ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Connexin43(Cx43) channels can be regulated by a variety of factors, including low pHi. Structure/function studies from this laboratory have demonstrated that pH gating follows a particle-receptor mechanism, similar to the "ball-and-chain" model of voltage-dependent inactivation of ion channels. The question whether the particle-receptor model is applicable only to pH gating or to other forms of Cx43 regulation as well remains. To address this question, we looked at the uncoupling effects of insulin and of insulin-like growth factor-1 (IGF) on Cx43 channels expressed in Xenopus oocytes. These agonists do not induce changes in pHi. Junctional conductance (Gj) was measured by the dual 2-electrode voltage-clamp technique. Control studies showed that relative Gj did not change spontaneously as a function of time. Continuous exposure of Cx43-expressing oocytes to insulin (10 [micro sign]/L) led to a decrease in Gj. After 80 minutes, Gj was 54 +/- 5% from control (n=12). Exposure of oocytes to IGF (10 nmol/L) caused an even more pronounced change in Gj (37 +/- 4% of control, n=6). The time course of the IGF-induced uncoupling was similar to that observed after insulin exposure. The effect of insulin was abolished by truncation of the carboxyl-terminal domain of Cx43 at amino acid 257 (M257). Interestingly, as in the case of pH gating, coexpression of the carboxyl-terminal domain (amino acids 258 to 282) together with M257 rescued the ability of insulin to reduce coupling (Gj, 39 +/- 12% from control; n=6). Structure/function experiments using various deletion mutants of the carboxyl-terminal domain showed that insulin treatment does not modify Gj if amino acids 261 to 280 are missing from the Cx43 sequence. Our results suggest that a particle-receptor (or ball-and-chain) mechanism, similar to that described for pH gating, also applies to chemical regulation of Cx43 by other factors. (Circ Res. 1998;83:27-32.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The whole-cell patch-clamp technique was used to monitor the effects of genistein, a tyrosine kinase inhibitor, on membrane currents recorded from isolated guinea pig ventricular myocytes. Under control conditions, genistein (50 [micro sign]mol/L) did not activate the latent cAMP-regulated Cl-current (ICl). However, in the presence of a subthreshold concentration (1 nmol/L) of the beta-adrenergic agonist isoproterenol (Iso), genistein caused a near-maximal activation of this current. In the absence of genistein, Iso activated IClwith an EC50of 5 nmol/L. In the presence of genistein, Iso activated IClwith an EC50of 0.3 nmol/L. This facilitatory effect was not observed in the presence of daidzein (50 [micro sign]mol/L), an analogue of genistein that only weakly inhibits tyrosine kinase activity. Furthermore, peroxovanadate, a potent inhibitor of phosphotyrosine phosphatase activity, inhibited IClactivated by Iso alone, and it blocked the stimulatory effect of genistein in the presence of Iso. To determine whether the stimulatory effect of genistein was specific for ICl, we also studied its action on the cAMP-regulated delayed rectifier K+current (IK) and L-type Ca2+current (ICa-L) present in these cells. Basal IKand ICa-Lwere partially ([=approximate]30% to 40%) inhibited by genistein. However, this inhibitory effect was mimicked by daidzein, suggesting that inhibition of tyrosine kinase activity is not involved. In addition to the nonspecific inhibitory effect, genistein also caused a significant increase in the beta-adrenergic sensitivity of the unblocked cationic currents. In the absence of genistein, 1 nmol/L Iso had no effect on either IKor ICa-L. However, in the presence of genistein, 1 nmol/L Iso significantly increased the magnitude of both currents. These results suggest that tyrosine kinase activity may play an important role in regulating beta-adrenergic responsiveness of the heart. (Circ Res. 1998;83:33-42.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

alpha1-Adrenergicreceptor stimulation induces cardiac myocytes to hypertrophy and reactivates many fetal genes, including beta-myosin heavy chain (beta MyHC) and skeletal alpha-actin (SKA), by signaling through myocyte-specific CAT (M-CAT) cis elements, binding sites of the transcriptional enhancer factor-1 (TEF-1) family of transcription factors. To examine functional differences between TEF-1 and related to TEF-1 (RTEF-1) in alpha1-adrenergicreactivation of the fetal program, expression constructs were cotransfected with beta MyHC and SKA promoter/reporter constructs in neonatal rat cardiac myocytes. TEF-1 overexpression tended to transactivate a minimal beta MyHC promoter but significantly interfered with a minimal SKA promoter. In contrast, RTEF-1 transactivated both the minimal beta MyHC and SKA promoters. TEF-1 and RTEF-1 also affected the alpha1-adrenergicresponse of the beta MyHC and SKA promoters differently. TEF-1 had no effect. In contrast, RTEF-1 potentiated the alpha1-adrenergicresponses of the SKA promoter and of a -3.3-kb beta MyHC promoter. To determine why the promoters responded differently to TEF-1 and RTEF-1, promoters with mutated M-CAT elements were tested in the same way. The beta MyHC promoter required an intact M-CAT element to respond to TEF-1 and RTEF-1, whereas the SKA promoter M-CAT was required for the TEF-1 response but not for the RTEF-1 response, suggesting that SKA promoter-specific cofactors may be involved. By competition gel shift assay, the M-CAT of the minimal beta MyHC promoter had a lower affinity than that of the SKA promoter, which partly explains the different responses of these promoters to TEF-1. These results highlight functional differences between TEF-1 and RTEF-1 and suggest a novel function of RTEF-1 in mediating the alpha1-adrenergicresponse in hypertrophic cardiac myocytes. (Circ Res. 1998;83:43-49.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

A growing body of evidence has been accumulated recently suggesting that growth hormone (GH) and insulin-like growth factor-1 (IGF-1) affect cardiac function, but their mechanism(s) of action is unclear. In the present study, GH and IGF-1 were administered to isolated isovolumic aequorin-loaded rat whole hearts and ferret papillary muscles. Although GH had no effect on the indices of cardiac function, IGF-1 increased isovolumic developed pressure by 24% above baseline. The aequorin transients were abbreviated and demonstrated decreased amplitude. The positive inotropic effects of IGF-1 were not associated with increased intracellular Ca2+availability to the contractile machinery but to a significant increase of myofilament Ca2+sensitivity. Accordingly, the Ca2+-forcerelationship obtained under steady-state conditions in tetanized muscle was shifted significantly to the left (EC50, 0.44 +/- 0.02 versus 0.52 +/- 0.03 [micro sign]mol/L with and without IGF-1 in the perfusate, respectively; P<0.05); maximal Ca2+-activatedtetanic pressure was increased significantly by 12% (211 +/- 3 versus 235 +/- 2 mm Hg in controls and IGF-1-treated hearts, respectively; P<0.01). The positive inotropic actions of IGF-1 were not associated with changes in either pHior high-energy phosphate content, as assessed by31P nuclear magnetic resonance spectroscopy, and were blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. Concomitant administration of IGF binding protein-3 blocked IGF-1-positive inotropic action in ferret papillary muscles. In conclusion, IGF-1 is an endogenous peptide that through a wortmannin-sensitive pathway displays distinct positive inotropic properties by sensitizing the myofilaments to Ca2+without increasing myocyte [Ca2+]i. (Circ Res. 1998;83:50-59.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

Cardiac myofilaments contain proteins that regulate the interaction between actin and myosin. In the thick filament, there are several proteins that may contribute to the regulation of the contraction. The myosin binding protein C, or C protein, has 4 sites that can be phosphorylated by a Ca2+-calmodulin-controlledkinase, protein kinase A or protein kinase C. Using electron microscopy and optical diffraction, we examined the structure of thick filaments isolated from rat ventricles with either the alpha or beta isoform of myosin heavy chain (MHC) and the effect of specific phosphorylation of C protein on the structure. In thick filaments with alpha-MHC, crossbridges were clearly visible. Phosphorylation of C protein by protein kinase A extended the crossbridges from the backbone of the filament, changed their orientation, increased the degree of order of the crossbridges, and decreased the flexibility of the crossbridges. Crossbridges in filaments with beta-MHC were less ordered and apparently more flexible. Phosphorylation of C protein in beta-MHC-containing filaments did not extend the crossbridges and did not alter degree of order or flexibility. The relative flexibility of the crossbridges inferred from the optical diffraction pattern correlated well with the rate of ATP hydrolysis by actomyosin. These results suggest that (1) crossbridge flexibility is an important parameter in setting the rate of crossbridge cycling, and (2) C protein-mediated control of the position and flexibility of crossbridges may regulate actomyosin ATPase activity by modifying the kinetics of crossbridge cycling. (Circ Res. 1998;83:60-72.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID

摘要:

The goal of this study was to test the hypothesis that the cardioprotective effects of the late phase of ischemic preconditioning (PC) can be mimicked by treatment with NO donors. In phase I (studies of myocardial stunning), conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days (days 1, 2, and 3). In group I (controls, n=6), the total deficit of systolic wall thickening (WTh) after the sixth reperfusion was reduced by 54% on days 2 and 3 compared with day 1 (P<0.05), indicating a late PC effect against myocardial stunning. When rabbits were given the NO donors diethylenetriamine/NO (DETA/NO, 0.1 mg/kg IV, 4 times [group II, n=5]) or S-nitroso-N-acetylpenicillamine (SNAP, 2.5 [micro sign]g [center dot] kg-1[center dot] min-1IV for 75 minutes [group III, n=5]) 24 hours before the first sequence of occlusion/reperfusion cycles, the deficit of WTh on day 1 was 60% (group II) and 54% (group III) less than that observed in controls (P<0.05 for both). In both groups II and III, there was no further improvement in the deficit of WTh on days 2 and 3 compared with day 1. The protective effect of DETA/NO was completely abrogated when this agent was given in conjunction with the ONOO-and [center dot]OH scavenger mercaptopropionyl glycine (MPG) (group IV, n=5). In phase II (studies of myocardial infarction), conscious rabbits underwent a 30-minute coronary occlusion followed by 3 days of reperfusion. When rabbits were preconditioned 24 hours earlier with six 4-minute occlusion/4-minute reperfusion cycles, infarct size was reduced by 43% (33.2 +/- 2.7% versus 58.3 +/- 4.1% of the region at risk in controls, P<0.05), indicating a late PC effect against myocardial infarction. When rabbits were pretreated with DETA/NO (group VII, n=8) or SNAP (group IX, n=7) 24 hours before the 30-minute occlusion, infarct size was reduced by a similar degree (29.3 +/- 3.6% and 32.0 +/- 3.3% of the region at risk, respectively; P<0.05 versus controls). The degree of protection could not be increased by doubling the dose of DETA/NO (group VIII, n=5). Coadministration of MPG completely abrogated the infarct-sparing action of DETA/NO (group X, n=7). Taken together, these results demonstrate that in conscious rabbits the administration of 2 structurally unrelated NO donors induces protection 24 hours later against both reversible (stunning) and irreversible (infarction) ischemia/reperfusion injury and that the magnitude of this protection is indistinguishable from that observed during the late phase of ischemic PC. The fact that the late phase of ischemic PC can be mimicked by NO donors provides direct evidence that NO in itself is sufficient to elicit this cardioprotective mechanism. The fact that NO donor-induced late PC was abrogated by MPG indicates that the mechanism whereby NO induces this phenomenon involves the generation of oxidant species, possibly ONOO-and/or [center dot]OH. Since a relatively brief treatment with hemodynamically inactive doses of NO donors can induce long-lasting protective effects, these agents could be useful for preconditioning the heart in patients. (Circ Res. 1998;83:73-84.)

ISSN:0009-7330    

出版商:OVID    

年代:1998

数据来源: OVID