PKB在人上皮性卵巢癌中的表达及P13K抑制剂Wortmannin对细胞增殖的影响

目的检测蛋白激酶B(PKB)在上皮性卵巢癌组织中的表达,以及磷脂酰肌醇3激酶(PI3K)特异性抑制剂wortmannin对卵巢癌细胞株CAOV3细胞增殖的影响,探讨PI3K/PKB信号传导通路在卵巢癌发生发展中的意义。方法应用RT-PCR和western印迹方法对50例上皮性卵巢癌组织PKB的表达进行了分析。通过MTT比色法,观察wortmannin对卵巢癌细胞增殖的影响。流式细胞仪测定wortmannin对细胞周期的影响。结果PKBmRNA在上皮性卵巢癌组织中表达,与正常卵巢、卵巢瘤样病变及卵巢良性肿瘤相比,无显著差异(P>0.05);western印迹检测见上皮性卵巢癌组织中磷酸化PKB蛋白条带深于正常卵巢、卵巢瘤样病变及卵巢良性肿瘤组织,量化后比较差异显著(P<0.05),且在Ⅲ～Ⅳ期卵巢癌中的表达水平明显高于Ⅰ～Ⅱ期(P<0.0 5)。加入wortmannin后,CAOV3细胞株增殖比明显下降,其增殖下降程度与wortmannin浓度呈显著正相关,在wortmannin的作用下,由GO+G1期进入S和G2+M期的细胞明显减少,并在一定浓度范围内成剂量依赖性,与对照组比较,差异显著。结论磷酸化PKB蛋白与上皮性卵巢癌的发生、发展、浸润呈正相关,wortmannin可有效阻滞此传导途径。     

激酶调控PKB/Akt活性的机制

PKB/Akt是胞内信号转导通路网络的中心分子。活化的PKB参与多种生物学效应的调控过程,调控PKB活性作用机制的研究一直是信号转导通路领域的难点。新近的研究结果确定了若干新的调控PKB活性的激酶,从多方面解释了PKB活化和作用的分子机制。其中mTORC2、ATM和DNA-PK均通过磷酸化PKB的Ser473位点以依赖于PI3K的方式全面活化PKB;此外,其它以不依赖PI3K的方式调控PKB活性的激酶包括,RET/PTC通过磷酸化PKB的Tyr315位点、JNK通过磷酸化PKB的Thr450位点以及CK2通过磷酸化PKB的Ser129位点活化PKB,Brk通过磷酸化PKB的Tyr474位点以及GRK2均可通过磷酸化PKB抑制其活性。     

Nitric oxide mediates the central sensitization of primate spinothalamic tract neurons

Nitric oxide (NO) has been proposed to contribute to the development of hyperalgesia by activating the NO/guanosine 3',5'-cyclic monophosphate (cGMP) signal transduction pathway in the spinal cord. We have examined the effects of NO on the responses of primate spinothalamic tract (STT) neurons to peripheral cutaneous stimuli and on the sensitization of STT cells following intradermal injection of capsaicin. The NO level within the spinal dorsal horn was increased by microdialysis of a NO donor, 3-morpholinosydnonimine (SIN-1). SIN-1 enhanced the responses of STT cells to both weak and strong mechanical stimulation of the skin. This effect was preferentially on deep wide dynamic range STT neurons. The responses of none of the neurons tested to noxious heat stimuli were significantly changed when SIN-1 was administered. Intradermal injection of capsaicin increased dramatically the content of NO metabolites, NO-2/NO-3, within the dorsal horn. This effect was attenuated by pretreatment of the spinal cord with a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). Sensitization of STT cells induced by intradermal injection of capsaicin was also prevented by pretreatment of the dorsal horn with the NOS inhibitors, L-NAME or 7-nitroindazole. Blockade of NOS did not significantly affect the responses of STT cells to peripheral stimulation in the absence of capsaicin injection. The data suggest that NO contributes to the development and maintenance of central sensitization of STT cells and the resultant mechanical hyperalgesia and allodynia after peripheral tissue damage or inflammation. NO seems to play little role in signaling peripheral stimuli under physiological conditions.     

Selective expansion of perforin-positive CD8+ T cells by immature dendritic cells infected with live Bacillus Calmette-Guérin mycobacteria

Tyrosine phosphorylation is thought to be critical in the regulation of neutrophil functioning, and members of the Src family of tyrosine kinases have recently been shown to be regulated in activated granulocytes. We have used a specific pharmacological inhibitor of Src kinases, pyrazolpyrimidine 1 (PP1), to evaluate the role of Src kinases in cytokine/chemoattractant-induced regulation of neutrophil function. PP1 inhibits PKB phosphorylation but not STAT5 phosphorylation or the activation of MAP kinases by fMLP or GM-CSF. Pretreatment of neutrophils with PP1 and with the PI3K inhibitor LY294002 resulted in a strong inhibition of fMLP-induced superoxide production and cytokine-mediated survival but not fMLP-induced migration. It is interesting that the kinetics of inhibition of actin polymerization and the respiratory burst are very similar. Although initiation of both processes was not affected, sustained activation was inhibited by PP1. Taken together, our results demonstrate a critical role for Src kinases in regulating neutrophil cytotoxic-effector functioning through PI3K-PKB.     

Intradermal injection of capsaicin induces acute substance P release from rat spinal cord dorsal horn

Increased release of substance P (SP) from the dorsal horn following noxious stimuli, such as spinal administration of capsaicin, has been demonstrated in previous studies. However, changes in the release of SP in response to intradermal injection of capsaicin still remain unknown. This study was designed to demonstrate in vivo spinal SP release following intradermal injection of capsaicin (3%, 50 μl), using polyimide tubing with a single hole introduced into the rat dorsal horn. The changes in the content of SP in the rat dorsal horn tissues before and after capsaicin (3%, 50 μl) injection were also investigated. The SP concentration in the samples was analyzed using an enzyme-linked immunosorbent assay (ELISA). We found that intradermal injection of capsaicin induced a quick SP release within the dorsal horn. The peak of the release appeared around 10 min after the injection. In contrast, intradermal injection of capsaicin had no significant effect on the SP content in the dorsal horn. This study has provided direct evidence of the effect of intradermal injection of capsaicin on SP release within the dorsal horn, with the major source being from the central terminals of primary afferents.     

丝胶对2型糖尿病大鼠胰腺胰岛素PI3K-Akt信号通路的调节作用

目的探讨丝胶是否通过影响胰腺胰岛素PI3K-Akt信号通路发挥降血糖的作用。方法 36只雄性SD大鼠随机分为正常对照组、糖尿病模型组和丝胶治疗组,每组12只。采用高脂高糖饲料喂养联合链脲佐菌素(35mg/kg,2次,1次/d)连续腹腔注射法制作2型糖尿病大鼠模型,模型成功标准是空腹血糖≥11.1mmol/L。模型成功建立后,丝胶治疗组大鼠给予丝胶灌胃35d。采用ELISA法检测大鼠血清脂联素水平,Western blotting法和Real-time PCR法分别检测大鼠胰腺胰岛素受体(IR)、胰岛素受体底物-1(IRS-1)、磷脂酰肌醇-3-激酶(PI3K)和Akt蛋白和mRNA的表达情况。结果与糖尿病模型组比较,丝胶治疗组大鼠血清脂联素水平,胰腺IR、IRS-1、PI3K、Akt蛋白和mRNA的表达明显升高(P0.01,P0.05)。结论丝胶可通过上调糖尿病模型大鼠胰腺IR、IRS-1、PI3K和Akt的表达,改善糖尿病时胰腺胰岛素PI3K-Akt信号转导通路的异常,从而发挥降低血糖的作用。     

Phosphoinositide 3-kinase and its targets in B-cell and T-cell signaling

Phosphoinositide 3-kinase (PI3K) activation is essential for lymphocyte proliferation driven by receptors for antigen, costimulatory ligands and cytokines. The lipid products of PI3K contribute to the assembly of membrane-associated signaling complexes by promoting recruitment of selected proteins from the cytoplasm. Many proteins possess domains that are able to bind selectively to PI3K products. Different 'PI3K effector' proteins are coupled to distinct biological responses, depending on cell type and on the receptor that is engaged. In B cells and T cells, Tec-family tyrosine kinases and Akt serine/threonine kinases are emerging as crucial mediators of proliferation and survival signals downstream of PI3K. Of particular interest is recent evidence that PI3K signaling controls increases in lymphocyte size and metabolic activity that accompany cell cycle progression.     

Developmental regulation of neuronal K(Ca) channels by TGFbeta1: an essential role for PI3 kinase signaling and membrane insertion

TGFbeta1 is a target-derived factor responsible for the developmental expression of large-conductance Ca(2+)-activated K(+) (K(Ca)) channels in ciliary neurons of the chick ciliary ganglion. The acute effects of TGFbeta1 on K(Ca) channels are mediated by posttranslational events and require activation of the MAP kinase Erk. Here we show that TGFbeta1 evokes robust phosphorylation of Akt/PKB, a protein kinase dependent on the products of phosphatidylinositol 3-OH kinase (PI3K). TGFbeta1-evoked stimulation of K(Ca) channels is blocked by the PI3K inhibitors wortmannin and LY294002. These drugs also inhibit TGFbeta1 effects on Akt/PKB phosphorylation but have no effect on TGFbeta1-evoked Erk activation. Application of the MEK1 inhibitor PD98059 blocked TGFbeta1 effects on Erk but had no effect on Akt/PKB phosphorylation. These results indicate that PI3K and Erk represent parallel signaling cascades activated by TGFbeta1 in ciliary neurons. The effects of TGFbeta1 on functional expression of K(Ca) are blocked by the microtubule inhibitors colchicine and nocodazole, by botulinum toxins A and E, and by brefeldin-A, an agent that disrupts the Golgi apparatus. These data indicate that translocation of a membrane protein, possibly Slowpoke (SLO), is required for the acute posttranslational effects of TGFbeta1 on K(Ca) channels. Confocal immunofluorescence studies with three different SLO antisera showed robust expression of SLO in multiple intracellular compartments of embryonic day 9-13 ciliary neurons, including the cell nucleus. These data suggest that TGFbeta1 evokes insertion of SLO channels into the plasma membrane as a result of signaling cascades that entail activation of Erk and PI3K.     

Inhibitors of G-proteins and protein kinases reduce the sensitization to mechanical stimulation and the desensitization to heat of spinothalamic tract neurons induced by intradermal injection of capsaicin in the primate

 Intradermal injection of capsaicin results in sensitization of spinothalamic tract cells to brushing and pressure applied to the cutaneous receptive field in anesthetized monkeys. A significant increase in background activity also occurs immediately after capsaicin injection that lasts for at least 2 h. A 40–50% decrease in the response to noxious heat stimuli is also observed following capsaicin injection. This study investigated the spinal role of second messengers by extracellularly recording from spinothalamic tract cells and delivering inhibitors of second messenger pathways to the spinal cord by microdialysis. Blockade of protein kinases with the general protein kinase inhibitor, H7 (5.0 mM, n = 6), reduced the sensitization of the cells to brush and pressure. Blockade of protein kinase C with NPC15437 (10.0 mM, n = 10) reduced the increased background activity and the increased responses to brush. Blockade of protein kinase A with H89 (0.01 mM, n = 9) was most effective. H89 reduced the background activity, the increased responses to brush and press, and reversed the decreased response to noxious heat stimuli. Blockade of G-proteins with the general G-protein inhibitor, GDP-β-S (1.0 mM, n = 9), reduced the background activity and the responses to brush and pressure without affecting the decreased response to heat. Thus, multiple intracellular messengers appear to be involved in the processing of central sensitization induced by activation of C-fibers following intradermal injection of capsaicin. Received: 5 June 1996 / Accepted: 29 November 1996     

蛋白激酶B的研究进展

Protein kinase B (Akt) is a Ser/Thr kinase, which in mammals comprise three highly ho-mologous members known as PKBα/Aktl, PKBβ/Akt2 and PKBγ/Akt3. PKB is activated by hormones,growth factor and extra cellular matrix. The activation occurs downstream of PI3K. PKB phosphorylates and regulates the function of many cellular protein involved in processes that include survival, apoptosis, proliferation,glycogen metabolism and cancer progression. Although many mechanisms remains to be fully characterized, the research of PKB is thought to have a useful profect.     

Interleukin-32α production is regulated by MyD88-dependent and independent pathways in IL-1β-stimulated human alveolar epithelial cells

Interleukin (IL)-32 is a recently described cytokine that appears to play a critical role in a variety of inflammatory diseases including chronic obstructive pulmonary disease (COPD). However, thus far, the regulation of IL-32 production has not been fully established. Here, we report on signaling pathways that regulate the production of IL-32α, the most abundant isoform, in the human alveolar epithelial cell line, A549. IL-32α was expressed and secreted by IL-1β. The IL-32 expression was attenuated by PP2 (a Src-family kinase [SFK] inhibitor), rottlerin (a protein kinase [PK] Cδ inhibitor), and LY294002 (a phosphatidylinositol 3-kinase [PI3K] inhibitor). Furthermore, the overexpression of Fgr rather than other SFKs upregulated IL-32α expression, while Fgr small interfering RNA (siRNA) transfection downregulated it. The suppression of Fgr with PP2 and Fgr siRNA inhibited activating phosphorylation of PKCδ and PI3K/Akt, but not IL-1 receptor-associated kinase (IRAK)1, a well-known MyD88-dependent signaling molecule, and Erk1/2, p38, and JNK. Rottlerin and PKCδ siRNA also inhibited expression of IL-32α and activation of PI3K/Akt, but not of IRAK1 and mitogen activation protein (MAP) kinases. MyD88 siRNA suppressed the expression of IL-32α and the phosphorylation of IRAK1, PI3K, and MAP kinases, but not of PKCδ. Of interest, both Fgr/PKCδ and MyD88-dependent signals regulated PI3K/Akt, suggesting that it is a crosstalk molecule. Among MyD88-dependent MAP kinases, only p38 regulated IL-32α expression and PI3K/Akt activation. With these results, we demonstrated that the expression and secretion of IL-32α are regulated by MyD88-dependent IRAK1/p38/PI3K and independent Fgr/PKCδ/PI3K pathways, and that Fgr and PKCδ are critical for the MyD88-independent IL-32α production.     

机械张应变诱导蛋白激酶B活化对血管平滑肌细胞迁移的影响

目的研究机械张应变诱导蛋白激酶B活化对大鼠血管平滑肌细胞迁移能力的影响。方法应用FX-4000T细胞应变加载系统,对大鼠血管平滑肌细胞施加牵拉幅度为15%、频率为1Hz的张应变。以Transwell和Westernblot等方法观察张应变作用下蛋白激酶B磷酸化和血管平滑肌细胞迁移能力的变化,以未加载张应变的血管平滑肌细胞为对照组。结果与对照组相比,机械张应变增加细胞中蛋白激酶B磷酸化水平,促进血管平滑肌细胞的迁移;PI3K的特异性抑制剂Wortmannin抑制张应变诱导的蛋白激酶B的磷酸化,降低了血管平滑肌细胞迁移能力。结论机械张应变通过上调蛋白激酶B磷酸化水平促进了血管平滑肌细胞迁移,提示蛋白激酶B信号通路参与机械张应变条件下血管平滑肌细胞迁移过程的信号传导。     

bFGF对卵巢癌CAOV3细胞Bcl-2、Bcl-xl、Bax、Bad表达的影响

目的研究bFGF调控卵巢癌CAOV3凋亡的信号通路及对Bcl-2、Bcl-xl、Bax、Bad表达的影响。方法无血清饥饿诱导细胞凋亡。分为饥饿对照、bFGF、bFGF PD98059、bFGF Wortmannin组。流式细胞术、DNA Ladder检测细胞凋亡;Western印迹法检测ERK、PKB、Bad活性以及Bcl-2、Bax表达,RT- PCR检测Bcl-2、Bcl-xl mRNA变化。结果bFGF促进p-ERK、p-PKB、p-Bad、Bcl-2表达,抑制Bax表达及饥饿诱导的细胞凋亡。激酶抑制剂PD98059可抑制bFGF对ERK、Bcl-2、Bax的调节作用,Wortmannin可抑制bFGF对PKB、Bad、Bax的调控作用,二者均可阻断bFGF对凋亡的抑制作用。bFGF对Bcl-xl表达无影响。结论bFGF可能通过激活MEK/ERK、P13K/PKB信号途径通路调节Bcl-2、Bax、Bad表达,抑制饥饿诱导的卵巢癌CAOV3细胞凋亡。     

磷脂酰肌醇-3激酶、蛋白激酶B及γ-谷氨酰半胱氨酸合成酶在慢性阻塞性肺疾病患者中的表达

目的观察慢性阻塞性肺疾病（COPD）患者中磷脂酰肌醇-3激酶（H3K）、蛋白激酶B（PKB）与γ-谷氨酰半胱氨酸合成酶（γ-GCS）的表达,研究P13K、PKB通路和γ-GCS的关系。方法将手术切馀的肺癌患者肺组织标本（16例）分为COPD组和对照组,每组8例,检测肺组织中γ—GCS活性,原位杂交检测肺组织中γ-GCSmRNA的表达,免疫组化分析肺组织中H3K、PKB与γ-GCS的蛋白水平。结果COPD组中1．GCS活性明显高于对照组,差异有统计学意义（P〈0．01）。原位杂交显示COPD组支气管、肺泡上皮细胞与小动脉平滑肌细胞γ-GCSmRNA广泛表达,与对照组比较差异有统计学意义（P〈0．01）。P13K、PKB与γ-GCS免疫组化在COPD组可见肺泡、支气管壁细胞及小血管平滑肌细胞胞浆中皆有蛋白阳性信号表达,图像定量分析显示COPD组H3K、PKB与γ—GCS蛋白表达显著高于对照组,差异有统计学意义（P〈0．01）。直线相关分析得出H3K、P-PKB蛋白表达与γ-GCS活性、mRNA及蛋白表达呈正相关。结论COPD患者肺组织γ-GCS蛋白和mRNA表达增高,P13K、p-PKB蛋白也有相应高表达,提示P13K、PKB和γ-GCS可能在COPD的发病机制中发挥作用,且可能参与了γ-GCS的信号传导通路。     

丙戊酸协同伊马替尼诱导K562细胞凋亡并下调Bcr/Abl mRNA和磷酸化蛋白激酶B的表达

 目的：探讨丙戊酸联合伊马替尼对慢性粒细胞白血病细胞株K562凋亡的影响,同时探索其可能的作用机制。方法：K562细胞分别用丙戊酸、伊马替尼以及两药联合处理。检测各组细胞在药物处理后的细胞周期、细胞凋亡、Bcr/Abl mRNA 水平、总蛋白激酶B（PKB）以及磷酸化PKB （p-PKB）水平。结果：丙戊酸组、伊马替尼组和两药联用组K562细胞凋亡率分别为(11.47±0.25)％、(28.43±1.70)％和(57.73±4.38)% (P<0.05),对于细胞周期各指标,两药联用组与单药组无明显差异。Bcr/Abl mRNA以及p-PKB在丙戊酸组、伊马替尼组和两药联用组的水平分别为(0.00±0.00)、(64.17±12.27)和(0.00±0.00)×10 9 copies/(g total mRNA)以及0.25±002、0.17±0.01和0.08±0.01(均P<0.05),总PKB 3组细胞间无明显差异。结论：丙戊酸能协同伊马替尼促进K562细胞凋亡,其作用机制可能与降低Bcr/Abl mRNA和p-PKB水平有关。     

Phosphorylation of extracellular signal-related protein kinase is required for rapid facilitation of heat-induced currents in rat dorsal root ganglion neurons

A subgroup of dorsal root ganglion (DRG) neurons responds to noxious heat with an influx of cations carried by specific ion channels such as the transient receptor potential channel of the vanilloid receptor type, subtype 1 (TRPV1). Application of capsaicin induces a reversible facilitation of these currents. This facilitation could be an interaction of two agonists at their common receptor or be caused by an influx of calcium ions into the cell. Calcium influx into the cell can activate protein kinases such as the extracellular signal-related protein kinase (ERK) pathway. This study explored the kinetics, calcium-dependency and intracellular signals following application of capsaicin and leading to facilitation of heat-induced currents (Iheat) in rat DRG neurons. Application of 0.5 microM capsaicin caused a 2.65-fold increase of Iheat within 2 s, which was significantly correlated to a small capsaicin-induced current. Intracellular application of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a fast calcium chelator, did not change capsaicin-induced currents or Iheat itself, but inhibited facilitation of Iheat by capsaicin. ERK is activated by calcium influx and membrane depolarization via the mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK). Application of the MEK inhibitor U0126 also inhibited facilitation of Iheat by capsaicin. We conclude that the MEK/ERK cascade is an intracellular signaling pathway playing a vital role in the regulation of nociceptive neurons' sensitivity. The very fast kinetics (less than two seconds) are only explainable with a membrane-attached or at least membrane-near localization of these kinases.     

BCAP: the tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation

Tyrosine phosphorylation of adaptor proteins permits the B cell antigen receptor (BCR)-associated protein tyrosine kinases to regulate downstream effector molecules. Here, we report the identification of a novel B cell adaptor for phosphoinositide 3-kinase (PI3K), termed BCAP. Tyrosine phosphorylation of BCAP is mediated by Syk and Btk, thereby providing binding site(s) for the p85 subunit of PI3K. Disruption of the BCAP gene in the DT40 B cell line inhibits BCR-mediated phosphatidylinositol 3,4,5-trisphosphate generation, leading to impaired Akt response. Moreover, recruitment of PI3K to glycolipid-enriched microdomains (GEMs) is significantly attenuated in the absence of BCAP. Hence, these data suggest that BCAP bridges BCR-associated kinases to the PI3K pathway by regulating PI3K localization.     

IL-5-induced integrin adhesion of human eosinophils caused by ERK1/2-mediated activation of cPLA2

We examined the mechanism by which interleukin (IL)-5 causes beta(2)-integrin adhesion of human eosinophils. IL-5 caused time-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38alpha in eosinophils as detected by their phosphorylation. Preincubation of eosinophils with U0126, a mitogen-activated protein kinase/ERK kinase inhibitor, suppressed IL-5-induced activation of cytosolic phospholipase A(2) (cPLA(2)) and eosinophil adhesion, and p38 inhibition by SB203580 had neither effect. ERK1/2 phosphorylation and eosinophil adhesion were blocked by inhibition of the src-family tyrosine kinase, Janus tyrosine kinase (JAK)2, or phosphoinositide-3 kinase (PI3K). Coimmunoprecipitation assay demonstrated that Lyn, a src-family tyrosine kinase, was constitutively associated with PI3K. Inhibition of src-tyrosine kinase but not JAK2 suppressed PI3K activation. Our data suggest that IL-5 induces beta(2)-integrin adhesion of human eosinophils by regulation of cPLA(2) activation caused by ERK1/2 phosphorylation. This phosphorylation results from activation of PI3K and protein tyrosine kinases. We also find that src-family tyrosine kinase, possibly Lyn, is the upstream kinase causing PI3K activation.     

Insulin facilitates repetitive spike firing in rat insular cortex via phosphoinositide 3-kinase but not mitogen activated protein kinase cascade

The insular cortex (IC) processes gustatory and visceral information, which functionally correlate to feeding behavior. Insulin, a well-known hormone controlling glucose metabolism, is released by elevation of blood glucose concentration following feeding behavior. The IC expresses dense insulin receptors and receives projection from the hypothalamus, which monitors changes in glucose concentration. Therefore, it is likely that insulin modulates neural properties in the IC. However, little is known about the effects of insulin on electrophysiological properties of the neocortex including the IC. To explore the effects of insulin on subthreshold responses and action potential properties in the IC, intracellular recording with sharp glass electrodes was performed from IC pyramidal cells using slice preparations. Although application of insulin (100 nM) had little effect on the resting membrane potential, input resistance and rheobase, insulin significantly increased the frequency of repetitive spike firing in response to a long depolarizing current pulse injection: the slope of the frequency-current curve was increased from 23.7±2.3 Hz/nA to 29.5±3.4 Hz/nA. Insulin slightly decreased the action potential threshold without affecting the amplitude of medium-duration and slow afterhyperpolarization (sAHP) s. The insulin-induced facilitation of repetitive spike firing was dose-dependent and blocked by pre-application of 200 nM lavendustin A, a tyrosine kinase inhibitor. Moreover, when combined with 200 nM wortmannin, a phosphoinositide 3-kinase (PI3-K) inhibitor, or 500 nM deguelin, an inhibitor of protein kinase B (PKB/Akt) downstream of PI3-K, insulin failed to increase the frequency of repetitive spike firing. In contrast, co-application of insulin and (10 μM) PD 98059, an inhibitor of mitogen activated protein kinase (MAPK), exerted facilitation of repetitive spike firing. These results suggest that acute insulin-induced facilitation of firing frequency is at least partially induced by hyperpolarizing effects on the action potential threshold, and that this facilitation is induced by activation of PI3-K but not MAPK cascade.