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Results for “Enhancer mapping”

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PubMed2003

Nardosinone, the first enhancer of neurite outgrowth-promoting activity of staurosporine and dibutyryl cyclic AMP in PC12D cells.

Nardosinone was isolated as an enhancer of nerve growth factor (NGF) from Nardostachys chinensis [Neurosci. Lett. 273 (1999) 53]. Nardosinone (0.1-100 microM) enhanced dibutyryl cyclic AMP (dbcAMP, 0.3 mM)- and staurosporine (10 nM)-induced neurite outgrowth from PC12D cells in a concentration-dependent manner. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, partially blocked enhancements of dbcAMP (0.3 mM)- or staurosporine (10 nM)-induced neurite outgrowth by nardosinone. Nardosinone alone had no effect on the phosphorylation of MAP kinase. The dbcAMP-induced increase in phosphorylation of MAP kinase was not affected by nardosinone. Staurosporine almost unaffected the phosphorylation of MAP kinase, and nardosinone potentiated the staurosporine-induced neurite outgrowth without stimulation of the phosphorylation of MAP kinase. Since it is known that MAP kinase signaling is required for neurite outgrowth in PC12D cells, these results suggest that nardosinone enhances staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying both the MAP kinase-dependent and -independent signaling pathways of dbcAMP and staurosporine. It is also suggested that nardosinone enhances a downstream step of MAP kinase in the MAP kinase-dependent signaling pathway. Nardosinone is the first enhancer of the neuritogenic action of dbcAMP and staurosporine and may become a useful pharmacological tool for studying the mechanism of action of not only NGF but also both the neuritogenic substances.

Animals↗

PubMed2002

Picrosides I and II, selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells.

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.

Animals↗

PubMed2006

Turboprop: improved PROPELLER imaging.

A variant of periodically rotated overlapping parallel lines with enhanced reconstruction (PROPELLER) MRI, called turboprop, is introduced. This method employs an oscillating readout gradient during each spin echo of the echo train to collect more lines of data per echo train, which reduces the minimum scan time, motion-related artifact, and specific absorption rate (SAR) while increasing sampling efficiency. It can be applied to conventional fast spin-echo (FSE) imaging; however, this article emphasizes its application in diffusion-weighted imaging (DWI). The method is described and compared with conventional PROPELLER imaging, and clinical images collected with this PROPELLER variant are shown.

Algorithms↗

PubMed2001

Regulation of an endogenous locus using a panel of designed zinc finger proteins targeted to accessible chromatin regions. Activation of vascular endothelial growth factor A.

We have mapped conserved regions of enhanced DNase I accessibility within the endogenous chromosomal locus of vascular endothelial growth factor A (VEGF-A). Synthetic zinc finger protein (ZFP) transcription factors were designed to target DNA sequences contained within the DNase I-hypersensitive regions. These ZFPs, when fused to either VP16 or p65 transcriptional activation domains, were able to activate expression of the VEGF-A gene as assayed by mRNA accumulation and VEGF-A protein secretion through a range exceeding that induced by hypoxic stress. Importantly, multiple splice variants of VEGF-A mRNA with defined physiological functions were induced by a single engineered ZFP transcription factor. We present evidence for an enhanced activation of VEGF-A gene transcription by ZFP transcription factors fused to VP16 and p65 targeted to two distinct chromosomal sites >500 base pairs upstream or downstream of the transcription start site. Our strategy provides a novel approach for dissecting the requirements for gene regulation at a distance without altering the DNA sequence of the endogenous target locus.

Cell Line, Transformed↗

PubMed1992

A survey of protein-DNA interaction sites within the murine immunoglobulin heavy chain locus reveals a particularly complex pattern around the DQ52 element.

The expression of immunoglobulin (Ig) genes is regulated at two levels: rearrangement of individual gene segments and transcription of continuous genes. To find transacting factors involved in mediating locus- and segment specific gene activation and expression, we surveyed a 3600-bp genomic region of the murine Ig heavy chain locus, spanning from the DQ52 element to the Ig heavy chain intron enhancer. We discovered nine, previously undescribed, protein-DNA complexes and estimated their individual binding-affinity preferences (Kr) by quantitative gel shift measurements. We observed one novel protein DNA interaction at the enhancer, two in the JH1 region and six within a 300-bp region immediately 5' to the DQ52 locus. The latter show a complex and specific binding pattern when comparing nuclear extracts derived from pre-B cells and fibroblasts. Further characterization of the interactions at the DQ52 locus by electron microscopy revealed the preferential formation of a protein complex binding to the DQ52 locus with pre B cell extracts. This behavior and the clustering of interaction sites 5' to the DQ52 element suggest that this region is involved in the regulation of heavy chain gene assembly.

Animals↗

PubMed2003

Single peptide-based protein identification in human proteome through MALDI-TOF MS coupled with amino acids coded mass tagging.

Identification of proteins with low sequence coverage using mass spectrometry (MS) requires tandem MS/MS peptide sequencing. It is very challenging to obtain a complete or to interpret an incomplete tandem MS/MS spectrum from fragmentation of a weak peptide ion signal for sequence assignment. Here, we have developed an effective and high-throughput MALDI-TOF-based method for the identification of membrane and other low-abundance proteins with a simple, one-dimensional separation step. In this approach, several stable isotope-labeled amino acid precursors were selected to mass-tag, in parallel, the human proteome of human skin fibroblast cells in a residue-specific manner during in vivo cell culturing. These labeled residues can be recognized by their characteristic isotope patterns in MALDI-TOF MS spectra. The isotope pattern of particular peptides induced by the different labeled precursors provides information about their amino acid compositions. The specificity of peptide signals in a peptide mass mapping is thus greatly enhanced, resolving a high degree of mass degeneracy of proteolytic peptides derived from the complex human proteome. Further, false positive matches in database searching can be eliminated. More importantly, proteins can be accurately identified through a single peptide with its m/z value and partial amino acid composition. With the increased solubility of hydrophobic proteins in SDS, we have demonstrated that our approach is effective for the identification of membrane and low-abundant proteins with low sequence coverage and weak signal intensity, which are often difficult for obtaining informative fragment patterns in tandem MS/MS peptide sequencing analysis.

Amino Acids↗

PubMed1990

Ganglioside-enhanced neurite growth: evidence for a selective induction of high-molecular-weight MAP-2.

Neuroblastoma cells maintained in serum-free medium exhibit a significant induction of MAP-1a and Tau proteins, but not MAP-2; the time course of these inductions is highly correlated with an increase in microtubule mass which parallels neurite growth. Bovine brain gangliosides (BBG) enhance the neurite outgrowth response of these cells. We report here that one effect of gangliosides is the selective and dramatic induction of MAP-2 expression. Our results also indicate a strong parallelism between this induction and the increase in microtubule mass which accompanies the appearance of more numerous, longer, and highly branched neurites. These observations suggest that MAP-2 induction in neuroblastoma cells may lead to a further differentiation of neurites equivalent to that observed in mature brain neurons. Finally, our results indicate that gangliosides per se are not neuritogenic factors but rather substances capable of enhancing cell-derived influences which affect the neurite outgrowth response of neuroblastoma cells and the type of MAP that they express.

Animals↗

PubMed2004

Diffusion-weighted imaging in the follow-up of treated high-grade gliomas: tumor recurrence versus radiation injury.

BACKGROUND AND PURPOSE: Diffusion-weighted (DW) MR imaging is a means to characterize and differentiate morphologic features, including edema, necrosis, and tumor tissue, by measuring differences in apparent diffusion coefficient (ADC). We hypothesized that DW imaging has the potential to differentiate recurrent or progressive tumor growth from treatment-induced damage to brain parenchyma in high-grade gliomas after radiation therapy. METHODS: We retrospectively reviewed follow-up conventional and DW MR images obtained starting 1 month after completion of radiation treatment with or without chemotherapy for histologically proved high-grade gliomas. Eighteen patients with areas of abnormal enhancing tissue were identified. ADC maps were calculated from echo-planar DW images, and mean ADC values and ADC ratios (ADC of enhancing lesion to ADC of contralateral white matter) were compared with final diagnosis. Recurrence was established by histologic examination or by clinical course and a combination of imaging studies. RESULTS: Recurrence and nonrecurrence could be differentiated by using mean ADC values and ADC ratios. ADC ratios in the recurrence group showed significantly lower values (mean +/- SD, 1.43 +/- 0.11) than those of the nonrecurrence group (1.82 +/- 0.07, P <.001). Mean ADCs of the recurrent tumors (mean +/- SD, 1.18 +/- 0.13 x 10(-3) mm/s(2)) were significantly lower than those of the nonrecurrence group (1.40 +/- 0.17 x 10(-3) mm/s(2), P <.006). CONCLUSION: Assessment of ADC ratios of enhancing regions in the follow-up of treated high-grade gliomas is useful in differentiating radiation effects from tumor recurrence or progression.

Adolescent↗

PubMed1996

[In vivo registration of monophasic action potentials--new possible applications in clinical electrophysiology].

Monophasic action potential (MAP) recordings have gained a more important role during the recent years. A new application for their use in the clinical laboratory is the precise differentiation between ventricular tachycardias and ventricular fibrillation. A correct diagnosis of the arrhythmic episode may be of relevance for the determination of the defibrillation threshold during implantation and testing of implantable cardioverter defibrillators. Diagnostic studies and ablation procedures of triggered arrhythmias represent another indication for MAP recordings. The focus of enhanced automaticity can be located by the detection of afterdepolarizations which allows a reliable ablation using a MAP-ablation combination catheter. Recently, a new phenomenon has been reported regarding the relationship between ventricular repolarization and excitability during programmed electrical stimulation in humans. Each additional extrastimulus was able to capture the myocardium at a less complete repolarization level than the previous one. This "facilitation of excitability" phenomenon or "encroachment" was correlated with the initiation of ventricular tachyarrhythmias. The involved mechanism can be modulated pharmacologically and might therefore play a role in the antiarrhythmic treatment of patients with ventricular arrhythmias.

Cardiac Pacing, Artificial↗

PubMed2005

Nuclear factor of activated T cells c1 induces osteoclast-associated receptor gene expression during tumor necrosis factor-related activation-induced cytokine-mediated osteoclastogenesis.

Osteoclast differentiation from hematopoietic precursors is controlled by the tumor necrosis factor family member tumor necrosis factor-related activation-induced cytokine (TRANCE) via induction of various transcription factors, including nuclear factor of activated T cells (NFAT) c1. During osteoclast differentiation, NFATc1 is further activated via calcium signaling when costimulatory receptors expressed on osteoclast precursors, such as osteoclast-associated receptor (OSCAR), are stimulated. Here we show that NFATc1 expression precedes that of OSCAR during TRANCE-mediated osteoclastogenesis and that inhibition of NFATc1 by cyclosporin A abolishes TRANCE-induced OSCAR expression and subsequent osteoclast differentiation. Moreover, we show that the 1.0-kb promoter region of the OSCAR gene contains three potential NFATc1-binding sites. Induction of an OSCAR promoter-luciferase reporter is significantly increased when transiently transfected into 293T cells in combination with NFATc1 expression plasmid. Deletion and site-directed mutant constructs confirmed that NFATc1-binding sites are both functional and NFATc1-specific. Furthermore, NFATc1 synergistically activates an OSCAR reporter construct together with microphthalmia transcription factor and PU.1, transcription factors previously shown to be critical for osteoclast differentiation. In addition, a plasmid expressing constitutively active MAP kinase kinase 6 enhances the transactivation activity of NFATc1/microphthalmia transcription factor/PU.1 on the OSCAR promoter. Taken together, our results indicate that NFATc1 is an important transcription factor in the induction of OSCAR during osteoclastogenesis. Elucidation of NFATc1 as a transcription factor for OSCAR expression implies the presence of a positive feedback circuit of TRANCE-induced activation of NFATc1, involving NFATc1-mediated OSCAR expression and its subsequent activation of NFATc1, necessary for efficient differentiation of osteoclasts.

Animals↗

PubMed2002

Unified sampling approach for multipoint linkage disequilibrium mapping of qualitative and quantitative traits.

Rapid development in biotechnology has enhanced the opportunity to deal with multipoint gene mapping for complex diseases, and association studies using quantitative traits have recently generated much attention. Unlike the conventional hypothesis-testing approach for fine mapping, we propose a unified multipoint method to localize a gene controlling a quantitative trait. We first calculate the sample size needed to detect linkage and linkage disequilibrium (LD) for a quantitative trait, categorized by decile, under three different modes of inheritance. Our results show that sampling trios of offspring and their parents from either extremely low (EL) or extremely high (EH) probands provides greater statistical power than sampling in the intermediate range. We next propose a unified sampling approach for multipoint LD mapping, where the goal is to estimate the map position (tau) of a trait locus and to calculate a confidence interval along with its sampling uncertainty. Our method builds upon a model for an expected preferential transmission statistic at an arbitrary locus conditional on the sampling scheme, such as sampling from EL and EH probands. This approach is valid regardless of the underlying genetic model. The one major assumption for this model is that no more than one quantitative trait locus (QTL) is linked to the region being mapped. Finally we illustrate the proposed method using family data on total serum IgE levels collected in multiplex asthmatic families from Barbados. An unobserved QTL appears to be located at tau; = 41.93 cM with 95% confidence interval of (40.84, 43.02) through the 20-cM region framed by markers D12S1052 and D12S1064 on chromosome 12. The test statistic shows strong evidence of linkage and LD (chi-square statistic = 18.39 with 2 df, P-value = 0.0001).

Asthma↗

PubMed1995

Mutational analysis of Ca(2+)-independent autophosphorylation of calcium/calmodulin-dependent protein kinase II.

Previous studies with synthetic peptides indicate that residues 290-309, corresponding to the calmodulin (CaM)-binding domain of Ca2+/CaM-dependent protein kinase II interact with the catalytic core of the enzyme as a pseudosubstrate (Colbran, R. J., Smith, M. K., Schworer, C. M., Fong, Y. L., and Soderling, T. R. (1989) J. Biol. Chem. 264, 4800-4804). In the present study, we attempted to locate the pseudosubstrate motif by generation or removal of potential substrate recognition sequences (RXXS/T) at selected positions using site-directed mutagenesis. Based on previous results, Arg297, Thr305/306, and Ser314 were selected as key residues. Single mutations such as N294S, K300S, A302R, A309R, and R311A were expressed, purified, and characterized. Several of the mutants exhibited decreased binding of and activation by CaM, not surprising since the mutations were within the CaM-binding domain. None of the mutants exhibited enhanced Ca(2+)-independent kinase activity toward exogenous substrate, but the K300S and N294S mutants showed a significant enhancement in the rate and stoichiometry of 32P incorporation during Ca(2+)-independent autophosphorylation. Using two-dimensional peptide mapping and phosphoamino acid analyses, enhanced phosphorylation of the introduced Ser residue was demonstrated in the K300S mutant but not in the N294S mutant. This specific Ca(2+)-independent autophosphorylation of Ser300 is consistent with the hypothesis that Arg297 may occupy the P (-3) position in a pseudosubstrate autoinhibitory interaction with the catalytic core in the nonactivated state of the kinase.

Amino Acids↗

PubMed2004

Radiolytic modification of acidic amino acid residues in peptides: probes for examining protein-protein interactions.

Hydroxyl radical-mediated footprinting coupled with mass spectroscopic analysis is a new technique for mapping protein surfaces, identifying structural changes modulated by protein-ligand binding, and mapping protein-ligand interfaces in solution. In this study, we examine the radiolytic oxidation of aspartic and glutamic acid residues to probe their potential use as structural probes in footprinting experiments. Model peptides containing Asp or Glu were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The radiolysis products were characterized by electrospray mass spectrometry including tandem mass spectrometry. Both Asp and Glu are susceptible to radiolytic oxidization by gamma-rays or synchrotron X-rays. Radiolysis results primarily in the oxidative decarboxylation of the side chain carboxyl group and formation of an aldehyde group at the carbon next to the original carboxyl group, giving rise to a characteristic product with a -30 Da mass change. A similar oxidative decarboxylation also takes place for amino acids with C-terminal carboxyl groups. The methylene groups in the Asp and Glu side chains also undergo oxygen addition forming ketone or alcohol groups with mass changes of +14 and +16 Da, respectively. Characterizing the oxidation reactions of these two acidic residues extends the number of useful side chain probes for hydroxyl radical-mediated protein footprinting from 10 (Cys, Met, Trp, Tyr, Phe, Arg, Leu, Pro, His, Lys) to 12 amino acid residues, thus enhancing our ability to map protein surface structure and in combination with previously identified basic amino acid probes can be used to examine molecular details of protein-protein interactions that are driven by electrostatics.

Aspartic Acid↗

PubMed1994

An upstream enhancer regulating brown-fat-specific expression of the mitochondrial uncoupling protein gene.

Previous studies on the regulation of a Ucp minigene in transgenic mice demonstrated that the sequences necessary for brown-fat-specific expression and inducibility by norepinephrine were located in the 5' flanking region between 1 and 2.8 kb from the transcriptional start site. We have investigated this region in more detail in cultured mouse brown adipocyte tumor cells. Deletion analysis of two types of chloramphenicol acetyltransferase reporter gene constructs under control of either the Ucp promoter or a heterologous herpes simplex virus-tk promoter defined an enhancer in a 220-bp HindIII-XbaI fragment which was essential for both brown fat specificity and norepinephrine inducibility. Site-directed mutagenesis of the reporter gene constructs established that independent mutations to a cyclic AMP-responsive element (CRE-2) or one of two TTCC motifs (BRE [brown fat regulatory element]), all within 17 bp, eliminated transient expression. Competitive DNA mobility shift assays with probes of the CRE and BRE motifs indicate that nuclear proteins interact with these motifs in a cooperative, synergistic manner. While these CRE-BRE probes do not show changes in binding which is dependent on norepinephrine treatment, a probe containing a third TTCC motif located 130 bp downstream of BRE-1 does show this dependency. The results indicate that a complex interaction of the CRE and BRE motifs, which cannot be functionally separated, control Ucp expression.

Adipose Tissue, Brown↗

PubMed2004

Electromechanical properties of perfusion/metabolism mismatch: comparison of nonfluoroscopic electroanatomic mapping with 18F-FDG PET.

UNLABELLED: The aim of this study was to compare nonfluoroscopic electroanatomic mapping (NOGA), SPECT perfusion imaging, and PET metabolic imaging for assessment of myocardial viability. In particular, we sought to elucidate differences of electromechanical properties between the perfusion/metabolism mismatch as an indicator of a potentially reversible ischemic injury and the perfusion/metabolism match indicating irreversibly damaged myocardial tissue. METHODS: Twenty-one patients with coronary artery disease underwent NOGA mapping of endocardial unipolar voltage, cardiac 18F-FDG PET of glucose utilization, and resting 201Tl SPECT of myocardial perfusion. RESULTS: Electrical activity was 10.8 +/- 4.6 mV (mean +/- SD) in normal myocardium and was unchanged in hypoperfused segments with maintained glucose metabolism (perfusion/metabolism mismatch), 9.3 +/- 3.4 mV (P = not significant). In contrast, hypoperfused segments with a perfusion/metabolism match and nonviable segments showed significantly lower voltage (6.9 +/- 3.1 mV, P < 0.0001 and 4.1 +/- 1.1 mV, P < 0.0001 vs. normal). In hypoperfused segments, metabolic activity was more closely related to endocardial voltage than was myocardial perfusion (201Tl vs. voltage: r = 0.38, SEE = 3.2, P < 0.001; 18F-FDG PET vs. voltage: r = 0.6, SEE = 2.8, P < 0.0001). CONCLUSION: In hypoperfused myocardium, electrical activity by NOGA mapping is more closely related to PET metabolic activity than to SPECT myocardial perfusion. As NOGA mapping does not differentiate hypoperfused myocardium with enhanced glucose utilization from normal myocardium, results from NOGA mapping need to be correlated with results from perfusion imaging to identify hypoperfused, yet viable, myocardium and to stratify patients for revascularization procedures.

Coronary Artery Disease↗

PubMed2003

Endocannabinoids protect the rat isolated heart against ischaemia.

1 The purpose of this study was to determine whether endocannabinoids can protect the heart against ischaemia and reperfusion. 2 Rat isolated hearts were exposed to low-flow ischaemia (0.5-0.6 ml min(-1)) and reperfusion. Functional recovery as well as CK and LDH overflow into the coronary effluent were monitored. Infarct size was determined at the end of the experiments. Phosphorylation levels of p38, ERK1/2, and JNK/SAPK kinases were measured by Western blots. 3 None of the untreated hearts recovered from ischaemia during the reperfusion period. Perfusion with either 300 nM palmitoylethanolamide (PEA) or 300 nM 2-arachidonoylglycerol (2-AG), but not anandamide (up to 1 micro M), 15 min before and throughout the ischaemic period, improved myocardial recovery and decreased the levels of coronary CK and LDH. PEA and 2-AG also reduced infarct size. 4 The CB(2)-receptor antagonist, SR144528, blocked completely the cardioprotective effect of both PEA and 2-AG, whereas the CB(1)-receptor antagonist, SR141716A, blocked partially the effect of 2-AG only. In contrast, both ACEA and JWH015, two selective agonists for CB(1)- and CB(2)- receptors, respectively, reduced infarct size at a concentration of 50 nM. 5 PEA enhanced the phosphorylation level of p38 MAP kinase during ischaemia. PEA perfusion doubled the baseline phosphorylation level of ERK1/2, and enhanced its increase upon reperfusion. The cardioprotective effect of PEA was completely blocked by the p38 MAP kinase inhibitor, SB203580, and significantly reduced by the ERK1/2 inhibitor, PD98059, and the PKC inhibitor, chelerythrine. 6 In conclusion, endocannabinoids exert a strong cardioprotective effect in a rat model of ischaemia-reperfusion that is mediated mainly through CB(2)-receptors, and involves p38, ERK1/2, as well as PKC activation.

Amides↗

PubMed1994

cAMP inhibits the insulin-stimulated mitogen-activated protein kinase pathway in rat hepatoma H4EII cells.

Effects of cAMP on insulin-stimulated mitogen-activated protein (MAP) kinase pathway were examined using rat hepatoma H4EII cells. MAP kinase was rapidly activated and reached a peak 3 min after the stimulation by insulin. Forskolin (1 microM) and 8(4-chlorophenylthio)cAMP (8-CPT-cAMP) (0.1 mM) inhibited the insulin-stimulated MAP kinase activity. Pretreatment of the cells with H-8 (50 microM), a cAMP-dependent protein kinase inhibitor, enhanced the insulin-stimulated MAP kinase activity and partially restored the inhibitory effect of cAMP. Furthermore, insulin-induced phosphorylation of MAP kinase was inhibited by 8-CPT-cAMP, and the inhibition was restored by H-8. 8-CPT-cAMP did not inhibit the autophosphorylation of insulin receptor. These data indicate that elevation of intracellular cAMP blocks the insulin-stimulated MAP kinase pathway downstream of insulin receptor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1996

Calcium/calmodulin-dependent protein kinase IIalpha mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells.

We have investigated the contribution of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and mitogen-activated protein kinase (MAP kinase) in norepinephrine (NE)-induced arachidonic acid (AA) release in rabbit aortic vascular smooth muscle cells (VSMC). NE enhanced release of AA via activation of cytosolic phospholipase A2 (cPLA2) but not secretory PLA2 in VSMC prelabeled with [3H]AA. NE (10 microM) enhanced CaM kinase II and MAP kinase activity. In cells transiently transfected with antisense oligonucleotides complementary to the translation initiation sites of CaM kinase II and MAP kinase, NE-induced AA release was inhibited by 100 and 35% respectively. Treatment of cells with PD-098059, a MAP kinase kinase inhibitor, or with MAP kinase antisense oligonucleotide reduced NE-induced activation of MAP kinase and cPLA2. NE-induced MAP kinase and cPLA2 activation was also inhibited in cells treated with a CaM kinase II inhibitor, KN-93, or with CaM kinase II antisense oligonucleotide. On the other hand, inhibition of MAP kinase kinase with PD-098059 or of MAP kinase with antisense oligonucleotides did not alter the NE-induced increase in CaM kinase II activity. Phosphorylation of MAP kinase and CaM kinase II by NE, studied by 32P incorporation and immune complex kinase assays, was inhibited by KN-93. Collectively, these data suggest that CaM kinase II can activate MAP kinase, which in turn activates cPLA2 to release AA for prostacyclin synthesis in the rabbit VSMC. This novel pathway for activation of MAP kinase by CaM kinase II appears to be mediated through stimulation of MAP kinase kinase. Activation of adrenergic receptors with NE in VSMC caused translocation of CaM kinase II, MAP kinase, and cPLA2 to the nuclear envelope only in the presence of extracellular Ca2+. Okadaic acid, which increased phosphorylation and activity, did not translocate these enzymes. Therefore, it appears that in rabbit VSMC, NE, by promoting extracellular Ca2+ influx, increases CaM kinase II activity, leading to activation of MAP kinase and cPLA2 and translocation to the nuclear envelope, resulting in release of AA from the nuclear envelope for prostacyclin synthesis.

Animals↗