ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Abstract—Nitration of unsaturated fatty acids such as linoleate by NO-derived reactive species forms novel derivatives (including nitrolinoleate [LNO2]) that can stimulate smooth muscle relaxation and block platelet activation by either NO/cGMP or cAMP-dependent mechanisms. Here, LNO2was observed to inhibit human neutrophil function. LNO2, but not linoleic acid or the nitrated amino acid 3-nitrotyrosine, dose-dependently (0.2 to 1 &mgr;mol/L) inhibited superoxide (O2·−) generation, Ca2+influx, elastase release, and CD11b expression in response to either phorbol 12-myristate 13-acetate orN-formyl-Met-Leu-Phe. LNO2did not elevate cGMP, and inhibition of guanylate cyclase by 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one did not restore neutrophil responses, ruling out a role for NO. In contrast, LNO2caused elevations in intracellular cAMP in the presence and absence of phosphodiesterase inhibition, suggesting activation of adenylate cyclase. Compared with phorbol 12-myristate 13-acetate–activated neutrophils,N-formyl-Met-Leu-Phe–activated neutrophils were more susceptible to the inhibitory effects of LNO2, indicating that LNO2may inhibit signaling both upstream and downstream of protein kinase C. These data suggest novel signaling actions for LNO2in mediating its potent inhibitory actions. Thus, nitration of lipids by NO-derived reactive species yields products with antiinflammatory properties, revealing a novel mechanism by which NO-derived nitrated biomolecules can influence the progression of vascular disease.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Abstract—Despite the importance of vascular smooth muscle cells in the regulation of blood vessel function, the molecular mechanisms governing their development and differentiation remain poorly understood. Using an in vitro system whereby a pluripotent neural crest cell line (MONC-1) can be induced to differentiate into smooth muscle cells, we isolated a cDNA fragment that was robustly induced during this differentiation process. Sequence analysis revealed high homology to a partial cDNA termed modulator recognition factor 2 (Mrf2). Because the full-length cDNA has not been reported, we cloned the full-length Mrf2 cDNA by cDNA library screening and 5′ rapid amplification of cDNA ends and identified two isoforms of Mrf2 (&agr; [3.0 kb] and &bgr; [3.7 kb]) that differ in the N-terminus but share the DNA-binding domain. Protein homology analysis suggests that Mrf2 is a member of the AT-rich interaction domain family of transcription factors, which are known to be critically involved in the regulation of development and cellular differentiation. Mrf2&agr; and Mrf2&bgr; are highly induced during in vitro differentiation of MONC-1 cells into smooth muscle cells, and Mrf2&agr; is expressed in adult mouse cardiac and vascular tissues. To define the function of Mrf2, we overexpressed both isoforms in 3T3 fibroblast cells and observed an induction of smooth muscle marker genes, including smooth muscle &agr;-actin and smooth muscle 22&agr;. Furthermore, Mrf2&agr; and Mrf2&bgr; retarded cellular proliferation. These data implicate Mrf2 as a novel regulator of smooth muscle cell differentiation and proliferation.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Abstract—We have previously reported that SHP-2 upregulation is necessary for NO-stimulated motility in differentiated rat aortic smooth muscle cells. We now test the hypothesis that upregulation of SHP-2 is necessary and sufficient to stimulate cell motility. Overexpression of SHP-2 via recombinant adenoviral vector stimulated motility to the same extent as NO, whereas the expression of C463S-SHP-2, the dominant-negative SHP-2 allele, blocked the motogenic effect of NO. On the basis of previous studies, we next tested the hypothesis that NO decreases RhoA activity and that this event is necessary and sufficient to explain NO-induced motogenesis. We found that NO decreased RhoA activity in a concentration-dependent manner. Moreover, a dominant-negative SHP-2 allele, DSH2, blocked the NO-induced inhibition of RhoA activity, indicating that upregulation of SHP-2 is necessary for this event. Expression of G14V-RhoA, the constitutively active RhoA allele, decreased cell motility and blocked the motogenic effect of NO, whereas the expression of T19N-RhoA, the dominant-negative RhoA allele, increased cell motility to an extent similar to that induced by NO. Dominant-negative RhoA reversed the effect of dominant-negative SHP-2, indicating that RhoA functions downstream from SHP-2. To investigate events downstream from RhoA, we treated cells with fasudil, a selective Rho kinase inhibitor, and found that it increased cell motility. These results indicate that upregulation of SHP-2, leading to downregulation of RhoA, which is followed by decreased Rho kinase activity, is a sequence of events necessary and sufficient to explain NO-induced cell motility in differentiated aortic smooth muscle cells. The results may be of relevance to in vivo events such as neointimal formation, angiogenesis, and vasculogenesis.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Abstract—As thrombin binding to the G protein–coupled proteinase activated receptor-1 (PAR-1) induces endothelial adhesivity to leukocytes through NF-&kgr;B activation and intercellular adhesion molecule-1 (ICAM-1) expression, we determined the signaling pathways mediating the response. Studies showed that the heterotrimeric G proteins, G&agr;q, and the G&bgr;&ggr; dimer were key determinants of the PAR-1 agonist peptide (TFLLRNPNDK)-induced NF-&kgr;B activation and ICAM-1 expression in endothelial cells. Cotransfection of RGS3T, a regulator of G-protein signaling that inhibits G&agr;q, or &agr;-transducin (G&agr;t), a scavenger of the G&bgr;&ggr;, markedly decreased NF-&kgr;B activity induced by PAR-1 activation. We determined the downstream signaling targets activated by G&agr;qand G&bgr;&ggr; that mediate NF-&kgr;B activation. Expression of the kinase-defective protein kinase C (PKC)-&dgr; mutant inhibited NF-&kgr;B activation induced by the constitutively active G&agr;qmutant, but had no effect on NF-&kgr;B activity induced by G&bgr;1&ggr;2. In related experiments, NF-&kgr;B as well as ICAM-1 promoter activation induced by G&bgr;1&ggr;2were inhibited by the expression of the dominant-negative mutant of 85-kDa regulatory subunit of PI 3-kinase; however, the expression of this mutant had no effect on the response induced by activated G&agr;q. Cotransfection of the catalytically inactive Akt mutant inhibited the NF-&kgr;B activation induced by the constitutively active PI 3-kinase mutant as well as that by the activated forms of G&agr;qand PKC-&dgr;. These results support a model in which ligation of PAR-1 induces NF-&kgr;B activation and ICAM-1 transcription by the engagement of parallel G&agr;q/PKC-&dgr; and G&bgr;&ggr;/PI3-kinase pathways that converge at Akt.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Abstract—Angiotensin II (Ang II)–stimulated hypertrophy of vascular smooth muscle cells is mediated by reactive oxygen species (ROS) derived from NAD(P)H oxidases. The upstream signaling mechanisms by which Ang II activates these oxidases are unclear but may include protein kinase C, tyrosine kinases, phosphatidylinositol-3-kinase, and Rac, a small molecular weight G protein. We found that Ang II–stimulated ROS production is biphasic. The first phase occurs rapidly (peak at 30 seconds) and is dependent on protein kinase C activation. The larger second phase of ROS generation (peak at 30 minutes) requires Rac activation, because inhibition of Rac by eitherClostridium difficiletoxin A or dominant-negative Rac significantly inhibits Ang II–induced ROS production. Phosphatidylinositol-3-kinase inhibitors (wortmannin or LY294002) and the epidermal growth factor (EGF) receptor kinase blocker AG1478 attenuate both Rac activation and ROS generation. The upstream activator of EGF receptor transactivation, c-Src, is also required for ROS generation, because PP1, an Src kinase inhibitor, abrogates the Ang II stimulation of both responses. These results suggest that c-Src, EGF receptor transactivation, phosphatidylinositol-3-kinase, and Rac play important roles in the sustained Ang II–mediated activation of vascular smooth muscle cell NAD(P)H oxidases and provide insight into the integrated signaling mechanisms whereby Ang II stimulation leads to activation of the growth-related NAD(P)H oxidases.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Abstract—Despite extensive research, the mechanisms responsible for the graded nature and early termination of Ca2+-induced Ca2+release (CICR) from the sarcoplasmic reticulum (SR) in cardiac muscle remain poorly understood. Suggested mechanisms include cytosolic Ca2+-dependent inactivation/adaptation and luminal Ca2+-dependent deactivtion of the SR Ca2+release channels/ryanodine receptors (RyRs). To explore the importance of cytosolic versus luminal Ca2+regulatory mechanisms in controlling CICR, we assessed the impact of intra-SR Ca2+buffering on global and local Ca2+release properties of patch-clamped or permeabilized rat ventricular myocytes. Exogenous, low-affinity Ca2+buffers (5 to 20 mmol/L ADA, citrate or maleate) were introduced into the SR by exposing the cells to “internal” solutions containing the buffers. Enhanced Ca2+buffering in the SR was confirmed by an increase in the total SR Ca2+content, as revealed by application of caffeine. At the whole-cell level, intra-SR [Ca2+] buffering dramatically increased the magnitude of Ca2+transients induced byICaand deranged the smoothly gradedICa-SR Ca2+release relationship. The amplitude and time-to-peak of local Ca2+release events, Ca2+sparks, as well as the duration of local Ca2+release fluxes underlying sparks were increased up to 2- to 3-fold. The exogenous Ca2+buffers in the SR also reduced the frequency of repetitive activity observed at individual release sites in the presence of the RyR activator Imperatoxin A. We conclude that regulation of RyR openings by local intra-SR [Ca2+] is responsible for termination of CICR and for the subsequent restitution behavior of Ca2+release sites in cardiac muscle.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Abstract—The aim of the present study was to investigate the single-channel properties of different gating modes in the native human cardiac Na+channel. Patch-clamp experiments were performed at low noise using ultrathick-walled pipettes. In 17 cell-attached patches containing only one channel, fast back and forth switching between five different Na+-channel gating modes (F-mode, M1-mode, M2-mode, S-mode, and P-mode) was identified, but no difference in the gating properties was found between normal and diseased cardiomyocytes from atrium or ventricle, respectively. Hodgkin-Huxley fits to the ensemble-averaged currents yielded the activation-time (&tgr;m) and inactivation-time (&tgr;h) constants. &tgr;mwas comparably fast in the F-mode, M1-mode, M2-mode, and S-mode (0.15 ms) and slow in the P-mode (0.3 ms). &tgr;hranged from 0.35 ms (F-mode) to 4.5 ms (S-mode and P-mode). The mean open-channel lifetime (&tgr;o) was shortest in the F-mode and P-mode (0.15 ms) and longest in the S-mode (1.25 ms). The time before which half of the first channel openings occurred (t0.5) was comparably short in the F-mode, M1-mode, M2-mode, and S-mode (0.3 ms) and long in the P-mode (0.9 ms). It is concluded that (1) a single native human cardiac Na+channel can be recorded at low noise, (2) this channel can change its gating properties at a time scale of milliseconds, (3) lifetimes of the observed gating modes are short ranging from milliseconds to seconds only, and (4) the gating modes are characterized by specific activation and inactivation kinetics and differ at least in their mean open time and first latency.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID

摘要:

Abstract—CCAAT/enhancer-binding proteins (C/EBPs) upregulate transcription of various inflammatory cytokines and acute phase proteins, such as interleukin (IL)-1&bgr;, IL-6, tumor necrosis factor-&agr;, and cyclooxygenase-2. Recent studies have demonstrated that peroxisome proliferator-activated receptor (PPAR)-&ggr; is present in atherosclerotic lesions, and negatively regulates expression of these genes. Interestingly, PPAR-&ggr; gene promoter has tandem repeats of C/EBP-binding motif, and C/EBP-&dgr; plays a pivotal role in transactivation of PPAR-&ggr; gene. It has been well known that the interaction between C/EBPs and PPAR-&ggr; plays a central role in maintaining adipocyte differentiation and glucometabolism; however, the relationship between PPAR-&ggr; and C/EBPs in the vessel wall remains unclear. In the present study, we showed that a high level of C/EBP-&dgr; expression induced by inflammation positively regulated transcription and protein expression of PPAR-&ggr; in vascular smooth muscle cells (VSMCs). On the other hand, PPAR-&ggr; ligands troglitazone, pioglitazone, and 15-deoxy-&Dgr;12,14-prostaglandin J2inhibited IL-1&bgr;-induced IL-6 expression at a transcriptional revel in VSMCs. Functional promoter analysis revealed that PPAR-&ggr; ligands inhibited IL-1&bgr;-induced transactivation of IL-6 gene via suppression of not only nuclear factor-&kgr;B but also C/EBP-DNA binding. Moreover, PPAR-&ggr; ligands suppressed protein expression and transcription of C/EBP-&dgr; through dephosphorylation of signal transducer and activator of transcription 3. These findings strongly suggest that C/EBP-&dgr; is negatively autoregulated via transactivation of PPAR-&ggr;. This feedback mechanism probably downregulates transcription of inflammatory cytokines and acute phase proteins, and modulates inflammatory responses in the early process of atherosclerosis.

ISSN:0009-7330    

出版商:OVID    

年代:2002

数据来源: OVID