Correlation Between Platelet Aggregation and Dephosphorylation of a 68 kDa Protein Revealed through the Use of Putative PKC Inhibitors

1993 ◽  
Vol 70 (04) ◽  
pp. 648-653 ◽  
Author(s):  
Marco E Turini ◽  
Douglas C Gaudette ◽  
Bruce J Holub ◽  
James B Kirkland
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Gel Electrophoresis ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Calphostin C ◽  
Kinase C ◽  
Protein Dephosphorylation ◽  
Pkc Inhibitors

SummaryThe efficacy of two structurally and functionally unrelated protein kinase C (PKC) inhibitors, chelerythrine and calphostin C, was assessed in intact human platelets by studying platelet aggregation in response to Stimulation with phorbol 12-myristate 13-acetate (PMA) or the thromboxane-A2 mimetic, U46619. Surprisingly, both inhibitors increased aggregation in response to PMA, but decreased aggregation in response to U46619. To further explore this phenomenon, gel electrophoresis of 32P-labelled proteins from PMA- or U46619-stimulated platelets in the presence and absence of the two putative PKC inhibitors was performed. Although neither chelerythrine nor calphostin C proved to be effective PKC inhibitors in intact human platelets, a strong correlation between the dephosphorylation of a 68 kDa protein and the rate of platelet aggregation was observed. From these results, the indiscriminate use of PKC inhibitors in whole platelets is questioned and attention is drawn to the role of protein dephosphorylation in platelet activation. The 68 kDa protein was the major phosphorylated substrate in resting platelets. Okadaic acid increased phosphorylation of this band, indicating active phosphate group turnover under resting conditions.

Mechanisms Involved in the Antiplatelet Activity of Midazolam in Human Platelets

2002 ◽  
Vol 96 (3) ◽  
pp. 651-658 ◽  
Author(s):  
Joen R. Sheu ◽  
George Hsiao ◽  
Hsiung N. Luk ◽  
Yi W. Chen ◽  
Ta L. Chen ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Antiplatelet Activity ◽  
Phosphoinositide Breakdown ◽  
Kinase C ◽  
Inhibitory Mechanisms ◽  
Intracellular Ca

Background Midazolam is widely used as a sedative and anesthetic induction agent. The aim of this study was to systematically examine the inhibitory mechanisms of midazolam in platelet aggregation. Methods The inhibitory mechanisms of midazolam in platelet aggregation were explored by means of analysis of the platelet glycoprotein IIb-IIIa complex, phosphoinositide breakdown, intracellular Ca+2 mobilization, measurement of membrane fluidity, thromboxane B2 formation, and protein kinase C activity. Results In this study, midazolam dose-dependently (6-26 microm) inhibited platelet aggregation in human platelets stimulated by agonists. Midazolam also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. Midazolam (6-26 mum) significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, midazolam (15 and 26 mum) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by collagen (2 microg/ml). This phosphorylation was markedly inhibited by midazolam (26 microm). Conclusions These results indicate that the antiplatelet activity of midazolam may be involved in the following pathways: the effects of midazolam may initially be caused by induction of conformational changes in platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein.

R59022, A DIACYLGLYCEROL (DG) KINASE INHIBITOR POTENTIATES THROMBIN-INDUCED PLATELET AGGREGATION AND GRANULE RELEASE

1987 ◽  
Author(s):  
S K Joseph ◽  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Kinase Inhibitor ◽  
Human Platelets ◽  
Maximal Concentration ◽  
Inhibitory Effects ◽  
Kinase C ◽  
Granule Secretion ◽  
Granule Release

R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosphatidic acid. While R59002 inhibits DG conversion in platelets resulting in enhanced protein kinase C (PrkC) activation [1], little is known on its effect on other platelet responses. In this study, we have examined the effect of R59022 on agonist-induced platelet aggregation and [14C]-5-hydroxytryptamine (5HT) release using washed human platelets. With a sub-maximal concentration of thrombin (T, 0.05U/ml) R59022 (10-30μM) significantly potentiated T-induced platelet aggregation and [14C]-5HT release eg % [14C]-5HT release:- 0.05U/ml T-52±5,30μM R59022+T-76±8. Removal of external Ca2+ (ImM) using EGTA (5mM) reduced T-induced 5HT release but not the potentiation of it by R59022 eg EGTA+ 0.05U/ml T-36±6%, EGTA+R59022+T- 72±5%. These results show that in the presence of EGTA and R59022 the increased DG levels can compensate for the diminished rise in T-induced Ca/2+ mobilisation thus re-emphasizing the importance of DG in promoting granule secretion. In addition to inhibiting DG phosphorylation, R59022 also inhibits the phosphorylation of the DG analogue 1-oleoyl 2-acetylglycerol (OAG) [1]. OAG (63μM) with pre-incubation times of 10-60 sec, significantly potentiated threshold T (0.03U/ml)-induced [l4C]-5HT release, though with longer incubation times, this potentiatory effect was gradually lost eg 0.03U/ml T-l±0.3%, OAG+T (10 sec)- 33±4%, OAG+T (1 min)-11±3%, 0AG+veh.-0%. However, in the presence of R59022 (30μM), OAG retained its potentiatory effect for longer periods eg R59022+0AG+T (1 min)-45+10%, R59022+T-2±l%. With incubation times > 5 min the potentiatory effects of OAG were lost even in the presence of R59022. This is possibly due to the metabolism of OAG by DG lipase. Our results demonstrate that R59022, which has been reported to inhibit DG kinase leading to enhanced PrkC activation, also enhances agonist-induced platelet aggregation and 5HT release. It may therefore be a useful compound in elucidating further the role of DG in terms of both stimulatory and inhibitory effects on platelet activation.[1]. de Chaffoy de Coucelles, D. et al (1985) J Biol Chem 260, 15762.

EXOGENOUSLY ADDED DIACYLGLYCEROL (DAG) AND ELEVATED INTRACELLULAR Ca2+ LEVELS CANNOT SUBSTITUTE FOR PROSTAGLANDIN END0PER0XIDES/ THROMBOXANE ki IN MEDIATING WEAK AGONIST-INDUCED PLATELET SECRETION

1987 ◽  
Author(s):  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Human Platelets ◽  
Similar Degree ◽  
Low Concentration ◽  
Kinase C ◽  
Platelet Secretion

We have compared the abilities of exogenously added U46619, the PG endoperoxide analogue and, sn-l-oleoyl 2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (diCg), the membrane-permeant DAG analogues, at restoring weak agonist-induced secretion in indomethacin (10μM)-treated platelets (I-PL) in the absence of endogenous PG/Tx synthesis. [14C]-5HT secretion from pre-loaded, washed human platelets was correlated with the levels of [Ca2+]i, using platelets loaded with quin 2. Concentrations of OAG (62-125μM) and diCg (15-30μM), which have previously been shown to be fully effective at activating protein kinase C, failed to significantly enhance [14C]-5HT secretion in combination with ADP (10μM), adrenaline (10μM) or PAF (0.2μM) although they potentiated platelet aggregation, when added 10-30 sec after these agonists to I-PL. eg ADP-0%, 30jiM diCg-9.8%, ADP+diCg-11.9%, 5HT release (p>O.05). In contrast, a low concentration of U46619 (0.2μM), that induced no aggregation, [14C]-5HT secretion or rise in [Ca2+]i levels on its own, was able to synergize strongly at potentiating secretion in combination with all three weak agonists examined, as well as in combination with OAG and diCg (U46619-0%, ADP+U46619-20.4%, U46619+30μM diC8-48% 5HT release) . The greater effectiveness of U46619 at potentiating secretion in combination with the weak agonists was not related to different degrees of [Ca2+]i mobilisation, as ADP and PAF-induced rise in [Ca2+]i occurred to a similar degree in the presence of U46619 and diCg. At a higher concentration of U46619 (0.6μM), which was maximally effective at inducing secretion and elevating [Ca2+]i levels on its own, addition of the weak agonists or OAG or diCg, along with U46619, resulted in a further enhancement of secretion which was independent of changes in [Ca2+]i levels. The results demonstrate that U46619 but not OAG or diCg, is able to fully restore weak agonist-induced secretion in indomethacin-treated platelets, suggesting that the actions of endogenously formed PG endoperoxides/TxA2 cannot be substituted by DAG and raised [Ca2+]i levels and, may be mediated via a mechanism additional to that involving these mediators.

Involvement of proline-rich tyrosine kinase 2 in platelet activation: tyrosine phosphorylation mostly dependent on αIIbβ3 integrin and protein kinase C, translocation to the cytoskeleton and association with Shc through Grb2

2000 ◽  
Vol 347 (2) ◽  
pp. 561-569 ◽  
Author(s):  
Tsukasa OHMORI ◽  
Yutaka YATOMI ◽  
Naoki ASAZUMA ◽  
Kaneo SATOH ◽  
Yukio OZAKI
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Human Platelets ◽  
Sh2 Domain ◽  
Kinase C ◽  
Tyrosine Kinase 2 ◽  
Αiibβ3 Integrin

Proline-rich tyrosine kinase 2 (Pyk2) (also known as RAFTK, CAKβ or CADTK) has been identified as a member of the focal adhesion kinase (FAK) family of protein-tyrosine kinases and it has been suggested that the mode of Pyk2 activation is distinct from that of FAK. In the present study we investigated the mode of Pyk2 activation in human platelets. When platelets were stimulated with thrombin, Pyk2, as well as FAK, was markedly tyrosine-phosphorylated, in a manner mostly dependent on αIIbβ3 integrin-mediated aggregation. The residual Pyk2 tyrosine phosphorylation observed in the absence of platelet aggregation was completely abolished by pretreatment with BAPTA/AM [bis-(o-aminophenoxy)ethane-N,N,Nʹ,Nʹ-tetra-acetic acid acetoxymethyl ester]. The Pyk2 phosphorylation was inhibited by protein kinase C (PKC) inhibitors at concentrations that inhibited platelet aggregation. In contrast, direct activation of PKC with the active phorbol ester PMA induced the tyrosine phosphorylation of Pyk2 and FAK but only when platelets were fully aggregated with the exogenous addition of fibrinogen (the ligand for αIIbβ3 integrin). Furthermore, PMA-induced Pyk2 (and FAK) tyrosine phosphorylation was also observed when platelets adhered to immobilized fibrinogen. The activation of the von Willebrand factor (vWF)--glycoprotein Ib pathway with botrocetin together with vWF failed to induce Pyk2 (and FAK) tyrosine phosphorylation. Most Pyk2 and FAK was present in the cytosol and membrane skeleton fractions in unstimulated platelets. When platelets were stimulated with thrombin, both Pyk2 and FAK were translocated to the cytoskeleton in an aggregation-dependent manner. In immunoprecipitation studies, Pyk2, as well as FAK, seemed to associate with Shc through Grb2. With the use of glutathione S-transferase fusion proteins containing Shc-SH2, Grb2-SH2, and Grb2 N-terminal and C-terminal SH3 domains, it was implied that the proline-rich region of Pyk2 (and FAK) binds to the N-terminal SH3 domain of Grb2 and that the phosphotyrosine residue of Shc binds to the SH2 domain of Grb2. Although Pyk2 and FAK have been reported to be differentially regulated in many cell types, our results suggest that, in human platelets, the mode of Pyk2 activation is mostly similar to that of FAK, in terms of αIIbβ3 integrin-dependent and PKC-dependent tyrosine phosphorylation. Furthermore, Pyk2, as well as FAK, might have one or more important roles in post-aggregation tyrosine phosphorylation events, in association with the cytoskeleton and through interaction with adapter proteins including Grb2 and Shc.

κ- but not δ-opioid receptors mediate effects of ischemic preconditioning on both infarct and arrhythmia in rats

2001 ◽  
Vol 280 (1) ◽  
pp. H384-H391 ◽  
Author(s):  
Guan-Ying Wang ◽  
Song Wu ◽  
Jian-Ming Pei ◽  
Xiao-Chun Yu ◽  
Tak-Ming Wong
Keyword(s):  
Infarct Size ◽  
Opioid Receptors ◽  
Ischemic Preconditioning ◽  
Antiarrhythmic Action ◽  
Perfused Rat Heart ◽  
Calphostin C ◽  
Kinase C ◽  
Series Of Experiments ◽  
Pkc Inhibitors

Two series of experiments were performed in the isolated perfused rat heart to determine the role of κ- and δ-opioid receptors (OR) in cardioprotection of ischemic preconditioning (IP). In the first series of experiments, it was found that IP with two cycles of 5-min regional ischemia followed by 5-min reperfusion each reduced infarct size induced by 30-min ischemia, and the ameliorating effect of IP on infarct was attenuated with blockade of either 5 × 10−6 mol/l nor-binaltorphimine (nor-BNI), a selective κ-OR antagonist, or 5 × 10−6 mol/l naltrindole (NTD), a selective δ-OR antagonist. The second series showed that U50,488H, a selective κ-OR agonist, ord-Ala2-d-leu5-enkephalin (DADLE), a selective δ-OR agonist, dose dependently reduced the infarct size induced by ischemia, which mimicked the effects of IP. The effect of 10−5 mol/l U50,488H on infarct was significantly attenuated by blockade of protein kinase C (PKC) with specific PKC inhibitors, 5 × 10−6 mol/l chelerythrine or 8 × 10−7 mol/l calphostin C, as well as by blockade of ATP-sensitive K+ (KATP) channels with blockers of the channel, 10−5 mol/l glibenclamide or 10−4 mol/l 5-hydroxydecanoate. IP also reduced arrhythmia induced by ischemia. Nor-BNI, but not NTD, attenuated, while U50,488H, but not DADLE, mimicked the antiarrhythmic action of IP. In conclusion, the present study has provided first evidence that κ-OR mediates the ameliorating effects of IP on infarct and arrhythmia induced by ischemia, whereas δ-OR mediates the effects only on infarct. Both PKC and KATP channels mediate the effect of activation of κ-OR on infarct.

scholarly journals Role of Specific Protein Kinase C Isozymes in Mediating Epidermal Growth Factor, Thyrotropin-Releasing Hormone, and Phorbol Ester Regulation of the Rat Prolactin Promoter in GH4/GH4C1 Pituitary Cells

2002 ◽  
Vol 16 (12) ◽  
pp. 2840-2852 ◽  
Author(s):  
Cheryl A. Pickett ◽  
Nicole Manning ◽  
Yoshiko Akita ◽  
Arthur Gutierrez-Hartmann
Keyword(s):  
Gene Transcription ◽  
Promoter Activity ◽  
The Novel ◽  
Calphostin C ◽  
Kinase C ◽  
Epidermal Growth ◽  
Pkc Isozymes ◽  
Pkc Inhibitors ◽  
Prl Gene

Abstract Epidermal growth factor (EGF) and TRH both produce enhanced prolactin (PRL) gene transcription and PRL secretion in GH4 rat pituitary tumor cell lines. These agents also activate protein kinase C (PKC) in these cells. Previous studies have implicated the PKCε isozyme in mediating TRH-induced PRL secretion. However, indirect studies using phorbol ester down-regulation to investigate the role of PKC in EGF- and TRH-induced PRL gene transcription have been inconclusive. In the present study, we examined the role of multiple PKC isozymes on EGF- and TRH-induced activation of the PRL promoter by utilizing general and selective PKC inhibitors and by expression of genes for wild-type and kinase-negative forms of the PKC isozymes. Multiple nonselective PKC inhibitors, including staurosporine, bisindolylmaleimide I, and Calphostin C, inhibited both EGF and TRH induced rat PRL promoter activity. TRH effects were more sensitive to Calphostin C, a competitive inhibitor of diacylglycerol, whereas Go 6976, a selective inhibitor of Ca2+-dependent PKCs, produced a modest inhibition of EGF but no inhibition of TRH effects. Rottlerin, a specific inhibitor of the novel nPKCδ isozyme, significantly blocked both EGF and TRH effects. Overexpression of genes encoding PKCs α, βΙ, βΙΙ, δ, γ, and λ failed to enhance either EGF or TRH responses, whereas overexpression of nPKCη enhanced the EGF response. Neither stable nor transient overexpression of nPKCε produced enhancement of EGF- or TRH-induced PRL promoter activity, suggesting that different processes regulate PRL transcription and hormone secretion. Expression of a kinase inactive nPKCδ construct produced modest inhibition of EGF-mediated rPRL promoter activity. Taken together, these data provide evidence for a role of multiple PKC isozymes in mediating both EGF and TRH stimulated PRL gene transcription. Both EGF and TRH responses appear to require the novel isozyme, nPKCδ, whereas nPKCη may also be able to transmit the EGF response. Inhibitor data suggest that the EGF response may also involve Ca2+-dependent isozymes, whereas the TRH response appears to be more dependent on diacylglycerol.

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

2010 ◽  
Vol 429 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Analia Garcia ◽  
Soochong Kim ◽  
Kamala Bhavaraju ◽  
Simone M. Schoenwaelder ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Functional Responses ◽  
Induce Platelet Aggregation ◽  
Erk Phosphorylation

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

scholarly journals Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽  
1993 ◽  
Vol 82 (9) ◽  
pp. 2704-2713 ◽  
Author(s):  
R Vezza ◽  
R Roberti ◽  
GG Nenci ◽  
P Gresele
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Prostaglandin E2 ◽  
Platelet Activation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Kinase C ◽  
Activated Platelets ◽  
Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

ADRENALINE (ADR) INTERACTIONS WITH THROMBIN AND COLLAGEN -EFFECTS ON PLATELET ARACHIDONATE RELEASE AND 5HT SECRETION AND ROLE OF ENDOGENOUSLY FORMED THROMBOXANE A2 (TxA2)

1987 ◽  
Author(s):  
Y Patel ◽  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Synergistic Effects ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Presence And Absence ◽  
Pre Treatment ◽  
Arachidonate Release

We have examined the effect of combinations of ADR + thrombin (T) and ADR + collagen (C) on platelet arachidonate release and 5HT secretion, and assessed the role of endogenously formed TxA2 on these responses using indomethacin (I). Washed, human platelets prelabelled with [3H]-arachidonic acid (AA) or [14C]-5HT were used, ADR was added 10 sec before T or C and the reaction was terminated 3 min later. In the range 1-100μM, ADR induced no detectable aggregation or 5HT secretion but potentiated platelet aggregation when added with sub-threshold concentrations of T or C, which on their own induced no aggregation. At 2-4 fold higher concentrations of T and C (threshold for 5HT secretion), 5HT secretion and AA/TXB2 release were also potentiated by ADR (1-10μM) by 30-50%. Pre-treatment of platelets with I (10μM) abolished threshold T and C-induced 5HT secretion, as well as its potentiation by ADR. However, approximately 2-fold and 5-fold higher concentrations of T and C respectively were able to induce 'I-insensitive'secretion, which was further potentiated by ADR. In I-treated platelets, C-induced AA release and its potentiation by ADR were also abolished suggesting a role for endogenously formed TxA2 This was confirmed by addition of the TxA2 mimetic, U46619 (0.3μM), which potentiated C-induced AA release in the presence and absence of ADR, even though it induced no AA release on its own or, in combination with ADR alone in the absence of collagen. The latter suggests agonist specificity regarding the ability of TxA2 to synergistically stimulate AA release. Finally, unstirred platelets in PRP pre-incubated with ADR (10μM) for 120 min lost their responsiveness to ADR, when eventually stirred; however, these 'ADR-desensitised' platelets when washed and resuspended, were able to demonstrate synergistic effects on secretion when stimulated with ADR+T or ADR+C. This is analogous to the previously demonstrated ability of ADR to inhibit adenylate cyclase even in 'ADR-desensitised' platelets and re-inforces the separation regarding the mechanisms underlying the various effects of ADR on platelets.

The role of protein kinase C in GH secretion induced by GH-releasing factor and GH-releasing peptides in cultured ovine somatotrophs

1997 ◽  
Vol 154 (2) ◽  
pp. 219-230 ◽  
Author(s):  
D Wu ◽  
I J Clarke ◽  
C Chen
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Adenylyl Cyclase Activity ◽  
Down Regulation ◽  
Gh Secretion ◽  
Calphostin C ◽  
Kinase C ◽  
Pka Pathway

Abstract The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P<0·001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP–protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP–PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP–PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP–PKA pathway in ovine somatotrophs during the action of GRF or GHRP. Journal of Endocrinology (1997) 154, 219–230

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