Takuji Machida

Docosahexaenoic acid potentiates interleukin-1β induction of nitric oxide synthase through mechanism involving p44/42 MAPK activation in rat vascular smooth muscle cells

The effect of docosahexaenoic acid (DHA) on nitric oxide (NO) production and inducible NO synthase (iNOS) expression induced by interleukin (IL)‐1β, and whether the effect of DHA is related to its effect on mitogen‐activated protein kinase (MAPK) activation were investigated in cultured rat vascular smooth muscle cells (VSMCs). DHA and eicosapentaenoic acid (EPA), although less potent, increased the NO production induced by IL‐1β (3 ng ml−1) in a concentration‐dependent manner (3–30 μM) Arachidonic acid had no significant effect. The stimulatory effect of DHA (30 μM) on the NO production was more obvious at lower concentrations of IL‐1β. IL‐1β induced iNOS protein and mRNA expressions, which were significantly potentiated by DHA. EPA (30 μM) had a tendency to increase the iNOS protein and mRNA expressions, but arachidonic acid had no effect. IL‐1β‐induced iNOS protein expression was significantly inhibited by PD 98059 (10 μM), a selective inhibitor of p44/42 MAPK kinase, both in the absence and the presence of DHA. SB 203580 (10 μM), a selective inhibitor of p38 MAPK activity, had no significant effect, although had a tendency to inhibit slightly. IL‐1β increased the phosphorylation of p44/42 MAPK, while it did not apparently increase the phosphorylation of p38 MAPK. DHA significantly potentiated the IL‐1β‐induced phosphorylation of p44/42 MAPK, while it had no significant effect on the phosphorylation of p38 MAPK. These results suggest that DHA increases NO production by potentiating iNOS expression induced by IL‐1β through mechanism involving p44/42 MAPK signalling cascade in rat VSMCs. The present study may contribute to the understanding of basic mechanisms underlying the beneficial effects of DHA on various cardiovascular disorders.

Reduced expressions of inducible nitric oxide synthase and cyclooxygenase-2 in vascular smooth muscle cells of stroke-prone spontaneously hypertensive rats

Inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 are expressed in vascular smooth muscle cells stimulated with interleukin-1beta (IL-1beta), resulting in the production of nitric oxide (NO) and prostaglandins (PGs) such as PGI2. The iNOS and COX-2 proteins and their mRNA expressions in cultured vascular smooth muscle cells isolated from 6-7 week-old stroke-prone spontaneously hypertensive rats (SHRSP) were compared with those in the cells isolated from age-matched normotensive Wistar Kyoto rats (WKY). The IL-1beta-induced NO production and iNOS expression in vascular smooth muscle cells of SHRSP were significantly lower than those in cells of WKY. Similarly, PGI2 production and COX-2 expression were significantly lower in vascular smooth muscle cells of SHRSP than WKY, whereas there was no difference in the COX-1 expression. There were no significant differences in iNOS and COX-2 mRNA expressions between the two strains, suggesting that these protein expression may be reduced at the post-transcriptional level in cells of SHRSP. These results further suggest that the reduction of iNOS and COX-2 expressions in vascular smooth muscle cells may have relevance to the pathophysiology in SHRSP.

5-Hydroxytryptaime induces cyclooxygenase-2 in rat vascular smooth muscle cells: Mechanisms involving Src, PKC and MAPK activation

Considering the importance of 5-hydroxytryptamine (5-HT) and cyclooxygenase (COX) products in vascular pathology, we investigated the effects of 5-HT on COX expression in rat vascular smooth muscle cells (VSMCs), and to provide mechanistic insights into these effects. VSMCs were enzymatically isolated from aortic media of Wistar rats. Incubation of VSMCs with 5-HT for 24h stimulated prostaglandin I(2) production, but this stimulation was completely suppressed by NS-398, a selective COX-2 inhibitor. 5-HT induced transient COX-2, but not COX-1, protein and mRNA expression in concentration- and time-dependent manners. This effect of 5-HT was completely inhibited by sarpogrelate, a 5-HT(2A) receptor antagonist. 5-HT-induced COX-2 expression was markedly blunted by Ca(2+) depletion; GF 109203X, a protein kinase C (PKC) inhibitor; PP2, an inhibitor of Src-family tyrosine kinase (Src); PD 98059, an inhibitor of extracellular signal-regulated kinase (ERK) activation; SB 203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK); and SP 600125, an inhibitor of c-Jun N-terminal kinase (JNK). 5-HT activated ERK and p38 MAPK, followed by JNK activation. PP2 inhibited these activations, while GF 109203X inhibited only JNK activation. Furthermore, PD 98059 inhibited JNK activation. These results suggest that 5-HT induces COX-2 expression in rat VSMCs, and that PKC, Src, and MAPK activation are each essential for the full expression of COX-2 pathways.

Pulsatile mechanical pressure promotes Angiotensin-converting enzyme expression in aortic smooth muscle cells.

BackgroundHypertension is a major risk factor for atherosclerosis, and elevated pressure (i.e., mechanical pressure stresses) has been known to modulate vascular remodeling, possibly by affecting the tissue renin-angiotensin system.MethodsIn the present study we applied pulsatile pressure to human aortic smooth muscle cells (HASMCs) and investigated whether mechanical pressure stress affects cell proliferation and/or the angiotensin-converting enzyme (ACE), and we tested whether the administration of an angiotensin II (AII) receptor blocker has a favorable effect.ResultsThree hours of pulsatile atmospheric pressure resulted in an approximately 8% increase in cell proliferation of human aortic smooth muscle cells. The cell surface ACE level, enzyme activity and mRNA expression were all elevated (37%, 110% and 17%, respectively) under pressurized conditions, and co-administration of AII reduced all these values. The reductions in these three parameters resulting from the administration of AII were all abolished by AII receptor blocker co-administration and values were increased (11%, 62% and12%, respectively) under pressurized conditions. Pulsatile atmospheric pressure increased the amount of phosphorylated extracellular signal-regulated kinase (ERK) by approximately 54% in HASMCs. The administration of PD98059 (10μM) resulted in a decrease in phosphorylated ERK and ACE activity in HASMCs compared to those of the pressurized control.ConclusionFrom these observations, we conclude that pulsatile mechanical pressure is one of the mediators of ACE production in vascular smooth muscle cells and that AII receptor blocking may prevent negative feedback. The present findings may provide a potential therapeutical target beyond lowering blood pressure in hypertensive patients.

Sphingosine 1-phosphate increases an intracellular Ca(2+) concentration via S1P3 receptor in cultured vascular smooth muscle cells.

We investigated the effect of sphingosine 1‐phosphate (S1P) on intracellular Ca2+ dynamics in rat vascular smooth muscle cells (VSMCs).

Anti-inflammatory drugs ameliorate opposite enzymatic changes in ileal 5-hydroxytryptamine metabolism in the delayed phase after cisplatin administration to rats.

The effects of anti-inflammatory drugs on ileal 5-hydroxytryptamine (5-HT) metabolic dynamics at 72 h after a single administration of cisplatin were investigated in rats. Cisplatin 5 mg/kg i.p. caused pathological changes, with an inflammatory response occurring 72 h after its administration. The inflammatory response was associated with the induction of cyclooxygenase-2, but not cyclooxygenase-1, in the ileal mucosa at 72 h after the cisplatin administration. Daily treatment with meloxicam 3 mg/kg s.c. ameliorated the cisplatin-induced mucosal damage, whereas dexamethasone 1 mg/kg s.c. did not. Cisplatin administration also caused a significant increase in cyclooxygenase-2 mRNA expression at 72 h after administration, which was blunted by dexamethasone, but not by meloxicam. Cisplatin increased the content of 5-HT and its metabolite, 5-hydroxyindoleacetic acid (5-HIAA), but had no effect on 5-HT turnover (5-HIAA/5-HT ratio). Meloxicam and dexamethasone did not significantly decrease 5-HT and 5-HIAA content. Cisplatin significantly decreased monoamine oxidase activity but increased tryptophan hydroxylase (TPH) activity and TPH(1) mRNA expression in ileal tissue. Meloxicam and dexamethasone significantly restored the decreased monoamine oxidase activity and inhibited the cisplatin-induced increase in tryptophan hydroxylase activity toward the control levels. These drugs also decreased the cisplatin-induced increase in TPH(1) mRNA expression. Neither cisplatin nor the anti-inflammatory drugs had significant effect on mRNA expression of the serotonin re-uptake transporter. These results suggest that the inflammatory response associated with cyclooxygenase-2 induction is involved in the opposite change in ileal tryptophan hydroxylase and monoamine oxidase activities in the delayed phase after single administration of cisplatin to rats.

Methotrexate causes a change in intestinal 5-hydroxytryptamine metabolism in rats.

The effects of methotrexate on 5-hydroxytryptamine (5-HT) metabolism in the intestinal tissue of rats were investigated during the delayed phase after a single administration. Rats were i.p. injected with methotrexate or with saline as a control, and kaolin and food intakes were measured by an automatic monitoring apparatus. At 96 h after administration, dissected-out ileal tissue was frozen rapidly in liquid nitrogen for further analysis or fixed for immunohistochemical staining. Methotrexate at a dose of 50 mg/kg caused a time-dependent increase in kaolin intake lasting up to 72 h after administration, which returned to the control level at 96 h after administration. This dose of methotrexate caused a gradual decrease in body weight, food intake, and water intake lasting up to 72 h, which approached the control level at 96 h. Methotrexate caused pathologic changes, including a moderate inflammatory response in the ileal tissue and an increase in the number of L-tryptophan hydroxylase (TPH)-expressing cells in the ileal mucosa. Methotrexate also caused a significant increase in 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) content and in TPH1 mRNA expression in the ileal tissues. It had no significant effects on mRNA expression of serotonin transporter, COX-1, or COX-2 or on myeloperoxidase activity. This study demonstrated, for the first time, that methotrexate caused a change in the ileal 5-HT metabolism associated with hyperplasia of mucosal enterochromaffin cells.

Enhanced inhibitory effect of 5-hydroxytryptamine on nitric oxide production by vascular smooth muscle cells derived from stroke-prone spontaneously hypertensive rats

We investigated the effect of 5-hydroxytryptamine (5-HT) on nitric oxide (NO) production by vascular smooth muscle cells derived from 7–8 weeks old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched normotensive Wistar Kyoto rats (WKY). 5-HT significantly inhibited NO production and inducible NO synthase (iNOS) expression induced by interleukin-1β (IL-1β), which effect was greater in SHRSP cells than in WKY cells. The inhibitory effect of 5-HT was mimicked by α-methyl-5-HT, a 5-HT2 receptor agonist, but not by 5-HT1, 5-HT3 or 5-HT4 receptor agonists. 5-HT inhibition of NO production was dose-dependently reversed by sarpogrelate, a 5-HT2 receptor antagonist. Staurosporin, a protein kinase C (PKC) inhibitor, dose-dependently reversed the inhibitory effect of 5-HT, and phorbol 12-myristate 13-acetate, a PKC activator, dose-dependently inhibited the NO production in both WKY and SHRSP cells. Thus, 5-HT had an inhibitory effect on NO production and iNOS expression by vascular smooth muscle cells via 5-HT2 receptor subtype involving PKC activation, which effect was greater in SHRSP cells than in WKY cells. The enhanced inhibitory effect of 5-HT may have a pathophysiological relevance to vascular diseases in SHRSP.

Cellular function and signaling pathways of vascular smooth muscle cells modulated by sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P) plays important roles in cardiovascular pathophysiology. S1P1 and/or S1P3, rather than S1P2 receptors, seem to be predominantly expressed in vascular endothelial cells, while S1P2 and/or S1P3, rather than S1P1 receptors, seem to be predominantly expressed in vascular smooth muscle cells (VSMCs). S1P has multiple actions, such as proliferation, inhibition or stimulation of migration, and vasoconstriction or release of vasoactive mediators. S1P induces an increase of the intracellular Ca2+ concentration in many cell types, including VSMCs. Activation of S1P3 seems to play an important role in Ca2+ mobilization. S1P induces cyclooxygenase-2 expression in VSMCs via both S1P2 and S1P3 receptors. S1P2 receptor activation in VSMCs inhibits inducible nitric oxide synthase (iNOS) expression. At the local site of vascular injury, vasoactive mediators such as prostaglandins and NO produced by VSMCs are considered primarily as a defensive and compensatory mechanism for the lack of endothelial function to prevent further pathology. Therefore, selective S1P2 receptor antagonists may have the potential to be therapeutic agents, in view of their antagonism of iNOS inhibition by S1P. Further progress in studies of the precise mechanisms of S1P may provide useful knowledge for the development of new S1P-related drugs for the treatment of cardiovascular diseases.

Pressure stress reduces inducible NO synthase expression by interleukin-1β stimulation in cultured rat vascular smooth muscle cells

Elevated mechanical stress applied to vascular walls is well known to modulate vascular remodeling. We investigated the effect of pulsatile pressure stress on nitric oxide (NO) production and inducible NO synthase (iNOS) expression by interleukin-1β (IL-1β) stimulation in rat vascular smooth muscle cells (VSMCs). VSMCs were enzymatically isolated from aortic media of Wistar rats. Pulsatile pressure applied to VSMCs was repeatedly given between 80 and 160 mm Hg at a frequency of 4 cycles per min using an original apparatus. Protein expression and activation were evaluated by Western blot analysis. mRNA expression was evaluated by real-time reverse transcription-polymerase chain reaction. The pulsatile pressure reduced IL-1β-induced NO production, iNOS protein, and mRNA expression. The pressure also reduced GTP cyclohydrolase I mRNA expression. Furthermore, the pressure reduced phosphorylation of IL-1β-induced extracellular signal-regulated kinase (ERK), nuclear factor-κB (NF-κB) p65, and I-κBα. The pressure had no effect on I-κBβ degradation by IL-1β stimulation. The present study shows for the first time that pressure stress reduces IL-1β-induced iNOS expression via a mechanism involving the ERK-NF-κB signaling pathway.