摘要:

DiGeorge syndrome (DGS) is a congenital disease characterized by defects in organs and tissues that depend on contributions by cell populations derived from neural crest for proper development. A number of candidate genes that lie within the q11 region of chromosome 22 commonly deleted in DGS patients have been identified. Orthologues of the DGS candidate gene HIRA are expressed in the neural crest and in neural crest-derived tissues in both chick and mouse embryos. By exposing a portion of the premigratory chick neural crest to phosphorothioate end-protected antisense oligonucleotides, ex ovo, followed by orthotopic backtransplantation to the untreated embryos, we have shown that the functional attenuation of cHIRA in the chick cardiac neural crest results in a significantly increased incidence of persistent truncus arteriosus, a phenotypic change characteristic of DGS, but does not affect the repatterning aortic arch arteries, the ventricular function, or the alignment of the outflow tract. (Circ Res. 1999;84:127-135.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

beta-Adrenergic modulation of the L-type Ca2+current (ICaL) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37[degree sign]C. Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bodies grown for 7 days (7d). ICaLwas insensitive to isoproterenol, forskolin, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocytes (from 7+1d to 7+2d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-gamma-S. At this stage, basal ICaLwas stimulated by the nonselective phosphodiesterase (PDE) inhibitor isobutylmethylxanthine, by PDE inhibitors selective for the PDE II, III, and IV isoforms, as well as by the phosphatase inhibitor okadaic acid. Stimulation of ICaLby the catalytic subunit of the cAMP-dependent protein kinase A (PKA) was found to be similar (about 3 times) throughout development and in adult mouse ventricular cardiomyocytes, indicating that no structural changes of the Ca2+channel related to phosphorylation occurred during development. ICaLwas stimulated by isoproterenol in the presence of a PKA inhibitor and GTP-gamma-S in LDS but not VEDS cardiomyocytes, suggesting the development of a membrane-delimited stimulatory pathway mediated through the stimulatory GTP binding protein, Gs. We conclude that uncoupling and/or low expression of Gsprotein accounted for the ICaLinsensitivity to beta-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 7+4d stage, the reduced beta-adrenergic response is due, at least in part, to high intrinsic PDE and phosphatase activities. (Circ Res. 1999;84:136-145.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The minK gene encodes a 129-amino acid peptide the expression of which modulates function of cardiac delayed rectifier currents (IKrand IKs), and mutations in minK are now recognized as one cause of the congenital long-QT syndrome. We have generated minK-deficient mice in which the bacterial lacZ gene has been substituted for the minK coding region such that beta-galactosidase expression is controlled by endogenous minK regulatory elements. In cardiac myocytes isolated from wild-type neonatal mice, IKsis rarely recorded, while IKris common. In minK (-/-) myocytes, IKsis absent and IKris significantly reduced and its deactivation slowed; these results further support a role for minK in modulating both IKsand IKr. Despite these changes, ECGs in (+/+) and (-/-) animals are no different at adult and at neonatal stages. ECG responses to isoproterenol are also similar in the 2 groups. beta-Galactosidase staining in postnatal minK (-/-) hearts is highly restricted, to the sinus-node region, caudal atrial septum, and proximal conducting system. Moreover, as early as embryonal day 11, segmentally restricted beta-galactosidase expression is observed in the portions of the sinoatrial and atrioventricular junctions that are thought to give rise to the conducting system, thereby implicating minK expression as an early event in conduction system development. More generally, the restricted nature of minK expression in the mouse heart suggests species-specific roles of this gene product in mediating the electrophysiological properties of the heart. (Circ Res. 1999;84:146-152.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Anodal stimulation by external pacemakers has been explained on the basis of bidomain models of cardiac tissue. Bidomain models predict that anodal stimuli will hyperpolarize the underlying tissue while adjacent regions become depolarized (virtual cathodes), initiating excitation. We investigated the contribution of active cellular properties to anode-break stimulation. A bidomain model was implemented in which each cell contained realistic ionic currents, including those recruited by hyperpolarization. Simulations reveal that anode-break excitation can originate at the site of stimulation itself and not only from adjacent regions of induced depolarization. The threshold for initiating excitation at the site of stimulation is lower than that for stimulation initiating from adjacent depolarized regions. Thus, incorporation of active cellular properties into a bidomain model predicts a novel mechanism for anode-break stimulation of the heart. The results will improve our understanding of anodal pacing and its risks and benefits in patients. (Circ Res. 1999;84:153-156.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The hypothesis that cellular hypertrophy in congestive heart failure (CHF) modulates mechanosensitive (ie, swelling- or stretch-activated) anion channels was tested. Digital video microscopy and amphotericin-perforated-patch voltage clamp were used to measure cell volume and ion currents in ventricular myocytes isolated from normal dogs and dogs with rapid ventricular pacing-induced CHF. In normal myocytes, osmotic swelling in 0.9T to 0.6T solution (T, relative osmolarity; isosmotic solution, 296 mOsmol/L) was required to elicit ICl,swell, an outwardly rectifying swelling-activated Cl-current that reversed near -33 mV and was inhibited by 1 mmol/L 9-anthracene carboxylic acid (9AC), an anion channel blocker. Block of ICl,swell by 9AC simultaneously increased the volume of normal cells in hyposmotic solutions by up to 7%, but 9AC had no effect on volume in isosmotic or hyperosmotic solutions. In contrast, ICl,swell was persistently activated under isosmotic conditions in CHF myocytes, and 9AC increased cell volume by 9%. Osmotic shrinkage in 1.1T to 1.5T solution inhibited both ICl,swell and 9AC-induced cell swelling in CHF cells, whereas osmotic swelling only slightly increased ICl,swell. The current density for fully activated 9AC-sensitive ICl,swell was 40% greater in CHF than normal myocytes. In both groups, 9AC-sensitive current and 9AC-induced cell swelling were proportional with changes in osmolarity and 9AC concentration, and the effects of 9AC on current and volume were blocked by replacing bath Cl-with methanesulfonate. CHF thus altered the set point and magnitude of ICl,swell and resulted in its persistent activation. We previously observed analogous regulation of mechanosensitive cation channels in the same CHF model. Mechanosensitive anion and cation channels may contribute to the electrophysiological and contractile derangements in CHF and may be novel targets for therapy. (Circ Res. 1999;84:157-165.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Calcification of vascular tissue is a common complication in aging, atherosclerosis, diabetes, renal failure, aortic stenosis, and prosthetic valve replacement. Osteopontin is a noncollagenous adhesive protein routinely found at sites of dystrophic calcification and synthesized at high levels by macrophages in calcified aortic valves and atherosclerotic plaques. In the present study, we have characterized the calcification of bovine aortic smooth muscle cell (BASMC) cultures in vitro and have studied the effects of exogenous osteopontin on mineral deposition. Induction of calcification in BASMC cultures was alkaline phosphatase-dependent and was characterized by a multilayer cell morphology. Mineral deposition occurred in the basal matrix of multilayered areas as indicated by von Kossa staining, and transmission electron microscopy and electron diffraction identified the mineral as apatite. Ultrastructural analysis of the cultures showed the presence of extracellular matrix vesicles, calcifying collagen fibrils, and nodular-type calcifications similar to those found in calcified heart valves and atherosclerotic plaques. Purified osteopontin (0.05 to 5 [micro sign]g/mL) dose dependently inhibited calcification of BASMC cultures, whereas vitronectin and fibronectin had no effect. In contrast to the inhibitory mechanism of levamisole on mineral deposition, osteopontin did not inhibit alkaline phosphatase activity or reduce phosphorus levels in the culture medium. Addition of calcium to the cultures overcame the inhibitory effect of osteopontin on BASMC culture calcification and resulted in decreased levels of calcium in the culture medium and increased levels in the cell layer. Moreover, using high-resolution, colloidal-gold immunocytochemistry, osteopontin was found intimately associated with growing apatite crystals. These data indicate that the effect of osteopontin, although calcium-dependent, was not mediated by simple calcium chelation but most likely by direct interaction of osteopontin with crystal surfaces. These studies suggest that BASMCs can be used to model vascular calcification in vitro and that soluble osteopontin released near sites of vascular calcification may represent an adaptive mechanism aimed at preventing vascular calcification. (Circ Res. 1999;84:166-178.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Many studies using small-animal models suggest that angiotensin II (Ang II) plays an important role in neointimal formation after vascular injury. In the present study, we examined whether Ang II type 1 receptor (AT1)-mediated Ang II signaling is indispensable for the development of injury-induced neointimal formation using AT1aknockout (KO) mice. Reverse transcriptase-polymerase chain reaction analysis revealed that AT1mRNA was not detectable in both uninjured and injured carotid arteries of KO mice, whereas the AT1gene was expressed in uninjured carotid arteries of wild-type (WT) mice. At 14 days after injury, AT (1) mRNA levels were increased by 1.5-fold in injured arteries of WT mice. Although AT2mRNA was not detectable in uninjured arteries, expression of AT2gene was induced in both animal groups at 2 weeks after injury. Vascular injury induced neointimal formation in KO mice as well as in WT mice. There were no significant differences between WT and KO mice in the extent of histological findings such as increased cross-sectional areas of the neointima and the media, the number of proliferating smooth muscle cells, and the amount of collagen and fibronectin. Treatment with subpressor doses of Ang II after injury enhanced the growth of neointima in WT mice but not in KO mice. Moreover, treatment with the selective AT1antagonist CV-11974 before injury significantly decreased the formation of neointima in only WT mice, whereas treatment with the selective AT2antagonist PD-123319 before injury had no effects in both animal groups. These results suggest that AT1-mediatedAng II signaling is not essential for the development of neointimal formation, although it may modify it. (Circ Res. 1999;84:179-185.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

This study aimed to characterize the interaction between nitric oxide (NO)- and cAMP-related pathways in the control of renal blood flow. Using the isolated perfused rat kidney model, we determined the effects of inhibition of NO formation by Nomega-nitro-L-argininemethyl ester (L-NAME; 1 mmol/L) and of NO administration by sodium nitroprusside (SNP, 10 [micro sign]mol/L) on renal vascular resistance under conditions of elevated vascular cAMP levels. cAMP levels were increased either by adenylate cyclase activation via isoproterenol or by inhibition of cAMP phosphodiesterases (PDEs) 1, 3, and 4. We found that L-NAME markedly increased vascular resistance and that this effect was completely reversed by SNP. Both isoproterenol and inhibitors of the cAMP PDEs lowered basal vascular resistance. In the presence of isoproterenol (3 nmol/L) and inhibitors of PDE-1 [8-methoxymethyl-1-methyl-3-(2-methylpropyl)-xanthine; 8-MM-IBMX, 20 [micro sign]mol/L] and PDE-4 (rolipram, 20 [micro sign]mol/L), L-NAME again substantially increased vascular resistance, and this effect of L-NAME was completely reversed by SNP. In the presence of the PDE-3 inhibitors milrinone (20 [micro sign]mol/L) and trequinsin (200 nmol/L), however, both L-NAME and SNP failed to exert any additional effects. Because PDE-3 is a cGMP-inhibited cAMP PDE and because the vasodilatory effect of SNP was abrogated by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) (20 [micro sign]mol/L), our findings are compatible with the idea that an action of NO on PDE-3 could account for the vasodilatory properties of NO on the renal vasculature. Moreover, our findings suggest that PDE-3 activity is an important determinant of renal vascular resistance. (Circ Res. 1999;84:186-192.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Prostacyclin (prostaglandin I2[PGI2]) is a key mediator of pulmonary vascular function during early postnatal life, and its production in the pulmonary vasculature rises markedly during that period because of increasing expression of cyclooxygenase type 1 (COX-1). The postnatal rise in COX-1 may be due to the release of inhibition by glucocorticoids, since plasma glucocorticoid levels fall after birth and glucocorticoids decrease PGI2synthesis in certain nonpulmonary cell types. We therefore studied the direct effects of dexamethasone (DEX) on COX-1 expression in early-passage ovine fetal pulmonary-artery endothelial cells (PAECs). DEX (10-10to 10-6mol/L) caused a dose-related decrease in COX-1 mRNA expression that was evident by 24 hours, was maximal at 10-6mol/L (50% inhibition), and was not due to changes in mRNA stability. There was a parallel decline in COX-1 protein expression. COX-1 protein rose following DEX withdrawal, and DEX blunted the stimulatory effect of 17 beta-estradiol on COX-1 expression. DEX alone (10-8mol/L for 48 hours) caused a 93% fall in basal PGI2production, and bradykinin- and A23187-stimulated PGI2were diminished 96% and 94%, respectively. Similarly, PGI2synthesis from arachidonic acid fell 86% with DEX; all of the above effects are consistent with COX-1 downregulation. The glucocorticoid receptor (GR) antagonist mifepristone (RU-486; 10-6mol/L) blocked the inhibitory effect of DEX, and GR expression was evident by immunoblot analysis. These findings indicate that glucocorticoids downregulate COX-1 expression and PGI2synthesis in fetal PAECs through the activation of PAEC GR and effects on COX-1 gene transcription. This mechanism may modulate pulmonary PGI2production in the perinatal period, and it may also play a role in the effects of glucocorticoids on the systemic circulation at a variety of ages. (Circ Res. 1999;84:193-200.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Nitric oxide (NO) is a potent inhibitor of thrombin-induced increase in cytoplasmic free Ca2+concentration and aggregation in platelets, but the precise mechanism of this inhibition is unclear. To measure Ca2+/Mn2+influx in intact platelets and to monitor Ca2+uptake into the stores in permeabilized platelets, fura-2 was used. In intact platelets, maximal capacitative Ca2+and Mn2+influx developed rapidly (within 30 s) after fast release of Ca2+from the stores with thrombin (0.5 U/mL) or slowly (within 5 to 10 minutes) following passive Ca2+leak caused by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase(SERCA) with 30 [micro sign]mol/L 2,5-di-(tert-butyl)-1,4-benzohydroquinone (BHQ). NO (1 [micro sign]mol/L) inhibited capacitative Ca2+and Mn2+influx independently of the time after thrombin application. In contrast, the effect of NO on BHQ-induced Ca2+and Mn2+influx was observed only during the first few minutes after BHQ application and completely disappeared when capacitative cation influx reached its maximum. In Ca2+-freemedium, NO reduced the peak Ca2+rise caused by thrombin and significantly promoted Ca2+back-sequestration into the stores. Both effects disappeared in the presence of BHQ. Inhibition of guanylate cyclase with H-(1,2,4) oxadiazolo(4,3-a) quinoxallin-1-one (10 [micro sign]mol/L) attenuated but did not prevent the effects of NO on cytoplasmic free Ca2+concentration. Inhibition of Ca2+uptake by mitochondria did not change the effects of NO. In permeabilized platelets, NO accelerated back-sequestration of Ca2+into the stores after inositol-1,4,5-trisphosphate-induced Ca2+release or after addition of Ca2+(1 [micro sign]mol/L) in the absence of inositol-1,4,5-trisphosphate. The effect of NO depended on the initial rate of Ca2+uptake and on the concentration of ATP and was abolished by BHQ, indicating the direct involvement of SERCA. These data strongly support the hypothesis that NO inhibits store-operated cation influx in human platelets indirectly via acceleration of SERCA-dependent refilling of Ca (2+) stores. (Circ Res. 1999;84:201-209.)

ISSN:0009-7330    

出版商:OVID    

年代:1999

数据来源: OVID