ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Antisense oligodeoxynucleotides (ODNs) have been used to modify gene expression in vitro and are also promising therapeutic agents. Although there are numerous reports of antisense ODN-mediated changes in protein expression of cultured cells, use of these compounds to achieve antisense regulation of specific proteins in intact tissue has been limited. The aims of this study were (1) to define organ culture conditions for ileum smooth muscle that would permit long-term maintenance of force-generating capabilities and normal ultrastructure and (2) to develop a method for efficient introduction of antisense ODNs into intact tissue. Sheets of ODN-containing, reversibly permeabilized rat outer longitudinal ileum were maintained in a serum-free organ culture medium for 1 week without significant decreases in tension response to membrane depolarization or carbachol stimulation; the G protein-coupled calcium sensitization pathway was also intact after 7 days. Reversible permeabilization, a method previously used to load smooth and cardiac muscle with aequorin and heparin, was effective for loading more than 95% of ileum smooth muscle cells with a fluorescein-conjugated antisense ODN (5 prime-AAGGGCCATTTTGTT-FITC-3 prime). Confocal microscopy of reversibly permeabilized smooth muscle loaded with fluorescent antisense ODNs revealed intense nuclear fluorescence and less intense, homogeneous, cytoplasmic fluorescence. Internally radiolabeled ODNs (homologous to the above sequence) showed complete degradation between 4 and 16 hours after introduction into the cells. In summary, we have demonstrated methods for long-term organ culture and high-efficiency introduction of antisense ODNs into intact smooth muscle sheets. Such methods have broad potential utility for investigating many questions in smooth muscle biology. At present, however, a major limitation of this approach is the short half-life of phosphorothioated ODNs.(Circ Res. 1995;77:220-230.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The intracellular signaling mechanisms that mediate basic fibroblast growth factor (bFGF)-induced angiogenesis have not been fully identified. In particular, whether activation of the intracellular enzyme protein kinase C (PKC) is necessary or sufficient for bFGF-induced mitogenesis of human endothelial cells is not clear. Accordingly, the effect of bFGF stimulation on the Ca2plus increase and PKC activity of normal human endothelial cells (HEC) was studied, as was the effect of inhibition of PKC and the distribution of PKC isoenzymes in these cells. The addition of bFGF to cultured HEC increased overall PKC activity in the absence of an increase in intracellular Ca2plus and markedly stimulated their proliferation, as did the addition of PKC-activating phorbol esters. bFGF-induced proliferation was prevented by the PKC inhibitors chelerythrine and H-7 and by downregulation of PKC after prolonged incubation with phorbol esters. In contrast, these inhibitors did not prevent HEC proliferation induced by epidermal growth factor. Because of the failure of bFGF to increase Ca2plus, we determined whether bFGF-induced proliferation could be mediated by novel or atypical PKC isoenzymes (which are not regulated by Ca2plus). Investigation of the isoenzyme distribution of confluent and subconfluent HEC by immunoblotting, Northern transfer analysis, and polymerase chain reaction of reversetranscribed RNA revealed the presence of several novel and atypical isoenzymes (PKC-delta, -eta, -theta, and -zeta) as well as small amounts of the conventional (Ca2plus-regulated) isoenzymes PKC-alpha and -beta. Activation of PKC by bFGF, in the absence of an increase in intracellular Ca2plus, suggests that one or more of these Ca2plus-independent PKC isoenzymes are both necessary and sufficient for HEC proliferation after bFGF.Circ Res. 1995;77:231-238.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The type 1B angiotensin II (AT sub 1B) receptor cloned from rat kidney was stably expressed in Chinese hamster ovary cells. The stably expressed receptor was characterized by radioligand binding studies and functional coupling to inositol 1,4,5-triphosphate (IP3) formation. Exposure of cells expressing the AT1Breceptor to angiotensin II (Ang II) resulted in a rapid and dose-dependent homologous desensitization of receptor-mediated production of IP3, with an essentially complete desensitization at an agonist concentration more than 10 nmol/L. Binding studies revealed no significant change in the number of AT1Breceptors in transfected cells exposed to 1 nmol/L Ang II, whereas exposure to 100 nmol/L Ang II caused a rapid decrease of cell surface receptors, with a 75% loss of receptor number seen at 1 hour. Rapid desensitization occurred in the absence of receptor internalization. Blockade of receptor internalization with concanavalin A had at most only a slight effect on the agonist-induced desensitization. This indicates that factors other than internalization are chiefly responsible for the rapid agonist-induced desensitization. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, caused rapid desensitization of the receptor-mediated IP3response. Neither tyrosine kinase inhibitors nor a protein kinase A activator affected the receptor-mediated IP3response. The specific PKC inhibitor GF109203X or PKC depletion by prolonged treatment with 1 mu mol/L PMA completely blocked the PMA-dependent desensitization. Desensitization evoked by a low Ang II agonist concentration (1 nmol/L) was reversed by the PKC-specific inhibitor GF109203X or PKC depletion, whereas the desensitizing effect at a high agonist concentration (100 nmol/L) is only partially prevented by PKC inhibitory treatment. These results demonstrate that PKC plays a crucial role in the desensitization of the AT1Breceptor. They also suggest that receptor internalization and an additional PKC-independent pathway also contribute to desensitization of the AT1Breceptor in transfected cells.(Circ Res. 1995;77:239-248.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Enhanced vascular responsiveness to angiotensin II at the AT sub 1 receptor has been considered one of the major contributing factors to vascular hypertrophy and high blood pressure. The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells (less than 12 passages). There are three putative glucocorticoid responsive elements (GREs) in the promoter. However, only one GRE was found to respond to dexamethasone (1 mu mol/L) and was located at positions minus 756 to minus 770 bp upstream from the transcription initiation site. When compared with the consensus sequence of GRE, 9 of 12 bases were identical. RU38486, a glucocorticoid antagonist, completely blocked the induction by dexamethasone, suggesting that the GRE was functional through a specific glucocorticoid receptor. The response to dexamethasone was lost in vascular smooth muscle cells at higher passage numbers (more than 8 passages) but was restored when the cells were transfected with a glucocorticoid-receptor expression construct. This finding provided additional support that the response to dexamethasone was mediated by the glucocorticoid receptor. The gel mobility supershift assay showed that the GRE binds in vitro-translated rat glucocorticoid receptors in a specific manner. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs. We conclude from these experiments that the dexamethasone-induced increase in the transcription of the angiotensin II type 1A receptor gene occurred through the binding of GRE to the glucocorticoid-specific receptor.(Circ Res. 1995;77:249-257.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

We have previously shown that mechanical stress induces activation of protein kinases and increases in specific gene expression and protein synthesis in cardiac myocytes, all of which are similar to those evoked by humoral factors such as growth factors and hormones. Many lines of evidence have suggested that angiotensin II (Ang II) plays a vital role in cardiac hypertrophy, and it has been reported that secretion of Ang II from cultured cardiac myocytes was induced by mechanical stretch. To examine the role of Ang II in mechanical stress-induced cardiac hypertrophy, we stretched neonatal rat cardiac myocytes in the absence or presence of the Ang II receptor antagonists saralasin (an antagonist of both type 1 and type 2 receptors), CV-11974 (a type 1 receptor-specific antagonist), and PD123319 (a type 2 receptor-specific antagonist). Stretching cardiac myocytes by 20% using deformable silicone dishes rapidly increased the activities of mitogen-activated protein (MAP) kinase kinase activators and MAP kinases. Both saralasin and CV-11974 partially inhibited the stretch-induced increases in the activities of both kinases, whereas PD123319 showed no inhibitory effects. Stretching cardiac myocytes increased amino acid incorporation, which was also inhibited by approximately 70% with the pretreatment by saralasin or CV-11974. When the culture medium conditioned by stretching cardiocytes was transferred to nonstretched cardiac myocytes, the increase in MAP kinase activity was observed, and this increase was completely suppressed by saralasin or CV-11974. These results suggest that Ang II plays an important role in mechanical stress-induced cardiac hypertrophy and that there are also other (possibly nonsecretory) factors to induce hypertrophic responses.(Circ Res. 1995;77:258-265.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Apoptosis of vascular smooth muscle cells has recently been described in culture and also in remodeling of the artery after birth. However, the genes that regulate apoptosis in smooth muscle cells are mostly unknown. We studied the regulation of apoptosis in rat smooth muscle cells stably infected with retrovirus constructs containing c-myc, adenovirus E1A, bcl-2, and a temperature-sensitive mutant of the tumor suppressor gene p53. Apoptosis was verified by electron microscopy and quantified by time-lapse videomicroscopy. Death was induced by c-myc and E1A when cells were deprived of serum survival factors. bcl-2 suppressed apoptosis of cells infected with c-myc and E1A and also normal smooth muscle cells. Overexpression of wild-type p53 induced apoptosis of cells infected with E1A and c-myc but not normal cells. In contrast, expression of mutant p53, which blocks wild-type p53 function, suppressed apoptosis of cells infected with E1A or c-myc but not normal cells. Both adenovirus E1A and c-myc increased the expression of endogenous p53 protein but not p53 mRNA. Although bcl-2 suppressed apoptosis induced by E1A and c-myc, upregulation of p53 protein induced by these agents was unaffected. We conclude that apoptosis of vascular smooth muscle cells is regulated by p53-dependent and -independent pathways. Death induced by c-myc and E1A is mediated by, and dependent on, p53. However, the suppression of apoptosis by bcl-2 is not mediated by changes in p53 expression, and the low level of apoptosis seen in normal VSMCs upon removal of survival factors is independent of p53.(Circ Res. 1995;77:266-273.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

The endothelium serves many functional roles, including the modulation of vascular smooth muscle tone through the release of vasoactive agents such as nitric oxide (NO) and the eicosanoids. We proposed that NO produced by endothelial cells would increase the production of eicosanoids through enhanced expression and/or activation of prostaglandin H synthase. NO and eicosanoid synthesis were stimulated in a bovine coronary microvessel endothelial cell line with the calcium ionophore A23187 (1 mu mol/L). Our data demonstrated the following: (1) A23187 stimulated NO synthesis along with prostacyclin and thromboxane production. (2) Inhibition of NO synthesis with NG-nitro-L-arginine methyl ester (0.1 mmol/L) significantly diminished both prostacyclin and thromboxane production. (3) Cells incubated with hemoglobin (2 mu g/mL), which inactivates NO, decreased A23187-stimulated prostacyclin production, whereas cells incubated with superoxide dismutase (20 U/mL), which protects NO from superoxide anions, enhanced prostacyclin production. (4) Exogenous NO stimulated prostacyclin production. (5) The interaction of NO with prostacyclin persisted in the presence of excess exogenous arachidonic acid (100 mu mol/L). (6) Cyclooxygenase activity in cell lysates increased in the first hour of NO stimulation. (7) NO stimulation of prostacyclin occurred within 1 hour and continued for 8 hours. (8) Neither constitutive nor inducible prostaglandin H synthase enzyme expression was altered by NO. (9) Cycloheximide (10 mu mol/L) had no effect on A23187 stimulation of prostacyclin production. (10) Exogenous cGMP (10 mu mol/L) or a phosphodiesterase inhibitor (1 mmol/L) did not affect prostacyclin production. These data indicate that stimulating synthesis of endogenous NO in cultured endothelial cells increased eicosanoid production through activation of prostaglandin H synthase.(Circ Res. 1995;77:274-283.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID

摘要:

Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca2plus dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca2plus] sub i and NO release due to shear stress or agonist (ATP or brominated Ca2plus ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 mu L). The microsensor was positioned approximate equal 100 micro meter above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm2. Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca2plus]i, which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133 plus minus 9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5 plus minus 2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 mu mol/L) or Br-A23187 (10 mu mol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 mu mol/L) chelation of extracellular Ca2plus blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress-induced NO release (2.23 plus minus 0.07 nmol/L per second) closely resembled the kinetics of Ca2plus flux but differed markedly from the kinetics of ATP-induced NO release (5.64 plus minus 0.32 nmol/L per second). These data argue that shear stress causes a Ca2plus-mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress. The transient nature of this response may be due to NO-induced inhibition of Ca2plus influx via a mechanism yet to be determined.(Circ Res. 1995;77:284-293.)

ISSN:0009-7330    

出版商:OVID    

年代:1995

数据来源: OVID