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N Akamatsu,
S Sawada,
S Komatsu,
T Tamagaki,
O Hiranuma,
T Kawahara,
Y Tsuda, Y Kono,
T Higaki,
Y Tada,
S Yamasaki,
H Imamura,
T Sato,
H Tsuji,
M Nakagawa
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ABSTRACT: 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-N(G)-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.
Journal of Cardiovascular Pharmacology 09/2001; 38(2):174-82. · 2.29 Impact Factor
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S Yamasaki,
S Sawada,
S Komatsu,
T Kawahara,
Y Tsuda,
T Sato,
A Toratani, Y Kono,
T Higaki,
H Imamura,
Y Tada,
N Akamatsu,
T Tamagaki,
H Tsuji,
M Nakagawa
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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/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.
Hypertension 09/2000; 36(2):201-7. · 6.21 Impact Factor
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T Higaki,
S Sawada, Y Kono,
H Imamura,
Y Tada,
S Yamasaki,
A Toratani,
T Sato,
S Komatsu,
N Akamatsu,
T Tamagaki,
Y Tsuda,
H Tsuji,
M Nakagawa
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ABSTRACT: The purpose of this study was to elucidate the mechanism by which bradykinin (BK) enhances prostacyclin (PGI(2)) production in human umbilical vein endothelial cells (HUVEC). BK-induced enhancement of PGI(2) synthesis was observed in a dose- and time-dependent manner, and it also increased [Ca(2+)](i) followed by enhancement of cytosolic phospholipase A(2) (cPLA(2)) activity. The PKC inhibitors GF109203X and H7 attenuated the BK-induced increase in [Ca(2+)](i) and inhibited the BK-induced PGI(2) synthesis. Phorbol 12-myristate 13-acetate increased cPLA(2) activity and PGI(2) synthesis but failed to alter [Ca(2+)](i). BK increased cPLA(2) mRNA eightfold by 15 min, and this increase was inhibited by pretreatment with the PKC inhibitors. In response to cycloheximide pretreatment, cPLA(2) mRNA was superinduced. These results suggest that BK stimulates PGI(2) synthesis in HUVEC by activation of cPLA(2) by dual mechanisms: an elevation of [Ca(2+)](i) and a PKC-dependent pathway. Moreover, changes in calcium kinetics and expression of cPLA(2) mRNA may underlie the BK-induced PGI(2) enhancement in these cells.
Microvascular Research 10/1999; 58(2):144-55. · 2.83 Impact Factor
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T Sato,
S Sawada,
Y Tsuda,
S Komatsu,
N Akamatsu, Y Kono,
T Higaki,
H Imamura,
Y Tada,
S Yamasaki,
T Tamagaki,
K Nakagawa,
H Tsuji,
M Nakagawa
[show abstract]
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ABSTRACT: 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.
Journal of Pharmacological and Toxicological Methods 09/1999; 41(4):173-82. · 2.32 Impact Factor
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A Toratani,
S Sawada, Y Kono,
T Higaki,
H Imamura,
Y Tada,
S Yamasaki,
T Sato,
S Komatsu,
N Akamatsu,
T Tamagaki,
K Nakagawa,
H Tsuji,
M Nakagawa
[show abstract]
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ABSTRACT: 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.
Journal of Cardiovascular Pharmacology 07/1999; 33(6):843-51. · 2.29 Impact Factor
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S Komatsu,
S Sawada,
T Tamagaki,
Y Tsuda, Y Kono,
T Higaki,
H Imamura,
Y Tada,
S Yamasaki,
A Toratani,
T Sato,
N Akamatsu,
H Tsuji,
M Nakagawa
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ABSTRACT: 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.
Journal of Pharmacological and Toxicological Methods 03/1999; 41(1):33-41. · 2.32 Impact Factor
