Rossella Monteforte

D-Aspartate affects secretory activity in rat Harderian gland: molecular mechanism and functional significance.

In this paper, the role of d-aspartate in the rat Harderian gland (HG) was investigated by histochemical, ultrastructural, and biochemical analyses. In this gland, substantial amounts of endogenous d-Asp were detected, along with aspartate racemases that convert d-Asp to l-Asp and vice versa. We found that the gland was capable of uptaking and accumulating exogenously administered d-Asp. d-Asp acute treatment markedly increased lipid and porphyrin secretion and induced a powerful hyperaemia in inter-acinar interstitial tissue. Since d-Asp is known to be recognized by NMDA receptors, the expression of such receptors in rat HG led us to the hypothesis that d-Asp acute treatment induced the activation of the extracellular signal-regulated protein kinase (ERK) and nitric oxide synthase (NOS) pathways mediated by NMDA. Interestingly, as a result of enhanced oxidative stress due to increased porphyrin secretion, the revealed activation of the stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JNK) pro-apoptotic pathway was probably triggered by the gland itself to preserve its cellular integrity.

Mast cells in Wallerian degeneration: Morphologic and ultrastructural changes

The morphologic and ultrastructural changes of mast cells were followed in degenerating distal and regenerating proximal stumps of frog brachial nerve during Wallerian degeneration. Quantitative analysis included determination of both number and size of mast cells. The mast cell response to injury consisted of an early and a late phase. In the early phase, there was an increase in mast cell numbers in the proximal site of the lesion and a release of Alcian blue material consistent with mediator release. This phase of mast cell activation may be related, through the secretion of biogenic agents such as heparin and histamine, to the increase of endoneurial vessel size and vascular permeability, providing access for macrophages and mast cell precursors. The later phase, which peaked at 40 days after transection in the degenerating distal stump, consisted in the degranulation of the mast cells. These mast cells, closely associated with macrophages and degenerating Schwann cells, released secretory granules into the endoneurial microenvironment. These degranulating mast cells, through the released acid hydrolases, may contribute along with macrophages and Schwann cells, to the degradation of myelin debris. At the same time, mast cells appeared filled with granular content in the regenerating proximal segment. Therefore, we suggest that mast cells in peripheral nerves may play an important role in nerve degenerating and regenerating mechanisms through the secretion of diffusible molecules. J. Comp. Neurol. 445:199–210, 2002.

Morphological changes in frog mast cells induced by nerve stimulation in vivo

We investigated at both histochemical and ultrastructural levels the effects of unilateral electrical stimulation in vivo of the frog hypoglossal nerve on the mast cells (MCs) within the nerve fascicles and among the axon terminals. The right ventral root of the hypoglossal nerve in different experiments was stimulated respectively for 1, 3, 5, 10 min with over-threshold stimuli (10 Hz; 2 ms duration). The stimulations at 3, 5 and 10 min caused a progressive degranulation and histochemical and ultrastructural changes of the MCs at the stimulated side. The morphological changes consisted of the loss of Alcian Blue secretory content and of a progressive release of safranin+ secretory granules, depending upon duration of stimulation. The ultrastructural study showed that granules are discharged whole into the microenvironment or may release their content through exocytosis. A functional relationship between nerve and MCs is also suggested by the close anatomical association between MCs and pre-terminal axons observed following 10 min of hypoglossal stimulation. No changes in MC morphology occurred after 1 min of electrical stimulation. The results suggest that active cholinergic fibres can modulate MC secretion.

Thyroid Status Can Influence Brain Mast Cell Population

Neuroanatomical mapping as well as the influence of thyroid status on brain mast cell distribution and detectable mast cell number in adult Rana esculenta is studied. Treatment with tizoxin (T4) does not modify number or activational state of brain mast cells, whereas administration of the antithyroid agent 6‐n‐propyl‐2‐thiouracil induces a significant increase (up to 40%) in the mast cell number within the telencephalon and diencephalon. Hypophysectomy induces a significant decrease (up to 65%) of mast cells in all brain regions, whereas the pituitary homogenate augments their number. The results suggest that the pituitary–thyroid axis may be involved in the regulation of brain mast cell population.

Morphological and biochemical changes in the Harderian gland of hypothyroid rats.

SUMMARY The secretory activity of the Harderian gland (HG) is influenced by both exogenous (such as light and temperature) and endogenous (such as prolactin, thyroid hormones and steroid hormones) factors, which vary among species. In the present study, the effects of hypothyroidism on the rat HG were examined at morphological and biochemical levels. The decrease in cytoplasmic lipoproteic vacuoles and the increase in mucosubstance secretion in the acinar lumina were the most notable histological effects elicited by hypothyroidism. The release of all granules with nuclei and cellular debris suggested the occurrence of holocrine secretion. Electron microscopy revealed in the glandular cells of hypothyroid rat an increased condensation of chromatin in the nuclei, mitochondria with decreased cristae and vacuolisation, decreased glycogen granules, autophagic vacuoles, and lipofuscins in the cytoplasm. TUNEL reaction indicated DNA fragmentation in hypothyroid HG, indicative of an underlying apoptotic process. Translocation of cytochrome c from mitochondria to cytosol strongly supported this hypothesis. In conclusion, these findings indicate that thyroid hormones play a pivotal role in preserving the structural integrity of the rat HG and, hence, its secretory activity.

Dimorphic expression of uncoupling protein-3 in golden hamster harderian gland: Effects of castration and testosterone administration

Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell‐types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein‐3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II‐cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post‐castration (when the type II‐cell percentage had fallen from 50 to 5%), although testosterone was already near zero at 15 days (when neither the type II‐cell percentage nor the UCP3 level had fallen), and testosterone‐replacement therapy prevented these changes. Testosterone‐treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the glands morphological and lipid dimorphism. Straight‐chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched‐chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome‐rich fraction contained α‐methylacyl‐CoA racemase (AMACR), an enzyme important in the β‐oxidation of branched‐chain fatty acids. J. Cell. Physiol. 215: 481–487, 2008.

Mast cell population in the frog brain: distribution and influence of thyroid status.

SUMMARY In the developing frog brain, the majority of mast cells (MC) are distributed in the pia mater, and some immature MC are located adjacent to the blood capillaries in and around the neuropil. In the adult brain, MC are more numerous than in pre- and pro-metamorphic tadpoles; they are mainly located within the pia mater and are particularly numerous in the choroid plexuses. Many MC are found within the brain ventricles juxtaposed to the ependymal lining. MC are rarely observed in the brain parenchyma. In the adult brain, MC number is much higher than in the brain of post-metamorphic froglets. In the latter, MC number is nearly 2-fold over that found in the pre-metamorphic brain. Treatment of pre- and pro-metamorphic tadpoles with 3,5,3′-triiodothyronine (T3) and thyroxine (T4) stimulates overall larval development but does not induce a significant change in MC population within the brain. By contrast, treatment with 6-n-propyl-2-thiouracil (PTU) delays larval development and leads to a significant numerical increase of brain MC. In the adult, PTU treatment also has a similar effect whereas hypophysectomy causes a drastic decrease of MC population. The negative effects of hypophysectomy are successfully counteracted by a two-week replacement therapy with homologous pars distalis homogenate. In the adult frog, MC population seems to be refractory to thyroid hormone treatment. The present study on frog brain suggests that pituitary–thyroid axis may be involved in the regulation of MC frequency.

Mast cells in the amphibian brain during development

This is the first descriptive study of ontogenesis and anatomical distribution of mast cells in the developing brain of three different amphibian species. In the toad and the green frog, mast cells are preferentially located in: (i) the meningeal lining (pia mater), (ii) the choroid plexuses, both anterior and posterior, and (iii) the neuropil, in close association with the epithelial cell lining of blood vessels. It is only in the perennially aquatic African clawed frog that mast cells never appear inside brain ventricles and within the neuropil. Mast cells first become identifiable in brain of different species in different stages of development. While there are differences in the number of mast cells in different species at different stages of development, the number nearly doubles in all three species during the transition from pro‐metamorphic stage of larval development to the peak of metamorphic climax. Furthermore, the number of mast cells is comparatively higher in the toad and remarkably lower in the fully aquatic Xenopus laevis, in which species the first appearance of identifiable mast cells during larval development occurs much later than in equivalent stages of development of the toad and the green frog. The secretory nature of mast cells can be assumed by the presence of cytoplasmic granules, which may show species‐specific texture. Further experimental analyses are required to unveil the usefulness of mast cells in the amphibian brain.

D-aspartic acid induces merocrine secretion in the frog harderian gland

High levels of D-aspartic acid (D-Asp) have been found in the harderian gland (hg) of the frog,Rana esculenta. This is the first report of D-aspartate in an exocrine gland. D-aspartate content is correlated with secretory activity: it is high in July when the hg shows the highest secretory activity, lower in February, in concomitance with the low secretory activity. The harderian gland has the capacity to uptake D-Asp injected i.p. In July, gland uptake is higher than in February. Administration of 2.0 µmol/g D-Asp in frogs during both periods induces the release of secretory granules in the hg. This effect is more evident in July, when the amino acid accumulates at the apex of the cells beneath the plasma membrane. Such evidence suggests a role of D-Asp in the exocytotic mechanism.RiassuntoRana esculenta. Alti livelli di acido D-aspartico (D-Asp) sono presenti nella ghiandola di Harder diRana esculenta. Si tratta della prima dimostrazione della presenza di D-Asp in una ghiandola esocrina. La concentrazione dell’amminoacido varia in funzione dell’attività secretoria: è alta a luglio (alta attività secretoria), bassa a febbraio (bassa attività secretoria). La ghiandola ha la capacità di accumulare il D-Asp iniettato i.p. Sia a febbraio che a luglio la somministrazione di 2.0 µmol/g di D-Asp determina il rilas cio di granuli secretori dalla ghiandola. Questo effetto è più evidente a luglio, quando l’amminoacido si accumula all’apice cellulare al di sotto della membrana plasmatica. Queste osservazioni suggeriscono un ruolo del D-Asp nel meccanismo di secrezione merocrina.