Hepatic autophagy and intracellular ATP. A morphometric study

In order to estimate the sensitivity of macroautophagy in liver toward changes in ATP we have analyzed the volume density of the autophagic/lysosomal system in isolated rat hepatocytes, incubated under conditions where intracellular ATP was partially depleted. (a) It appeared that reduction of the intracellular ATP concentration by 30-50% decreased the volume density of autophagic vacuoles by 70%. (b) Partial ATP depletion did not involve significant changes in the volume density of dense bodies. Together with studies showing that the rate of overall proteolysis via macroautophagy decreases with decreasing ATP concentration (P.J.A.M. Plomp, E.J. Wolvetang, A.K. Groen, A.J. Meijer, P.B. Gordon, and P.O. Seglen (1987) Eur. J. Biochem. 164, 197-203) our data indicate that changes in intracellular ATP primarily affect early steps in the autophagic/proteolytic pathway.     

Inhibition of cellular autophagy in proximal tubular cells of the kidney in streptozotocin-diabetic and uninephrectomized rats.

To examine the significance of anti-catabolism in renal hypertrophy, cellular autophagy was investigated by electron microscopic morphometry in proximal tubular cells (PTCs) of the outer cortex of the rat kidney after the induction of diabetes mellitus by streptozotocin (STZ) and after unilateral nephrectomy. Adult male Sprague-Dawley rats were divided into three groups and killed by retrograde perfusion fixation, 1, 2 and 3 days after the induction of diabetes (group D; n = 24), after unilateral nephrectomy (group N; n = 24) and after combined treatment (group DN; n = 24). Untreated, age-matched litter mates served as controls (group C; n = 24). By comparison with these controls, the left kidney to initial body weight ratio was increased by 8, 23, and 15% in group D animals, by 8, 23, and 24% in group N animals, and by 10, 21, and 25% in group DN animals at the first, second and third day, respectively. Quantitative evaluation of large test areas showed that the volume and numerical densities of autophagic vacuoles (AVs) in PTCs were significantly lower in these hypertrophed kidneys than in the controls. The average reduction in AV volume density was about 65% in group D animals, about 50% in group N animals and about 75% in group DN animals. These data show that autophagic degradation of cytoplasmic components in PTCs is inhibited in renal hypertrophy independently of the growth stimulus, i.e. uninephrectomy or diabetes. Since insulin per se inhibits cellular autophagy in PTCs, the expected effect of insulin dificiency seems to be counteracted by as yet undefined stimuli that may be related to metabolic work load.     

睾丸间质细胞—研究自体吞噬的一种正常细胞模型

In the present study, we tried to estimate, in a semiquantitative way, the relative frequency of the autophagic activity in various cell types under physiological condition. The results indicated that the highest activity appeared to be in the Leydig cells of rat testes. Autophagosomes were frequently observed in electron microscope photographs of Leydig cells, which provide a good model to study the autophagocytosis in normal cells. The autophagic process in Leydig cells was observed with the electron microscope in preparations treated to show CMPase activity. The mode of formation of autophagosomes in Leydig cells can be divided into three steps. Step 1, flattened membranous elements expand to enclose a small cytoplasmic territory to form pre-autophagosome. Step 2, The double membrane profile of the pre-autophagosome then completely encloses the cytoplasmic territory to form early autophagosome in which structurally normal organelles are contained. Step 3, the transformation of an early autophagosome into a late one is accompanied by the loss of one of the two delimiting membranes, the partial disintegration of the enclosed content and simultaneous acquisition of acid phosphatase activity. The enzymatic reactivity is acquired following a close association with the lysosomes. The late autophagosome then reaches the cell surface and appear to exocytose their residual content.     

Beta-adrenoceptors in kidney tubules of spontaneously hypertensive and normotensive rats

Beta-adrenoceptor binding characteristics were determined in different fractions of rat kidney tubules using a [125Iodo]-(-)-cyanopindolol (ICYP) binding assay. The highest amount of binding sites was found in a fraction containing predominantly distal tubular fragments. In a separate series of experiments the ICYP binding characteristics were compared in whole tubular fractions from spontaneously hypertensive (SHR) and normotensive Wistar Kyoto rats (WKY) of different ages. The maximum number of binding sites was significantly higher both in young (3 weeks) and adult (14 weeks) SHR when compared to age-matched WKY. These studies showed the presence of beta-adrenoceptor binding sites in rat kidney tubules and support the potential importance of tubular beta-adrenoceptors in the development of spontaneous hypertension and in the mechanism of antihypertensive action of beta-blockers.     

A morphometric study of the carotid body in chronically hypoxic rats

We performed morphometric studies of carotid body in acutely and chronically hypoxic rats (inspired PO2 = 70 Torr, at sea level). Acute exposure was for the duration of about 10 min, and chronic exposure lasted for 28 days. We confirmed that the total volume of the organ increased by severalfold. At the light-microscopy level we found an enlargement of the volume density of the blood sinuses from 14 to 31% due to chronic hypoxia. The morphometric hematocrit increased from 39 to 70% paralleling changes in the conventionally measured venous hematocrit. These data do not show any specific plasma skimming in the carotid body blood vessels. With the electron microscope we found that the mean average volume of type I cells increased from 320 micron3 in controls to 1,120 micron3 in the chronically hypoxic rats without hyperplasia, whereas type II cells had increased in number without alteration in size. Qualitative observations revealed that the normal appearance of clusters of ovoid type I cells interspersed by capillaries had been transformed into a pattern of individual cells forming plates between expanded blood vessels with a large increase of contact area between the cells and vessels. Type II cells appeared to have proliferated without changes in individual size to cover the enlarged periphery of type I cells. The observed structural changes in the carotid body parenchyma and vasculature appear to be physiologically adaptive and provide further support for the idea that various elements in the organ are particularly sensitive to hypoxia.     

Computerized morphometric quantitation of cellular features in splenocytes from normal and nutrient-restricted mice

The morphometric analysis of splenocytes reveals quantitative changes in the chromatin and cytoplasm that may be used to distinguish between cells from normally fed animals and cells from subjects fed restricted diets. Analysis of Feulgen-stained and Papanicolaou-stained cells from mice fed normal diets and mice fed either calorie-restricted diets or isocaloric but protein-restricted diets showed an approximately 10% reduction in the nuclear area and in the total optical density (TOD) of stained chromatin in cells from diet-restricted mice. Some changes in chromatin texture features were also observed. Utilization of nuclear area, TOD and one textural feature in a linear discriminant analysis produced a distinct separation of the cells from dietary-restricted mice and the cells from normally fed subjects; this was observed with both Feulgen-stained and Papanicolaou-stained cells. The cellular effect of dietary restriction was more noticeable in the cytoplasm than in the nucleoplasm; Papanicolaou-stained cells from diet-restricted animals showed a 23% reduction in the cytoplasmic TOD and a 10% reduction in the nuclear TOD. This study shows that computerized morphometric analysis may be used in place of or in conjunction with other measurement procedures and chemical tests to quantitate and differentiate cells subjected to different types and levels of nutritional stress.     

A kallikrein-specific inhibitor in rat kidney tubules.

A kallikrein inhibitor was found in tubules of the rat kidney and purified by chromatography on Sephadex G-100. The molecular weight of the inhibitor, estimated by gel filtration and dodecylsulfate electrophoresis, is about 4700. It inhibits the following kallikreins: porcine submanidbular and pancreatic kallikrein, rat kidney and urine kallikrein, and human urine and plasma kallikrein. An inhibition of bovine trypsin was not observed.     

A freeze-etch study of angular marginal-plate-containing peroxisomes in the proximal tubules of bovine kidney

Summary The ultrastructure of peroxisomes in the proximal nephron tubules of bovine kidney cortex was studied using ultrathin-sectioning, diaminobenzidine cytochemistry for the visualization of catalase, and by freeze-fracture. Peroxisomes in this nephron segment are up to 1.5 m in diameter and exhibit a peculiar angular shape, which is probably related to the occurrence of multiple straight plate-like inclusions (marginal plates) in the matrix of peroxisomes; they lie directly underneath the peroxisomal membranes. The peroxisomal membrane in such regions follows the outline of the marginal plate. The peculiar shape of peroxisomes allows their unequivocal identification in freeze-fracture preparations. Peroxisomal membranes are recognized by their flat, often rectangular appearance. Intramembrane particles are much more numerous on P-fracture faces than on E-fracture faces. A crystalline lattice-structure with a periodicity of approximately 10 nm can be observed on the flat rectangular areas of E-fracture faces. This lattice structure is intensified after prolonged freeze-etching. Intramembranous particles seem to be superimposed over this pattern. The crystalline pattern on the E-fracture faces of peroxisomal membranes is probably not a membrane structure but it reveals the structure of the membrane-associated marginal plates. A cast of the marginal-plate surface may be generated by a collapse of the peroxisomal membrane half onto the immediately underlying matrix inclusion.     

Correlations of intracellular and cellular regeneration of the epithelium of the kidney tubules in necronephrosis

The study of radioautographs in ultrathin and semithin sections extracted from animal tissues and following the impulsive as well as long-term injection of 3H-thymidine was made. Intracellular regeneration processes (external cell membrane synthesis, DNA reparation, restoration of the lost albumin-synthesizing complex and cytoskeleton) turned to develop in proximal canals of epithelial cells. The processes were aimed at the liquidation of distortions preventing cell from entering mitosis.     

A new method of a morphometric study of the kidney using a computer-assisted morphometric complex "Diamorph"]

The use of a computer--based morphometric complex "DIAMORPH" for automated morphometric analysis of renal glomeruli is described. By combining the principles of morphological and functional approaches to diagnosis with the capabilities of present--day research equipment, a new informative morphometric parameter--the extent of capillary--mesangium contact--has been identified. With this parameter and its derivatives, it has been possible to differentiate, in a statistically significant way (p less than 0.01), healthy subjects (controls) from patients with manifest diabetes mellitus of up to I year in duration who could not be distinguished through light microscopic or conventional morphometric studies.     

Cellular autophagy in proximal tubules of early diabetic rats following insulin treatment and islet transplantation

The influence of insulin treatment (group 1) and allogenic islet transplantation (group 2) on renal cellular autophagy were evaluated in adult Lewis rats in the early phase of streptozotocin-induced diabetes mellitus—a condition in which autophagy is inhibited and renal mass is increased. Three days after insulin treatment or islet transplantation (IT), the right kidney was resected and cortical tubular tissue was examined by quantitative electron microscopy. In group 1, the volume and numerical densities of autophagic vacuoles (AVs) increased by 70% and 80% respectively in the proximal tubular cells compared with saline-injected controls. The additive effect of unilateral nephrectomy (Ux) on cellular autophagy was investigated 1 or 2 days after Ux. Compared with the resected right kidney, the volume and numerical densities of AVs in the remnant left kidney decreased by 49% and 43% in the insulin-treated rats, and by 43% and 39% in the saline-injected diabetic animals. In group 2, the volume and numerical densities of AVs increased by 45% and 44% in parenchyma regressing from diabetic hypertrophy after IT, compared with sham-operated controls. After Ux, the volume and numerical densities of AVs decreased by 49% and 43% in IT rats, and by 41% and 53% in the still diabetic sham-operated animals. The data show that inhibition of cellular autophagy in the proximal tubules of the early diabetic kidney can be reversed by insulin replacement, despite the fact that insulin per se inhibits cellular autophagy in the nondiabetic kidney. Thus the stimulation of cellular autophagy in the diabetic kidney by insulin replacement may be an important mechanism in the regression of diabetic renal hypertrophy. Both the diabetic kidney and the kidney regressing under the influence of insulin respond to the additional growth stimulus of Ux by inhibition of cellular autophagy.     

A morphometric study of 24-hour variations in subcellular structures of the rat pancreatic acinar cell

Summary Subcellular structures of pancreatic acinar cells were examined at six evenly spaced time points in the 24-h period (light cycle: 06.00 h–18.00 h) in four Wistar male rats at each time point. At each sampling point, the area and circumference of acinar cell bodies and the area, number and circumference of their cytoplasmic organelles were measured using a semiautomatic computer system for morphometry and a point-counting method.The area, number and circumference-area ratio of the cytoplasmic organelles were subject to strong circadian variations, and the cellular area and circumference exhibited weak circadian variations. Variation pattern of the cytoplasmic organelles suggested an intracellular route for secretory proteins during a 24-h span. From the results it was possible to divide the 24-h period into three stages. 1. The resting or protein synthetic stage (00.00 h to 08.00h): the area of the rough surfaced endoplasmic reticulum (rER) was strongly increased, and that of zymogen granules was clearly decreased. 2. The granule accumulation stage (08.00h to 16.00h): the area of the rER was markedly decreased; that of zymogen granules was increased. 3. The secretion stage (16.00 h to 00.00): as a result of the release of zymogen granules from the acinar cell, the area of zymogen granules decreased, and that of the rER increased. The relationship between the area of the rER and zymogen granules varied in a reciprocal manner. Other cytoplasmic organelles, namely the Golgi complex, condensing vacuoles, mitochondria and lysosomes also varied prominently during the 24-h span, corresponding to variations in the rER and zymogen granules.     

A morphometric study of sub-cellular organization in mouse mammary cancers and normal lactating tissue

Summary Four normal lactating glands of Mice and nine mammary cancers have been studied by quantitative electron microscopy.Normal lactating cells are characterized by a high organelle development: the mitochondria occupy 11.2% and the Golgi apparatus 7.4% of the cytoplasm (without lipid droplets). The ergastoplasm attains a surface of 4.7 square micron per cubic micron of cytoplasm.All mammary cancers examined had a lower organelle development than normal lactating gland cells and this independently from the fact whether the tumour cells were secreting or not.A relationship between the number of virus particles in the section area and the degree of organelle development does not exist.

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Résumé L'étude quantitative au microscope électronique de quatre glandes mammaires lactantes de souris et de neuf cancer mammaires a été effectuée.Les cellules lactantes normales sont caractérisées par un grand développement des organites cytoplasmiques: les mitochondries occupent 11,2% et l'appareil de Golgi 7,4% du cytoplasme (gouttelettes lipidiques exclues); 'ergastoplasme atteind une surface de 4,7 microns carrés par micron cubique de cytoplasme.Tous les cancers mammaires examinés, que les cellules tumorales soient sécrétantes ou non, ont des organites cellulaires moins développés que les cellules lactantes normales.Il n'existe pas de relation entre le nombre de particules virales dans une section donnée et le degré de développement des organites cellulaires.