Characterization and partial purification of rat submandibular gland protein kinase C

1988 ◽  
Vol 66 (7) ◽  
pp. 683-690
Author(s):  
D. J. Pon ◽  
S. P. Lintlop ◽  
A. K. Sen
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Submandibular Gland ◽  
Kinase Activity ◽  
Partial Purification ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Membrane Bound ◽  
Rat Submandibular Gland

Membrane-bound protein kinase C of rat submandibular gland was characterized and the cytosolic kinase C of the tissue was partially purified. The membrane-bound kinase could be activated by Triton X-100 but not EGTA in the presence of both Ca2+ and phosphatidylserine (PS). The Km values for Ca2+ and PS were 150 μM and 5 μg, respectively. Addition of 10−6 M diacylglycerol resulted in an increased affinity of the kinase for Ca2+ (Km = 10 μM). Phorbol 12,13-dibutyrate activated the kinase in the absence of exogenous Ca2+ and PS, suggesting that adequate amounts of each activator are present in the membrane itself. Polymyxin B inhibited the stimulated kinase C activity in a concentration-dependent manner. This inhibition could be overcome by addition of PS. The cytosolic kinase was partially purified 133-fold by chromatography on columns of DEAE-Sephacel and S-300 Sephacryl. The total kinase activity increased with respect to the kinase activity measured in the starting material with column chromatography, suggesting that an inhibitor is present in the cytosolic fraction of the tissue.

Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+

1993 ◽  
Vol 264 (5) ◽  
pp. F845-F853
Author(s):  
M. M. Friedlaender ◽  
D. Jain ◽  
Z. Ahmed ◽  
D. Hart ◽  
R. L. Barnett ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase D ◽  
Intracellular Signaling ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Eta Receptor ◽  
Ca2 Channels ◽  
Stimulation Of

Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.

Carvedilol-induced elevation in cytosolic free Ca2+ level and apoptosis in SIRC corneal epithelial cells

2009 ◽  
Vol 29 (6) ◽  
pp. 477-487
Author(s):  
Pochuen Shieh ◽  
Chih-Hung Lee ◽  
Ng Ling Yi ◽  
Chung-Ren Jan
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Phospholipase C ◽  
Dependent Manner ◽  
Corneal Epithelial Cells ◽  
Concentration Dependent Manner ◽  
Corneal Epithelial ◽  
Kinase C

The effect of the cardiovascular drug carvedilol on cytosolic free Ca2+ concentrations ([Ca 2+]i) and viability was examined in Statens Seruminstitut rabbit cornea (SIRC) corneal epithelial cells. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1), respectively. Carvedilol at concentrations between 1 and 30 μM increased [Ca 2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Carvedilol induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was inhibited by suppression of protein kinase C activity. In Ca2+-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor), carvedilol-induced [Ca2+]i rise was reduced; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca 2+]i rise. Addition of the phospholipase C inhibitor 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17-yl]amino] hexyl]-1H-pyrrole-2,5-dione (U73122; 2 μM) did not change carvedilol-induced [Ca2+]i rise. At concentrations between 5 and 70 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 20 μM carvedilol was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5—70 μM carvedilol. Collectively, in SIRC corneal epithelial cells, carvedilol-induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca 2+ influx via protein kinase C-regulated Ca2+ channels. Carvedilol-caused cytotoxicity was mediated by Ca2+-independent apoptosis in a concentration-dependent manner.

scholarly journals G-protein βγ subunits antagonize protein kinase C-dependent phosphorylation and inhibition of phospholipase C-β1

1997 ◽  
Vol 326 (3) ◽  
pp. 701-707 ◽  
Author(s):  
Irene LITOSCH
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
G Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Negative Feedback Regulation ◽  
Kinase C ◽  
Stimulation Of

Protein kinase C (PKC) isoforms phosphorylated phospholipase C-β1 (PLC-β1) in vitro as follows: PKCα ≫ PKCϵ; not PKCζ. PLC-β3 was not phosphorylated by PKCα. G-protein βγ subunits inhibited the PKCα phosphorylation of PLC-β1 in a concentration-dependent manner. Half-maximal inhibition occurred with 500 nM βγ. G-protein βγ subunits also antagonized the PKCα-mediated inhibition of PLC-β1 enzymic activity. PKCα, in turn, inhibited the stimulation of PLC-β1 activity by βγ. There was little effect of PKCα on the stimulation of PLC-β1 by αq/11–guanosine 5′[γ-thio]triphosphate (GTP[S]). These findings demonstrate that G protein βγ subunits antagonize PKCα regulation of PLC-β1. Thus βγ subunits might have a role in modulating the negative feedback regulation of this signalling system by PKC.

The role of protein kinase C in LHRH-induced LH and FSH release and LHRH self-priming in rat anterior pituitary glands in vitro

1988 ◽  
Vol 116 (2) ◽  
pp. 231-239 ◽  
Author(s):  
M. S. Johnson ◽  
R. Mitchell ◽  
G. Fink
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Polymyxin B ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Pituitary Glands

ABSTRACT We have investigated the role of protein kinase C (PKC) in LHRH-induced LH and FSH secretion and LHRH priming. Hemipituitary glands from pro-oestrous rats were incubated with agents known to affect PKC and with or without LHRH, during which time the secretion of gonadotrophins was measured. Phorbol esters and phospholipase C, activators of PKC, released LH and FSH in a concentration-dependent manner and potentiated the LHRH-induced secretion of gonadotrophins in parallel with their ability to release these hormones alone. Inhibitors of PKC had either no effect on LH release (1-(5-isoquinolinesulphonyl)-2-methylpiperazine hydrochloride) or they augmented LHRH-induced gonadotrophin release (polymyxin B and 8-(N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate). Neither the activators nor the inhibitors of PKC, when present with LHRH, caused any change in LHRH priming, even though the activators alone produced a release of gonadotrophins that showed a temporal pattern similar to that produced by LHRH priming. The profiles of effects on LH and FSH secretion were always qualitatively similar. These results show that PKC may be involved in general regulation of gonadotrophin release but that it is not important in acute responses to LHRH nor in LHRH self-priming. J. Endocr. (1988) 116, 231–239

scholarly journals Modulation of Calmodulin and Protein Kinase C Activities by Pencillium Mycotoxins

1999 ◽  
Vol 18 (2) ◽  
pp. 91-96 ◽  
Author(s):  
I. Pala ◽  
A. Srinivasan ◽  
P. J. S. Vig ◽  
D. Desaiah
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Complex Formation ◽  
Mammalian Brain ◽  
Dependent Manner ◽  
Active Conformation ◽  
Cellular Functions ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Ic50 Values

Calmodulin (CaM), a calcium-binding protein, is found in high concentrations in mammalian brain where it plays a pivotal role in a large number of cellular functions. Protein kinase C (PKC), a multifunctional cytosolic enzyme, in the presence of both Ca2+ and phospholipids, transduce extracellular signals into intracellu-lar events. Both CaM and PKC are partially involved in maintaining Ca2+ homeostasis in the cell. Any fluctuations in the intracel-lular Ca2+ can modulate cellular functions and may contribute to neuronal dysfunction. Hence, the present investigation was initiated to study the effects of some selected penicillium (naturally occurring tremorgenic) mycotoxins like secalonic acid, citreoviridin, and verruculogen on CaM activity, active conformation of CaM and PKC activity. Stimulation of CaM-deflcient bovine brain 3′-5′ phosphodieste rase (PDE) indicated CaM activity. The modification of CaM active conformation was studied by the binding of fluorescent probe N-phenyl-1-napthylamine (NPN) to CaM. Alterations in the fluorescence of dansyl-CaM was used to study the effect of these compounds on complex formation between CaM and PDE. Rat brain cytosolic PKC was studied using 32P-ATP as a measure of altered protein phosphorylation. The concentrations of mycotoxins used were in the range of 10 to 50 μM. All three mycotoxins inhibited CaM-stimulated PDE activity in a concentration-dependent manner. Citreoviridin and secalonic acid inhibited NPN fluorescence and Ca2+-dependent complex formation of dansyl-CaM and PDE. The IC50 values for NPN fluorescence of citreoviridin and secalonic acid were 13 μM and 19 μM respectively. However, verruculogen showed little effect on NPN fluorescence and the Ca2+-dependent complex formation of dansyl-CaM and PDE. These mycotoxins also inhibited PKC activity in a concentration-dependent manner with IC50 values of 19.8, 25.7, and 38.4 μM for secalonic acid, citreoviridin, and verruculogen, respectively. The results of our study suggest that these mycotoxins at very low concentrations are interacting with CaM and PKC. Such an effect could lead to impairment of neurotransmission and result in neurotoxicity.

scholarly journals The diacylglycerols dioctanoylglycerol and oleoylacetylglycerol enhance prostaglandin synthesis by inhibition of the lysophosphatide acyltransferase

1987 ◽  
Vol 247 (3) ◽  
pp. 773-777 ◽  
Author(s):  
M Goppelt-Strübe ◽  
H J Pfannkuche ◽  
D Gemsa ◽  
K Resch
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Direct Interaction ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Key Enzyme ◽  
Free Arachidonic Acid

Prostanoids are synthesized by resident macrophages upon stimulation with diacylglycerols. Oleoylacetylglycerol and dioctanoylglycerol induced prostaglandin E and thromboxane synthesis in a time- and concentration-dependent manner. Both diacylglycerols inhibited the lysophosphatide acyltransferase, which is the key enzyme in the reacylation of arachidonic acid. By this mechanism the pool of free arachidonic acid available for prostanoid synthesis is increased. Both diacylglycerols were able to inhibit the membrane-bound lysophosphatide acyltransferase by a direct interaction independent of protein kinase C. Thus lysophosphatide acyltransferase could be shown to be a new target of these diacylglycerols, known as activators of protein kinase C.

scholarly journals Differential effect of phorbol esters and interleukin-3 on protein kinase C isoform content and kinase activity in the FDC-P1 cell line

Blood ◽  
1991 ◽  
Vol 78 (10) ◽  
pp. 2633-2641
Author(s):  
DK Ways ◽  
W Qin ◽  
RS Riddle ◽  
TD Garris ◽  
TE Bennett ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Line ◽  
Kinase Activity ◽  
Phorbol Esters ◽  
Dependent Manner ◽  
Interleukin 3 ◽  
Kinase C ◽  
Endogenous Substrates ◽  
Cells Cultured

FD/PMA is a subclone of the interleukin-3 (IL-3)-dependent, FDC-P1 cell line, which proliferates in response to either 12-O- tetradecanoylphorbol-13 acetate (PMA) or IL-3. While several endogenous substrates were phosphorylated in response to protein kinase C (PKC) activation in FDC-P1, phospholipid-dependent phosphorylation in the FD/PMA grown in PMA was not observed. Basal, phosphatidylserine- independent, and diolein-independent phosphorylation of cytosolic substrates with molecular weights of 17, 52, 57, and 105 Kd were enhanced in FD/PMA cells grown in PMA as compared with FDC-P1 cells cultured in IL-3. Phosphorylation of a 105-Kd substrate was enhanced in the particulate fraction of FD/PMA cells maintained in PMA. The 17-Kd substrate in FD/PMA cells comigrated with a substrate phosphorylated in a PKC-dependent manner in FDC-P1 cells. Phosphorylation of the 52- and 57-Kd substrates, but not of the 17-Kd substrate, was inhibited by H-7 and staurosporine. A portion of the PMA-induced cytosolic kinase activity coeluted with PKC on diethyl aminoethyl chromatography. While FD/PMA cells cultured in PMA contained negligible PKC-dependent phosphorylation of endogenous substrates or histone, alpha and epsilon PKC isoforms were detected by Western blot analysis. PKC phosphotransferase activity was observed in FD/PMA cells grown in PMA when peptides corresponding to residues 720 to 737 of PKC-epsilon or residues 4 to 14 of myelin basic protein were used as substrates. These data indicate that maintenance of FD/PMA cells in PMA stimulates proliferation and markedly alters PKC substrate specificity. Generation of at least two phospholipid-independent kinases occurs in PMA-treated cells.

Resveratrol Attenuates Adenosine Diphosphate-Induced Platelet Activation by Reducing Protein Kinase C Activity

2008 ◽  
Vol 36 (03) ◽  
pp. 603-613 ◽  
Author(s):  
Yu-Min Yang ◽  
Xing-Xiang Wang ◽  
Jun-Zhu Chen ◽  
Shi-Jun Wang ◽  
Hu Hu ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Adenosine Diphosphate ◽  
Dependent Manner ◽  
Chinese Medicinal Herb ◽  
Concentration Dependent Manner ◽  
Kinase C

Inappropriate platelet activation is the key point of thrombogenesis. The aim of the present study was to investigate the effects of resveratrol (RESV), a compound extracted from the Chinese medicinal herb Polygonum cuspidatum sieb et Zucc, on the platelet activation induced by adenosine diphosphate (ADP) and its possible mechanism. The percentage of platelet aggregation and surface P-selectin-positive platelets, and the activity of protein kinase C (PKC) of platelet were observed with platelet aggregometer, flow cytometry and phosphorimaging system, respectively. RESV at 25, 50 and 100 μM showed anti-platelet aggregation and inhibition of surface P-selectin-positive platelets in a concentration-dependent manner. RESV (50 μM) inhibited the activity of PKC in the membrane fraction of platelets and decreased the percentage of membrane associated PKC activity in total PKC activity. Moreover, DL-erythro-1,3-Dihydroxy-2-aminooctadecane, an elective protein kinase C inhibitor (PKCI), and RESV had additive effects of inhibiting the percentage of platelet aggregation and surface P-selectin-positive platelets. It is suggested that RESV may inhibit platelet aggregation, the percentage of surface P-selectin-positive platelets and subsequent thrombus formation. The mechanisms may be partly relative to the decrease of the activity of PKC of platelets.

PGE2 regulates cAMP production in cultured rabbit CCD cells: evidence for dual inhibitory mechanisms

1992 ◽  
Vol 263 (6) ◽  
pp. C1208-C1215 ◽  
Author(s):  
T. D. Noland ◽  
C. E. Carter ◽  
H. R. Jacobson ◽  
M. D. Breyer
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Collecting Duct ◽  
Dependent Manner ◽  
Cortical Collecting Duct ◽  
Camp Production ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Inhibitory Mechanisms ◽  
Protein Kinase C Inhibitor

In cultured cortical collecting duct (CCD) cells, exogenous prostaglandin E2 (PGE2) inhibited arginine vasopressin (AVP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in a concentration-dependent manner. Although pertussis toxin (PT, 500 ng/ml) alone did not reverse the PGE2-dependent inhibition, PT and staurosporine, a protein kinase C inhibitor, together partially reversed the effect of exogenous PGE2. In contrast, PT completely reversed the inhibition of AVP-dependent cAMP production by sulprostone. These data suggest that exogenous PGE2 can inhibit AVP-stimulated cAMP production and that the inhibitory effects of PGE2 are mediated by staurosporine- and PT-sensitive component(s). Short-term (15-240 min) incubation with phorbol 12-myristate 13-acetate (PMA, 10(-7) M) inhibited PGE2-stimulated cAMP production. Long-term (20 h) incubation with PMA augmented PGE2-stimulated cAMP production. These data provide evidence for the maintenance of a PT-sensitive PGE2-dependent inhibitory pathway of cAMP production in cultured CCD cells. In addition, data are presented that support an inhibitory role for protein kinase C in the effects of PGE2 on the metabolism of cAMP in these cells.

Sign in / Sign up

Export Citation Format

Share Document