Ca-dependent K channels in exocrine salivary glands

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca²⁺-dependent K⁺ channels take part in key functions including membrane potential regulation, fluid movement and K⁺ secretion in exocrine glands. Two K⁺ channels have been identified in exocrine salivary glands: (1) a Ca²⁺-activated K⁺ channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca²⁺-dependent K⁺ channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca²⁺-dependent K⁺ channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca²⁺-dependent K⁺ channels by protein–protein interactions that may significantly impact exocrine gland physiology.     

Simultaneous Combined Pancreatic Test

A simultaneous combined pancreatic test can be performed in one morning with only one intubation of the duodenum. The test includes the measurement of exocrine pancreatic secretion of bicarbonate, enzymes, and radioactive selenium, pancreatic scanning, hypotonic duodenography, and cytology of the duodenal aspirate. In the first 70 patients it was found that a single secretion test was of only limited value in detecting pancreatic disease; cytology was the most reliable and scanning the least reliable single test; and that the combined test provided near-complete discrimination between patients with no pancreatic disease, with chronic pancreatitis, and with carcinoma of the pancreas.     

Gut integrity and duodenal enteropathogen burden in undernourished children with environmental enteric dysfunction

Environmental enteric dysfunction (EED) is a subclinical condition of intestinal inflammation, barrier dysfunction and malabsorption associated with growth faltering in children living in poverty. This study explores association of altered duodenal permeability (lactulose, rhamnose and their ratio) with higher burden of enteropathogen in the duodenal aspirate, altered histopathological findings and higher morbidity (diarrhea) that is collectively associated with linear growth faltering in children living in EED endemic setting. In a longitudinal birth cohort, 51 controls (WHZ > 0, HAZ > −1.0) and 63 cases (WHZ< -2.0, refractory to nutritional intervention) were recruited. Anthropometry and morbidity were recorded on monthly bases up to 24 months of age. Dual sugar assay of urine collected after oral administration of lactulose and rhamnose was assessed in 96 children from both the groups. Duodenal histopathology (n = 63) and enteropathogen analysis of aspirate via Taqman array card (n = 60) was assessed in only cases. Giardia was the most frequent pathogen and was associated with raised L:R ratio (p = 0.068). Gastric microscopy was more sensitive than duodenal aspirate in H. pylori detection. Microscopically confirmed H. pylori negatively correlated with HAZ at 24 months (r = −0.313, p = 0.013). Regarding histopathological parameters, goblet cell reduction significantly correlated with decline in dual sugar excretion (p< 0.05). Between cases and controls, there were no significant differences in the median (25^th, 75^th percentile) of urinary concentrations (μg/ml) of lactulose [27.0 (11.50, 59.50) for cases vs. 38.0 (12.0, 61.0) for controls], rhamnose [66.0 (28.0, 178.0) vs. 86.5 (29.5, 190.5)] and L:R ratio [0.47 (0.24, 0.90) vs. 0.51 (0.31, 0.71)] respectively. In multivariable regression model, 31% of variability in HAZ at 24 months of age among cases and controls was explained by final model including dual sugars. In conclusion, enteropathogen burden is associated with altered histopathological features and intestinal permeability. In cases and controls living in settings of endemic enteropathy, intestinal permeability test may predict linear growth. However, for adoption as a screening tool for EED, further validation is required due to its complex intestinal pathophysiology.     

The volume and area of the capillaries in the endocrine and exocrine pancreas of the rat

Summary The capillary volumes in the endocrine and exocrine parenchyma of the pancreas were compared with a point-sampling technique. The islets were found to have a capillary volume of approximately 3.5%, while the value for the exocrine pancreas was significantly (P<0.001) lower at 2%. When the capillary wall area was measured, however, both types of parenchyma had a similar value of approximately 20 mm²/mm³ tissue. The reason for the discrepancy between these parameters is probably the lack of lymphatic capillarics, with their relatively small lumen in the islets.     

Quantification of metronidazole in small-volume biological samples using narrow-bore high-performance liquid chromatography

A rapid, selective and sensitive HPLC assay has been developed for the routine analysis of metronidazole in small volumes of rat plasma, gastric aspirate and gastric tissue. The extraction procedure involves liquid–liquid extraction and a protein precipitation step. A microbore Hypersil ODS 3 μm (150×2.1 mm I.D.) column was used with a mobile phase consisting of acetonitrile–aqueous 0.05 M potassium phosphate buffer (pH 7) containing 0.1% triethylamine (10:90). The column temperature was at 25°C and the detection was by UV absorbance at 317 nm. The limit of detection was 0.015 μg ml⁻¹ for gastric juice aspirate and plasma and 0.010 μg g⁻¹ for gastric tissue (equivalent to 0.75 ng on-column). The method was linear up to a concentration of 200 μg ml⁻¹ for plasma and gastric juice aspirate and up to 40 μg g⁻¹ for tissue, with inter- and intra-day relative standard deviations less than 14%. The measured recovery was at least 78% in all sample matrices. The method proved robust and reliable when applied to the measurement of metronidazole in rat plasma, gastric juice aspirate and gastric tissue for pharmacokinetic studies in individual rats.     

Role of peptide YY in regulation of duodenal motility during the interdigestive period in sheep

Temporal coordination between duodenal migrating myoelectric complexes (MMC) and pancreatic exocrine secretion, and the effects of porcine peptide YY (PYY) on gastroduodenal motility and pancreatic exocrine secretion were examined during the interdigestive period in conscious mature sheep. Fluid and enzyme secretions from the exocrine pancreas showed a periodic pattern corresponding to the phases of duodenal MMC, although these secretion rates were maintained at a high level during phase II in sheep. Intravenous continuous infusion of PYY at doses ranging from 50 to 200 pmol · kg⁻¹ · h⁻¹ or intravenous bolus infusion of PYY at doses ranging from 50 to 200 pmol · kg⁻¹ showed a tendency to prolong the first cycle of the duodenal MMC and significantly shorten the second cycle. However, there was almost no effect on ruminal contractions from the PYY administration. In the pancreatic exocrine secretion, PYY could inhibit only bicarbonate secretion at only the highest dose of 200 pmol · kg⁻¹. These results imply that endogenous PYY may play a physiological role in the regulation of the duodenal MMC cycles in sheep but not in ruminal contractions. PYY seems unlikely to regulate the pancreatic exocrine secretion in normal sheep, because a supraphysiological dose of PYY was required to inhibit the pancreatic exocrine secretion. Accepted: 3 March 1997     

Duck Pancreatic Acinar Cell as a Unique Model for Independent Cholinergic Stimulation–Secretion Coupling

This paper investigated the role of acetylcholine (ACh) in physiological regulation of amylase secretion in avian exocrine pancreas. In the isolated duck pancreatic acini, ACh dose dependently stimulated amylase secretion, with a maximal effective concentration at 10 μM. The cAMP-mobilizing compounds forskolin, vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase activating peptide (PACAP) receptor (VPAC) agonists PACAP-38 and PACAP-27 had no effect on the dose–response curve. ACh dose dependently induced increases in cytosolic Ca²⁺ concentration ([Ca²⁺] _c ), with increasing concentrations transforming oscillations into plateau increases. Forskolin (10 μM), PACAP-38 (1 nM), PACAP-27 (1 nM), or VIP (10 nM) alone did not stimulate [Ca²⁺] _c increase; neither did they modulate ACh-induced oscillations, nor made ACh low concentration effective. These data indicate that ACh-stimulated zymogen secretion in duck pancreatic acinar cells is not subject to modulation from the cAMP signaling pathway; whereas it has been widely reported in the rodents that ACh-stimulated exocrine pancreatic secretion is significantly enhanced by cAMP-mobilizing agents. This makes the duck exocrine pancreas unique in that cholinergic stimulus-secretion coupling is not subject to cAMP regulation.     

Localization and regulation of Ca entry and exit pathways in exocrine gland cells

Studies of Ca²⁺ transport pathways in exocrine gland cells have been useful, chiefly because of the polarized nature of the secretory epithelial cells. In pancreatic acinar cells, for example, Ca²⁺ reloading of empty intracellular stores can occur solely via Ca²⁺ entry through the basal part of the plasma membrane. On the other hand, the principal site for intracellular Ca²⁺ release—with the highest concentration of inositol 1,4,5-trisphosphate (IP₃) receptors—is in the apical secretory pole close to the apical plasma membrane. This apical part of the plasma membrane contains the highest density of Ca²⁺ pumps and is therefore the principal site for Ca²⁺ extrusion. On the basis of the known properties of Ca²⁺ entry and exit pathways in exocrine gland cells, the mechanisms controlling Ca²⁺ exit and entry are discussed in relation to recent direct information about Ca²⁺ transport into and out of the endoplasmic reticulum (ER) and the mitochondria in these cells.     

Calium, prostaglandins and the phosphatidylinositol effect in exocrine gland cells

The hypothesis that arachidonic acid metabolism might be involved in Ca-mobilization mechanisms in exocrine gland cells was investigated. Arachidonate (10⁻⁴M) failed to stimulate protein secretion from slices of pancreas, parotid or lacrimal glands and failed to stimulate ⁸⁶Rb efflux from parotid or lacrimal glands. The stimulation of protein secretion (all three glands) or ⁸⁶Rb efflux (parotid and lacrimal glands) by appropriate secretagogues was unaffected by 10⁻⁵M indomethacin. Eicosatetraynoic acid (2×10⁻⁵M) inhibited ⁸⁶Rb efflux due to carbachol but not that due to physalaemin or ionomycin. Nordihydroguaiaretic acid inhibited lacrimal and parotid gland responses only at high (10⁻⁴M) concentration. Collectively, these results argue against an obligatory role for arachidonate metabolites in Ca-mediated responses of these exocrine glands.In the exocrine glands activation by neurotransmitters (or analogs) of receptors that mobilize cellular Ca also stimulates the incorporation of ³²PO₄ into phosphatidylinositol (1–3). Michell (4,5) has suggested that in some manner this alteration in phospholipid metabolism may be functionally responsible for the opening of surface membrane Ca gates which presumably precedes the expression of a number of Ca-mediated responses by the exocrine cell. That this reaction probably preceeds Ca mobilization is deduced primarily from two experimental observations. First, receptor activation of phosphatidylinositol turnover is not prevented by Ca omission (6–8). Second, the effect is not mimicked by the divalent cationophore A-23187, while other effects of receptor activation are mimicked by this compound (7–9).There has also been some speculation as to the manner in which altered phosphatidylinositol metabolism might be involved in the Ca-gating mechanism (10–14). One such hypothesis suggests that receptor activation may lead to phosphatidylinositol breakdown which in turn leads to the release of free arachidonate (13, 14). As free arachidonate is generally believed to be the rate-limiting substrate for prostaglandin synthesis (15), the resulting prostaglandins might act to mobilize Ca or might act in concert with Ca (13, 14). There is evidence for this hypothesis for the mouse pancreas, where exogenous arachidonate and prostaglandins can stimulate amylase release (13). The effects of arachidonate, carbachol, caerulein and pancreozmin were all antagonized by sub-micromolar concentrations of indomethacin (13), a potent cyclooxygenase inhibitor (15). Additionally, recent reports have demonstrated stimulation by acetylcholine of prostaglandin E synthesis in mouse pancreas (16, 17).The purpose of this study was to examine the general applicability of this hypothesis by investigating the effects of arachidonate and substances that inhibit prostaglandin formation in two other exocrine tissues that show a prominent phosphatidylinositol turnover — the rat parotid and lacrimal glands.     

Mypt1 regulates Bmp signaling to promote embryonic exocrine pancreas growth in zebrafish

Myosin phosphatase targeting subunit 1 (Mypt1) is the regulatory subunit of myosin phosphatase which dephosphorylates the light chain of myosin II to inhibit its contraction. Although biochemical properties of Mypt1 have been characterized in detail, its biological functions in organisms are not well understood. The zebrafish mypt1 ^sq181 allele was found defective in the ventral pancreatic bud and extrapancreatic duct development, resulting in dysplasia of exocrine pancreas. In mypt1 ^sq181 mutant, the early growth of the ventral pancreatic bud was initiated but failed to expand due to impaired cell proliferation and increased cell apoptosis. As Mypt1 is essential for cell migration, the loss‐of‐function of Mypt1 in the mutant disrupted the lateral plate mesoderm migration during gut looping, therefore, altering the Bmp2a expression pattern within it, and eventually leading to impaired Bmp signaling in the adjacent exocrine pancreas. Overexpression of bmp2a could rescue the development of exocrine pancreas, suggesting that the impaired Bmp2a signaling is responsible for the pancreatic development defects. Bmp2a has been reported to promote the early specification of the ventral pancreatic bud, and our study reveals that it continues to serve as a cell proliferation/survival signal to ensure pancreatic bud growth properly in zebrafish.     

Calcium release and internal calcium regulation in acinar cells of exocrine glands

Conclusion Recent results call for a reinterpretation of the mechanisms underlying the recruitment of intracellular Ca²⁺ in exocrine glands. One new hypothesis suggested by these developments is that InsP₃-sensitive channels liberate Ca²⁺ ions from secretory vesicles, as illustrated in Fig. 5.     

Immunoscintigraphy with biosynthetically-labeled 75Se-antibodies—I. Biodistribution of 75Se-monoclonal antibodies and of free radionuclide

Biosynthetically-radiolabeled MoAbs provide a tool to test whether structural modifications of the MoAbs influence the results of conventional immunoscintigraphy. When biosynthetically-labeled ⁷⁵Se-MoAbs from the Mel-14 hybridoma were injected into mice with melanoma xenografts, the high tumor recovery supported the hypothesis of a structural advantage. The increased excretion of ⁷⁵Se obtained by supplementing the diet of the mice with cold selenium did not reduce the tumor recovery, demonstrating an accumulation of the free radionuclide in normal tissue.     

Some characteristics of 75Se-P,a selenoprotein found in rat liver and plasma,and comparison of it with selenoglutathione peroxidase

The selenoenzyme glutathione peroxidase cannot account for all the physiological effects of selenium in rat liver. Therefore, a study was carried out with the ultimate aim of identifying selenoproteins other than glutathione peroxidase. The incorporation of ⁷⁵Se, given as ⁷⁵SeO₃^2?, into centrifugally separated fractions of selenium-deficient and control rat livers was determined. In selenium-deficient liver much less ⁷⁵Se was incorporated into the 105,000g supernatant fraction than in controls, so this fraction was studied further by gel filtration, ion-exchange, and hydroxylapatite chromatography. Selenoglutathione peroxidase and another selenoprotein, called ⁷⁵Se-P, were separated and identified. Both these selenoproteins were also found in plasma. Selenium deficiency had opposite effects on incorporation of ⁷⁵Se by these proteins. It decreased ⁷⁵Se incorporation by glutathione peroxidase at 3 and 72 h after ⁷⁵Se injection but increased ⁷⁵Se incorporation by ⁷⁵Se-P. This suggests that ⁷⁵Se-P competes for available selenium better than does glutathione peroxidase when the element is in short supply. Apparent molecular weights of ⁷⁵Se-P from liver and plasma determined by gel filtration were, respectively, 83,000 and 79,000, which indicate proteins smaller than glutathione peroxidase. Cycloheximide pretreatment of the rat blocked ⁷⁵Se incorporation into plasma ⁷⁵Se-P. These experiments establish the existence of a selenoprotein, ⁷⁵Se-P, in rat liver and plasma which is chromatographically distinct from glutathione peroxidase and which incorporates ⁷⁵Se differently from glutathione peroxidase. ⁷⁵Se-P may account for some of the physiological effects of selenium.     

p75 NTR -Immunoreactivity in the subventricular zone of adult male rats: Expression by cycling cells

While the study of in vitro regulation of neural stem cell lineage from both embryonic and adult neurospheres is greatly advanced, much less is known about factors acting in situ for neural stem cell lineage in adult brain. We reported that neurotrophin low affinity receptor p75^NTR is present in the subventricular zone (SVZ) in adult male rats. We then characterized co-distribution of markers associated with precursor cells (nestin and PSA-NCAM) with growth factor receptors (p75^NTR, trkA, EGFr) and proliferation-associated antigens (Ki67 and BrDU-uptake) in adult male rat by immunocytochemistry and confocal laser scan microscopy. Distribution of p75^NTR-immunoreactivity (IR) was investigated using different mono- and polyclonal antisera. p75^NTR– is not co-distributed with glial fibrillary acid protein. It was found to be co-distributed with a small number of nestin-IR cells, whereas no coexistence with PSA-NCAM-IR was observed. Conversely, p75^NTR-IR was present in numerous dividing cells (Ki-67-positive) and co-distributed with EGFr. In order to verify the possible association between p75^NTR and cell death, we investigated co-distribution of p75^NTR-IR with nuclear condensation images as visualized by Hoechst 33258 staining. While few images indicating nuclear condensation were observed in the SVZ, no coexistence with p75^NTR was found. TrkA- and trkB-IR was not found in the SVZ. We also investigated p75^NTR immunostaining on post-natal day 1 and day 16, because of the dramatic reduction of proliferating cells in SVZ over this time-interval. p75^NTR-IR was not increased in the early post-natal phase. Thus, p75^NTR seems to be associated with cell cycle regulation in SVZ in adult rat brain.     

p75NTR optimizes the osteogenic potential of human periodontal ligament stem cells by up‐regulating α1 integrin expression

Human periodontal ligament stem cells (hPDLSCs) are a promising source in regenerative medicine. Due to the complexity and heterogeneity of hPDLSCs, it is critical to isolate homogeneous hPDLSCs with high regenerative potential. In this study, p75 neurotrophin receptor (p75NTR) was used to isolate p75NTR⁺ and p75NTR^? hPDLSCs by fluorescence‐activated cell sorting. Differences in osteogenic differentiation among p75NTR⁺, p75NTR^? and unsorted hPDLSCs were observed. Differential gene expression profiles between p75NTR⁺ and p75NTR^? hPDLSCs were analysed by RNA sequencing. α1 Integrin (ITGA1) small interfering RNA and ITGA1‐overexpressing adenovirus were used to transfect p75NTR⁺ and p75NTR^? hPDLSCs. The results showed that p75NTR⁺ hPDLSCs demonstrated superior osteogenic capacity than p75NTR^? and unsorted hPDLSCs. Differentially expressed genes between p75NTR⁺ and p75NTR^? hPDLSCs were highly involved in the extracellular matrix‐receptor interaction signalling pathway, and p75NTR⁺ hPDLSCs expressed higher ITGA1 levels than p75NTR^? hPDLSCs. ITGA1 silencing inhibited the osteogenic differentiation of p75NTR⁺ hPDLSCs, while ITGA1 overexpression enhanced the osteogenic differentiation of p75NTR^? hPDLSCs . These findings indicate that p75NTR optimizes the osteogenic potential of hPDLSCs by up‐regulating ITGA1 expression, suggesting that p75NTR can be used as a novel cell surface marker to identify and purify hPDLSCs to promote their applications in regenerative medicine.     

A Role for the p75 Neurotrophin Receptor in Axonal Degeneration and Apoptosis Induced by Oxidative Stress

The p75 neurotrophin receptor (p75^NTR) mediates the death of specific populations of neurons during the development of the nervous system or after cellular injury. The receptor has also been implicated as a contributor to neurodegeneration caused by numerous pathological conditions. Because many of these conditions are associated with increases in reactive oxygen species, we investigated whether p75^NTR has a role in neurodegeneration in response to oxidative stress. Here we demonstrate that p75^NTR signaling is activated by 4-hydroxynonenal (HNE), a lipid peroxidation product generated naturally during oxidative stress. Exposure of sympathetic neurons to HNE resulted in neurite degeneration and apoptosis. However, these effects were reduced markedly in neurons from p75^NTR−/− mice. The neurodegenerative effects of HNE were not associated with production of neurotrophins and were unaffected by pretreatment with a receptor-blocking antibody, suggesting that oxidative stress activates p75^NTR via a ligand-independent mechanism. Previous studies have established that proteolysis of p75^NTR by the metalloprotease TNFα-converting enzyme and γ-secretase is necessary for p75^NTR-mediated apoptotic signaling. Exposure of sympathetic neurons to HNE resulted in metalloprotease- and γ-secretase-dependent cleavage of p75^NTR. Pharmacological blockade of p75^NTR proteolysis protected sympathetic neurons from HNE-induced neurite degeneration and apoptosis, suggesting that cleavage of p75^NTR is necessary for oxidant-induced neurodegeneration. In vivo, p75^NTR−/− mice exhibited resistance to axonal degeneration associated with oxidative injury following administration of the neurotoxin 6-hydroxydopamine. Together, these data suggest a novel mechanism linking oxidative stress to ligand-independent cleavage of p75^NTR, resulting in axonal fragmentation and neuronal death.     

Expression and function of Xenopus laevis p75NTR suggest evolution of developmental regulatory mechanisms

Neurotrophins signal through two different classes of receptors, members of the trk family of receptor tyrosine kinases, and p75 neurotrophin receptor (p75^NTR), a member of the tumor necrosis factor receptor family. While neurotrophin binding to trks results in, among other things, increased cell survival, p75^NTR has enigmatically been implicated in promoting both survival and cell death. Which of these two signals p75^NTR imparts depends on the specific cellular context. Xenopus laevis is an excellent system in which to study p75^NTR function in vivo because of its amenability to experimental manipulation. We therefore cloned partial cDNAs of two p75^NTR genes from Xenopus, which we have termed p75^NTRa and p75^NTRb. We then cloned two different cDNAs, both of which encompass the full coding region of p75^NTRa. Early in development both p75^NTRa and p75^NTRb are expressed in developing cranial ganglia and presumptive spinal sensory neurons, similar to what is observed in other species. Later, p75^NTRa expression largely continues to parallel p75^NTR expression in other species. However, Xenopus p75^NTRa is additionally expressed in the neuroepithelium of the anterior telencephalon, all layers of the retina including the photoreceptor layer, and functioning axial skeletal muscle. Finally, misexpression of full length p75^NTR and each of two truncated mutants in developing retina reveal that p75^NTR probably signals for cell survival in this system. This result contrasts with the reported role of p75^NTR in developing retinae of other species, and the possible implications of this difference are discussed. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 79–98, 2001     

Gamma-Secretase Represents a Therapeutic Target for the Treatment of Invasive Glioma Mediated by the p75 Neurotrophin Receptor

The multifunctional signaling protein p75 neurotrophin receptor (p75^NTR) is a central regulator and major contributor to the highly invasive nature of malignant gliomas. Here, we show that neurotrophin-dependent regulated intramembrane proteolysis (RIP) of p75^NTR is required for p75^NTR-mediated glioma invasion, and identify a previously unnamed process for targeted glioma therapy. Expression of cleavage-resistant chimeras of p75^NTR or treatment of animals bearing p75^NTR-positive intracranial tumors with clinically applicable γ-secretase inhibitors resulted in dramatically decreased glioma invasion and prolonged survival. Importantly, proteolytic processing of p75^NTR was observed in p75^NTR-positive patient tumor specimens and brain tumor initiating cells. This work highlights the importance of p75^NTR as a therapeutic target, suggesting that γ-secretase inhibitors may have direct clinical application for the treatment of malignant glioma.