ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. Because mitogen-activated protein (MAP) kinases have been shown to be activated by physical forces, we measured the phosphorylation and enzyme activity of MAP kinase to identify the signal events involved in the endothelial cell response to fluid shear stress. Flow at physiological shear stress (3.5 to 117 dynes/cm2) activated 42-kD and 44-kD MAP kinases present in cultured bovine aortic endothelial cells, with maximal effect at 12 dynes/cm2. Activation of a G protein was necessary, as demonstrated by complete inhibition by the nonhydrolyzable GDP analog GDP-beta S. Activation of protein kinase C (PKC) was required, as shown by inhibiting PKC with staurosporine or downregulating PKC with phorbol 12,13-dibutyrate. Both Ca2plus-dependent and -independent PKC activity, measured by translocation and substrate phosphorylation, increased in response to flow. However, MAP kinase activation was not dependent on Ca2plus mobilization, since Ca2plus chelation had no inhibitory effect. On the basis of these findings, it is proposed that flow activates two signal-transduction pathways in endothelial cells. One pathway is Ca2plus dependent and involves activation of phospholipase C and increases in intracellular Ca2plus. A new pathway, described in the present study, is Ca2plus independent and involves a G protein and increases in PKC and MAP kinase activity.

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The objective of this study was to assess the effect of leukotriene C sub 4 (LTC sub 4) on the flux of rolling leukocytes, leukocyte rolling velocity, and leukocyte adhesion in postcapillary venules in vivo and to study the underlying molecular mechanisms involved.LTC4(20 nmol/L) induced a rapid and significant increase in leukocyte rolling flux that was inhibitable by an anti-P-selectin antibody and soluble sialyl Lewisx(sLex). LTC4also induced a significant reduction in leukocyte rolling velocity, an event that was independent of P-selectin but entirely dependent on sLex. This LTC4-induced reduction in leukocyte rolling velocity was independent of any hemodynamic alterations. Another P-selectin effector, histamine, did not affect leukocyte rolling velocity even at more than 5000 times the concentration of LTC4. Treatment with an anti-L-selectin antibody had no effect on the LTC sub 4-induced increase in leukocyte rolling or reduction in rolling velocity. Inhibition of LTC4bioconversion to LTD4by pretreatment with L-serine (100 mu mol/L) prevented the LTC4-induced increase in leukocyte rolling flux and the LTC4-induced reduction in leukocyte rolling velocity. A subtle, yet significant, increase in leukocyte adhesion was also observed with LTC4. Pretreatment with a platelet-activating factor receptor antagonist returned the LTC4-induced leukocyte rolling velocity to baseline levels. The addition of a very low concentration of platelet-activating factor (1 nmol/L) induced significant leukocyte adhesion in the presence of LTC4but not histamine. This study demonstrates that LTC4, via bioconversion to leukotriene D4, induces a P-selectin-dependent and sLex-dependent increase in leukocyte rolling flux and a P-selectin-independent but sLex-dependent reduction in leukocyte rolling velocity, a parameter that may play an essential role in subsequent leukocyte adhesion.

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Lysophosphatidic acid (LPA), the smallest and structurally simplest phospholipid, has the potential to modulate cellular signaling in diverse tissues and cell types, including fibroblasts.In the present study, we assessed the effects of LPA on cultured rat glomerular mesangial cells. Quantitative changes of [Ca2plus]iin response to LPA were measured in monolayers of mesangial cells loaded with the fluorescent Ca2plus indicator fura 2. LPA (10 nmol/L to 100 mu mol/L) increased [Ca2plus]iin a dose-dependent manner and evoked inositol trisphosphate formation. LPA (1 mu mol/L to 30 mu mol/L) also elicited a marked, albeit transient, contractile response in mesangial cells cultured on collagen gel, as assessed by a decrease in cell surface area (CSA). The contraction persisted for 5 minutes (CSA decreased by 31%), whereupon the mesangial cells gradually relaxed with a consequent increase in CSA. Pretreatment of mesangial cells with isradipine (1 mu mol/L), a dihydropyridine-sensitive Ca2plus channel blocker, completely blocked LPA-induced contraction. Isradipine also decreased resting [Ca2plus]ilevels as well as the peak and the subsequently sustained [Ca2plus]ilevels induced by LPA, suggesting that the contractile effects of LPA are dependent on Ca2plus entry through voltage-gated Ca2plus channels. Finally, LPA stimulated an increase in both prostaglandin E2synthesis and cAMP accumulation, indicating that these mediators may modulate the contractile effects of LPA. Our study is the first demonstration that exogenous LPA induces mesangial cell contraction and suggests that the contraction is mediated by mobilization of intracellular Ca2plus by activation of the phosphoinositide cascade as well as by promotion of Ca2plus entry across the plasma membrane.(Circ Res. 1995;77:888-896.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Previous studies demonstrated that the thrombin-induced permeability of endothelial cell monolayers is reduced by the elevation of cGMP. In the present study, the presence of cGMP-dependent protein kinase (cGMP-PK) immunoreactivity and activity in various types of human endothelial cells (ECs) and the role of cGMP-PK in the reduction of thrombin-induced endothelial permeability was investigated. cGMP-PK type I was demonstrated in freshly isolated ECs from human aorta and iliac artery as well as in cultured ECs from human aorta, iliac vein, and foreskin microvessels. Addition of the selective cGMP-PK activator 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) to these ECs caused phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), an established cGMP-PK substrate, which is localized at cell-cell contact sites of confluent ECs. cGMP-PK type I expression decreased during serial passage of ECs, which correlated with a diminished ability of 8-pCPT-cGMP to induce VASP phosphorylation. Preincubation of aorta and microvascular EC monolayers with 8-pCPT-cGMP caused a 50% reduction of the thrombin-stimulated permeability, as determined by measuring the peroxidase passage through EC monolayers on porous filters. Furthermore, the thrombin-induced rise in cytoplasmic [Ca2plus]iwas strongly attenuated by the cGMP-PK activator in fura 2-loaded aorta ECs. In contrast, cGMP-PK could not be demonstrated in freshly isolated and cultured human umbilical vein ECs. Incubation of umbilical vein ECs with 8-pCPT-cGMP did not cause VASP phosphorylation and had no effect on the thrombin-induced increases in cytoplasmic Ca2plus and endothelial permeability. These data indicate that cGMP-PK type I is expressed in various types of human macrovascular and microvascular ECs but is absent or expressed in very low amounts in umbilical vein ECs. cGMP-PK type I expression in ECs may be important in the regulation of endothelial permeability and the release of factors involved in vasoregulation and hemostasis.(Circ Res. 1995;77:897-906.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

In vitro evidence suggests that natriuretic peptide receptors (NPR)-B and NPR-C inhibit vascular smooth muscle (VSM) proliferation. NPR-B is guanylate cyclase-coupled and selectively activated by C-type natriuretic peptide (CNP)-(1-22). NPR-C is not guanylate cyclase-coupled and, unlike NPR-B, avidly binds atrial natriuretic peptide (ANP)-(1-28) as well as CNP-(1-22). Here, we investigate these receptors during the VSM proliferation and neointimal formation found 5, 7, and 20 days after compressing the central ear artery of the rabbit. Receptors were mapped autoradiographically using [sup 125 I-Tyr0]CNP-(1-22), which binds NPR-B and NPR-C, and125I-ANP-(1-28), which binds NPR-C and NPR-A, another guanylate cyclase-coupled receptor. Normal tunica media had NPR-B-like binding sites, and the level of these did not change significantly after compression. Consistent with this, CNP-(1-22) stimulated cGMP production equally with membranes from normal or damaged arteries and was more effective than ANP-(1-28). Neointima, which became evident 5 to 7 days after arterial damage, expressed NPR-C-like sites and no detectable NPR-B-like binding. NPR-C-like sites also appeared on the media for the first time between 5 and 7 days after compression. Immunohistochemistry for proliferating cell nuclear antigen revealed widespread mitosis in VSM at 5 days after compression, but mitosis was virtually restricted to the neointima at and beyond 7 days after compression. Thus, whereas levels of NPR-B did not change significantly after arterial injury and NPR-A was not detected, NPR-C-like receptors were upregulated as mitosis declined in the media and as a prominent neointima formed.(Circ Res. 1995;77:906-918.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

4-Methylumbelliferyl beta-D-xylosides (beta-D-xylosides) inhibit proteoglycan synthesis, and this is associated with reduced proliferation and extracellular matrix production by vascular smooth muscle cells. This study evaluated whether treatment with naroparcil, a beta-D-xyloside analogue, reduced intimal hyperplasia after arterial injury in the hypercholesterolemic rabbit. Forty-two rabbits were assigned to three groups that received either a 1% cholesterol-enriched diet (group 1, n equals 15) or the same diet with added 100 mg centered dot kgminus1 naroparcil (group 2, n equals 15) or 300 mg centered dot kgminus1 naroparcil (group 3, n equals 12). All animals underwent iliac artery endothelial abrasion at day 14 and were killed at day 56. At the time of death, the angiographic minimal luminal diameter was significantly larger in both treated groups. Morphometric analysis showed a larger luminal area in treated rabbits (groups 2 and 3) compared with control rabbits (group 1) (0.75 plus minus 0.54 and 0.85 plus minus 0.61 mm2versus 0.32 plus minus 0.25 mm sup 2, respectively; P less than .05), with a decreased intimal thickness in groups 2 and 3 (average reduction of 37% and 39%, respectively, compared with group 1; P less than .05) but without changes in medial area. Total vessel area was comparable among all groups. In the media, immunohistochemistry suggested reduced infiltration by macrophages and a larger fractional area of smooth muscle cells. There were no differences in plasma or arterial wall cholesterol content between groups. Plasma levels of glycosaminoglycans and dermatan sulfate content were increased only in group 3. In this model, oral treatment with naroparcil appears to preserve the arterial lumen and reduce intimal thickness after arterial injury.(Circ Res. 1995;77:919-926.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Intracellular Ca sup 2 plus pools contribute to changes in cytosolic [Ca sup 2 plus] ([Ca sup 2 plus] sub i), which play an important role in endothelial cell signaling.Recently, endothelial ryanodine-sensitive Ca2plus stores were shown to regulate agonist-sensitive intracellular Ca2plus pools. Since caffeine binds the ryanodine Ca2plus release channel on the endoplasmic reticulum in a variety of cell types, we examined the effect of caffeine on [Ca2plus]iin human aortic endothelial cell monolayers loaded with the fluorescent probe indo 1. Under baseline conditions, 10 mmol/L caffeine induced a small increase in [Ca2plus] sub i from 86 plus minus 10 to 115 plus minus 17 nmol/L (mean plus minus SEM); this effect was similar to that of 5 mu mol/L ryanodine and was unaffected by buffer Ca sup 2 plus removal. After depletion of an intracellular Ca2plus store by the irreversible endoplasmic reticulum Ca2plus-ATPase inhibitor thapsigargin (1 mu mol/L), ryanodine did not affect [Ca2plus]i. In contrast, caffeine induced a large rapid increase in [Ca2plus]i(176 plus minus 19 to 338 plus minus 35 nmol/L, P less than .001) after thapsigargin exposure; this effect of caffeine was only observed when extracellular Ca2plus was present. A similar increase in [Ca2plus]iwas induced by caffeine after depletion of ryanodine- and histamine-sensitive Ca2plus stores or after pretreatment with the endoplasmic reticulum Ca2plus-ATPase inhibitor cyclopiazonic acid (10 mu mol/L). Thus, under baseline conditions the effect of caffeine on [Ca2plus]iis similar to that of ryanodine and appears to be due to the release of an intracellular store. However, after depletion of an endoplasmic reticulum Ca2plus store, caffeine, but not ryanodine, stimulates Ca2plus influx, resulting in a large increase in [Ca2plus]i. The data suggest that caffeine-induced Ca2plus influx is controlled by the status of Ca2plus loading of intracellular Ca2plus stores in human aortic endothelial cells.(Circ Res. 1995;77:927-935.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Women rarely suffer cardiovascular dysfunction before menopause, but by the age of 65 a woman becomes as vulnerable to cardiovascular mortality as a man.It has been proposed that estrogens protect against cardiovascular disease; however, the physiological basis of estrogen protection is unknown. In the present study the mechanism of estrogen-induced relaxation of coronary arteries was investigated at the tissue, cellular, and molecular levels. Tissue studies demonstrated that 17 beta-estradiol relaxes porcine coronary arteries by an endothelium-independent mechanism involving Kplusefflux, and subsequent studies employing the patch-clamp technique confirmed that estrogen stimulates Kpluschannel gating in coronary smooth muscle. Perforated-patch recordings from metabolically intact coronary myocytes revealed that 17 beta-estradiol more than doubles steady state outward currents in these cells at positive voltages. Studies of on-cell patches demonstrated a potent stimulatory effect of 17 beta-estradiol on the gating of the large-conductance, Ca2plus- and voltage-activated Kplus(BKCa) channels, while 17 alpha-estradiol had no effect. Furthermore, blocking BKCachannels in intact arteries inhibited estrogeninduced relaxation. The effect of 17 beta-estradiol on BKCachannels was blocked by inhibiting cGMP-dependent protein kinase (PKG) activity and was mimicked by exogenous cGMP or by stimulating PKG activity. Therefore, we propose that 17 beta-estradiol relaxes coronary arteries by opening BKCachannels via cGMP-dependent phosphorylation. This novel mechanism could account for the hypotensive effect of estrogens and help explain, at least in part, why postmenopausal estrogen therapy lowers the risk of cardiovascular disease.(Circ Res. 1995;77:936-942.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Effects on Ca sup 2 plus-induced Ca2plus release due to phosphorylation of sarcoplasmic reticulum (SR) proteins were investigated in isoproterenol-treated saponin-permeabilized trabeculae from rat ventricles. In these experiments, Ca2plus release from the SR was induced by a rapid change in concentration of free Ca2plus (ie, trigger Ca2plus) achieved by flash photolysis of nitr-5, and the amount of Ca2plus released was assessed by measuring isometric tension. Ca2plus uptake by the SR was more rapid, and the amount of Ca2plus released by a given concentration of trigger Ca2plus was greater in isoproterenol-treated trabeculae compared with control trabeculae. However, under the same conditions of Ca2plus loading, the amplitudes of caffeine-elicited tension transients in control trabeculae were similar to those in isoproterenol-treated trabeculae, suggesting that the Ca2plus available for release was similar in the two cases. Control experiments showed that there were no significant differences in Ca2plus sensitivity of tension between isoproterenol-treated and control trabeculae. Also, application of alkaline phosphatase to trabeculae that had previously been treated with isoproterenol returned SR Ca2plus release to control levels. We conclude that the greater release of Ca2plus in isoproterenol-treated trabeculae in response to a given concentration of trigger Ca2plus is due to phosphorylation of SR proteins, most likely the Ca2plus release channel.(Circ Res. 1995;77:943-949.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID