摘要:

Gap junctions are formed by oligomerization of a protein called connexin. Most cells express more than one connexin isotype. Atrial myocytes, for example, coexpress connexin (Cx) 40 and Cx43. The consequence of connexin coexpression on the regulation of gap junctions is not well understood. In the present study, we show that cells coexpressing Cx40 and Cx43 are more susceptible to acidification-induced uncoupling than those cells expressing only one connexin isotype.Xenopusoocytes were injected with mRNA for Cx40, Cx43, or a combination of both. Intracellular pH and junctional conductance were simultaneously measured while cells were progressively acidified by superfusion with a bicarbonate-buffered solution gassed with increasing concentrations of carbon dioxide. The data show that the pKa (ie, the pH at which junctional conductance decreased to 50% from maximum) shifted from ≈6.7 when cells expressed only Cx40 or only Cx43 to ≈7.0 when one of the oocytes was coexpressing both connexins. Truncation of the carboxyl terminal domains of the connexins caused the loss of pH sensitivity even after coexpression. The data are interpreted on the basis of previous studies from our laboratory that demonstrated heterodomain interactions in the regulation of Cx40 and Cx43 gap junctions. The possible implications of these findings on the regulation of native gap junctions that express both connexins remain to be determined. The full text of this article is available at http://www.circresaha.org.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

The small (21 kDa) guanine nucleotide-binding protein (small G protein) superfamily comprises 5 subfamilies (Ras, Rho, ADP ribosylation factors [ARFs], Rab, and Ran) that act as molecular switches to regulate numerous cellular responses. Cardiac myocyte hypertrophy is associated with cell growth and changes in the cytoskeleton and myofibrillar apparatus. In other cells, the Ras subfamily regulates cell growth whereas the Rho subfamily (RhoA, Rac1, and Cdc42) regulates cell morphology. Thus, the involvement of small G proteins in hypertrophy has become an area of significant interest. Hearts from transgenic mice expressing activated Ras develop features consistent with hypertrophy, whereas mice overexpressing RhoA develop lethal heart failure. In isolated neonatal rat cardiac myocytes, transfection or infection with activated Ras, RhoA, or Rac1 induces many of the features of hypertrophy. We discuss the mechanisms of activation of the small G proteins and the downstream signaling pathways involved. The latter may include protein kinases, particularly the mitogen-activated or Rho-activated protein kinases. We conclude that although there is significant evidence implicating Ras, RhoA, and Rac1 in hypertrophy, the mechanisms are not fully understood.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Transforming growth factor-&bgr;1(TGF-&bgr;1) is expressed in the adult and embryonic vasculature; however, the biological consequences of increased vascular TGF-&bgr;1expression remain controversial. To establish an experimental setting for investigating the role of increased TGF-&bgr;1in vascular development and disease, we generated transgenic mice in which a cDNA encoding a constitutively active form of TGF-&bgr;1is expressed from the SM22&agr; promoter. This promoter fragment directs transgene expression to smooth muscle cells of large arteries in late-term embryos and postnatal mice. We confirmed the anticipated pattern of SM22&agr;-directed transgene expression (heart, somites, and vasculature of the embryo and yolk sac) in embryos carrying an SM22&agr;–&bgr;-galactosidase transgene. SM22&agr;– &bgr;-galactosidase transgenic mice were born at the expected frequency (13%); however, nearly all SM22&agr;–TGF-&bgr;1transgenic mice died before E11.5. SM22&agr;–TGF-&bgr;1transgenic embryos identified at E8.5 to E10.5 had growth retardation and both gross and microscopic abnormalities of the yolk sac vasculature. Overexpression of TGF-&bgr;1from the SM22&agr; promoter is lethal at E8.5 to E10.5, most likely because of yolk sac insufficiency. Investigation of the consequences of increased vascular TGF-&bgr;1expression in adults may require a conditional transgenic approach. Moreover, because the SM22&agr; promoter drives transgene expression in the yolk sac vasculature at a time when embryonic survival is dependent on yolk sac function, use of the SM22&agr; promoter to drive expression of “vasculoactive” transgenes may be particularly likely to cause embryonic death.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

This study aimed to examine molecular mechanisms for endotoxin-induced adhesive changes in platelets in vivo. Platelets labeled with carboxyfluorescein diacetate succinimidyl ester were visualized in rat mesenteric venules through intravital microscopy assisted by a high-speed fluorescence video imager at 1000 frames per second or by a normal-speed intensifier under monitoring of erythrocyte velocity. Leukocyte rolling was examined by normal-speed transmission video images. The velocity of platelets traveling along the centerline of venules followed that of erythrocytes, whereas that measured at the periendothelial space was significantly smaller than the erythrocyte velocity; a majority of these cells exhibited transient but notable rolling with endothelium. Administration of endotoxin increased the density of periendothelial platelets and reduced the rolling velocities of platelets and leukocytes in venules: All events were attenuated by anti–rat P-selectin monoclonal antibody s789G or by anti–human glycoprotein (GP) Ib&agr; monoclonal antibody GUR83/35, which blocks ristocetin-induced aggregation of rat platelets. Isolated rat platelets injected into endotoxin-pretreated rats were able to roll on the venules. This event was attenuated by pretreatment of platelets in vitro with GUR83/35 but not with s789G, suggesting involvement of endothelial P-selectin and platelet GP Ib&agr; in the endotoxin-induced responses. Furthermore, isolated human platelets showed similar rolling interactions with endotoxin-preexposed rat venules, and pretreatment of the platelets with GUR83/35, but not with s789G, significantly reduced such interactions. Our results provide the first evidence for involvement of GP Ib&agr; in endotoxin-induced microvascular rolling of platelets and leukocytes, and this system serves as a potentially useful tool to examine GP Ib&agr;–associated function of human platelets in vivo.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1±2.3% viable, n=4,P=NS) and Fas-sensitive (31.3±2.6% viable, n=3,P<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Genetically altered mouse models constitute unique systems to delineate the role of adrenergic receptor (AR) signaling mechanisms as modulators of cardiomyocyte function. The interpretation of results from these models depends on knowledge of the signaling properties of endogenous ARs in mouse cardiomyocytes. In the present study, we identify for the first time several defects in AR signaling in cardiomyocytes cultured from mouse ventricles. &bgr;1-ARs induce robust increases in cAMP accumulation and the amplitude of the calcium and cell motion transients in mouse cardiomyocytes. Selective &bgr;2-AR stimulation increases the amplitude of calcium and motion transients, with only a trivial rise in cAMP accumulation in comparison. &bgr;2-AR responses are not influenced by pertussis toxin in cultured mouse cardiomyocytes. &agr;1-ARs fail to activate phospholipase C, the extracellular signal–regulated protein kinase, p38-MAPK, or stimulate hypertrophy in mouse cardiomyocytes. Control experiments establish that this is not due to a lesion in distal elements in the signaling machinery, because these responses are induced by protease-activated receptor-1 agonists and phospholipase C is activated byPasteurella multocidatoxin (a Gq&agr;-subunit agonist). Surprisingly, norepinephrine activates p38-MAPK via &bgr;-ARs in mouse cardiomyocytes, but &bgr;-AR activation of p38-MAPK alone is not sufficient to induce cardiomyocyte hypertrophy. Collectively, these results identify a generalized defect in &agr;1-AR signaling and a defect in &bgr;2-AR linkage to cAMP (although not to an inotropic response) in cultured mouse cardiomyocytes. These naturally occurring vagaries in AR signaling in mouse cardiomyocytes provide informative insights into the requirements for hypertrophic signaling and impact on the value of mouse cardiomyocytes as a reconstitution system to investigate AR signaling in the heart.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID

摘要:

Previous studies have established that cardiomyocytes express protease-activated receptor (PAR)-1, a high-affinity receptor for thrombin, which is also activated by the tethered-ligand domain sequence (SFLLRN) and which promotes inositol trisphosphate accumulation, stimulates extracellular signal–regulated protein kinase, and modulates contractile function. A single previous report identified PAR-1 as a hypertrophic stimulus, but there have been no subsequent investigations of the mechanism. This study reveals the coexpression of PAR-1 and PAR-2 (a second PAR, which is activated by trypsin/tryptase but not thrombin) by Northern blot analysis and compares their signaling properties in neonatal rat ventricular cardiomyocytes. SFLLRN and SLIGRL (an agonist peptide for PAR-2) promote inositol trisphosphate accumulation, stimulate mitogen-activated protein kinases (extracellular signal–regulated protein kinase and p38-mitogen-activated protein kinase), elevate calcium concentration, and increase spontaneous automaticity. SFLLRN (but not SLIGRL) also activates c-Jun NH2-terminal kinase and AKT. In keeping with their linkage to pathways that have been associated with growth and/or survival, SFLLRN and SLIGRL both induce hypertrophy. However, PAR agonists promote cell elongation, a morphology that is distinct from the uniform increase in cell dimension induced by &agr;1-adrenergic receptor activation. These studies provide novel evidence that cardiomyocytes coexpress 2 functional PARs, which link to a common set of signals that culminate in changes in contractile function and hypertrophic growth. PAR actions may assume clinical importance in the border zone surrounding an infarction, where local proteolysis of PARs by serine proteases generated during inflammatory or thrombogenic pathways would elevate calcium concentration (setting the stage for arrhythmias), promote hypertrophic growth, and/or influence cardiomyocyte survival.

ISSN:0009-7330    

出版商:OVID    

年代:2000

数据来源: OVID