scholarly journals Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3106
Author(s):  
Syevda Tagirova Sirenko ◽  
Ihor Zahanich ◽  
Yue Li ◽  
Yevgeniya O. Lukyanenko ◽  
Alexey E. Lyashkov ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Firing Rate ◽  
Protein Phosphorylation ◽  
Cardiac Pacemaker ◽  
Dependent Manner ◽  
Pacemaker Cells ◽  
Action Potential Firing ◽  
Concentration Dependent Manner ◽  
Dependent Protein ◽  
The Impact

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

The roles of adenosine 3′,5′-cyclic monophosphate-dependent protein kinase A and protein kinase C in stimulus-secretion coupling in AtT-20 cells

1996 ◽  
Vol 16 (2) ◽  
pp. 133-140 ◽  
Author(s):  
B W McFerran ◽  
S B Guild
Keyword(s):  
Protein Kinase ◽  
Calcium Ions ◽  
Dependent Manner ◽  
Acth Secretion ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Dependent Protein ◽  
Messenger System ◽  
Stimulus Secretion Coupling ◽  
Dependent Pathway

ABSTRACT The ACTH-secreting mouse AtT-20/D16–16 anterior pituitary tumour cell line was used to study adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent protein kinase A (PKA) and protein kinase C (PKC) involvement in stimulus-secretion coupling pathways. In permeabilised AtT-20 cells under calcium ion-free conditions, forskolin (10 μm), CRH-41 (100 nm), guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S; 100 μm) but not mastoparan (10 μm) stimulated cAMP accumulation. Measurement of ACTH secretion under identical incubation conditions revealed that GTP-γ-S and mastoparan significantly stimulated ACTH secretion but forskolin and CRH-41 did not. This dissociates cAMP accumulation from ACTH secretion under calcium ion-free conditions and indicated that the effects of mastoparan and GTP-γ-S on ACTH secretion are not mediated by cAMP production. Calcium ions (1 nm to 1 mm) stimulated ACTH secretion from electrically permeabilised cells in a concentration-dependent manner. cAMP (100μm) and phorbol 12-myristate 13-acetate (PMA; 100 nm) synergistically enhanced the response to calcium ions. cAMP did not stimulate ACTH secretion in the absence of calcium ions nor did it alter the concentrations at which calcium stimulated ACTH secretion. This suggests that stimulation of ACTH secretion via the calcium-dependent pathway is necessary before any cAMP-mediated enhancement of secretion is manifest. PMA, however, did stimulate ACTH secretion in the absence of calcium ions, indicating distinct mechanisms for PKC-evoked secretion. Co-incubation with cAMP and PMA did not exceed the secretory response obtained with the combination of PMA and calcium ions. CRH-41 (1 pm to 100 nm) and forskolin (1 nm to 100μm) stimulated ACTH secretion from intact cells in a concentration-dependent manner. Co-incubation with PMA (100 nm) further enhanced the ACTH response to CRH-41 and forskolin; the effects were simply additive. The present study indicates that there are distinct roles for PKA and PKC in stimulussecretion coupling in AtT-20 cells. The PKA-dependent pathway, acting in concert with the calcium messenger system, serves as part of the stimulus-secretion coupling pathway by which activation of CRH-41 receptors control ACTH secretion. The PKC-dependent pathway, in contrast, seems to be independent of the calcium messenger system and may represent a separate control mechanism of ACTH secretion.

Normal spontaneous firing of cardiac pacemaker cells is regulated by basal pkc delta activation

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T Vinogradova ◽  
K Tarasov ◽  
D Riordon ◽  
Y Tarasova ◽  
E Lakatta
Keyword(s):  
Protein Kinase ◽  
Cycle Length ◽  
Cardiac Pacemaker ◽  
Funding Source ◽  
Spontaneous Firing ◽  
Pacemaker Cells ◽  
Patch Clamp Technique ◽  
Sa Node ◽  
Pkc Delta ◽  
Pkc Isoforms

Abstract   The spontaneous beating rate of rabbit sinoatrial node cells (SANC) is regulated by local subsarcolemmal calcium releases (LCRs) from sarcoplasmic reticulum (SR). LCRs appear during diastolic depolarization (DD) and activate an inward sodium/calcium exchange current which increases DD rate and thus accelerates spontaneous SANC firing. High basal level of protein kinase A and calcium/calmodulin-dependent protein kinase II phosphorylation are required to sustain basal LCRs and normal spontaneous SANC firing. Recently we discovered that basal PKC activation is also obligatory for cardiac pacemaker function: inhibition of PKC activity by broad spectrum PKC inhibitors Bis I or calphostin C markedly suppressed SR calcium cycling and decreased or abolished spontaneous beating of freshly isolated rabbit SANC. Here we studied which PKC isoforms mediate PKC-dependent effects on cardiac pacemaker cell automaticity. The PKC superfamily consists of 3 major subgroups: conventional, novel and atypical. All PKC isoforms were detected at the RNA level (RT-qPCR) in the rabbit SA node and ventricle, and expression levels were comparable in both tissues. Expression of PKCβ, however, was markedly higher in the rabbit SA node, compared to other PKC isoenzymes in either tissue. We verified expression of conventional PKC (α, β) and novel PKC-delta at the protein level in SANC and ventricular myocytes (VM). Western blot confirmed RNA results, showing a 6-fold higher PKCβ protein abundance in SANC compared to VM. Expression of PKCα protein was similar in both cell types, while PKC-delta protein was more abundant in VM. To study whether PKCβ regulates spontaneous beating of SANC we employed selective inhibitor of conventional (α, β, gamma) PKC isoforms Go6976 (10 μmol/L), which had no effects on either LCR characteristics (confocal microscopy, calcium indicator Fluo-3AM) or spontaneous beating of freshly isolated rabbit SANC (perforated patch-clamp technique). Because selective PKC-delta inhibitors are not available, we explored effects of PKC-delta inhibition comparing effects of Go6976 (the inhibitor of conventional PKCs) and Go6983, which inhibits conventional PKCs and PKC-delta. In contrast to Go6976, Go6983 (5 μmol/L) markedly decreased the LCR size (from 7.1±0.4 to 4.5±0.3 μm) and number per each spontaneous cycle (from 1.3±0.1 to 0.8±0.1). It also markedly increased the LCR period (time from the prior AP-induced calcium transient to the subsequent LCR) which was paralleled by an increase in the spontaneous SANC cycle length. Rottlerin, another PKC-delta inhibitor, produced similar effects on LCR characteristics, and markedly and time-dependently decreased DD rate, leading to an increase in the spontaneous cycle length, and finally abrogated the spontaneous SANC firing. Thus, our data indicate that basal activity of PKC-delta, but not that of PKCβ, is essential for generation of LCRs and normal spontaneous firing of cardiac pacemaker cells. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Intramural Research Program, National Institute on Aging, National Institute of Health, USA

Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 309-316 ◽  
Author(s):  
Carsten Krischek ◽  
Burkhard Meinecke
Keyword(s):  
Map Kinase ◽  
Protein Kinases ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Histone H1 ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

scholarly journals Ca2+-induced insulin secretion from electrically permeabilized islets. Loss of the Ca2+-induced secretory response is accompanied by loss of Ca2+-induced protein phosphorylation

1992 ◽  
Vol 285 (3) ◽  
pp. 973-978 ◽  
Author(s):  
P M Jones ◽  
S J Persaud ◽  
S L Howell
Keyword(s):  
Protein Kinase C ◽  
Insulin Secretion ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Protein Phosphorylation ◽  
Prolonged Exposure ◽  
Kinase C ◽  
Dependent Protein ◽  
32P Incorporation ◽  
Rat Islets Of Langerhans

Increasing the cytosolic Ca2+ concentration of electrically permeabilized rat islets of Langerhans caused rapid increases in insulin secretion and in 32P incorporation into islet proteins. However, the secretory responsiveness of permeabilized islets was relatively transient, with insulin secretion approaching basal levels within 20-30 min despite the continued presence of stimulatory concentrations of Ca2+. The loss of Ca2(+)-induced insulin secretion was accompanied by a marked reduction in Ca2(+)-dependent protein phosphorylation, but not in cyclic AMP-dependent protein phosphorylation. Similarly, permeabilized islets which were no longer responsive to Ca2+ were able to mount appropriate secretory responses to cyclic AMP and to a protein kinase C-activating phorbol ester. These results suggest that prolonged exposure to elevated cytosolic Ca2+ concentrations results in a specific desensitization of the secretory mechanism to Ca2+, perhaps as a result of a decrease in Ca2(+)-dependent kinase activity. Furthermore, these studies suggest that secretory responses of B-cells to cyclic AMP and activators of protein kinase C are not dependent upon the responsiveness of the cells to changes in cytosolic Ca2+.

scholarly journals Complex effects of kinase localization revealed by compartment-specific regulation of protein kinase A activity

2021 ◽  
Author(s):  
Rebecca LaCroix ◽  
Benjamin Lin ◽  
Andre Levchenko
Keyword(s):  
Cell Migration ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Kinase Activity ◽  
Single Cells ◽  
Regulatory Subunit ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Specific Regulation ◽  
Kinase A

SummaryKinase activity in signaling networks frequently depends on regulatory subunits that can both inhibit activity by interacting with the catalytic subunits and target the kinase to distinct molecular partners and subcellular compartments. Here, using a new synthetic molecular interaction system, we show that translocation of a regulatory subunit of the protein kinase A (PKA-R) to the plasma membrane has a paradoxical effect on the membrane kinase activity. It can both enhance it at lower translocation levels, even in the absence of signaling inputs, and inhibit it at higher translocation levels, suggesting its role as a linker that can both couple and decouple signaling processes in a concentration-dependent manner. We further demonstrate that superposition of gradients of PKA-R abundance across single cells can control the directionality of cell migration, reversing it at high enough input levels. Thus complex in vivo patterns of PKA-R localization can drive complex phenotypes, including cell migration.

scholarly journals Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 810-817
Author(s):  
KJ Balazovich ◽  
JE Smolen ◽  
LA Boxer
Keyword(s):  
Protein Kinase ◽  
Phorbol Esters ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Soluble Form ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Dose Dependent

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the “translocation” of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.

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