scholarly journals Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion

1992 ◽  
Vol 281 (2) ◽  
pp. 465-472 ◽  
Author(s):  
C P D Wheeler-Jones ◽  
T Saermark ◽  
V V Kakkar ◽  
K S Authi
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
G Protein ◽  
Human Platelets ◽  
Dependent Manner ◽  
Kinase C ◽  
Reversible Electroporation ◽  
Dose Dependent ◽  
Stimulation Of

Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5′-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.

Regulation of oxytocin secretion by the ovine corpus luteum: effect of activators of protein kinase C

1990 ◽  
Vol 124 (2) ◽  
pp. 225-232 ◽  
Author(s):  
J. J. Hirst ◽  
G. E. Rice ◽  
G. Jenkin ◽  
G. D. Thorburn
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Corpus Luteum ◽  
Phospholipase C ◽  
Tissue Slices ◽  
Kinase C ◽  
Luteal Tissue ◽  
Dose Dependent ◽  
Stimulation Of

ABSTRACT The effect of protein kinase C activation and dibutyryl cyclic AMP on oxytocin secretion by ovine luteal tissue slices was investigated. Several putative regulators of luteal oxytocin secretion were also examined. Oxytocin was secreted by luteal tissue slices at a basal rate of 234·4 ± 32·8 pmol/g per h (n = 24) during 60-min incubations.Activators of protein kinase C: phorbol 12,13-dibutyrate (n = 8), phorbol 12-myristate,13-acetate (n = 4) and 1,2-didecanoylglycerol (n = 5), caused a dose-dependent stimulation of oxytocin secretion in the presence of a calcium ionophore (A23187; 0·2 μmol/l). Phospholipase C (PLC; 50–250 units/l) also caused a dose-dependent stimulation of oxytocin secretion by luteal slices. Phospholipase C-stimulated oxytocin secretion was potentiated by the addition of an inhibitor of diacylglycerol kinase (R59 022; n = 4). These data suggest that the activation of protein kinase C has a role in the stimulation of luteal oxytocin secretion. The results are also consistent with the involvement of protein kinase C in PLC-stimulated oxytocin secretion. The cyclic AMP second messenger system does not appear to be involved in the control of oxytocin secretion by the corpus luteum. Journal of Endocrinology (1990) 124, 225–232

Mechanisms of PTH-induced rise in cytosolic calcium in adult rat hepatocytes

1994 ◽  
Vol 267 (5) ◽  
pp. G754-G763 ◽  
Author(s):  
M. Klin ◽  
M. Smogorzewski ◽  
H. Khilnani ◽  
M. Michnowska ◽  
S. G. Massry
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
G Protein ◽  
Cytosolic Calcium ◽  
Amino Terminal ◽  
Kinase C ◽  
Dose Dependent ◽  
Kinase A ◽  
Stimulation Of

Available data indicate that the liver is a target organ for parathyroid hormone (PTH) and that this effect is most likely mediated by PTH-induced calcium entry into hepatocytes. The present study examined the effects of both PTH-(1-84) and its amino-terminal fragment [PTH-(1-34)] on cytosolic calcium concentration ([Ca2+]i) of hepatocytes and explored the cellular pathways that mediate this potential action of PTH. Both moieties of PTH produced a dose-dependent rise in [Ca2+]i, but the effect of PTH-(1-84) was greater (P < 0.01) than an equimolar amount of PTH-(1-34). This effect required calcium in the medium and was totally [PTH-(1-34)] or partially [PTH-(1-84)] blocked by PTH antagonist ([Nle8,18,Tyr34]bPTH-(7-34)-NH2] and by verapamil or nifedipine. Sodium or chloride channel blockers did not modify this effect. 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), and G protein activator also produced a dose-dependent rise in [Ca2+]i. Staurosporine abolished the effect of TPA, and both staurosporine and calphostin C partially inhibited the effect of PTH. Staurosporine and verapamil together produced greater inhibition of PTH action than each alone. Rp-cAMP, a competitive inhibitor of cAMP binding to the R subunit of protein kinase A, and N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a protein kinase A inhibitor, blocked the effect of both DBcAMP and PTH, but the effect of these agents was greater (P < 0.01) on DBcAMP action. G protein inhibitor and pertussis toxin partially blocked the action of PTH. The data indicate that 1) PTH increases [Ca2+]i of hepatocytes; 2) this action of the hormone is receptor mediated; 3) the predominant pathway for this PTH action is the stimulation of a G protein-adenylate cyclase-cAMP system, which then leads to stimulation of a calcium transport system inhibitable by verapamil or nifedipine or activation of L-type calcium channels; 4) activation of protein kinase C is also involved; and 5) the PTH-induced rise in [Ca2+]i is due, in major parts, to movement of extracellular calcium into the cell.

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.

Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2

1994 ◽  
Vol 131 (5) ◽  
pp. 510-515 ◽  
Author(s):  
Osamu Kozawa ◽  
Haruhiko Tokuda ◽  
Atsushi Suzuki ◽  
Jun Kotoyori ◽  
Yoshiaki Ito ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Phospholipase A ◽  
Phosphoinositide Hydrolysis ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Kinase C ◽  
Dose Dependent

Kozawa O, Tokuda H, Suzuki A, Kotoyori J, Ito Y, Oiso Y. Effect of glucocorticoid on prostaglandin F2α-induced prostaglandin E2 synthesis in osteoblast-like cells: inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2. Eur J Endocrinol 1994;131:510–15. ISSN 0804–4643 It is well known that osteoporosis is a common complication of patients with glucocorticoid excess. We showed previously that prostaglandin (PG) F2α stimulates the synthesis of PGE2, a potent bone resorbing agent, and that the activation of protein kinase C amplifies the PGF2α-induced PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of dexamethasone on PGE2 synthesis induced by PGF2α in MC3T3-E1 cells. The pretreatment with dexamethasone significantly inhibited the PGE2 synthesis in a dose-dependent manner in the range between 0.1 and 10 nmol/l in these cells. This effect of dexamethasone was dependent on the time of pretreatment up to 8 h. Dexamethasone also inhibited PGE2 synthesis induced by melittin, known as a phospholipase A2 activator. Furthermore, dexamethasone significantly inhibited the enhancement of PGF2α- or melittin-induced PGE2 synthesis by 12-O-tetradecanoylphorbol-13-acetate, known as a protein kinase C activator. In addition, dexamethasone significantly inhibited PGF2α-induced formation of inositol phosphates in a dose-dependent manner between 0.1 and 10 nmol/l in MC3T3-E1 cells. These results strongly suggest that glucocorticoid inhibits PGF2α-induced PGE2 synthesis through the inhibition of phosphoinositide hydrolysis by phospholipase C as well as phospholipase A2 in osteoblast-like cells. Osamu Kozawa, Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Kasugai, Aichi 480-03, Japan

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.

scholarly journals Phosphatidylinositol 3-Kinase-mediated Endocytosis of Renal Na+,K+-ATPase α Subunit in Response to Dopamine

1998 ◽  
Vol 9 (5) ◽  
pp. 1209-1220 ◽  
Author(s):  
Alexander V. Chibalin ◽  
Juleen R. Zierath ◽  
Adrian I. Katz ◽  
Per-Olof Berggren ◽  
Alejandro M. Bertorello
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
Α Subunit ◽  
Kinase C ◽  
Dose Dependent ◽  
Α Subunits ◽  
K Activity ◽  
Phosphatidylinositol 3

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its α and β subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase α subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+,K+-ATPase α subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase α subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.

scholarly journals Regulation of prostaglandin synthesis by protein kinase C in mouse peritoneal macrophages

1989 ◽  
Vol 260 (2) ◽  
pp. 471-478 ◽  
Author(s):  
H J Pfannkuche ◽  
V Kaever ◽  
D Gemsa ◽  
K Resch
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Membrane Phospholipids ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Dependent Protein ◽  
Dose Dependent ◽  
Mouse Peritoneal Macrophages

Resident mouse peritoneal macrophages synthesized and released prostaglandins (PGs) when challenged with 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (DiC8). Both stimuli were found to activate Ca2+/phospholipid-dependent protein kinase C (PKC). 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine (‘H-7’) and D-sphingosine, known to inhibit PKC by different mechanisms, were able to decrease the PKC activity of macrophages in a dose-dependent manner. Addition of either PKC inhibitor decreased PG synthesis and also the release of arachidonic acid (AA) from phospholipids induced by TPA or DiC8. Simultaneously TPA or DiC8 also decreased incorporation of free AA into membrane phospholipids of macrophages. AA incorporation could be restored, however, by pretreatment with the PKC inhibitors. Our results demonstrate an involvement of PKC in the regulation of PG synthesis in mouse peritoneal macrophages and provide further evidence that reacylation of released fatty acids may be an important regulatory step.

On the signal transducing mechanisms involved in the synergistic interaction between interleukin-1 and bradykinin on prostaglandin biosynthesis in human gingival fibroblasts

1992 ◽  
Vol 12 (4) ◽  
pp. 263-271 ◽  
Author(s):  
Ulf H. Lerner ◽  
Gustaf Brunius ◽  
Thomas Modeer
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Phorbol Esters ◽  
Synergistic Interaction ◽  
Human Gingival Fibroblasts ◽  
Gingival Fibroblasts ◽  
Kinase C ◽  
Stimulation Of

Recombinant human interleukin-1β (IL-1β) and bradykinin (BK) synergistically stimulate prostaglandin E2 (PGE2) formation in human gingival fibroblasts cultured for 24 h. Neither BK or IL-1β per se, nor their combinations, caused any acute stimulation of cellular cyclic AMP accumulation. BK, but not IL-1β, caused a rapid, transient rise of intracellular Ca2+ concentration ([Ca2+]i), as assessed by recordings of fura-2 fluorescence in monolayers of prelabelled gingival fibroblasts. IL-1β did not change the effect of BK on [Ca2+]i. Ionomycin and A 23187, two calcium ionophores, synergistically potentiated the stimulatory effect of IL-1β on PGE2 formation. Three different phorbol esters known to activate protein kinase C also synergistically potentiated the action of IL-1β on PGE2 formation. Exogenously added arachidonic acid significantly enhanced the basal formation of PGE2. In IL-1β treated cells, the enhancement of PGE2 formation seen after addition of arachidonic acid, was synergistically upregulated by IL-1β. These data show that i) the synergistic interaction between IL-1β and BK on PGE2 formation is not due to an effect linked to an upregulation of cyclic AMP or [Ca2+]i; ii) the signal transducing mechanism by which BK interacts with IL-1β, however, may be linked to a BK induced stimulation of [Ca2+]i and/or protein kinase C; iii) the mechanism involved in the action of IL-1β may, at least partly, be due to enhancement of the biosynthesis of prostanoids mediated by an upregulation of cyclooxygenase activity.

scholarly journals Nesfatin-1 Suppresses Cardiac L-type Ca2+ Channels Through Melanocortin Type 4 Receptor and the Novel Protein Kinase C Theta Isoform Pathway

2015 ◽  
Vol 36 (2) ◽  
pp. 555-568 ◽  
Author(s):  
Jiaoqian Ying ◽  
Yuan Zhang ◽  
Shan Gong ◽  
Zhigang Chang ◽  
Xiaofeng Zhou ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Dependent Manner ◽  
The Novel ◽  
Inotropic Effects ◽  
Kinase C ◽  
Melanocortin 4 Receptor ◽  
Dose Dependent ◽  
Ca2 Channels

Background/Aims: Nesfatin-1 (NF-1), an anorexic nucleobindin-2 (NUCB2)-derived hypothalamic peptide, acts as a peripheral cardiac modulator and it can induce negative inotropic effects. However, the mechanisms underlying these effects in cardiomyocytes remain unclear. Methods: Using patch clamp, protein kinase assays, and western blot analysis, we studied the effect of NF-1 on L-type Ca2+ currents (ICa,L) and to explore the regulatory mechanisms of this effect in adult ventricular myocytes. Results: NF-1 reversibly decreased ICa,L in a dose-dependent manner. This effect was mediated by melanocortin 4 receptor (MC4-R) and was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Dialysis of cells with GDP-β-S or anti-Gβ antibody as well as pertussis toxin pretreatment abolished the inhibitory effects of NF-1 on ICa,L. Protein kinase C (PKC) antagonists abolished NF-1-induced responses, whereas inhibition of PKA activity or intracellular application of the fast Ca2+-chelator BAPTA elicited no such effects. Application of NF-1 increased membrane abundance of PKC theta isoform (PKCθ), and PKCθ inhibition abolished the decrease in ICa,L induced by NF-1. Conclusion: These data suggest that NF-1 suppresses L-type Ca2+ channels via the MC4-R that couples sequentially to the βγ subunits of Gi/o-protein and the novel PKCθ isoform in adult ventricular myocytes.

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