Enhancement of Potency and Efficacy of NADA by PKC-Mediated Phosphorylation of Vanilloid Receptor

2004 ◽  
Vol 91 (3) ◽  
pp. 1442-1449 ◽  
Author(s):  
Louis S. Premkumar ◽  
Zhan-Heng Qi ◽  
Jeremy Van Buren ◽  
Manish Raisinghani
Keyword(s):  
Action Potentials ◽  
Vanilloid Receptor ◽  
Dependent Manner ◽  
Induced Current ◽  
Repeated Application ◽  
Drg Neurons ◽  
Kinase C ◽  
Potent Agonist ◽  
Intracellular Binding

The search for an endogenous ligand for the vanilloid receptor (VR or TRPV1) has led to the identification of N-arachidonyl dopamine (NADA). This study investigates the role of protein kinase C (PKC)-mediated phosphorylation on NADA-induced membrane currents in Xenopus oocytes heterologously expressing TRPV1 and in dorsal root ganglion (DRG) neurons. In basal state, current induced by 10 μM NADA is 5-10% of the current induced by 1 μM capsaicin or protons at pH 5. However, PKC activator, phorbol 12,13-dibutyrate (PDBu) strongly potentiated (∼15-fold) the NADA-induced current. Repeated application of NADA at short intervals potentiated its own response approximately fivefold in a PKC-dependent manner. PKC inhibitor, bisindolylmaleimide (BIM, 500 nM), a mutant TRPV1 (S800A/S502A), and maximal activation of PKC abolished the potentiation induced by repeated application of NADA. As a further confirmation that NADA could stimulate PKC, pretreatment with NADA potentiated the response of protons at pH 5 (∼20 fold), which was dramatically reduced in the mutant TRPV1. In DRG neurons, capsaicin (100 nM) induced a ∼15 mV depolarization and initiated a train of action potentials compared with 1 μM NADA that produced a ∼5 mV response. Pretreatment with PDBu induced significantly larger depolarization and potentiated NADA-induced current. Furthermore, exposure of NADA to the intracellular surface of the membrane-induced larger currents suggesting inaccessibility to the intracellular binding site might contribute to its weaker action. These results indicate that NADA is a potent agonist of VR when the receptor is in the PKC-mediated phosphorylation state.

PKCϵ has an alcohol-binding site in its second cysteine-rich regulatory domain

2009 ◽  
Vol 421 (3) ◽  
pp. 405-413 ◽  
Author(s):  
Joydip Das ◽  
Satyabrata Pany ◽  
Ghazi M. Rahman ◽  
Simon J. Slater
Keyword(s):  
Protein Kinase ◽  
Binding Site ◽  
Maximum Effect ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
C1 Domain ◽  
Pkc Protein Kinase C ◽  
And Function

Alcohols regulate the expression and function of PKC (protein kinase C), and it has been proposed that an alcohol-binding site is present in PKCα in its C1 domain, which consists of two cysteine-rich subdomains, C1A and C1B. A PKCϵ-knockout mouse showed a significant decrease in alcohol consumption compared with the wild-type. The aim of the present study was to investigate whether an alcohol-binding site could be present in PKCϵ. Here we show that ethanol inhibited PKCϵ activity in a concentration-dependent manner with an EC50 (equilibrium ligand concentration at half-maximum effect) of 43 mM. Ethanol, butanol and octanol increased the binding affinity of a fluorescent phorbol ester SAPD (sapintoxin-D) to PKCϵC1B in a concentration-dependent manner with EC50 values of 78 mM, 8 mM and 340 μM respectively, suggesting the presence of an allosteric alcohol-binding site in this subdomain. To identify this site, PKCϵC1B was photolabelled with 3-azibutanol and 3-azioctanol and analysed by MS. Whereas azibutanol preferentially labelled His236, Tyr238 was the preferred site for azioctanol. Inspection of the model structure of PKCϵC1B reveals that these residues are 3.46 Å (1 Å=0.1 nm) apart from each other and form a groove where His236 is surface-exposed and Tyr238 is buried inside. When these residues were replaced by alanine, it significantly decreased alcohol binding in terms of both photolabelling and alcohol-induced SAPD binding in the mutant H236A/Y238A. Whereas Tyr238 was labelled in mutant H236A, His236 was labelled in mutant Y238A. The present results provide direct evidence for the presence of an allosteric alcohol-binding site on protein kinase Cϵ and underscore the role of His236 and Tyr238 residues in alcohol binding.

Erythropoietin and testicular steroidogenesis: the role of second messengers

1995 ◽  
Vol 132 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Carlo Foresta ◽  
Roberto Mioni ◽  
Paola Bordon ◽  
Francesco Gottardello ◽  
Andrea Nogara ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Leydig Cells ◽  
Second Messengers ◽  
Indirect Evidence ◽  
Calcium Flux ◽  
Dependent Manner ◽  
Kinase C ◽  
Testicular Steroidogenesis

Foresta C, Mioni R, Bordon P, Gottardello F, Nogara A, Rossato M. Erythropoietin and testicular steroidogenesis: the role of second messengers. Eur J Endocrinol 1995;132:103–8. ISSN 0804–4643 It has been demonstrated that erythropoietin (EPO) influences rat and human Leydig cell steroidogenesis, stimulating testosterone production through a direct and specific receptor-mediated mechanism. The aim of this study was to investigate the mechanism by which recombinant human erythropoietin (rHuEPO) exerts its stimulatory effect on rat Leydig cells. Recombinant human EPO did not induce, at any dose tested (10−10 to 10−13 mol/l), an increase in either cAMP or cGMP, suggesting that in Leydig cells the effect of rHuEPO does not involve the adenylate or guanylate–cyclase systems. The role of transmembrane calcium flux in rHuEPO-stimulated steroidogenesis was studied by evaluating the effect of calcium channel blocker, verapamil, and by the 45Ca2+ uptake method. Verapamil did not influence rHuEPO-induced testosterone secretion and rHuEPO did not modify calcium recycling, indicating that calcium transmembrane flux is not involved in the rHuEPO effect. The protein kinase C inhibitor staurosporine (10, 30, 100 and 300 nmol/l) inhibited rHuEPO-stimulated testicular steroidogenesis in a dose-dependent manner. This indirect evidence suggests that the stimulatory effect of rHuEPO on rat Leydig cells may involve protein kinase C activation. Carlo Foresta, Institute of Internal Medicine, Via Ospedale Civile 105, 35128 Padova, Italy

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

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

Role of protein kinase C in the pigment cell of the lizard (Anolis carolinensis)

1987 ◽  
Vol 112 (2) ◽  
pp. 283-287 ◽  
Author(s):  
A. M. Lucas ◽  
A. J. Thody ◽  
S. Shuster
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Pigment Cell ◽  
Anolis Carolinensis ◽  
Regulatory Subunit ◽  
Dependent Manner ◽  
Kinase C

ABSTRACT The role of protein kinase C in melanosome dispersion was examined using the melanophores of the lizard Anolis carolinensis and an in-vitro rate method of bioassay. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which directly activates protein kinase C, was able to potentiate the melanophore response to α-MSH in a dose-dependent manner. Similarly, the stimulatory response to forskolin, which activates the adenylate cyclase catalytic subunit, was also potentiated by TPA. The response of the melanophore to cyclic AMP, however, remained unaltered by any dose of TPA. We thus propose that the potentiation of α-MSH potency by TPA is through an interaction of protein kinase C with adenylate cyclase and, more specifically, that this interaction may be at the level of the linkage of the nucleotide regulatory subunit Ns with the catalytic moiety C of adenylate cyclase. J. Endocr. (1987) 112, 283–287

Long-term regulation of Na(+)-H+ exchange in vascular smooth muscle cells: role of protein kinase C

1991 ◽  
Vol 260 (3) ◽  
pp. C562-C569 ◽  
Author(s):  
M. Mitsuka ◽  
B. C. Berk
Keyword(s):  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Kinetic Properties ◽  
Dependent Manner ◽  
Kinase C ◽  
Measured Exposure

Regulation of intracellular pH (pHi) plays an important role in vascular smooth muscle cell (VSMC) contractile tone and growth. We have shown that pHi in proliferating VSMC is more alkaline (7.25) than in growth-arrested cells (7.10). To study the Na(+)-H+ exchanger in the growth-dependent regulation of VSMC pHi, ethylisopropylamiloride (EIPA)-sensitive Na+ influx was measured. Exposure of growth-arrested VSMC to 10% serum initially increased Na+ influx (145% of baseline at 30 min), which then decreased (52% of baseline at 24 h). Serum-induced alterations in the kinetic properties of the Na(+)-H+ exchanger were studied by analysis of its external Na+ binding site properties. Exposure of growth-arrested VSMC to 10% serum for 24 h increased the Km for external Na+ from 54 to 380 mM, with a change in the Vmax from 155 to 199 nmol Na+.mg protein-1.min-1. The change in Km was due to activation of protein kinase C (PKC). Phorbol 12,13-dibutyrate caused a 48% decrease in EIPA-sensitive influx, the inactive 4 alpha-phorbol 12,13-didecanoate had no effect, and the PKC inhibitor sphingosine reversed the effect. Therefore, the Na(+)-H+ exchanger in VSMC is regulated in a growth-dependent manner via PKC.

Cross talk between serine/threonine and tyrosine kinases regulates ADP-induced thromboxane generation in platelets

2015 ◽  
Vol 114 (09) ◽  
pp. 558-568 ◽  
Author(s):  
Dheeraj Bhavanasi ◽  
Rachit Badolia ◽  
Bhanu Kanth Manne ◽  
Sumalaxmi Janapati ◽  
Carol Dangelmaier ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Functional Role ◽  
Human Platelets ◽  
Src Family Kinases ◽  
Dependent Manner ◽  
Kinase C ◽  
Pkc Isoforms ◽  
First Time ◽  
Stimulation Of

SummaryADP-induced thromboxane generation depends on Src family kinases (SFKs) and is enhanced with pan-protein kinase C (PKC) inhibitors, but it is not clear how these two events are linked. The aim of the current study is to investigate the role of Y311 phosphorylated PKCδ in regulating ADP-induced platelet activation. In the current study, we employed various inhibitors and murine platelets from mice deficient in specific molecules to evaluate the role of PKCδ in ADP-induced platelet responses. We show that, upon stimulation of platelets with 2MeSADP, Y311 on PKCδ is phosphorylated in a P2Y1/Gq and Lyn-dependent manner. By using PKCδ and Lyn knockout murine platelets, we also show that tyrosine phosphorylated PKCδ plays a functional role in mediating 2MeSADP-induced thromboxane generation. 2MeSADP-induced PKCδ Y311 phosphorylation and thromboxane generation were potentiated in human platelets pre-treated with either a pan-PKC inhibitor, GF109203X or a PKC α/β inhibitor and in PKC α or β knockout murine platelets compared to controls. Furthermore, we show that PKC α/β inhibition potentiates the activity of SFK, which further hyper-phosphorylates PKCδ and potentiates thromboxane generation. These results show for the first time that tyrosine phosphorylated PKCδ regulates ADP-induced thromboxane generation independent of its catalytic activity and that classical PKC isoforms α/β regulate the tyrosine phosphorylation on PKCδ and subsequent thromboxane generation through tyrosine kinase, Lyn, in platelets.

scholarly journals Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

2010 ◽  
Vol 2010 ◽  
pp. 1-9 ◽  
Author(s):  
Takehiro Ko ◽  
Yutaka Kakizoe ◽  
Naoki Wakida ◽  
Manabu Hayata ◽  
Kohei Uchimura ◽  
...  
Keyword(s):  
Serine Protease ◽  
Channel Blocker ◽  
Systemic Circulation ◽  
Luciferase Assay ◽  
Dependent Manner ◽  
Kinase C ◽  
Aldosterone Production ◽  
H295r Cells ◽  
Dose Dependent

A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells). Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC) inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

Inhibition of Rapid Heat Responses in Nociceptive Primary Sensory Neurons of Rats by Vanilloid Receptor Antagonists

1999 ◽  
Vol 82 (6) ◽  
pp. 2853-2860 ◽  
Author(s):  
Timo Kirschstein ◽  
Wolfgang Greffrath ◽  
Dietrich Büsselberg ◽  
Rolf-Detlef Treede
Keyword(s):  
Sensory Neurons ◽  
Reversal Potential ◽  
Vanilloid Receptor ◽  
Maximum Effect ◽  
Dependent Manner ◽  
Primary Sensory Neurons ◽  
Drg Neurons ◽  
Patch Clamp Technique ◽  
Noxious Heat ◽  
Duration Of Action

Recent studies demonstrated that heat-sensitive nociceptive primary sensory neurons respond to the vanilloid receptor (VR) agonist capsaicin, and the first cloned VR is a heat-sensitive ion channel. Therefore we studied to what extent heat-evoked currents in nociceptive dorsal root ganglion (DRG) neurons can be attributed to the activation of native vanilloid receptors. Heat-evoked currents were investigated in 89 neurons acutely dissociated from adult rat DRGs as models for their own terminals using the whole cell patch-clamp technique. Locally applied heated extracellular solution (effective temperature ∼53°C) rapidly activated reversible and reproducible inward currents in 80% (62/80) of small neurons (≤32.5 μm), but in none of nine large neurons ( P < 0.001, χ2 test). Heat and capsaicin sensitivity were significantly coexpressed in this subpopulation of small DRG neurons ( P < 0.001, χ2 test). Heat-evoked currents were accompanied by an increase of membrane conductance (320 ± 115%; mean ± SE, n = 7), had a reversal potential of 5 ± 2 mV ( n = 5), which did not differ from that of capsaicin-induced currents in the same neurons (4 ± 3 mV), and were carried at least by Na+ and Ca2+(pCa2+ > pNa+). These observations are consistent with the opening of temperature-operated nonselective cation channels. The duration of action potentials was significantly higher in heat-sensitive (10–90% decay time: 4.45 ± 0.39 ms, n = 12) compared with heat-insensitive neurons (2.18 ± 0.19 ms, n = 6; P< 0.005, Student's t-test), due to an inflection in the repolarizing phase. This property as well as capsaicin sensitivity and small cell size are characteristics of nociceptive DRG neurons. When coadministered with heat stimuli, the competitive VR antagonist capsazepine (1 μM to 1 mM) significantly reduced heat-evoked currents in a dose-dependent manner (IC50 13 μM, Hill slope −0.58, maximum effect 75%). Preincubation for 12–15 s shifted the IC50 by ∼0.5 log10 units to an estimated IC50 of ∼4 μM. The noncompetitive VR antagonist ruthenium red (5 μM) significantly reduced heat-evoked currents by 33 ± 6%. The effects of both VR antagonists were rapidly reversible. Our results provide evidence for a specific activation of native VRs in nociceptive primary sensory neurons by noxious heat. The major proportion of the rapid heat-evoked currents can be attributed to the activation of these temperature-operated channels, and noxious heat may be the signal detected by VRs under physiological conditions.

Sign in / Sign up

Export Citation Format

Share Document