Thomas M. Devlin

A novel treatment of global cerebral ischaemia with a glycine partial agonist

Chronic treatment of gerbils with 1-aminocyclopropanecarboxylic acid (a high affinity, partial agonist at strychnine-insensitive glycine receptors) resulted in a 3-fold increase in survival, a significant improvement in neurological status, and an extensive protection of vulnerable brain regions following severe forebrain ischaemia. A bolus of 1-aminocyclopropanecarboxylic acid 30 min prior to ischaemia did not further improve outcome compared to gerbils receiving their last injection 24 h prior to ischaemia. These findings are consistent with the hypothesis that chronic treatment with a glycine partial agonist desensitizes the N-methyl-D-aspartate receptor complex. Pharmacological intervention at the strychnine-insensitive glycine receptor may be an effective means of ameliorating the consequences of neuronal degeneration caused by excitotoxic phenomena.

Calcium ionophore activity of a prostaglandin B1 derivative (PGBX)

Abstract A water soluble derivative of PGB1, designated PGBX, has been found to stimulate the release of Ca2+ from fragmented sarcoplasmic reticulum and heart mitochondria; its activity is almost two orders of magnitude greater than other prostaglandins. PGBX demonstrates ionophoretic activity in its ability to transfer Ca2+ from an aqueous to an organic phase.

Control of mitochondrial matrix calcium: Studies using fluo-3 as a fluorescent calcium indicator

Fluo-3, a fluorescent Ca2+ indicator, is sequestered by isolated rat liver mitochondria and is an effective probe for evaluating the concentration and kinetics of change of mitochondrial matrix ionized calcium ([Ca2+]m) under a variety of conditions. At the wavelengths employed, there is no significant interference by auto-fluorescence. There is an insignificant release of the indicator over four hours and the loading and presence of fluo-3 has no effect on respiratory rate or oxidative phosphorylation. The [Ca2+]m steady state can be altered by the assay conditions, i.e. the presence of extra-mitochondrial Ca2+, Mg2+ phosphate and respiratory inhibitors. The total matrix ionized calcium represents a small percent (less than 0.01%) of the total mitochondrial calcium.

Extracellular ATP inhibits agonist-induced mobilization of internal calcium in human platelets

Our previous studies have demonstrated that platelets possess ATP purinergic receptors in addition to the ADP, P2T, receptor. Occupancy of the P2 receptor by ATP inhibited agonist-induced platelet aggregation. This study demonstrated that the mechanism of inhibition may involve ATP inhibition of agonist-induced mobilization of internal calcium. Within the cardiovascular system, the ATP inhibition of calcium mobilization is unique to platelets. All other cell types in the cardiovascular system, where calcium mobilization is affected by extracellular ATP, responded with an increased mobilization as opposed to inhibition. The platelet inhibitory response to ATP was enhanced by the addition of an ATP generating system, creatine phosphate/phosphocreatine kinase. ATP and ATP analogues were found to inhibit calcium mobilization with a rank order of alpha beta-methylene ATP, beta gamma-methylene ATP approximately ATP > benzoyl ATP > 2 methylthio ATP which is a characteristic of P2x-like receptors. The inhibitory effect of ATP could be abrogated by prolonged treatment of platelets with the P2x desensitizing agent, alpha beta-methylene ATP. Also, UTP and CTP were approximately as effective inhibitors as ATP while GTP was not. ATP competition with ADP for the P2T receptor was excluded in studies with platelets derived from an aspirin-treated individual which were essentially insensitive to ADP. The agonist-induced calcium mobilization and inhibition by ATP occurred with the thromboxane A2 mimetic, U46619, collagen and thrombin; however, the kinetics of mobilization varied somewhat with the different agonists. The responses to extracellular ATP were independent of extracellular Ca2+, where 1 mM calcium or 0.3 mM EGTA was added to the reaction mixture. The inhibition of calcium mobilization coupled to inhibition of platelet aggregation by extracellular ATP may serve an important physiologic role. ATP, released from activated platelets at localized sites of vascular injury, may help to limit the size of the platelet plug-clot that, if left unregulated, could occlude the injured blood vessel.

Activation and deactivation of aflatoxin B1 in isolated rat hepatocytes.

Isolated rat hepatocytes took up [3H]-aflatoxin B1 during incubation with fifty percent of the aflatoxin B1 covalantly bound to cellular macromolecules. The amount of bound-aflatoxin B1 was proportional to the medium concentration of aflatoxin B1. The specific radioactivity (pmole/mg) of aflatoxin B1 found in the DNA fraction was 20 fold greater than that associated with protein. Metyrapone (0.75 mM) inhibited significantly the uptake and binding whereas 1,2-epoxy-3,3,3-trichloropropane (0.5 mM) enhanced 2-3 fold both the uptake and binding. Glutathione (0.25 mM) reduced these processes. Results indicate that a transformation of aflatoxin B1 is catalyzed by cytochrome P-450 mixed function oxidase and aflatoxin B1-2,3-epoxide so formed is primarily deactivated by epoxide hydrolase. In the isolated hepatocyte depletion of the epoxide by glutathione apparently has an insignificant role in aflatoxin detoxication.

Effect of inhibitors of microsomal enzymes on aflatoxin B1-induced cytotoxicity and inhibition of RNA synthesis in isolated rat hepatocytes.

Previous studies conducted in this laboratory demonstrated that AFB1 activation and deactivation was effectively inhibited by metyrapone and TCPO in isolated hepatocytes. The present study was undertaken to study the toxic effect of AFB1 on hepatocyte and RNA synthesis, and to assess the influence of the inhibitors on AFB1-induced cytotoxicity and AFB1-inhibited RNA synthesis. AFB1 at 50 microM was toxic and inhibited macromolecular synthesis by greater than 70% at 180 min of incubation whereas at lower concentrations of AFB1 (0.05-10 microM) dose-and time-dependent decreases in cell viability, protein and RNA synthesis were observed. Using [3H]-AFB1 (0.1.5 microM), the uptake and covalent binding of the toxin were also dose-and time-dependent. Initial rates of these processes to reach half-maximum was found to be 0.25 microM AFB1. In cells treated with AFB1 (5 microM) and metyrapone (1.0 mM) or SKF-525A (10 microM), the cell viability was similar to the control and [3H]-uridine incorporation was significantly higher than AFB1 treated cells. AFB1 and TCPO (0.5 mM) treated cells exhibited further decreases in cell viability and RNA synthesis. Results suggest that the binding of AFB1 to DNA and impairment of transcriptional activity may lead to cell death.

MK-801 is neuroprotective but does not improve survival in severe forebrain ischemia

The effects of MK-801 on postischemic recovery, survival and neuronal preservation in the cortex, hippocampus and striatum were studied in Mongolian gerbils. The drug was administered 30 min prior to 20 of min forebrain ischemia induced by bilateral ligation of the carotids. Neurological recovery and survival were monitored for 7 days. At the end of the monitoring period neuronal damage was analyzed in the brains of the survivors in both groups. Treatment with MK-801 did not improve either neurological recovery or end-point survival. However, significant (P < 0.01) neuronal protection was observed in the hippocampi and striata of the drug treated animals while cortical neurons were not significantly protected. These findings demonstrate that protection against ischemic neuronal damage can be observed without concomitant improvement in either postischemic neurological recovery or survival. Protection of selectively vulnerable brain regions, often used as the predictor of the therapeutic potential of an agent, does not appear to correlate well with postischemic survival in this animal model of ischemia.

19F-NMR and fluorescence polarization of yeast plasma membrane and isolated lipids

Summary 16-Fluoropalmitic acid was incorporated biosynthetically into the plasma membrane of yeast and the F-NMR spectra of the intact membrane and of aqueous dispersions of membrane lipids were compared. The greater degree of resonance broadening in the intact membrane reveals a lesser degree of mobility of the acyl chains. Fluorescence polarization studies with a probe that monitors the polar environment and one that detects viscosity changes in the inner core of the bilayer indicate hindered motions of the polar head groups and acyl chains in the intact membrane as compared to liposomes prepared from membrane lipids. The observations indicate that the bulk of lipids in the yeast membrane experiences immobilization by proteins and that membrane proteins influence lipid-lipid interactions and inhibit lipid phase transition.

The distribution and intracellular translocation of aflatoxin B1 in isolated hepatocytes

The distribution and intracellular translocation of AFB1 in various subcellular fractions was investigated in isolated hepatocytes by pulse-chase experiments. After labeling the hepatocytes with [3H]-AFB1 (14.5 nM) for 15 min, the highest concentration of [3H]-AFB1 was found in the cytosolic fraction where 66% was bound noncovalently and 1.5% covalently. The lowest concentration of [3H]-AFB1 was found in the nuclear fraction; 36% and 4.9% were bound noncovalently and covalently respectively. When the [3H]-AFB1 loaded cells were chased with unlabeled AFB1 (1 microM), the radioactivity of [3H]-AFB1 in the cell lysate and cytosolic fraction decreased in time with an apparent rate of elimination (t1/2) of 93 min and 66 min, respectively. The levels of covalently bound AFB1 increased with time and reached a maximum at 60 min in nuclei (270%), and at 120 min in mitochondria (220%) and cytosol (430%) as compared to the zero time. Only in the microsomal fraction was there no significant increase with time in covalently bound AFB1. These results suggest that the toxin after activation by the microsomal mixed function oxidases was either detoxified or transported to other cellular organelles where covalent binding of macromolecules occurred.

The in vivo disposition of aflatoxin B1 in rat liver

The disposition of a non-toxic i.p. dose of [3H]-aflatoxin B1 (0.70 micrograms/kg) in the blood, plasma, and liver was studied in male Wistar rats. Uptake into the blood, plasma, and liver was biphasic; there was an initial rapid rise (0-2 hr) followed by a second phase (2-12 hr) of a gradual increase. Most of the radioactivity in the blood was bound noncovalently to albumin. Distribution of radioactivity in the subcellular fractions of liver showed that the microsomes exhibited the highest labeling which increased over the time course; labeling of the cytosol reached a maximum at 2 hr then decreased to a new steady state, whereas the mitochondria and nuclei reached a plateau. When the content of aflatoxin B1 in the nuclear subfractions was examined, greater than 92% of the total radioactivity was found in the deoxyribonucleoprotein fraction, and 84% of this was bound noncovalently. These results suggest that aflatoxin B1 is transported from the site of injection through the blood to the liver and its subcellular and subnuclear fractions primarily in a noncovalent form.