Low level hyperbaric antagonism of diazepam's locomotor depressant and anticonvulsant properties in mice.

Exposure to 12 atmospheres absolute (12 ATA) helium oxygen gas (heliox) (low level hyperbaric exposure) antagonizes the behavioral effects of ethanol and n-propanol, but not morphine. These and other results indicate that the mechanism of the antagonism is direct (pharmacodynamic) and selective. Our study further investigates the selectivity of low level hyperbaric antagonism by testing its effectiveness against diazepam, a high affinity binding drug that acts via allosteric modulation of GABAA receptors. C57BL/6J mice received injections i.p. of vehicle or diazepam, and were then exposed to 1 ATA air, 1 ATA heliox or 12 ATA heliox. Exposure to 12 ATA heliox antagonized the locomotor depressant effect of 4 and 6 mg/kg, but not 8 mg/kg diazepam. Hyperbaric exposure also antagonized the anticonvulsant effect of 8 and 24 mg/kg, but not 4 mg/kg, diazepam vs. 300 mg/kg isoniazid. Exposure to 12 ATA heliox did not significantly affect blood concentrations of diazepam or its metabolite n-desmethyl diazepam. The pharmacological characteristics of the antagonism (direct, surmountable, rightward shift in diazepam's dose-response curve) closely matched those seen in previous studies for hyperbaric antagonism of ethanol. The results add to the evidence that low level hyperbaric exposure is a direct, mechanistic antagonist that selectively antagonizes drugs that act via perturbation or allosteric modulation of receptor function. Moreover, the results suggest that allosteric coupling pathways, which transduce binding events on ligand-gated ion channels, may represent initial sites of action for ethanol.

Low-level hyperbaric exposure antagonizes locomotor effects of ethanol and n-propanol but not morphine in C57BL mice.

Low-level hyperbaric exposure antagonizes a broad range of behavioral effects of ethanol in a direct, reversible, and competitive manner. This study investigates the selectivity of the antagonism across other drugs. C57BL/6 mice were injected with saline, ethanol, n-propanol, or morphine sulfate, and then were exposed to 1 atmosphere absolute (ATA) air, 1 ATA helium-oxygen gas mixture (heliox), or 12 ATA heliox. Locomotor activity was measured from 10 to 40 min following injection. N-propanol produced a dose-dependent depression of locomotor activity from 1.0 g/kg. Morphine produced a dose-dependent stimulation of locomotor activity at doses of 3.75-12.0 mg/kg. Exposure to 12 ATA heliox significantly antagonized the locomotor depressant effects of 1.0 g/kg n-propanol and 2.5 g/kg ethanol, without significantly affecting blood concentrations of these drugs measured at 40 min postinjection. Exposure to 12 ATA heliox did not significantly antagonize the locomotor-stimulating effects of the two morphine doses tested (3.75 and 7.5 mg/kg). These findings suggest that exposure to 12 ATA heliox antagonizes the behavioral effects of intoxicant-anesthetic drugs like ethanol and n-propanol, which are believed to act via perturbation or allosteric modulation of functional proteins, but does not antagonize the effects of drugs like morphine, which act via more direct mechanisms. This demonstration of selective antagonism adds important support for the hypothesis that low-level hyperbaric exposure is a direct mechanistic ethanol antagonist, with characteristics similar to a competitive pharmacological antagonist.

Temperature dependence of ethanol depression in mice: dose response.

Manipulation of body temperature during intoxication significantly alters brain sensitivity to ethanol. The current study tested the generality of this effect within the hypnotic dose range. Drug naive, male C57BL/6J mice were injected with 3.2, 3.6, or 4.0 g/kg ethanol (20% w/v) and were exposed to 1 of 7 designated temperatures from 13 degrees to 34 degrees C to manipulate body temperature during intoxication. Rectal temperature at return of righting reflex (RORR) was significantly, positively correlated with loss of righting reflex (LORR) duration and significantly, negatively correlated with blood ethanol concentration (BEC) at RORR at all three doses. These results indicate that increasing body temperature during intoxication increased ethanol sensitivity in C57 mice at all three doses tested and demonstrate the generality of temperature dependence across hypnotic doses in these animals. Interestingly, the LORR duration was dose-dependent at each ambient temperature, but the degree of body temperature change and the BEC at RORR were not dose-dependent. Overall, these results emphasize the importance of body temperature as a variable in ethanol research.

Low-level hyperbaric antagonism of ethanol-induced locomotor depression in C57BL/6J mice: dose response.

This study characterized the antagonistic effects of hyperbaric exposure on the dose-response curve for ethanol-induced depression of locomotor activity. Drug-naive, male C57BL/6 mice were injected intraperitoneally with saline, 1.5, 2.0, 2.5, or 3.0 g/kg ethanol, and were exposed to 1 atmosphere absolute (ATA) air or 12 ATA helium-oxygen gas mixtures (heliox) at temperatures that offset the hypothermic effects of ethanol and helium. Locomotor activity was measured 10-30 min after injection. In addition, the effects of exposure to 12 ATA heliox on blood ethanol concentrations were tested in a separate group of mice injected with 2.5 g/kg ethanol. Ethanol produced a dose-dependent depression of locomotor activity beginning at 2.0 g/kg. Exposure to 12 ATA heliox completely antagonized the locomotor depressant effects of 2.0 and 2.5 g/kg ethanol and partially blocked the effects of 3.0 g/kg. Activity in mice given 1.5 g/kg ethanol was not significantly affected at 1 ATA air, but was significantly increased at 12 ATA heliox. Low-level hyperbaric exposure shifted the ethanol dose-response curve to the right with a resultant increase in the ED50 of ethanol for locomotor depression from 2.6 to 3.3 g/kg. Exposure to 12 ATA heliox did not alter blood ethanol concentrations in mice injected with 2.5 g/kg ethanol. These findings with 12 ATA heliox present key new evidence for the hypothesis that low-level hyperbaric exposure acts directly, with a pattern analogous to a competitive, mechanistic antagonist of ethanol.

Genetically determined differences in the antagonistic effect of pressure on ethanol-induced loss of righting reflex in mice.

Hyperbaric exposure antagonizes ethanol's behavioral effects in a wide variety of species. Recent studies indicating that there are genetically determined differences in the effects of body temperature manipulation on ethanol sensitivity suggested that genotype might also influence the effects of hyperbaric exposure on ethanol intoxication. To investigate this possibility, ethanol injected long sleep (LS)/Ibg (2.7 g/kg), short sleep (SS)/Ibg (4.8 g/kg), 129/J (2.9 g/kg), and C57BL/6J (3.6 g/kg) mice were exposed to one atmosphere absolute (ATA) air or to one or 12 ATA helium-oxygen (heliox) at ambient temperatures selected to offset ethanol and helium-induced hypothermia. Hyperbaric exposure significantly reduced loss of righting reflex (LORR) duration in LS, 129, and C57 mice, but not in SS mice. A second experiment found that hyperbaric exposure significantly reduced LORR duration and increased the blood ethanol concentration (BEC) at return of righting reflex (RORR) in LS mice, but did not significantly affect either measure in SS mice. These results indicate that exposure to 12 ATA heliox antagonizes ethanol-induced LORR in LS, 129 and C57 mice, but not in SS mice. Taken with previous results, the present findings suggest that the antagonism in LS, 129, and C57 mice reflects a pressure-induced decrease in brain sensitivity to ethanol and that the lack of antagonism in SS mice cannot be explained by pressure-induced or genotypic differences in ethanol pharmacokinetics.(ABSTRACT TRUNCATED AT 250 WORDS)

The relationship between brain temperature during intoxication and ethanol sensitivity in LS and SS mice.

The present study characterized the relationship between brain temperature, rectal temperature, and ethanol sensitivity in the selectivity bred long-sleep (LS) and short-sleep (SS) mice. Radiotelemetric brain probe implanted and nonimplanted LS/lbg and SS/lbg male mice were injected with 2.5 and 4.9 g/kg ethanol, respectively, before exposure to ambient temperatures of 15 degrees C, 22 degrees C, or 34 degrees C. Ambient temperature significantly affected rectal temperature, brain temperature, and ethanol sensitivity, measured by impairment of righting reflex. Brain and rectal temperatures at return of righting reflex (RORR) were highly correlated. In SS mice brain and rectal temperatures at RORR were significantly positively correlated with loss of righting reflex (LORR) duration and significantly negatively correlated with blood ethanol concentration (BEC) at RORR. In LS mice rectal temperature at RORR was significantly negatively correlated with LORR duration, while both brain and rectal temperature at RORR were significantly positively correlated with BEC at RORR. The strength of the correlations and r2 values generated from linear regression analysis indicates that body temperature during intoxication can explain up to 52% of the variability in ethanol sensitivity in SS mice, but only 19% of the variability in ethanol sensitivity in LS mice. The correlational analyses are consistent with previous results based on comparisons between rectal temperature and ethanol sensitivity and extend to direct brain temperature measurement the evidence that decreasing temperature during intoxication decreases ethanol sensitivity in SS mice and increases ethanol sensitivity in LS mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of subcutaneous and intracerebroventricular administration of the sigma receptor ligand 1,3-Di-o-tolylguanidine on body temperature in the rat: interactions with BMY 14802 and rimcazole.

The current study investigated the effects of the acute s.c. and i.c.v. administration of 1,3-di-o-tolylguanidine (DTG) on body temperature in rats. The effects of putative sigma receptor antagonists BMY 14802 and rimcazole on DTG-induced changes in body temperature also were evaluated. The acute s.c. administration of DTG (10.0 and 20.0 mg/kg) produced hypothermia but no observable behavioral effects. Similarly, the acute i.c.v. administration of DTG (12.0-100.0 micrograms/rat) produced hypothermia, but ataxia occurred after this route of administration. The s.c. administration of BMY 14802 alone (25.0 mg/kg) decreased body temperature and enhanced the DTG-induced hypothermia, whereas the administration of rimcazole (25.0 mg/kg) neither altered body temperature nor affected the hypothermia produced by DTG. Neither BMY 14802 nor rimcazole produced any behavioral effects when administered alone. The inability of the putative sigma receptor antagonists BMY 14802 and rimcazole to antagonize DTG-induced hypothermia suggests that either these compounds at the dose used have little sigma receptor antagonist activity, or that the DTG-induced hypothermia is not due to specific interactions with sigma receptors.

Brain temperature and ethanol sensitivity in C57 mice: a radiotelemetric study.

This study investigated the relationship between ethanol sensitivity and brain temperature using radiotelemetric techniques. Radiotelemetric brain probes were implanted in the lateral cerebral ventricle of C57BL/6 mice. Rectal and brain temperatures, duration of loss of righting reflex (LORR), and blood and brain ethanol concentrations at the return of righting reflex (RORR) were measured following intraperitoneal (IP) injection with 3.6 g/kg ethanol and exposure to 12, 15, 22 or 34 degrees C. Rectal and brain temperatures were significantly correlated in untreated and intoxicated mice. Brain temperatures were lower than rectal temperatures in untreated mice, but were not different than rectal temperatures in intoxicated mice. Ethanol sensitivity, measured by the duration of LORR and ethanol concentrations at RORR, was significantly correlated with brain as well as rectal temperatures at RORR. Brain probe implantations did not significantly affect ethanol sensitivity. The direct positive relationship between brain temperature and ethanol sensitivity in C57 mice fits predictions based on membrane actions of ethanol and supports the hypothesis that temperature-induced changes in behavioral sensitivity to ethanol are mediated through changes in brain membrane temperature.

Temperature alters ethanol-induced fluidization of C57 mouse brain membranes.

The interaction between temperature and ethanol-induced fluidization was investigated in brain synaptic plasma membranes from C57BL/6 mice. Changes in fluidity were measured using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Fluorescence polarization was tested in the presence and absence of ethanol at 25, 32 and 37 degrees C. An increase in temperature resulted in a significant increase in the baseline fluidity of the membranes and an increase in the magnitude of ethanol-induced fluidization of brain membranes. The combined effect of temperature on baseline fluidity and the magnitude of the response to ethanol resulted in a significant temperature-related increase in the relative response to ethanol (% change in polarization). The minimum concentration of ethanol required to cause a significant increase in the fluidity of the membranes was 170.7 mM at 25 degrees C and 85.3 mM at both 32 and 37 degrees C. The present results indicate that temperature-related changes in the effects of ethanol on membrane properties may underlie the effects of temperature on ethanol sensitivity in C57 mice.

Body temperature influences ethanol and ethanol/pentobarbital lethality in mice.

The effect of body temperature on ethanol (ETOH) or ethanol plus pentobarbital (ETOH/PB) lethality was investigated in C57BL/6J mice. Decreasing ambient temperatures from 35-20 degrees C, decreased rectal temperatures from 38-20 degrees C and increased 8-hour survival from 0-93% for ETOH-treated and from 0-100% for ETOH/PB-treated mice. ETOH and ETOH/PB animals with no hypothermia (body temperatures = 38 degrees C) had the highest lethality. Those with body temperatures between 30-32 degrees C had the highest 24-hour survival. These results suggest that controlled hypothermia may be useful in reducing lethality from ethanol or ethanol/combination overdoses.

Body temperature and ethanol pharmacokinetics in temperature-challenged mice.

The relationships between ambient temperature, body temperature, ethanol pharmacokinetics and behavioral sensitivity to ethanol were examined in C57BL/6 mice. Mice were injected intraperitoneally with 3.6 or 2.0 g/kg ethanol. Exposure to increasing ambient temperatures of 4-34 degrees C immediately after ethanol injection resulted in a significant increase in body temperature, ethanol elimination rate and brain:blood ethanol concentration ratios in 3.6 g/kg ethanol-injected mice, but not in mice injected with 2.0 g/kg ethanol. As the mean body temperature increased from 26.0 to 38.2 degrees C in the 3.6 g/kg mice, there was a 50% increase in ethanol elimination rate. Delayed (30 min) exposure to increasing ambient temperatures following injection of 3.6 g/kg ethanol resulted in a significant increase in ethanol elimination rate, a marked increase in the duration of loss of righting reflex and a decrease in ethanol concentration at the regain of righting reflex. The results indicate that temperature-induced changes in the absorption, distribution and elimination of ethanol do not appear to mediate the effects of temperature on behavioral sensitivity to ethanol in C57 mice.

Body temperature differentially affects ethanol sensitivity in both inbred strains and selected lines of mice.

Offsetting ethanol-induced hypothermia in five inbred strains of mice changed ethanol sensitivity within strains and markedly reduced differences between strains in brain sensitivity to hypnotic ethanol doses. The present study extended this work to mice selectively bred for sensitivity and resistance to ethanol-induced loss of righting reflex (LORR) and hypothermia. In all experiments LORR duration and ethanol concentrations at return of righting reflex were measured after i.p. hypnotic ethanol doses and exposure to 22 or 34 degrees C. In experiment 1, C57BL/6J, A/HeJ, 129/J, LS/lbg and SS/lbg mice were given 4.2 g/kg ethanol. In experiment 2, the same mouse genotypes were tested with different ethanol doses (2.5-4.9 g/kg) selected to produce an equivalent degree of impairment (60 min LORR duration). In experiment 3, HOT and COLD lines of mice were given 4.0 g/kg ethanol. In agreement with previous work, offsetting hypothermia reduced differences between genotypes in ethanol sensitivity. Comparisons within genotypes indicated that ethanol sensitivity in C57, A/He, SS, HOT and COLD mice increased as body temperature increased. In contrast, ethanol sensitivity in 129 and LS mice decreased as body temperature increased. These results extend previous findings indicating that body temperature during intoxication contributes to differences between genotypes in ethanol sensitivity. The present findings also suggest that there are qualitative differences in the effects of temperature on ethanol sensitivity within genotypes.

Effects of acute and chronic administration of (+)-SKF 10,047 on body temperature in the rat: cross-sensitization with phencyclidine.

The current study investigated the effects of acute and chronic administration of (+)-SKF 10,047 on body temperature in rats. The effect of phencyclidine on body temperature in chronically (+)-SKF 10,047-treated rats was also investigated. The acute administration of (+)-SKF 10,047 at doses of 5 to 40 mg/kg s.c. did not alter body temperature; however, 80 mg/kg produced hypothermia. In contrast, chronic administration of (+)-SKF 10,047 (5-20 mg/kg) produced dose-dependent hyperthermia when tested on day 7 and 10 of chronic treatment. Moreover, sensitization to the hyperthermic effects occurred as the degree of hyperthermia was greater on day 10 compared to day 7. Phencyclidine (20 mg/kg s.c.) produced hypothermia in rats chronically treated with saline for 13 days, but hyperthermia in rats chronically treated with 20 mg/kg of (+)-SKF 10,047 for the same duration. The hyperthermic effect of chronic (+)-SKF 10,047 treatment is similar to the previously reported dose-dependent hyperthermia in chronically phencyclidine-treated animals. The cross-sensitization of chronically (+)-SKF 10,047-treated rats to the hyperthermic effects of phencyclidine supports the hypothesis that there may be common mechanisms underlying the chronic effects of these drugs on body temperature.

Temperature affects ethanol lethality in C57BL/6, 129, LS and SS mice.

The effect of ambient and body temperature on ethanol lethality in inbred strains and selected lines of mice was investigated. C57BL/6J, 129/J, LS/Ibg and SS/Ibg mice were exposed to 23 or 34 degrees C following IP injection of lethal ethanol doses (8.2, 6.0, 6.5 or 7.0 g/kg ethanol, respectively). All mice exposed to 23 degrees C during intoxication became markedly hypothermic, with mean body temperatures dropping to lows of 27.9, 30.3, 33.0 and 33.3 degrees C in C57, LS, SS and 129 animals, respectively. Compared to the 23 degrees C groups, exposure to 34 degrees C offset the ethanol-induced hypothermia and significantly increased percent mortality in all four mouse genotypes. Exposure to 34 degrees C increased mortality at 24 hours postinjection from 15% to 95% in SS mice, from 37.5% to 100% in 129 mice and from 50% to 100% in LS and C57 mice. Blood ethanol data suggest that the present results cannot be explained by temperature-related changes in ethanol elimination. These results provide further evidence that body temperature during intoxication can have major effects on mortality rates in mice.

Genetically determined differences in ethanol sensitivity influenced by body temperature during intoxication.

The present study investigated the importance of body temperature during intoxication in mediating differences between five inbred strains of mice (C57BL/6J; BALB/cJ; DBA/2J; A/HeJ; 129/J) in their acute sensitivity to the hypnotic effects of ethanol. Mice exposed to 22 degrees C after ethanol injection became hypothermic and exhibited statistically significant differences between strains in rectal temperatures at the return of the righting reflex (RORR), duration of loss of the righting reflex (LORR), and blood and brain ethanol concentrations at RORR. Exposure to 34 degrees C after injection offset ethanol-hypothermia and markedly reduced strain-related differences in rectal temperatures and blood and brain ethanol concentrations at RORR. Brain ethanol concentrations at RORR were significantly lower in C57, BALB, DBA and A/He mice exposed to 34 degrees C compared to mice exposed to 22 degrees C during intoxication suggesting that offsetting hypothermia increased ethanol sensitivity in these strains. Taken with previous in vitro studies, these results suggest that genetically determined differences in acute sensitivity to the behavioral effects of ethanol reflect differences in body temperature during intoxication as well as differences in sensitivity to the initial actions of ethanol at the cellular level.

Chronic functional ethanol tolerance in mice influenced by body temperature during acquisition.

Previous studies have found that body temperature during intoxication influences brain sensitivity to ethanol with the sensitivity being less at cool than at warm body temperatures. If this effect of temperature reflects alterations in the acute membrane perturbing action of ethanol, as suggested by in vitro studies, then body temperature reduction (hypothermia) during tolerance acquisition should reduce the effectiveness of a given ethanol concentration and, in turn, should reduce the development of chronic functional ethanol tolerance. To test this hypothesis, adult drug-naive C57BL/6J mice were injected i.p. once daily for five days with 3.6 g/kg ethanol (20% w/v) and were exposed to 34 degrees C or 25 degrees C for five hours following injection. On day 6, both ethanol acquisition groups and naive mice were injected i.p. with 4.0 g/kg ethanol and exposed to 25 degrees C. During acquisition, the group exposed to 34 degrees C had significantly higher body temperatures than the mice exposed to 25 degrees C, and there were no statistically significant differences in blood ethanol concentrations between treatment conditions. The extent of tolerance on day 6, measured by sleep-times and wake-up blood and brain ethanol concentrations versus naive mice, was significantly greater in the 34 degrees C acquisition group than in the 25 degrees C acquisition group. The results demonstrate that body temperature influences tolerance development in the manner predicted by membrane perturbation theories of anesthesia and adaptation based tolerance theories.

Rectal and brain temperatures in ethanol intoxicated mice.

The present study tested the assumption that deep rectal temperature reflects brain temperature in ethanol-intoxicated mice exposed to a range of ambient temperatures. Adult C57BL/6J mice were injected IP with one of three hypnotic doses of ethanol (3.2, 3.6, or 4.0 g/kg, 20% w/v) or with normal saline and were exposed to ambient temperatures of 15, 22, 32, or 34 degrees C. Thirty minutes post-injection, the mice were killed by cervical dislocation, decapitated and their rectal and brain temperatures were recorded simultaneously. Rectal and brain temperatures in the intoxicated mice increased significantly as the ambient temperature increased and were highly correlated and linearly related with each other. Although correlated, brain and rectal temperatures in these mice did not change in parallel, with brain temperatures increasing less rapidly than rectal temperatures. Additional studies indicated that similar relationships (correlated, but non-parallel) exist between the brain and rectal temperatures at 60, 120, and 180 min after injection of 3.6 g/kg ethanol. These findings suggest that rectal temperature can be used to quantify brain temperature in intoxicated mice, and extend to intoxicated animals evidence that brain temperature is controlled independently from rectal temperature.

Temperature modulates ethanol sensitivity in mice: generality across strain and sex.

Findings in our laboratory indicate that body temperature alters ethanol potency as measured by sleep-time, wake-up brain ethanol concentration and mortality in male C57BL/6J mice. The current studies tested the generality of these results. Experiment one tested age-matched, drug-naive, male C57BL/6S and BALB/cS mice. Experiment two tested age-matched, drug-naive, male and female C57BL/6J mice. Each mouse was injected IP with 3.6 g/kg ethanol (20% w/v). After losing its righting reflex, the mouse was placed into a v-shaped sleep tray within a chamber kept at a designated temperature from 12 to 36 degrees C. Upon awakening, rectal temperature was measured and blood and brain samples were taken for gas chromatographic analysis. As in previous studies, ambient temperature modulated the body temperatures and sleep-times of intoxicated animals. More important, wake-up rectal temperatures were positively correlated with sleep-times and negatively correlated with wake-up ethanol concentrations in all animals tested. These results support the hypothesis that brain sensitivity to ethanol depression varies with body temperature in accordance with membrane perturbation theories of anesthesia.

A study of the structure of fibronectin.

The structure of a fibronectin molecule has been studied by circular dichroism, infrared spectroscopy, scanning microcalorimetry and electron microscopy. It has been shown that the secondary structure of fibronectin is formed exclusively by the antiparallel beta-form -- 35%; the fibronectin molecule consists of several domains; the protein has a compact structure, the length of the molecule is 15.5 +/- 1.3 nm, the width is 8.8 +/- 1.7 nm, the axial ratio is approximately 2:1.1.