摘要:

Wehypothesized that a redox-sensitive transcription factor, redox factor-1(Ref-1) (HAP1, APE, and APEX), was critical in the regulation of endothelialcell survival in response to hypoxia and cytokines, including tumor necrosisfactor (TNF)-&agr;. Hypoxia resulted in a significant decrease in Ref-1protein expression in both human umbilical vein endothelial cells and calfpulmonary artery endothelial cells. The hypoxia-induced decrease in Ref-1expression was followed by a significant induction of apoptosis as measured bycaspase 3 activity and nuclear morphology. Transient upregulation of Ref-1significantly inhibited hypoxia-induced apoptosis. However, deletion of theredox-sensitive domain of Ref-1 abolished the antiapoptotic effect. Wepostulated that the antiapoptotic effects of Ref-1 were mediated throughnuclear factor-&kgr;B (NF-&kgr;B). However, blockade of NF-&kgr;B with adominant-negative I&kgr;B (S32A/S36A) expression vector had no effect onRef-1-mediated survival under hypoxic conditions. The second aim of this studywas to test the cytoprotective ability of Ref-1 upregulation in response toTNF-induced apoptosis. Ref-1 inhibition of TNF-induced death was associatedwith a significant potentiation of NF-&kgr;B activity. Deletion of theredox-sensitive domain of Ref-1 significantly inhibited TNF-induced NF-&kgr;Bactivation. Moreover, loss of the redox-sensitive domain also abolished theantiapoptotic effect of Ref-1 in response to TNF. To test whether Ref-1induced activation of NF-&kgr;B was necessary to promote survival, we blockedNF-&kgr;B activity with a dominant-negative I&kgr;B (S32A/S36A). Indeed,blockade of NF-&kgr;B activity abolished the ability of Ref-1 to rescueTNF-induced apoptosis. In conclusion, upregulation of Ref-1 promotesendothelial cell survival in response to hypoxia and TNF throughNF-&kgr;B-independent and NF-&kgr;B-dependent signaling cascades,respectively. Moreover, it seems that Ref-1 may act as a critical cofactor,mediating the TNF-induced NF-&kgr;B response in the vascularendothelium.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Therate of spontaneous diastolic depolarization (DD) of sinoatrial nodal cells(SANCs) that triggers recurrent action potentials (APs) is a fundamentalaspect of the heart’s pacemaker. Here, in experiments on isolated SANCs,using confocal microscopy combined with a patch clamp technique, we show thatryanodine receptor Ca2+release during the DDproduces a localized subsarcolemmal Ca2+increase that spreads in a wavelike manner byCa2+-inducedCa2+release and produces an inward currentvia theNa+-Ca2+exchanger (NCX). Ryanodine, a blocker of the sarcoplasmic reticulumCa2+release channel, in a dose-dependentmanner reduces the SANC beating rate with an IC50of2.6 &mgr;mol/L and abolishes the local Ca2+transients that precede the AP upstroke. In voltage-clamped cells in which theDD was simulated by voltage ramp, 3 &mgr;mol/L ryanodine decreased an inwardcurrent during the voltage ramp by 1.6±0.3 pA/pF (SEM, n=4)leaving the peak of L-type Ca2+currentunchanged. Likewise, acute blockade of the NCX (via rapid substitution of bathNa+by Li+)abolished SANC beating and reduced the inward current to a similar extent(1.7±0.4 pA/pF, n=4), as did ryanodine. Thus, in addition toactivation/inactivation of multiple ion channels,Ca2+activation of the NCX, because oflocalized sarcoplasmic reticulum Ca2+release,is a critical element in a chain of molecular interactions that permits theheartbeat to occur and determines its beatingrate.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Wetested whether mitochondria function as the O2sensorunderlying hypoxic pulmonary vasoconstriction (HPV). In buffer-perfused ratlungs, rotenone, myxothiazol, and diphenyleneiodonium, which inhibitmitochondria in the proximal region of the electron transport chain (ETC),abolished HPV without attenuating the response to U46619. Cyanide andantimycin A inhibit electron transfer in the distal region of the ETC, butthey did not abolish HPV. Cultured pulmonary artery (PA) myocytes contract inresponse to hypoxia or to U46619. The hypoxic response was abolished while theresponse to U46619 was maintained in mutant(&rgr;0) PA myocytes lacking a mitochondrial ETC.To test whether reactive oxygen species (ROS) derived from mitochondria act assignaling agents in HPV, the antioxidants pyrrolidinedithiocarbamate andebselen and the Cu,Zn superoxide dismutase inhibitor diethyldithiocarbamatewere used. These abolished HPV without affecting contraction to U46619,suggesting that ROS act as second messengers. In cultured PA myocytes,oxidation of intracellular 2′,7′-dichlorofluorescin diacetate(DCFH) dye increased under 2% O2, indicating thatmyocytes increase their generation ofH2O2during hypoxia. This wasattenuated by myxothiazol, implicating mitochondria as the source of increasedROS during HPV. These results indicate that mitochondrial ATP is not requiredfor HPV, that mitochondria function as O2sensorsduring hypoxia, and that ROS generated in the proximal region of the ETC actas second messengers in theresponse.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Mitochondriacan either enhance or suppress cell death. Cytochromecrelease from mitochondria anddepolarization of the mitochondrial membrane potential (&Dgr;&PSgr;) arecrucial events in triggering apoptosis. In contrast, activation ofmitochondrial ATP-sensitive potassium (mitoKATP)channels prevents lethal ischemic injury in vivo, implicating these channelsas key players in the process of ischemic preconditioning. We probed therelationship between mitoKATPchannels and apoptosis incultured neonatal rat cardiac ventricular myocytes. Incubation with 200&mgr;mol/L hydrogen peroxide induced TUNEL positivity, cytochromectranslocation, caspase-3 activation,poly(ADP-ribose) polymerase cleavage, and dissipation of &Dgr;&PSgr;.Pharmacological opening of mitoKATPchannels bydiazoxide (100 &mgr;mol/L) preserved mitochondrial integrity and suppressedall markers of apoptosis. Diazoxide prevented &Dgr;&PSgr; depolarization in aconcentration-dependent manner (EC50≈40 &mgr;mol/L,with saturation by 100 &mgr;mol/L), as shown by both flow cytometry andquantitative image analysis of cells stained with fluorescent &Dgr;&PSgr;indicators. These cytoprotective effects of diazoxide were reproduced bypinacidil, another mitoKATPagonist, and blocked by themitoKATPchannel antagonist 5-hydroxydecanoate (500&mgr;mol/L). Our findings identify a novel mitochondrial pathway that isprotective against apoptosis. The results also pinpointmitoKATPchannels as logical therapeutic targets indiseases of enhanced apoptosis and oxidativestress.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Itis generally accepted that endothelial cells generate most of their ATP byanaerobic glycolysis and that very little ATP is derived from the oxidation offatty acids or glucose. Previously, we have reported that, in cultured humanumbilical vein endothelial cells (HUVECs), activation of AMP-activated proteinkinase (AMPK) by the cell-permeable activator 5-aminoimidazole-4-carboximideriboside (AICAR) is associated with an increase in the oxidation of3H-palmitate. In the present study, experimentscarried out with cultured HUVECs revealed the following: (1) AICAR-inducedincreases in palmitate oxidation during a 2-hour incubation are associatedwith a decrease in the concentration of malonyl coenzyme A (CoA) (an inhibitorof carnitine palmitoyl transferase 1), which temporally parallels the increasein AMPK activity and a decrease in the activity of acetyl CoA carboxylase(ACC). (2) AICAR does not stimulate either palmitate oxidation when carnitineis omitted from the medium or oxidation of the medium-chain fatty acidoctanoate. (3) When intracellular lipid pools are prelabeled with3H-palmitate, the measured rate of palmitateoxidation is 3-fold higher, and in the presence of AICAR, it accounts fornearly 40% of calculated ATP generation. (4) Incubation of HUVECs in aglucose-free medium for 2 hours causes the same changes in AMPK, ACC, malonylCoA, and palmitate oxidation as does AICAR. (5) Under all conditions studied,the contribution of glucose oxidation to ATP production is minimal. Theresults indicate that the AMPK-ACC-malonyl CoA-carnitine palmitoyl transferase1 mechanism plays a key role in the physiological regulation of fatty acidoxidation in HUVECs. They also indicate that HUVECs oxidize fatty acids fromboth intracellular and extracellular sources, and that when this is taken intoaccount, fatty acids can be a major substrate for ATP generation. Finally,they suggest that AMPK is likely to be a major factor in modulating theresponse of the endothelium to stresses that alter its energystate.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Alterationsof the Ca2+sensitivity of contraction havebeen reported for porcine coronary artery, but the mechanisms are not clearlyunderstood. We investigated the mechanism(s) ofCa2+sensitization in response to thethromboxane A2analogue (U46619). Our hypothesis isthat different mechanisms of Ca2+sensitization could be distinguished by their distinct time courses.Therefore, we measured the time course of[Ca2+]iandisometric force simultaneously in an intact artery after a single addition ofU46619. The initial transient phase was associated withCa2+release from the sarcoplasmic reticulum,whereas the maintained phase was associated withCa2+influx. Two distinct types ofCa2+sensitization characterized these phaseswith either protein kinase C (PKC)-mediated or Rho-kinase-mediated mechanisms.Their effects were quite distinct on the basis of the time courses over whichthe sensitization was effective. PKC inhibition (1 &mgr;mol/L calphostin C)had a much greater effect in the initial phase, diminishing the size of thetransient and prolonging the rise in force and the decline in[Ca2+]i.There were limited effects on the sustained force. Rho-kinase inhibition (10&mgr;mol/L Y27632), in contrast, nearly abolished the sustained force but hada lesser effect on the transient phase. Neither inhibitor had any effect onthe force versus[Ca2+]irelations for KCl contractures. Our evidence suggests that both PKC-mediatedand Rho-kinase-mediated Ca2+sensitizationsare present in coronary arteries, but the latter is dominant in thromboxaneA2receptor-mediatedcontraction.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Diabetesmellitus (DM) is a primary risk factor for cardiovascular disease. Althoughrecent studies have demonstrated an important role for extracellular matrixmetalloproteinases (MMPs) in atherosclerosis, little is known about theeffects of hyperglycemia on MMP regulation in vascular cells. Gelatinzymography and Western blot analysis revealed that the activity and expressionof 92-kDa (MMP-9) gelatinase, but not of 72 kDa (MMP-2) gelatinase, weresignificantly increased in vascular tissue and plasma of two distinct rodentmodels of DM. Bovine aortic endothelial cells (BAECs) grown in culture did notexpress MMP-9 constitutively; however, chronic (2-week) incubation with highglucose medium induced MMP-9 promoter activity, mRNA and protein expression,and gelatinase activity in BAECs. On the other hand, high glucose culture didnot change MMP-9 activity from vascular smooth muscle cells or macrophages.Electron paramagnetic resonance studies indicate that BAECs chronically grownin high glucose conditions produce 70% more ROS than do control cells.Enhanced MMP-9 activity was significantly reduced by treatment with theantioxidants polyethylene glycol-superoxide dismutase andN-acetyl- l -cysteinebut not by inhibitors of protein kinase C. In conclusion, vascular MMP-9activity is increased in DM, in part because of enhanced elaboration fromvascular endothelial cells, and oxidative stress plays an important role. Thisnovel mechanism of redox-sensitive MMP-9 expression by hyperglycemia mayprovide a rationale for antioxidant therapy to modulate diabetic vascularcomplications.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—Adecreased exercise tolerance is a common symptom in patients with congestiveheart failure (CHF). This decrease has been suggested to be partly due toaltered skeletal muscle function. Therefore, we have studied contractilefunction and cytoplasmic free Ca2+concentration([Ca2+]i,measured with the fluorescent dye indo 1) in isolated muscles from rats inwhich CHF was induced by ligation of the left coronary artery. The resultsshow no major changes of the contractile function and[Ca2+]ihandling in unfatigued intact fast-twitch fibers isolated from flexordigitorum brevis muscles of CHF rats, but these fibers were markedly moresusceptible to damage during microdissection. Furthermore, CHF fibersdisplayed a marked increase of baseline[Ca2+]iduring fatigue. Isolated slow-twitch soleus muscles of CHF rats displayedslower twitch contraction and tetanic relaxation than did muscles fromsham-operated rats; the slowing of relaxation became more pronounced duringfatigue in CHF muscles. Immunoblot analyses of sarcoplasmic reticulum proteinsand sarcolemmaNa+,K+-ATPaseshowed no difference in flexor digitorum brevis muscles of sham-operatedversus CHF rats. In conclusion, functional impairments can be observed in limbmuscle isolated from rats with CHF. These impairments seem to mainly involvestructures surrounding the muscle cells and sarcoplasmic reticulumCa2+pumps, the dysfunction of which becomesobvious duringfatigue.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Abstract—Anessential role for protein kinase C &egr; (PKC&egr;) has been shown inmultiple forms of cardioprotection; however, there is a distinct paucity ofinformation concerning the signaling architecture that is responsible for themanifestation of a protective phenotype. We and others have recently shownthat signal transduction may proceed via the formation of signaling complexes(Circ Res. 2001;88:59–62). Inorder to understand if the assembly of multiprotein complexes is the manner bywhich signaling is conducted in cardioprotection, we designed a series ofexperiments to characterize the associations of Src tyrosine kinase withPKC&egr; in a conscious rabbit model of nitric oxide (NO)-induced latepreconditioning. Our data demonstrate that PKC&egr; and Src can formfunctional signaling modules in vitro: PKC&egr; interacts with Src; theassociation with PKC&egr; activates Src; and adult cardiac cells receivingrecombinant adenoviruses encoding PKC&egr; exhibit increased Src activity.Furthermore, our results show that NO-induced late preconditioning involvedPKC&egr;-Src module formation and enhanced the enzymatic activity ofPKC&egr;-associated Src. Inhibition of PKC blocked cardioprotection, moduleformation, and PKC&egr;-associated Src activity, providing direct evidence fora functional role of the PKC&egr;-Src module in the orchestration ofNO-induced cardioprotection in consciousrabbits.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID