U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation

1-[6-[[17a-3-Methoxyestra-1,3,5(10)-trien17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca²⁺ concentration ([Ca 2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca²⁺]i elevation of neutrophils suspended in Ca²⁺-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC₅₀ values 4.06 ± 0.27 µM and 4.04 ± 0.44 µM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC₅₀ value 0.62 ± 0.04 µM) U-73122 also reduced the intracellular Ca²⁺ mobilization of neutrophils suspended in Ca ²⁺-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC₅₀ values 0.52 ± 0.02 µM and 6.82 ± 0.74 µM, respectively. 1-[6-[[17f3-Methoxyestra-1,3,5(10)-trien-l7-yl]amino]hexyl]2,5-pyrrolidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca²⁺]_i elevation of neutrophils challenged by fMLP in Ca²⁺-containing medium, but not in Ca²⁺-free medium, with IC₅₀ value 22.30 ± 1.61 µM. In Mn²⁺-quench studies, U-73122 suppressed the Mn²⁺ influx in CPA-activated neutrophils (IC₅₀ value was 7.16 ± 0.28 µM) as well as in resting neutrophils (IC₅₀ value was 6.72 ± 0.30 M). U-73343 also suppressed the Mn²⁺ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca²⁺]i of activated neutrophils is attributed partly to the suppression of Ca²⁺ release from the intracellular Ca²⁺ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca²⁺ entry from the extracellular space through PLC-independent processes.     

Inhibition of signaling from Type 1 receptor tyrosine kinases via intracellular expression of single-chain antibodies

Summary Members of the Type I / epidermal growth factor receptor (EGFR)-related family of receptor tyrosine kinases have been implicated in the development of human cancer. We have taken a novel approach using the intracellular expression of single chain antibodies (scFv) to specifically inhibit thein vivo action of these receptors. A scFv is a recombinant protein analogous to an Fv domain which is the smallest high affinity binding portion of an antibody. We report here on the expression in mammalian cells of cDNAs encoding scFv-225 and scFv-FRP5 directed against the extracellular domain of, respectively, human EGFR and human ErbB-2. The scFvs were provided with a signal peptide which directs them to the secretory pathway of the cell. scFv-225, which competes with EGF for binding, functions in an autocrine fashion to inhibit EGF-dependent cell growth. scFv-FRP5 was also provided with an endoplasmic reticulum (ER) retention signal and inactivates ErbB-2 in an intracrine fashion, by preventing its appearance on the cell surface.Presented at the symposium "New Approaches in the Therapy of Breast Cancer", Georgetown University Medical Center, Washington DC, October 1994, generously supported by an education grant from Bristol-Myers Squibb.     

Cumulative biochemical effects of repeated subclinical hydrogen sulfide intoxication in mouse brain

Summary Adult female NMRI mice exposed four times to 100 ppm hydrogen sulfide vapour for 2 h at 4-day intervals showed increasing inhibition of the cerebral cytochrome oxidase activity. Cerebral RNA decreased significantly after the fourth exposure. This change was accompanied by the reduced orotic acid uptake in the RNA fraction. At the same time, 2,3-cyclic nucleotide 3-phosphohydrolase activity used as a marker for glia increased. Acetylcholine esterase activity remained unchanged. The initial exposures also caused an increase in the superoxide dismutase activity and an increase in the glutathione concentration. The latter effects were abolished in the third and fourth exposures. The present data seem to indicate that the biochemical effects of repeated subclinical hydrogen sulfide intoxications are cumulative.     

Comparison of immunochemical and biological properties of human anti thrombin during blood clotting and upon interaction with exogenous thrombin

Modification of immunological and biological properties of human antithrombin were studied in plasma-serum pairs and in defibrinated plasma supplemented with human thrombin. Modified antithrombin obtained through whole-blood clotting or upon addition of exogenous thrombin appeared the same with regards to its electrophoretic or biological properties. However, amounts of thrombin higher than that physiologically available, had to be used to obtain a "serum-like" antithrombin in thrombin supplemented plasma suggesting different pathways for this transformation. This was in agreement with the observation in plasma of a modification of antithrombin antigenic properties upon thrombin addition whereas no difference was demonstrated when comparing serum to normal plasma. It may be concluded that the inactivation of antithrombin and the appearance of electrophoretically modified forms in normal serum is not mainly due to the formation of enzyme-inhibitor complexes and therefore that proteolytically modified, enzyme-free forms of antithrombin demonstrated in purified systems (Fish et al. 1979) could be of physiological relevance.     

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

Summary The effects of three different opioid agonists on contractions and [³H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine₃ (5-HT₃) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu ()-opioid receptor agonist (d-Ala²,NMe-Phe⁴,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa ()-opioid receptor agonist U50488 (10 nM -1 M) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC₅₀ estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta ()-opioid receptor agonist [d-Pen^2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 M). ³H-overflow from LMMP preparations preincubated with [³H]-choline was measured as an indicator of [³H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 M) inhibited the increases in release of [³H]-ACh evoked by 5-HT (10 M) and by senktide (10 nM) in a concentration-dependant manner. IC₅₀ estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [³H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 M). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [³H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 M).These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT₃ and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by - and -, but not -, opioid receptors.     

Regionally selective effects of intracerebral dopamine infusion on sensorimotor gating of the startle reflex in rats

Systemic administration of dopamine (DA) agonists markedly disrupts sensorimotor gating in rats as measured by prepulse inhibition (PPI) of the acoustic startle response. A qualitatively similar, but quantitatively weaker disruption of PPI follows DA infusion into the nucleus accumbens (NAC). The present study was designed to determine whether forebrain DA terminal fields other than the NAC contribute to the DAergic modulation of PPI. PPI was impaired significantly after infusion of DA (0–40 µg) into the NAC or anteromedial striatum, but not after DA infusion into the orbital cortex or posterolateral striatum. DA infusion into the amygdala also disrupted PPI, but this disruption was accompanied by a dose-dependent decrease in startle amplitude. These results suggest that DA overactivity in the both NAC and anteromedial striatum contribute to the gating-disruptive effects of systemically administered DA agonists, and that DA overactivity in mesocortical, mesoamygdaloid and non-limbic mesostriatal DA systems are not major substrates for a DAergic modulation of PPI.     

Depression by neuropeptide Y of noradrenergic inhibitory postsynaptic potentials of locus coeruleus neurones

Summary Intracellular recordings were performed in a pontine slice preparation of the rat brain containing the locus coeruleus (LC). The spontaneous firing of action potentials was prevented by passing continuous hyperpolarizing current via the recording electrode. Focal electrical stimulation evoked a synaptic depolarization (PSP) followed by a hyperpolarization (IPSP). Neuropeptide Y (NPY; 0.1 mol/l) inhibited the IPSP only. Pressure ejection of noradrenaline produced hyperpolarization which was potentiated in the presence of NPY (0.1 mol/l). Hence, NPY appears to inhibit the release of noradrenaline from dendrites or recurrent axon collaterals of LC neurones. Correspondence to: P. Illes at the above address     

Enhanced Delivery of 5-Iodo-2′-Deoxyuridine to the Brain Parenchyma

5-Ester derivatives of 5-iodo-2-deoxyuridine (IDU) with varying degrees of lipophilicity were examined to evaluate the effectiveness of lipophilic ester prodrugs for enhanced and sustained delivery of IDU to the brain parenchyma. Approximately 1.0% (1.0 ± 0.19; n = 4) of the total radioactivity was found in the brain at 30 min following intravenous administration of the lipophilic benzoyl-5-ester of ¹²⁵I-labeled IDU, whereas IDU per se yielded only 0.01% (0.01 ± 0.06; n = 4). Since the IDU 5-esters generated significantly higher levels of IDU in the brain, an HPLC analysis of IDU in the presence of 5-esters and the metabolite 5-iodouracil was developed to characterize IDU uptake in the brain. The drug was detected at levels of 6.6 and 9.5 µg/g of brain tissue at 3 hr following intravenous administration of valeryl and benzoyl IDU, respectively, at a dose level of 40 mg/kg IDU equivalent each. IDU, on the other hand, when injected at a similar dose level, produced concentration levels below 0.01 µg/g of brain tissue, which was too low to be detected accurately by the HPLC assay. These results suggest that the 5-ester derivatives cross the blood-brain barrier effectively and generate significantly higher brain levels of the parent drug in the brain parenchyma. The regenerated hydrophilic drug because of its polarity is locked in the brain and is subsequently metabolized by pyrimidine phosphorylase to 5-iodouracil. A higher concentration of IDU was generated following administration of the benzoyl ester probably because the ester itself is slowly hydrolyzed by the brain cholinesterases, thereby competitively inhibiting the metabolism of IDU to 5-iodouracil by brain pyrimidine phosphorylase. 5-Benzoyl IDU appears to be a promising bioreversible analogue which can provide enhanced and sustained delivery of IDU to the brain parenchyma.     

Cycloheximide Reduces the Effects of Anoxic Insult In Vivo and In Vitro

In vivo and in vitro techniques were utilized to examine the influence of a protein synthesis blocker, cycloheximide (CHX), on the damaging effects of anoxia in the rat. CHX administered 1 h before transient (30 min) forebrain ischaemia increased the survival of animals, decreased body weight loss and reduced the occurrence of delayed degeneration in the CA1 pyramidal region. The same dose of CHX injected 1 h after ischaemia induced status epilepticus, a decrease in survival rate, and did not reduce weight loss or CA1 damage in any of the surviving rats. Electrophysiological techniques were then used to determine the effects of various periods of anoxia and aglycaemia (AA) on CA1 field excitatory postsynaptic potentials (EPSPs) in hippocampal slices incubated in the presence or absence of CHX. In CHX-treated slices, recuperation of EPSP amplitude (45±16%) was significantly greater than in control slices (9±9%) following an AA episode of 3 min 45 s. No difference was seen in the percent recuperation of EPSPs in the control and CHX-treated slices after shorter or longer episodes of AA. From these studies, it appears that CHX protects against the damaging effect of ischaemia in vivo or AA in vitro.     

Monosynaptic Interjoint Reflexes and their Central Modulation During Fictive Locomotion in Crayfish

An in vitro preparation of the crayfish nervous system has been utilized to study an interjoint reflex pathway and its variability during rhythmic locomotor activity. The coxo-basal chordotonal organ (CBCO) is a joint stretch receptor spanning the second joint of walking legs in crayfish, where it encodes joint movements and position. Mechanical stimulation (stretch and release) of the CBCO and electrical stimulation of the CBCO nerve elicits reflex responses in promotor and remotor motor neurons innervating muscles moving the basal thoraco-coxal (TC) leg joint. Promotor and remotor motor neurons receive monosynaptic excitatory inputs from at least four CBCO afferents, including both stretch- and release-sensitive CBCO afferents. In a tonic preparation, in which there is no tendency to produce alternating bursts of activity in antagonistic motor neurons, the reflex responses were evoked during each cycle of imposed movement. However, when the preparation became rhythmic and produced bouts of fictive locomotion, the reflex responses were unstable and their gain was phasically modulated. Paired recordings indicate that such a modulation of the monosynaptic interjoint reflex could be due to both a phasic change in the excitability of the motor neurons and presynaptic inhibition that reduces the excitatory input from CBCO primary afferents.