scholarly journals Dual mechanism for cAMP-dependent modulation of Ca2+ signalling in articular chondrocytes

1996 ◽  
Vol 318 (2) ◽  
pp. 569-573 ◽  
Author(s):  
Paola D'ANDREA ◽  
Valentina PASCHINI ◽  
Franco VITTUR
Keyword(s):  
Protein Kinase ◽  
Primary Culture ◽  
Cell Population ◽  
Articular Chondrocytes ◽  
Kinase Inhibitor ◽  
Free Medium ◽  
Dependent Protein Kinase ◽  
Fura 2 ◽  
Dependent Protein ◽  
Dual Mechanism

The ability of cAMP to modulate the actions of Ca2+-mobilizing agonists was studied in single Fura-2-loaded pig articular chondrocytes in primary culture. Forskolin and 8-Br-cAMP increased both the frequency and amplitude of Ca2+ oscillations induced by ATP, and, in unstimulated cells, induced single Ca2+ transients or even Ca2+ oscillations. The cAMP-dependent protein kinase inhibitor H89 totally prevented the effect of cAMP-elevating agents on Ca2+ signalling. Forskolin and 8-Br-cAMP promptly increased the rate of Mn2+ quenching, when administered in the presence of ATP, suggesting a potentiation of receptor-mediated Ca2+ influx. In Ca2+-free medium, ATP-induced Ca2+ oscillations decreased and stopped after a few cycles: subsequent ATP additions temporarily resumed the activity, an effect that could be mimicked by forskolin. The same agent induced single Ca2+ transients in 42% of the cell population maintained in Ca2+-free medium. Thapsigargin prevented Ca2+ responses to both ATP and forskolin. The results indicate a dual mechanism for cAMP-induced potentiation of Ca2+ signalling in articular chondrocytes: an increase of receptor-mediated Ca2+ influx and a positive modulation of intracellular Ca2+ release.

scholarly journals ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

1989 ◽  
Vol 94 (4) ◽  
pp. 693-717 ◽  
Author(s):  
B Ribalet ◽  
S Ciani ◽  
G T Eddlestone
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Insulin Secreting Cells ◽  
Dependent Protein ◽  
Inside Out

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

scholarly journals The Malaria Parasite Cyclic GMP-Dependent Protein Kinase Plays a Central Role in Blood-Stage Schizogony

2009 ◽  
Vol 9 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Helen M. Taylor ◽  
Louisa McRobert ◽  
Munira Grainger ◽  
Audrey Sicard ◽  
Anton R. Dluzewski ◽  
...  
Keyword(s):  
Life Cycle ◽  
Protein Kinase ◽  
Cyclic Gmp ◽  
Malaria Parasite ◽  
Kinase Inhibitor ◽  
Blood Stage ◽  
Parasite Life Cycle ◽  
Dependent Protein Kinase ◽  
Asexual Blood Stage ◽  
Dependent Protein

ABSTRACT A role for the Plasmodium falciparum cyclic GMP (cGMP)-dependent protein kinase (PfPKG) in gametogenesis in the malaria parasite was elucidated previously. In the present study we examined the role of PfPKG in the asexual blood-stage of the parasite life cycle, the stage that causes malaria pathology. A specific PKG inhibitor (compound 1, a trisubstituted pyrrole) prevented the progression of P. falciparum schizonts through to ring stages in erythrocyte invasion assays. Addition of compound 1 to ring-stage parasites allowed normal development up to 30 h postinvasion, and segmented schizonts were able to form. However, synchronized schizonts treated with compound 1 for ≥6 h became large and dysmorphic and were unable to rupture or liberate merozoites. To conclusively demonstrate that the effect of compound 1 on schizogony was due to its selective action on PfPKG, we utilized genetically manipulated P. falciparum parasites expressing a compound 1-insensitive PfPKG. The mutant parasites were able to complete schizogony in the presence of compound 1 but not in the presence of the broad-spectrum protein kinase inhibitor staurosporine. This shows that PfPKG is the primary target of compound 1 during schizogony and provides direct evidence of a role for PfPKG in this process. Discovery of essential roles for the P. falciparum PKG in both asexual and sexual development demonstrates that cGMP signaling is a key regulator of both of these crucial life cycle phases and defines this molecule as an exciting potential drug target for both therapeutic and transmission blocking action against malaria.

Relaxin stimulates atrial natriuretic peptide secretion in perfused rat heart

1996 ◽  
Vol 150 (3) ◽  
pp. 487-495 ◽  
Author(s):  
M Toth ◽  
P Taskinen ◽  
H Ruskoaho
Keyword(s):  
Heart Rate ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Rat Heart ◽  
Perfusion Pressure ◽  
Kinase Inhibitor ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Heart Preparation

Abstract Relaxin, a reproductive hormone of the insulin-like growth factor family, increases heart rate in experimental animals but its other actions on cardiac function and cellular mechanisms responsible for the positive chronotrophic effect remain unknown. We have studied the actions of human recombinant gene-2 relaxin on the release of atrial natriuretic peptide (ANP) and cardiac function (heart rate, contractile force, perfusion pressure) as well as the underlying signal transduction mechanisms by using the isolated perfused spontaneously beating rat heart preparation. The administration of relaxin into the perfusion fluid at concentrations of 1·5, 3 or 10 nm for 30 min caused a dose-dependent sustained increase in heart rate, while contractile force and perfusion pressure remained unchanged. In addition, infusion of relaxin at a concentration of 10 nm into the perfusate produced a gradual 1·5-fold increase in immunoreactive ANP (IR-ANP) secretion (from 456 ± 76 to 701 ± 124 pg/ml, F=4·5, P<0·001). The ANP secretory and chronotrophic effects of relaxin appear to involve the activation of protein kinase C, since administration of a protein kinase C inhibitor staurosporine at a concentration of 30 nm completely blocked the effect of relaxin (10 nm) on IR-ANP secretion P<0·001) and heart rate (P<0·001). A cAMP-dependent protein kinase inhibitor, H-89 (100 nm), also substantially reduced the ANP secretory effect of relaxin and attenuated the increase in heart rate during the sustained phase of the relaxin infusion (P<0·001). KN-62 (3 μm), a Ca2+/calmodulin-dependent protein kinase inhibitor, decreased the positive chronotrophic effect of relaxin (P<0·001) but did not influence significantly the effect of relaxin on IR-ANP release in isolated perfused rat heart preparation. These results provide the first evidence that relaxin stimulates the secretion of ANP from isolated perfused rat hearts. Our results also suggest that relaxin modulates ANP secretion by activation of protein kinase C and cAMP-dependent protein kinase pathways. Journal of Endocrinology (1996) 150, 487–495

scholarly journals Downregulation of cAMP-Dependent Protein Kinase Inhibitor-b Promotes Preeclampsia by Decreasing Phosphorylated Akt

2020 ◽  
Vol 28 (1) ◽  
pp. 178-185
Author(s):  
Chunfeng Liu ◽  
Hao Wang ◽  
Mo Yang ◽  
Yiheng Liang ◽  
Li Jiang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Matrix Metalloproteinase ◽  
Signaling Pathway ◽  
Kinase Inhibitor ◽  
Akt Signaling ◽  
Migration And Invasion ◽  
Akt Signaling Pathway ◽  
Dependent Protein Kinase ◽  
Phosphorylated Akt ◽  
Dependent Protein

AbstractPreeclampsia is a multi-system disease that is unique to human pregnancy. Impaired extravillous trophoblast migration and invasion accompanied by poor spiral vascular remodeling is thought to be the initial reason. This study investigated cAMP-dependent protein kinase inhibitor-b(PKIB) expression in placentas and its involvement in the pathogenesis of PE. We used immunohistochemistry and western blotting to calculate PKIB levels in the placentas. Then we knocked down PKIB by siRNA and used real-time cell analysis to assess the invasion and migration ability of trophoblasts. Tube formation assay and spheroid sprouting assay were utilized to identify the ability to form vessels of trophoblasts. At last, western blotting was used to demonstrate the level of phosphorylated Akt, as well as downstream-related genes of Akt signaling pathway in trophoblasts. We first found that PKIB expression level was lower in the PE placentas than in the normal placentas. In addition, we found that downregulation of PKIB can inhibit the migration, invasion, and the ability to form vessels of HTR8/SVneo cells. Downregulation of PKIB leaded to a decrease in phosphorylated Akt, as well as downstream proteins such as matrix metalloproteinase 2, matrix metalloproteinase 9, and glycogen synthase kinase 3β, which are related to migration and invasion. Our study revealed that the downregulation of PKIB expression resulted in decreased migration, invasion, and vessel formation ability by regulating Akt signaling pathway in placental trophoblasts in PE.

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