Controlled osmotic lysis (water-washing) of rat liver mitochondria results in a mixed population of small vesicles derived mainly from the outer mitochondrial membrane and of larger bodies containing a few cristae derived from the inner membrane. These elements have been separated on Ficoll and sucrose gradients. The small vesicles were rich in monoamine oxidase, and the large bodies were rich in cytochrome oxidase. Separation of the inner and outer membranes has also been accomplished by treating mitochondria with digitonin in an isotonic medium and fractionating the treated mitochondria by differential centrifugation. Treatment with low digitonin concentrations released monoamine oxidase activity from low speed mitochondrial pellets, and this release of enzymatic activity was correlated with the loss of the outer membrane as seen in the electron microscope. The low speed mitochondrial pellet contained most of the cytochrome oxidase and malate dehydrogenase activities of the intact mitochondria, while the monoamine oxidase activity could be recovered in the form of small vesicles by high speed centrifugation of the low speed supernatant. The results indicate that monoamine oxidase is found only in the outer mitochondrial membrane and that cytochrome oxidase is found only in the inner membrane. Digitonin treatment released more monoamine oxidase than cytochrome oxidase from sonic particles, thus indicating that digitonin preferentially degrades the outer mitochondrial membrane.

Animals↗

PubMed1966

Brain aldehyde dehydrogenase. Localization, purification, and properties.

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Acetaldehyde↗

PubMedDate not supplied

Comparison of neurotensin levels, receptors and actions in LS/Ibg and SS/Ibg mice.

Neurotensin (NT), injected centrally, markedly enhances sensitivity to ethanol-induced anesthesia in SS but not in LS mice (4). Since LS and SS mice were bred selectively for differential sensitivity to ethanol, these findings suggest that neurotensinergic neuronal processes mediate some of ethanol's actions and that LS and SS mice might differ genetically in neurotensinergic systems. Indeed, in biochemical studies it was shown that LS and SS mice differ in NT-like immunoreactivity in specific brain regions, i.e., hypothalamus, and in NT receptor densities (Bmax) in frontal cortex and striatum. In other experiments LS and SS mice differed in behavioral responses to centrally administered NT. Intracerebroventricular (ICV) administration of NT produced dose-dependent changes in motor activity, hypothermia, and analgesia in both LS and SS mice. SS mice appeared to be more sensitive than LS to NT-induced analgesia but not hypothermia. Neurotensin increased or decreased locomotor activity in both SS and LS mice following intraventral tegmental area or ICV administration, respectively. The results indicate that LS and SS mice, which were selectively bred for differences in ethanol sensitivity, differ genetically in NT concentrations, receptor densities in specific brain regions, and in some receptor-mediated behavioral responses to NT.

Analgesia↗

PubMedDate not supplied

Genetic-based differences in neurotensin levels and receptors in brains of LS x SS mice.

Levels of endogenous neurotensin (NT-IR) in the LS x SS RI strains differed by 3.0-, 4.7-, 5.4-, and 6.9-fold in the ventral midbrain (VMB), hypothalamus (HY), nucleus accumbens (NA), and caudate putamen (CP), respectively. Frequency distributions and estimates of the number of genes indicate that differences in NT-IR are polygenically influenced. The NT-IR levels in NA and CP were significantly correlated, but levels in the VMB did not correlate with those in the NA or CP. Specific binding to either low (NTL)- or high (NTH)-affinity receptors as measured in the absence or presence of levocabastine differed significantly in brain regions from among LS X SS mouse strains. Results indicate a polygenic influence mediating the differences in receptor densities and suggest differences in genetic regulation of NTL and NTH receptors.

Animals↗

PubMedDate not supplied

Brain neurotensin receptors in mice bred for differences in sensitivity to ethanol.

The hypothesis that some of ethanol's acute effects are mediated via neurotensinergic systems was investigated by characterizing neurotensin (NT) receptors in mice (LS and SS) selectively bred for differences in sensitivity to ethanol. [3H]Neurotensin binding in brain membranes from both mouse lines was specific, saturable, reversible, and linear with protein concentrations. Subcellular localization studies showed specific NT binding to be concentrated in the microsomal/synaptosomal fractions. Scatchard analyses of [3H]NT binding indicated similar KD values for membranes from various brain regions of LS and SS mice. However, Bmax values in frontal cortex, cerebellum, and striatum were greater in SS than in LS mice. In competitive binding studies IC50 values were lower for NT8-13 than for NT1-13, and IC50 values for NT1-8, NT1-11, D-Trp11-NT, and D-Tyr11-NT were greater than 1000 nM. Association and dissociation rate constants for [3H]NT and resulting KD values (0.8 nM) were similar for LS and SS brain membranes. Ethanol, in vitro, had no effect on NT binding characteristics, but as expected various cations markedly increased KD values.

Animals↗

PubMedDate not supplied

Ethanol differentially enhances adrenocortical response in LS and SS mice.

The stimulatory effect of ethanol on the hypothalamo-pituitary-adrenal (HPA) axis was investigated in the long-sleep (LS) and short-sleep (SS) lines of mice. Plasma corticosterone concentrations peaked 30 min after IP administration of ethanol in both lines of mice. Ethanol produced dose-dependent elevations in plasma corticosterone in both LS and SS mice; however, at low doses of ethanol (0.25 to 1 g/kg) the adrenocortical response observed in LS mice was markedly greater than in SS mice. Passive immunoneutralization of circulating corticotropin releasing factor (CRF) completely abolished ethanol-induced elevation in plasma corticosterone in LS mice. CRF or ACTH (adrenocorticotropin) produced dose-dependent elevations in plasma corticosterone in the two lines of mice. Epinephrine co-administered with CRF did not potentiate the adrenocortical response obtained with CRF in either line of mice, and hexamethonium only slightly attenuated ethanol-induced elevations in plasma corticosterone in both lines of mice, suggesting that differentially elevated plasma catecholamines are not responsible for differences in ethanol-induced adrenocortical response. The results suggest that differential adrenocortical response to ethanol exhibited by LS and SS mice is due primarily to differential ethanol-induced CRF release.

Adrenocorticotropic Hormone↗

PubMedDate not supplied

Neurotensin modulates K(+)-stimulated dopamine release from the caudate-putamen but not the nucleus accumbens of mice with differential sensitivity to ethanol.

Slices of caudate-putamen (CP) and nucleus accumbens (NA) prepared from Long-Sleep (LS) and Short-Sleep (SS) mice were used to determine the effects of neurotensin (NT) and ethanol on K(+)-stimulated 3H-dopamine (3H-DA) release and to test the hypothesis that ethanol acts, in part, via NT receptor-mediated processes. Slices prepared from either LS or SS CP or NA did not differ in submaximal (25 mM) K(+)-stimulated 3H-DA release but 60 mM K+ induced significantly greater 3H-DA release from LS CP slices compared with SS CP slices. NT had no effect on unstimulated 3H-DA overflow but enhanced 25 mM K(+)-stimulated 3H-DA release from slices of the CP of both lines of mice. Augmentation of DA release by NT from caudate slices was concentration dependent and tetrodotoxin (TTX) insensitive, implicating a role of presynaptic neurotensin receptors located on nigrostriatal DA neurones. In contrast, NT did not enhance K(+)-stimulated 3H-DA release from NA slices from either line of mice. The absence of an NT effect in NA slices was not due to a rapid desensitization of NT receptors but the data were consistent with the absence of presynaptic NT receptors on dopaminergic terminals in the NA. Between-line differences were observed in the effect of ethanol on NT enhancement of 25 mM K(+)-stimulated 3H-DA release from CP slices. Ethanol (100 mM) applied concomitantly with NT blocked the NT enhancement of 3H-DA release from CP slices of LS but not SS mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMedDate not supplied

Genetic influences on acquisition of tolerance to alcohol.

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Alcoholic Intoxication↗

PubMedDate not supplied

Effects of ethanol: II. Behavioral sensitivity and acute behavioral tolerance.

Two doses of ethyl alcohol were administered to 48 young male subjects (24 brother-pairs), and placebo doses were administered to five brother-pairs (controls). Dose 1 amount was calculated to result in a peak blood alcohol content (BAC) of 100 mg/100 ml; dose 2 was given when BAC had fallen to one-half the peak BAC resulting from dose 1, in an attempt to raise BAC back to 100 mg/100 ml. Prior to alcohol dosage, baseline measurements were made on a number of behavioral tests. Behavioral performances during metabolism of doses 1 and 2 were compared to baseline performance. Sensitivity to ethanol was indicated by performance decrements on nearly all tests after ethanol dosing. Acute behavioral tolerance to ethanol (ABTE) was indicated for some tests by improved performance after dose 2 compared to performance at a time after dose 1 when BACs were about equivalent. A preliminary search for possible genetic components of sensitivity and ABTE was undertaken by comparing the scores of the brothers.

Adolescent↗

PubMedDate not supplied

Effects of ethanol: I. Acute metabolic tolerance and ethnic differences.

Forty-five young men were given an initial dose of ethanol calculated to achieve a blood alcohol concentration (BAC) of 100 mg of ethanol/100 ml of blood. Using breath analysis, the rates of ethanol clearance were calculated over a period of a few hours. When each individual reached a BAC level exactly one-half (about 50 mg%) that seen at his peak BAC, he was given a second dose of ethanol amounting to 45% of his initial dose, and the clearance rate was again determined. Estimates of absorption time, peak BAC, volume of distribution, clearance rate, and other metabolic parameters were made separately following each dose of ethanol. As intended, there was no significant difference between peak 1 and peak 2; however, there was a significant increase in clearance rate (beta 60) after dose 2. We term this phenomenon acute metabolic tolerance to ethanol ( AMTE ). When the sample was stratified by ethnicity, there was a trend for men of Oriental ancestry to clear the drug at a faster rate than was the case for men of Caucasian or Polynesian ancestry. The data also indicated that prior drinking history may have had an effect on clearance rate, though this differed by ethnicity. Two different methods for estimating volume of distribution (V) were used. The correlation between the separate estimates obtained was poor, and is probably an indication of the need for development of more accurate methods for estimating V.

Adolescent↗