Accumulation Of Secretory Granules Research Articles

Loss of CAPS2/Cadps2 leads to exocrine pancreatic cell injury and intracellular accumulation of secretory granules in mice.

The type 2 Ca2+-dependent activator protein for secretion (CAPS2/CADPS2) regulates dense-core vesicle trafficking and exocytosis and is involved in the regulated release of catecholamines, peptidergic hormones, and neuromodulators. CAPS2 is expressed in the pancreatic exocrine acinar cells that produce and secrete digestive enzymes. However, the functional role of CAPS2 in vesicular trafficking and/or exocytosis of non-regulatory proteins in the exocrine pancreas remains to be determined. Here, we analyzed the morpho-pathological indicators of the pancreatic exocrine pathway in Cadps2-deficient mouse models using histochemistry, biochemistry, and electron microscopy. We used whole exosome sequencing to identify CADPS2 variants in patients with chronic pancreatitis (CP). Caps2/Cadps2-knockout (KO) mice exhibited morphophysiological abnormalities in the exocrine pancreas, including excessive accumulation of secretory granules (zymogen granules) and their amylase content in the cytoplasm, deterioration of the fine intracellular membrane structures (disorganized rough endoplasmic reticulum, dilated Golgi cisternae, and the appearance of empty vesicles and autophagic-like vacuoles), as well as exocrine pancreatic cell injury, including acinar cell atrophy, increased fibrosis, and inflammatory cell infiltration. Pancreas-specific Cadps2 conditional KO mice exhibited pathological abnormalities in the exocrine pancreas similar to the global Cadps2 KO mice, indicating that these phenotypes were caused either directly or indirectly by CAPS2 deficiency in the pancreas. Furthermore, we identified a rare variant in the exon3 coding region of CADPS2 in a non-alcoholic patient with CP and showed that Cadps2-dex3 mice lacking CAPS2 exon3 exhibited symptoms similar to those exhibited by the Cadps2 KO and cKO mice. These results suggest that CAPS2 is critical for the proper functioning of the pancreatic exocrine pathway, and its deficiency is associated with a risk of pancreatic acinar cell pathology.

Hypothalamic remodeling of thyroid hormone signaling during hibernation in the arctic ground squirrel

Hibernation involves prolonged intervals of profound metabolic suppression periodically interrupted by brief arousals to euthermy, the function of which is unknown. Annual cycles in mammals are timed by a photoperiodically-regulated thyroid-hormone-dependent mechanism in hypothalamic tanycytes, driven by thyrotropin (TSH) in the pars tuberalis (PT), which regulates local TH-converting deiodinases and triggers remodeling of neuroendocrine pathways. We demonstrate that over the course of hibernation in continuous darkness, arctic ground squirrels (Urocitellus parryii) up-regulate the retrograde TSH/Deiodinase/TH pathway, remodel hypothalamic tanycytes, and activate the reproductive axis. Forcing the premature termination of hibernation by warming animals induced hypothalamic deiodinase expression and the accumulation of secretory granules in PT thyrotrophs and pituitary gonadotrophs, but did not further activate the reproductive axis. We suggest that periodic arousals may allow for the transient activation of hypothalamic thyroid hormone signaling, cellular remodeling, and re-programming of brain circuits in preparation for the short Arctic summer.

Ультраструктурні зміни пінеальних клітин епіфіза за умов тривалої дії нітратів питної води та засобів корекції

According to modern data, the epiphyse (pineal gland) is an organ that combines the processes of adaptogenesis and immunogenesis, takes part in triggering stress reactions and determines the sequence of disorders in the body at different stages of stress development. Researchers consider the pineal gland to be the most important organizer of biological rhythms associated with photoperiodism and the organ that determines the stereotype of the organism. Its individual functions are rhythmically variable under the influence of the environment and age. The purpose of the study was to study the structural and functional changes of pineal cells of the pineal gland in rats at different stages of normal development, under the action of nitrates and the simultaneous action of nitrates and methylene blue. Materials and methods. In accordance with the purpose of the work, the study was carried out on 90 nonlinear white male rats of different ages. The animals were kept in the vivarium in equivalent conditions. Long-term exposure to nitrates on the body of animals was achieved by daily introduction into the drinking ration, starting from the 7th day of postnatal development of rats (after preliminary water purification), 120 mg/l sodium nitrate, that is, in a dose that is typical for many regions of Ukraine. When simulating the action of methylene blue, this substance was daily orally administered to the animals in doses: 0.1-0.15 ml of a 1% aqueous solution per 1 kg of body weight. Results and discussion. As a result of the 7-day action of nitrates in 14-day-old rats, structural changes were observed in the pineal gland, which corresponded to a decrease in the function of light cells and an increase in the functional activity of type II pinealocytes. The ultrastructure of the cytoplasm of type I pinealocytes contained poorly developed organelles and single secretory granules. In 45-day-old animals exposed to nitrates in light pinealocytes, pronounced disturbances in membrane organelles, primarily in mitochondria and the granular endoplasmic reticulum, were noted. The functional activity of dark pinealocytes increased during this period of the study. In the pineal parenchyma of 90-day-old rats after exposure to nitrates, the functional activity of type I pinealocytes was at a low level. The functional activity of dark pinealocytes was also weakened. Thus, as a result of the simultaneous action of nitrates and methylene blue in the pineal gland of 14-day-old rats, a tendency to gradual restoration of the structural and functional parameters of cells was observed. In 45-day-old animals, after the simultaneous action of nitrates and methylene blue, the ultrastructural data of pineal cells indicated numerous mitochondria and secretory granules in the cytoplasm. In the parenchyma of the pineal gland of 90-day-old rats after chronic action of nitrates and methylene blue at the ultrastructural level, no sharp changes in the cytoplasm and nucleus of light and dark pinealocytes were found in comparison with the control. Conclusion. The intake of nitrates in 14-day-old rats causes the development of a stress reaction, poorly developed organelles and signs of degranulation appear in the ultrastructure of light pinealocytes, however, the cytoplasm and nuclei of dark cells indicated an increase in function. In 45-day-old rats after exposure to nitrates, the signs of the stress reaction are enhanced. In the ultrastructure of the cytoplasm of light cells, pronounced violations of membrane organelles are determined. Enhanced function continues in dark pinealocytes. After the action of nitrates in 90-day-old rats, changes occur that are characteristic of the stage of depletion of the general adaptation syndrome, the result of which is a deep imbalance in the work of the pineal gland. The combined action of nitrates and methylene blue in 14-day-old animals helps to reduce the toxic effect and the strength of stress reactions in the pineal gland. In the ultrastructure of pinealocytes, the number of ribosomes, small secretory granules and mitochondria increases in comparison with the action of nitrates alone. In 45-day-old animals with the simultaneous intake of nitrates and methylene blue in the ultrastructure of melanotropic cells, the accumulation of secretory granules of the same size and electron density, an increase in the number of organelles and signs of restoration of the structure of the cytoplasm and nucleus are noted. The use of methylene blue against the background of long-term intake of nitrates in 90-day-old rats at the ultrastructural level of abrupt changes in the cytoplasm and nucleus of light and dark pinealocytes is not manifested in comparison with the control

Ультраструктурні зміни меланотропних клітин проміжної частки аденогіпофіза у віковому аспекті

In modern theoretical and practical biology and medicine, the key problem of research is to reveal the patterns of structural and functional organization of the human and animal body at different stages of development. Literature sources provide very limited data on the organization of the intermediate lobe of the adenohypophysis in humans, leaving insufficiently studied the ultrastructural state and activity of melanotropic cells of the intermediate lobe of the adenohypophysis in animals of different ages, including rats. The issues of the intermediate pituitary gland functioning remain important and little studied, especially in the period of the beginning of the optic-thalamic system. The purpose of the research was to study the ultrastructural changes of melanotropic cells of the intermediate lobe of the adenohypophysis in rats of different ages in the norm. Material and methods. In accordance with the purpose of the study we conducted the experiment on 30 nonlinear white male rats of different ages: 14-, 45- and 90-day-old. The animals were kept in the vivarium in equivalent conditions. The keeping and using of animals was carried out in accordance with the provisions of the "General Ethical Principles of Animal Experiments", approved by the IV National Congress of Bioethics. While examining the intermediate pituitary gland of intact rats on the electron microscope, the material was fixed in 2.5% solution of glutaraldehyde on phosphate buffer with fixation in 1% solution of osmium tetroxide according to Caulfield. It was dehydrated in alcohols of increasing concentration (70%, 80%, 90%, 100%) and acetone, poured into a mixture of epon-araldite. Semi-thin sections were made from the obtained blocks, which were stained with toluidine blue. Results and discussion. At the ultrastructural level in the intermediate lobe of the adenohypophysis of 14-day-old intact rats, several cell types could be identified that differed in the number and size of secretory granules. The ultrastructure of glandular cells of 45-day-old rats had no significant differences compared with 14-day-old animals. Secretory granules of different sizes and electron densities were observed in the cytoplasm of melanotropic cells. It was often possible to see a glandular cell with numerous secretory granules in one part of the cytoplasm, while in another they were virtually absent. Most melanotropocytes were characterized by slightly compacted mitochondria, but their numbers were slightly higher than in 45-day-old rats. The nuclei of most cells were large, oval in shape with a clear structure of nucleoli and their ribosomal component. The latter represented groups of ribosomes that were collected in osmophilic complexes. The amount of heterochromatin exceeded euchromatin and it was located mainly in the membrane with areas of rarefaction in the pore area Conclusion. In 14-day-old intact male rats, the intermediate lobe of the adenohypophysis was presented as a formed functionally active organ. In the ultrastructure of the cytoplasm of melanotropes there was a moderate development of organelles, and judging by the number and size of secretory granules, as well as the density of their content, we can assume that all these cells differed from each other in their functional activity. In 45-day-old intact rats, accumulation of secretory granules was observed in the cytoplasm of melanotropes, especially near the nucleus, which indicated an increase in melanocyte-stimulating hormones synthesis with age. The ultrastructural state of the cytoplasm and nucleus also indicated an increase in functional activity. The ultrastructure of the intermediate lobe cells of the adenohypophysis of rats at the age of 90 days differed from that of younger animals by signs of different functional activity of individual melanotropic cells

The Protective Effects of Jamun Seeds and Orange Peels Extracts on Calcitonin Cells and Parathyroid Glands against Cypermethrin Toxicity

Background: This study aimed to investigate changes in Calcitonin Cells (C cells) and Parathyroid Glands (PTG) induced by cypermethrin exposure in rats and evaluate the protective effects of Jamun Seeds (Syzygium cumini; JSE) and orange peels (Citrus sinensis; OPE) extracts. Methods: Wistar rats (N=120) were treated in groups of 20 each as: A. Controls; B. Cypermethrin (CY); C. Cypermethrin and JSE; D. Cypermethrin and OPE; E. OPE; F. JSE. Thyro-parathyroid tissue samples were fixed on days 15 or 30 following each treatment. Results: Cypermethrin treatment in rats either alone or combined with JSE or OPE caused degranulation of calcitonin cells and mitochondrial enlargement on day 15 of exposure. Nuclear volume of C cells in groups B, C and D remained unchanged. On day 30, we found increased nuclear volumes, accumulation of secretory granules and degeneration of C cells. In groups E and F no changes in the morphology of C cells were observed. The PTG cells treated with cypermethrin or CY+JSE or CY+OPE over 15 days exhibited hyperchromatic, elongated and increased in volume of the nuclei. The nuclei of PTG cells treated with cypermethrin, CY+JSE or CY+OPE were hyperchromatic and elongated on day 30. Degenerate nuclei were detected after cypermethrin treatment, and the nuclear volumes were increased on day 30. In groups E and F there were no changes in PTG cells on days 15 and 30 post treatment. Conclusion: Cypermethrin provoked alterations in the calcitonin and PTG cells, with microscopic evidence of protection after treatment with JSE and OPE.

Ameliorative effects of jamun seed and orange peel extracts on microcystin LR induced alterations in calcitonin cells and parathyroid gland of rats.

This study investigated changes in calcitonin cells (C-cells) and parathyroid glands (PTG) induced by microcystin LR (MCLR) exposure to rats and evaluated ameliorative effects of jamun (Syzygium cumini) seed (JSE) and orange (Citrus sinensis) peel (OPE) extracts. Wistar rats were treated as-Group A (control), Group B (MCLR), Group C (MCLR + JSE), Group D (MCLT + OPE), Group E (OPE) and Group F (JSE). Microcystin dose was (10 μg/kg body wt/day whereas OPE and JSE dose was 200 mg/kg body wt/day. Thyroid and PTG were fixed on 15 and 30 days following the treatment. C-cells of treated rats for 15 days with MCLR; MCLR + JSE and MCLR + OPE exhibit degranulation, mitochondrial swelling and prominent RER. In MCLR treated rats few cells completely lack secretory granules. After 30 days MCLR treatment accumulation of secretory granules and degeneration were noticed in C-cells. C-cell nuclear volume (NV) of MCLR, MCLR + JSE and MCLT + OPE treated rats show an increase. In MCLR, MCLR + JSE and MCLR + OPE treated rats PTG exhibit hyperchromatic nuclei, nuclear elongation and increased NV after 15 days. After 30 days MCLR treatment nuclei of PTG become more hyperchromatic, more elongated, show degeneration of nuclei and increase in NV. NV is increased in Group C and Group D. PTG remain unaltered 30 days following treatment with OPE and JSE. Microcystin LR provoke physiological effects on the blood calcium and alterations in C cells and PTG, which cause serious threat to organism. These changes can be protected by JSE and OPE.

Recent advances in understanding and managing malabsorption: focus on microvillus inclusion disease.

Microvillus inclusion disease (MVID) is a rare congenital severe malabsorptive and secretory diarrheal disease characterized by blunted or absent microvilli with accumulation of secretory granules and inclusion bodies in enterocytes. The typical clinical presentation of the disease is severe chronic diarrhea that rapidly leads to dehydration and metabolic acidosis. Despite significant advances in our understanding of the causative factors, to date, no curative therapy for MVID and associated diarrhea exists. Prognosis mainly relies on life-long total parenteral nutrition (TPN) and eventual small bowel and/or liver transplantation. Both TPN and intestinal transplantation are challenging and present with many side effects. A breakthrough in the understanding of MVID emanated from seminal findings revealing mutations in MYO5B as a cause for MVID. During the last decade, many studies have thus utilized cell lines and animal models with knockdown of MYO5B to closely recapitulate the human disease and investigate potential therapeutic options in disease management. We will review the most recent advances made in the research pertaining to MVID. We will also highlight the tools and models developed that can be utilized for basic and applied research to increase our understanding of MVID and develop novel and effective targeted therapies.

Three-dimensional ultrastructural analyses of anterior pituitary gland expose spatial relationships between endocrine cell secretory granule localization and capillary distribution.

Endocrine and endothelial cells of the anterior pituitary gland frequently make close appositions or contacts, and the secretory granules of each endocrine cell tend to accumulate at the perivascular regions, which is generally considered to facilitate secretory functions of these cells. However, three-dimensional relationships between the localization pattern of secretory granules and blood vessels are not fully understood. To define and characterize these spatial relationships, we used scanning electron microscopy (SEM) three-dimensional reconstruction method based on focused ion-beam slicing and scanning electron microscopy (FIB/SEM). Full three-dimensional cellular architectures of the anterior pituitary tissue at ultrastructural resolution revealed that about 70% of endocrine cells were in apposition to the endothelial cells, while almost 30% of endocrine cells were entirely isolated from perivascular space in the tissue. Our three-dimensional analyses also visualized the distribution pattern of secretory granules in individual endocrine cells, showing an accumulation of secretory granules in regions in close apposition to the blood vessels in many cases. However, secretory granules in cells isolated from the perivascular region tended to distribute uniformly in the cytoplasm of these cells. These data suggest that the cellular interactions between the endocrine and endothelial cells promote an uneven cytoplasmic distribution of the secretory granules.

An inducible mouse model for microvillus inclusion disease reveals a role for myosin Vb in apical and basolateral trafficking

Microvillus inclusion disease (MVID) is a rare intestinal enteropathy with an onset within a few days to months after birth, resulting in persistent watery diarrhea. Mutations in the myosin Vb gene (MYO5B) have been identified in the majority of MVID patients. However, the exact pathophysiology of MVID still remains unclear. To address the specific role of MYO5B in the intestine, we generated an intestine-specific conditional Myo5b-deficient (Myo5bfl/fl;Vil-CreERT2) mouse model. We analyzed intestinal tissues and cultured organoids of Myo5bfl/fl;Vil-CreERT2 mice by electron microscopy, immunofluorescence, and immunohistochemistry. Our data showed that Myo5bfl/fl;Vil-CreERT2 mice developed severe diarrhea within 4 d after tamoxifen induction. Periodic Acid Schiff and alkaline phosphatase staining revealed subapical accumulation of intracellular vesicles in villus enterocytes. Analysis by electron microscopy confirmed an almost complete absence of apical microvilli, the appearance of microvillus inclusions, and enlarged intercellular spaces in induced Myo5bfl/fl;Vil-CreERT2 intestines. In addition, we determined that MYO5B is involved not only in apical but also basolateral trafficking of proteins. The analysis of the intestine during the early onset of the disease revealed that subapical accumulation of secretory granules precedes occurrence of microvillus inclusions, indicating involvement of MYO5B in early differentiation of epithelial cells. By comparing our data with a novel MVID patient, we conclude that our mouse model completely recapitulates the intestinal phenotype of human MVID. This includes severe diarrhea, loss of microvilli, occurrence of microvillus inclusions, and subapical secretory granules. Thus, loss of MYO5B disturbs both apical and basolateral trafficking of proteins and causes MVID in mice.

Light and electron microscopy of the European beaver (Castor fiber) stomach reveal unique morphological features with possible general biological significance.

Anatomical, histological, and ultrastructural studies of the European beaver stomach revealed several unique morphological features. The prominent attribute of its gross morphology was the cardiogastric gland (CGG), located near the oesophageal entrance. Light microscopy showed that the CGG was formed by invaginations of the mucosa into the submucosa, which contained densely packed proper gastric glands comprised primarily of parietal and chief cells. Mucous neck cells represented <0.1% of cells in the CGG gastric glands and 22–32% of cells in the proper gastric glands of the mucosa lining the stomach lumen. These data suggest that chief cells in the CGG develop from undifferentiated cells that migrate through the gastric gland neck rather than from mucous neck cells. Classical chief cell formation (i.e., arising from mucous neck cells) occurred in the mucosa lining the stomach lumen, however. The muscularis around the CGG consisted primarily of skeletal muscle tissue. The cardiac region was rudimentary while the fundus/corpus and pyloric regions were equally developed. Another unusual feature of the beaver stomach was the presence of specific mucus with a thickness up to 950 µm (in frozen, unfixed sections) that coated the mucosa. Our observations suggest that the formation of this mucus is complex and includes the secretory granule accumulation in the cytoplasm of pit cells, the granule aggregation inside cells, and the incorporation of degenerating cells into the mucus.

Cytosolic Dynamics of Annexin A6 Trigger Feedback Regulation of Hypertrophy via Atrial Natriuretic Peptide in Cardiomyocytes

Malfunctions in regulatory pathways that control cell size are prominent in pathological cardiac hypertrophy. Here, we show annexin A6 (Anxa6) to be a crucial regulator of atrial natriuretic peptide (ANP)-mediated counterhypertrophic responses in cardiomyocytes. Adrenergic stimulation of H9c2 cardiomyocytes by phenylephrine (PE) increased the cell size with enhanced expression of biochemical markers of hypertrophy, concomitant with elevated expression and subcellular redistribution of Anxa6. Stable cell lines with controlled increase in Anxa6 levels were protected against PE-induced adverse changes, whereas Anxa6 knockdown augmented the hypertrophic responses. Strikingly, Anxa6 knockdown also abrogated PE-induced juxtanuclear accumulation of secretory granules (SG) containing ANP propeptides (pro-ANP), a signature of maladaptive hypertrophy having counteractive functions. Mechanistically, PE treatment prompted a dynamic association of Anxa6 with pro-ANP-SG, parallel to their participation in anterograde traffic, in an isoform-specific fashion. Moreover, Anxa6 mutants that failed to associate with pro-ANP hindered ANP-mediated protection against hypertrophy, which was rescued, at least partially, by WT Anxa6. Additionally, elevated intracellular calcium (Ca(2+)) stimulated Anxa6-pro-ANP colocalization and membrane association. It also rescued pro-ANP translocation in cells expressing an Anxa6 mutant (Anxa6(ΔC)). Furthermore, stable overexpression of Anxa6(T356D), a mutant with superior flexibility, provided enhanced protection against PE, compared with WT, presumably due to enhanced membrane-binding capacity. Together, the present study delivers a cooperative mechanism where Anxa6 potentiates ANP-dependent counterhypertrophic responses in cardiomyocytes by facilitating regulated traffic of pro-ANP.

Botulinum toxin A inhibits salivary secretion of rabbit submandibular gland

Botulinum toxin A (BTXA) has been used in several clinical trials to treat excessive glandular secretion; however, the precise mechanism of its action on the secretory function of salivary gland has not been fully elucidated. In this study, we aimed to investigate the effect of BTXA on secretion of submandibular gland in rabbits and to identify its mechanism of action on the secretory function of salivary gland. At 12 weeks after injection with 5 units of BTXA, we found a significant decrease in the saliva flow from submandibular glands, while the salivary amylase concentration increased. Morphological analysis revealed reduction in the size of acinar cells with intracellular accumulation of secretory granules that coalesced to form a large ovoid structure. Expression of M3-muscarinic acetylcholine receptor (M3 receptor) and aquaporin-5 (AQP5) mRNA decreased after BTXA treatment, and distribution of AQP5 in the apical membrane was reduced at 1, 2 and 4 weeks after BTXA injection. Furthermore, BTXA injection was found to induce apoptosis of acini. These results indicate that BTXA decreases the fluid secretion of submandibular glands and increases the concentration of amylase in saliva. Decreased expression of M3 receptor and AQP5, inhibition of AQP5 translocation, and cell apoptosis might involve in BTXA-reduced fluid secretion of submandibular glands.

Возрастные структурно-функциональные особенности миоэндокринных клеток сердца у крыс в норме и при наследственной гипертензии

A qualitative and quantitative ultrastructural study of right atrial cardiomyocytes in WAG (normotensive control) and ISIAH (inherited stress-induced arterial hypertension) rats of different age (on day 18 of embryogenesis, on days 12 and 21 after birth, and at an age of 6 and 13 months) was performed. It was shown that, in embryos with an as yet incomplete atrial morphogenesis, secretory granules containing natriuretic peptides are actively formed, accumulated, and dissolved. In postnatal ontogeny, the secretory product is accumulated in atrial cells. In all ontogeny stages studied, the numerical density of secretory granules in the myoendocrine cells of hypertensive rats is greater and the qualitative composition of these granules is more diverse than in the control. It was established that, in atrial myocytes of ISIAH rats, the morphological signs of natriuretic peptide hypersecretion precede the development of genetically programmed high blood pressure. In adult hypertensive rats, hypertrophic and degenerative changes in myocytes are accompanied by excessive accumulation of secretory granules, some of which undergo intracellular degradation.

Molecular Mechanism of Myosin Va Recruitment to Dense Core Secretory Granules

The brain-spliced isoform of Myosin Va (BR-MyoVa) plays an important role in the transport of dense core secretory granules (SGs) to the plasma membrane in hormone and neuropeptide-producing cells. The molecular composition of the protein complex that recruits BR-MyoVa to SGs and regulates its function has not been identified to date. We have identified interaction between SG-associated proteins granuphilin-a/b (Gran-a/b), BR-MyoVa and Rab27a, a member of the Rab family of GTPases. Gran-a/b-BR-MyoVa interaction is direct, involves regions downstream of the Rab27-binding domain, and the C-terminal part of Gran-a determines exon specificity. MyoVa and Gran-a/b are partially colocalised on SGs and disruption of Gran-a/b-BR-MyoVa binding results in a perinuclear accumulation of SGs which augments nutrient-stimulated hormone secretion in pancreatic beta-cells. These results indicate the existence of at least another binding partner of BR-MyoVa that was identified as rabphilin-3A (Rph-3A). BR-MyoVa-Rph-3A interaction is also direct and enhanced when secretion is activated. The BR-MyoVa-Rph-3A and BR-MyoVa-Gran-a/b complexes are linked to a different subset of SGs, and simultaneous inhibition of these complexes nearly completely blocks stimulated hormone release. This study demonstrates that multiple binding partners of BR-MyoVa regulate SG transport, and this molecular mechanism is universally used by neuronal, endocrine and neuroendocrine cells.

Evaluation of Novel Dry Eye Model: Preganglionic Parasympathetic Denervation in Rabbit

To evaluate ocular surface status after interruption of preganglionic, parasympathetic neural control after surgical removal of the greater superficial petrosal nerve (GSPN). New Zealand White rabbits underwent unilateral section and removal of a 5-mm portion of the GSPN by a route through the inner ear; no ocular or orbital tissue was involved. Before and 7 days after surgery, all animals underwent preliminary examination, including fluorescein staining, rose bengal instillation, blink rate, tear breakup time (BUT), tear flow, and impression cytology. Total tarsorrhaphy was carried out in four additional rabbits, and another four animals underwent unilateral sham procedures. The GSPN, pterygopalatine ganglion, lacrimal gland, and conjunctiva were evaluated by light and transmission electron microscopy (TEM). GSPN sectioning resulted in significant changes of the ocular surface after 7 days: intense rose bengal staining of the conjunctiva, fluorescein staining of the cornea, increased blink rate (P < 0.05), decreased BUT (P < 0.005), decreased tear flow by 26% (P < 0.005), and decreased goblet cell density (P < 0.01). TEM revealed massive accumulation of secretory granules in lacrimal acinar cells. The changes were also seen after tarsorrhaphy. Neither the contralateral control nor the sham eyes were affected. The effects of GSPN nerve section led to the rapid onset of a dry eye condition in the rabbits that continued for at least 1 week. The authors suggest that continuous neural drive of the pterygopalatine ganglion is necessary to maintain adequate tear flow and mucin secretion. It is likely the trigeminal system is the afferent origin of this continuous neural tone.

Ultrastructural features of the uterus in the sexually immature ostrich (&lt;i&gt;Struthio camelus&lt;/i&gt;) during periods of ovarian inactivity and activity

The ultrastructure of the surface epithelium and tubular glands of the uterus in the immature ostrich is described. In ostriches with inactive ovaries the uterus is lined by a non-ciliated simple columnar epithelium, with basally located heterochromatic nuclei. Scanning electron microscopy revealed that these non-ciliated cells have a dense microvillous cover. A simple columnar to pseudostratified columnar epithelium, comprised of non-ciliated and ciliated cells, lines the uterus in birds with active ovaries. The ciliated cells possess a wide luminal region, which contains a nucleus and various organelles. An accumulation of secretory granules was observed in the apical regions of the non-ciliated cells, as well as in a few ciliated cells. In addition to non-ciliated and ciliated cells, a cell type with rarefied cytoplasm was also identified. These cells appear to correspond to calcium secreting cells identified in other avian species. The results of this study indicate that, although uterine differentiation is present in immature ostriches with active ovaries, the production of secretory product appears to occur mainly in non-ciliated epithelial cells.

VAMP8/endobrevin as a general vesicular SNARE for regulated exocytosis of the exocrine system.

The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.

Cellular expression of Noc2, a Rab effector protein, in endocrine and exocrine tissues in the mouse

Noc2 is a Rab effector which participates in regulated exocytosis. It is expressed abundantly in endocrine cells but at low levels in exocrine tissues. Noc2-deficient mice, however, exhibit marked accumulation of secretory granules in exocrine cells rather than endocrine cells. In the present study, we investigated localization of Noc2 immunohistochemically in various endocrine and exocrine tissues in normal mice. Western blotting detected a Noc2-immunoreactive band of 38 kDa in isolated pancreatic islets, the adrenal gland, pituitary gland, and thyroid gland. Immunostaining for Noc2 labeled endocrine cells in the adrenal medulla and adenohypophysis, pancreatic islet cells, thyroid parafollicular cells, and gut endocrine cells, supporting the notion that Noc2 is a Rab effector protein shared by amine/peptide-secreting endocrine cells. Besides endocrine tissues, granular ducts in salivary glands contained Noc2. Although immunostaining failed to detect Noc2 in acinar cells of all exocrine glands examined, reverse transcriptase-polymerase chain reaction analysis detected the mRNA expression in exocrine pancreas. Ultrastructurally, Noc2 immunoreactivity was associated with the limiting membrane of granules in both pancreatic endocrine and salivary duct exocrine cells. The cellular and subcellular localizations of Noc2 should yield key information on its functional significance as well as account for the phenotype in Noc2-deficient mice.

Noc-king out exocrine and endocrine secretion

The Rab GTPase effector Noc2 was brought into the limelight by a recent publication that demonstrated its requirements at different stages of regulated exocytosis. Noc2 knockout resulted in distinct abnormalities in endocrine and exocrine cells, ranging from the accumulation of secretory granules of increased size to impairments in the regulated release of their secretory products. Explanations for these defects are beginning to emerge and they promise to reveal some of the most jealously kept secrets of regulated exocytosis.

Noc2 is essential in normal regulation of exocytosis in endocrine and exocrine cells.

Rab3 is a subfamily of the small GTP-binding protein Rab family and plays an important role in exocytosis. Several potential effectors of Rab3, including rabphilin3 and Rims (Rim1 and Rim2), have been isolated and characterized. Noc2 was identified originally in endocrine pancreas as a molecule homologous to rabphilin3, but its role in exocytosis is unclear. To clarify the physiological function of Noc2 directly, we have generated Noc2 knockout (Noc2(-/-)) mice. Glucose intolerance with impaired insulin secretion was induced in vivo by acute stress in Noc2(-/-) mice, but not in wild-type (Noc2(+/+)) mice. Ca(2+)-triggered insulin secretion from pancreatic isles of Noc2(-/-) mice was markedly impaired, but was completely restored by treatment with pertussis toxin, which inhibits inhibitory G protein Gi/o signaling. In addition, the inhibitory effect of clonidine, an alpha(2)-adrenoreceptor agonist, on insulin secretion was significantly greater in Noc2(-/-) islets than in Noc2(+/+) islets. Impaired Ca(2+)-triggered insulin secretion was rescued by adenovirus gene transfer of wild-type Noc2 but not by that of mutant Noc2, which does not bind to Rab3. Accordingly, Noc2 positively regulates insulin secretion from endocrine pancreas by inhibiting Gi/o signaling, and the interaction of Noc2 and Rab3 is required for the effect. Interestingly, we also found a marked accumulation of secretory granules in various exocrine cells of Noc2(-/-) mice, especially in exocrine pancreas with no amylase response to stimuli. Thus, Noc2, a critical effector of Rab3, is essential in normal regulation of exocytosis in both endocrine and exocrine cells.