cGMP-dependent protein kinase regulation of a chloride channel in T84 cells

1992 ◽  
Vol 262 (5) ◽  
pp. C1304-C1312 ◽  
Author(s):  
M. Lin ◽  
A. C. Nairn ◽  
S. E. Guggino
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Chloride Channel ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
T84 Cells ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Chloride channels at the apical membrane of intestinal epithelial cells are involved in the excessive fluid secretion in diarrhea and diminished secretion in cystic fibrosis (CF). Diarrhea induced by heat-stable toxin from Escherichia coli is associated with elevated guanosine 3',5'-cyclic monophosphate (cGMP) in intestinal epithelial cells, but it is unknown whether chloride secretion is regulated by cGMP directly or via cGMP-dependent protein kinase (PKG). Single-channel recordings (inside-out excised patches) from the apical membrane of T84 cells reveal a 10-pS chloride channel with a linear current-voltage relationship, which is opened when an endogenous membrane-bound PKG is activated with ATP (1 mM) and cGMP (100 microM). Soluble PKG (200 nM) isolated from bovine lung, added to the intracellular face of patches, also opens this channel. No activation occurs with Ringer solution alone or only ATP or cGMP. Addition of nonhydrolyzable forms of ATP (AMP-PNP, 1 mM) or a combination of ATP, cGMP, plus H-8 (5 microM), an inhibitor of PKG, also does not stimulate the channel. The catalytic subunit of adenosine 3',5'-cyclic mono-phosphate-dependent protein kinase (PKA, 200 nM, with 1 mM ATP) activates a channel with similar characteristics. The 10 pS channel has a PNa/PCl ratio of 0.06, an anion selectivity of Br- (1.2) greater than Cl- (1.0) greater than I- (0.8) greater than F- (0.4), and a low affinity for the chloride channel blockers, 4,4-dinitrostilbene-2,2-disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Estrogen-Induced Apoptosis of Breast Epithelial Cells Is Blocked by NO/cGMP and Mediated by Extranuclear Estrogen Receptors

Endocrinology ◽  
2010 ◽  
Vol 151 (12) ◽  
pp. 5602-5616 ◽  
Author(s):  
Irida Kastrati ◽  
Praneeth D. Edirisinghe ◽  
Gihani T. Wijewickrama ◽  
Gregory R. J. Thatcher
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Estrogen Receptors ◽  
Cell Line ◽  
Mammary Epithelial ◽  
Dependent Protein Kinase ◽  
No Donors ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Induced Apoptosis

Estrogen action, via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cGMP. The action of 17β-estradiol (E2) at ER is known to elicit NO signaling via activation of NO synthase (NOS) in many tissues. The MCF-10A nontumorigenic, mammary epithelial cell line is genetically stable and insensitive to estrogenic proliferation. In this cell line, estrogens or NOS inhibitors alone had no significant effect, whereas in combination, apoptosis was induced rapidly in the absence of serum; the presence of inducible NOS was confirmed by proteomic analysis. The application of pharmacological agents determined that apoptosis was dependent upon NO/cGMP signaling via cyclic GMP (cGMP)-dependent protein kinase and could be replicated by inhibition of the phosphatidylinositol 3 kinase/serine-threonine kinase pathway prior to addition of E2. Apoptosis was confirmed by nuclear staining and increased caspase-3 activity in E2 + NOS inhibitor-treated cells. Apoptosis was partially inhibited by a pure ER antagonist and replicated by agonists selective for extranuclear ER. Cells were rescued from E2-induced apoptosis after NOS blockade, by NO-donors and cGMP pathway agonists; preincubation with NO donors was required. The NOS and ER status of breast cancer tissues is significant in etiology, prognosis, and therapy. In this study, apoptosis of preneoplastic mammary epithelial cells was triggered by estrogens via a rapid, extranuclear ER-mediated response, after removal of an antiapoptotic NO/cGMP/cGMP-dependent protein kinase signal.

scholarly journals Vibrio cholerae Flagellins Induce Toll-Like Receptor 5-Mediated Interleukin-8 Production through Mitogen-Activated Protein Kinase and NF-κB Activation

2008 ◽  
Vol 76 (12) ◽  
pp. 5524-5534 ◽  
Author(s):  
Lisa M. Harrison ◽  
Prasad Rallabhandi ◽  
Jane Michalski ◽  
Xin Zhou ◽  
Susan R. Steyert ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Intestinal Epithelial Cells ◽  
Mitogen Activated Protein Kinase ◽  
Intestinal Epithelial ◽  
Toll Like Receptor ◽  
T84 Cells ◽  
Mitogen Activated Protein ◽  
Toll Like Receptor 5 ◽  
Culture Supernatants

ABSTRACT Vaccine reactogenicity has complicated the development of safe and effective live, oral cholera vaccines. Δctx Vibrio cholerae mutants have been shown to induce inflammatory diarrhea in volunteers and interleukin-8 (IL-8) production in cultured intestinal epithelial cells. Bacterial flagellins are known to induce IL-8 production through Toll-like receptor 5 (TLR5). Since the V. cholerae genome encodes five distinct flagellin proteins, FlaA to FlaE, with homology to conserved TLR5 recognition regions of Salmonella FliC, we hypothesized that V. cholerae flagellins may contribute to IL-8 induction through TLR5 and mitogen-activated protein kinase (MAPK) signaling. Each purified recombinant V. cholerae flagellin induced IL-8 production in T84 intestinal epithelial cells and also induced nuclear factor kappa B (NF-κB) activation in HEK293T/TLR5 transfectants, which was blocked by cotransfection with a TLR5 dominant-negative construct, demonstrating TLR5 specificity. Supernatants derived from ΔflaAC and ΔflaEDB mutants induced IL-8 production in HT-29 intestinal epithelial cells and in HEK293T cells overexpressing TLR5, whereas ΔflaABCDE supernatants induced significantly less IL-8 production, demonstrating the contribution of multiple flagellins in IL-8 induction. NF-κB activation by ΔflaABCDE supernatants was partially restored by flaA or flaAC complementation. Western analysis confirmed the presence of V. cholerae flagellins in culture supernatants. Purified recombinant V. cholerae FlaA activated the MAPKs p38, c-jun N-terminal kinase (JNK), and extracellular regulated kinase (ERK) in T84 cells. FlaA-induced IL-8 production in T84 cells was inhibited by the p38 inhibitor in combination with either the JNK or ERK inhibitors. Collectively, these data suggest that V. cholerae flagellins are present in culture supernatants and can induce TLR5- and MAPK-dependent IL-8 secretion in host cells.

cDNA Cloning and Gene Expression of Human Type Iα cGMP-Dependent Protein Kinase

Hypertension ◽  
1996 ◽  
Vol 27 (3) ◽  
pp. 552-557 ◽  
Author(s):  
Naohisa Tamura ◽  
Hiroshi Itoh ◽  
Yoshihiro Ogawa ◽  
Osamu Nakagawa ◽  
Masaki Harada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cdna Cloning ◽  
Dependent Protein Kinase ◽  
Human Type ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia

2020 ◽  
Vol 22 (1) ◽  
pp. 52
Author(s):  
Mirja Koch ◽  
Constanze Scheel ◽  
Hongwei Ma ◽  
Fan Yang ◽  
Michael Stadlmeier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mouse Model ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Cone Photoreceptor ◽  
Photoreceptor Degeneration ◽  
Dependent Protein Kinase ◽  
Genetic Ablation ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.

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