Toshiyuki Sato

Effects of high-density lipoproteins on intracellular pH and proliferation of human vascular endothelial cells.

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 (PGI2). The [pH]i was slightly acidified when extracellular Ca2+ 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 Ca2+ 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 PGI2 generation resulted from an increase in the release of Ca2+ from storage sites, due not only to an increased production of inositol 1,4,5-trisphosphate (IP3), but also to the alkalinization of [pH]i. These effects may be involved in the mechanism of HDLs anti-atherosclerotic action.

Effects of Bradykinin on Prostaglandin I2 Synthesis in Human Vascular Endothelial Cells

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 mechanism of thrombin-induced prostacyclin synthesis in human endothelial cells with reference to the gene transcription of prostacyclin-related enzymes and Ca2+ kinetics.

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.

Effect of probucol on intracellular pH and proliferation of human vascular endothelial cells

We investigated the effect of probucol on the intracellular pH ([pH]i) and proliferation of human umbilical vein endothelial cells (HUVEC), as well as their production of prostacyclin (PGI2). The addition of probucol produced a biphasic shift in [pH]i, with a brief initial acidification followed by a rapid alkaline shift. After pretreatment with EGTA, the initial decrease in [pH]i was abolished, and the subsequent increase was inhibited. After pretreatment with amiloride, only the increase of [pH]i was abolished. These results suggest that the probucol-induced increase of [pH]i was mainly dependent on Na+/H+ exchange and partly on extracellular Ca2+. In contrast, the addition of LDL produced a decrease of [pH]i. Under Ca2+-free condition, [pH]i was further decreased by LDL. In cells pretreated with amiloride, however, [pH]i was not further decreased by LDL. It was found that probucol promoted cell proliferation, and LDL inhibited cell proliferation. Addition of probucol also enhanced prostacyclin generation by HUVEC. This enhancement of PGI2 generation resulted from increased release of Ca2+ from the storage sites, due not only to increased production of inositol 1,4,5-triphosphate (IP3) but also to the increase of [pH]i. These findings may help to explain the antiatherosclerotic action of probucol.

Interleukin-1α stimulated prostacyclin release by increasing gene transcription of prostaglandin H synthase and phospholipase A2 in human vascular endothelial cells

This study was conducted to evaluate the effects of interleukin-1alpha (IL-1alpha) on prostacyclin (PGI2) production in cultured human vascular endothelial cells in association with intracellular Ca2+, 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-1alpha 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- 1alpha had no effect on intracellular Ca2+ 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-1alpha, 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-1alpha-induced PGI2 production, which involve PGHS and PLA2 gene transcription.

Bradykinin inhibits serum-depletion-induced apoptosis of human vascular endothelial cells by inducing nitric oxide via calcium ion kinetics.

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 2+ 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 G -monomethyl- l -arginine ( l -NMMA) and consequently restored by combined treatment with l -NMMA and l -arginine. Bradykinin increased influx of extracellular Ca 2+, generation of inositol 1,4,5-trisphosphate, and release of Ca 2+ from intracellular storage sites, thus increasing the total intracellular Ca 2+ concentration ([Ca 2+ ]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 −5M 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 2+ ]i.

Effect of cicletanine on the nitric oxide pathway in human umbilical vein endothelial cells

Summary: 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 ([Ca2+]i), cyclic nucleotide, inositol 1,4,5‐trisphosphate (IP3), and prostacyclin generation. Primary cultured human umbilical vein endothelial cells were used in this study. [Ca2+]i 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 [Ca2+]i. Cicletanine (10−6‐10−4 M) increased cyclic GMP but decreased prostacyclin generation. Cicletanine had no stimulating effect on cyclic AMP or IP3 generation. IP3 increased 45Ca 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‐NG‐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.

Hypokalemic alkalosis with hypocalciuria and normomagnesemia: A subgroup of Gitelman's syndrome?

Abstract We report a normomagnesemic patient with low normal blood pressure, hypokalemic alkalosis, hyperreninemia, hyperaldosteronism, and hypocalciuria. In renal clearance studies, distal delivery increased well and fractional distal solute reabsorption was dramatically diminished after the administration of furosemide, whereas thiazide produced no change in distal delivery and a moderate decrease in fractional distal solute reabsorption. These findings suggested that there may have been a defective locus in some part of the thiazide-sensitive segment of the distal convoluted tubule. As the features in this patient appear to be similar to those in Gitelmans syndrome, it is appropriate to designate this case, characterized by lack of hypomagnesemia, as a subgroup of Gitelmans syndrome.