Propofol Inhibits Phosphorylation of N-methyl-d-aspartate Receptor NR1 Subunits in Neurons

Background: Anesthetics may interact with ionotropic glutamate receptors to produce some of their biologic actions. Cellular studies reveal that the ionotropic glutamate receptors, N-methyl-d-aspartate receptors (NMDARs), can be phosphorylated on their NR1 subunits at the C-terminal serine residues, which is a major mechanism for the regulation of NMDAR functions. It is currently unknown whether anesthetics have any modulatory effects on NMDAR NR1 subunit phosphorylation.

Methods: The possible effect of a general anesthetic propofol on phosphorylation of NR1 subunits at serine 897 (pNR1S897) and 896 (pNR1S896) was detected in cultured rat cortical neurons.

Results: Propofol consistently reduced basal levels of pNR1S897 and pNR1S896 in a concentration-dependent manner. This reduction was rapid as the reliable reduction of pNR1S896 developed 1 min after propofol administration. Pretreatment of cultures with the protein phosphatase 2A inhibitors okadaic acid or calyculin A blocked the effect of propofol on the NR1 phosphorylation, whereas okadaic acid or calyculin A alone did not alter basal pNR1S897 and pNR1S896 levels. In addition, propofol decreased tyrosine phosphorylation of protein phosphatase 2A at tyrosine 307, resulting in an increase in protein phosphatase 2A activity. In the presence of propofol, the NMDAR agonist-induced intracellular Ca2+ increase was impaired in neurons with dephosphorylated NR1 subunits.     

Modulation of AMPA receptor GluR1 subunit phosphorylation in neurons by the intravenous anaesthetic propofol

Background: The ionotropic glutamate receptor is a potential molecular sitein the central nervous system that general anaesthetics mayinteract with to produce some of their biological actions. Proteinphosphorylation has been well documented to occur in the intracellularC-terminal domain of -amino-3-hydroxy-5-methylisoxazole-4-propionicacid (AMPA) subtype of glutamate receptors, which representsa pivotal mechanism for the post-translational modulation ofAMPA receptor functions. In this study, we investigated a possibleinfluence of an i.v. anaesthetic agent propofol on the phosphorylationof AMPA receptor GluR1 subunits in cultured neurons. Methods: The effect of propofol on phosphorylation of GluR1 subunitsat serine 831 and 845 was assayed in cultured rat striatal andcortical neurons by western blot with phospho- and site-specificantibodies. Results: Propofol consistently elevated phosphorylation of GluR1 subunitsat the C-terminal serine 845 site in both striatal and corticalneurons. The elevation in phosphorylation was concentration-dependentand started at a low concentration (3 µM). This increasein serine 845 phosphorylation was rapid and sustained duringthe entire course of propofol exposure. In contrast to serine845, phosphorylation of GluR1 at serine 831 was not alteredby propofol in striatal and cortical neurons. Total GluR1 abundanceremained unchanged in response to propofol incubation. Conclusions: These data indicate that propofol possesses the ability to upregulateAMPA receptor GluR1 subunit phosphorylation at a specific serine845 site in neurons and provide evidence supporting the AMPAreceptor as a molecular target for general anaesthetics.     

Mechanical loading modulates glutamate receptor subunit expression in bone

The cellular mechanisms coupling mechanical loading with bone remodeling remain unclear. In the CNS, the excitatory amino acid glutamate (Glu) serves as a potent neurotransmitter exerting its effects via various membrane Glu receptors (GluR). Nerves containing Glu exist close to bone cells expressing functional GluRs. Demonstration of a mechanically sensitive glutamate/aspartate transporter protein and the ability of glutamate to stimulate bone resorption in vitro suggest a role for glutamate linking mechanical load and bone remodeling. We used immunohistochemical techniques to identify the expression of N-methyl-d-aspartate acid (NMDA) and non-NMDA (AMPA or kainate) ionotropic GluR subunits on bone cells in vivo. In bone sections from young adult rats, osteoclasts expressed numerous GluR subunits including AMPA (GluR2/3 and GluR4), kainic acid (GluR567) and NMDA (NMDAR2A, NMDAR2B and NMDAR2C) receptor subtypes. Bone lining cells demonstrated immunoexpression for NMDAR2A, NMDAR2B, NMDAR2C, GluR567, GluR23, GluR2 and GluR4 subunits. Immunoexpression was not evident on osteocytes, chondrocytes or vascular channels. To investigate the effects of mechanical loading on GluR expression, we used a Materials Testing System (MTS) to apply 10 N sinusoidal axial compressive loads percutaneously to the right limbs (radius/ulna, tibia/fibula) of rats. Each limb underwent 300-load cycles/day (cycle rate, 1 Hz) for 4 consecutive days. Contralateral, non-loaded limbs served as controls. Mechanically loaded limbs revealed a load-induced loss of immunoexpression for GluR2/3, GluR4, GluR567 and NMDAR2A on osteoclasts and NMDAR2A, NMDAR2B, GluR2/3 and GluR4 on bone lining cells. Both neonatal rabbit and rat osteoclasts were cultured on bone slices to investigate the effect of the NMDA receptor antagonist, MK801, and the AMPA/kainic acid receptor antagonist, NBQX, on osteoclast resorptive activity in vitro. The inhibition of resorptive function seen suggested that both NMDAR and kainic acid receptor function are required for normal osteoclast function. While the exact role of ionotropic GluRs in skeletal tissue remains unclear, the modulation of GluR subunit expression by mechanical loading lends further support for participation of Glu as a mechanical loading effector. These ionotropic receptors appear to be functionally relevant to normal osteoclast resorptive activity.     

Neuronal and glial localization of NR1 and NR2A/B subunits of the NMDA receptor in the human cerebral cortex

N-Methyl-D-aspartate (NMDA) receptors play a critical role in many cortical functions and are implicated in several neuropsychiatric diseases. In this study, the cellular expression of the NMDAR1 (NR1) and NMDAR2A and B (NR2A and B) subunits was investigated in the human cerebral cortex by immunocytochemistry with antibodies that recognize the NR1 or the NR2A and B subunits of the NMDA receptor. In frontal (areas 10 and 46) and temporal (area 21) association cortices and the cingulofrontal transition cortex (area 32), NR1 and NR2A/B immunoreactivity (ir) were similar and were localized to numerous neurons in all cortical layers. NR1- and NR2A/B-positive neurons were mostly pyramidal cells, but some nonpyramidal neurons were also labeled. Electron-microscopic observations showed that NR1 and NR2A/B ir were similar. In all cases, labeling of dendrites and dendritic spines was intense. In addition, both NR1 and NR2A/B were consistently found in the axoplasm of some axon terminals and in distal astrocytic processes. This investigation revealed that numerous NMDA receptors are localized to dendritic spines, and that they are also localized to axon terminals and astrocytic processes. These findings suggest that the effects of cortical NMDA activation in the human cortex do not depend exclusively on the opening of NMDA channels located at postsynaptic sites, and that the localization of NMDA receptors is similar in a variety of mammalian species.     

Activity-dependent regulation of neuronal apoptosis in neonatal mouse cerebral cortex

A massive neuronal loss during early postnatal development has been well documented in the murine cerebral cortex, but the factors that drive cells into apoptosis are largely unknown. The role of neuronal activity in developmental apoptosis was studied in organotypic neocortical slice cultures of newborn mice. Multielectrode array and whole-cell patch-clamp recordings revealed spontaneous network activity characterized by synchronized burst discharges, which could be blocked by tetrodotoxin and ionotropic glutamate receptor antagonists. The identical neuropharmacological manipulations also caused a significant increase in the number of apoptotic neurons as early as 6 h after the start of drug treatment. Moreover, inhibition of the NMDA receptor subunit NR2A or NR2B induced a differential short-term versus delayed increase in the apoptosis rate, respectively. Activation of L-type, voltage-dependent calcium channels was neuroprotective and could prevent activity-dependent apoptosis during NMDA receptor blockade. Furthermore, this effect involved phosphorylation of cAMP response element-binding protein and activation of the tropomyosin-related kinase (Trk) receptors. Inhibition of electrical synapses and blockade of ionotropic gamma-aminobutyric acid receptors induced specific changes in spontaneous electrical activity patterns, which caused an increase in caspase-3-dependent cell death. Our results demonstrate that synchronized spontaneous network bursts activating ionotropic glutamate receptors promote neuronal survival in the neonatal mouse cerebral cortex.     

Bupivacaine Inhibits Activation of Neuronal Spinal Extracellular Receptor-activated Kinase through Selective Effects on Ionotropic Receptors

Background: Central terminals of primary nociceptors release neurotransmitters glutamate and substance P, which bind to ionotropic or metabotropic receptors on spinal neurons to induce cellular responses. Extracellular signal-regulated kinases are activated by these receptors and are important modulators of pain at the dorsal horn. The authors investigated these pathways as potential targets for antinociceptive actions of local anesthetics.

Methods: The effects of bupivacaine on the activation of extracellular receptor-activated kinase (phosphorylation to pERK) in rat spinal cord slices, induced by presynaptic release (capsaicin), by presynaptic or postsynaptic ionotropic or metabotropic receptor activation, or by activation of intracellular protein kinase C or protein kinase A and also by a receptor-independent Ca2+ ionophore, were quantitated by immunohistochemistry, counting pERK-positive neurons in the superficial dorsal horn.

Results: Capsaicin (3 [mu]m, 10 min)-stimulated pERK was reduced by bupivacaine (IC50 approximately 2 mm, approximately 0.05%), which similarly suppressed pERK induced by the ionotropic glutamate receptors for N-methyl-d-aspartate and (S)-[alpha]-amino-3-hydroxy-5-methyle-4-isoxazole propionic acid but not that induced by the metabotropic receptors for glutamate, bradykinin, or substance P. Extracellular receptor-activated kinase activation by the Ca+2 ionophore ionomycin was also sensitive to bupivacaine, but direct activation by protein kinase A or protein kinase C was not.     

Bupivacaine inhibits activation of neuronal spinal extracellular receptor-activated kinase through selective effects on ionotropic receptors

BACKGROUND: Central terminals of primary nociceptors release neurotransmitters glutamate and substance P, which bind to ionotropic or metabotropic receptors on spinal neurons to induce cellular responses. Extracellular signal-regulated kinases are activated by these receptors and are important modulators of pain at the dorsal horn. The authors investigated these pathways as potential targets for antinociceptive actions of local anesthetics. METHODS: The effects of bupivacaine on the activation of extracellular receptor-activated kinase (phosphorylation to pERK) in rat spinal cord slices, induced by presynaptic release (capsaicin), by presynaptic or postsynaptic ionotropic or metabotropic receptor activation, or by activation of intracellular protein kinase C or protein kinase A and also by a receptor-independent Ca2+ ionophore, were quantitated by immunohistochemistry, counting pERK-positive neurons in the superficial dorsal horn. RESULTS: Capsaicin (3 microm, 10 min)-stimulated pERK was reduced by bupivacaine (IC50 approximately 2 mm, approximately 0.05%), which similarly suppressed pERK induced by the ionotropic glutamate receptors for N-methyl-D-aspartate and (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid but not that induced by the metabotropic receptors for glutamate, bradykinin, or substance P. Extracellular receptor-activated kinase activation by the Ca2+ ionophore ionomycin was also sensitive to bupivacaine, but direct activation by protein kinase A or protein kinase C was not. CONCLUSIONS: Bupivacaine inhibits pERK activation resulting from different modes of Ca2+ influx through the plasma membrane. This represents a postsynaptic mechanism of analgesia that occurs in parallel with impulse inhibition during neuraxial blockade.     

Spinal axonal injury induces brief downregulation of ionotropic glutamate receptors and no stripping of synapses in cord-projection central neurons

Spinal cord injury often damages the axons of cord-projecting central neurons. To determine whether their excitatory inputs are altered following axonal injury, we used rat rubrospinal neurons as a model and examined their excitatory input following upper cervical axotomy. Anterograde tracing showed that the primary afferents from the cerebellum terminated in a pattern similar to that of control animals. Ultrastructurally, neurons in the injured nucleus were contacted by excitatory synapses of normal appearance, with no sign of glial stripping. Since cerebellar fibers are glutamatergic, we examined the expression of ionotropic receptor subunits GluR1-4 and NR1 for AMPA and NMDA receptors, respectively, in control and injured neurons using immunolabeling methods. In control neurons, GluR2 appeared to be low as compared to GluR1, GluR3, and GluR4, while NR1 labeling was intense. Following unilateral tractotomy, the levels of expression of each subunit in axotomized neurons appeared to be normal, with the exception that they were lower than those of control neurons of the nonlesioned side at 2-6 days postinjury. These findings suggest that axotomized neurons are only temporarily protected from excitotoxicity. This is in sharp contrast to the responses of central neurons that innervate peripheral targets, in which both synaptic stripping and reduction of their ionotropic glutamate receptor subunits persist following axotomy. The absence of an injury-induced trimming of afferents and stripping of synapses and the lack of a persistent downregulation of postsynaptic receptors might enable injured cord-projection neurons to continue to control their supraspinal targets during most of their postinjury survival. Although this may support neurons by providing trophic influences, it nevertheless may subject them to excitotoxicity and ultimately lead to their degenerative fate.     

Inhibition of NR2B phosphorylation restores alterations in NMDA receptor expression and improves functional recovery following traumatic brain injury in mice

Traumatic brain injury (TBI) triggers a massive glutamate efflux, hyperactivation of N-methyl-D-aspartate receptors (NMDARs) and neuronal cell death. Previously it was demonstrated that, 15 min following experimentally induced closed head injury (CHI), the density of activated NMDARs increases in the hippocampus, and decreases in the cortex at the impact site. Here we show that CHI-induced alterations in activated NMDARs correlate with changes in the expression levels of the major NMDARs subunits. In the hippocampus, the expression of NR1, NR2A, and NR2B subunits as well as the GluR1 subunit of the AMPA receptor (AMPAR) were increased, while in the cortex at the impact site, we found a decrease in the expression of these subunits. We demonstrate that CHI-induced increase in the expression of NMDAR subunits and GluR1 in the hippocampus, but not in the cortex, is associated with an increase in NR2B tyrosine phosphorylation. Furthermore, inhibition of NR2B-phosphorylation by the tyrosine kinase inhibitor PP2 restores the expression of this subunit to its normal levels. Finally, a single injection of PP2, prior to the induction of CHI, resulted in a significant improvement in long-term recovery of motor functions observed in CHI mice. These results provide a new mechanism by which acute trauma contributes to the development of secondary damage and functional deficits in the brain, and suggests a possible role for Src tyrosine kinase inhibitors as preoperative therapy for planned neurosurgical procedures.     

Modulation of NMDA receptor function by ketamine and magnesium. Part II: interactions with volatile anesthetics

Mg2+ and ketamine interact superadditively at N- methyl-D-aspartate (NMDA) receptors, which may explain the clinical efficacy of the combination. Because patients are usually exposed concomitantly to volatile anesthetics, we tested the hypothesis that volatile anesthetics interact with ketamine and/or Mg2+ at recombinantly expressed NMDA receptors. NR1/NR2A or NR1/NR2B receptors were expressed in Xenopus oocytes. We determined the effects of isoflurane, sevoflurane, and desflurane on NMDA receptor signaling, alone and in combination with S(+)-ketamine (4.1 microM on NR1/NR2A, 3.0 microM on NR2/NR2B) and/or Mg2+ (416 microM on NR1/NR2A, 629 microM on NR1/NR2B). Volatile anesthetics inhibited NR1/NR2A and NR1/NR2B glutamate receptor function in a reversible, concentration-dependent, voltage-insensitive and noncompetitive manner (half-maximal inhibitory concentration at NR1/NR2A receptors: 1.30 +/- 0.02 minimum alveolar anesthetic concentration [MAC] for isoflurane, 1.18 +/- 0.03 MAC for desflurane, 1.24 +/- 0.06 MAC for sevoflurane; at NR1/NR2B receptors: 1.33 +/- 0.12 MAC for isoflurane, 1.22 +/- 0.08 MAC for desflurane, and 1.28 +/- 0.08 MAC for sevoflurane). On both NR1/NR2A and NR1/NR2B receptors, 50% inhibitory concentration for volatile anesthetics was reduced approximately 20% by Mg2+, approximately 30% by S(+)-ketamine, and approximately 50% by the compounds in combination. Volatile anesthetic effects on NMDA receptors can be potentiated significantly by Mg2+, S(+)-ketamine, or-most profoundly-both. Therefore, the analgesic effects of ketamine and Mg2+, are likely to be enhanced in the presence of volatile anesthetics. IMPLICATIONS: Clinically relevant concentrations of volatile anesthetics inhibit functioning of N-methyl-D-aspartate receptors expressed recombinantly in Xenopus oocytes. This inhibition is reversible, concentration-dependent and voltage-insensitive, and results from noncompetitive antagonism of glutamate/glycine signaling. In addition, these effects can be potentiated significantly by co-application of either Mg2+, S(+)-ketamine, or--most profoundly--both.     

Reduced sensitivity to ketamine and pentobarbital in mice lacking the N-methyl-D-aspartate receptor GluRepsilon1 subunit

Ketamine is an IV anesthetic with N-methyl-d-aspartate receptor (NMDAR)-blocking properties. However, it is still unclear whether ketamine's general anesthetic actions are mediated primarily via blockade of NMDAR. Functional NMDARs are composed by the assembly of a GluRzeta1 (NR1) subunit with GluRepsilon (GluRepsilon1-4; NR2A-D) subunits, which confer unique properties on native NMDARs. We hypothesized that animals deficient in GluRepsilon1, an abundant and ubiquitously postnatally expressed NMDAR subunit, might be resistant to the effects of ketamine. Here, we evaluated a righting reflex to determine the general anesthetic/hypnotic potency of ketamine administered intraperitoneally to GluRepsilon1 knockout mice and compared these results with those for wild-type mice. Mutant mice were more resistant to ketamine than control mice. Unexpectedly, mutant mice were also more resistant to pentobarbital, which is thought not to interact with NMDAR at clinically relevant concentrations. Although these data in no way eliminate the possibility of the involvement of the NMDAR GluRepsilon1 subunit in mediation of ketamine anesthesia/hypnosis, they suggest the difficulties with interpretation of altered anesthetic sensitivity in knockout animal models.     

Effects of intravenous anesthetic agents on glutamate release: a role for GABAA receptor-mediated inhibition

BACKGROUND: Many anesthetic agents are known to enhance the alpha1beta2gamma2S gamma-aminobutyric acid type A (GABAA) chloride current; however, they also depress excitatory neurotransmission. The authors evaluated two hypotheses: intravenous anesthetic agents inhibit glutamate release and any observed inhibition may be secondary to GABAA receptor activation. METHODS: Cerebrocortical slices were prepared from Wistar rats. After perfusion in oxygenated Krebs buffer for 60 min at 37 degrees C, samples for glutamate assay were obtained at 2-nmin intervals. After 6 min, a 2-min pulse of 46 mM K+ was applied to the slices (S1); this was repeated after 30 min (S2). Bicuculline (1-100 microM) was applied when the S1 response returned to basal level, and 10 min later, thiopental (1-300 micro/M), propofol (10 microM), or ketamine (30 microM) were also applied until the end of S2. Perfusate glutamate concentrations were measured fluorometrically, and the area under the glutamate release curves was expressed as a ratio (S2/S1). RESULTS: Potassium (46 mM) evoked a monophasic release of glutamate during S1 and S2, with a mean control S2/S1 ratio of 1.07 +/- 0.33 (mean +/- SD, n = 96). Ketamine and thiopental produced a concentration-dependent inhibition of K+-evoked glutamate release with half-maximum inhibition of release values of 18.2 and 10.9 /microM, respectively. Release was also inhibited by propofol. Bicuculline produced a concentration dependent reversal of thiopental inhibition of glutamate release with a half-maximum reversal of the agonist effect of 10.3 microM. Bicuculline also reversed the effects of propofol but not those of ketamine. CONCLUSIONS: The authors' data indicate that thiopental, propofol, and ketamine inhibit K+-evoked glutamate release from rat cerebrocortical slices. The inhibition produced by thiopental and propofol is mediated by activation of GABAA receptors, revealing a subtle interplay between GABA-releasing (GABAergic) and glutamatergic transmission in anesthetic action.     

切口痛-瑞芬太尼痛觉过敏大鼠脊髓总蛋白及膜蛋白NMDA受体NRl 、NR2A及NR2B亚基表达的变化

目的 评价切口痛-瑞芬太尼痛觉过敏大鼠脊髓总蛋白及膜蛋白NMDA受体NR1、NR2A及NR2B亚基表达的变化.方法 尾静脉置管成功的雄性SD大鼠32只,体重240～260 g,月龄2～3月,采用随机数字表法,将大鼠随机分为4组(n=8):对照组(C组)静脉输注等容量生理盐水60 min,瑞芬太尼组(R组)静脉输注瑞芬太尼1.2μg·kg-1·min-1 60 min;切口痛组(I组)建立切口痛模型,同时静脉输注等容量生理盐水60 min;瑞芬太尼+切口痛组(R+I组)建立切口痛模型,同时静脉输注瑞芬太尼1.2 μg· kg-1 ·min-1 60 min.于生理盐水或瑞芬太尼给药前24h、给药后2、6、24和48 h时测定机械刺激缩足阈值(PWT)和热刺激缩足潜伏期(PWL),最后一次测定痛阈后处死取脊髓L4～6节段,采用Western blot法测定脊髓总蛋白及膜蛋白NMDA受体NR1、NR2A及NR2B亚基的表达,并计算膜蛋白中NR2B/NR2A比值.结果 与C组比较,I组、R组和R+I组PWT降低,PWL缩短,总蛋白及膜蛋白NMDA受体NR1和NR2B亚基表达上调,膜蛋白中NR2B/NR2A比值增加(P＜0.05).与R组和I组比较,R+I组PWT降低,PWL缩短,总蛋白及膜蛋白NMDA受体NR1和NR2B亚基表达上调,膜蛋白中NR2B/NR2A比值增加(P＜0.05).各组总蛋白及膜蛋白NMDA受体NR2A亚基表达差异无统计学意义(P＞0.05).结论 大鼠切口痛-瑞芬太尼痛觉过敏的形成可能与脊髓总蛋白及膜蛋白NMDA受体NR1和NR2B亚基表达上调和膜蛋白中NMDA受体NR2B亚基组成比例的增加有关.     

The NR3B subunit does not alter the anesthetic sensitivities of recombinant N-methyl-D-aspartate receptors

The N-methyl-D-aspartate (NMDA) receptor NR3B subunit co-assembles with NR1 and NR2 subunits to form a receptor complex with distinct channel properties. In the present study, we investigated the effects of co-expression of the NR3B subunit on the anesthetic sensitivities of NMDA receptors for NR1/NR2 channels expressed in Xenopus oocytes. Although the NR3B subunit prominently reduced the current amplitude of NR1/NR2A-B channels, the sensitivities of NR1/NR2A-B channels to Mg2+, ketamine, isoflurane, nitrous oxide, and ethanol were not altered by coexpression of the NR3B subunit. These results suggest that the anesthetic sensitivities of NMDA receptors do not depend on the presence or absence of the NR3 subunit. Mutations of two amino acid residues in the NR3B subunit at positions homologous to the N and N + 1 sites in the NR1 and NR2 subunits, which constitute the blocking sites for Mg2+ and ketamine, did not affect the sensitivities of NR1/NR2B/NR3B channels to Mg2+, ketamine and isoflurane. Thus, the amino acid residues at the N and N + 1 sites in NR3 subunits are unlikely to be involved in the formation of channel blocking sites in NR1/NR2/NR3 channels.     

烫伤大鼠海马N-甲基-D-天冬氨酸受体亚型基因表达的变化

目的　观察严重烫伤应激后大鼠海马N 甲基 D 天冬氨酸受体 (N methyl D aspartatere ceptorsNMDAR)亚型基因表达的变化。　方法　制作 30 %TBSAⅢ度烫伤大鼠应激模型 ,以大鼠背部剃毛 ,浸入温水 10s为对照 ,利用RT PCR技术 ,分别检测对照组及烫伤后 0 .5、1、2、4h各时相点海马NMDAR的主要亚型NMDAR1、NMDAR2A、NMDAR2B、NMDAR2DmRNA的表达。　结果　伤后 0 .5h及1hNMDAR各亚单位mRNA表达无显著变化 ;伤后 2h及 4hNMDAR1mRNA表达比对照组增加 2 4 .3%及 2 0 .9% (P <0 .0 5 ) ,NMDAR2AmRNA表达比对照组增加 2 7.8%及 2 7.6 % (P <0 0 5 ) ,NM DAR2B及NMDAR2D的mRNA表达无明显变化。　结论　海马NMDAR1/NMDAR2A构成的受体通道在烫伤 2h后表达增强 ,这种变化可能导致了烫伤应激时NMDAR的进一步开放 ,从而可能参与烫伤应激HPA轴失调及其后续的病理生理改变     

Serine/threonine phosphorylation of IRS-1 triggers its degradation: possible regulation by tyrosine phosphorylation

Insulin receptor substrate (IRS)-1 protein expression is markedly reduced in many insulin-resistant states, although the mechanism for this downregulation is unclear. In this study, we have investigated the early events in the insulin pathway that trigger the degradation of IRS-1. Incubation of the adipocytes with insulin induced a fast electrophoretic mobility shift of IRS-1 and a subsequent degradation of the protein. Wortmannin and rapamycin blocked this mobility shift of IRS-1, maintained the insulin-induced tyrosine phosphorylation of IRS-1, and blocked its degradation. In contrast, a glycogen synthase kinase 3 inhibitor, a mitogen-activated protein kinase/extracellular-regulated kinase inhibitor, and various protein kinase C inhibitors had no effect. Incubation with okadaic acid increased the serine/threonine phosphorylation of IRS-1 and its degradation, mimicking insulin, and its effect was prevented by the proteasome inhibitor lactacystin, as well as by rapamycin. Treatment of the cells with the tyrosine phosphatase inhibitor orthovanadate in the presence of insulin or okadaic acid partially inhibited the degradation of IRS-1. We propose that a rapamycin-dependent pathway participates as a negative regulator of IRS-1, increasing its serine/threonine phosphorylation, which triggers degradation. Thus, regulation of serine/threonine versus tyrosine phosphorylation may modulate IRS-1 degradation, affecting insulin sensitivity.