James F. Brien

Does carbon monoxide have a physiological function

Recently endothelium-derived relaxing factor (EDRF) has been identified as nitric oxide. The source of the nitric oxide is L-arginine, and the L-arginine-nitric oxide pathway has been proposed to function as a widespread transduction mechanism for the regulation of cell function and communication. Gerald Marks and colleagues suggest that carbon monoxide, which is formed endogenously from heme catabolism and which shares some of the chemical and biological properties of nitric oxide, may play a similar role. This would be achieved by carbon monoxide binding to the iron atom of the heme moiety of soluble guanylyl cyclase and to the iron-sulfur centers of macrophage enzymes.

Amiodarone hepatotoxicity simulating alcoholic liver disease

AMIODARONE, an iodine-containing benzofuran derivative, has been widely available in Europe for over 15 years, but its use in North America is still experimental. The drug has potent antiarrhythmic...

Impaired acquisition in the water maze and hippocampal long‐term potentiation after chronic prenatal ethanol exposure in the guinea‐pig

In the hippocampus, the CA1 region is selectively vulnerable to the effects of chronic prenatal ethanol exposure. In the guinea‐pig, the number of CA1 pyramidal cells is decreased after chronic prenatal ethanol exposure. We tested the hypotheses that chronic prenatal ethanol exposure (through maternal ethanol ingestion) results in impairments in spatial learning and short‐ and long‐term plasticity in the CA1 region of the postnatal guinea‐pig hippocampus. Timed, pregnant guinea‐pigs were treated with ethanol (4 g/kg maternal body weight/day), isocaloric sucrose/pair‐feeding, or water throughout gestation. Offspring were studied between postnatal days 40 and 80. In the Morris water maze, animals exposed to ethanol prenatally showed slower acquisition of an escape response to a hidden platform over 5 days of training. The amplitude of the field excitatory postsynaptic potential in the CA1 region in response to contralateral CA3 stimulation was decreased in offspring exposed to ethanol prenatally. Two forms of short‐term plasticity (paired‐pulse and frequency facilitation) were unaffected by chronic prenatal ethanol exposure. Long‐term potentiation (LTP) in response to high‐frequency CA3 stimulation was induced reliably and maintained over 60 min in isocaloric‐sucrose and water control animals. However, LTP failed to be induced in the CA1 area of the hippocampus in prenatal ethanol‐exposed offspring. These data show that chronic prenatal ethanol exposure, through maternal ethanol administration, impairs spatial performance and LTP in CA1 neurons. Hippocampal dysfunction could contribute importantly to the cognitive and behavioural deficits resulting from chronic prenatal ethanol exposure.

Chronic prenatal ethanol exposure alters hippocampal GABAA receptors and impairs spatial learning in the guinea pig

Chronic prenatal ethanol exposure (CPEE) can injure the developing brain, and may lead to the fetal alcohol syndrome (FAS). Previous studies have demonstrated that CPEE upregulates gamma-aminobutyric acid type A (GABA(A)) receptor expression in the cerebral cortex, and decreases functional synaptic plasticity in the hippocampus, in the adult guinea pig. This study tested the hypothesis that CPEE increases GABA(A) receptor expression in the hippocampus of guinea pig offspring that exhibit cognitive deficits in a hippocampal-dependent spatial learning task. Timed, pregnant guinea pigs were treated with ethanol (4 g/kg maternal body weight per day), isocaloric-sucrose/pair-feeding, or water throughout gestation. GABA(A) receptor subunit protein expression in the hippocampus was measured at two development ages: near-term fetus and young adult. In young adult guinea pig offspring, CPEE increased spontaneous locomotor activity in the open-field and impaired task acquisition in the Morris water maze. CPEE did not change GABA(A) receptor subunit protein expression in the near-term fetal hippocampus, but increased expression of the beta2/3-subunit of the GABA(A) receptor in the hippocampus of young adult offspring. CPEE did not change either [(3)H]flunitrazepam binding or GABA potentiation of [(3)H]flunitrazepam binding, but decreased the efficacy of allopregnanolone potentiation of [(3)H]flunitrazepam binding, to hippocampal GABA(A) receptors in adult offspring. Correlational analysis revealed a relationship between increased spontaneous locomotor activity and growth restriction in the hippocampus induced by CPEE. Similarly, an inverse relationship was found between performance in the water maze and the efficacy of allopregnanolone potentiation of [(3)H]flunitrazepam binding in the hippocampus. These data suggest that alterations in hippocampal GABA(A) receptor expression and pharmacological properties contribute to hippocampal-related behavioral and cognitive deficits associated with CPEE.

Gas-liquid chromatographic analysis of ethanol and acetaldehyde in blood with minimal artifactual acetaldehyde formation

A gas-liquid chromatographic procedure utilizing headspace gas analysis is described for the determination of ethanol and its metabolite, acetaldehyde, in a 100-microliters sample of blood from the rat, guinea pig, sheep, or human. Artifactual formation of ethanol-derived acetaldehyde is minimized during sample preparation by using a chemical solution containing perchloric acid and sodium azide in saline, and thiourea. Aqueous standards of ethanol and acetaldehyde are used to calibrate the procedure, and 1-propanol is used as the internal standard of the method. The recovery of ethanol and acetaldehyde from spiked blood samples is quantitative and reproducible, with a within-day coefficient of variation of less than 7% for ethanol and less than 9% for acetaldehyde. The lower limit of quantitative sensitivity is 0.006 mg/ml ethanol and 0.10 microgram/ml acetaldehyde. The instrumental analysis time is less than 3 min, which enables high sample throughput.

Effects of chronic prenatal ethanol exposure on locomotor activity, and hippocampal weight, neurons, and nitric oxide synthase activity of the young postnatal guinea pig

Decreased nitric oxide synthase (NOS)-catalyzed formation of NO from L-arginine may be involved in ethanol teratogenesis involving the hippocampus. This hypothesis was tested by determining the effects of chronic prenatal ethanol exposure on locomotor activity and on hippocampal weight, number of CA1 and CA3 pyramidal cells and dentate gyrus granule cells, and NOS activity of the postnatal guinea pig. Timed, pregnant guinea pigs received one of the following chronic oral regimens throughout gestation: 4 g ethanol/kg maternal body weight/day, isocaloric-sucrose/pair-feeding, or water. At postnatal day (PD) 10, spontaneous locomotor activity was measured. At PD 12, histological analysis was performed on the hippocampal formation, in which hippocampal CA1 and CA3 pyramidal cells and dentate gyrus granule cells were counted; body, brain, and hippocampal weights were measured; and hippocampal NOS enzymatic activity was determined using a radiometric assay. Chronic prenatal ethanol exposure produced hyperactivity, decreased the brain and hippocampal weights with no change in body weight, decreased the number of hippocampal CA1 pyramidal cells by 25-30%, and had no effect on hippocampal NOS activity compared with the two control groups. These data, together with our previous findings in the fetal guinea pig, demonstrate that chronic prenatal ethanol exposure decreases hippocampal NOS activity in near-term fetal life that temporally precedes the selective loss of hippocampal CA1 pyramidal cells in postnatal life.

Selectivity of imidazole–dioxolane compounds for in vitro inhibition of microsomal haem oxygenase isoforms

Haem oxygenases (HO) are involved in the catalytic breakdown of haem to generate carbon monoxide (CO), iron and biliverdin. It is widely accepted that products of haem catabolism are involved in biological signaling in many physiological processes. Conclusions to most studies in this field have gained support from the judicious use of synthetic metalloporphyrins such as chromium mesoporphyrin (CrMP) to selectively inhibit HO. However, metalloporphyrins have also been found to inhibit other haem‐dependent enzymes, such as nitric oxide synthase (NOS), cytochromes P‐450 (CYPs) and soluble guanylyl cyclase (sGC), induce the expression of HO‐1 or exhibit varied toxic effects. To obviate some of these problems, we have been examining non‐porphyrin HO inhibitors and the present study describes imidazole–dioxolane compounds with high selectivity for inhibition of HO‐1 (rat spleen microsomes) compared to HO‐2 (rat brain microsomes) in vitro. (2R,4R)‐2‐[2‐(4‐chlorophenyl)ethyl]‐2‐[(1H‐imidazol‐1‐yl)methyl]‐4‐methyl‐1,3‐dioxolane hydrochloride) was identified as the most selective inhibitor with a concentration of 0.6 μM inhibiting HO‐1(inducible) by 50% compared with 394 μM for HO‐2 (constitutive). These compounds were found to have no effects on the catalytic activities of rat brain NOS and lung sGC, but were potent inhibitors of microsomal CYP2E1 and CYP3A1/3A2 activities. In conclusion, we have identified imidazole–dioxolanes that are able to inhibit microsomal HO in vitro with high selectivity for HO‐1 compared to HO‐2, and little or no effect on the activities of neuronal NOS and sGC. These molecules could be used to facilitate studies on the elucidation of physiological roles of HO/CO in biological systems.

Quantitation of nitric oxide formation from nitrovasodilator drugs by chemiluminescence analysis of headspace gas

A rapid and reliable method has been developed for the quantitation of nitric oxide by chemiluminescence analysis of headspace gas. Aqueous nitric oxide standards are used to calibrate the method. There is a linear relationship between the amount of nitric oxide and the redox chemiluminescence detector response over the range of 52-2445 pmol of nitric oxide in 2.0 mL of deionized water contained in a sealed 6.2-mL flask. The intra-day and inter-day coefficient of variation values of the method do not exceed 4% and 9%, respectively. The lower limit of quantitative sensitivity and the lower limit of qualitative detection are 52 pmol and 26 pmol of nitric oxide, respectively. This method has been used to measure nitric oxide formation during the incubation of glyceryl trinitrate or sodium nitroprusside in the presence of cysteine. This method has two major advantages over the currently available procedures for the quantitation of nitric oxide, viz., no artifactual formation of nitric oxide during sample preparation and decreased instrumental contamination.

Dose-dependent effects of prenatal ethanol exposure in the guinea pig

The guinea pig is an appropriate animal for studying ethanol central nervous system (CNS) teratogenesis due to its extensive prenatal CNS development. In order to establish an ethanol dosage regimen that produces CNS teratogenesis, the objective of this study was to characterize the dose-dependent effects of chronic ethanol administration on pregnancy outcome and locomotor activity of the offspring. Pregnant guinea pigs received one of the following oral treatments, via intubation into the oral cavity, throughout gestation: 3, 4, 5 or 6 g ethanol/kg maternal body weight/day; isocaloric sucrose and pair feeding; or water. The 5 and 6 g ethanol/kg/day regimens produced maternal death, spontaneous abortion, and perinatal death with at least 75% incidence; the 3 and 4 g ethanol/kg/day regimens produced little or no maternal, embryonic/fetal, or perinatal lethality. The 3 and 4 g ethanol/kg/day regimens did not affect other indices of pregnancy outcome compared with the respective isocaloric-sucrose pair-fed control animals and water-treated animals. The 3, 4, and 5 g ethanol/kg/day regimens increased spontaneous locomotor activity in the offspring, and there was a direct relationship between the magnitude of hyperactivity at days 10 and 60 of age and each of the ethanol dosage regimens and the maternal blood ethanol concentration on day 56 of gestation. The data demonstrate that, in the guinea pig, chronic oral administration of ethanol produces: (a) dose-dependent effects on pregnancy outcome, (b) hyperactivity in the offspring that is dose- (and maternal blood ethanol concentration-) and age-related, and (c) persistent hyperactivity into adulthood with minimal toxicity on pregnancy outcome for the 4 g ethanol/kg/day regimen.

Measurement of endogenous carbon monoxide formation in biological systems.

Endogenous carbon monoxide (CO) formation has been measured in different biological systems using a variety of analytical procedures. The methods include gas chromatography-reduction gas detection, gas chromatography-mass spectroscopic detection, laser sensor-infrared absorption, UV-visible spectrophotometric measurement of CO-hemoglobin or CO-myoglobin complex, and formation of (14)CO from (14)C-heme formed following [2-(14)C]glycine administration. CO formation ranged from a low of 0.029 nmol/mg of protein/h in chorionic villi of term human placenta to a high of 0.28 nmol/mg of protein/h in rat olfactory receptor neurons in culture and rat liver perfusate.