional strain increments. Profound insights into the local deformation and restoration processes within the steady-state regime were gained. The observations lead to the conclusion that grain boundary migration perpendicular to the rolling direction leads to the disappearance of certain grains, enabling the occurrence of a steady state.

Loss of LIN9, a member of the DREAM complex, cooperates with SV40 large T antigen to induce genomic instability and anchorage-independent growth.

The DREAM complex is an important regulator of mitotic gene expression during the cell cycle. Here we report that inactivation of LIN9, a subunit of DREAM, results in premature senescence, which can be overcome by the SV40 large T (LT) antigen. Together with the observation that p16(INK4a) and p21(Waf1) are upregulated upon loss of LIN9, these results indicate that senescence is triggered by the pRB and p53 tumor suppressor pathways. We also find that LIN9-null cells that escape senescence are chromosomally instable because of compromised mitotic fidelity. SV40 LT-expressing cells that adapt to the loss of LIN9 can grow anchorage-independently in soft agar, a hallmark of oncogenic transformation. Taken together, these results suggest an important role of mitotic gene regulation in the maintenance of genomic stability and tumor suppression.

Human kisspeptins activate neuropeptide FF2 receptor.

We studied the possible activation of a neuropeptide FF2 receptor (NPFF2R) by kisspeptins, neuropeptides derived from the mouse and human metastin or Kiss-1 precursor. The hypothesis was that the human kisspeptins, which share the C-terminal dipeptide RF-NH(2) with NPFF, might activate the NPFF2R, as has previously been shown for two related peptides, prolactin-releasing peptide and RF-amide-related peptide. Using two-electrode voltage clamp of Xenopus oocytes, we found that 100 nM NPFF strongly activated the human NPFF2R expressed together with rat GIRK1/4 inward rectifier potassium channels, and that 100 nM hKisspeptin-13 and hKisspeptin-8 had about 25% relative efficacy to that of NPFF. The current response induced by hKisspeptin-13 was proportional to its concentration (1-500 nM). The corresponding mouse peptides resulted in low activation only. When hNPFF2R was expressed in Chinese hamster ovary (CHO) cells, NPFF and its stable analog (1DMe)Y8Fa induced guanosine 5'-(gamma-[(35)S]thio)-triphosphate (GTP-gamma-[(35)S]) binding with EC(50) values of 13+/-4 and 16+/-4 nM, respectively. hKisspeptin-13 induced the binding with an EC(50) value of 110+/-50 nM, whereas mKisspeptin-13 induced very modestly activation with an EC(50) value>2 microM. The results suggest that, besides regulation of reproduction, kisspeptins have a potential to mediate physiological effects on, for example autonomic regulation and nociception in man via the NPFF2R pathways.

Pharmacological characterization and CNS effects of a novel highly selective alpha2C-adrenoceptor antagonist JP-1302.

BACKGROUND AND PURPOSE: Pharmacological validation of novel functions for the alpha2A-, alpha2B-, and alpha2C-adrenoceptor (AR) subtypes has been hampered by the limited specificity and subtype-selectivity of available ligands. The current study describes a novel highly selective alpha2C-adrenoceptor antagonist, JP-1302 (acridin-9-yl-[4-(4-methylpiperazin-1-yl)-phenyl]amine). EXPERIMENTAL APPROACH: Standard in vitro binding and antagonism assays were employed to demonstrate the alpha2C-AR specificity of JP-1302. In addition, JP-1302 was tested in the forced swimming test (FST) and the prepulse-inhibition of startle reflex (PPI) model because mice with genetically altered alpha2C-adrenoceptors have previously been shown to exhibit different reactivity in these tests when compared to wild-type controls. KEY RESULTS: JP-1302 displayed antagonism potencies (KB values) of 1,500, 2,200 and 16 nM at the human alpha2A-, alpha2B-, and alpha2C-adrenoceptor subtypes, respectively. JP-1302 produced antidepressant and antipsychotic-like effects, i.e. it effectively reduced immobility in the FST and reversed the phencyclidine-induced PPI deficit. Unlike the alpha2-subtype non-selective antagonist atipamezole, JP-1302 was not able to antagonize alpha2-agonist-induced sedation (measured as inhibition of spontaneous locomotor activity), hypothermia, alpha2-agonist-induced mydriasis or inhibition of vas deferens contractions, effects that have been generally attributed to the alpha2A-adrenoceptor subtype. In contrast to JP-1302, atipamezole did not antagonize the PCP-induced prepulse-inhibition deficit. CONCLUSIONS AND IMPLICATIONS: The results provide further support for the hypothesis that specific antagonism of the alpha2C-adrenoceptor may have therapeutic potential as a novel mechanism for the treatment of neuropsychiatric disorders.

Effects of the somatostatin receptor subtype 4 selective agonist J-2156 on sensory neuropeptide release and inflammatory reactions in rodents.

BACKGROUND AND PURPOSE: Substance P (SP) and calcitonin gene-related peptide (CGRP) released from capsaicin-sensitive sensory nerves induce local neurogenic inflammation; somatostatin exerts systemic anti-inflammatory actions presumably via sst4/sst1 receptors. This study investigates the effects of a high affinity, sst4-selective, synthetic agonist, J-2156, on sensory neuropeptide release in vitro and inflammatory processes in vivo. EXPERIMENTAL APPROACH: Electrically-induced SP, CGRP and somatostatin release from isolated rat tracheae was measured with radioimmunoassay. Mustard oil-induced neurogenic inflammation in rat hindpaw skin was determined by Evans blue leakage and in the mouse ear with micrometry. Dextran-, carrageenan- or bradykinin-induced non-neurogenic inflammation was examined with plethysmometry or Evans blue, respectively. Adjuvant-induced chronic arthritis was assessed by plethysmometry and histological scoring. Granulocyte accumulation was determined with myeloperoxidase assay and IL-1beta with ELISA. KEY RESULTS: J-2156 (10-2000 nM) diminished electrically-evoked neuropeptide release in a concentration-dependent manner. EC50 for the inhibition of substance P, CGRP and somatostatin release were 11.6 nM, 14.3 nM and 110.7 nM, respectively. J-2156 (1-100 microg kg(-1) i.p.) significantly, but not dose-dependently, inhibited neurogenic and non-neurogenic acute inflammatory processes and adjuvant-induced chronic oedema and arthritic changes. Endotoxin-evoked myeloperoxidase activity and IL-1beta production in the lung, but not IL-1beta- or zymosan-induced leukocyte accumulation in the skin were significantly diminished by J-2156. CONCLUSIONS AND IMPLICATIONS: J-2156 acting on sst4 receptors inhibits neuropeptide release, vascular components of acute inflammatory processes, endotoxin-induced granulocyte accumulation and IL-1beta synthesis in the lung and synovial and inflammatory cells in chronic arthritis. Therefore it might be a promising lead for the development of novel anti-inflammatory drugs.

Identification and characterization of the imidazoline I2b-binding sites in the hamster brown adipose tissue as a study model for imidazoline receptors.

The imidazoline-type compound, MPV-1743, has been found to activate nonshivering thermogenesis (NST) in brown adipose tissue (BAT) of the genetically obese Zucker rats. The regulation of NST in BAT is linked to the catecholamine metabolism, and the imidazoline I2-binding sites have been found on the monoamine oxidase, a catecholamine metabolising enzyme. In this study, the I2-binding sites of hamster BAT have been characterised using a receptor binding assay with 3H-idazoxan as a radioligand, and the interaction of MPV-1743 with these I2-binding sites has been studied using the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089. Cirazoline was used to determine the specific binding of 3H-idazoxan to the imidazoline I2-binding sites. Rauwolscine was added in the 3H-idazoxan binding assay in order to inhibit any binding to potential alpha2-adrenergic sites. In the presence of rauwolscine mask 3H-Idazoxan labelled a population of non-adrenergic binding sites expressing the properties of the imidazoline I2b-receptor subtype similar to that found in the rat liver (cirazoline >> guanabenz = amiloride >> clonidine). The binding of 3H-idazoxan to the I2b-binding sites could be displaced by the imidazole compounds with the following affinities: detomidine (KiHigh 9.2 nM; KiLow 3200 nM), MPV-2088 (KiHigh 19 nM; IKiLow 760 nM) and MPV-2089 (KiHigh 190 nM; KiLow 1300 nM), atipamezole (3500 nM) and dexmedetomidine (Ki 8400 nM). These results have shown that the hamster BAT contains the imidazoline I2b-binding sites with heterogeneous binding properties for some test compounds. In addition, the enantiomers of MPV 1743, that is, MPV 2088 and MPV 2089, had high affinity to these BAT imidazoline I2b-binding sites. Therefore, it is suggested that the regulation of NST in the hamster BAT may be an attractive model to study the role of imidazoline I2b-binding sites.

Molecular mechanism for agonist-promoted alpha(2A)-adrenoceptor activation by norepinephrine and epinephrine.

We present a mechanism for agonist-promoted alpha(2A)-adrenergic receptor (alpha(2A)-AR) activation based on structural, pharmacological, and theoretical evidence of the interactions between phenethylamine ligands and alpha(2A)-AR. In this study, we have: 1) isolated enantiomerically pure phenethylamines that differ both in their chirality about the beta-carbon, and in the presence/absence of one or more hydroxyl groups: the beta-OH and the catecholic meta- and para-OH groups; 2) used [(3)H]UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; agonist] and [(3)H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; antagonist] competition binding assays to determine binding affinities of these ligands to the high- and low-affinity forms of alpha(2A)-AR; 3) tested the ability of the ligands to promote receptor activation by measuring agonist-induced stimulation of [(35)S]GTPgammaS binding in isolated cell membranes; and 4) used automated docking methods and our alpha(2A)-AR model to predict the binding modes of the ligands inside the alpha(2A)-AR binding site. The ligand molecules are sequentially missing different functional groups, and we have correlated the structural features of the ligands and ligand-receptor interactions with experimental ligand binding and receptor activation data. Based on the analysis, we show that structural rearrangements in transmembrane helix (TM) 5 could take place upon binding and subsequent activation of alpha(2A)-AR by phenethylamine agonists. We suggest that the following residues are important in phenethylamine interactions with alpha(2A)-AR: Asp113 (D(3.32)), Val114 (V(3.33)), and Thr118 (T(3.37)) in TM3; Ser200 (S(5.42)), Cys201 (C(5.43)), and Ser204 (S(5.46)) in TM5; Phe391 (F(6.52)) and Tyr394 (Y(6.55)) in TM6; and Phe411 (F(7.38)) and Phe412 (F(7.39)) in TM7.

Alpha2-adrenoceptor agonists stimulate high-affinity GTPase activity in a receptor subtype-selective manner.

Transfected Chinese hamster ovary cells expressing human alpha2A-, alpha2B- and alpha2C-adrenoceptor subtypes were used to monitor alpha2-adrenoceptor-stimulated GTP hydrolysis. Incubation with 100 microM (-)-adrenaline resulted in stimulation of pertussis toxin-sensitive GTPase by 380% after activation of the alpha2A-subtype, by 320% after activation of the alpha2B-subtype and by 110% after activation of the alpha2C-subtype. The agonists dexmedetomidine, UK14,304 (5-bromo-6-[2-imidazoline-2-ylamino]quinoxaline) and oxymetazoline showed subtype-dependent efficacy. Dexmedetomidine was a full agonist at the alpha2B-subtype and a partial agonist at the alpha2A- and the alpha2C-subtypes. UK14,304 was a full agonist at the alpha2A-subtype and a partial agonist at the other two. Oxymetazoline showed strong partial agonism at the alpha2B-subtype (63% of adrenaline), but did not significantly activate the alpha2A- and the alpha2C-subtypes. These results agreed with cAMP accumulation experiments carried out with cell lines endogenously expressing the alpha2A-subtype (human erythroleukemia, HEL) or the alpha2B-subtype (neuroblastoma-glioma, NG108-15). The GTPase assay may thus provide a valuable tool for the identification of subtype-selective alpha2-adrenoceptor agonists.

Modulation of agonist binding to recombinant human alpha2-adrenoceptors by sodium ions.

Agonist binding to alpha2-adrenoceptors is modulated by a number of factors such as Mg2+ and Na+ ions and by experimental manipulations which interfere with receptor-G-protein-coupling such as pertussis toxin pre-treatment or the presence of guanine nucleotides. Agonist binding assays may therefore offer an opportunity to make inferences, albeit indirect, about receptor states or conformations and about the molecular nature of the processes involved in receptor activation. We have investigated possible differences in the effects of Na+ ions on the binding of agonists to the three human alpha2-adrenoceptor subtypes, alpha2A, alpha2B and alpha2C, recombinantly expressed in S115 mouse mammary tumour cells. NaCl (40 mM) influenced the apparent affinity of a panel of alpha2-adrenoceptor ligands in a complex compound- and subtype-dependent manner. Sodium ions affected both high- and low-affinity conformations of the receptors, as defined by co-incubation with 10 microM 5'-guanylylimidodiphosphate (Gpp(NH)p). The effects of NaCl and Gpp(NH)p on agonist binding were additive indicating different modes of action for the two allosteric modulators. Thus, quite marked differences between closely related receptor subtypes were noted in the molecular details of agonist-receptor interactions and in the integration of allosteric modulation by Na+ ions. Possible explanations for the experimental findings are discussed within the theoretical framework of multi-state models, and a proposal is presented for a potential physiological role of the modulatory effect of Na+ ions, where intracellular Na+ concentrations would direct the activating influence of receptors to different G-proteins.

Expression of inducible nitric oxide synthase in human burn wounds.

Nitric oxide is produced by various cell types and can initiate either beneficial or deleterious effects. Because cultured human keratinocytes express an inducible isoform of nitric oxide synthase, it was postulated that keratinocytes within a burn wound would express increased levels of inducible nitric oxide synthase following the injury. Immunohistochemical staining identified the sites of cellular expression and temporal sequence of inducible nitric oxide synthase protein within partial- and full-thickness burns excised from 29 patients. While migrating keratinocytes at the immediate edge of the wounds showed decreased or undetectable levels of inducible nitric oxide synthase, the immediately adjacent proliferative population and upwardly growing keratinocytes from surviving hair follicles showed increasingly greater cytoplasmic staining for inducible nitric oxide synthase at 4-21 days after injury. Noninjured skin showed minimal inducible nitric oxide synthase staining. Within the wound, detectable inducible nitric oxide synthase protein appeared to decrease as keratinocytes assumed a differentiated phenotype in the outer newly resurfaced epidermis, in inner root sheath layers of hair follicles, or in epithelium of eccrine sweat ducts. Within granulation tissue, immunoreactive inducible nitric oxide synthase was detected in capillary endothelium and in arterial smooth muscle layer. Focal increases in inducible nitric oxide synthase expression were noted in association with inflammatory infiltrates. In conclusion, the cellular and temporal distributions of immunoreactive inducible nitric oxide synthase suggest that nitric oxide may play a role in the regulation of wound repair processes beyond the acute burn injury.

[Explant test with skin and peritoneum of the neonatal rat as a predictive test of tolerance of local anti-infective agents in wounds and body cavities]. / Explantationstest mit Haut und Peritoneum der neonatalen Ratte als Voraussagetest zur Verträglichkeit lokaler Antiinfektiva für Wunden und Körperhöhlen.

In vitro culture of peritoneal explants of neonatal rats after previous application of agents simulating wound antisepsis is a sensitive screening method for the determination of the tissue compatibility of local wound antiinfectives. Two test models are differentiated: (1) separated peritoneal explants as a model for chronic or deep wounds and (2) peritoneum in situ in the experimental animal with subsequent extraction and cultivation of the explants. Considering the present state of knowledge the following conclusions can be drawn regarding antisepsis of wounds: Lavasept (0.1%) may be classified as the agent of choice for deep and chronic wounds, for drip-suck irrigation and for antiinfective lavage of body cavities inclusively for peritoneal lavage (0.05%). Taurolidin is antiseptically effective in long term application (> 6 h), and because of its antitoxic effect as well as lack of cytotoxicity it is especially suitable for peritoneal lavage. Betaisodona solution is very well suited for superficial contaminated wounds and can be used in a dilution of 1:10 for short-term rinsing of deep wounds, including body cavities but not for peritoneal lavage. Ethanol causes no inhibition of explant growth and therefore retains its importance in wound antisepsis.

Protean agonism at alpha2A-adrenoceptors.

The coupling of the endogenously expressed alpha2A-adrenoceptors in human erythroleukemia cells (HEL 92.1.7) to Ca2+ mobilization and inhibition of forskolin-stimulated cAMP production was investigated. The two enantiomers of medetomidine [(+/-)-[4-(1-[2, 3-dimethylphenyl]ethyl)-1H-imidazole]HCl] produced opposite responses. Dexmedetomidine behaved as an agonist in both assays (i.e. , it caused Ca2+ mobilization and depressed forskolin-stimulated cAMP production). Levomedetomidine, which is a weak agonist in some test systems, reduced intracellular Ca2+ levels and further increased forskolin-stimulated cAMP production and therefore can be classified as an inverse agonist. A neutral ligand, MPV-2088, antagonized responses to both ligands. Several other, chemically diverse alpha2-adrenergic ligands also were tested. Ligands that could promote increases in Ca2+ levels and inhibition of cAMP production could be classified as full or partial agonists. Their effects could be blocked by the alpha2-adrenoceptor antagonist rauwolscine and by pertussis toxin treatment. Some typical antagonists such as rauwolscine, idazoxan, and atipamezole had inverse agonist activity like levomedetomidine. The results suggest that the alpha2A-adrenoceptors in HEL 92.1.7 cells exist in a precoupled state with pertussis toxin-sensitive G proteins, resulting in a constitutive mobilization of intracellular Ca2+ and inhibition of cAMP production in the absence of agonist. This constitutive activity can be antagonized by inverse agonists such as levomedetomidine and rauwolscine. Levomedetomidine can be termed a "protean agonist" because it is capable of activating uncoupled alpha2-adrenoceptors in other systems and inhibiting the constitutive activity of precoupled alpha2-adrenoceptors in HEL 92.1. 7 cells. With this class of compounds, the inherent receptor "tone" could be adjusted, which should provide a new therapeutic principle in receptor dysfunction.

Different apparent modes of inhibition of alpha2A-adrenoceptor by alpha2-adrenoceptor antagonists.

The inhibition of alpha2A-adrenoceptor-mediated Ca2+ elevation by alpha2-adrenoceptor antagonists was measured in HEL human erythroleukemia cells. The antagonists could be divided in two classes: those that displayed surmountable inhibition (right-shift of the agonist dose-response curve), and those that displayed different degrees of insurmountable inhibition (depression of the maximum signal and a possible right-shift of the agonist dose-response curve). The degree of surmountability of the inhibition correlated well with the measured antagonist dissociation rates, suggesting that the hypothesis of the antagonist dissociation rate governing the mode of inhibition of fast responses, holds true. HEL cells thus provide a useful model system for the investigation of physiological consequences of different dissociation rates. Also, the dissociation rates of antagonists not available in radiolabelled form can be predicted from the functional data. The data stresses the importance of measurement of kinetic parameters of the drug-receptor interaction in addition to the equilibrium binding constants.

Intraperitoneal sodium carboxymethylcellulose administration prevents reformation of peritoneal adhesions following surgical lysis.

Although intraperitoneal administration of sodium carboxymethylcellulose (SCMC) prevents the formation of adhesions following laparotomy in rats, it remains unknown whether SCMC treatment prevents the recurrence of preformed peritoneal adhesions following surgical lysis. Additionally, the optimal amount of SCMC required for adhesion prevention, as well as the effects of SCMC upon the healing of bowel anastomoses, has yet to be determined. To study this, 114 male rats underwent laparotomy and adhesion induction via peeling of the cecal serosa with a gauze sponge. Two weeks later, all animals again underwent laparotomy, the adhesions were graded, and surgical lysis of adhesions was performed. Following this, 3 to 12 ml of either normal saline or 1% SCMC solution was instilled into the peritoneal cavity prior to closure. A segment of small bowel was transected and reanastomosed prior to administration of SCMC or saline in another group of 70 rats. After an additional 2 weeks, the animals were sacrificed, the adhesions graded, and all the abdominal contents removed for fixation. The results show that treatment with high volume (i.e., 12 ml) intraperitoneal SCMC prevents reformation of adhesions following surgical lysis. This effect is demonstrated by a proportionate and significant decrease in the incidence of intraabdominal adhesions associated with administration of increasing amounts of SCMC (P < 0.05). While high volume SCMC did prevent adhesion of peritoneal structures to newly formed small bowel anastomoses, SCMC did not impair anastomotic healing.

Vascular smooth muscle contractile function is impaired during early and late stages of sepsis.

Although impairment of vascular smooth muscle contractility occurs during the late stages of polymicrobial sepsis, it is not known whether this also occurs in early stages of sepsis and, if so, whether different mechanisms are responsible for such smooth muscle dysfunction. To determine this, rats were subjected to sepsis by cecal ligation and puncture (CLP). Immediately following CLP or sham operation, all animals received 3 ml/100 g body wt normal saline. Septic and sham rats were then sacrificed at 5, 10, or 35 hr after CLP (5-10 hr post-CLP, early sepsis; 35 hr post-CLP, late sepsis), and aortic rings were prepared for contraction studies using organ chamber technique. Dose-response contractions to norepinephrine (NE, 10(-9) to 10(-5) M; receptor-mediated process) and KCl (7.5 to 90 mM; non-receptor mediated) were determined in rings with or without intact endothelium. Endothelial cell removal was confirmed by the absence of relaxation in response to an endothelium-dependent vasodilator, acetylcholine. The results indicate that NE- and KCl-induced vascular contractions were not altered at 5 hr after CLP. At 10 hr post-CLP, however, vascular contractility decreased markedly in the endothelium intact rings. Endothelium removal at 10 hr after CLP restored the contraction induced by NE and KCl to sham levels. In contrast, the smooth muscle contractile dysfunction, observed during late sepsis (35 hr post-CLP), was not restored by the removal of endothelium. Thus, the smooth muscle impairment, observed in early sepsis, is due to mediators released from septic endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

The voltage-sensitive Ca2+ channel (VSCC) antagonists omega-Aga-IVA and omega-CTX-MVIIC inhibit spontaneous epileptiform discharges in the rat cortical wedge.

The ability of VSCC antagonists to modulate excitatory amino acid (EAA) release was evaluated by measuring N-methyl-D-aspartate (NMDA) receptor-dependent spontaneous epileptiform discharges in rat cortical wedges. The N-type channel blocker omega-CTX-GVIA (300 nM) was ineffective. The P-type channel blocker omega-Aga-IVA at 300 nM reduced the frequency of discharges by 63%, while 300 nM omega-CTX-MVIIC reduced the frequency by 35%. These results coupled with the absence of NMDA antagonism by omega-Aga-IVA or omega-CTX-MVIIC in the cortical wedge suggest that the VSCCs blocked by these toxins are primarily responsible for mediating impulse dependent EAA release in the rat neocortex.

Effect of fluoride, pertussis and cholera toxin on the release of arachidonic acid and the formation of prostaglandin E2, D2, superoxide and inositol phosphates in rat liver macrophages.

Fluoride elicited in liver macrophages a release of arachidonic acid and prostaglandins but not formation of inositol phosphates or superoxide. The effects of fluoride required extracellular calcium and were inhibited by staurosporine and by phorbol ester treatment of the cells. Furthermore, fluoride led to a translocation of protein kinase C from the cytosol to membranes. This indicates that the calcium-dependent protein kinase C is involved in the action of fluoride. Cholera toxin decreased the zymosan-induced release of arachidonic acid and prostaglandins but not of inositol phosphates or superoxide. Pertussis toxin ADP-ribosylated a 41,000 molecular weight membrane protein; enhanced specifically the zymosan-induced formation of prostaglandin(PG)E2 but did not affect the zymosan-induced release of arachidonic acid, PGD2, inositol phosphates or superoxide. These data suggest that activation of phospholipase (PL)A2, phosphoinositide (PI)-specific PLC and NADPH oxidase in liver macrophages is most probably not mediated by activation of guanine nucleotide binding (G)-proteins coupled directly to these enzymes.