Ethanol-induced c-fos expression in catecholamine- and neuropeptide Y-producing neurons in rat brainstem.

BACKGROUND: Previous studies have used c-Fos-like immunoreactivity (cFLI) to examine the neuroanatomical location of cells that are activated in response to ethanol administration. However, the use of cFLI alone fails to reveal the phenotypical identity of cells. In the present study we used double-labeling procedures to identify the neurochemical phenotype of neurons that showed ethanol-induced cFLI in the rat brainstem. METHODS: Individual groups of rats received intraperitoneal injection of ethanol (1.5 g/kg or 3.5 g/kg) or isotonic saline (23 ml/kg). To assess the specificity of cFLI induced by ethanol, we injected other rats with the drug lithium chloride (LiCl; 76 mg/kg). Two hours after injection, rats were killed and their brains were processed for immunohistochemistry. RESULTS: Both doses of ethanol promoted cFLI in several brainstem regions, including the nucleus of the solitary tract (NTS), the locus coeruleus (LC), and the ventrolateral medulla (VLM). Although LiCl caused significant cFLI in the NTS, this drug promoted only minimal cFLI in the VLM and no significant activation in the LC. We found that a significant proportion of tyrosine hydroxylase (TH)-positive neurons coexpressed ethanol-induced cFLI in the VLM (approximately 75-85%), the NTS (approximately 65-75%), and the LC (approximately 30-65%). Additionally, a significant proportion of neuropeptide Y (NPY)-producing neurons in the VLM coexpressed ethanol-induced cFLI (approximately 60-75%). On the other hand, LiCl promoted activation of TH-positive neurons in the VLM and the NTS but failed to stimulate cFLI in TH-producing neurons in the LC or in NPY-producing neurons of the VLM. CONCLUSIONS: Neurons in the rat brainstem that show ethanol-induced c-Fos expression produce catecholamines and NPY. This research demonstrates the usefulness of double-labeling immunohistochemistry procedures for identifying the neurochemical identity of neurons that are activated after ethanol administration.

[Validation of half-night polysomnography for sleep apnea/hypoapnea syndrome]. / Validación de estudios polisomnográficos de mitad de la noche en el síndrome de apneas/hipoapneas durante el sueño.

UNLABELLED: A diagnosis of sleep apnea/hypopnea syndrome (SAHS) is based on clinical signs and nighttime polysomnograms. Brief polysomnography has been proposed as an alternative to all-night recording. OBJECTIVES: The aim of this study was to determine whether a polysomnograms obtained during the first half of the night is sufficient for establishing a diagnosis of SAHS and to determine the correlation between polysomnographic variables recorded during the first four hours (half the study time) with those recorded over the full eight hours (full study time), as well as to determine diagnostic agreement. DESIGN: Thirty-five patients suspected of having SAHS were studied prospectively. Baseline polysomnograms were scored blindly by two independent observers following standard methods. A diagnosis of SAHS was made according to guidelines of the Spanish Society of Pneumology and Chest Surgery. During the first half of the night and up to the end of each recording period we gathered neurophysiological and respiratory variables and diagnostic impressions. RESULTS: The correlation between variables (sleep stage, overall AHI, REM-AHI, non-REM-AHI and sleep efficiency) recorded in the first half of the night and throughout the night was significant (p < 0.05) by both Pearson's correlation coefficient (r) and by the intraclass correlation coefficient (ICC). In 33 of 35 patients (94.3%) diagnostic agreement was achieved (95% CI 80.84-99.30); when SAHS was severe, agreement was 100%. CONCLUSION: Based on these results, we conclude that for patients with a diagnosis of severe SAHS during the first half of the night, data recorded during the second half can be considered supplementary.

Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification.

In order to determine whether disruption of mitochondrial function could trigger apoptosis in murine haematopoietic cells, we used the potassium ionophore valinomycin. Valinomycin induces apoptosis in the murine pre-B cell line BAF3, which cannot be inhibited by interleukin-3 addition or Bcl-2 over-expression. Valinomycin triggers rapid loss of mitochondrial membrane potential. This precedes cytoplasmic acidification, which leads to cysteine-active-site protease activation, DNA fragmentation and cell death. Bongkrekic acid, an inhibitor of the mitochondrial permeability transition, prevents acidification and subsequent induction of apoptosis by valinomycin.

Thyroid hormones regulate the onset of osmotic activity of rat liver mitochondria after birth.

The effect of thyroid hormone deprivation on the osmotic activity of liver mitochondria from early newborn rats was studied. Experimentally induced hypothyroidism prevented the increase in the osmotic activity of mitochondria observed immediately after birth. Osmotic activity was restored by T4 and T3 treatment to hypothyroid newborns but not when this treatment was supplemented with cycloheximide. Under the same circumstances, streptomycin had no effect. Hypothyroidism abolished the change in the slope of the osmotic curve (plot of inverse absorbance of mitochondrial suspensions incubated in sucrose solutions vs. inverse sucrose concentration) observed in mitochondria from euthyroid newborns at 110-120 mOsm sucrose, suggesting that hypothyroidism prevents the formation of tight physical connections between mitochondrial outer and inner membranes. Thyroid hormone deprivation increased the passive permeability of the mitochondrial inner membrane to protons, resulting in a decreased respiratory control ratio. Hypothyroidism prevented the sharp decrease in the affinity of mitochondria for ATP observed in euthyroid newborns immediately after birth. These results corroborate our previous suggestion (Endocrinology, 1995, 136:4448) that, during the early neonatal period, thyroid hormones control the synthesis of some nucleus-coded protein(s) involved in the assembly of F0,F1-ATPase.

cAMP and Ca2+ involvement in the mitochondrial response of cultured fetal rat hepatocytes to adrenaline.

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+ concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+ concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+ concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+ act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both alpha- and beta-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.

Hypothyroidism prevents postnatal changes in rat liver mitochondrial populations defined by rhodamine-123 staining.

The effect of hypothyroidism on the percentages of low fluorescence population (LFP) and high fluorescence population (HFP) rhodamine-123-stained mitochondria, respiratory parameters, and ATPase activity were studied in liver mitochondria from early newborn rats. Hypothyroidism prevented the decrease in the percentage of HFP and the subsequent increase in LFP that occurs immediately after birth. This effect coincides with the impairment of mitochondrial respiratory function, as shown by the low respiratory control ratio and the low activity of F0,F1-ATPase found in hypothyroid newborns. All of these changes were reversed by the administration of thyroid hormones. ATP in vitro promotes the conversion of HFP into LFP and increases the respiratory control ratio in hypothyroid newborns, although this effect was not observed after thyroid hormone treatment. The effect of thyroid hormones on both the postnatal changes in mitochondrial populations and in F0,F1-ATPase activity was prevented by cycloheximide, but not by streptomycin. Thus, the observed effects of thyroid hormones on neonatal mitochondria must be accomplished by the induction of the synthesis of some nuclei-coded protein, possibly involved in F0,F1-ATPase assembly.

Postnatal changes in rhodamine-123 stained mitochondrial populations are sensitive to protein synthesis inhibitors but mimicked in vitro by ATP.

The incubation of term fetus mitochondria with ATP mimicked in vitro the increase in the respiratory control index and in the percentage of the rhodamine-123-low fluorescence population that occurred in vivo immediately after birth, suggesting that both phenomena are closely associated. The administration of streptomycin inhibited the increase in the percentage of the low fluorescence population that occurred immediately after birth, while the administration of cycloheximide even reversed these changes. These results suggest that the in vivo interconversion between mitochondrial forms depends on both cytosolic and mitochondrial protein synthesis.

Docking the mitochondrial inhibitor protein IF1 to a membrane receptor different from the F1-ATPase beta subunit.

Monoclonal antibodies reacting with the inhibitor protein (IF1) of the mitochondrial ATPase/ATP synthase complex did not modify the IF1-induced inhibition of soluble F1 ATPase activity. On the contrary, they increased the ATPase activity of inverted electron-transport particles without inducing a significant release of IF1 from these particles. This suggested that IF1 could be linked to a membrane protein when it was not inhibiting the ATPase activity. IF1 antibodies have been used to show that IF1 can bind not only to the beta subunit of F1-ATPase [Klein, G., Satre, M., Dianoux, A. C. & Vignais, P. V. (1981) Biochemistry 20, 1339-1344] but also to a protein present in the inner-mitochondrial membrane. The cross-linking of IF1 to this membrane protein gave a product of M(r) 15000-16000 that migrated differently from IF1 and from the dimer of IF1 using SDS/PAGE. When the cross-linked product was obtained by using a cleavable cross-linking reagent, the complex between IF1 and the docking protein was partly dissociated and free IF1 was recovered. Considering the molecular mass of IF1, this docking protein for IF1 has apparent M(r) 5000-6000. The complex between IF1 and this receptor protein can be detected in low amounts by antibodies against IF1 in the absence of cross-linking reagent. Since this complex remained in the pellet after treatment of the membrane with Triton X-100, it should be a membrane protein. Therefore, IF1 can bind not only to its inhibitory-binding site at the beta subunit of F1, but also to a non inhibitory site which is a membrane protein of approximate M(r) 5000-6000.

2-Ketoglutarate dehydrogenase deficiency, a rare cause of primary hyperlactataemia: report of a new case.

Two new familial cases of 2-ketoglutarate dehydrogenase (2-KGD) deficiency are reported: a girl who died at 10 years and a boy, still alive at 4 years, born to consanguineous parents. The cases developed progressively severe encephalopathy with axial hypotonia, psychotic behaviour, pyramidal symptoms and failure to thrive. Both children exhibited permanent lactic acidosis with acute episodes during emotional stress and various infections, associated with elevated lactate/pyruvate (L/P) ratio and slightly decreased ketone body ratio in plasma. In fibroblasts, the L/P ratio was greatly increased in the boy. No respiratory chain complex deficiency could be demonstrated in cultured fibroblasts or in mitochondria isolated from a muscle biopsy performed on the boy. In muscle isolated mitochondria, a progressive decrease of the rate of glutamate oxidation was observed after ADP addition; the rate of 2-ketoglutarate oxidation was low in the absence of ADP and did not increase after ADP addition. 2-KGD deficiency was demonstrated in fibroblasts from both children and in the boy's muscle and myoblasts. The 2-KGD complex is composed of three separate enzymes: E1, E2 and E3. We could demonstrate in our patient that the E1 and E3 subunits were normal, suggesting that the E2 component could be responsible for the defect.