PKC-dependent regulation of transepithelial resistance: roles of MLC and MLC kinase

1999 ◽  
Vol 277 (3) ◽  
pp. C554-C562 ◽  
Author(s):  
J. R. Turner ◽  
J. M. Angle ◽  
E. D. Black ◽  
J. L. Joyal ◽  
D. B. Sacks ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Myosin Ii ◽  
Transepithelial Resistance ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
H 51 ◽  
Mlc Phosphorylation ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Pkc Activation

The mechanisms by which protein kinase C (PKC) activation results in increased transepithelial resistance (TER) are unknown [G. Hecht, B. Robinson, and A. Koutsouris. Am. J. Physiol. 266 ( Gastrointest. Liver Physiol. 29): G214–G221, 1994]. We have previously shown that phosphorylation of the regulatory light chain of myosin II (MLC) is associated with decreases in TER and have suggested that contraction of the perijunctional actomyosin ring (PAMR) increases tight junction (TJ) permeability [J. R. Turner, B. K. Rill, S. L. Carlson, D. Carnes, R. Kerner, R. J. Mrsny, and J. L. Madara. Am. J. Physiol. 273 ( Cell Physiol. 42): C1378–C1385, 1997]. We therefore hypothesized that PKC activation alters TER via relaxation of the PAMR. Activation of PKC by the phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a progressive dose-dependent increase in TER that was apparent within 15 min (111% of controls) and maximal within 2 h (142% of controls). Similar increases were induced by a diacylglycerol analog, and the effects of both PMA and the diacylglycerol analog were prevented by the PKC inhibitor bisindolylmaleimide I. PMA treatment caused progressive decreases in MLC phosphorylation, by 12% at 15 min and 41% at 2 h. Phosphorylation of MLC kinase (MLCK) increased by 64% within 15 min of PMA treatment and was stable over 2 h (51% greater than that of controls). Thus increases in MLCK phosphorylation preceded decreases in MLC phosphorylation. These data suggest that PKC regulates TER via decreased phosphorylation of MLC, possibly due to inhibitory phosphorylation of MLCK. The decreased phosphorylation of MLC likely reduces PAMR tension, leading to decreased TJ permeability.

scholarly journals P2-purinoceptor regulation of surfactant phosphatidylcholine secretion. Relative roles of calcium and protein kinase C

1990 ◽  
Vol 266 (2) ◽  
pp. 407-413 ◽  
Author(s):  
W R Rice ◽  
C C Dorn ◽  
F M Singleton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Kinase C ◽  
Dose Dependent Increase ◽  
Dose Dependent ◽  
Phosphatidylcholine Secretion

Ca2+ and protein kinase C have both been proposed as intracellular signals for subsequent phosphatidylcholine secretion by alveolar Type II cells. We have determined the relative roles of Ca2+ and protein kinase C in regulating surfactant phosphatidylcholine secretion by utilizing exogenous ATP and the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) as secretagogues, along with MAPTAM to chelate intracellular Ca2+ and sphingosine to inhibit endogenous protein kinase C. Exposure of Type II cells to the P2-purinoceptor agonist, ATP, results in a dose-dependent increase in surfactant phosphatidylcholine secretion from isolated alveolar Type II cells with an EC50 (concn. producing 50% of maximal response) of 2 microM. Administration of exogenous ATP to Type II cells also results in a dose-dependent increase in inositol trisphosphate production, Ca2+ mobilization and [3H]phorbol 12,13-dibutyrate ([3H]PDBu) binding as a measure of protein kinase C translocation. The EC50 in each case is 1-5 microM, indicating association of these events with surfactant phosphatidylcholine secretion. Loading Type II cells with non-hydrolysable GTP analogue (GTP[S]) inhibited ATP-induced Ca2+ mobilization, supporting the hypothesis that Type II cell P2-purinoceptors are coupled to phospholipase C via a GTP-binding protein. The ATP-induced elevation of cytosolic Ca2+ was also inhibited by MAPTAM (a cell-permeant EGTA analogue) by 90%, but MAPTAM was without effect on surfactant phosphatidylcholine secretion induced by ATP. Sphingosine inhibited both ATP- and TPA-induced surfactant phosphatidylcholine secretion as well as [3H]PDBu binding with a similar IC50 (concn. producing 50% of maximal inhibition) (10 microM). Sphingosine did not affect surfactant phosphatidylcholine secretion induced by terbutaline and did not have a significant effect on Ca2+ mobilization induced by exogenous ATP. These results are consistent with a prominent role for protein kinase C in regulation of P2-purinoceptor-induced surfactant phosphatidylcholine secretion, and indicate that Ca2+ mobilization is not a necessary step for ATP-induced surfactant phosphatidylcholine secretion.

Modulation of ventricular action potential by alpha 1-adrenoceptors and protein kinase C

1990 ◽  
Vol 258 (3) ◽  
pp. H907-H911 ◽  
Author(s):  
R. T. Dirksen ◽  
S. S. Sheu
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Guinea Pig ◽  
Action Potential ◽  
Kinase C ◽  
Adrenoceptor Agonist ◽  
Lower Temperature ◽  
Cardiac Excitation ◽  
Dose Dependent ◽  
Pkc Activation

The effects of the alpha 1-adrenoceptor agonist methoxamine (MTX) and the direct protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBU) on action potentials from guinea pig papillary muscles were studied. Measured with conventional microelectrodes, MTX (1 x 10(-7) to 3 x 10(-4) M) and PDBU (1 x 10(-9) to 1 x 10(-6) M) both caused a dose-dependent and reversible decrease in the action potential duration measured at 90% repolarization (APD90) at 36.5 degrees C. The MTX-mediated response was blocked by both prazosin (3 x 10(-6) M) and phentolamine (1 x 10(-6) M) and mimicked by phenylephrine. Maximal concentrations of the two agents together resulted in only a partial (50%) additive decrease in the APD90. At a lower temperature (27.5 degrees C), PDBU no longer produced a shortening in the APD90 and MTX produced a prolongation in the APD90. These results demonstrate that although alpha 1-adrenoceptor stimulation and PKC activation in guinea pig papillary muscle both lead to a decrease in the APD90, the differences in their effects as related to the magnitude, partial additivity, and temperature dependence suggest that the mechanism of action are not identical. These subtle differences may help to delineate the exact physiological implications of alpha 1-adrenoceptors in cardiac excitation and contraction.

scholarly journals Suppression of c-fos precursor RNA splicing by the protein kinase C inhibitor H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]

1991 ◽  
Vol 278 (1) ◽  
pp. 305-308 ◽  
Author(s):  
M Harbers ◽  
H Hilz
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Rna Splicing ◽  
Epidermal Cells ◽  
Pkc Inhibitor ◽  
Oncogene Expression ◽  
Kinase C ◽  
Protein Kinase C Inhibitor ◽  
Precursor Rna ◽  
Dose Dependent

In JB6 epidermal cells, induction of fos proto-oncogene expression by phorbol 12-myristate 13-acetate can be inhibited by the protein kinase C (PKC) inhibitor H7 [1-5(isoquinolinesulphonyl)-2-methylpiperazine]. The compound causes also a dose-dependent suppression of fos precursor RNA splicing which, however, appears to react somewhat less sensitively to H7 than does PKC activity. This indicates that H7-induced accumulation of fos precursor RNA is not due to inhibition of PKC. Support for this interpretation comes from the finding that other inhibitors of PKC, such as N-(2-guanidinoethyl)-5-isoquinolinesulphonamide dihydrochloride, sphingosine, staurosporine or N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide, do not suppress splicing when applied at PKC-inhibiting concentrations.

Dose-dependent heterogenous actions of vasopressin in rabbit cortical collecting ducts

1989 ◽  
Vol 256 (4) ◽  
pp. F556-F562 ◽  
Author(s):  
Y. Ando ◽  
M. D. Breyer ◽  
H. R. Jacobson
Keyword(s):  
Protein Kinase C ◽  
Negative Feedback ◽  
Cyclooxygenase Inhibitor ◽  
Pkc Inhibitor ◽  
Kinase C ◽  
Collecting Ducts ◽  
High Concentrations ◽  
Pi Hydrolysis ◽  
Dose Dependent ◽  
Stimulation Of

In cortical collecting ducts (CCD), arginine vasopressin (AVP) has been proposed to autoinhibit its own hydrosmotic effect through stimulation of prostaglandin (PG) synthesis or binding to a receptor coupled to phosphatidylinositol (PI) hydrolysis, the so-called V1-receptor, with resultant elevation of intracellular Ca2+ concentration [( Ca2+]i) and activation of protein kinase C (PKC). Using isolated perfused rabbit CCD, we examined whether blocking the negative feedback by a PKC inhibitor, staurosporine (SSP), or a cyclooxygenase inhibitor, indomethacin (IND), enhances AVP-induced increase in hydraulic conductivity (Lp). The Lp induced by a pharmacological concentration (23 nM) of AVP was lower than that induced by 230 pM AVP. This blunted Lp response to 23 nM AVP was significantly restored by SSP or IND pretreatment. In contrast, both SSP and IND did not affect the Lp induced by 23 pM or 230 pM AVP. Fluorescence microscopy of isolated perfused CCD using fura-2 showed a spike-like increase in [Ca2+]i only by 23 nM but not by 23 or 230 pM AVP. We conclude that 1) AVP can increase [Ca2+]i, activate PKC, and stimulate PG synthesis in CCD with resultant autoregulation of its own hydrosmotic effect and 2) importantly, however, this negative feedback occurs only with pharmacologically high concentrations of AVP. Therefore it is unlikely that circulating AVP, via binding to receptors on CCD, autoregulates water transport through activating PG synthesis and/or PI breakdown.

Phosphatidates inhibit vasopressin-induced water transport via protein kinase C activation

1989 ◽  
Vol 257 (4) ◽  
pp. F524-F530 ◽  
Author(s):  
Y. Ando ◽  
H. R. Jacobson ◽  
M. D. Breyer
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Pkc Inhibitor ◽  
Phosphatidylinositol Bisphosphate ◽  
Kinase C ◽  
Collecting Ducts ◽  
Arachidonate Metabolites ◽  
Pkc Activation ◽  
Protein Kinase C Activation

Phosphatidic acid (PA), best known as an intermediate of phosphatidylinositol bisphosphate (PIP2) turnover, inhibits vasopressin (AVP)-induced increase in hydraulic conductivity (Lp) in rabbit cortical collecting ducts (CCD) perfused in vitro (Ando, Y., H. R. Jacobson, and M. D. Breyer, J. Clin. Invest. 80: 590, 1987). The present study addresses the mechanism(s) responsible for this action of PA. In control experiments, 10 microU/ml AVP (23 pM) increased Lp of CCDs from a basal of 4.9 +/- 0.4 X 10(-7) cm.atm-1.s-1 to a peak of 171.2 +/- 4.6 X 10(-7) cm.atm-1.s-1. Basolateral pretreatment of the tubule with PA (25 micrograms/ml) suppressed AVP-induced increase in peak Lp by 45.0%. This suppression was not attenuated by 5 microM indomethacin pretreatment. L-alpha-dipalmitoyl(C16) PA (DPPA, 25 micrograms/ml), an arachidonate-free synthetic PA, inhibited peak Lp by 79.0%, whereas another synthetic PA with shorter fatty acid (C12), L-alpha-dilauroyl PA (DLPA, 25 micrograms/ml), had no significant effect on AVP-induced peak Lp. In the presence of 100 nM staurosporine, a protein kinase C (PKC) inhibitor, the inhibition by PA and DPPA on AVP-induced peak Lp were abolished. Furthermore, another PKC inhibitor, 100 microM 1-(5-isoquiniline-sulfonyl)-2-methylpiperzine, also reversed the DPPA-induced inhibition of AVP action. In separate experiments using fura-2-loaded isolated perfused CCDs, however, neither PA nor DPPA caused a significant increase in intracellular free Ca2+ concentration [( Ca2+]i). Taken together, in CCD, PA-induced inhibition of AVP action is primarily mediated by PKC but not by an increased [Ca2+]i or the production of arachidonate metabolites, such as prostaglandins. Thus the PA-induced activation of PKC does not seem to involve the classic pathway for PKC activation, breakdown of PIP2.

scholarly journals Protein kinase C activation decreases peripheral actin network density and increases central nonmuscle myosin II contractility in neuronal growth cones

2013 ◽  
Vol 24 (19) ◽  
pp. 3097-3114 ◽  
Author(s):  
Qing Yang ◽  
Xiao-Feng Zhang ◽  
David Van Goor ◽  
Ashleigh P. Dunn ◽  
Callen Hyland ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Myosin Ii ◽  
Growth Cones ◽  
Network Density ◽  
Actin Network ◽  
Neuronal Growth ◽  
Kinase C ◽  
Neuronal Growth Cones ◽  
Pkc Activation

Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase–potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.

Role of calcium and protein kinase C in ANP secretion by cultured rat cardiocytes

1988 ◽  
Vol 255 (3) ◽  
pp. H405-H409 ◽  
Author(s):  
H. Matsubara ◽  
Y. Hirata ◽  
H. Yoshimi ◽  
S. Takata ◽  
Y. Takagi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Direct Action ◽  
Atrial Myocytes ◽  
Kinase C ◽  
Muscarinic Cholinergic ◽  
Dose Dependent Increase ◽  
Dose Dependent

The secretory mechanism of rat atrial natriuretic peptide (rANP) was studied in vitro with the use of primary culture of atrial myocytes from neonatal rats. Norepinephrine, phenylephrine, and carbamylcholine stimulated immunoreactive (IR) rANP secretion, whereas neither angiotensin II, arginine vasopressin, nor isoproterenol affected its secretion. The stimulatory effects of carbamylcholine and phenylephrine were blocked by atropine and prazosin, respectively. 12-O-tetradecanoylphorbol-beta-acetate (TPA), protein kinase C activator, induced a dose-dependent increase in IR rANP secretion, and TPA combined with Ca2+ ionophore ionomycin produced a synergistic effect. Ca2+-channel agonist BAY K 8644 also stimulated IR rANP secretion, the effect of which was blocked by Ca2+-channel antagonist nifedipine. These data suggest that alpha 1-adrenergic and muscarinic cholinergic agonists have direct action on rat cardiocytes to stimulate ANP secretion that involves receptor-mediated mobilization of intracellular Ca2+ and activation of protein kinase C.

Endotoxin-Induced Tissue Factor in Human Monocytes is Dependent upon Protein Kinase C Activation

1993 ◽  
Vol 70 (05) ◽  
pp. 800-806 ◽  
Author(s):  
C Ternisien ◽  
M Ramani ◽  
V Ollivier ◽  
F Khechai ◽  
T Vu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tissue Factor ◽  
Factor Ix ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Human Monocytes ◽  
Kinase C ◽  
Pkc Activation

SummaryTissue factor (TF) is a transmembrane receptor which, in association with factors VII and Vila, activates factor IX and X, thereby activating the coagulation protease cascades. In response to bacterial lipopolysaccharide (LPS) monocytes transcribe, synthesize and express TF on their surface. We investigated whether LPS-induced TF in human monocytes is mediated by protein kinase C (PKC) activation. The PKC agonists phorbol 12- myristate 13-acetate (PMA) and phorbol 12, 13 dibutyrate (PdBu) were both potent inducers of TF in human monocytes, whereas 4 alpha-12, 13 didecanoate (4 a-Pdd) had no such effect. Both LPS- and PMA-induced TF activity were inhibited, in a concentration dependent manner, by three different PKC inhibitors: H7, staurosporine and calphostin C. TF antigen determination confirmed that LPS-induced cell-surface TF protein levels decreased in parallel to TF functional activity under staurosporine treatment. Moreover, Northern blot analysis of total RNA from LPS- or PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to H7 and staurosporine. The decay rate of LPS-induced TF mRNA evaluated after the arrest of transcription by actinomycin D was not affected by the addition of staurosporine, suggesting that its inhibitory effect occurred at a transcriptional level. We conclude that LPS-induced production of TF and its mRNA by human monocytes are dependent on PKC activation.

Phosphorylation by protein kinase C stabilizes ABCG1 and increases cholesterol efflux

2019 ◽  
Vol 166 (4) ◽  
pp. 309-315 ◽  
Author(s):  
Taro Watanabe ◽  
Noriyuki Kioka ◽  
Kazumitsu Ueda ◽  
Michinori Matsuo
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Translational Regulation ◽  
Cholesterol Efflux ◽  
Degradation Rate ◽  
Active Form ◽  
Density Lipoprotein ◽  
Protein Levels ◽  
Kinase C ◽  
Pkc Activation

Abstract ATP-binding cassette protein G1 (ABCG1) plays an important role in eliminating excess cholesterol from macrophages and in the formation of high-density lipoprotein (HDL), which contributes to the prevention and regression of atherosclerosis. The post-translational regulation of ABCG1 remains elusive, although phosphorylation by protein kinase A destabilizes ABCG1 proteins. We examined the phosphorylation of ABCG1 using HEK293 and Raw264.7 cells. ABCG1 phosphorylation was enhanced by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator. PKC activation by TPA increased ABCG1 protein levels and promoted ABCG1-dependent cholesterol efflux to HDL. This activity was suppressed by Go6976, a PKCα/βI inhibitor, suggesting that PKC activation stabilizes ABCG1. To confirm this, the degradation rate of ABCG1 was analysed; ABCG1 degradation was suppressed upon PKC activation, suggesting that PKC phosphorylation regulates ABCG1 levels. To confirm this involvement, we co-expressed ABCG1 and a constitutively active form of PKCα in HEK cells. ABCG1 was increased upon co-expression. These results suggest that PKC-mediated phosphorylation, probably PKCα, stabilizes ABCG1, consequently increasing ABCG1-mediated cholesterol efflux, by suppressing ABCG1 degradation. PKC activation could thus be a therapeutic target to suppress the development of atherosclerosis.

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