SK4/IK1-like channels mediate TEA-insensitive, Ca2+-activated K+ currents in bovine parotid acinar cells

2003 ◽  
Vol 284 (1) ◽  
pp. C127-C144 ◽  
Author(s):  
T. Takahata ◽  
M. Hayashi ◽  
T. Ishikawa
Keyword(s):  
Channel Blocker ◽  
Acinar Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Parotid Acinar Cells ◽  
Whole Cell Recordings ◽  
Pcr Techniques ◽  
Inside Out ◽  
Molecular Nature ◽  
K Currents

Although Ca2+-activated K+ (KCa) channels distinct from maxi-K+ channels have been suggested to contribute to muscarinically stimulated K+ currents in salivary acinar cells, the molecular nature of the channels is unclear. Using electrophysiological and RT-PCR techniques, we have now investigated the involvement of SK4/IK1-like channels in native KCacurrents in bovine parotid acinar (BPA) cells. Ca2+-dependent K+ efflux from perfused bovine parotid tissues was not inhibited by a maxi-K+ channel blocker, tetraethylammonium (TEA). Whole cell recordings from BPA cells showed a TEA-insensitive KCa conductance, which was highly permeable to Rb+. In inside-out macropatches, TEA-insensitive Rb+ currents were activated by Ca2+ with half-maximal values of 0.4 μM. 1-Ethyl-2-benzimidazolinone (1-EBIO) increased the Ca2+sensitivity of the currents. The calmodulin antagonists trifluoperazine, calmidazolium, and W-7 inhibited the Ca2+-activated Rb+ currents. In outside-out macropatches, Ca2+-activated Rb+ currents were inhibited by Ba2+, quinine, clotrimazole, and charybdotoxin but not by d-tubocrarine or apamin. RT-PCR analysis showed transcripts of SK4/IK1 in BPA cells. These results collectively suggest that SK4/IK1-like channels mediate the native KCa currents in BPA cells.

Molecular identification of Ca2+-activated K+ channels in parotid acinar cells

2003 ◽  
Vol 284 (2) ◽  
pp. C535-C546 ◽  
Author(s):  
Keith Nehrke ◽  
Claire C. Quinn ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Acinar Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Parotid Acinar Cells ◽  
Large Conductance Channel ◽  
Intracellular Ca

We used molecular biological and patch-clamp techniques to identify the Ca2+-activated K+ channel genes in mouse parotid acinar cells. Two types of K+ channels were activated by intracellular Ca2+ with single-channel conductance values of 22 and 140 pS (in 135 mM external K+), consistent with the intermediate and maxi-K classes of Ca2+-activated K+ channels, typified by the mIK1 ( Kcnn4) and mSlo ( Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo β-subunits ( Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each β-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from β1 knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the β4-subunit.

Pancreatic acinar cells express IL-6, KC, MCP-1, and MIP-2: Evidence from RT-PCR analysis of microscopically isolated acinar cells

1998 ◽  
Vol 114 ◽  
pp. A443
Author(s):  
T. Blinman ◽  
A. Gukovskaya ◽  
I. Gukovsky ◽  
V. Zaninovic ◽  
E. Livingston ◽  
...  
Keyword(s):  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Pcr Analysis ◽  
Rt Pcr

Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells

2002 ◽  
Vol 363 (1) ◽  
pp. 59-66 ◽  
Author(s):  
Akihiro NEZU ◽  
Akihiko TANIMURA ◽  
Takao MORITA ◽  
Kazuharu IRIE ◽  
Toshihiko YAJIMA ◽  
...  
Keyword(s):  
Ryanodine Receptors ◽  
Critical Role ◽  
Acinar Cells ◽  
Ion Concentration ◽  
Pcr Analysis ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Weak Band ◽  
Parotid Acinar Cells ◽  
Rat Parotid

Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the β-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca2+ signalling as intracellular Ca2+ stores, changes in cytosolic free Ca2+ ion concentration ([Ca2+]i) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca2+]i induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca2+ wave towards the basolateral region. The magnitude of the [Ca2+]i response and the speed of the Ca2+ wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP3R) was performed on zymogen granule membranes and microsomes using anti-IP3R antibodies. The immunoreactivity of all three IP3Rs was clearly observed in the microsomal preparations. Although a weak band of IP3R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca2+ signalling in rat parotid acinar cells.

Single cell RT-PCR analysis of ClC-2 mRNA expression in ureteric bud tip

1998 ◽  
Vol 274 (5) ◽  
pp. F951-F957 ◽  
Author(s):  
Stephan Huber ◽  
Bernd Schröppel ◽  
Matthias Kretzler ◽  
Detlef Schlöndorff ◽  
Michael Horster
Keyword(s):  
Patch Clamp ◽  
Single Cell ◽  
Collecting Duct ◽  
Pcr Analysis ◽  
Ureteric Bud ◽  
Rt Pcr ◽  
Outer Cortex ◽  
Mrna Isoforms ◽  
Pcr Products ◽  
Pcr Techniques

Embryonic epithelia at the tip of the ureteric bud (UB) face the interspace between epithelial and mesenchymal cells and are fundamentally involved in reciprocal signaling during early nephrogenesis. To characterize their membrane conductive proteins, patch-clamp and single cell RT-PCR techniques were applied to embryonic rat UBs [embryonic day 17( day E17)] microdissected from the outer cortex. Cells at the UB tip had a high whole cell conductance (14 ± 2 nS/10 pF, n = 8). The main fractional conductance resembled that of Ca-activated Cl channels in nonepithelial cells, with its time-dependent activation at depolarizing and inactivation at hyperpolarizing voltages. A second Cl-selective current fraction, by contrast, activated slowly during strong hyperpolarization, suggestive of a ClC-2-mediated conductance. To determine the origin of this current, cytoplasm was harvested into the patch pipette, RNA was reverse transcribed, and cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeper gene or the ClC-2 Cl channel was amplified by polymerase chain reaction (PCR). GAPDH and ClC-2 PCR products were identified in 23 and 8 (out of a total of 57) single cell cDNA samples, respectively. ClC-2 PCR products with two different lengths were obtained, which might be due to two alternatively spliced ClC-2 mRNA isoforms. This first and combined approach by patch-clamp and single cell RT-PCR techniques to embryonic epithelia indicates that 1) cells at the UB tip express a phenotype remarkably different from that of postembryonic collecting duct principal cells and that 2) ClC-2 is likely to have a key role in early nephrogenesis.

scholarly journals Apical Ca2+-activated potassium channels in mouse parotid acinar cells

2012 ◽  
Vol 139 (2) ◽  
pp. 121-133 ◽  
Author(s):  
Janos Almassy ◽  
Jong Hak Won ◽  
Ted B. Begenisich ◽  
David I. Yule
Keyword(s):  
Plasma Membrane ◽  
Digital Imaging ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
Bk Channels ◽  
Apical Plasma Membrane ◽  
K Channels ◽  
Parotid Acinar Cells ◽  
Cl Channels ◽  
K Currents

Ca2+ activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca2+] was used to investigate if Ca2+-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca2+-buffering conditions that produced brief, localized increases in [Ca2+] after focal laser photolysis of caged Ca2+. Conditions were used to isolate K+ and Cl− conductances. Photolysis at the apical PM resulted in a robust increase in K+ and Cl− currents. A localized reduction in [Ca2+] at the apical PM after photolysis of Diazo-2, a caged Ca2+ chelator, resulted in a decrease in both K+ and Cl− currents. The K+ currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance “maxi-K” (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34–sensitive K+ currents were also observed in BK-null parotid acini. In contrast, when the [Ca2+] was increased at the basal or lateral PM, no increase in either K+ or Cl− currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.

scholarly journals Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

2021 ◽  
Vol 11 (13) ◽  
pp. 5776
Author(s):  
Varvara G. Blinova ◽  
Natalia S. Novachly ◽  
Sofya N. Gippius ◽  
Abdullah Hilal ◽  
Yulia A. Gladilina ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Inflammatory Reactions ◽  
Effector Cells ◽  
Cycle Regulation ◽  
Further Development

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

scholarly journals Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption

2021 ◽  
Vol 9 (1) ◽  
pp. 7
Author(s):  
Yusuke Makino ◽  
Kaoru Fujikawa ◽  
Miwako Matsuki-Fukushima ◽  
Satoshi Inoue ◽  
Masanori Nakamura
Keyword(s):  
Bacterial Infections ◽  
Tooth Movement ◽  
Tooth Eruption ◽  
Intercellular Adhesion Molecule ◽  
Enamel Organ ◽  
Pcr Analysis ◽  
Maturation Stage ◽  
Oral Epithelium ◽  
Rt Pcr ◽  
Inflammatory Reactions

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1β, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

scholarly journals RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)

2005 ◽  
Vol 86 (12) ◽  
pp. 3419-3424 ◽  
Author(s):  
Constanze Yue ◽  
Elke Genersch
Keyword(s):  
Varroa Destructor ◽  
Pcr Analysis ◽  
Body Parts ◽  
Rt Pcr ◽  
Larval Food ◽  
Viral Pathogen ◽  
Deformed Wing Virus ◽  
Significant Difference ◽  
Almost All ◽  
Viral Sequences

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

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