A role for protein kinase C-mediated phosphorylation in eliciting glucagon desensitization in rat hepatocytes.
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ABSTRACT:
SUBMITTER: Savage A
PROVIDER: S-EPMC1136774 | biostudies-other | 1995 Apr
REPOSITORIES: biostudies-other
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Project description:Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5'-[beta-thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.
| S-EPMC1144160 | biostudies-other
Project description:In morphine tolerance a key question that remains to be answered is whether mu-opioid receptor (MOPr) desensitization contributes to morphine tolerance, and if so by what cellular mechanisms. Here we demonstrate that MOPr desensitization can be observed in single rat brainstem locus coeruleus (LC) neurons following either prolonged (> 4 h) exposure to morphine in vitro or following treatment of animals with morphine in vivo for 3 days. Analysis of receptor function by an operational model indicated that with either treatment morphine could induce a profound degree (70-80%) of loss of receptor function. Ongoing PKC activity in the MOPr-expressing neurons themselves, primarily by PKCalpha, was required to maintain morphine-induced MOPr desensitization, because exposure to PKC inhibitors for only the last 30-50 min of exposure to morphine reduced the MOPr desensitization that was induced both in vitro and in vivo. The presence of morphine was also required for maintenance of desensitization, as washout of morphine for > 2 h reversed MOPr desensitization. MOPr desensitization was homologous, as there was no change in alpha(2)-adrenoceptor or ORL1 receptor function. These results demonstrate that prolonged morphine treatment induces extensive homologous desensitization of MOPrs in mature neurons, that this desensitization has a significant PKC-dependent component and that this desensitization underlies the maintenance of morphine tolerance.
| S-EPMC2695152 | biostudies-literature
Project description:This report investigates acute changes in the sensitivity of 5-HT(1A) receptors in dorsal raphe (dr) neurons in response to elevated serotonin. DR neurons were isolated from adult rats and measurements of inhibition of Ca(2+) current by 5-HT were obtained using the whole cell patch clamp technique. During a 10-min application of 5-HT (with normal [Ca(2+)](i) approximately 100 nM) a desensitization occurred. The response to 20 nM 5-HT decreased by 66% relative to control and remained depressed for about 30 min. When the internal [Ca(2+)] was buffered to <1 nM only a weak transient desensitization occurred that was surmountable with higher [5-HT]. Adenylyl cyclase activation with forskolin mimicked the desensitization and selective inhibition of protein kinase A (PKA), but not protein kinase C (PKC), partially antagonized the desensitization induced by 5-HT. To measure the activity of PKA and phosphatase enzymes, dr slices were incubated with the selective agonist dipropyl-5-carboxamidotryptamine (DP-5-CT, 1 microM) for 10 min and the phosphorylation of the PKA substrate Kemptide was followed using ATP-gamma(32)P. DP-5-CT inhibited the cAMP stimulated maximal activity of PKA but raised basal PKA activity, thus increasing the percentage of PKA in the active state (activity ratio), an effect that was prevented by the selective 5-HT(1A) antagonist WAY100635. DP-5-CT also caused a significant inhibition of phosphatase activity. These data support a model in the dr where 5-HT(1A)-receptor stimulation of PKA promotes phosphorylation of a target and phosphatase inhibition leading to heterologous desensitization. The effect would be expected to have physiological consequences for 5-HT-mediated inhibitory post synaptic potentials and the Ca(2+) component of the action potentials of dr neurons.
| S-EPMC2900410 | biostudies-literature
Project description:1. Receptor phosphorylation and down-regulation by protein kinases may be a key event initiating desensitization. The present studies were designed to investigate the effect of a potent protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), on antinociception induced by intrathecal (i.t.) administration of a selective delta-opioid receptor agonist [D-Ala2] deltorphin II in the male ICR mouse and on the specific binding of [3H]-[D-Ser2, Leu5]enkephalin-Thr6 (DSLET), a delta-opioid receptor ligand, in the crude synaptic membrane of the spinal cord. 2. Intrathecal (i.t.) pretreatment with PDBu at low doses, which injected alone did not affect the basal tail-flick latency, dose-dependently attenuated the antinociception induced by i.t. administration of [D-Ala2]deltorphin II. The attenuation of i.t.-administered [D-Ala2] deltorphin II-induced antinociception by PDBu was reversed in a dose-dependent manner by i.t. concomitant pretreatment with a specific PKC inhibitor, calphostin C. 3. In the binding experiment, incubation of the crude synaptic membrane of the spinal cord for 2 h at 25 degrees C with PDBu (0.03 to 10 microM) caused a dose-dependent inhibition of the [3H]-DSLET binding. Scatchard analysis of [3H]-DSLET binding revealed that PDBu at 10 microM displayed a 30.7% reduction in the number of [3H]-DSLET binding sites with no significant change in affinity, compared with the non-treatment control, indicating that the activation of membrane-bound PKC by PDBu causes a decrease in the number of specific delta-opioid agonist binding sites. 4. An i.t. injection of [D-Ala2]deltorphin II produced an acute antinociceptive tolerance to the antinociceptive effect of a subsequent i.t. challenge of [D-Ala2]deltorphin II. Concomitant pretreatment with calphostin C markedly prevented the development of acute tolerance to the i.t.-administered [D-Ala2]deltorphin II-induced antinociception. On the other hand, a highly selective protein kinase A (PKA) inhibitor, KT5720, did not have any effect on the development of acute tolerance to [D-Ala2]deltorphin II antinociception. 5. These findings suggest that a loss of specific delta-agonist binding by the activation of PKC by PDBu is involved in the PDBu-induced antinociceptive unresponsiveness to delta-opioid receptor agonist in the mouse spinal cord. Based on the acute tolerance studies, we propose that PKC, but not PKA, plays an important role in the process of homologous desensitization of the spinal delta-opioid receptor-mediated antinociception.
| S-EPMC1909849 | biostudies-other
Project description:This SuperSeries is composed of the SubSeries listed below.
2020-06-30 | GSE147801 | GEO
Project description:We found that glucagon stimulated membrane protein kinase C (PKC) activity and phosphatidylcholine hydrolysis in 24 h-cultured rat hepatocytes. Phorbol myristate acetate, 8-bromo cyclic AMP, vasopressin, noradrenaline and the Ca2+ ionophore A23187 also stimulated membrane PKC activity. However, only vasopressin and noradrenaline stimulated inositol phosphate accumulation, whereas all agonists stimulated the rate of release of water-soluble choline metabolites into the medium. Choline, and to a much lesser extent phosphocholine, were released, suggesting predominantly phospholipase D activation. This was supported by the finding that the accumulation of phosphatidate and diacylglycerol was enhanced by the agents in [3H]myristate-labelled hepatocytes, as was [32P]phosphatidylethanol formation. Since the time courses for the release of choline into the medium and the accumulation of phosphatidate and diacylglycerol caused by vasopressin and glucagon were similar, the more rapid activation of PKC by vasopressin probably reflects diacylglycerol formation from phosphoinositide breakdown. The inability of glucagon to stimulate inositol phosphate production was not due to the prolonged culture, since similar results were obtained in 4 h cultures. We conclude that the stimulation of membrane PKC activity by glucagon correlates with accumulation of diacylglycerol and phosphatidate derived from the hydrolysis of phosphatidylcholine.
| S-EPMC1151243 | biostudies-other
Project description:BackgroundPhosphorylative desensitization of G-protein-coupled receptors contributes significantly to post-myocardial infarction (MI) remodeling and heart failure (HF). Here, we determined whether adiponectin receptors (AdipoRs) 1 and 2 (the 7-transmembrane domain-containing receptors mediating adiponectin functions) are phosphorylatively modified and functionally impaired after MI.Methods and resultsPost-MI HF was induced by coronary artery occlusion. Receptor phosphorylation, kinase expression, and adiponectin function were determined via in vivo, ex vivo, and in vitro models. AdipoR1 and AdipoR2 are not phosphorylated in the normal heart. However, AdipoR1 was significantly phosphorylated after MI, peaking at 7 days and remaining significantly phosphorylated thereafter. The extent of post-MI AdipoR1 phosphorylation positively correlated with the expression level of GPCR kinase (GRK) 2, the predominant GRK isoform upregulated in the failing heart. Cardiac-specific GRK2 knockout virtually abolished post-MI AdipoR1 phosphorylation, whereas virus-mediated GRK2 overexpression significantly phosphorylated AdipoR1 and blocked adiponectin metabolic-regulatory/anti-inflammatory signaling. Mass spectrometry identified serine-7, threonine-24, and threonine-53 (residues located in the n-terminal intracellular AdipoR1 region) as the GRK2 phosphorylation sites. Ex vivo experiments demonstrated that adenosine monophosphate-activated protein kinase activation and the anti-tumor necrosis factor-α effect of adiponectin were significantly inhibited in cardiomyocytes isolated from nonischemic area 7 days after MI. In vivo experiments demonstrated that acute adiponectin administration-induced cardiac GLUT4 translocation and endothelial nitric oxide synthase phosphorylation were blunted 7 days after MI. Continuous adiponectin administration beginning 7 days after MI failed to protect the heart from adverse remodeling and HF progression. Finally, cardiac-specific GRK2 knockdown restored the cardioprotective effect of adiponectin.ConclusionAdipoR1 is phosphorylatively modified and desensitized by GRK2 in failing cardiomyocytes, contributing to post-MI remodeling and HF progression.
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Project description:Inflammatory hyperalgesia represents a nociceptive phenotype that can become persistent in nature through dynamic protein modifications. However, a large gap in knowledge exists concerning how the integration of intracellular signaling molecules coordinates a persistent inflammatory phenotype. Herein, we demonstrate that Raf Kinase Anchoring Protein (RKIP) interrupts a vital canonical desensitization pathway to maintain bradykinin (BK) receptor activation in primary afferent neurons. Biochemical analyses of primary neuronal cultures indicate bradykinin-stimulated PKC phosphorylation of RKIP at Ser153. Furthermore, BK exposure increases G-protein Receptor Kinase 2 (GRK2) binding to RKIP, inhibiting pharmacological desensitization of the BK receptor. Additional studies found that molecular RKIP down-regulation increases BK receptor desensitization in real-time imaging of primary afferent neurons, identifying a key pathway integrator in the desensitization process that controls multiple GRK2-sensitive G-protein coupled receptors. Therefore, RKIP serves as an integral scaffolding protein that inhibits BK receptor desensitization.
| S-EPMC9283312 | biostudies-literature
Project description:In this study the possible role of Na+ influx, arachidonate mediators and alpha-subunit phosphorylation in the stimulatory response of hepatic Na+/K(+)-ATPase to glucagon was examined. Glucagon stimulation of ouabain-sensitive 86Rb+ uptake in freshly isolated rat hepatocytes reached maximal levels in less than 1 min after hormone addition and was half-maximal (EC50) at a concentration of 2.4( +/- 1.3) x 10(-10) M. Analysis of the K(+)-dependence of this response indicates an effect on the apparent Vmax. for K+ with no significant change in the apparent kappa 0.5. Unlike monensin, glucagon stimulation of Na+/K(+)-ATPase-mediated transport activity was not associated with an increase in 22Na+ influx. This indicates that the stimulation of Na+/K(+)-ATPase by glucagon is not secondary to an increase in Na+ influx. A role for arachidonate mediators in this effect also appears unlikely because neither basal nor glucagon-stimulated ouabain-sensitive 86Rb+ uptake was significantly affected by supramaximal concentrations of cyclo-oxygenase, lipoxygenase, cytochrome p-450 or phospholipase A2 inhibitors. To study the possible role of protein kinase-mediated phosphorylation in the stimulation of ouabain-sensitive 86Rb uptake, hepatocytes were metabolically radiolabelled with [32P]P(i), Glucagon stimulated incorporation of 32P into a 95 kDa phosphoprotein that comigrates with Na+/K(+)-ATPase alpha-subunit immunoreactivity in two-dimensional gel electrophoresis. The alpha-subunit could be immunoprecipitated from detergent-solubilized particulate fractions of hepatocytes using an anti-(rat kidney Na+/K(+)-ATPase) serum. When hepatocytes were metabolically radiolabelled with [32P]P(i), the immunoprecipitated alpha-subunit contained 32P. Glucagon increased the incorporation of 32P into the immunoprecipitated subunit by 197 +/- 21% (n = 6). Similar results were observed with a rabbit anti-peptide serum ('anti-LEAVE' serum) prepared against an amino acid sequence in the alpha-subunit. The EC50 for glucagon-stimulated phosphorylation of the alpha-subunit (approximately 1 x 10(-10) M) was very close to that for glucagon stimulation of ouabain-sensitive 86Rb+ uptake. In conclusion, it appears that glucagon stimulation of hepatic Na+/K(+)-ATPase-mediated transport activity is not secondary to increases in Na+ influx or changes in the levels of an arachidonate mediator. The data provide support for the hypothesis that glucagon stimulation of Na(+)-pump activity in hepatocytes may be related to protein kinase-mediated changes in the phosphorylation state of the alpha-subunit.
| S-EPMC1217007 | biostudies-other
Project description:Oxidative stress plays a critical role in liver tissue damage and in hepatocellular carcinoma (HCC) initiation and progression. However, the mechanisms that regulate autophagy and metabolic reprogramming during the generation of reactive oxygen species (ROS), and how ROS promotes tumorigenesis, still need to be fully understood. We show that protein kinase C (PKC) / loss in hepatocytes promotes autophagy and oxidative phosphorylation. This results in ROS generation, which through NRF2 drives HCC cell autonomously and non-autonomously. Although PKC / promotes tumorigenesis in oncogene-driven cancer models, emerging evidence demonstrate that it is a tumor suppressor in more complex carcinogenic processes. Consistently, PKC / levels negatively correlate with HCC histological tumor grade, establishing this kinase as a tumor suppressor in liver cancer.
2020-06-30 | GSE147800 | GEO