We investigated the effects of high-density lipoprotein (HDL) on the intracellular pH (pH
i), and on the proliferation of human vascular endothelial cells (HUVEC), as well as on their production of ...prostacyclin (PGI
2). The pH
i was slightly acidified when extracellular Ca
2+ was chelated with EGTA. Pretreatment of HUVEC with amiloride, the Na
+/H
+ exchange inhibitor, caused the pH
i to become strongly acidic. The addition of HDL produced a biphasic shift in pH
i, with a brief initial acidification followed by a rapid alkaline shift. The initial decrease in pH
i was abolished in the cells pretreated with EGTA, and subsequent alkalinization was inhibited. The alkalinization of pH
i disappeared in the cells pretreated with amiloride. These results suggest that pH
i depends mainly on Na
+/H
+ exchange and partially on the extracellular Ca
2+ of the HUVEC either in the resting unstimulated state or during HDL stimulation. In contrast, the addition of LDL produced an acidification of pH
i, which was increased by LDL in the Ca
2+-free condition. In the cells pretreated with amiloride, pH
i was not further acidified by LDL. As a result, HDL promoted the proliferation of cells, an action that was inhibited by pretreatment with EGTA. However LDL inhibited cell proliferation, an action unaffected by EGTA pretreatment. The addition of HDL also enhanced the generation of prostacyclin in endothelial cells, the enhancement of PGI
2 generation resulted from an increase in the release of Ca
2+ from storage sites, due not only to an increased production of inositol 1,4,5-trisphosphate (IP
3), but also to the alkalinization of pH
i. These effects may be involved in the mechanism of HDL's anti-atherosclerotic action.
The purpose of this study was to elucidate the mechanism by which bradykinin (BK) enhances prostacyclin (PGI2) production in human umbilical vein endothelial cells (HUVEC). BK-induced enhancement of ...PGI2 synthesis was observed in a dose- and time-dependent manner, and it also increased Ca2+i followed by enhancement of cytosolic phospholipase A2 (cPLA2) activity. The PKC inhibitors GF109203X and H7 attenuated the BK-induced increase in Ca2+i and inhibited the BK-induced PGI2 synthesis. Phorbol 12-myristate 13-acetate increased cPLA2 activity and PGI2 synthesis but failed to alter Ca2+i. BK increased cPLA2 mRNA eightfold by 15 min, and this increase was inhibited by pretreatment with the PKC inhibitors. In response to cycloheximide pretreatment, cPLA2 mRNA was superinduced. These results suggest that BK stimulates PGI2 synthesis in HUVEC by activation of cPLA2 by dual mechanisms: an elevation of Ca2+i and a PKC-dependent pathway. Moreover, changes in calcium kinetics and expression of cPLA2 mRNA may underlie the BK-induced PGI2 enhancement in these cells.
The effects of bradykinin on the regulatory mechanisms of prostacyclin synthesis in endothelial cells were investigated in association with intracellular Ca kinetics, cytosolic phospholipase A2 ...(cPLA2) activity, and mRNA expression of cPLA2 and prostaglandin H synthase (PGHS) isoforms. Bradykinin enhanced prostacyclin release from endothelial cells time-dependently, but pretreatment with EGTA H-7 or HOE 140 inhibited bradykinin-induced prostacyclin release. Bradykinin increased both the influx of extracellular Ca and Ca release from the intracellular Ca storage sites. These reactions occurred within 5 minutes after bradykinin stimulation. Within 15 minutes, bradykinin activated cPLA2 to 1.3-fold the control level. The constitutive expressions of mRNA of cPLA2, PGHS-1, and PGHS-2 was 87, 562, and 47 amol/μg RNA, respectively. With the stimulation of bradykinin, cPLA2 mRNA increased to 746 amol/μg RNA in 15 minutes, PGHS-1 mRNA increased to 10 608 amol/μg RNA, and PGHS-2 mRNA increased to 22 400 amol/μg RNA in 180 minutes. Pretreatment with cycloheximide superinduced cPLA2 and PGHS-2 mRNA expression but almost completely inhibited PGHS-1. Pretreatment with EGTA had effects similar to pretreatment with cycloheximide in the case of cPLA2 and PGHS-1 but did not affect PGHS-2. These findings suggest that the elevation of cPLA2 activity caused by the increase of intracellular Ca concentration is important in the early phase of bradykinin-induced prostacyclin synthesis and that the mechanisms regulating cPLA2 are different from those regulating PGHS isoforms in endothelial cells.
Abstract —The effects of bradykinin on the regulatory mechanisms of prostacyclin synthesis in endothelial cells were investigated in association with intracellular Ca 2+ kinetics, cytosolic ...phospholipase A 2 (cPLA 2 ) activity, and mRNA expression of cPLA 2 and prostaglandin H synthase (PGHS) isoforms. Bradykinin enhanced prostacyclin release from endothelial cells time-dependently, but pretreatment with EGTA H-7 or HOE 140 inhibited bradykinin-induced prostacyclin release. Bradykinin increased both the influx of extracellular Ca 2+ and Ca 2+ release from the intracellular Ca 2+ storage sites. These reactions occurred within 5 minutes after bradykinin stimulation. Within 15 minutes, bradykinin activated cPLA 2 to 1.3-fold the control level. The constitutive expressions of mRNA of cPLA 2 , PGHS-1, and PGHS-2 was 87, 562, and 47 amol/μg RNA, respectively. With the stimulation of bradykinin, cPLA 2 mRNA increased to 746 amol/μg RNA in 15 minutes, PGHS-1 mRNA increased to 10 608 amol/μg RNA, and PGHS-2 mRNA increased to 22 400 amol/μg RNA in 180 minutes. Pretreatment with cycloheximide superinduced cPLA 2 and PGHS-2 mRNA expression but almost completely inhibited PGHS-1. Pretreatment with EGTA had effects similar to pretreatment with cycloheximide in the case of cPLA 2 and PGHS-1 but did not affect PGHS-2. These findings suggest that the elevation of cPLA 2 activity caused by the increase of intracellular Ca 2+ concentration is important in the early phase of bradykinin-induced prostacyclin synthesis and that the mechanisms regulating cPLA 2 are different from those regulating PGHS isoforms in endothelial cells.
Vascular endothelial cells play important roles in atherogenesis, and bradykinin is associated with atherosclerosis. The effect of bradykinin on apoptosis in human umbilical vein endothelial cells ...(HUVECs) was investigated, with a focus on Ca kinetics and nitric oxide production. In serum-free conditions, the number of apoptotic cells increased in a time-dependent manner, but this increase was inhibited by bradykinin in a dose-dependent manner. The apoptosis inhibited by bradykinin was reduced by nitric oxide inhibitor N -monomethyl- l -arginine ( l -NMMA) and consequently restored by combined treatment with l -NMMA and l -arginine. Bradykinin increased influx of extracellular Ca , generation of inositol 1,4,5-trisphosphate, and release of Ca from intracellular storage sites, thus increasing the total intracellular Ca concentration (Ca i). Bradykinin increased nitric oxide production, which was inhibited by l -NMMA and restored by combined treatment with l -NMMA and l -arginine. Sodium nitroprusside (SNP) dose-dependently increased nitric oxide production and inhibited apoptosis; however, 10 M SNP did not inhibit apoptosis. Caspase-3 inhibitor, acetyl-Asp-Met-Gln-Asp-aldehyde, enhanced bradykinin-induced inhibition of apoptosis but did not effect bradykinin-induced nitric oxide production. These findings suggest that bradykinin inhibits serum-depletion-induced apoptosis in HUVECs by enhancing nitric oxide production via an increase in Ca i.
The effects of bradykinin on the regulatory mechanisms of prostacyclin synthesis in endothelial cells were investigated in association with intracellular Ca(2+) kinetics, cytosolic phospholipase A(2) ...(cPLA(2)) activity, and mRNA expression of cPLA(2) and prostaglandin H synthase (PGHS) isoforms. Bradykinin enhanced prostacyclin release from endothelial cells time-dependently, but pretreatment with EGTA H-7 or HOE 140 inhibited bradykinin-induced prostacyclin release. Bradykinin increased both the influx of extracellular Ca(2+) and Ca(2+) release from the intracellular Ca(2+) storage sites. These reactions occurred within 5 minutes after bradykinin stimulation. Within 15 minutes, bradykinin activated cPLA(2) to 1.3-fold the control level. The constitutive expressions of mRNA of cPLA(2), PGHS-1, and PGHS-2 was 87, 562, and 47 amol/microg RNA, respectively. With the stimulation of bradykinin, cPLA(2) mRNA increased to 746 amol/microg RNA in 15 minutes, PGHS-1 mRNA increased to 10 608 amol/microg RNA, and PGHS-2 mRNA increased to 22 400 amol/microg RNA in 180 minutes. Pretreatment with cycloheximide superinduced cPLA(2) and PGHS-2 mRNA expression but almost completely inhibited PGHS-1. Pretreatment with EGTA had effects similar to pretreatment with cycloheximide in the case of cPLA(2) and PGHS-1 but did not affect PGHS-2. These findings suggest that the elevation of cPLA(2) activity caused by the increase of intracellular Ca(2+) concentration is important in the early phase of bradykinin-induced prostacyclin synthesis and that the mechanisms regulating cPLA(2) are different from those regulating PGHS isoforms in endothelial cells.
The purpose of this study was to evaluate the effects of cicletanine, a slightly diuretic antihypertensive drug, on human vascular endothelial cells with regard to nitric oxide, intracellular calcium ...concentration (Cai), cyclic nucleotide, inositol 1,4,5-trisphosphate (IP3), and prostacyclin generation. Primary cultured human umbilical vein endothelial cells were used in this study. Cai was measured by fura-2/AM. Cyclic adenosine monophosphate (AMP), cyclic guanosine monophosphate (GMP), IP3, and prostacyclin were measured by radioimmunoassay. Nitric oxide was measured by the Griess method. Cicletanine had no effect on Cai. Cicletanine (10-10 M) increased cyclic GMP but decreased prostacyclin generation. Cicletanine had no stimulating effect on cyclic AMP or IP3 generation. IP3 increased Ca release from storage sites. Cicletanine decreased prostacyclin generation via increase in cyclic GMP. Cicletanine had no stimulating effect on nitrogen oxides for 2 h after incubation but increased it after 3-24 h. Pretreatment with L-N-monomethyl-arginine (L-NMMA) prevented this increase. The inhibitory effect of L-NMMA was prevented by pretreatment with L-arginine. These results indicate that nitric oxide and cyclic GMP may contribute to the antihypertensive action of cicletanine.
This study was designed to evaluate the effect of thrombin on prostacyclin (PGI2) production in cultured human vascular endothelial cells in association with intracellular Ca2+ and with the gene ...expression of prostaglandin H2 synthase (PGHS) and phospholipase A2 (PLA2) using competitive polymerase chain reaction. Thrombin enhanced the PGI2 synthesis dependent with time. Additionally, thrombin increased the intracellular Ca2+, which stimulates PLA2, resulting in arachidonic acid cleavage from membrane phospholipids and its subsequent conversion into PGI2 through the PGHS pathway. The elevation of intracellular Ca2+ was a result of Ca2+ influx and Ca2+ release from its intracellular storage sites. In this study, PGHS-1 mRNA was constitutively expressed, whereas PGHS-2 mRNA was not. With the stimulation of thrombin, cytosolic PLA2 (cPLA2) mRNA increased 9-fold at 15 min, PGHS-1 mRNA increased 3.4-fold at 180 min, and PGHS-2 mRNA increased 38-fold at 60 min. These results suggest that the elevation of intracellular Ca2+ and the expression of cPLA2, PGHS-1, and PGHS-2 mRNA cause PGI2 generation.
This study was conducted to evaluate the effects of interleukin-1α (IL-1α) on prostacyclin (PGI2) production in cultured human vascular endothelial cells in association with intracellular Ca, ...inositol 1,4,5-trisphosphate (IP3), and with prostaglandin H synthase (PGHS) and phospholipase A2 (PLA2) gene expression by using the competitive polymerase chain reaction (PCR) method. IL-1α did not increase PGI2 production for 15 min, but induced an increase of about three-fold relative to that in controls at 60 and 180 min. IL-1α had no effect on intracellular Ca levels throughout the experimental period. In this study, consistent with previous reports, PGHS-1 messenger RNA (mRNA) was constitutively expressed, whereas PLA2 mRNA was not. After stimulation with IL-1α, PLA2 mRNA level showed an eightfold increase within 15 min, and PGHS-2 mRNA level increased by 76-fold within 180 min. PGHS-1 mRNA level was increased 1.6-fold at 180 min. These results suggest the existence of regulatory mechanisms of IL-1α-induced PGI2 production, which involve PGHS and PLA2 gene transcription.
During the past ten years, we have performed combined resections of the lung and the heart or a great vessel in eleven patients with advanced lung carcinoma. In four of the nine, cardiopulmonary ...bypass was used. Three underwent right pneumonectomy with partial resection of the left atrium; in one, the right atrium was resected partially because of carcinomatous invasion to the atrial septum. One underwent left lobectomy with partial resection of the aorta. A 71-year-old patient had a cardiac infarction during the operation and multiple brain infarctions after the operation. He died 57 days after surgery. Of the other three, two are alive and one died of brain metastasis 11 months after surgery. The clinical findings of the four patients are described, and the indications for cardiopulmonary bypass in extended operations for lung carcinoma are discussed.
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