Ethanol exposure decreases mitochondrial outer membrane permeability in cultured rat hepatocytes

Mitochondrial metabolism depends on movement of hydrophilic metabolites through the mitochondrial outer membrane via the voltage-dependent anion channel (VDAC). Here we assessed VDAC permeability of intracellular mitochondria in cultured hepatocytes after plasma membrane permeabilization with 8 μM digitonin. Blockade of VDAC with Koenig’s polyanion inhibited uncoupled and ADP-stimulated respiration of permeabilized hepatocytes by 33% and 41%, respectively. Tenfold greater digitonin (80 μM) relieved KPA-induced inhibition and also released cytochrome c, signifying mitochondrial outer membrane permeabilization. Acute ethanol exposure also decreased respiration and accessibility of mitochondrial adenylate kinase (AK) of permeabilized hepatocytes membranes by 40% and 32%, respectively. This inhibition was reversed by high digitonin. Outer membrane permeability was independently assessed by confocal microscopy from entrapment of 3 kDa tetramethylrhodamine-conjugated dextran (RhoDex) in mitochondria of mechanically permeabilized hepatocytes. Ethanol decreased RhoDex entrapment in mitochondria by 35% of that observed in control cells. Overall, these results demonstrate that acute ethanol exposure decreases mitochondrial outer membrane permeability most likely by inhibition of VDAC.     

Immunohistochemical identification and localization of pancreatic polypeptide cells in the pancreas and gastrointestinal tract of the human fetus and adult man

Summary Pancreatic polypeptide (PP)-containing cells were detected by using anti-bovine PP (BPP) serum in the pancreas and gastrointestinal tract of human fetuses, premature infants and in the pancreas, antrum and jejunum of adult man obtained by biopsy from patients with normal gastroduodenal endoscopy. The localization was established by studying the distribution of PP cells in comparison to the distribution of glucagon-, somatostatin- and insulin cells. The first PP cells are seen in the pancreas at 10 weeks of gestation. They are located preferentially in the lower part of the head of the pancreas. The specificity of immunocytological reaction was ascertained by the inhibition of the reaction by bovine pancreatic polypeptide, glucagon and insulin did not modify the immunocytological reaction.     

Immunocytological localization of LH,FSH, TSH and their subunits in the pituitary of normal and anencephalic human fetuses

Summary Immunostaining with antisera to oLH, hCG, hLH, pLH, hFSH, hFSH, hTSH and bTSH was used to delineate the gonadotropic and thyrotropic cells of the human fetal anterior pituitary. Hypophyses from 29 normal fetuses, 3 newborn infants, and 5 totally ancencephalic fetuses were used. Several controls to check for the specificity of the immunocytological reaction were made.In normal fetuses, observations showed that: 1) the subunit was detected from the eighth week and throughout gestation without sex differences; 2) intact LH was detected during the third month, however, age and sex differences were observed during the fourth and fifth months; 3) intact FSH was detected in female fetuses from the beginning of the fourth month, a sex difference was observed; 4) LH and FSH were detected in the same cells; 5) the thyrotropic cells were detectable from 15 weeks of gestation and their number increased during gestation without sex difference; 6) at birth the gonadotropic cells were scarce and were located in the ventromedian zone of the anterior pituitary, while the thyrotropic cells remained numerous and were located in the dorsomedian zone.In anencephalic fetuses: 1) the subunit existed at each stage studied; 2) the reaction induced by anti-pLH and anti-hFSH sera was always very weak regardless of sex or age; 3) the thyrotropic cells were more numerous in comparison to the gonadotropic cells. These data are discussed in terms of the relationship of the hypophysiotropic hypothalamic factors to the appearance and evolution of the glycoprotein hormones and their subunits.     

Properties of channels in the mitochondrial outer membrane

Patch-clamping studies with native outer mitochondrial membranes show a complex behavior. In the range of potentials in which the polarity of the pipette is positive, the behavior resembles that of VDAC incorporated into bilayers. Accordingly, there is a decrease in conductance with voltage. An involvement of VDAC is also supported by responses of the patches to the presence of polyanion or treatment with succinic anhydride, both of which affect VDAC. In contrast, in the negative range of potential, the conductance of the patches generally increases with the magnitude of the voltage. The increase in conductance shows a biphasic time course which is consistent with a model in which channels are first activated (first phase) and then assembled into larger high-conductance channels (second phase). A variety of experiments support the notion that an assembly takes place. The time course of the conductance increase is consistent with formation of the second-phase channels from 6±1 subunits.     

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).     

Ontogeny and distribution of certain endocrine cells in the human fetal large intestine

Summary In 9 fetuses, 9 to 24 weeks-old, the occurrence and relative distribution of argentaffin cells, as well as of cells immunoreactive to somatostatin (SRIF), glucagon-like polypeptide (GLI), pancreatic polypeptide (PP) and substance P (SP) were studied in five segments of the colon (appendix, cecum, ascending colon, descending colon, and rectosigmoid). For each colonic segment, data concerned with the occurrence of endocrine cells were expressed either as mean absolute numbers of specific cells per entire mucosal section, or as cell densities per mm³ of mucosa after calculation of the mucosal volume of the sections. Argentaffin, GLI, SRIF and PP immunoreactive cells are all present in relatively large numbers, scattered along the entire length of the colonic mucosa as early as the 9th–10th week of gestation, whereas substance P-containing cells occur sporadically and first appear during the 14th–17th week. Until the 20th week, with progressing embryonic development, an increase was determined in absolute numbers per section of all types of endocrine cells in all segments of the colon. This observation is clearly related to the general growth of the colonic mucosa, since cell densities per mm³ of mucosa do not greatly change or even decrease during gestation. However, it is possible that densities of argentaffin, GLI and BPP cells increase in the appendix around the 14th–17th week of gestation. Between the 20th and 24th week, absolute numbers of cells per section remain stable or slightly increase, while cell densities tend rather to decrease in all segments. These data demonstrate that some endocrine cells are present very early in the human fetal colon, but their functional significance remains to be elucidated.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM)     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Secretin in the rat hypothalamo-pituitary system: Localization,identification and characterization

Secretin-like immunoreactivity (SLI) has been identified and characterized in the pituitary of the rat. The concentration in the neurointermediate lobe is about 45 fold higher than the concentration of SLI observed in the anterior lobe. Transections of the pituitary stalk of the rat caused a significant depletion of SLI in the neurointermediate lobe without affecting the content in the anterior lobe. In view of the relatively high concentration of SLI reported to occur in the hypothalamus, it appears that there may be a secretinergic pathway between the brain and the neurointermediate lobe of the pituitary.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

Transient assembly of F-actin on the outer mitochondrial membrane contributes to mitochondrial fission

In addition to established membrane remodeling roles in various cellular locations, actin has recently emerged as a participant in mitochondrial fission. However, the underlying mechanisms of its participation remain largely unknown. We report that transient de novo F-actin assembly on the mitochondria occurs upon induction of mitochondrial fission and F-actin accumulates on the mitochondria without forming detectable submitochondrial foci. Impairing mitochondrial division through Drp1 knockout or inhibition prolonged the time of mitochondrial accumulation of F-actin and also led to abnormal mitochondrial accumulation of the actin regulatory factors cortactin, cofilin, and Arp2/3 complexes, suggesting that disassembly of mitochondrial F-actin depends on Drp1 activity. Furthermore, down-regulation of actin regulatory proteins led to elongation of mitochondria, associated with mitochondrial accumulation of Drp1. In addition, depletion of cortactin inhibited Mfn2 down-regulation– or FCCP-induced mitochondrial fragmentation. These data indicate that the dynamic assembly and disassembly of F-actin on the mitochondria participates in Drp1-mediated mitochondrial fission.     

Effect of the outer mitochondrial membrane fraction on mitoplast respiration

Additions of the fraction of outer mitochondrial membranes to the mitoplast suspension is shown to bring about an increase of the ADP-stimulated respiration rate, indices of respiration control and uncoupled respiration. This effect is not a result of the cytochrome c presence in the fraction of outer membranes. In the glycerol-containing medium which causes dissociation of intermembrane contacts the coupling effect of outer membranes on mitoplast respiration is not revealed. It is concluded that the outer membrane in contact with the inner one takes part in realization of the mitochondrial coupling.     

Effect of neutral salts on the interaction of rat brain hexokinase with the outer mitochondrial membrane.

The glucose 6-phosphate (Glc-6-P)-induced solubilization of mitochondrial hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) from rat brain can be reversed by low concentrations (ionic strength <~0.02 m) of neutral salts. When compared to the original particulate enzyme (i.e., enzyme found on the particles prior to solubilization by Glc-6-P), the rebound enzyme is similar in distribution on sucrose gradients, K_m for ATP, inhibition by antiserum to purified brain hexokinase, and resistance to removal by exhaustive washing of the particles. The effectiveness of chloride salts at promoting rebinding increases in the order Cs⁺< Rb⁺< K⁺≤ Na⁺< Li⁺< Mg²⁺. This salt-induced rebinding is attributed to the screening of negative charges on the enzyme and/or membrane by cations, thereby decreasing repulsive forces and enhancing attractive interactions between enzyme and membrane. Solubilization of the enzyme, both in the presence and absence of Glc-6-P, is increased at alkaline pH, as would be expected due to increasing repulsive interactions between negative charges on membrane and enzyme as the pH is increased beyond the pI of the enzyme (pI = 6.3). In contrast to previous interpretations, P_i displayed no special efficacy at reversing Glc-6-P-induced solubilization, being comparable to other neutral salts on an ionic strength basis. However, P_i and its structural analog, arsenate, were shown to counteract specifically the Glc-6-P-induced inhibition and conformational change in the enzyme. At higher concentrations (ionic strength >~ 0.02 m) neutral salts themselves lead to reversible dissociation of the enzyme from the mitochondria. The efficacy of the salts depends primarily on the pH and on the position of the anion in the Hofmeister series, with salts of chaotropic anions (SCN^?, I^?, Br^?) being most effective. At pH 6, both chaotropic and nonchaotropic salts solubilize the enzyme, while at pH 8.5, only the chaotropes retain this ability. Neutral salts also have a reversible effect on the conformation of the enzyme, as reflected by enzymatic activity, with chaotropic salts again being most effective; there is no pronounced influence of pH (in the range of pH 6–8.5) on the ability of the salts to cause conformational change in the enzyme. Based on a lack of correlation between saltinduced solubilization and conformational changes affecting activity, it is concluded that the latter are not directly responsible for release of the enzyme from the membrane. In the presence of KSCN, the extent of solubilization decreased with increase in temperature, indicating a negative enthalpy for solubilization. In contrast, in the absence of salt, the enthalpy for solubilization was positive. These temperature effects and the effects of neutral salts on the hexokinase-membrane interaction are interpreted in terms of a model in which electrostatic forces are considered to be of major importance. At low ionic strength, repulsive forces between negative charges on enzyme and membrane predominate; screening of these charges by cations diminishes the repulsion, effectively enhancing attractive electrostatic forces between enzyme and membrane and thus promoting their interaction. At higher ionic strengths, the attractive electrostatic forces are themselves disrupted, resulting in dissociation of the enzyme from the membrane. It is proposed that the greater effectiveness of chaotropic salts at disrupting these attractive forces is due to their increased ability to penetrate through hydrophobic regions of enzyme and membrane to relatively inaccessible sites of electrostatic-interaction.     

Studies on the flavins in rat liver mitochondrial outer membranes

The incorporation of radioactivity derived from [2-¹⁴C] riboflavin into the flavins of rat liver mitochondrial outer membranes was studied. These membranes were found to contain about 0.6 nmol of non-covalently bound flavins per mg protein; the majority is in the form of FAD (73%) and FMN (24%). The membranes also contain about 1.5 nmol per mg of covalently bound flavins.After labeling, radioactive flavins appeared in the non-covalently bound flavins for about 4 h. Most of this radioactivity was in FAD (77%). Neither the rate nor extent of this labelling was affected by cycloheximide (1 mg/kg) administered 30 min prior to the radioactive riboflavin. With the covalently bound flavins, radioactivity was incorporated into the coenzymes for at least 18 h, but the rate of incorporation was much slower. After cycloheximide, radioactive flavins continued to appear in covalently bound flavins for about 2 h, but then stopped. Labeling of both types of flavins after [¹⁴C] riboflavin was considerably slower than the incorporation of [³H] leucine into outer membrane proteins. These results suggest that with flavoproteins from the mitochondrial outer membranes, the incorporation of flavins occurs after synthesis of the various apoenyzmes is complete.     

Regulation of the mitochondrial outer membrane channel,VDAC

The channel-forming protein, VDAC, located in the mitochondrial outer membrane, is probably responsible for the high permeability of the outer membrane to small molecules. The ability to regulate this channelin vitro raises the possibility that VDAC may perform a regulatory rolein vivo. VDAC exists in multiple, quasi-degenerate conformations with different permeability properties. Therefore a modest input of energy can change VDAC's conformation. The ability to use a membrane potential to convert VDAC from a high (open) to a low (closed) conducting form indicates the presence of a sensor in the protein that allows it to respond to the electric field. Titration and modification experiments point to a polyvalent, positively charged sensor. Soluble, polyvalent anions such as dextran sulfate and Konig's polyanion seem to be able to interact with the sensor to induce channel closure. Thus there are multiple ways of applying a force on the sensor so as to induce a conformational change in VDAC. Perhaps cells use one or more of these methods.     

Specializations of the outer mitochondrial membrane during oocyte development

Summary A coating of electron dense material is present on the cytoplasmic surface of outer mitochondrial membranes in medium-sized hamster oocytes. The coating is not present at earlier or later stages of oocyte development. Its possible relationship to the synthesis and transport of mitochondrial protein is discussed. Associations between endoplasmic reticulum, ribosomes, glycogen and the outer mitochondrial membrane are also described and discussed.     

Non-ohmic proton conductance of the mitochondrial inner membrane in hepatocytes

The mitochondrial membrane potential in isolated hepatocytes was measured using the distribution of the lipophilic cation triphenylmethylphosphonium (TPMP+) with appropriate corrections for plasma membrane potential, cytoplasmic and mitochondrial binding of TPMP+, and other factors. The relationship between mitochondrial membrane potential and respiration rate in hepatocytes was examined as the respiratory chain was titrated with myxothiazol in the presence of oligomycin. This relationship was nonproportional and similar to results with isolated mitochondria respiring on succinate. This shows that there is an increased proton conductance of the mitochondrial inner membrane in situ at high values of membrane potential. From the respiration rate and mitochondrial membrane potential of hepatocytes in the absence of oligomycin, we estimate that the passive proton permeability of the mitochondrial inner membrane accounts for 20-40% of the basal respiration rate of hepatocytes. The relationship between log[TPMP+]tot/[TPMP+]e and respiration rate in thymocytes was also nonproportional suggesting that the phenomenon is not peculiar to hepatocytes. There is less mitochondrial proton leak in hepatocytes from hypothyroid rats. A large proportion of the difference in basal respiration rate between hepatocytes from normal and hypothyroid rats can be accounted for by differences in the proton permeability characteristics of the mitochondrial inner membrane.     

Ultrastructural and functional development of macrophages in the dermal tissue of rat fetuses

Summary After about 12 days of gestation, fetal macrophages begin to appear in the subepidermal mesenchyme of rat fetuses. The macrophages are ultrastructurally characterized by cytoplasmic vacuoles, abundant polyribosomes and long filopodia. Immunocytologically, they possess Fc and complement (C3) receptors on the cell surface and are capable of immune phagocytosis, Latex or carbon phagocytosis, and glass adherence. From 15 days of gestation, lysosomal granules and miropinocytic vesicles gradually develop, together with an enlargement of Golgi complexes, whereas the number of polysomes and the number and size of cytoplasmic vacuoles are gradually reduced when gestation ends. Finally, the macrophages become amoeboid. Non-specific esterase and endogenous peroxidase activities are always absent in these macrophages. In culture experiments with cell suspensions prepared from the mesenchyme, fetal macrophages show a similar maturation process. Autoradiography with ³H-thymidine demonstrates a high proliferative capacity of the macrophages, particularly during the fetal stage.Supported by Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture, Japan (No. 401057)