PEROXISOMES IN INNER ADRENOCORTICAL CELLS OF FETAL AND ADULT GUINEA PIGS

Abundant membrane-bounded granules, 0.1–0.45 µm in diameter, occur among the elements of the smooth-surfaced endoplasmic reticulum in zona fasciculata and zona reticularis adrenocortical cells of guinea pigs. Acid phosphatase cannot be cytochemically demonstrated in them, and they are therefore distinct from lysosomes. Incubation in medium containing 3,3'-diaminobenzidine results in dense staining of the granules, identifying them as peroxisomes. These small peroxisomes increase in number as fetal adrenocortical cells differentiate, and they appear to arise from dilated regions of endoplasmic reticulum. They maintain interconnections with the smooth endoplasmic reticulum and with one another.     

INTRACISTERNAL GRANULES IN THE EXOCRINE CELLS OF THE PANCREAS

Dense, homogeneous granules of 250 to 350 mµ in diameter have been found inside the cavities of the endoplasmic reticulum in the acinar cells of the pancreas of the guinea pig. They apparently correspond to the fine granules described in the same material in light microscopy by Mankowski and Bensley. The location of these granules indicates that the endoplasmic reticulum is a cavitary system and suggests its participation in secretory processes.     

Catalase in guinea pig hepatocytes is localized in cytoplasm, nuclear matrix and peroxisomes

We have compared the intracellular localization of catalase and another peroxisomal marker enzyme, alpha-hydroxy acid oxidase (HAOX), in the livers of guinea pig and rat using immunoelectron microscopy and subcellular fractionation combined with immunoblotting and enzyme activity determination. Antibodies against both enzymes were raised in rabbits and their specificities established by immunoblotting. By immunoelectron microscopy, gold particles representing antigenic sites for catalase were found in guinea pig hepatocytes not only in peroxisomes but also in the cytoplasm and the nuclear matrix. In rat liver, however, catalase was localized exclusively in peroxisomes with no cytoplasmic labeling. Moreover, in both species HAOX was found only in peroxisomes. Subcellular fractionation revealed that purified peroxisomes from both species contained comparable levels of each, catalase and HAOX activities. The total catalase activity, however, was substantially higher in guinea pig and most of this excess catalase was in the cytosolic fraction with some activity also in nuclei. In rat liver, 30 to 40% of both enzymes and in guinea pig liver 30% of HAOX were recovered in the supernatant fraction implying that the fragility of peroxisomes in both species is quite comparable. These observations establish the occurrence of extraperoxisomal catalase in guinea pig liver. The catalase in the cytoplasm and nucleus of liver parenchymal cells is most probably involved in scavenging of H2O2, protecting the cell against toxic and mutagenic effects of this noxious agent.     

Alanine: glyoxylate aminotransferase 1 is present in the peroxisomes of guinea pig kidney

The subcellular distribution of alanine: glyoxylate aminotransferase 1 in guinea pig and rabbit kidneys was examined by centrifugation in a sucrose density gradient. The enzyme was located in the peroxisomes of guinea pig kidney and cross-reactive with the antibody against rat liver alanine: glyoxylate aminotransferase 1. This is the first report on the presence of the enzyme in the peroxisomes of mammalian kidney. The enzyme was found to be located in the mitochondria but not in the peroxisomes in rabbit kidney.     

PANCREATIC MICROSOMES : AN INTEGRATED MORPHOLOGICAL AND BIOCHEMICAL STUDY

The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its orderly disposition in the basal region of the cell. In addition to the small, (~15 mµ), dense particles attached to the limiting membrane of the endoplasmic reticulum, numerous particles of similar appearance are found freely scattered in the cytoplasmic matrix. The various cell structures of pancreatic exocrine cells can be satisfactorily identified in pancreatic homogenates. The microsome fraction consists primarily of spherical vesicles (80 to 300 mµ), limited by a thin membrane (7 mµ) which bears small (~15 mµ) dense particles attached on its outer surface. The content of the microsomal vesicles is usually of high density. Pancreatic microsomes derive by extensive fragmentation mainly from the rough surfaced parts of the endoplasmic reticula of exocrine cells. A few damaged mitochondria and certain dense granules (~150 mµ) originating probably from islet cells, contaminate the microsome fraction. Pancreatic microsomes contain RNA, protein, and a relatively small amount of phospholipide and hemochromogen. They do not have DPNH-cytochrome c reductase activity. In six experiments the RNA/protein N ratios were found grouped around two different means, namely 0.6 and 1.3. Pancreatic microsomes are more labile than liver microsomes but react in a similar way to RN-ase-(loss of the particulate component and RNA), and deoxycholate treatment (loss of the membranous component and of phospholipide, hemochromogen, and most of the protein). Postmicrosomal fractions consisting primarly of small (~15 mµ), dense particles of ribonucleoprotein (RNA/protein N ratio = 1 to 2) were obtained by further centrifugation of the microsomal supernatant. The small nucleoprotein particles of these fractions are frequently found associated in chains or clusters.     

Investigation of peroxisomal lipid beta-oxidation enzymes in guinea pig liver peroxisomes by immunoblotting and immunocytochemistry.

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.     

Peroxisomes in guinea pig liver: their peculiar morphological features may reflect certain aspects of lipoprotein metabolism in this species

Summary We have studied the ultrastructural characteristics and the distribution of peroxisomes in guinea pig liver using electron-microscopic cytochemistry for catalase and morphometry. By light microscopy, peroxisomes appear as dark 0.2–0.5 m granules in the cytoplasm of liver parenchymal cells, often forming large clusters that measure up to 5 m across. Rows of single peroxisomes or their aggregates line the sinusoidal surface of hepatocytes. Electron microscopy reveals that clusters of up to 25 individual peroxisomes are usually located in the subsinusoidal region of parenchymal cells. The mean diameter and the volume density of peroxisomes are larger in pericentral than in periportal regions of the liver lobule. Whereas large amounts of lipoprotein particles with a mean diameter of 160 nm (chylomicrons) are present in the Disse space, the cytoplasm of parenchymal cells contains multivesicular bodies and abundant lipid droplets. In addition, the Golgi complexes show distended lipoprotein-filled vesicles suggesting active biosynthesis of lipoproteins. We propose that the unique features of peroxisomes in guinea pig liver, such as cluster formation and alignment along the sinusoidal surface, may be related to the high levels of lipoproteins in the portal circulation and their hepatic catabolism in this species.     

Preparative isolation of peroxisomes from liver and kidney using metrizamide density gradient centrifugation in a vertical rotor

A method for the preparative isolation of peroxisomes from the livers of rat, guinea pig, and mouse, and also from rat kidney is described. The light mitochondrial fraction, i.e., particles sedimenting between 33,000 and 250,000g-min, or the postnuclear supernatant of liver or kidney, is subjected to a 20-50% Metrizamide density gradient ultracentrifugation in a vertical rotor. After centrifugation, the peroxisomes (marker enzyme catalase and dihydroxyacetone phosphate acyltransferase) sedimented as a band near the bottom of the tube (rho = 1.22 g/ml). From the distribution of different marker enzymes and also from the morphometric examinations, it was demonstrated that the isolated peroxisomes are not contaminated with lysosomes, mitochondria, or microsomes.     

Cytochemical studies on peroxisomes in rat and guinea pig myocardium

The enzymatic activity and distribution of peroxisomes (microbodies) in rat and guinea pig hearts were studied cytochemically, by means of oxidation of 3-3'-diaminobenzidine (DAB) and by using B-glycerophosphate and cytidine-5'-monophosphate as substrates. Peroxisomes were localized in proximity to mitochondria and sarcoplasmic reticulum and measured from 0.2 micrometers to 0.5 micrometers in diameter in both animal species. DAB positive bodies were seen both at pH 9.0 and pH 5.0 in rat myocardial cells. However, in guinea pig myocardial cells the reaction was observed only at pH 9.0, or very faintly at pH 5.0. Acid and alkaline phosphatases were not demonstrated in the peroxisomes. Lipid droplets were surrounded by a ring of dense granular reaction product for enzymes, such as acid and alkaline phosphatase, and lipofuscin granules were limited by acid phosphatase or DAB reaction products. The pathophysiological function of peroxisomes is discussed.     

Subcellular distribution and properties of acyl/alkyl dihydroxyacetone phosphate reductase in rodent livers

On subcellular fractionation, the enzyme acyl/alkyl dihydroxyacetone phosphate (DHAP) reductase (EC 1.1.1.101) in guinea pig and rat liver was found to be present in both the light mitochondrial (L) and microsomal fractions. By using metrizamide density gradient centrifugation, it was shown that the alkyl DHAP reductase activity in the "L" fraction is localized mainly in peroxisomes. From the distribution of the marker enzymes it was calculated that about two-thirds of the liver reductase activity is in the peroxisomes and the rest in the microsomes. The properties of this enzyme in peroxisomes and microsomes are similar with respect to heat inactivation, pH optima, sensitivity to trypsin, and inhibition by NADP+ and acyl CoA. The enzyme activity in the peroxisomes and microsomes from mouse liver is increased to the same extent by chronically feeding the animals clofibrate, a hypolipidemic drug. The kinetic properties of this enzyme in these two different organelles are also similar. From these results it is concluded that the same enzyme is present in two different subcellular compartments of liver.     

Neurally Mediated Airway Constriction in Human and Other Species: A Comparative Study Using Precision-Cut Lung Slices (PCLS)

The peripheral airway innervation of the lower respiratory tract of mammals is not completely functionally characterized. Recently, we have shown in rats that precision-cut lung slices (PCLS) respond to electric field stimulation (EFS) and provide a useful model to study neural airway responses in distal airways. Since airway responses are known to exhibit considerable species differences, here we examined the neural responses of PCLS prepared from mice, rats, guinea pigs, sheep, marmosets and humans. Peripheral neurons were activated either by EFS or by capsaicin. Bronchoconstriction in response to identical EFS conditions varied between species in magnitude. Frequency response curves did reveal further species-dependent differences of nerve activation in PCLS. Atropine antagonized the EFS-induced bronchoconstriction in human, guinea pig, sheep, rat and marmoset PCLS, showing cholinergic responses. Capsaicin (10 µM) caused bronchoconstriction in human (4 from 7) and guinea pig lungs only, indicating excitatory non-adrenergic non-cholinergic responses (eNANC). However, this effect was notably smaller in human responder (30±7.1%) than in guinea pig (79±5.1%) PCLS. The transient receptor potential (TRP) channel blockers {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 and ruthenium red antagonized airway contractions after exposure to EFS or capsaicin in guinea pigs. In conclusion, the different species show distinct patterns of nerve-mediated bronchoconstriction. In the most common experimental animals, i.e. in mice and rats, these responses differ considerably from those in humans. On the other hand, guinea pig and marmoset monkey mimic human responses well and may thus serve as clinically relevant models to study neural airway responses.     

Immunohistochemical localization of neurotensin in endocrine cells of the gut

Summary Endocrine cells displaying neurotensin immunoreactivity are found scattered in the jejuno-ileum of all mammals studied, including man. They are rather scarce in rat, guinea pig, rabbit and pig and fairly numerous in cat, dog and man. In most mammals the neurotensin cells predominate on the villi. Only in the dog are they more numerous in the crypts. In the chicken, neurotensin cells occur all along the intestinal tract. They are particularly numerous in the zone that joins the gizzard with the duodenum. The ontogeny of the neurotensin cells in the gut was studied in rats and chickens. In the rat, the cells are first observed in the jejuno-ileum immediately before birth. The adult frequency is reached 4–5 days later. In the chicken, neurotensin cells first appear in the colon in the 18 day old embryo and in the small intestine two days later (i.e. one or two days before hatching). A few days after hatching, the gut has achieved the adult number of neurotensin cells per unit area.     

A CRYSTALLOGRAPHIC STUDY OF PURE CARBONMON-OXIDE HEMOGLOBIN

1. Photomicrographs of crystals of pure carbonmonoxide hemoglobin of the following species are presented; ox, sheep, hog, dog, turkey, rat, horse, chicken and guinea pig. Photomicrographs of the oxyhemoglobin crystals of the following species are also shown: ox, sheep, hog, dog, rat, horse and guinea pig. The crystals were formed from the pure protein by adding a suitable amount of ethyl alcohol and maintaining a temperature of 0°C., or lower. 2. In some species a sufficient difference is shown between the carbonmonoxide hemoglobin and oxyhemoglobin crystals to distinguish these compounds, but the photographs of crystals of carbonmonoxide hemoglobin and oxyhemoglobin of some species, such as guinea pig, show no appreciable difference. 3. Differences between the carbonmonoxide hemoglobins, as well as between the oxyhemoglobins, of the different species studied are indicated. 4. The carbonmonoxide hemoglobin crystals from the bloods studied are species specific in their nature, and, in many cases, can be distinguished from the analogous oxyhemoglobin by crystallographic study.     

THE DEVELOPMENT OF MICROBODIES AND PEROXISOMAL ENZYMES IN GREENING BEAN LEAVES

The ontogeny of leaf microbodies (peroxisomes) has been followed by (a) fixing primary bean leaves at various stages of greening and examining them ultrastructurally, and (b) homogenizing leaves at the same stages and assaying them for three peroxisomal enzymes. A study employing light-grown seedlings showed that when the leaves are still below ground and achlorophyllous, microbodies are present as small organelles (e.g., 0.3 µm in diameter) associated with endoplasmic reticulum, and that after the leaves have turned green and expanded fully, the microbodies occur as much larger organelles (e.g., 1.5 µm in diameter) associated with chloroplasts. Specific activities of the peroxisomal enzymes increase 3- to 10-fold during this period. A second study showed that when etiolated seedlings are transferred to light, the microbodies do not appear to undergo any immediate morphological change, but that by 72 h they have attained approximately the size and enzymatic activity possessed by microbodies in the mature primary leaves of light-grown plants. It is concluded from the ultrastructural observations that leaf microbodies form as small particles and gradually develop into larger ones through contributions from smooth portions of endoplasmic reticulum. In certain aspects, the development of peroxisomes appears analogous to that of chloroplasts. The possibility is examined that microbodies in green leaves may be relatively long-lived organelles.     

Bradykinin receptors in isolated rat duodenum

Pharmacological properties of the bradykinin receptors in the isolated rat duodenum were investigated by examining the relaxant and contractile responses to bradykinin and [des-Arg9]-bradykinin, an agonist of B1 receptors. A specific desensitization and de novo formation for B1 receptors were observed. Changes in medium pH caused a decrease in the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum. Urea incubation in test tube inhibited the responses to bradykinin and [des-Arg9]-bradykinin of rat duodenum, while urea in bathing medium was ineffective. These findings strongly suggested that (a) ionic bonds are important in the interaction between bradykinin and its receptors, and (b) B2 receptors in rat duodenum are different from those in guinea pig ileum.     

Isolation of spinach leaf peroxisomes in 0.25 molar sucrose solution by percoll density gradient centrifugation

A procedure for isolating spinach (Spinacia oleracea L.) leaf peroxisomes in 0.25 molar sucrose solution by Percoll density gradient centrifugation followed by removal of the Percoll by washing and centrifugation was established. The preparation contains more than 90% peroxisomes as intact organelles with no detectable chlorophyll or cytochrome oxidase contamination. The peroxisomes are stable at 0 to 4°C or 25°C for at least 2 hours.     

Inactivation of Venezuelan Equine Encephalomyelitis Virus by gamma-Radiation

Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 10⁶ r γ-radiation (⁶⁰Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 10⁶ r but not in suspensions exposed to 8 × 10⁶ r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage.     

Inactivation of Venezuelan Equine Encephalomyelitis Virus by γ-Radiation

Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 10⁶ r γ-radiation (⁶⁰Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 10⁶ r but not in suspensions exposed to 8 × 10⁶ r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage.     

Oxaliplatin Neurotoxicity Involves Peroxisome Alterations. PPARγ Agonism as Preventive Pharmacological Approach

The development of neuropathic syndromes is an important, dose limiting side effect of anticancer agents like platinum derivates, taxanes and vinca alkaloids. The causes of neurotoxicity are still unclear but the impairment of the oxidative equilibrium is strictly related to pain. Two intracellular organelles, mitochondria and peroxisomes cooperate to the maintaining of the redox cellular state. Whereas a relationship between chemotherapy-dependent mitochondrial alteration and neuropathy has been established, the role of peroxisome is poor explored. In order to study the mechanisms of oxaliplatin-induced neurotoxicity, peroxisomal involvement was evaluated in vitro and in vivo. In primary rat astrocyte cell culture, oxaliplatin (10 µM for 48 h or 1 µM for 5 days) increased the number of peroxisomes, nevertheless expression and functionality of catalase, the most important antioxidant defense enzyme in mammalian peroxisomes, were significantly reduced. Five day incubation with the selective Peroxisome Proliferator Activated Receptor-γ (PPAR-γ) antagonist G3335 (30 µM) induced a similar peroxisomal impairment suggesting a relationship between PPARγ signaling and oxaliplatin neurotoxicity. The PPARγ agonist rosiglitazone (10 µM) reduced the harmful effects induced both by G3335 and oxaliplatin. In vivo, in a rat model of oxaliplatin induced neuropathy, a repeated treatment with rosiglitazone (3 and 10 mg kg⁻¹ per os) significantly reduced neuropathic pain evoked by noxious (Paw pressure test) and non-noxious (Cold plate test) stimuli. The behavioral effect paralleled with the prevention of catalase impairment induced by oxaliplatin in dorsal root ganglia. In the spinal cord, catalase protection was showed by the lower rosiglitazone dosage without effect on the astrocyte density increase induced by oxaliplatin. Rosiglitazone did not alter the oxaliplatin-induced mortality of the human colon cancer cell line HT-29. These results highlight the role of peroxisomes in oxaliplatin-dependent nervous damage and suggest PPARγ stimulation as a candidate to counteract oxaliplatin neurotoxicity.     

A QUANTITATIVE DESCRIPTION OF CORTISONE-INDUCED ALTERATIONS IN THE ULTRASTRUCTURE OF RAT LIVER PARENCHYMAL CELLS

A stereological comparison of the hepatic parenchymal cells from 125-g male rats given a daily injection for 6 days of either 5 mg of cortisone acetate or saline (controls) was carried out with both light and electron microscopy. Cortisone treatment results in an increase in average parenchymal cell cytoplasmic volume from 5100 to 5800 µ³ and a decrease in average nuclear diameter from 7.1 to 6.5 µ. The volume of the average mitochondrion is increased fourfold in midzonal and peripheral regions of hepatic lobules, and there is a decrease in the number of mitochondria per cell such that the total mitochondrial volume per cell remains approximately unchanged. The numbers of peroxisomes are reduced, while the numbers of lysosomes and lipid droplets are increased in all parts of the lobules. The average volume of glycogen is doubled in all cells. The areas of membranes of the smooth- and rough-surfaced endoplasmic reticulum are decreased to one-half and two-thirds of their control values, respectively. The effects of cortisone on these various structural elements is discussed with respect to steroid-related alterations in biochemical processes.