Immunolocalization of AQP-5 in rat parotid and submandibular salivary glands after stimulation or inhibition of secretion in vivo

2004 ◽  
Vol 287 (1) ◽  
pp. G151-G161 ◽  
Author(s):  
Veronika Gresz ◽  
Tae-Hwan Kwon ◽  
Hong Gong ◽  
Peter Agre ◽  
Martin C. Steward ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Salivary Glands ◽  
Salivary Secretion ◽  
Apical Plasma Membrane ◽  
Secretory Cells ◽  
Muscarinic Agonist ◽  
Rat Parotid

In vitro studies of cultured salivary gland cells and gland slices have indicated that there may be regulated translocation of aquaporin (AQP)-5 between the apical plasma membrane and intracellular compartments of the secretory cells. However, it remains unknown whether AQP-5 in salivary glands is subject to regulated trafficking in vivo. To examine this possibility, we have investigated the subcellular localization of AQP-5 in rat parotid and submandibular glands fixed in vivo under conditions of stimulated or inhibited salivary secretion. Immunofluorescence and immunoelectron microscopy was used to determine the subcellular distribution of AQP-5 in control conditions following the stimulation of secretion with pilocarpine (a muscarinic agonist) or epinephrine (an α-adrenoceptor agonist) or during inhibition of basal secretion with atropine (a muscarinic antagonist) or phentolamine (an α-adrenoceptor antagonist). Under control conditions, >90% of AQP-5 was associated with the apical plasma membrane of acinar and intercalated duct cells, with only rare gold particles associated with intracellular membrane domains. Pilocarpine treatment dramatically increased saliva production but had no discernible effect on AQP-5 distribution. However, the increased salivary secretion was associated with luminal dilation and the appearance of a markedly punctate AQP-5 labeling pattern due to clustering of AQP-5 at the microvilli (especially evident in the parotid gland) after 10 min of drug injection. No changes in the subcellular localization of AQP-5 were seen in response to epinephrine, atropine, or phentolamine treatment compared with control tissues. Thus AQP-5 is localized predominantly in the apical plasma membrane under control conditions, and neither the onset nor the cessation of secretion is associated in vivo with any significant short-term translocation of AQP-5 between intracellular structures and the apical plasma membrane.

5-Hydroxytryptamine Modulation of Rat Parotid Salivary Gland Secretion

1989 ◽  
Vol 68 (1) ◽  
pp. 59-63 ◽  
Author(s):  
W. Chernick ◽  
E. Bobyock ◽  
P. Bradford
Keyword(s):  
Salivary Glands ◽  
Salivary Secretion ◽  
Threshold Concentration ◽  
Amylase Secretion ◽  
Cell Aggregates ◽  
Salivary Gland Secretion ◽  
Combined Use ◽  
Rat Parotid

5-Hydroxytryptamine (5-HT) has been reported to produce significant responses in blowfly salivary glands, but little information is available concerning its action on mammalian salivary glands. When 5-HT (0.1 μmol/L to 10 μmol/L) is infused i. a. into anesthetized rats, no salivary secretion is obtained from either parotid or submandibular glands. However, when 5-HT is infused along with a threshold concentration of acetylcholine (0-1 mmol/L), potentiation of parotid secretory response is seen with 5-HT (1 μmol/L, 260% increase; 10 μmol/L, 146% increase). Substance P (0.3 μmol/L) combined with 5-HT (1 μmol/L) also resulted in a potentiation of parotid secretion (160% increase). Protein and calcium concentrations were not altered during such treatments. No potentiation of submandibular secretion was noted. Experiments in vitro with parotid cell aggregates exhibited no potentiation associated with the combined use of 5-HT and carbachol, as measured by amylase secretion and inositol trisphosphate accumulation. The experiments indicate that 5-HT substantially modulates parotid salivary secretion in vivo; however, the in vitro findings suggest that 5-HT does not act directly on surface glandular receptors. The magnitude of the in vivo potentiation could very well implicate circulating or released 5-HT as a physiological modulator of endogenous neurotransmitter action.

Morphology of isolated crustacean larval salt glands

1985 ◽  
Vol 248 (6) ◽  
pp. R709-R716
Author(s):  
R. J. Lowy ◽  
F. P. Conte
Keyword(s):  
Plasma Membrane ◽  
Salt Gland ◽  
Artemia Salina ◽  
Apical Plasma Membrane ◽  
Secretory Cells ◽  
Cell Contact ◽  
Salt Glands ◽  
Transmission Electron

Larval salt glands isolated from the naupliar brine shrimp (Artemia salina) were examined using light microscopy and scanning and transmission electron microscopy. These methods demonstrated that most cellular and subcellular features of the in vitro organ compared favorably with those seen in vivo. This salt gland measures 130 micron in diameter and is comprised of 50-70 secretory cells, which are of a single epithelial cell type. Characteristic ultrastructural features that are well preserved include apical to basal cell polarity, apical plasma membrane projections, and the extent of the basolateral tubular labyrinth and its association with numerous mitochondria. Some features that have been altered are a decrease in cell-cell contact, separation of septate junctions, and expansion of tubular labyrinth lumens and mitochondrial cristae. Use of this preparation has allowed examination of the salt gland cell's hemocoelic surface for the first time and provided information about the ultrastructure of the tufts formed by the apical plasma membrane.

scholarly journals Membrane differentiation markers of airway epithelial secretory cells.

1988 ◽  
Vol 36 (2) ◽  
pp. 167-178 ◽  
Author(s):  
K Wasano ◽  
K C Kim ◽  
R M Niles ◽  
J S Brody
Keyword(s):  
Secretory Cell ◽  
Helix Pomatia ◽  
Apical Plasma Membrane ◽  
Secretory Cells ◽  
Membrane Glycoproteins ◽  
Differentiation Markers ◽  
Tracheal Epithelial ◽  
Spent Media

We describe here a system for culturing epithelial cells isolated from hamster trachea, which results in a highly enriched population of mucus-secreting cells. The culture system has enabled us to study the process of secretory cell differentiation in vitro. We found that epithelial secretory cells, in vivo and after 5 days in vitro, selectively bind the lectin Helix pomatia agglutinin (HPA) to apical and, to a lesser extent, basolateral surfaces as well as to mucin granules and intracellular secretory organelles. SDS-PAGE gels of detergent extracts of secretory cells cultured for 5 days reveal three HPA-binding glycoproteins with MW of 120 KD, 220 KD, and greater than 400 KD. The high-MW glycoprotein appears identical to mucin, since it is found in secretions from intact trachea and in spent media from 5-day cultures. It does not appear in spent media from 3-day cultures when cells contain few mucous granules and secrete little mucin. The 220 KD HPA-binding glycoprotein is also present in 5-day but not in 3-day cultures. In contrast, the 120 KD glycoprotein is present at both times. HPA-gp120 is a hydrophobic integral membrane protein, whereas HPA-gp220 and mucin are hydrophilic and are membrane associated. These studies define three membrane glycoproteins, one of which is specific for the tracheal epithelial secretory cell regardless of its mucous content, whereas the other two glycoproteins correlate with mucin secretion. They also demonstrate that, in the fully differentiated state, mucin is bound in a non-covalent fashion to the apical plasma membrane of the tracheal epithelial secretory cell.

312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION

2007 ◽  
Vol 19 (1) ◽  
pp. 272
Author(s):  
N. Satake ◽  
A. K. Alhaider ◽  
W. V. Holt ◽  
P. F. Watson
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Plasma Membranes ◽  
Fertilization Rate ◽  
Apical Plasma Membrane ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fertilization Rates

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P < 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P < 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.

scholarly journals The Variable Domain of a Plant Calcium-dependent Protein Kinase (CDPK) Confers Subcellular Localization and Substrate Recognition for NADPH Oxidase

2013 ◽  
Vol 288 (20) ◽  
pp. 14332-14340 ◽  
Author(s):  
Shuta Asai ◽  
Tatsushi Ichikawa ◽  
Hironari Nomura ◽  
Michie Kobayashi ◽  
Yusuke Kamiyoshihara ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Subcellular Localization ◽  
Expression System ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Golgi Network ◽  
Respiratory Burst Oxidase

Calcium-dependent protein kinases (CDPKs) are Ca2+ sensors that regulate diverse biological processes in plants and apicomplexans. However, how CDPKs discriminate specific substrates in vivo is still largely unknown. Previously, we found that a potato StCDPK5 is dominantly localized to the plasma membrane and activates the plasma membrane NADPH oxidase (RBOH; for respiratory burst oxidase homolog) StRBOHB by direct phosphorylation of the N-terminal region. Here, we report the contribution of the StCDPK5 N-terminal variable (V) domain to activation of StRBOHB in vivo using heterologous expression system in Nicotiana benthamiana. Mutations of N-terminal myristoylation and palmitoylation sites in the V domain eliminated the predominantly plasma membrane localization and the capacity of StCDPK5 to activate StRBOHB in vivo. A tomato SlCDPK2, which also contains myristoylation and palmitoylation sites in its N terminus, phosphorylated StRBOHB in vitro but not in vivo. Functional domains responsible for activation and phosphorylation of StRBOHB were identified by swapping regions for each domain between StCDPK5 and SlCDPK2. The substitution of the V domain of StCDPK5 with that of SlCDPK2 abolished the activation and phosphorylation abilities of StRBOHB in vivo and relocalized the chimeric CDPK to the trans-Golgi network, as observed for SlCDPK2. Conversely, SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB. These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs.

scholarly journals RNF141 interacts with KRAS to promote colorectal cancer progression

Oncogene ◽  
2021 ◽  
Author(s):  
Jiuna Zhang ◽  
Xiaoyu Jiang ◽  
Jie Yin ◽  
Shiying Dou ◽  
Xiaoli Xie ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Plasma Membrane ◽  
Tumor Growth ◽  
Cancer Progression ◽  
Critical Role ◽  
Ring Finger ◽  
Immunohistochemical Analysis ◽  
Bimolecular Fluorescence Complementation

AbstractRING finger proteins (RNFs) play a critical role in cancer initiation and progression. RNF141 is a member of RNFs family; however, its clinical significance, roles, and mechanism in colorectal cancer (CRC) remain poorly understood. Here, we examined the expression of RNF141 in 64 pairs of CRC and adjacent normal tissues by real-time PCR, Western blot, and immunohistochemical analysis. We found that there was more expression of RNF141 in CRC tissue compared with its adjacent normal tissue and high RNF141 expression associated with T stage. In vivo and in vitro functional experiments were conducted and revealed the oncogenic role of RNF141 in CRC. RNF141 knockdown suppressed proliferation, arrested the cell cycle in the G1 phase, inhibited migration, invasion and HUVEC tube formation but promoted apoptosis, whereas RNF141 overexpression exerted the opposite effects in CRC cells. The subcutaneous xenograft models showed that RNF141 knockdown reduced tumor growth, but its overexpression promoted tumor growth. Mechanistically, liquid chromatography-tandem mass spectrometry indicated RNF141 interacted with KRAS, which was confirmed by Co-immunoprecipitation, Immunofluorescence assay. Further analysis with bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assays showed that RNF141 could directly bind to KRAS. Importantly, the upregulation of RNF141 increased GTP-bound KRAS, but its knockdown resulted in a reduction accordingly. Next, we demonstrated that RNF141 induced KRAS activation via increasing its enrichment on the plasma membrane not altering total KRAS expression, which was facilitated by the interaction with LYPLA1. Moreover, KRAS silencing partially abolished the effect of RNF141 on cell proliferation and apoptosis. In addition, our findings presented that RNF141 functioned as an oncogene by upregulating KRAS activity in a manner of promoting KRAS enrichment on the plasma membrane in CRC.

scholarly journals Norepinephrine Protects against Methamphetamine Toxicity through β2-Adrenergic Receptors Promoting LC3 Compartmentalization

2021 ◽  
Vol 22 (13) ◽  
pp. 7232
Author(s):  
Gloria Lazzeri ◽  
Carla L. Busceti ◽  
Francesca Biagioni ◽  
Cinzia Fabrizi ◽  
Gabriele Morucci ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Light Chain ◽  
Adrenergic Receptors ◽  
Cell Damage ◽  
Protective Effects ◽  
Autophagy Flux ◽  
Brain Areas ◽  
Indirect Consequence

Norepinephrine (NE) neurons and extracellular NE exert some protective effects against a variety of insults, including methamphetamine (Meth)-induced cell damage. The intimate mechanism of protection remains difficult to be analyzed in vivo. In fact, this may occur directly on target neurons or as the indirect consequence of NE-induced alterations in the activity of trans-synaptic loops. Therefore, to elude neuronal networks, which may contribute to these effects in vivo, the present study investigates whether NE still protects when directly applied to Meth-treated PC12 cells. Meth was selected based on its detrimental effects along various specific brain areas. The study shows that NE directly protects in vitro against Meth-induced cell damage. The present study indicates that such an effect fully depends on the activation of plasma membrane β2-adrenergic receptors (ARs). Evidence indicates that β2-ARs activation restores autophagy, which is impaired by Meth administration. This occurs via restoration of the autophagy flux and, as assessed by ultrastructural morphometry, by preventing the dissipation of microtubule-associated protein 1 light chain 3 (LC3) from autophagy vacuoles to the cytosol, which is produced instead during Meth toxicity. These findings may have an impact in a variety of degenerative conditions characterized by NE deficiency along with autophagy impairment.

scholarly journals Impact of Membrane Lipids on UapA and AzgA Transporter Subcellular Localization and Activity in Aspergillus nidulans

2021 ◽  
Vol 7 (7) ◽  
pp. 514
Author(s):  
Mariangela Dionysopoulou ◽  
George Diallinas
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Aspergillus Nidulans ◽  
Membrane Lipids ◽  
Membrane Lipid ◽  
Lipid Biosynthesis ◽  
Transport Kinetics ◽  
Negative Effect ◽  
And Function

Recent biochemical and biophysical evidence have established that membrane lipids, namely phospholipids, sphingolipids and sterols, are critical for the function of eukaryotic plasma membrane transporters. Here, we study the effect of selected membrane lipid biosynthesis mutations and of the ergosterol-related antifungal itraconazole on the subcellular localization, stability and transport kinetics of two well-studied purine transporters, UapA and AzgA, in Aspergillus nidulans. We show that genetic reduction in biosynthesis of ergosterol, sphingolipids or phosphoinositides arrest A. nidulans growth after germling formation, but solely blocks in early steps of ergosterol (Erg11) or sphingolipid (BasA) synthesis have a negative effect on plasma membrane (PM) localization and stability of transporters before growth arrest. Surprisingly, the fraction of UapA or AzgA that reaches the PM in lipid biosynthesis mutants is shown to conserve normal apparent transport kinetics. We further show that turnover of UapA, which is the transporter mostly sensitive to membrane lipid content modification, occurs during its trafficking and by enhanced endocytosis, and is partly dependent on autophagy and Hect-type HulARsp5 ubiquitination. Our results point out that the role of specific membrane lipids on transporter biogenesis and function in vivo is complex, combinatorial and transporter-dependent.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

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