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1.
Cell Tissue Res ; 379(1): 157-167, 2020 Jan.
Article in English | MEDLINE | ID: mdl-31673758

ABSTRACT

REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Adrenal Medulla/physiology , Secretory Vesicles/physiology , Adaptor Proteins, Signal Transducing/genetics , Adrenal Medulla/cytology , Adrenal Medulla/ultrastructure , Animals , Chromogranin A/metabolism , Down-Regulation , Female , In Situ Hybridization , Mice , Mice, Knockout , Neuropeptide Y/metabolism , RNA, Messenger , RNA-Seq , Secretory Vesicles/ultrastructure , Transcriptome
2.
Acta Histochem ; 120(4): 363-372, 2018 May.
Article in English | MEDLINE | ID: mdl-29628120

ABSTRACT

The adrenal medulla is crucial for the survival of species facing significant environmental changes. The parenchyma is composed mainly of chromaffin cells, ganglion cells and sustentacular cells (SC). The male viscacha exhibits seasonal variations of gonadal activity and other metabolic functions. The aim of this work was to investigate the influence of the reproductive conditions on the morphology of SC of this rodent. In addition, the effects of testosterone and melatonin on these cells were studied. Immunoexpression of S100 protein, GFAP and vimentin were analyzed. Furthermore, the distribution of adrenergic and noradrenergic chromaffin cells subpopulations was studied for the first time in this species. SC present long cytoplasmic processes in contact with chromaffin cells, probably generating an intraglandular communication network. Significant differences (p < 0.05) in the %IA (percentage of immunopositive area) for the S100 protein were observed according to winter (4.21 ±â€¯0.34) and summer (3.51 ±â€¯0.15) values. In castrated animals, the %IA (6.05 ±â€¯0.35) was significantly higher in relation to intact animals (3.95 ±â€¯0.40). In melatonin-treated animals the %IA (3.62 ±â€¯0.23) was significantly higher compared to control animals (2.65 ±â€¯0.26). GFAP immunoexpression was negative and no noradrenergic chromaffin cells were detected suggesting an adrenergic phenotype predominance. Vimentin was observed in SC, endothelial cells and connective tissue. Results indicate that SC exhibit variations along the annual reproductive cycle, along with castration and the melatonin administration. Our results suggest that in this rodent SC are not only support elements, but also participate in the modulation of the activity of the adrenal medulla; probably through paracrine effects.


Subject(s)
Adrenal Medulla/drug effects , Androgens/pharmacology , Melatonin/pharmacology , Reproduction/drug effects , Adrenal Medulla/ultrastructure , Animals , Immunohistochemistry , Male , Seasons
3.
Folia Histochem Cytobiol ; 55(2): 74-85, 2017.
Article in English | MEDLINE | ID: mdl-28691730

ABSTRACT

INTRODUCTION: In traditional medicine, Citrullus colocynthis is used to treat diabetes, hyperlipidemia, cardiovascular diseases, inflammation, and oxidative stress, all of which can appear when a diet rich in vegetable fats, such as palm oil, is continuously consumed. Such high-fat diets are chronic stressors of the hypothalamic-pituitary-adrenal axis. The objective of our study was to analyze and evaluate the effects of colocynth total alkaloids and glycosides on metabolic, hormonal, and structural disorders of the adrenal medulla in Wistar rats fed a high-fat diet. MATERIAL AND METHODS: Twenty six Wistar rats were distributed as follows: six control animals received a standard laboratory diet; twenty experimental rats received the standard laboratory diet supplemented with palm oil - the high-fat diet (HFD). After seven months of this diet, the HFD group was subdivided into rats treated for the next 2 months with either alkaloid extract (HFD-ALk group) or ethanol extract of glycosides (HFD-GLc) or animals on HFD only. Plasma metabolites and ACTH concentrations were measured by standard methods. Sections of adrenal medulla were stained by Heidenhain-Azan method and Sudan Black. RESULTS: The adrenal medulla of the HFD rats showed prominent structural changes, such as hypertrophy of chromaffin and ganglion cells, vacuolation, inflammatory foci, and fibrosis. The biochemical and hormonal parameters were significantly improved in the HFD rats treated with alkaloid and glycoside extracts of Citrullus colocynthis. Moreover, the morphological changes of the adrenal medulla were attenuated in HFD-ALk and HFD-Glc rats. CONCLUSIONS: The results of the study indicate that phytotherapy using Citrullus colocynthis alkaloids may correct metabolic and hormonal perturbations as well as adrenal medulla structure of rats maintained on HFD.


Subject(s)
Adrenal Medulla/drug effects , Alkaloids/pharmacology , Citrullus colocynthis/chemistry , Diet, High-Fat , Glycosides/pharmacology , Adrenal Medulla/ultrastructure , Adrenocorticotropic Hormone/blood , Animals , Glycosides/chemistry , Pituitary-Adrenal System/drug effects , Rats , Rats, Wistar
4.
Tissue Cell ; 49(2 Pt B): 285-295, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28320513

ABSTRACT

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances.


Subject(s)
Adrenal Glands/ultrastructure , Adrenal Medulla/ultrastructure , Mitochondria/ultrastructure , Adrenal Glands/anatomy & histology , Adrenal Medulla/anatomy & histology , Animals , Camelus/anatomy & histology , Endoplasmic Reticulum/ultrastructure , Female , Male , Microscopy, Electron
5.
Acta Histochem ; 119(1): 48-56, 2017 Jan.
Article in English | MEDLINE | ID: mdl-27889068

ABSTRACT

Diosgenin, a steroidal sapogenin of natural origin, has demonstrated benefits when it comes to the treatment of malignancies, cardiovascular issues and menopausal symptoms. In this study, we investigated the histological changes of the adrenal gland after diosgenin application in a rat model of the menopause. Middle-aged, acyclic female Wistar rats were divided into control (C; n=6) and diosgenin treated (D; n=6) groups. Diosgenin (100mg/kg b.w./day) was orally administered for four weeks, while C group received the vehicle alone. A histological approach included design-based stereology, histochemistry and immunohistochemistry. The adrenal cortex volume decreased in D females by 15% (p<0.05) while the volume of adrenal medulla increased (p<0.05) by 64%, compared to the same parameters in C group. Volume density of the zona glomerulosa (expressed per absolute adrenal gland volume) in D rats increased (p<0.05) by 22% in comparison with C animals. Diosgenin treatment decreased (p<0.05) the volume density of the zona fasciculata (expressed per volume of adrenal cortex) by 15% when compared to C females. Absolute volume of the zona reticularis in D group decreased (p<0.05) by 38% in comparison with the same parameter in C rats. Also, after diosgenin application, the volume density of the zona reticularis (expressed per volume of adrenal cortex) and the zona reticularis cell volume were decreased by 51% and 20% (p<0.05) respectively, compared to C animals. Our results, reflecting a decrease in many stereological parameters of the adrenal cortex, indicate that diosgenin took over the role of corticosteroid precursors and became incorporated into steroidogenesis.


Subject(s)
Adrenal Cortex/drug effects , Adrenal Medulla/drug effects , Diosgenin/pharmacology , Menopause/drug effects , Steroids/pharmacology , Administration, Oral , Adrenal Cortex/ultrastructure , Adrenal Medulla/ultrastructure , Animals , Female , Humans , Menopause/physiology , Models, Animal , Rats , Rats, Wistar
6.
Anat Rec (Hoboken) ; 296(7): 1089-95, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23630194

ABSTRACT

Animals living in nontropical climates modify their physiology and behavior to adapt to seasonal environmental changes. Part of this adaptation involves the release of catecholamine from sympathetic nerve endings and the adrenal medulla, which play a major role in regulating energy balance. The aim of this work was to investigate whether adult male viscachas in their natural habitat exhibits structural changes in the adrenal medulla during the annual seasonal cycle. In August-September, chromaffin granules revealed ultrastructural changes suggestive of piecemeal degranulation. Quantitative morphometric analysis by transmission electron microscopy showed a significantly lower percentage of resting chromaffin granules and a higher percentage of altered granules and empty containers in August-September (late winter) compared to February-March (late summer), suggesting an increased secretory process of catecholamines in August-September. The mechanism of piecemeal degranulation might amplify this process, encouraging the adaptive response to winter environmental conditions. Tissue levels of epinephrine, norepinephrine, and dopamine (analyzed by high-performance liquid chromatography) changed throughout the year, reaching maximum values in February-March and minimum values in August-September. These results demonstrate morphological and biochemical seasonal variations of the adrenal medulla, suggesting that epinephrine might promote energy mobilization, which allow the Lagostomus to cope with adverse environmental conditions and thus to survive during winter season.


Subject(s)
Adrenal Medulla/metabolism , Catecholamines/metabolism , Chromaffin Granules/metabolism , Rodentia/metabolism , Seasons , Adaptation, Physiological , Adrenal Medulla/ultrastructure , Animals , Cell Degranulation , Chromaffin Granules/ultrastructure , Chromatography, High Pressure Liquid , Dopamine/metabolism , Energy Metabolism , Epinephrine/metabolism , Male , Microscopy, Electron, Transmission , Norepinephrine/metabolism , Rain , Sunlight , Temperature , Time Factors
7.
Ann Diagn Pathol ; 17(1): 123-6, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22056038

ABSTRACT

We report an unprecedented case of an oncocytoma of the adrenal gland medulla in a 61-year-old woman. The patient presented with right flank pain and hematuria. Computed tomographic studies revealed a right adrenal gland mass that measured 2 cm, which was subsequently excised laparoscopically. Grossly, the tumor in the medulla measured 1.9 × 1.2 cm, weighed 5 g, and had a solid tan-brown cut surface. Histologically, it consisted of large tumor cells containing eosinophilic granular cytoplasm arranged in trabecular and nodular patterns. Electron microscopy revealed closely packed mitochondria in the cytoplasm of almost all tumor cells. The tumor cells were immunohistochemically positive for vimentin. The patient resumed usual activities 2 weeks after surgery, and at 6-month follow-up, she is doing well.


Subject(s)
Adenoma, Oxyphilic/diagnostic imaging , Adenoma, Oxyphilic/pathology , Adrenal Gland Neoplasms/diagnostic imaging , Adrenal Gland Neoplasms/pathology , Adenoma, Oxyphilic/surgery , Adrenal Gland Neoplasms/surgery , Adrenal Medulla/metabolism , Adrenal Medulla/pathology , Adrenal Medulla/ultrastructure , Female , Four-Dimensional Computed Tomography , Humans , Laparoscopy , Middle Aged , Mitochondria/pathology , Treatment Outcome , Vimentin/metabolism
8.
Genet Mol Res ; 11(3): 2780-9, 2012 Aug 24.
Article in English | MEDLINE | ID: mdl-23007973

ABSTRACT

We examined the underlying neural-endocrine mechanisms of asthma associated with respiratory syncytial virus infection. Thirty Sprague-Dawley rats were randomly divided into control group, respiratory syncytial virus (RSV) group, and anti-nerve growth factor (NGF) IgG group. An RSV infection model was established by nasal drip once a week. In the anti-NGF antibody intervention group, each rat was given an intraperitoneal injection of anti-NGF IgG 3 h before RSV infection. Optical microscopy and transmission electron microscopy were used to observe the structural changes in adrenal medulla cells. Changes in adrenaline and norepinephrine in serum were detected by ELISA. NGF expression was assayed by immunohistochemistry. Expression differences in synaptophysin mRNA were detected by RT-PCR. Transmission electron microscopy displayed widened adrenal medulla intercellular spaces, reduced chromaffin particle concentration, and increased mitochondria in the RSV infection group. At the same time, NGF expression was increased in the RSV infection group significantly. In addition, the adrenaline concentration was significantly decreased compared with the control and anti-NGF antibody groups. Synaptophysin mRNA expression was significantly increased in the RSV infection and anti-NGF antibody groups. However, compared with the RSV infection group, synaptophysin mRNA expression was significantly decreased in the anti-NGF antibody group. We conclude that RSV infection could induce adrenal medulla cell differentiation to nerve cells by over-expression of NGF, resulting in the decreased endocrine function found in asthma progression.


Subject(s)
Asthma/complications , Asthma/virology , Endocrine System/metabolism , Nervous System/metabolism , Respiratory Syncytial Virus Infections/complications , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/physiology , Adrenal Medulla/pathology , Adrenal Medulla/ultrastructure , Animals , Asthma/physiopathology , Bronchial Hyperreactivity/complications , Bronchial Hyperreactivity/physiopathology , Bronchial Hyperreactivity/virology , Disease Models, Animal , Epinephrine/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Immunohistochemistry , In Situ Hybridization , Lung/metabolism , Lung/pathology , Lung/virology , Nerve Growth Factor/metabolism , Norepinephrine/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Respiratory Syncytial Virus Infections/physiopathology , Reverse Transcriptase Polymerase Chain Reaction , Synaptophysin/genetics , Synaptophysin/metabolism
9.
Acta Histochem ; 113(1): 24-31, 2011 Jan.
Article in English | MEDLINE | ID: mdl-19767061

ABSTRACT

Mitochondria are organelles that produce ATP and reactive oxygen species, which are thought to be responsible for a decline in physiological function with aging. In this study, we morphologically and biochemically examined mitochondria in the rat adrenal gland. Immunohistochemistry showed that the rank order for intensity of immunolabelling for complex IV was zona reticularis > zona fasciculata >> adrenal medulla, whereas for complex V α and ß subunits, it was zona fasciculata > zona reticularis and adrenal medulla. The immunolabelling for complex I was homogeneous in the adrenal gland. The difference in immunolabelling between complexes I and IV indicates that the ratio of levels of complex I to that of complex IV in the zona reticularis was smaller than that in the zona fasciculata and the adrenal medulla. Electron microscopy revealed that aging rats had zona reticularis cells with many lysosomes and irregular nuclei. The result suggests that the level of proteins involved in oxidative phosphorylation is coordinated within the complex, but differs between the complexes. This might be responsible for degeneration of zona reticularis cells with aging.


Subject(s)
Adrenal Medulla/ultrastructure , Aging , Cell Nucleus/ultrastructure , Cytoplasm/ultrastructure , Mitochondria/ultrastructure , Mitochondrial Proteins , Multienzyme Complexes/metabolism , Oxidative Phosphorylation , Zona Fasciculata/ultrastructure , Zona Reticularis/ultrastructure , Adrenal Medulla/enzymology , Animals , Cell Nucleus/enzymology , Cytoplasm/enzymology , Immunohistochemistry , Male , Microscopy, Electron , Mitochondria/enzymology , Mitochondrial Proteins/metabolism , Rats , Rats, Wistar , Zona Fasciculata/enzymology , Zona Reticularis/enzymology
10.
Chin Med J (Engl) ; 123(10): 1333-7, 2010 May 20.
Article in English | MEDLINE | ID: mdl-20529591

ABSTRACT

BACKGROUND: Airway symptoms in asthma are related to decrease of epinephrine secretion, which may be ascribed to elevated nerve growth factor (NGF) in the organism. The aim of this study was to monitor the neuroendocrine alteration in the adrenal medulla of asthmatic rats. METHODS: Sixteen rats were randomly divided into two groups (n = 8), control group and asthma group, and the asthmatic rats were sensitized and challenged with ovalbumin (OVA). The levels of NGF, epinephrine and norepinephrine in serum were detected by enzyme linked immunosorbent assay (ELISA), the NGF expression in adrenal medulla was detected by immunohistochemistry, and the changes in the ultrastructure of the adrenal medulla was observed by electron microscopy. RESULTS: The NGF expression was increased in asthmatic rats compared with control rats. Compared with control rats, the results indicated that the epinephrine level was decreased in asthmatic rats, but no significant difference was found in norepinephrine levels. We found more ganglion cells in the adrenal medulla of asthmatic rats than in control rats, with NGF immunostaining mainly located in these ganglion cells. Electron microscopic images showed the density of chromaffin granula decreased and there was shrunken nucleolemma in the adrenal medullary cells of asthmatic rats. CONCLUSION: The innervation of the adrenal medulla is changed in asthmatic rats, and it may contribute to the epinephrine decrease in asthma.


Subject(s)
Adrenal Medulla/metabolism , Asthma/blood , Asthma/metabolism , Adrenal Medulla/ultrastructure , Animals , Bronchoalveolar Lavage , Enzyme-Linked Immunosorbent Assay , Epinephrine/blood , Male , Microscopy, Electron, Transmission , Nerve Growth Factor/blood , Nerve Growth Factor/metabolism , Norepinephrine/blood , Random Allocation , Rats , Rats, Sprague-Dawley
11.
Neuroendocrinology ; 89(2): 210-6, 2009.
Article in English | MEDLINE | ID: mdl-18840998

ABSTRACT

BACKGROUND/AIMS: The endopin serpin protease inhibitors have been identified by molecular studies as components of secretory vesicles that produce neuropeptides. Endopin 1 inhibits trypsin-like serine proteases, and endopin 2 inhibits cathepsin L that produces neuropeptides in secretory vesicles. To assess the secretory vesicle and neuroendocrine tissue distribution of these endopins, the goal of this study was to define specific antisera for each endopin isoform and to examine their localization with neuropeptides and in neuroendocrine tissues. METHODS: This study utilized methods consisting of Western blots, immunoelectron microscopy, and immunofluorescence microscopy for evaluation of the localization of endopin protease inhibitors in neuroendocrine tissues. RESULTS: Immunoelectron microscopy with these selective antisera demonstrated the localization of endopins 1 and 2 within secretory vesicles of adrenal medulla (bovine). Cellular immunofluorescence confocal microscopy illustrated the high level of colocalization of endopins 1 and 2 with enkephalin and NPY neuropeptides that are present in secretory vesicles of adrenal medullary chromaffin cells in primary culture. Tissue distribution studies (by Western blots) showed the expression of endopins 1 and 2 in bovine brain, pituitary, adrenal medulla, and other neuroendocrine tissues. CONCLUSIONS: These results implicate endopins 1 and 2 as endogenous protease inhibitors in neuropeptide-containing secretory vesicles and neuroendocrine tissues.


Subject(s)
Adrenal Medulla/chemistry , Neuroendocrine Cells/chemistry , Neuropeptides/analysis , Neurosecretory Systems/chemistry , Protease Inhibitors/analysis , Secretory Vesicles/chemistry , Serpins/analysis , Adrenal Medulla/ultrastructure , Animals , Cattle , Neuroendocrine Cells/ultrastructure , Secretory Vesicles/ultrastructure , Tissue Distribution
12.
Arch Histol Cytol ; 71(2): 115-22, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18974603

ABSTRACT

Effects of sham-pinealectomy and pinealectomy on preganglionic nerve endings on adrenomedullary adrenaline cells were investigated electron microscopically. Adult male golden hamsters from the normal, sham-pinealectomy and pinealectomy groups maintained under 24 h light-dark cycle and constant temperature were used at 28 days after surgery. From conventional electron microscopic specimens, montage photographs made of the adrenaline cell region at a magnification of x 11,000 were used for qualitative and quantitative electron microscopic analyses in 14 animals in each experimental group. The preganglionic nerve endings were localized mainly in the following three sites: the basal lamina part, the follicular lumen-junctional intercellular part, and the adrenaline cell-invaginated part. In the latter two parts, nerve endings and fibers had no envelope frequently, and in the former two parts, nerve endings sometimes showed the invagination complex. The frequency of nerve endings was highest in the follicular lumen-intercellular part, next highest in the basal lamina part and lowest in the A cell-invaginated part. The frequency of nerve endings in the basal lamina part was lower in the pinealectomy group than in the sham-pinealectomy group (P < 0.021), and those in the other two parts showed opposite changes, more evidently in the A cell-invaginated part. Nerve ending profiles in the adrenaline cell-invaginated part--which displayed a more rounded shape--increased in size in the pinealectomy group (longer diameter: P < 0.04; shorter diameter: P < 0.05). In conclusion, preganglionic nerve endings in the adrenal medulla of the golden hamster show differential morphological changes following PX depending on the intracellular part of A cells.


Subject(s)
Adrenal Medulla/innervation , Adrenal Medulla/ultrastructure , Epinephrine/metabolism , Nerve Endings/ultrastructure , Pineal Gland/surgery , Adrenal Medulla/metabolism , Animals , Chromaffin System/ultrastructure , Cricetinae , Male , Mesocricetus , Models, Biological
13.
Pak J Biol Sci ; 11(7): 1032-6, 2008 Apr 01.
Article in English | MEDLINE | ID: mdl-18810974

ABSTRACT

In C. auratus the adrenal chramaffin tissue is situated around the posterior cardinal veins, in the head kidney. Chromaffin tissue consists of two types of cells containing secretory granules, adrenaline and nor adrenaline cells. The cells produced catecholamine hormones. Adrenaline cell contains electron-lucent granules, whereas nor adrenaline cells possesses electron-dense granules. Cholinergic fibers embedded in the head kidney innervated the chromaffin cell. Two types of secretory structures, synaptic vesicles and secretory granules are found within the presynaptic terminal. Secretory granules discharge their contests, as neuropeptide in non synaptic area of nerve terminal by exocytosis, whereas synaptic vesicles discharge their contents as neurotransmitters at the synaptic thickening (active zone) in the presynaptic terminal by exocytosis.


Subject(s)
Adrenal Medulla/cytology , Adrenal Medulla/metabolism , Chromaffin System/cytology , Chromaffin System/innervation , Goldfish/metabolism , Adrenal Medulla/ultrastructure , Animals , Chromaffin Cells/metabolism , Chromaffin Cells/ultrastructure , Chromaffin System/metabolism , Chromaffin System/ultrastructure , Cytoplasmic Granules/ultrastructure , Epinephrine/metabolism , Exocytosis , Norepinephrine/metabolism , Secretory Vesicles/metabolism , Secretory Vesicles/ultrastructure , Synapses/ultrastructure , Synaptic Vesicles/metabolism , Synaptic Vesicles/ultrastructure
14.
J Neurosci ; 28(26): 6616-26, 2008 Jun 25.
Article in English | MEDLINE | ID: mdl-18579734

ABSTRACT

An increase in circulating catecholamine levels represents one of the mechanisms whereby organisms cope with stress. In the periphery, catecholamines mainly originate from the sympathoadrenal system. As we reported, in addition to the central control through cholinergic innervation, a local gap junction-delineated route between adrenal chromaffin cells contributes to catecholamine exocytosis. Here, we investigated whether this intercellular communication is modified when the hormonal demand is increased as observed during cold stress. Our results show that in cold exposed rats, gap-junctional communication undergoes a functional plasticity, as evidenced by an increased number of dye-coupled cells. Of a physiological interest is that this upregulation of gap-junctional coupling results in the appearance of a robust electrical coupling between chromaffin cells that allows the transmission of action potentials between coupled cells. This enhancement of gap-junctional communication parallels an increase in expression levels of connexin36 (Cx36) and connexin43 (Cx43) proteins. Both transcriptional and posttranslational mechanisms are involved because Cx36 transcripts are increased in stressed rats and the expression of the scaffolding protein zonula occludens-1, known to interact with both Cx36 and Cx43, is also upregulated. Consistent with an upregulated coupling extent in stressed rats, the cytosolic Ca(2+) concentration rises triggered in a single cell by an iontophoretic application of nicotine occur simultaneously in several neighboring cells. These results describe for the first time a functional plasticity of junctional coupling between adult chromaffin cells that should be crucial for adaptation to stress or sensitization to subsequent stressors.


Subject(s)
Adrenal Medulla/metabolism , Catecholamines/metabolism , Cell Communication/physiology , Chromaffin Cells/metabolism , Gap Junctions/metabolism , Stress, Psychological/metabolism , Action Potentials/physiology , Adrenal Medulla/ultrastructure , Animals , Calcium/metabolism , Calcium Signaling/physiology , Chromaffin Cells/ultrastructure , Cold Temperature/adverse effects , Connexin 43/genetics , Connexin 43/metabolism , Connexins/genetics , Connexins/metabolism , Gap Junctions/ultrastructure , Male , Membrane Potentials/physiology , Membrane Proteins/genetics , Membrane Proteins/metabolism , Neuronal Plasticity/physiology , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Processing, Post-Translational/physiology , Rats , Rats, Wistar , Stress, Psychological/physiopathology , Up-Regulation/physiology , Zonula Occludens-1 Protein , Gap Junction delta-2 Protein
15.
J Neurochem ; 106(1): 384-91, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18410501

ABSTRACT

Neuropeptide Y (NPY) functions as a peptide neurotransmitter and as a neuroendocrine hormone. The active NPY peptide is generated in secretory vesicles by proteolytic processing of proNPY. Novel findings from this study show that cathepsin L participates as a key proteolytic enzyme for NPY production in secretory vesicles. Notably, NPY levels in cathepsin L knockout (KO) mice were substantially reduced in brain and adrenal medulla by 80% and 90%, respectively. Participation of cathepsin L in producing NPY predicts their colocalization in secretory vesicles, a primary site of NPY production. Indeed, cathepsin L was colocalized with NPY in brain cortical neurons and in chromaffin cells of adrenal medulla, demonstrated by immunofluorescence confocal microscopy. Immunoelectron microscopy confirmed the localization of cathepsin L with NPY in regulated secretory vesicles of chromaffin cells. Functional studies showed that coexpression of proNPY with cathepsin L in neuroendocrine PC12 cells resulted in increased production of NPY. Furthermore, in vitro processing indicated cathepsin L processing of proNPY at paired basic residues. These findings demonstrate a role for cathepsin L in the production of NPY from its proNPY precursor. These studies illustrate the novel biological role of cathepsin L in the production of NPY, a peptide neurotransmitter, and neuroendocrine hormone.


Subject(s)
Adrenal Medulla/enzymology , Brain/enzymology , Cathepsins/genetics , Chromaffin Cells/enzymology , Cysteine Endopeptidases/genetics , Neuropeptide Y/biosynthesis , Secretory Vesicles/enzymology , Adrenal Medulla/ultrastructure , Amino Acid Sequence/physiology , Animals , Brain/ultrastructure , Cathepsin L , Cathepsins/physiology , Cells, Cultured , Chromaffin Cells/ultrastructure , Cysteine Endopeptidases/physiology , Fluorescent Antibody Technique , Gene Expression Regulation, Enzymologic/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Confocal , Microscopy, Immunoelectron , Neuropeptide Y/metabolism , Neurosecretory Systems/enzymology , Neurosecretory Systems/ultrastructure , PC12 Cells , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Rats , Secretory Vesicles/metabolism , Secretory Vesicles/ultrastructure
16.
Arch Histol Cytol ; 70(3): 147-61, 2007 Oct.
Article in English | MEDLINE | ID: mdl-18079584

ABSTRACT

Ultrastructural features of adrenomedullary adrenaline (A) cells in golden hamsters and rats were investigated qualitatively and quantitatively with special reference to the Golgi apparatus. The A cells displayed a characteristic follicular arrangement, with each cell showing structural polarity in hamsters, but not apparently in rats. In hamsters, the Golgi apparatus of A cells was larger (t-test: P<0.001) and more frequently showed large and complexly organized structures (chi(2)-test: P<0.005) compared with that of rats. Quantitative analysis of the Golgi apparatus revealed differences in the size and numerical density of Golgi vesicles in relation to the animal species and region. Two-way analysis of variance (ANOVA) confirmed species difference in the size of coated vesicles (P<0.005) and interaction between species and region concerning the size of smooth-clear vesicles (P<0.01) and numerical density of granular vesicles (P<0.05). One-way ANOVA revealed regional differences in the size and numerical density of smooth-clear vesicles in rats and hamsters (P<0.01 approximately 0.001), and in the numerical density of coated vesicles in hamsters (P<0.05). Data were further analyzed by Tukey-Kramer's method. These and other reported results suggest that, in hamster A cells, the Golgi apparatus has different structural, molecular, and functional mechanisms which are at least partly related to the distinct cellular polarity and higher concentration of peptide hormones in secretory vesicles, and that in rat A cells, in contrast, loading secretory vesicles with A during the post-Golgi stage is predominant. In conclusion, the Golgi apparatus in hamster A cells shows markedly different ultrastructural features compared with that in rat A cells.


Subject(s)
Adrenal Medulla/cytology , Adrenal Medulla/ultrastructure , Golgi Apparatus/ultrastructure , Animals , Cricetinae , Male , Mesocricetus , Microscopy, Electron , Rats
17.
J Neurochem ; 102(3): 944-56, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17630986

ABSTRACT

Bioactive lysophospholipids (LPLs) are released by blood cells and can modulate many cellular activities such as angiogenesis and cell survival. In this study, the effects of sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) on excitability and exocytosis in bovine chromaffin cells were investigated using the whole-cell configuration of the patch-clamp. Voltage-gated Ca(2+) current was inhibited by S1P and LPA pre-treatment in a concentration-dependent manner with IC(50)s of 0.46 and 0.79 mumol/L, respectively. Inhibition was mostly reversible upon washout and prevented by suramin, an inhibitor of G-protein signaling. Na(+) current was inhibited by S1P, but not by LPA. However, recovery of Na(+) channels from inactivation was slowed by both LPLs. The outward K(+) current was also significantly reduced by both LPLs. Chromaffin cells fired repetitive action potentials in response to minimal injections of depolarizing current. Repetitive activity was dramatically reduced by LPLs. Consistent with the reduction in Ca(2+) current, exocytosis elicited by a train of depolarizations and the ensuing endocytosis were both inhibited by LPL pre-treatments. These data demonstrate the interaction between immune and endocrine systems mediated by the inhibitory effects of LPLs on the excitability of adrenal chromaffin cells.


Subject(s)
Adrenal Medulla/metabolism , Cell Membrane/metabolism , Chromaffin Cells/metabolism , Lysophospholipids/metabolism , Action Potentials/drug effects , Action Potentials/physiology , Adrenal Medulla/ultrastructure , Animals , Cattle , Cell Membrane/drug effects , Cells, Cultured , Chromaffin Cells/drug effects , Dose-Response Relationship, Drug , Electric Stimulation , Endocytosis/drug effects , Endocytosis/physiology , Exocytosis/drug effects , Exocytosis/physiology , Immunologic Factors/metabolism , Immunologic Factors/pharmacology , Lysophospholipids/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Neurosecretory Systems/drug effects , Neurosecretory Systems/metabolism , Patch-Clamp Techniques , Potassium Channels/drug effects , Potassium Channels/metabolism , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/metabolism , Sodium Channels/drug effects , Sodium Channels/metabolism , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine/pharmacology , Suramin/pharmacology
18.
J Neurochem ; 100(5): 1340-50, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17241125

ABSTRACT

Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.


Subject(s)
Aminopeptidases/physiology , Chromaffin Granules/metabolism , Enkephalins/metabolism , Neuropeptide Y/metabolism , Secretory Vesicles/metabolism , Adrenal Medulla/ultrastructure , Amino Acid Sequence , Amino Acids/chemistry , Aminopeptidases/chemistry , Aminopeptidases/genetics , Animals , Base Sequence , Cathepsin L , Cathepsins/metabolism , Cattle , Chromaffin Granules/enzymology , Cloning, Molecular , Cysteine Endopeptidases/metabolism , Hydrogen-Ion Concentration , In Vitro Techniques , Kinetics , Molecular Sequence Data , Pituitary Gland/ultrastructure , Protease Inhibitors/pharmacology , Rats , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Secretory Vesicles/enzymology , Substrate Specificity
19.
Brain Res ; 1129(1): 174-90, 2007 Jan 19.
Article in English | MEDLINE | ID: mdl-17157274

ABSTRACT

The PC12 cell line is commonly used as a tool to understand the biochemical mechanisms underlying the physiology and degeneration of central dopamine neurons. Despite the broad use of this cell line, there are a number of points differing between PC12 cells and dopamine neurons in vivo which are missed out when translating in vitro data into in vivo systems. This led us to compare the PC12 cells with central dopamine neurons, aiming at those features which are predictors of in vivo physiology and degeneration of central dopamine neurons. We carried out this comparison, either in baseline conditions, following releasing or neurotoxic stimuli (i.e. acute or chronic methamphetamine), to end up with therapeutic agents which are suspected to produce neurotoxicity (l-DOPA). Although the neurotransmitter pattern of PC12 cells is close to dopamine neurons, ultrastructural morphometry demonstrates that, in baseline conditions, PC12 cells possess very low vesicles density, which parallels low catecholamine levels. Again, compartmentalization of secretory elements in PC12 cells is already pronounced in baseline conditions, while it is only slightly affected following catecholamine-releasing stimuli. This low flexibility is caused by the low ability of PC12 cells to compensate for sustained catecholamine release, due both to non-sufficient dopamine synthesis and poor dopamine storage mechanisms. This contrasts markedly with dopamine-containing neurons in vivo lending substance to opposite findings between these compartments concerning the sensitivity to a number of neurotoxins.


Subject(s)
Dopamine/metabolism , Mesencephalon/metabolism , Neurons/metabolism , Adrenal Medulla/metabolism , Adrenal Medulla/ultrastructure , Animals , Catecholamines/metabolism , Cell Compartmentation/drug effects , Cell Compartmentation/physiology , Chromaffin Cells/drug effects , Chromaffin Cells/metabolism , Chromaffin Cells/ultrastructure , Dopamine Agents/toxicity , Dopamine Uptake Inhibitors/toxicity , Immunohistochemistry , Levodopa/toxicity , Male , Mesencephalon/ultrastructure , Methamphetamine/toxicity , Microscopy, Electron, Transmission , Neurons/drug effects , Neurons/ultrastructure , Neurotoxins/toxicity , PC12 Cells , Presynaptic Terminals/metabolism , Presynaptic Terminals/ultrastructure , Rats , Rats, Sprague-Dawley , Secretory Vesicles/drug effects , Secretory Vesicles/metabolism , Secretory Vesicles/ultrastructure , Synaptic Transmission/drug effects , Synaptic Transmission/physiology
20.
Morfologiia ; 129(3): 59-62, 2006.
Article in Russian | MEDLINE | ID: mdl-17111663

ABSTRACT

Using light, electron microscopy and cytochemistry, the early (embryonic week 4-8) stages of adrenal gland (AG) development were studied in domestic pig. The interrelations between the cells of the fetal cortex (FC) and chromaffin cells (CC) were traced. At week 5, AG primordium is represented by FC, which consists of the epithelioid cells, with the ingrowing neural cords containing CC islets. Starting at the early embryonic period and up to fetal period, CC and interrenal cells of FC are closely interrelated with each other and sinusoidal capillaries. Both cellular types are at different stages of differentiation, including the functionally active elements. At weeks 7-8, FC cells undergo involution, while those ones, left at periphery, form definitive cortex. CC are located in the central part of the organ and form suprarenal tissue. Authors hypothesize, that CC, migrating into AG primordium, initially induce the development of interrenal primordium, and later cause the involution of FC. This, possibly, may be explained by the fact that further antenatal and postnatal development of the organism requires more corticosteroids than the amount produced by FC.


Subject(s)
Adrenal Glands/ultrastructure , Sus scrofa/anatomy & histology , Adrenal Cortex/embryology , Adrenal Cortex/ultrastructure , Adrenal Glands/embryology , Adrenal Medulla/embryology , Adrenal Medulla/ultrastructure , Animals , Chromaffin Cells/ultrastructure , Epithelial Cells/ultrastructure , Sus scrofa/embryology
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