Phagocytosis of cells in the gastric surface epithelium of the rat

Summary Gastric surface epithelial cells (SEC) from fed rats, from rats fasted for 16 h and from mucosae exposed in an ex-vivo chamber to 16 mM aspirin for 5 min were examined by transmission electron microscopy. SEC have the capability to phagocytose adjacent epithelial cells and parietal cells. Phagocytosis is rare in mucosae from fasted animals but common in fed animals or after brief exposure to aspirin. Phagocytic capabilities are not restricted to the progenitor zone but exist throughout the surface epithelium. Phagocytosis may provide a mechanism for the removal of damaged or senescent cells from the surface epithelium.Suported by the Medical Research Council of Canada     

Myelin-like structures in heart muscle cells following exposure to adrenaline]

The state of the muscle cells of the heart was studied experimentally in 36 albino rats the next day after a single administration of adrenaline hydrochloride (3mg/kg), intramuscularly into the hip. Areas of overcontraction and overextension of myofibrills, numerous myelin-like figures not infrequently containing mitochondria and glycogen, drops of lipids were observed by electron microscopy. It is suggested that the affecting action of adrenaline is expressed through the activation of lipolysis which results in dissociation of the protein-lipid components of the mitochondrial membranes, accumulation of fatty acids in the cytoplasm and resynthesis of the new unusual membranous formations.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Fasting induced alterations in mitochondrial palmitoyl-CoA metabolism may inhibit adipocyte pyruvate dehydrogenase activity.

1. Adipocytes from fed and fasted (24 hr) groups of rats were fractionated into mitochondria, microsomes and plasma membranes. 2. Fasting significantly decreased the mitochondrial activity of palmitoyl-CoA synthetase, palmitoyl-CoA hydrolase, beta-oxidation and pyruvate dehydrogenase. 3. Fasting elevated intramitochondrial long-chain acyl-CoA. 4. Pyruvate dehydrogenase was inhibited 50% by addition of 30 microM palmitoyl-CoA. 5. Fasting-induced changes in palmitoyl-CoA metabolism may modulate pyruvate dehydrogenase activity in adipocyte mitochondria.     

Mitochondrial migration and Ca-ATPase modulation in secretory ameloblasts of fasted and calcium-loaded rats

Migration of mitochondria and modulation of Ca-ATPase activity in secretory ameloblasts were investigated ultrastructurally and ultracytochemically by using lower incisors taken from normally fed, 30-hr-fasted, and calcium (Ca)-loaded rats. In normally fed rats, almost all mitochondria were localized in a narrow infranuclear compartment between the nucleus and proximal cell webs of secretory ameloblasts. In 30-hr-fasted rats, a prominent migration of many mitochondria into the supranuclear region of the cells was noted. Mitochondria returned to the infranuclear compartment and seldom appeared in the supranuclear region when fasted rats were Ca-loaded by transcardiac perfusion with physiological Ca solution. Normally, the mitochondria of secretory ameloblast exhibited moderate Ca-ATPase activity along their inner membranes. This mitochondrial Ca-ATPase was decreased by a 30-hr fast and became prominent again after Ca loading. Plasma-membrane Ca-ATPase was demonstrated in the entire cell surface of secretory ameloblasts. An especially abundant reaction was found along the invaginated cell surface of the Tomes process. This Ca-ATPase also became very weak and was almost abolished from the Tomes process after fasting, but Ca loading caused reappearance of an intense Ca-ATPase activity on the entire cell surface, including along Tomes's processes. These results suggest that 1) mitochondrial localization in secretory ameloblasts is influenced by the Ca concentration of the extracellular milieu, and 2) the level of mitochondrial and cell-membrane ATPase activity is responsive to the concentration of extracellular calcium.     

Effects of fasting on oxidative stress in rat liver mitochondria

While moderate caloric restriction has beneficial effects on animal health state, fasting may be harmful. The present investigation was designed to test how fasting affects oxidative stress, and to find out whether the effects are opposite to those previously found in caloric restriction studies. We have focused on one of the main determinants of aging rate: the rate of mitochondrial free radical generation. Different parameters related to lipid and protein oxidative damage were also analyzed. Liver mitochondria from rats subjected to 72 h of fasting leaked more electrons per unit of O₂ consumed at complex III, than mitochondria from ad libitum fed rats. This increased leak led to a higher free radical generation under state 3 respiration using succinate as substrate. Regarding lipids, fasting altered fatty acid composition of hepatic membranes, increasing the double bond and the peroxidizability indexes. In accordance with this, we observed that hepatic membranes from the fasted animals were more sensitive to lipid peroxidation. Hepatic protein oxidative damage was also increased in fasted rats. Thus, the levels of oxidative modifications, produced either indirectly by reactive carbonyl compounds (N^epsilon- malondialdehyde-lysine), or directly through amino acid oxidation (glutamic and aminoadipic semialdehydes) were elevated due to the fasting treatment in both liver tissue and liver mitochondria. The current study shows that severe food deprivation increases oxidative stress in rat liver, at least in part, by increasing mitochondrial free radical generation during state 3 respiration and by increasing the sensitivity of hepatic membranes to oxidative damage, suggesting that fasting and caloric restriction have different effects on liver mitochondrial oxidative stress.     

Differences in the sensitivity of carnitine palmitoyltransferase to inhibition by malonyl-CoA are due to differences in Ki values

The hepatic carnitine palmitoyltransferase that is present on the outer surface of the mitochondrial inner membrane demonstrates hyperbolic substrate saturation curves with oleoyl-CoA in both fasted and fed rats. However, the addition of malonyl-CoA resulted in sigmoid substrate saturation curves, suggesting that malonyl-CoA induced the cooperative behavior. There was more of the outer carnitine palmitoyltransferase in liver mitochondria derived from fasted rats and that enzyme had a much greater Ki for malonyl-CoA than the enzyme from fed rats, but the Km values were apparently not different. The Dixon plot with mitochondria from fed rats, but not fasted rats, was curved upward, indicating cooperative inhibition by malonyl-CoA. Carnitine palmitoyltransferase of heart mitochondria had a Ki for malonyl-CoA that was much less than that of the liver enzyme and it did not change on fasting. Furthermore, no evidence for cooperative inhibition was found in the heart. The results of these studies indicate that carnitine palmitoyltransferase is not subject to substrate cooperativity and that malonyl-CoA is not a simple competitive inhibitor of this enzyme but inhibits by a mechanism involving cooperative inhibition. The fasting-feeding cycle induces changes in the liver enzyme that alter its affinity for malonyl-CoA without changing its affinity for its acyl-CoA substrate. Carnitine palmitoyltransferase from heart appears to be different from that of liver and is apparently not subject to the same control mechanisms.     

The morphological relationship between mitochondria and cytoplasmic membranes of the follicular oocyte in domestic pig

Summary An ultrastructural study of the mature follicular oocytes in domestic pig demonstrate a morphological relationship between the mitochondria and the cytoplasmic membranes immediately surrounding the yolk globules of the cells. Frequently, the cytoplasmic membranes are observed to be in close proximity of the mitochondria or are found to be continuous with the outer mitochondrial membrane. Sometimes the cytoplasmic membranes are found to display the formation of one or more oval loops of different diameter located at their presumed ends or free in the nearby cytoplasm. The significance of these observations is discussed in the light of the available informations, which suggest that the cytomembrane system in certain phases of development may take part in the formation of mitochondria.This work was supported by the Agricultural Research Council of Norway.     

Non-uniform distribution of mitochondria in pancreatic acinar cells

The distribution of mitochondria in pancreatic acinar cells was investigated using confocal fluorescence microscopy and transmission electron microscopy (EM). Acinar cells were studied either after enzymatic isolation or in small segments of undisassociated pancreatic tissue. Loading of isolated acinar cells with Mito Tracker Green or Red, a fluorescence mitochondrial probe, showed that mitochondria are predominantly situated in the perigranular, subplasmalemmal and perinuclear regions. Subsequent applications of EM fixatives induced a leak of the fluorescent indicator to the cytosol but did not change the distribution of mitochondria. EM was then performed on isolated acinar cells and on acinar cells of pancreatic tissue segments. The intracellular distribution of mitochondria was quantified by calculating the percentage of the cross-sectional area that was occupied by mitochondria. In isolated acinar cells the highest density of mitochondria was seen in the perigranular region, where mitochondria occupied 25.69±1.58% of the area, then the subplasmalemmal region with 12.61±0.77% and the perinuclear region with 9.07±0.97% (n=26). Similar results were obtained from acinar cells of pancreatic tissue segments: the perigranular 22.9±1.95%, subplasmalemmal 12.45±0.78% and perinuclear regions 9.07±0.97% (n=26). The outer mitochondrial membranes were frequently positioned close to membranes of the ER, which followed the outer contour of mitochondria. Mitochondria were never found in direct contact with the nuclear envelope: there were usually layers of ER between the mitochondrial and nuclear membranes. Subplasmalemmal mitochondria were found in a very close proximity to the plasma membrane with no ER layers between the mitochondrial and the corresponding plasma membranes. We conclude that in pancreatic acinar cells mitochondria are preferentially distributed to perigranular, subplasmalemmal and perinuclear regions and this distribution is not affected by isolation or fixation procedures.P.R. Johnson and N.J. Dolman contributed equally to the study. This work was supported by a Medical Research Council programme grant. P.R.J. is a Medical Research Council PhD student and N.J.D. is a Wellcome Trust PhD student.     

Ultrastructure of juvenile hormone-induced degenerating flight muscles in a bark beetle,Ips paraconfusus

Summary Topical application of 5 g of a juvenile hormone analogue (JHA), ZR-615, to female callow adults of Ips paraconfusus induced degeneration of the dorsoventral flight muscles. Within 24h after JHA-treatment the diameter of the myofibrils was reduced to almost half due to the lysis of the peripheral myofilaments. Mitochondria showed conspicuous degenerative changes like swelling, dissolution of the matrix or presence in the matrix of dense filamentous material or myelin-like figures. Degeneration of the mitochondria seemed to take place inside isolation membranes derived from sarcoplasmic reticulum. A number of granular osmiophilic bodies appeared in the sarcoplasm. Three days after JHA-treatment the muscles were very thin and sheath-like. Most of the mitochondria had already degenerated. The dense sarcoplasm contained numerous crystalline bodies. The granular dense bodies were also more frequent. The myofibrils were comprised of only occasional small bundles of myofilaments. The tubules of the T system enclosed an amorphous material. The nuclei and the tracheal system remained intact but they were crowded due to the decreased volume of the muscle. In some specimens degeneration of the myofibrils and mitochondria was completed by the third day. No sign of degeneration was observed in the flight muscles of acetone treated control insects.Supported by the National Research Council of Canada grant A4669.We thank Dr. G.B. Staal, Zoecon Corp., Palo Alto, California, for the generous gift of ZR 615, and Messrs A. Syed and M. Horta for rearing the insects     

Effects of differentiation of embryonal carcinoma cells (P19) on mitochondrial DNA content in vitro

Summary The embryonal carcinoma cell line P19 is derived from mouse teratocarcinomas. These pluripotent cells can be induced to differentiate into a variety of cell types by exposure to various drugs. We used retinoic acid to induce embryonal carcinoma cells to differentiate into neuronlike cells. In this study, we show that changes occur in mitochondria during differentiation of embryonal carcinoma cells to neuronlike cells. We found that various morphologic parameters such as mitochondrial fractional area and mitochondrial size decrease as embryonal carcinoma cells differentiate into neuronlike cells. Similar changes were also observed in mitochondrial DNA content. Stereologic analysis of cell preparations provided a measure of mitochondrial fractional area per cell and mtDNA content was assessed by radiolabeled mtDNA probe. This study establishes that mitochondria are regulated as cells differentiate. This study was financially supported by the Medical Research Council of Canada.     

A possible localization of α-glycerophosphate dehydrogenase to the inner boundary membrane of mitochondria in livers from rats fed with ethanol

Summary The morphology and respiration of liver mitochondria were studied in rats fed with ethanol for eight months. Morphometric analysis shows an increase of the volume fraction of mitochondria, a decrease of the density of crista membranes but the density of surface area remained unaltered in the ethanol treated rats as compared to the controls. The ethanol treatment caused a reduced capacity of the mitochondria to oxidize succinate. The capacity to oxidize -glycerophosphate remained unchanged.As it is known that succinate dehydrogenase as well as -glycerophosphate dehydrogenase are bound to the inner mitochondrial membrane, different localization is proposed of the two enzymes in this membrane. Thus, there is a good correlation between the reduction of inner mitochondrial membrane and succinate oxidation. As the activity of -glycerophosphate dehydrogenase remained unchanged, this enzyme should be localized to that part of the inner mitochondrial membrane, which is called inner boundary membrane, and which is not altered by the ethanol treatment.The work is a part of investigations made possible by financial support from the Swedish Medical Research Council, Project No. B71-12Y-2364-04.     

The effect of iron overload on the mitochondrial porphyrin level in the hexachlorobenzene induced experimental porphyria

Liver mitochondria isolated from rats treated with hexachlorobenzene plus iron, present a lower content of total porphyrin in respect to that of mitochondria from rats fed hexachlorobenzene alone. The in vitro mitochondrial porphyrin accumulation processes have been studied in mitochondria from iron loaded rats. It has been found that under these conditions the active porphyrin uptake process, which is driven by the K+ transmembrane gradient, is maximally inhibited in the presence of pentachlorophenol at a concentration similar to that found in vivo in the hexachlorobenzene experimental porphyria. By contrast the same degree of inhibition is presented by control mitochondria only in the presence of pentachlorophenol plus valinomycin, a condition which collapses the transmembrane K+ gradient. A strict correlation between porphyrin uptake and K+ concentration has been found in control as well as in iron treated mitochondria. A possible involvement of peroxidative reactions in the mitochondrial membranes has been proposed as a cause of the changes in the permeability properties of the mitochondrial membranes in the experimental chronic hepatic porphyria under conditions of iron overload.     

Isolation and initial characterization of myelin-like membrane fractions from the nerve cord of earthworms (Lumbricus terrestris L)

We report here the isolation of fractions enriched in components of the myelin-like membranes surrounding the giant axons of the earthworm.Lumbricus terrestris L. The composition and purity of the fractions have been assessed using SDS-protein electrophoresis, Western immunoblots, and electron microscopy. Preliminary enzyme assays indicated that the mitochondrial marker, succinate dehydrogenase, has a similar specific activity distribution in earthworm nerve cord and in mouse liver sedimentation velocity fractions, however, the distribution of the total units of activity among the fractions seems to indicate the existence of smaller mitochondria in earthworm nerve cord compared with mouse liver mitochondria. In earthworm nerve cord fractions, Na⁺/K⁺ ATPase and Ca²⁺/Mg²⁺ ATPase were found to be enriched exclusively in the fraction containing large plasma and myelin-like membranes, while in the mouse liver fractions, the total units of these two enzymes were found to be distributed broadly among fractions. 5-Nucleotidase activity in the earthworm nerve cord seemed to be restricted to the microsomal fractions (endomembrane network), with a very low activity associated with the large plasma and myelin-like membrane fraction. We have established the presence of keratins or prekeratins in the myelin-like membranes, probably in the form of tonofilaments. However, we could not show that the desmosome-like structures, characteristic of these membranes, are composed of those proteins described for vertebrate epithelial desmosomes.     

Stereological studies on the small intestinal epithelium of the rat

Summary Quantitative macroscopic, light-microscopic and electron-microscopic studies were performed on the small intestine of fasted and non-fasted adult, male Sprague-Dawley rats. In non-fasted rats the small intestine was longer than in fasted rats. Due to the presence of villi the surface area in the duodenum and the jejunum was enlarged about six times. The microvilli on the villous crests caused a surface enlargement by 13 times in the duodenum (value corrected for overestimation due to section thickness), and 19 times in the jejunum of the fasted rats. At the base of the villi these values were about 50% lower. It was calculated that, in the fasted rats, the total enlargement of the luminal surface area — due to villi and microvilli — was 63 times in the duodenum and 81 times in the jejunum (corrected for section thickness).Differences between the villous crest epithelium and the villous base epithelium were also found with regard to the mean cell height, and the volume densities of the absorptive cell nuclei, the mitochondria, and the paracellular channels.Supported by grants from the Swedish Medical Research Council (Project No. 12X-2298), from the Swedish Group-Insurance Co. Förenade Liv, from Tore Nilson's Fund for Medical Research and from the Medical Faculty, University of Umeå     

The role of malate in exercise-induced enhancement of mitochondrial respiration

Liver mitochondria isolated from rats immediately after exercise oxidize substrates more rapidly than do mitochondria from resting animals. In both fed and fasted rats, a 1-h period of exercise resulted in increased concentrations of malate in their livers and in the mitochondria isolated therefrom. This increase occurred in both untrained and exercise-trained rats. Because mitochondrial malate is known to facilitate mitochondrial uptake of other carboxylic substrates, it seems likely that the increased mitochondrial malate is responsible for the increased rate of oxidation. Rats injected with small amounts of malate (4.6 mumol/100 g body wt) yielded liver mitochondria with increased malate concentration and increased rates of oxidation of citrate, alpha-ketoglutarate, and succinate. The beta adrenergic antagonist propranolol (0.25 mg/100 g body wt) and the alpha 1 antagonist prazosin (same dose) did not abolish the effect of exercise on mitochondrial malate concentration or substrate oxidation.     

Transfer and reoxidation of reducing equivalents as the rate-limiting steps in the oxidation of ethanol by liver cells isolated from fed and fasted rats

A study was made of factors regulating the oxidation of ethanol in liver cells isolated from fed and fasted rats. The rate of ethanol oxidation was greater in liver cells from fed rats than from fasted rats. Inhibitors of the malate-aspartate shuttle decreased the rate of ethanol oxidation, suggesting that this shuttle contributes to the reoxidation of cytosolic NADH produced during the oxidation of ethanol. The greater inhibition of ethanol oxidation by antimycin than by rotenone suggests that the α-glycerophosphate shuttle also plays an important role in transporting reducing equivalents. The components of the malate-aspartate and α-glycerophosphate shuttles stimulated ethanol oxidation to a greater extent in liver cells from fasted rats than those from fed rats, suggesting that in the fasted state, ethanol oxidation is regulated by the intracellular concentrations of substrate shuttle components which transfer reducing equivalents into the mitochondria. Therefore, uncoupling agents, which stimulate oxygen consumption, do not stimulate ethanol oxidation, and concentrations of antimycin which depress oxygen uptake are much less effective in decreasing ethanol oxidation. By contrast, in liver cells from fed rats, the rate of ethanol oxidation was increased by uncoupling agents. Such stimulation was not observed when cells were prepared in the absence of albumin, probably due to leakage of shuttle substrates which leads to abnormally low intracellular levels. Indeed, when the shuttle substrates were added back to these preparations, uncouplers were effective in stimulating the rate of ethanol oxidation beyond the stimulation produced by the shuttle substrates alone. Thus, under conditions of sufficient intracellular levels of the intermediates of the substrate shuttles, ethanol oxidation is regulated by the capacity of the mitochondrial respiratory chain to reoxidize reducing equivalents generated by the alcohol dehydrogenase reaction.     

Dietary dependent distribution of acetyl CoA carboxylase between cytoplasm and mitochondria of rat liver

Biotinyl proteins in cytoplasm and mitochondria of rat liver were examined by fluorography and the quantity of acetyl CoA carboxylase was determined after sodium dodecyl sulfate-denatured proteins were incubated with [14C] methyl avidin and separated by polyacrylamide gel electrophoresis. Results show that one-half of the total acetyl CoA carboxylase in liver of fed rats was associated with mitochondria in a relatively inactive form. Fasting shifted the distribution of the enzyme toward the mitochondrial fraction and refeeding previously fasted rats shifted the distribution towards cytoplasm. Thus, acetyl CoA carboxylase can be added to the list of ambiquitous enzymes whose subcellular distribution varies with physiological conditions.     

Physiological state and the sensitivity of liver mitochondrial outer membrane carnitine palmitoyltransferase to malonyl-CoA. Correlations with assay temperature, salt concentration and membrane lipid composition.

1. Liver mitochondrial outer membranes were pre-exposed to media of low (20 mM phosphate) or high salt concentration (20 mM phosphate + 0.3 M KCl) before assay of carnitine palmitoyltransferase (CPT) at 25 degrees C. 2. With membranes from fed rats, exposure to high salt decreased sensitivity of CPT to malonyl-CoA whereas high salt increased sensitivity of CPT to malonyl-CoA in membranes from 48 hr-fasted rats. These changes were paralleled by alterations in the KD for high affinity binding of [14C]malonyl-CoA to outer membranes. 3. Decreasing the CPT assay temperatures from 25 to 10 degrees C caused qualitatively similar changes to those seen on exposure to high salt. 4. The relative content of sphingomyelin was increased 2-fold and 4-fold in liver mitochondrial outer membranes from fasted and diabetic rats respectively. Fasting had no effect on the content of cholesterol whereas diabetes decreased this by a third.     

Absorption of horse-radish peroxidase by the mucosal cells of the duodenum of mouse

Summary Horse-radish peroxidase was injected intraperitonealy to fasted and fed newborns. The protein was rapidly absorbed by the fasting animals and more slowly by the fed newborns. The tracer was progressively segregated into the supranuclear cluster of dense tubules and dense bodies. The cellular sap of several cells was deeply stained after the intraperitoneal injection. Control experiments showed that peroxidase has a high affinity for the matrix of the dense bodies in the first days of neonatal life.This work was supported by grants no. MA-3994 Medical Research Council of Canada, and by no. 9340-06 National Defense Board.The author is greatly indebted to Mr. Michel Couture for his skillful technical aid.