NO increases permeability of cultured human cervical epithelia by cGMP-mediated increase in G-actin

2000 ◽  
Vol 278 (5) ◽  
pp. C942-C952 ◽  
Author(s):  
George I. Gorodeski
Keyword(s):  
Protein Kinase ◽  
Cell Size ◽  
Guanylate Cyclase ◽  
Intercellular Space ◽  
Paracellular Permeability ◽  
Dependent Protein Kinase ◽  
Lateral Intercellular Space ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Ly 83583

Human cervical epithelial cells express mRNA for the nitric oxide (NO) synthase (NOS) isoforms ecNOS, bNOS, and iNOS and release NO into the extracellular medium. NG-nitro-l-arginine methyl ester (l-NAME), an NOS inhibitor, and Hb, an NO scavenger, decreased paracellular permeability; in contrast, the NO donors sodium nitroprusside (SNP) and N-(ethoxycarbonyl)-3-(4-morpholinyl)sydnonimine increased paracellular permeability across cultured human cervical epithelia on filters, suggesting that NO increases cervical paracellular permeability. The objective of the study was to understand the mechanisms of NO action on cervical paracellular permeability. 8-Bromo-cGMP (8-BrcGMP) also increased permeability, and the effect was blocked by KT-5823 (a blocker of cGMP-dependent protein kinase), but not by LY-83583 (a blocker of guanylate cyclase). In contrast, LY-83583 and KT-5823 blocked the SNP-induced increase in permeability. Treatment with SNP increased cellular cGMP, and the effect was blocked by Hb and LY-83583, but not by KT-5823. Neither SNP nor 8-BrcGMP had modulated cervical cation selectivity. In contrast, both agents increased fluorescence from fura 2-loaded cells in the Ca2+-insensitive wavelengths, indicating that SNP and 8-BrcGMP stimulate a decrease in cell size and in the resistance of the lateral intercellular space. Neither SNP nor 8-BrcGMP had an effect on total cellular actin, but both agents increased the fraction of G-actin. Hb blocked the SNP-induced increase in G-actin, and KT-5823 blocked the 8-BrcGMP-induced increase in G-actin. On the basis of these results, it is suggested that NO acts on guanylate cyclase and stimulates an increase in cGMP; cGMP, acting via cGMP-dependent protein kinase, shifts actin steady-state toward G-actin; this fragments the cytoskeleton and renders cells more sensitive to decreases in cell size and resistance of the lateral intercellular space and, hence, to increases in permeability. These results may be important for understanding NO regulation of transcervical paracellular permeability and secretion of cervical mucus in the woman.

Regulation of human neutrophil degranulation by LY-83583 and L-arginine: role of cGMP-dependent protein kinase

1993 ◽  
Vol 265 (1) ◽  
pp. C201-C211 ◽  
Author(s):  
T. A. Wyatt ◽  
T. M. Lincoln ◽  
K. B. Pryzwansky
Keyword(s):  
Protein Kinase ◽  
Soluble Guanylate Cyclase ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
A 23187 ◽  
Neutrophil Degranulation ◽  
Dependent Protein ◽  
Ly 83583

The effects of guanosine 3',5'-cyclic monophosphate (cGMP) on the secretory response of activated human neutrophils were investigated using LY-83583, an inhibitor of soluble guanylate cyclase, and L-arginine, the precursor of nitric oxide formation. A 30% release of myeloperoxidase (MPO) and lactoferrin (LF) from the primary and specific granules, respectively, was detected by enzyme-linked immunosorbent assay in adhered neutrophils stimulated with 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 20 microM A-23187. LY-83583 (100 microM) inhibited the release of both LF and MPO after stimulation with FMLP or A-23187. Conversely, preincubation of neutrophils with 0.5 mM L-arginine augmented the release of LF and MPO in FMLP- and A-23187-stimulated cells. Concurrent with the increase in the degranulation response was an elevation of cGMP levels in L-arginine-treated cells, while stimulated cGMP levels were reduced in LY-83583-treated cells. Furthermore, cGMP-dependent protein kinase (G-kinase) activity was reduced in LY-83583-treated cells, as determined by the delay in G-kinase translocation to intermediate filaments and the inhibition of vimentin phosphorylation. Degranulation, elevation of cGMP levels, and targeting of G-kinase were also dependent on the concentration of A-23187 or FMLP. These data suggest that activators of neutrophil degranulation mediate this response through a cGMP-dependent protein kinase mechanism.

scholarly journals Relaxin Affects Smooth Muscle Biophysical Properties and Mechanical Activity of the Female Mouse Colon

Endocrinology ◽  
2015 ◽  
Vol 156 (12) ◽  
pp. 4398-4410 ◽  
Author(s):  
Roberta Squecco ◽  
Rachele Garella ◽  
Eglantina Idrizaj ◽  
Silvia Nistri ◽  
Fabio Francini ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Guanylate Cyclase ◽  
Membrane Properties ◽  
Mechanical Activity ◽  
Biophysical Properties ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

The hormone relaxin (RLX) has been reported to influence gastrointestinal motility in mice. However, at present, nothing is known about the effects of RLX on the biophysical properties of the gastrointestinal smooth muscle cells (SMCs). Other than extending previous knowledge of RLX on colonic motility, the purpose of this study was to investigate the ability of the hormone to induce changes in resting membrane potential (RMP) and on sarcolemmal ion channels of colonic SMCs of mice that are related to its mechanical activity. To this aim, we used a combined mechanical and electrophysiological approach. In the mechanical experiments, we observed that RLX caused a decay of the basal tone coupled to an increase of the spontaneous contractions, completely abolished by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one (ODQ). The electrophysiological results indicate for the first time that RLX directly affects the SMC biophysical properties inducing hyperpolarization of RMP and cycles of slow hyperpolarization/depolarization oscillations. The effects of RLX on RMP were abolished by ODQ as well as by a specific inhibitor of the cGMP-dependent protein kinase, KT5823. RLX reduced Ca2+ entry through the voltage-dependent L-type channels and modulated either voltage- or ATP-dependent K+ channels. These effects were abolished by ODQ, suggesting the involvement of the nitric oxide/guanylate cyclase pathway in the effects of RLX on RMP and ion channel modulation. These actions of RLX on membrane properties may contribute to the regulation of the proximal colon motility by the nitric oxide/cGMP/cGMP-dependent protein kinase pathway.

cGMP-dependent ADP depolymerization of actin mediates estrogen increase in cervical epithelial permeability

2000 ◽  
Vol 279 (6) ◽  
pp. C2028-C2036 ◽  
Author(s):  
George I. Gorodeski
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Epithelial Permeability ◽  
Estrogen Action ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
17Β Estradiol ◽  
Ly 83583

Estrogen increases secretion of cervical mucus in women, and the effect depends on fragmentation of the cytoskeleton. The objective of the present study was to understand the molecular mechanism of estrogen action. Treatment of human cervical epithelial cells with 17β-estradiol, sodium nitroprusside (SNP), or 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) increased cellular monomeric G-actin and decreased polymerized F-actin. The effects of estradiol were blocked by tamoxifen, by the guanylate cyclase inhibitor LY-83583, and by the cGMP-dependent protein kinase inhibitor KT-5823. The effects of SNP were blocked by LY-83583 and KT-5823, while the effects of 8-Br-cGMP were blocked only by KT-5823. Treatment with phalloidin decreased paracellular permeability and G-actin. Treatment with 17β-estradiol, SNP, or 8-Br-cGMP attenuated SNP-induced phosphorylation of [32P]adenylate NAD in vitro: tamoxifen blocked the effect of estrogen; LY-83583 blocked the effect of SNP but not that of 8-Br-cGMP, while KT-5823 blocked effects of both SNP and 8-Br-cGMP. These results indicate that estrogen, nitric oxide (NO), and cGMP stimulate actin depolymerization. A possible mechanism is NO-induced, cGMP-dependent protein kinase augmentation of ADP-ribosylation of monomeric actin.

scholarly journals Nitric Oxide Regulation of Gene Transcription via Soluble Guanylate Cyclase and Type I cGMP-dependent Protein Kinase

1999 ◽  
Vol 274 (14) ◽  
pp. 9489-9493 ◽  
Author(s):  
Soha D. Idriss ◽  
Tanima Gudi ◽  
Dareen E. Casteel ◽  
Vladimir G. Kharitonov ◽  
Renate B. Pilz ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
Gene Transcription ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Type I ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

scholarly journals Nitric oxide reduces flow-induced superoxide production via cGMP-dependent protein kinase in thick ascending limbs

2009 ◽  
Vol 296 (5) ◽  
pp. F1061-F1066 ◽  
Author(s):  
Nancy J. Hong ◽  
Jeffrey L. Garvin
Keyword(s):  
Nitric Oxide ◽  
Protein Kinase ◽  
No Synthase ◽  
Minor Role ◽  
No Production ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Ly 83583 ◽  
Reduced Flow

We have shown that increased luminal flow induces O2− and nitric oxide (NO) production in thick ascending limbs (TALs). However, the interaction of flow-stimulated NO and O2− in TALs is unclear. We hypothesized that NO inhibits flow-induced O2− production in TALs via cGMP-dependent protein kinase (PKG). We measured flow-stimulated O2− production in rat TALs using dihydroethidium in the absence and presence of l-arginine (0.3 mM), the substrate for NO synthase. The addition of l-arginine reduced flow-induced net O2− production from 68 ± 9 to 17 ± 4 AU/s ( P < 0.002). The addition of the NO synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 5 mM) in the presence of l-arginine stimulated production (l-arginine: 15 ± 4 AU/s vs. l-arginine + l-NAME: 63 ± 7 AU/s; P < 0.002). The guanylate cyclase inhibitor LY-83583 (10 μM) also enhanced flow-induced net O2− production in the presence of l-arginine (l-arginine: 7 ± 4 AU/s vs. l-arginine + LY-83583: 53 ± 7 AU/s; P < 0.01). In the presence of LY-83583, l-arginine only reduced flow-induced net O2− by 36% (LY-83583: 80 ± 7 AU/s vs. LY-83583 + l-arginine: 51 ± 3 AU/s; P < 0.006). The cGMP analog dibutyryl (db)-cGMP reduced flow-induced net O2− from 39 ± 9 to 7 ± 3 AU/s ( P < 0.03). The PKG inhibitor KT-5823 (5 μM) partially restored flow-induced net O2− in the presence of l-arginine (l-arginine: 4 ± 4 AU/s vs. l-arginine + KT-5823: 32 ± 9 AU/s; P < 0.03) and db-cGMP (db-cGMP: 9 ± 7 AU/s vs. db-cGMP + KT-5823: 54 ± 5 AU/s; P < 0.01). Phosphodiesterase II inhibition had no effect on arginine-inhibited O2− production. We conclude that 1) NO reduces flow-stimulated O2− production, 2) this occurs primarily via the cGMP/PKG pathway, and 3) O2− scavenging by NO plays a minor role.

scholarly journals Aortic disease in Marfan syndrome is caused by overactivation of sGC-PRKG signaling by NO

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Andrea de la Fuente-Alonso ◽  
Marta Toral ◽  
Alvaro Alfayate ◽  
María Jesús Ruiz-Rodríguez ◽  
Elena Bonzón-Kulichenko ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Marfan Syndrome ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Aortic Tissue ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
No Donors ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein

AbstractThoracic aortic aneurysm, as occurs in Marfan syndrome, is generally asymptomatic until dissection or rupture, requiring surgical intervention as the only available treatment. Here, we show that nitric oxide (NO) signaling dysregulates actin cytoskeleton dynamics in Marfan Syndrome smooth muscle cells and that NO-donors induce Marfan-like aortopathy in wild-type mice, indicating that a marked increase in NO suffices to induce aortopathy. Levels of nitrated proteins are higher in plasma from Marfan patients and mice and in aortic tissue from Marfan mice than in control samples, indicating elevated circulating and tissue NO. Soluble guanylate cyclase and cGMP-dependent protein kinase are both activated in Marfan patients and mice and in wild-type mice treated with NO-donors, as shown by increased plasma cGMP and pVASP-S239 staining in aortic tissue. Marfan aortopathy in mice is reverted by pharmacological inhibition of soluble guanylate cyclase and cGMP-dependent protein kinase and lentiviral-mediated Prkg1 silencing. These findings identify potential biomarkers for monitoring Marfan Syndrome in patients and urge evaluation of cGMP-dependent protein kinase and soluble guanylate cyclase as therapeutic targets.

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