E. Bácsy

Neurotoxin induced nerve cell degeneration: Possible involvement of calcium

Neurotoxin induced nerve cell degeneration has been studied in sensory ganglia of newborn and in the area postrema of adult rats following the administration of the selective sensory neurotoxin, capsaicin and the amino acid excitotoxin, glutamic acid, respectively. Light microscopic histochemical, autoradiographic, electroncytochemical and X-ray microanalytical studies revealed that degeneration of certain small-sized, type B primary sensory neurons, induced by capsaicin, was associated with a marked accumulation of calcium predominantly in mitochondria of the damaged ganglion cells. Similarly, monosodium glutamate treatment resulted in the appearance of calcium-containing electron-dense granules in mitochondria of degenerating area postrema neurons. In addition, after a combined administration of 45Ca2+ and capsaicin or monosodium glutamate, significantly higher levels of radioactivity have been detected by liquid scintillation spectroscopy in the Gasserian ganglia and the area postrema, respectively. It is concluded that an enhancement in intracellular calcium level may be intimately involved in the process of neuronal cell death and may represent a common basic mechanism responsible for the development of cellular events leading ultimately to the degeneration of nerve cells.

Ecto-ATPases and 5'-nucleotidases in the caveolae of smooth muscle. Enzyme-histochemical evidence may indicate a role for caveolae in neurotransmission.

We have demonstrated the localization of ecto‐Ca‐ATPase and 5′‐nucleotidase activity in the caveolae of smooth muscle cells of guinea pig vas deferens and the ileum longitudinal muscle strips with a cerium‐precipitation enzyme‐cytochemical method. The activities seemed to be strongest in the caveolae. Since the simultaneous presence of the 5′‐nucleotidase activity supports the hypothesis that this ecto‐Ca‐ATPase activity does not have a pump function, but, together with 5′‐nucleotidase, may play a role in neurotransmission, these specific membrane invaginations, the caveolae, have a functional relationship with transverse tubules of striated muscle.

A glutamate-sensitive neuronal system originating from the area postrema terminates in and transports acetylcholinesterase to the nucleus of the solitary tract

SummaryThe possible cellular mechanism of action of systemically administered monosodium-l-glutamate and the projections of glutamate-sensitive area postrema neurons have been studied in rats. Parenteral administration of monosodium-l-glutamate induced a selective degeneration of a particular population of AChE-containing area postrema neurons. Electron microscopic cytochemistry and X-ray microanalysis revealed the presence of calcium-containing electron-dense deposits in the mitochondria of degenerating area postrema neurons indicating the possible pathogenetic role of an enhanced intracellular calcium level in the mechanism of monosodium-l-glutamate-induced nerve cell degeneration. Degeneration of area postrema neurons was followed by the appearance of degenerating axon terminals in a well-defined region of the nucleus of the solitary tract, the area subpostrema. Degenerating area postrema neurons and axon terminals were rapidly engulfed by phagocytes predominantly of microglial character. AChE activity, localized to the basal lamina of the capillaries of the area subpostrema under normal conditions, could no longer be detected in rats treated with monosodium-l-glutamate 3–4 weeks previously.These findings provide evidence for the existence of a particular population of glutamate-sensitive, AChE-containing area postrema neurons which project and transport AChE to the nucleus of the solitary tract. This specific neuronal pathway connecting the area postrema with the nucleus of the solitary tract may play an important role in some of the functions attributed to the area postrema. The results also strengthen the hypothesis that brain capillary AChE activity may be of neuronal origin.

Inhibition of growth hormone and prolactin secretion by a serine proteinase inhibitor

The action of the tripeptide aldehyde t-butyloxycarbonyl-DPhe-Pro-Arg-H (boc-fPR-H), belonging to a family of serine proteinase inhibitors, on the release of immunoreactive prolactin (iPRL) and growth hormone (iGH) has been studied. In rat anterior pituitary cell cultures and pituitary quarters 1 mM boc-fPR-H inhibited basal iPRL and iGH release. Thyroliberin-induced iPRL release by cultured cells was also markedly inhibited with a concomitant accumulation of intra-cellular iPRL. During the short- and long-term exposure of cells to boc-fPR-H there no changes in total cell protein contents and in activities of some lysosomal marker enzymes. A wide scale of unchanged parameters characteristic for cellular metabolism indicated that the tripeptide aldehyde has no cytotoxic effect. The marked inhibition of basal as well as stimulated hormone release in the presence of the enzyme inhibitor might suggest that at least a portion of the hormones is released via a proteolytic enzyme-dependent process.

Intracellular distribution of arylsulphatase activity in adrenal cortical cells of the rat

SummaryArylsulphatase (B) activity has been demonstrated by means of an electronmicroscopic cytochemical method (substrate: p-nitrocatechol sulphate; capturing ion: barium, pH 5.5) in the lysosomes, in many Golgi elements, and occasionally in the endoplasmic reticulum of rat adrenal cortical cells.

Lysosomal enzyme activities in hypo- and hypersecretory anterior pituitary cells

SummaryThe involvement of lysosomes in ACTH and prolactin secretion was studied. Lysosomes were visualized in the anterior pituitary by their non-specific esterase (gold thioacetic acid technique) or acid phosphatase (Gomori technique) activity. Corticotrophs and mammotrophs were identified by postembedding immunocytochemistry for their respective hormones. Corticotrophs were rendered hypersecretory by bilateral adrenalectomy (7 or 12 days prior to examination), hyposecretory by dexamethasone administration. Prolactin secretion was enhanced by 17-beta-estradiol, prolactin release was inhibited by bromoergocriptine administration. Long-term hypersecretion of ACTH was accompanied by the presence of numerous autophagic vacuoles often containing secretory granules in the corticotrophs. Lysosomal enzyme-containing tubules and small lysosomes were abundant in the cytoplasm near the cell membrane, among the mature secretory granules. Feed-back inhibition of ACTH release by dexamethasone resulted in the extension of enzyme-containing tubules, continuous with cisternae and small lysosomes anywhere in the cytoplasm and in the appearance of numerous crinophagic vacuoles. A higher frequency of tubular lysosomes was described at the periphery of mammotrophs stimulated by 17-beta-estradiol. Bromoergocriptine caused a high incidence of characteristic crinophagic vacuoles in the prolactin cells. The concept of crinophagy has been extended to the corticotrophs. Morphological phenomena were attributed to the traffic and increased turnover of membranes, ligands and cytoplasmic organelles during stimulated secretion.

Microspectrophotometric data on the kinetics of acid phosphatase activity in cells removed from the peritoneal cavity of the rat

SynopsisThe extinctions (optical densities) of cells incubated for acid phosphatase in a histochemical azo-coupling procedure (naphthol AS-BI prosphate-hexazotized pararosaniline) have been measured microspectrophotometrically as a function of the incubation time and substrate concentration. A microcuvette was designed for the incubation. The amount of the reaction product in the cells at 23±1°C was found to be proportional to the incubation time for at least 40 min. Lineweaver-Burk plots were linear for some cells while in others nonlinear plots suggested the presence of two or more enzymes. This suggestion was supported by results obtained from polyacrylamide gel electrophoresis experiments.

Enzymic heterogeneity of adrenocortical lysosomes: an X-ray microanalytical study.

SummaryThe enzymic heterogeneity of the lysosomal system has been demonstrated earlier. This study was concerned whether or not the lysosomes of the two outer zones of the rat adrenal cortex reveal any population characteristics on the basis of the activity of two enzymes. A double incubation method was used for the simultaneous electron cytochemical demonstration of acid phosphatase and arylsulphatase activity in the zonae glomerulosa and fasciculata of the adrenal. Lysosomes in semi-thin (0.5μm) or ultra-thin sections were analysed with an ORTEC energy-dispersive X-ray microanalyser mounted on a JEOL TEMSCAN-100 C electron microscope.Linearity of the amount of reaction deposit with incubation time was found for both enzymes. On the basis of the proportion of the two reaction products in individual lysosomes, three populations were distinguished. One with high acid-phosphatase and low, if any, arylsulphatase activity was only present in the zona glomerulosa. The other two populations exhibited stronger or weaker prevalence of arylsulphatase activity and were common in both zones. The values of the Ba/Pb ratio characteristic of each population changed with the reaction sequence but the principal distribution pattern did not. The nature of the interaction between the two reactions, as well as the possible functional significance of the lysosomal populations in the adrenal cortex, are discussed but are not yet clarified.

Morphological evidence of lysosomal uptake of high-density lipoproteins by rat adrenocortical cells in vitro

The high-density lipoprotein (HDL) pathway in rat adrenocortical cells was studied at the electron microscopic level in vitro via colloidal gold labelling. Steroid hormone assays were performed to confirm that the cells remained intact, viable, responsive to ACTH under the applied conditions, and to reveal the steroidogenic effect of HDL. The gold-labelled HDL particles (HDL-Au) were observed on the surface of the parenchymal cells, often attached to the membranes of the microvilli, but rarely in coated pits and coated vesicles. HDL-Au was accumulated by non-coated vesicles, multivesicular bodies and lysosomes. The lysosomes were identified by means of a non-specific esterase reaction. It is concluded that HDL particles are internalized by both zona glomerulosa and zona fasciculata cells. HDL is required for the enhanced functional activity of these cells in long-term incubation, and the lysosomes are involved in the process.

Lysosomes in anterior pituitary corticotrophs of the rat

SummaryE600 resistent non-specific esterase activity or acid phosphatase activity were localized in corticotrophic cells identified by postembedding immunocytochemistry (PAP or protein A-immunogold techniques). The lysosomal system of this cell type consists of dense bodies, of a population of small lysosomes mostly situated at the cell periphery in the vicinity of secretory granules as well as of tubular structures. These latter were located either in the central part of the cytoplasm and probably belonged to the Golgi apparatus or at the cell periphery, partly in the extensions. Small lysosomes occurred to be in continuity with enzyme-containing tubules. In a few structures lysosomal enzyme activity and ACTH immunoreactivity overlapped. Some autophagic vacuoles seemed to contain secretory granule matrix. It is suggested that the concept of crinophagy can be extended to the corticotrophs, though the lysosomal system may be involved in the specific function of this cell type by other mechanisms as well.