Apoptogenic effect of the lipophilic o-naphthoquinone CG 10-248 on rat hepatocytes: light and electron microscopy studies

CG 10-248 (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione; CG-NQ), a beta-lapachone analogue, modified the ultrastructure of rat hepatocytes, as demonstrated by light and electron microscopy. After 4 h incubation with 100 microM CG-NQ, the following effects were observed: (a) nuclear chromatin condensation; (b) chromatin fragmentation; (c) displacement of mitochondria, concentrated around the nucleus; (d) disruption or expansion of mitochondrial outer or inner membranes, respectively; (e) displacement and alteration of endoplasmic reticulum (rough and smooth); (f) decrease of microvilli; (g) blebbing of plasma membrane and production of apoptotic bodies formed by folding of plasma membrane fragments around mitochondria or peroxysomes; and (h) production of hydrogen peroxide. Expression of such effects varied according to hepatocyte samples and taken together strongly support an apoptotic action of CG-NQ dependent on reactive oxygen species. (AU)

Estudio histoquimico y ultraestructural de la celula de Merkel utilizando un modelo tumoral. / Histochemical and ultrastructural study of Merkels cell using a tumoral model

Se realiza el estudio histoquimico y ultraestructural de un tumor de celulas de Merkel. El material fue dividido en tres partes que se sometieron a las siguientes fijaciones: 1) glutaraldehido-OsO4; 2) glutaraldehido-dicromato (GD);3) formaldehido-glutaraldehido-dicromato (FGD). GD y FGD fueron hechas de acuerdo a la tecnica histoquimica de Wood. GD para la deteccion de catecolaminas (CA) y serotonina (5HT) y FGD para la deteccion especifica de 5HT.En los tres casos el material fue procesado para microscopia electronica. Morfologicamente se observo en 1) que las celulas tumorales contenian en su citoplasma las vesiculas granuladas caracteristicas de las celulas de Merkel. Con GD y FGD se observo una reaccion positiva a nivel de los granulos, mas intensa con GD que con FGD. Los resultados expuestos indican, por la positividad de FGD, que las vesiculas granuladas de las celulas estudiadas contienen 5HT y sugieren la presencia simultanea de CA por ser GD notablemente mas intensa que FGD

The release of catecholamines by an endogenous factor that inhibits neuronal Na+,K(+)-ATPase.

It is known that the inhibition of Na+,K(+)-ATPase induces neurotransmitter release in several experimental models. In this laboratory it was previously observed that a brain soluble fraction separated by Sephadex G-50 (peak II) is able to inhibit Na+,K(+)-ATPase but not other membrane-bound enzymes. The object of the present study was to test the effect of brain peak II fraction on neurotransmitter content of the pineal nerves synaptic vesicles. Uninjected rats and rats injected 30 min before with 5-hydroxydopamine (30 mg per kg,i.p.) were used. 5-hydroxydopamine produces a false neurotransmitter whose presence in the synaptic vesicles is visualized after fixation with glutaraldehyde-osmium as an electron dense material with the electron microscope which fills totally or partially the vesicles. In uninjected rats the osmiophilia and the chromaffin reaction of the electron dense core were studied. The pineal glands were incubated in Tyrode solution without calcium in the presence and absence of peak II at room temperature and processed for electron microscopy. When the glands from rats pretreated with 5-hydroxydopamine were incubated with peak II a significant decrease in the number of vesicles totally stained was observed. This indicates a reduction in false neurotransmitter content, specially in the matrix of the synaptic vesicles. In the presence of an aged peak II, which does not inhibit Na+,K(+)-ATPase, this effect on the synaptic vesicles was not observed. When the glands from uninjected rats were incubated with peak II no changes in the osmiophilia and the chromaffin reaction of the synaptic vesicles were found. The osmiophilia and the chromaffin reaction of the cores marks the monoamines storage site (catechol and indoleamines in the pineal nerves). These results are coherent with the idea of a relationship between the inhibition of Na+,K(+)-ATPase activity and the release of a pool of neurotransmitter stored in the nerve endings.

Effect of illumination on different lipid fractions of rat pinealocytes. A histochemical study.

Total lipids and phospholipids of pineal glands of female rats maintained under a diurnal rhythm of 12 hr of light and 12 hr of darkness, continuous illumination for 2 days or 4 weeks and constant darkness for 2 days or 4 weeks were studied histochemically. The controlled chromation of Elftman followed by Sudan black B to visualize total lipids and by hematoxylin to reveal phospholipids was used. Staining was made on paraffin embedding and vibratome sections. Total lipids showed a significant decrease under constant illumination for 2 days or 4 weeks, but phospholipids, which suffered a dramatic decrease in rats maintained under constant light for 4 weeks, remained without significant variation after 2 days of treatment. Thus, the lipid decrement after 2 days of constant light seems to be due to another lipid fraction, probably triglycerides. Total lipids and phospholipids remain almost unchanged after 2 days or 4 weeks of constant darkness. The solubility in lipid solvents was considerably increased for phospholipids after 4 weeks and for the non-phospholipidic fraction after 2 days of permanent light. This suggests that continuous illumination induces a change in the solubility of lipid droplets components affecting different kinds of lipids depending on the light stimulation.

Immunocytochemical localization of sorotonin in the pineal gland

Se estudió la localización de serotonina (5HT) en la glándula pineal de ratas en condiciones de luz diurna por medio de microscopía óptica y electrónica. Se usó un anticuerpo policlonal para 5 HT y el método inmunocitoquímico de peroxidasa antiperoxidas (PAP). La glándula mostró fibras varicosas inmunomarcadas distribuidas por toda su extensión. Las observaciones ultraestructurales revelaron la presencia de depósitos electrodensos de diaminobencidina (DAB) en terminales localizados principalmente en los espacios perivasculares (AU)

Immunocytochemical localization of serotonin in the pineal gland.

The localization of serotonin (5HT) in the rat pineal gland during daylight conditions was investigated by light and electron microscopy using a polyclonal antibody to 5HT and the immunocytochemical method of peroxidase-antiperoxidase (PAP). Thin varicose 5HT fibers were found distributed in the gland at light microscopy level. Ultrastructural observations revealed the presence of terminal endings containing 5HT and localized mainly in the perivascular spaces, labeled with dense diaminobenzidine (DAB) deposits.

Effect of dyskinetoplastic agents on ultrastructure and oxidative phosphorylation in Crithidia fasciculata.

Ethidium bromide (EB) is an intercalating agent which binds specifically to the kinetoplast (mitochondrial) DNA (kDNA) of trypanosomatids. Accordingly, EB inhibits DNA replication, thus inducing dyskinetoplasty. Since in eukariotic organisms mitochondrial DNA encodes the genetic information for cytochromes b, aa3 and F0F1 ATPase, it seemed of interest to establish whether a similar effect occurs in Crithidia fasciculata, a trypanosomatid used for assay of potential trypanocidal drugs. Culturing of C. fasciculata in the presence of EB inhibited growth and induced dyskinetoplasty, as confirmed by electron microscopy. The kinetoplast of EB-cultured crithidia lost its characteristic arc shape, it was misplaced in the cell cytoplasm its matrix structure and membrane differentiation were specifically modified. Dyskinetoplasty decreased crithidia respiration and oxidative phosphorylation, as indicated by the lower ATP level, ATP/ADP ratio and adenylate energy charge. The interference of EB with kinetoplastic constituents synthesis was confirmed by the lack of action of EB on crithidia in the stationary phase of growth, that ruled out direct inhibition of oxidative phosphorylation enzymes. The lipophilic o-naphthoquinone beta-lapachone produced structural alterations in kinetoplast membranes, that correlated with inhibition of oxidative phosphorylation. These latter effects involved free radicals since they were prevented by free radical scavengers.

Ultrastructural study of atrial specific granules in experimental renovascular hypertension elicited by the constriction of both renal arteries.

The heart has an endocrine activity which depends on the secretion of a natriuretic, diuretic and hypotensive factor contained in osmophilic, secretory granules localized in the myocardiocytes and called [quot ]atrial specific granules[quot ] (the atrial natriuretic factor, ANF). In this paper, the relationship between these specific granules and renovascular hypertension elicited by the constriction of both renal arteries was investigated at the electron microscope level during the acute, subacute and chronic phases of hypertension. Male Wistar CHbb THOM rats were divided in three groups: 1) clipped rats; 2) sham operated rats; 3) ether anesthesia as unique manoeuver 48 h before decapitation. Blood pressure increased progressively after the constriction of both renal arteries. The atrial specific granules were not affected by ether anesthesia alone; 48-72 h after clipping the granules almost disappeared and this situation persisted up to the 6th week. In sham operated rats the picture was very similar to the clip rats 48 and 72 h after surgery (severe granule disappearance); in contrast, at one, two and six weeks after surgery, the granularity of cardiomyocytes in sham rats was absolutely restored. It is concluded that: 1) similarities in morphology of atrial specific granules in sham and clip rats 48 and 72 h after surgery would suggest that stress plays a primary role in determining the observed images; 2) thereafter, the contrast between sham and clip rats 1, 2 and 6 weeks after surgery would indicate that the ANF is linked to the subacute and chronic regulation of renovascular hypertension.