摘要:

Aldosterone upregulates the Na+-K+pump in kidney and colon, classical target organs for the hormone. An effect on pump function in the heart is not firmly established. Because the myocardium contains mineralocorticoid receptors, we examined whether aldosterone has an effect on Na+-K+pump function in cardiac myocytes. Myocytes were isolated from rabbits given aldosterone via osmotic minipumps and from controls. Electrogenic Na+-K+pump current, arising from the 3:2 Na+:K+exchange ratio, was measured in single myocytes using the whole-cell patch clamp technique. Treatment with aldosterone induced a decrease in pump current measured when myocytes were dialyzed with patch pipette solution containing Na+in a concentration of 10 mmol/L, whereas there was no effect measured when the solution contained 80 mmol/L Na+. Aldosterone had no effect on myocardial Na+-K+pump concentration evaluated by vanadate-facilitated [3H]ouabain binding or by K+-dependent paranitrophenylphosphatase activity in crude homogenates. Aldosterone induced an increase in intracellular Na+activity. The aldosterone-induced decrease in pump current and increased intracellular Na+were prevented by cotreatment with the mineralocorticoid receptor antagonist spironolactone. Our results indicate that hyperaldosteronemia decreases the apparent Na+affinity of the Na+-K+pump, whereas it has no effect on maximal pump capacity.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

G protein–coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including &agr;1B-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the &bgr;-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type &agr;1BAR. Transgenic mice with cardiac CAM&agr;1BAR overexpression had enhanced myocardial &agr;1AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAM&agr;1BARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAM&agr;1BAR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type &agr;1BAR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type &agr;1BAR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial &agr;1AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo &agr;1AR mitogen–activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with &agr;1BARs in vivo such that GRK3 desensitizes all &agr;1BAR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo &agr;1BAR signaling in the heart.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Myosin binding protein C (MyBP-C) is one of the major sarcomeric proteins involved in the pathophysiology of familial hypertrophic cardiomyopathy (FHC). The cardiac isoform istris-phosphorylated by cAMP-dependent protein kinase (cAPK) on &bgr;-adrenergic stimulation at a conserved N-terminal domain (MyBP-C motif), suggesting a role in regulating positive inotropy mediated by cAPK. Recent data show that the MyBP-C motif binds to a conserved segment of sarcomeric myosin S2 in a phosphorylation-regulated way. Given that most MyBP-C mutations that cause FHC are predicted to result in N-terminal fragments of the protein, we investigated the specific effects of the MyBP-C motif on contractility and its modulation by cAPK phosphorylation. The diffusion of proteins into skinned fibers allows the investigation of effects of defined molecular regions of MyBP-C, because the endogenous MyBP-C is associated with few myosin heads. Furthermore, the effect of phosphorylation of cardiac MyBP-C can be studied in a defined unphosphorylated background in skeletal muscle fibers only. Triton skinned fibers were tested for maximal isometric force, Ca2+/force relation, rigor force, and stiffness in the absence and presence of the recombinant cardiac MyBP-C motif. The presence of unphosphorylated MyBP-C motif resulted in a significant (1) depression of Ca2+-activated maximal force with no effect on dynamic stiffness, (2) increase of the Ca2+sensitivity of active force (leftward shift of the Ca2+/force relation), (3) increase of maximal rigor force, and (4) an acceleration of rigor force and rigor stiffness development.Tris-phosphorylation of the MyBP-C motif by cAPK abolished these effects. This is the first demonstration that the S2 binding domain of MyBP-C is a modulator of contractility. The anchorage of the MyBP-C motif to the myosin filament is not needed for the observed effects, arguing that the mechanism of MyBP-C regulation is at least partly independent of a “tether,” in agreement with a modulation of the head-tail mobility. Soluble fragments occurring in FHC, lacking the spatial specificity, might therefore lead to altered contraction regulation without affecting sarcomere structure directly.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Extension of the I-band segment of titin gives rise to part of the diastolic force of cardiac muscle. Previous studies of human cardiac titin transcripts suggested a series of differential splicing events in the I-band segment of titin leading to the so-called N2A and N2B isoform transcripts. Here we investigated titin expression at the protein level in a wide range of mammalian species. Results indicate that the myocardium coexpresses 2 distinct titin isoforms: a smaller isoform containing the N2B element only (N2B titin) and a larger isoform with both the N2B and N2A elements (N2BA titin). The expression ratio of large N2BA to small N2B titin isoforms was found to vary greatly in different species; eg, in the left ventricle the ratio is ≈0.05 in mouse and ≈1.5 in pig. Differences in the expression ratio were also found between atria and ventricles and between different layers of the ventricular wall. Immunofluorescence experiments with isoform-specific antibodies suggest that coexpression of these isoforms takes place at the single-myocyte level. The diastolic properties of single cardiac myocytes isolated from various species expressing high levels of the small (rat and mouse) or large (pig) titin isoform were studied. On average, pig myocytes are significantly less stiff than mouse and rat myocytes. Gel analysis indicates that this result cannot be explained by varying amounts of titin in mouse and pig myocardium. Rather, low stiffness of pig myocytes can be explained by its high expression level of the large isoform: the longer extensible region of this isoform results in a lower fractional extension for a given sarcomere length and hence a lower force. Implications of our findings to cardiac function are discussed.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Differentiation-inducing factor-1 (DIF-1) is a morphogen that induces differentiation ofDictyostelium. Recently, DIF-1 has been shown to inhibit proliferation and induce differentiation in tumor cells, although the underlying mechanisms remain unknown. In this study, we examined the effects of DIF-1 on the proliferation and differentiation of vascular smooth muscle cells, to explore novel therapeutic strategies for atherosclerosis. DIF-1 nearly completely inhibited DNA synthesis and cell division in mitogen-stimulated cells. DIF-1 inhibited the phosphorylation of the retinoblastoma protein and the activities of cyclin-dependent kinase (Cdk) 4, Cdk6, and Cdk2, which phosphorylate the retinoblastoma protein. DIF-1 strongly suppressed the expression of cyclins D1, D2, and D3, as well as those of cyclins E and A, which normally began after that of the D-type cyclins. The mRNAs for the smooth muscle myosin heavy chains SM1 and SM2 were expressed in quiescent cells in primary culture, and these expression levels decreased after mitogenic stimulation. In the presence of DIF-1, the rate of the reduction was significantly decelerated. Moreover, the addition of DIF-1 to dedifferentiated cells induced the expressions of SM1 and SM2, accompanied by a reduction in the level of SMemb, a nonmuscle-type myosin heavy chain. Therefore, DIF-1 seemed to interrupt a very early stage of G1probably by suppressing the expressions of the D-type cyclins. Furthermore, this compound may prevent phenotypic modulation and induce differentiation of vascular smooth muscle cells.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Apoptosis of arterial smooth muscle cells (ASMCs) could play an important role in the pathogenesis of atherosclerosis and restenosis. Recent studies have demonstrated that extracellular adenosine induces apoptosis in various cell types. Our aim was to delineate the capacity of this nucleoside to induce ASMC apoptosis in arterial diseases. We demonstrate that adenosine dose-dependently triggers apoptosis of cultured human ASMCs. Apoptotic cell death was quantified by analysis of nuclear chromatin morphology and characterized by DNA laddering. The involvement of adenosine receptors was suggested, because neither an adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, nor an inhibitor of cellular nucleoside transport, dipyridamole, was able to inhibit adenosine-induced ASMC apoptosis. In contrast, an A1/A2-adenosine receptor antagonist, xanthine amine congener, totally inhibited adenosine-induced apoptosis. Furthermore, among more selective inhibitors of P1purinoceptor subtypes, only alloxazine, an antagonist of A1- and A2-adenosine receptors, completely inhibited adenosine-induced ASMC apoptosis, suggesting that adenosine triggers ASMC apoptosis via either 1 or both of these receptors. However, 8-cyclopentyl-1,3-dipropylxanthine, 8-(3-chlorostyryl) caffeine, and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate, which are A1-, A2a-, and A3-adenosine receptor antagonists, did not inhibit adenosine-induced apoptosis, suggesting an involvement of the A2b-receptor in this process. Moreover, the cAMP increase followed by cAMP-dependent protein kinase activation appears essential to mediate adenosine-induced ASMC apoptosis, thus confirming the previous hypothesis. These results indicate that adenosine-induced apoptosis of ASMCs is essentially mediated via A2b-adenosine receptor and involves a cAMP-dependent pathway.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The mechanism(s) underlying ventricular fibrillation (VF) remain unclear. We hypothesized that at least some forms of VF are not random and that high-frequency periodic sources of activity manifest themselves as spatiotemporal periodicities, which drive VF. Twenty-four VF episodes from 8 Langendorff-perfused rabbit hearts were studied using high-resolution video imaging in conjunction with ECG recordings and spectral analysis. Sequential wavefronts that activated the ventricles in a spatially and temporally periodic fashion were identified. In addition, we analyzed the lifespan and dynamics of wavelets in VF, using a new method of phase mapping that enables identification of phase singularity points (PSs), which flank individual wavelets. Spatiotemporal periodicity was found in 21 of 24 episodes. Complete reentry on the epicardial surface was observed in 3 of 24 episodes. The cycle length of discrete regions of spatiotemporal periodicity correlated highly with the dominant frequency of the optical pseudo-ECG (R2=0.75) and with the global bipolar electrogram (R2=0.79). The lifespan of PSs was short (14.7±14.4 ms); 98% of PSs existed for <1 rotation. The mean number of waves entering (6.50±0.69) exceeded the mean number of waves that exited our mapping field (4.25±0.56;P<0.05). These results strongly suggest that ongoing stable sources are responsible for the majority of the frequency content of VF and therefore play a role in its maintenance. In this model, multiple wavelets resulting from wavebreaks do not appear to be responsible for the sustenance of this arrhythmia, but are rather the consequence of breakup of high-frequency activation from a dominant reentrant source.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Acetylcholine (ACh) evokes the conduction of vasodilation along resistance microvessels. However, it is not known which cell layer (endothelium or smooth muscle) serves as the conduction pathway. In isolated, cannulated feed arteries (≈70 &mgr;m in diameter at 75 mm Hg; length ≈4 mm) of the hamster retractor muscle, we tested the hypothesis that endothelial cells provide the pathway for conduction. Microiontophoresis of ACh (500 ms, 500 nA) onto the distal end of a feed artery evoked hyperpolarization (−13±2 mV) of both cell layers with vasodilation (15±1 &mgr;m) along the entire vessel. To selectively damage endothelial cells (confirmed by loss of vasodilation to ACh and labeling of disrupted cells with propidium iodide), an air bubble was perfused through a portion of the vessel lumen, or a 70-kDa fluorescein-conjugated dextran (FCD) was illuminated within a segment (300 &mgr;m) of the lumen. After endothelial cell damage, hyperpolarization and vasodilation conducted up to, but not through, the treated segment. To selectively damage smooth muscle cells (confirmed by loss of vasoconstriction to phenylephrine and labeling with propidium iodide), FCD was perifused around the vessel and illuminated. Vasodilation and hyperpolarization conducted past the disrupted smooth muscle cells without attenuation. We conclude that endothelial cells provide the pathway for conducting hyperpolarization and vasodilation along feed arteries in response to ACh.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Hypercholesterolemia may render atherosclerotic plaques prone to rupture. To test this hypothesis, catheters with matrix-covered balloons were implanted into the aorta of rabbits fed standard or 0.5% cholesterol chow (n=70). In 1 month, fibrous plaques developed around the balloon. Time-dependent accumulation of cholesteryl esters and free cholesterol was detected in the plaques of the cholesterol-fed group only. The pressure needed to rupture the plaque by balloon inflation was used as an index of plaque strength. Three months after the catheter implantation, the breaking pressure was 2.1 times lower (P<0.05) in cholesterol-fed rabbits. It was accompanied by collagen loss, as measured by plaque hydroxyproline content, but not with deficiency of collagen cross-linking. Sirius red staining showed preservation of collagen originally covering the balloon and accumulation of nascent collagen in the lesions of standard chow-fed rabbits. In the cholesterol-fed group, both mature and new collagen underwent degradation predominantly in the plaque shoulders. Collagen breakdown was associated with local accumulation of foamy macrophages. Gel zymography demonstrated relative enhancement of gelatinolytic activity at 92 and 72 kDa, as well as caseinolytic activity at 57, 45, and 19 kDa in the lipid-laden plaques. Lipid accumulation in the plaque was also associated with a loss of smooth muscle cells, the cellular source of the collagen fibers. The remaining smooth muscle cells showed increased collagen synthesis, although it was insufficient to counterbalance collagen degradation and cell loss. Thus, we have obtained direct evidence that hypercholesterolemia is accompanied by enhanced local collagen degradation, which is potentially responsible for plaque weakening.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID