Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells

Poronnik, Philip; O’Mullane, Lauren M; Harding, E. A.; Greger, R.; Cook, David I.

doi:10.1016/s0143-4160(98)90077-x

Cited by 16 publications

(32 citation statements)

References 33 publications

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“…The replication-deficient adenovirus, MX-17 (the gift of G. W. Both, Commonwealth Scientific and Industrial Research Organization, Division of Molecular Sciences, North Ryde, New South Wales), was grown and titered as described previously (23). It contains a null insert and does not express a transgene.…”

Section: Methodsmentioning

confidence: 99%

Influenza virus inhibits amiloride-sensitive Na⁺channels in respiratory epithelia

Kunzelmann

Beesley

King

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

109

124

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Many pathogens causing diarrhea do so by modulating ion transport in the gut. Respiratory pathogens are similarly associated with disturbances of fluid balance in the respiratory tract, although it is not known whether they too act by altering epithelial ion transport. Here we show that influenza virus A͞PR͞8͞34 inhibits the amiloride-sensitive Na ؉ current across mouse tracheal epithelium with a half-time of about 60 min. We further show that the inhibitory effect of the influenza virus is caused by the binding of viral hemagglutinin to a cell-surface receptor, which then activates phospholipase C and protein kinase C. Given the importance of epithelial Na ؉ channels in controlling the amount of fluid in the respiratory tract, we suggest that down-regulation of Na ؉ channels induced by influenza virus may play a role in the fluid transport abnormalities that are associated with influenza infections.hemagglutinin ͉ protein kinase C ͉ ENaC ͉ phospholipase C

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Section: Methodsmentioning

confidence: 99%

Influenza virus inhibits amiloride-sensitive Na⁺channels in respiratory epithelia

Kunzelmann

Beesley

King

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

109

124

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“…After a cytopathic effect was observed, the crude viral lysate was harvested and the viruses characterized. CsCl-purified virus stocks were subsequently prepared and cells infected prior to experimentation as described previously (18).…”

Section: Methodsmentioning

confidence: 99%

The Distribution of P2X Receptor Clusters on Individual Neurons in Sympathetic Ganglia and Their Redistribution on Agonist Activation

Li¹,

Lee²,

Blair³

et al. 2000

Journal of Biological Chemistry

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The distribution of P2X receptors on neurons in rat superior cervical ganglia and lability of P2X receptors on exposure to agonists were determined. Antibody labeling of each P2X subtype P2X 1 -P2X 7 showed neurons isolated into culture possessed primarily P2X 2 subunits with others occurring in order P2X 7 > P2X 6 > P2X 3 > P2X 1 > P2X 5 > P2X 4 . Application of ATP and ␣,␤؊meATP to neurons showed they possessed a predominantly nondesensitizing P2X receptor type insensitive to ␣,␤؊ meATP, consistent with immunohistochemical observations. P2X 1 -green fluorescent protein (GFP) was used to study the time course of P2X 1 receptor clustering in plasma membranes of neurons and internalization of receptors following prolonged exposure to ATP. At 12-24 h after adenoviral infection, P2X 1 -GFP formed clusters about 1 m diameter in the neuron membrane. Application of ATP and ␣,␤؊meATP showed these neurons possessed a predominantly desensitizing P2X receptor type sensitive to ␣,␤؊meATP. Infection converted the major functional P2X receptor type in the membrane to P2X 1 . Exposure of infected neurons to ␣,␤؊meATP for less than 60 s led to the disappearance of P2X 1 -GFP fluorescence from the cell surface that was blocked by monensin, indicating the chimera is normally endocytosed into these organelles on exposure to agonist.P2X-type purinergic receptors consist of seven subtypes, classified as P2X 1 to P2X 7 , which form ligand-gated cation channels that contain cytoplasmic N and C termini and a large extracellular domain separating the two transmembrane segments (1). Antibodies against peptides from the C-terminal portion of the P2X 1 subtype have been used to determine the distribution of P2X 1 receptors in the vas deferens and urinary bladder, where they are found on the smooth muscle cells (2). Recently it has been shown that different P2X receptor subtypes form clusters of two different sizes on the smooth muscle cells of the rat urinary bladder, vas deferens, and blood vessels: large numbers of small clusters of about 0.4-m diameter distributed over the smooth muscle and small numbers of large clusters greater than 1-m diameter positioned under junctional varicosities (3-5). The large junctional P2X clusters can be observed during normal development to be formed from small clusters of a number of different subtypes, which gather in large numbers in the vicinity of the nerve varicosities (6). Sympathetic ganglion cells were reported to possess P2X receptors, primarily of the P2X 2 , P2X 4 , and P2X 6 subtypes (7), and transcripts for P2X 1 , P2X 2 , P2X 4 , and P2X 6 are present (8), although it is not known whether these receptors are organized into large and small clusters and if so whether the large clusters are found beneath boutons as expected if they mediate some aspect of transmission. In the present work, both large and small clusters of different P2X subunit types are shown to exist on ganglion cells, and their spatial disposition with respect to preganglionic boutons has been ascertained.The existence of P2...

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“…Once the recombinant adenoviruses are made and purified, the major advantages of this system are the high yields of protein expression over a shorter time period and small volumes of culture media to process which leads to a more time-and cost-effective procedure. The high efficiency of adenoviruses in infecting all target cells results in the expression of the transgene in at least 95% of the cell population (13). Another advantage of the system is that 911 cells do not secrete any IGFBPs, as judged by a lack of signal when medium conditioned by uninfected cells was analyzed by 125 I-IGF-II ligand blot (data not shown), which could present as contaminants during the purification process.…”

Section: Discussionmentioning

confidence: 98%

“…Although this study has concentrated solely on the overexpression of IGFBP-3, the availability of IGFBP-3-expressing adenoviruses will enable the infection of nonpermissive cell lines resulting in a highly efficient transient expression system. Cell function can be altered by the expression of exogenous genes introduced by the infection of replication-deficient, recombinant adenoviruses, for example, the expression of aquaporin-5 in epithelial cells (39) and the expression of a dominant negative G protein subunit in salivary gland cells (13). Such a system could be exploited for studies into the cellular actions of IGFBP-3.…”

Section: Fig 6 Comparison Of Binding To T47d Human Breast Cancer Cementioning

confidence: 99%

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Adenoviral-Mediated Expression of Human Insulin-like Growth Factor-Binding Protein-3

Firth

Ganeshprasad

Poronnik

et al. 1999

Protein Expression and Purification

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Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells

Cited by 16 publications

References 33 publications

Influenza virus inhibits amiloride-sensitive Na⁺channels in respiratory epithelia

Influenza virus inhibits amiloride-sensitive Na⁺channels in respiratory epithelia

The Distribution of P2X Receptor Clusters on Individual Neurons in Sympathetic Ganglia and Their Redistribution on Agonist Activation

Adenoviral-Mediated Expression of Human Insulin-like Growth Factor-Binding Protein-3

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Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells

Cited by 16 publications

References 33 publications

Influenza virus inhibits amiloride-sensitive Na+channels in respiratory epithelia

Influenza virus inhibits amiloride-sensitive Na+channels in respiratory epithelia

The Distribution of P2X Receptor Clusters on Individual Neurons in Sympathetic Ganglia and Their Redistribution on Agonist Activation

Adenoviral-Mediated Expression of Human Insulin-like Growth Factor-Binding Protein-3

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Influenza virus inhibits amiloride-sensitive Na⁺channels in respiratory epithelia

Influenza virus inhibits amiloride-sensitive Na⁺channels in respiratory epithelia