摘要:

Using the whole-cell patch-clamp configuration in rat ventricular myocytes, we recently reported that microtubule disruption increases calcium current (ICa) and [Ca2+]itransient and accelerates their kinetics by adenylyl cyclase activation. In the present report, we further analyzed the effects of microtubule disruption by 1 &mgr;mol/L colchicine on Ca2+signaling in cardiac myocytes with intact sarcolemma. In quiescent intact cells, it is possible to investigate ryanodine receptor (RyR) activity by analyzing the characteristics of spontaneous Ca2+sparks. Colchicine treatment decreased Ca2+spark amplitude (F/F0: 1.78±0.01, n=983, versus 1.64±0.01, n=1660, recorded in control versus colchicine-treated cells;P<0.0001) without modifying the sarcoplasmic reticulum Ca2+load and enhanced their time to peak (in ms: 6.85±0.09, n=1185, versus 7.33±0.13, n=1647;P<0.0001). Microtubule disruption also induced the appearance of Ca2+sparks in doublets. These alterations may reflect RyR phosphorylation. To further investigate Ca2+signaling in cardiac myocytes with intact sarcolemma, we analyzed [Ca2+]itransient evoked by field stimulation. Cells were loaded with the fluorescence Ca2+indicator, Fluo-3 cell permeant, and stimulated at 1 Hz. [Ca2+]itransient amplitude was greater and its decay was accelerated in colchicine-treated, field-stimulated myocytes. This effect is reversible. When colchicine-treated myocytes were placed in a colchicine-free solution for 30 minutes, tubulin was repolymerized into microtubules, as shown by immunofluorescence, and the increase in [Ca2+]itransient was reversed. In summary, we demonstrate that microtubule disruption by colchicine reversibly modulates Ca2+signaling in cardiac cells with intact sarcolemma. The full text of this article is available at http://www.circresaha.org.

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

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

摘要:

There are strong data favoring the pathogenic role of angiotensin II type 1 receptor (AT1) activation with subsequent promotion of myocyte growth and cardiac fibrosis in the development of cardiac hypertrophy and heart failure. An emerging hypothesis suggests that the activity of the angiotensin II type 2 receptor (AT2) may counterregulate AT1receptor effects during cardiac development and during the evolution of cardiac hypertrophy and heart failure. In this review, we examine the potential role of AT2activity in the context of this hypothesis. In contrast to the counterregulatory hypothesis, studies in mice with an overabundance of, or a deficiency in, the AT2receptor do not suggest that AT2signaling is essential for cardiac development. Moreover, the proposed antigrowth effects of AT2receptor signaling in pathological cardiac hypertrophy could not be shown in two mice models both deficient in AT2receptors. The role of AT2receptor signaling in cardiac fibrosis is, however, still debatable because of conflicting data in the same two studies. In angiotensin II–evoked apoptosis in cardiomyocytes, the proposed proapoptotic role of AT2activity could not be confirmed. Furthermore, in the progression from the bench to bedside, the results of two large clinical trials in heart failure, namely ELITE II and Val-HeFT, can be explained without ascribing a major protective role to the unopposed activity of the AT2receptor in the failing myocardium. In this review, we conclude that the collective evidence does not strongly support a net beneficial effect of AT2stimulation in the diseased myocardium.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

The lipoxygenase (LO) pathway has been implicated as an important mediator of chronic glucose and platelet-derived growth factor (PDGF)-induced effects in the vascular system. Endothelial cells treated with 12LO products or cultured in high glucose showed enhanced monocyte adhesion, an important step in atherogenesis. We have previously reported that PDGF increased HETE levels in porcine aortic smooth muscle cells. Although several pharmacological inhibitors to the LO pathway are available, most lack specificity and may harbor undesirable side effects. Therefore, we developed a recombinant adenovirus expressing a hammerhead ribozyme (AdRZ) targeted against the porcine leukocyte-type 12LO mRNA to investigate the involvement of LO in glucose- and PDGF-mediated effects in vascular cells. Infection of porcine aortic endothelial cells with AdRZ reduced the level of glucose-enhanced 12LO mRNA expression as determined by quantitative, real-time reverse transcriptase–polymerase chain reaction. Reverse-phase HPLC and RIA analysis also revealed a corresponding decrease in glucose-stimulated 12HETE production in both the cellular and supernatant fractions. In the ribozyme-treated porcine aortic endothelial cells, there was marked inhibition of high glucose-stimulated monocyte adhesion. Infection with AdRZ also reduced PDGF-induced porcine aortic smooth muscle cell migration by approximately 50%. These studies demonstrate the efficacy of recombinant adenovirus expressing 12LO ribozyme in studying the effects of 12LO in vascular wall cells. They document an important role for the 12LO pathway in regulating inflammatory changes in endothelial cells and smooth muscle cells.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Chemical regulation of connexin (Cx) 40 and Cx43 follows a ball-and-chain model, in which the carboxyl terminal (CT) domain acts as a gating particle that binds to a receptor affiliated with the pore. Moreover, Cx40 channels can be closed by a heterodomain interaction with the CT domain of Cx43 and vice versa. Here, we report similar interactions in the establishment of the unitary conductance and voltage-dependent profile of Cx40 in N2A cells. Two mean unitary conductance values (“lower conductance” and “main”) were detected in wild-type Cx40. Truncation of the CT domain at amino acid 248 (Cx40tr248) caused the disappearance of the lower-conductance state. Coexpression of Cx40tr248 with the CT fragment of either Cx40 (homodomain interactions) or Cx43 (heterodomain interactions) rescued the unitary conductance profile of Cx40. In the N2A cells, the time course of macroscopic junctional current relaxation was best described by a biexponential function in the wild-type Cx40 channels, but it was reduced to a single-exponential function after truncation. However, macroscopic junctional currents recorded in the oocyte expression system were not significantly different between the wild-type and mutant channels. Concatenation of the CT domain of Cx43 to amino acids 1 to 248 of Cx40 yielded a chimeric channel with unitary conductance and voltage-gating profile indistinguishable from that of wild-type Cx40. We conclude that residence of Cx40 channels in the lower-conductance state involves a ball-and-chain type of interaction between the CT domain and the pore-forming region. This interaction can be either homologous (Cx40 truncation with Cx40CT) or heterologous (with the Cx43CT).

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

Blood vessels are continuously exposed to mechanical forces that lead to adaptive remodeling and atherosclerosis. Although there have been many studies characterizing the responses of vascular cells to mechanical stimuli, the precise mechanical characteristics of the forces applied to cells to elicit these responses are not clear. We designed a magnetic exposure system capable of producing a defined normal force on ferromagnetic beads that are specifically bound to cultured cells coated with extracellular matrix proteins or integrin-specific antibodies. Rat aortic smooth muscle cells were incubated with engineered fibronectin–coated ferromagnetic beads and then exposed to a magnetic field. With activation of extracellular signal–regulated mitogen-activated protein kinase 1/2 (ERK 1/2MAPK) used as a prototypical marker for cell responsiveness to mechanical forces, Western blot analysis demonstrated an increase in phosphorylated ERK 1/2MAPKexpression reaching a maximal response of a 3.5-fold increase at a total force of ≈2.5 pN per cell. The peak response occurred after 5 minutes of exposure and slowly decreased to baseline after 30 minutes. A cyclic, rather than static, force was required for this activation, and the frequency-response curve increased ≈2-fold between 0.5 and 2.0 Hz. Vitronectin- and &bgr;3antibody–coated beads showed a response nearly identical to those coated with engineered fibronectin, whereas forces applied to beads coated with &agr;2and &bgr;1antibodies did not significantly activate ERK 1/2MAPK. Mechanical activation of the ERK 1/2MAPKsystem in rat aortic smooth muscle cells occurs through specific integrin receptors and requires a cyclic force with a magnitude estimated to be in the piconewton range.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal–regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor–activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID

摘要:

In heart failure, reduced cardiac contractility is accompanied by blunted cAMP responses to &bgr;-adrenergic stimulation. Parathyroid hormone (PTH)–related peptide and arginine vasopressin are released from the myocardium in response to increased wall stress but do not stimulate contractility or adenylyl cyclase at physiological concentrations. To bypass the defective &bgr;-adrenergic signaling cascade, recombinant P1 PTH/PTH-related peptide receptors (rPTH1-Rs) and V2vasopressin receptors (rV2-Rs), which are normally not expressed in the myocardium and which are both strongly coupled to adenylyl cyclase, and recombinant &bgr;2-adrenergic receptors (r&bgr;2-ARs) were overexpressed in cardiomyocytes by viral gene transfer. The capacity of endogenous hormones to increase contractility via the heterologous, recombinant receptors was compared. Whereas V2-Rs are uniquely coupled to Gs, PTH1-Rs and &bgr;2-ARs are also coupled to other G proteins. Gene transfer of rPTH1-Rs or r&bgr;2-ARs to adult cardiomyocytes resulted in maximally increased basal contractility, which could not be further stimulated by adding receptor agonists. Agonists at rPTH1-Rs induced increased cAMP formation and phospholipase C activity. In contrast, healthy or failing rV2-R–expressing cardiomyocytes showed unaltered basal contractility. Their contractility and cAMP formation increased only at agonist exposure, which did not activate phospholipase C. In summary, we found that gene transfer of PTH1-Rs to cardiomyocytes results in constitutive activity of the transgene, as does that of &bgr;2-ARs. In the absence of receptor agonists, rPTH1-Rs and r&bgr;2-ARs increase basal contractility, coupling to 2 G proteins simultaneously. In contrast, rV2-Rs are uniquely coupled to Gs and are not constitutively active, retaining their property to be activated exclusively on agonist stimulation. Therefore, gene transfer of V2-Rs might be more suited to test the effects of cAMP-stimulating receptors in heart failure than that of PTH1-Rs or &bgr;2-ARs.

ISSN:0009-7330    

出版商:OVID    

年代:2001

数据来源: OVID