Activation of a Ca2+-dependent K+ current in mouse fibroblasts by lysophosphatidic acid requires a pertussis toxin-sensitive G protein and Ras

Lysophosphatidic acid (LPA) is a bioactive lipid that acts through G protein-coupled plasma membrane receptors and mediates a wide range of cellular responses. Here we report that LPA activates a K⁺ current in NIH3T3 mouse fibroblasts that leads to membrane hyperpolarization. The activation occurs with an EC₅₀ value of 1.7 nM LPA. The K⁺ current is Ca²⁺-dependent, voltage-independent, and completely blocked by the K⁺ channel blockers charybdotoxin, margatoxin, and iberiotoxin with IC₅₀ values of 1.7, 16, and 62 nM, respectively. The underlying K⁺ channels possess a single channel conductance of 33 pS in symmetrical K⁺ solution. Pretreatment of cells with pertussis toxin (PTX), Clostridium sordellii lethal toxin, or a farnesyl protein transferase inhibitor reduced the K⁺ current amplitude in response to LPA to about 25% of the control value. Incubation of cells with the protein tyrosine kinase inhibitor genistein or microinjection of the neutralizing anti-Ras monoclonal antibody Y13–259 reduced it by more than 50%. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase A activator 8-bromo-cAMP had no effect. These results indicate that the K⁺ channel activation by LPA is mediated by a signal transduction pathway involving a PTX-sensitive G protein, a protein tyrosine kinase, and Ras. LPA is already known to activate Cl^– channels in various cell types, thereby leading to membrane depolarization. In conjunction with our results that demonstrate LPA-induced membrane hyperpolarization by activation of K⁺ channels, LPA appears to be significantly involved in the regulation of the cellular membrane potential. Received: 20 May 1998 / Accepted: 10 August 1998     

Activation of a Ca2+-dependent K+ current in mouse fibroblasts by sphingosine-1-phosphate involves the protein tyrosine kinase c-Src

Sphingosine-1-phosphate (S1P) is a phospholipid that acts through G-protein-coupled plasma membrane receptors and induces a broad spectrum of cellular responses, including proliferation, migration, differentiation and apoptosis. Here we report that in NIH3T3 and C3H10T1/2 mouse fibroblasts S1P activates a Ca2+-dependent, voltage-independent K+ current (EC50-value 113 nM) that is blocked by the K+ channel blockers charybdotoxin, margatoxin, and iberiotoxin. The K+ current activation by S1P is transient and leads to a large membrane hyperpolarization. Recently, we showed that lysophosphatidic acid (LPA), a serum lipid with similar biological effects compared to those of S1P, can activate a Ca2+-dependent K+ current in NIH3T3 cells that has identical properties compared to the one that is activated by S1P. When applied consecutively, both S1P and LPA induced a K+ current response in NIH3T3 cells, which indicates that the K+ current activation is not subjected to cross-desensitization between S1P and LPA. In C3H10T1/2 mouse fibroblasts that overexpress the nonreceptor protein tyrosine kinase c-Src, the amplitude of the S1P-induced K+ current was almost doubled compared to the one that we found in control cells. Expression of a non-myristylated c-Src mutant led to a further increase in the K+ current response to S1P, whereas expression of a kinase-defective c-Src mutant reduced it to about 40% compared to the control value. Our data show that S1P activates Ca2+-dependent K+ channels in mouse fibroblasts via an intracellular signalling pathway that involves the non-receptor protein tyrosine kinase c-Src.     

谷氨酸触发大鼠大脑皮质神经元Ca~(2+)内流与PTK的关系

目的　研究谷氨酸 (glutamate,Glu)触发大鼠大脑皮质神经元Ca2 + 内流特性 ,蛋白酪氨酸激酶 (PTK)抑制剂genistein及蛋白酪氨酸磷酸酶 (PTP)抑制剂vanadate对其影响 ,揭示PTK与Glu触发大鼠大脑皮质神经元Ca2 + 内流的内在联系。方法　采用Fura 2 /AM荧光测定胞浆Ca2 + 变化技术 ,在原代培养的大鼠大脑皮质神经元上观察药物对Glu触发Ca2 + 内流的影响。结果　Glu触发的Ca2 + 内流不受电压依赖性钙通道 (VDCC)阻断剂尼莫地平影响 ,亦不受非VDCC阻断剂SK&F96 36 5影响 ,但可被PTK抑制剂genis tein抑制 ,被PTP抑制剂vanadate增强。genistein(1～ 30μmol·L-1)呈浓度依赖性抑制Glu触发的Ca2 + 内流。vana date则浓度依赖性增强Glu触发的Ca2 + 内流。结论　对尼莫地平敏感的VDCC及对SK&F96 36 5敏感的非VDCC没有参与Glu触发的Ca2 + 内流。PTK激活参与了Glu触发的Ca2 + 内流     

蛋白酪氨酸激酶抑制剂对脑血管平滑肌细胞Ca~(2+)池操纵性Ca~(2+)内流的影响

目的　研究蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶抑制剂对牛脑血管平滑肌细胞 (CSMC)Ca2 + 池操纵性Ca2 + 内流的影响。方法　采用培养的CSMC ,在生物荧光双波长影像分析系统用Fura 2 /Am荧光探针测定单个细胞内游离Ca2 + 浓度。结果　 (1)蛋白酪氨酸激酶抑制剂 (genistein ,2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低内皮素 1(ET 1,10 -7mol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为5 6%± 2 .9%、2 5 6%± 3 9%、48 9%± 3 7% ;蛋白酪氨酸磷酸酶抑制剂 (vanadate ,2 ,4,8μmol·L-1)能浓度依赖性升高CPA刺激引起的CSMCCa2 + 内流 ,增加比率分别为8 2 %± 3 9%、18 8%± 4 9%、46 6%± 6 9% ;(2 ) genistein(2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低ATP(10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 7%±2 6%、2 4 6%± 6 5 %、5 1 3 %± 6 9% ;vanadate (2 ,4,8μmol·L-1)能浓度依赖性升高ATP刺激引起的CSMCCa2 +内流 ,增加比率分别为 4 8%± 2 0 %、2 8 5 %± 4 6%、49 6%± 3 3 % ;(3 ) genistein (2 5 ,5 ,10 μmol·L-1)能浓度依赖性降低环匹阿尼酸 (Cyclopiazonicacid ,CPA ,10 μmol·L-1)刺激引起的CSMCCa2 + 内流 ,抑制率分别为 6 5 %± 3 0 %、2 2 5 %± 5 2 %、     

Epidermal growth factor receptor tyrosine kinase regulates the human inward rectifier potassium K(IR)2.3 channel, stably expressed in HEK 293 cells

BACKGROUND AND PURPOSE

The detailed molecular modulation of inward rectifier potassium channels (including the K_IR2.3 channel) is not fully understood. The present study was designed to determine whether human K_IR2.3 (K_IR2.3) channels were regulated by protein tyrosine kinases (PTKs).

EXPERIMENTAL APPROACH

Whole-cell patch voltage-clamp, immunoprecipitation, Western blot analysis and site-directed mutagenesis were employed to determine the potential PTK phosphorylation of Kir2.3 current in HEK 293 cells stably expressing Kir2.3 gene.

KEY RESULTS

The broad-spectrum PTK inhibitor genistein (10 µM) and the selective epidermal growth factor (EGF) kinase inhibitor AG556 (10 µM) reversibly decreased K_IR2.3 current and the effect was reversed by the protein tyrosine phosphatase inhibitor, orthovanadate (1 mM). Although EGF (100 ng·mL⁻¹) and orthovanadate enhanced K_IR2.3 current, this effect was antagonized by AG556. However, the Src-family tyrosine kinase inhibitor PP2 (10 µM) did not inhibit K_IR2.3 current. Tyrosine phosphorylation of K_IR2.3 channels was decreased by genistein or AG556, and was increased by EGF or orthovanadate. The decrease of tyrosine phosphorylation of K_IR2.3 channels by genistein or AG556 was reversed by orthovanadate or EGF. Interestingly, the response of K_IR2.3 channels to EGF or AG556 was lost in the K_IR2.3 Y234A mutant channel.

CONCLUSION AND IMPLICATIONS

These results demonstrate that the EGF receptor tyrosine kinase up-regulates the K_IR2.3 channel via phosphorylation of the Y234 residue of the WT protein. This effect may be involved in the endogenous regulation of cellular electrical activity.     

Regulation of two-pore-domain (K2P) potassium leak channels by the tyrosine kinase inhibitor genistein

BACKGROUND AND PURPOSE: Two-pore-domain potassium (K2P) channels mediate potassium background (or 'leak') currents, controlling excitability by stabilizing membrane potential below firing threshold and expediting repolarization. Inhibition of K2P currents permits membrane potential depolarization and excitation. As expected for key regulators of excitability, leak channels are under tight control from a plethora of stimuli. Recently, signalling via protein tyrosine kinases (TKs) has been implicated in ion channel modulation. The objective of this study was to investigate TK regulation of K2P channels. EXPERIMENTAL APPROACH: The two-electrode voltage clamp technique was used to record K2P currents in Xenopus oocytes. In addition, K2P channels were studied in Chinese hamster ovary (CHO) cells using the whole-cell patch clamp technique. KEY RESULTS: Here, we report inhibition of human K2P3.1 (TASK-1) currents by the TK antagonist, genistein, in Xenopus oocytes (IC50=10.7 microM) and in CHO cells (IC50=12.3 microM). The underlying molecular mechanism was studied in detail. hK2P3.1 was not affected by genistin, an inactive analogue of genistein. Perorthovanadate, an inhibitor of tyrosine phosphatase activity, reduced the inhibitory effect of genistein. Current reduction was voltage independent and did not require channel protonation at position H98 or phosphorylation at the single TK phosphorylation site, Y323. Among functional hK2P family members, genistein also reduced K2P6.1 (TWIK-2), K2P9.1 (TASK-3) and K2P13.1 (THIK-1) currents, respectively. CONCLUSIONS AND IMPLICATIONS: Modulation of K2P channels by the TK inhibitor, genistein, represents a novel molecular mechanism to alter background K+ currents.     

bFGF激活tyrosine kinase/PI3K/PLCγ增加血管内皮细胞[Mg~(2+)]_i的研究

目的探讨碱性成纤维细胞生长因子(basic fibroblastgrowth factor,bFGF)对人脐带静脉内皮细胞(human umbilicalvein endothelial cells,HUVECs)内游离镁离子浓度([Mg2+]i)的调节机制研究。方法我们采用荧光指示剂mag-fura-2,运用PTi阳离子测定系统动态测HUVECs的[Mg2+]i。结果经酪氨酸激酶阻断剂(tyrphostin A23和genistein)、3-磷脂酰肌醇激酶阻断剂(wortmannin和LY294002)、磷脂酶Cγ阻断剂(U73122)预处理,能阻断bF-GF诱导的[Mg2+]i增加。但经磷脂酶Cγ阻断剂无活性的类似物(U73343)和丝裂原活化蛋白激酶阻断剂(SB202190和PD98059)预处理,不能阻断bFGF诱导的[Mg2+]i增加。结论bFGF通过酪氨酸激酶/3-磷脂酰肌醇激酶/磷脂酶Cγ信号传递途径使细胞内的Mg2+库释放Mg2+,从而增加HUVECs的[Mg2+]i。     

Involvement of protein kinase C and Src tyrosine kinase in acute tolerance to ethanol inhibition of spinal NMDA-induced pressor responses in rats

Background and purpose:

The present study was carried out to examine the role of protein kinases in the development of acute tolerance to the effects of ethanol on spinal N-methyl-D-aspartate (NMDA) receptor-mediated pressor responses during prolonged ethanol exposure.

Experimental approach:

Blood pressure responses induced by intrathecal injection of NMDA were recorded. The levels of several phosphorylated residues on NMDA receptor NR1 (GluN1) (NR1) and NMDA receptor NR2B (GluN2B) (NR2B) subunits were determined by immunohistochemistry and Western blot analysis.

Key results:

Ethanol inhibited spinal NMDA-induced pressor responses at 10 min, but the inhibition was significantly reduced at 40 min following continuous infusion. This effect was dose-dependently blocked by chelerythrine [a protein kinase C (PKC) inhibitor, 1–1000 pmol] or PP2 (a Src family tyrosine kinase inhibitor, 1–100 pmol) administered intrathecally 10 min following ethanol infusion. A significant increase in the immunoreactivity of phosphoserine 896 of NR1 subunits (pNR1-Ser896) and phosphotyrosine 1336 of NR2B subunits (pNR2B-Tyr1336) was found in neurons of intermediolateral cell column during the development of tolerance. Levels of pNR1-Ser896 and pNR2B-Tyr1336 were also significantly increased in lateral horn regions of the spinal cord slices incubated with ethanol for 40 min in vitro. The increases in pNR1-Ser896 and pNR2B-Tyr1336 levels were inhibited by post-treatment with chelerythrine and PP2, respectively, both in the in vivo and in vitro studies.

Conclusions and implications:

The results suggest that activation of PKC and Src tyrosine kinase during prolonged ethanol exposure leading to increases in the levels of pNR1-Ser896 and pNR2B-Tyr1336 may contribute to acute tolerance to inhibition by ethanol of NMDA receptor function.     

Activation of deoxycytidine kinase by protein kinase inhibitors and okadaic acid in leukemic cells

Deoxycytidine kinase (dCK) is a key enzyme in the deoxynucleoside salvage pathway and in the activation of numerous nucleoside analogues used in cancer and antiviral chemotherapy. Recent studies indicate that dCK activity might be regulated through reversible phosphorylation. Here, we report the effects of a large panel of protein kinase inhibitors on dCK activity in the B-leukemia cell line EHEB, both in basal conditions and in the presence of the nucleoside analogue 2-chloro-2'-deoxyadenosine (CdA) which induces activation of dCK. Except staurosporine and H-7 that significantly reduced the activation of dCK by CdA, no specific protein kinase inhibitor diminished basal dCK activity or its activation by CdA. In contrast, genistein, a general protein tyrosine kinase inhibitor, and AG-490, an inhibitor of JAK2 and JAK3, increased basal dCK activity more than two-fold. Two specific inhibitors of the MAPK/ERK pathway, PD-98059 and U-0126, also enhanced dCK activity. These data suggest that the JAK/MAPK pathway could be involved in the regulation of dCK. Moreover, we show that the activity of dCK, raised by CdA, can return to its initial level by treatment with protein phosphatase-2A (PP2A). Accordingly, dCK activity in intact cells increased upon incubation with okadaic acid (OA) at concentrations that should inhibit PP2A, but not protein phosphatase-1. Activation of dCK by protein kinase inhibitors and OA was also observed in CCRF-CEM cells and in chronic lymphocytic leukemia B-lymphocytes, suggesting a general mechanism of post-translational regulation of dCK, which could be exploited to enhance the activation of antileukemic nucleoside analogues.     

New retinoids and protein tyrosine kinase inhibitors for the treatment of disseminated cancer: selective induction of apoptosis in malignant cells

Enhanced cell growth and diminished programmed cell death (or an imbalance of these two processes in favour of mitosis) are believed to be decisive players in cancer development. Thus, the major aim of potentially successful therapeutic settings for disseminated cancer is the induction of enhanced programmed cell death (often referred to as apoptosis) rates within the malignant tissue in order to re-establish cell homeostasis and respectively eradicate malignant cells. This might be achieved either by activation of cell death programs as a consequence of cell injury (e.g., conventional chemotherapy) or of differentiation processes (e.g., retinoid therapy), or by suppression of tumour cell-specific signal pathways transmitted by survival factors or other effector molecules (many of them are in part executed by protein kinase cascades). The discussed patents lay claims on the inhibition of tumour cell growth and/or the induction of apoptosis in cancer cells by adamantyl or adamantyl group derivative-containing retinoids or by substituted heteroaromatic compounds which are protein tyrosine kinase inhibitors.     

Activation of protein kinase C via the T-cell receptor complex potentiates cyclic AMP responses in T-cells

Summary We have recently shown that activation of protein kinase C by tumour promoting phorbolesters, such as 4--phorbol-12,13-dibutyrate, stimulates adenosine-induced accumulation of cAMP in 7urkat cells, a human T-leukaemia line. Activating the CD3 complex associated with the T-cell receptor by means of the monoclonal antibody OKT3 caused a concentration-dependent accumulation of inositol phosphates and an increase in the phosphorylation of an endogenous protein kinase C substrate. OKT3 also mimicked the previously reported effects of protein kinase C since it potentiated the cAMP stimulation by either an adenosine analogue, NECA, or cholera toxin. Thus, our results indicate that stimulation of a receptor activating phospholipase C and protein kinase C can secondarily enhance the action of agonists that act on adenylate cyclase-coupled receptors.Abbreviations cAMP cyclic adenosine-monophosphate - NECA 5-N-ethylcarboxyamido adenosine The present study was supported by grants from The Swedish Cancer Association, The Swedish Medical Research Council (project no. 2553), by Gustaf V 80 Years Fund, by Nanna Swartz foundation and by Karolinska Institutet     

慢性染铅对小鼠海马Ca~(2+)-钙调蛋白依赖性蛋白激酶Ⅱ表达的影响

目的通过检测海马Ca2+钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)基因表达来探讨慢性铅中毒影响学习记忆的分子机制。方法小鼠交配后,通过饮水饲以2.4,4.8和9.6mmol·L-1醋酸铅。小鼠子代自胚胎期始即暴露于醋酸铅。幼鼠出生后,先通过哺乳接触铅,断乳后则自行饮用与母鼠饮用浓度相同的含铅水。6周后用逆转录聚合酶链反应法观察各组小鼠海马CaMKⅡmRNA的表达。结果3个染铅组小鼠CaMKⅡmRNA水平均明显降低,并具有浓度效应关系。结论铅致CaMKⅡ基因表达水平下降。     

Inhibition of the muscarinic receptor-activated K+ current by N-ethylmaleimide in rabbit heart

Summary The effects of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on the ACh-activated K⁺ current were examined in single cells from rabbit hearts using whole-cell and single channel patch clamp techniques. Bath application of NEM (50 M) or the muscarinic antagonist, atropine (1 M) completely inhibited the ACh-activated K⁺ current in whole-cell recordings. In cell-attached patch conditions, the inhibitory effect of NEM was still observed; in contrast, atropine was ineffective when the agents were bath applied, indicating that the site of action of NEM is different from that of atropine. Inside-out patch recordings confirmed that GTP was required on the internal side of the membrane for activation of this K⁺ channel when ACh was present in the pipette. NEM abolished this GTP-activated K⁺ channel activity. GTPS, a non-hydrolysable GTP analogue, was able to activate this K⁺ channel in the absence of a muscarinic agonist, an effect thought to be due to the direct activation of GTP-binding proteins. Pretreatment with NEM almost completely prevented this effect of GTPS. In contrast, after the activation of the K⁺ channel by GTPS had reached a steady-state, NEM failed to show a significant inhibitory effect. These results demonstrate that NEM prevents the activation of muscarinic receptor-regulated K⁺ channel and suggest an involvement of alkylation of the GTP-binding proteins which are coupled to this type of K⁺ channel. Send offprint requests to W. Giles at the above address     

Competitive binding of postsynaptic density 95 and Ca^2＋-calmodulin dependent protein kinase Ⅱ to N-methyl-D-aspartate receptor subunit 2B in rat brain

AIM: To investigate the interactions among postsynaptic density 95 (PSD-95), Ca^2 -calmodulin dependent protein kinase Ⅱα (CaMKⅡα), and N-methyl-D-aspartate receptor subunit 2B (NR2B) during ischemia and reperfusion in hippocampus of rats. METHODS: Brain ischemia was induced by four-vessel occlusion procedure in rats. Immunoprecipitation and immunoblotting were performed to study the interactions and phosphorylation of proteins. The association-dissociation of PSD-95 and CaMKⅡα to and from N-methyl-D-aspartate (NMDA) receptor induced by ischemia and reperfusion and the effects of 1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenyl-piperazine (KN-62, a selective inhibitor of CaMKⅡ) on these protein interactions were investigated. Coimmunoprecipitation and immunoblotting were performed for the studies of interactions among proteins. RESULTS: The alternations of the binding level of PSD-95 and CaMKⅡα to NR2B during ischemia and reperfusion demonstrated the negative correlation to each other. Pre-administration of KN62 through both cerebral ventricles inhibited the 10min ischemia-induced increase of the binding of PSD-95 to NR2B and, on the contrary, promoted the binding of CaMKⅡα to NR2B. CONCLUSION: PSD-95 competes with CaMKⅡ to bind to NR2B during ischemia and reperfusion in rat hippocampus.     

Triphenyltin impairs a protein kinase A (PKA)-dependent increase of cytosolic Na+ and Ca2+ and PKA-independent increase of cytosolic Ca2+ associated with insulin secretion in hamster pancreatic beta-cells

Oral administration of triphenyltin chloride (TPT) (60 mg/kg body weight) inhibits the insulin secretion by decreasing the cytoplasmic Ca2+ concentration ([Ca2+]i) induced by glucose-dependent insulinotropic polypeptide (GIP) in pancreatic beta-cells of the hamster. To test the possibility that the abnormal level of [Ca2+]i induced by TPT administration could be due to a defect in the cAMP-dependent cytoplasmic Na+ concentration ([Na+]i) in the beta-cells, we investigated the effects of TPT administration on the changes of [Na+]i induced by GIP, glucagon-like peptide-1 (GLP-1), or forskolin, an activator of adenylyl cyclase, and on the changes of [Na+]i or [Ca2+]i induced by 6-Bnz-cAMP, an activator of protein kinase A (PKA), and 8-pCPT-2'-O-Me-cAMP, an activator of Epac. The [Na+]i and [Ca2+]i were measured in islet cells loaded with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. In the presence of 135 mM Na+, TPT administration significantly reduced the rise in [Na+]i by 10 nM GLP-1, 10 microM forskolin, and 50 microM 6-Bnz-cAMP, but had not effect in a Na+-free medium. In the presence of 135 mM Na+, TPT administration also reduced the rise in [Ca2+]i by 8-pCPT-2'-O-Me-cAMP plus10 microM H-89, a inhibitor of PKA, and 6-Bnz-cAMP. Moreover, TPT administration significantly reduced the insulin secretion by 2 mM db-cAMP, GLP-1, GIP, and 8-pCPT-2'-O-Me-cAMP with and without H-89, and that by 6-Bnz-cAMP and forskolin. Our study suggested that TPT has inhibitory effects on the cellular Ca2+ response due to a reduced Na+ permeability through PKA-dependent mechanisms in hamster islet cells. Also TPT has the reduction of [Ca2+]i related to Na+-dependent insulin secretion after an activation of Epac.     

Veratridine causes the Ca2+-dependent increase in diacylglycerol formation and translocation of protein kinase C to membranes in cultured bovine adrenal medullary cells

Summary Our previous studies suggested that protein kinase C is involved in the veratridine (an activator of voltage-dependent Na⁺ channels)-induced phosphorylation and activation of tyrosine hydroxylase as well as the synthesis of catecholamines in adrenal medulla (Uezono et al. 1989). In the present study, we investigated whether treatment of cultured bovine adrenal medullary cells with veratridine causes the accumulation of diacylglycerol, a physiological activator of protein kinase C and the translocation of protein kinase C from cytosol to membrane, a process required for protein kinase C activation. Veratridine (100 mol/l) increased diacylglycerol level about 2.2 fold in a monophasic manner, with peaking at 5 min and declining toward the basal level within 20 min. Veratridine also increased membrane protein kinase C from 15.6% to 26.9% of total protein kinase C in a timecourse similar to that of diacylglycerol accumulation. Both stimulatory effects of veratridine were inhibited by tetrodotoxin and not observed in Ca²⁺-free, EGTA-containing medium. Amiloride, an inhibitor of Na⁺/Ca²⁺ and Na⁺/H⁺ exchange, did not alter veratridine-induced events. These results suggest that veratridine-induced Ca²⁺ influx contributes to the accumulation of diacylglycerol and the activation of protein kinase C in adrenal medullary cells. Send offprint requests to Y. Uezono at the above address     

Activation of microsomal cytochrome P450 mono-oxygenase by Ca2+ store depletion and its contribution to Ca2+ entry in porcine aortic endothelial cells

1. We investigated how microsomal cytochrome P450 mono-oxygenase (Cyp450 MO) is regulated in cultured porcine aortic endothelial cells. The hypothesis that a Cyp450 MO-derived metabolite links Ca²⁺ store depletion and Ca²⁺ entry was studied further.
2. Microsomal Cyp450 MO was monitored fluorometrically by dealkylation of 1-ethoxypyrene-3,6,8-tris-(dimethyl-sulphonamide; EPSA) in saponin permeabilized cells or in subcellular compartments. Endothelial Ca²⁺ signalling was measured by a standard fura-2 technique, membrane potential was determined with the potential-sensitive fluorescence dye, bis-(1,3-dibutylbarbituric acid) pentamethine oxonol (DiBAC₄(5)) and tyrosine kinase was quantified by measuring the phosphorylation of a immobilized substrate with a horseradish peroxidase labelled phosphotyrosine specific antibody.
3. Depletion of cellular Ca²⁺ pools with inositol 1,4,5-trisphosphate (IP₃), thapsigargin or cyclopiazonic acid activated microsomal Cyp450 MO. Similar to direct Ca²⁺ store depletion, chelating of intra-microsomal Ca²⁺ with oxalate stimulated Cyp450 MO activity, while changing cytosolic free Ca²⁺ failed to influence Cyp450 MO activity. These data indicate that microsomal Cyp450 MO is activated by depletion of IP₃-sensitive stores.
4. Besides the common cytochrome P450 inhibitors, econazole, proadifen and miconazole, thiopentone sodium and methohexitone inhibited Cyp450 MO in a concentration-dependent manner. The physiological substrate of Cyp450 MO, arachidonic acid, inhibited EPSA dealkylation. In contrast to most other cytochrome P450 inhibitors used in this study, thiopentone sodium did not directly interfere with Ca²⁺ entry pathways, membrane hyperpolarization due to K⁺ channel activation or tyrosine kinase activity.
5. Inhibition of Cyp450 MO by thiopentone sodium diminished Ca²⁺/Mn²⁺ entry to Ca²⁺ store depletion by 43%, while it did not interfere with intracellular Ca²⁺ release by IP₃ or thapsigargin.
6. Cyp450 MO inhibition with thiopentone sodium diminished autacoid-induced membrane hyperpolarization.
7. Induction of Cyp450 MO with dexamethasone/clofibrate for 72 h yielded increases in thapsigargin-induced Cyp450 MO activity (by 35%), Ca²⁺/Mn²⁺ entry (by 105%) and membrane hyperpolarization (by 40%).
8. The Cyp450 MO-derived compounds, 11,12 and 5,6-epoxyeicosatrienoic acids (EETs) yielded membrane hyperpolarization, insensitive to thiopentone sodium.
9. These data demonstrate that endothelial Cyp450 MO is activated by Ca²⁺ store depletion and Cyp450 MO produced compounds that hyperpolarize endothelial cells.
10. The data presented and our previous findings indicate that Cyp450 MO plays a crucial role in the regulation of store-operated Ca²⁺ influx. We propose that Cyp450 MO-derived EETs constitute a signal for Ca²⁺ entry activation and increase the driving force for Ca²⁺ entry by membrane hyperpolarization in porcine aortic endothelial cells.

     

A combinatorial in silico and cellular approach to identify a new class of compounds that target VEGFR2 receptor tyrosine kinase activity and angiogenesis

BACKGROUND AND PURPOSE

Vascular endothelial growth factor receptor 2 (VEGFR2) is an attractive therapeutic target for the treatment of diseases such as cancer. Small-molecule VEGFR2 inhibitors of a variety of chemical classes are currently under development or in clinical use. In this study, we describe the de novo design of a new generation pyrazole-based molecule (JK-P3) that targets VEGFR2 kinase activity and angiogenesis.

EXPERIMENTAL APPROACH

JK-P compound series were designed using de novo structure-based identification methods. Compounds were tested in an in vitro VEGFR2 kinase assay. Using primary endothelial cells, JK-P compounds were assessed for their ability to inhibit VEGF-A-stimulated VEGFR2 activation and intracellular signalling. We tested these compounds in cell migration, proliferation and angiogenesis assays.

KEY RESULTS

JK-P3 and JK-P5 were predicted to bind the VEGFR2 kinase domain with high affinity, and both compounds showed pronounced inhibition of endogenous VEGFR2 kinase activity in primary human endothelial cells. Only JK-P3 inhibited VEGF-A-stimulated VEGFR2 activation and intracellular signalling. Interestingly, JK-P3 inhibited endothelial monolayer wound closure and angiogenesis but not endothelial cell proliferation. Both compounds inhibited fibroblast growth factor receptor kinase activity in vitro, but not basic fibroblast growth factor-mediated signalling in endothelial cells.

CONCLUSIONS AND IMPLICATIONS

This is the first report that describes an anti-angiogenic inhibitor based on such a pyrazole core. Using a de novo structure-based identification approach is an attractive method to aid such drug discovery. These results thus provide an important basis for the development of multi-tyrosine kinase inhibitors for clinical use in the near future.