Prevention of the expression of inducible nitric oxide synthase by a novel positive inotropic agent, YS 49, in rat vascular smooth muscle and RAW 264.7 macrophages

Abstract

1 The effects of a novel positive inotropic isoquinoline compound, YS 49, on NO production and iNOS protein expression were investigated in cultured rat aortic vascular smooth muscle cells (RAVSMC) and RAW 264.7 cells exposed to lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma). In addition, the effects of YS 49 on vascular hyporeactivity in vitro and ex vivo, and on survival rate (mice) and serum NOx (rat) levels, were also investigated in LPS-treated animals. 2 Pre- or co-treatment of YS 49 with LPS plus IFN-gamma, concentration-dependently reduced NO production in RAVSMC and RAW 264.7 cells (IC50 values, 22 and 30 microM, respectively). Although the inhibitory effect on NO production was reduced when YS 49 was applied 2 and 4 h after cytokine in RAW 264.7 cells, it was still statistically significant (P<0.05). 3 YS 49 reduced iNOS mRNA expression in LPS-treated rat aorta in vitro, an effect which was associated with restoration of contractility to the vasoconstrictor, phenylephrine (PE), and reduction in L-arginine-induced relaxation. 4 Serum NOx levels were significantly (P<0.01) reduced by YS 49 (5 mg kg-1, i.p.) in LPS-treated rats (10 mg kg-1, i.p.). Administration of YS 49 (10 and 20 mg kg-1) 30 min prior to LPS (10 mg kg-1) also significantly (P<0.01) increased the subsequent survival rates in mice. 5 Finally, expression of iNOS protein induced by LPS plus IFN-gamma in RAVSMC and RAW 264.7 cells was suppressed by YS 49, in a concentration-dependent manner. 6 These data strongly suggest that YS 49 suppresses iNOS gene expression induced by LPS and/or cytokines in RAVSMC and RAW 264.7 cells at the transcriptional level. YS 49 could therefore be beneficial in septic shock and other diseases associated with iNOS over-expression.

Figure 1

Chemical structure of YS 49.

Figure 2

Concentration-dependent inhibition of production of nitrite by YS 49 in RAVSMC and RAW 264.7 cells exposed to LPS+INF-γ. LPS (300 ng ml⁻¹ for RAVSMC and 10 ng ⁻¹ for RAW 264.7 macrophages)+INF-γ (10  u  ml⁻¹) were added simultaneously (RAW 264.7 cells) or 1 h before (RAVSMC) YS 49 (1–100  μM), and then incubated for further 18 h. Nitrite was measured by the Griess reaction in 0.5 ml of conditioned medium. Each value represents the mean±s.e.mean of triplicate determinations from a representative experiment performed three separate times with comparable results. ^*Significantly different from all other groups at P<0.05.

Figure 3

The time-dependent effects of YS 49 on NO production in RAW 264.7 cells stimulated with LPS+INFγ. Cells were incubated with LPS (10 ng ml⁻¹)+INF-γ (10 u ml⁻¹), together with YS 49 (30 μM) added at the indicated times, for a total of 18 h. Nitrite was measured by the Griess reagent in 0.5 ml of the conditioned medium. Each value represents the mean±s.e.mean of triplicate determinations from a representative experiment performed three separate times with comparable results. ^*Significantly different from all other groups at P<0.05.

Figure 4

Preventive effect of YS 49 on LPS-induced vascular hyporeactivity ex vivo. Rats were injected (i.p.) with either LPS (10 mg kg⁻¹), YS 49 (5 mg kg⁻¹)+LPS, YS 49 (5 mg kg⁻¹) or saline, and then sacrificed 8 h after injection. Thoracic aortas were removed and isometric responses recorded. Maximum contractile forces (g) of rat thoracic aorta exposed to 10 μM PE are shown (a) The contractile response to PE in aortas taken from LPS-treated rats was significantly (P<0.01) lower than controls, an effect that was reversed by treatment with YS 49 (P<0.01). (b) L-arginine, concentration-dependently relaxed aortas taken from LPS-treated rats. This response was almost entirely absent in aortas taken from YS 49+LPS-treated rats. Aortas from control rats did not relax in response to L-arginine. All aortas were contracted with U 46619. Values are expressed as mean±s.e.mean of three to five separate experiments. ^* and ^**Significantly different from corresponding controls at P<0.05 and P<0.01, respectively.

Figure 5

Concentration-response curves to PE in endothelium-denuded aortic rings. Rings were incubated either with Krebs' solution (control), LPS (300 ng ml⁻¹), or different concentration of YS 49+LPS for 8 h. Values are expressed as contractile force in grams and are means±s.e.mean of three to five separate experiments. ^*Significantly different from all other groups at P<0.05.

Figure 6

Effects of YS 49 on iNOS mRNA expression in rat aortic smooth muscle incubated with LPS (300 ng ml⁻¹). Aortic rings were incubated for 8 h at 37°C with: 1, Krebs' solution; 2, LPS; 3, YS 49 (10 μM)+LPS; 4, YS 49 (30 μM)+LPS; and 5, YS 49 (100 μM)+LPS. After completion of incubation, isometric force to PE was measured and then iNOS mRNA expression analysed in the same tissues. Upper panel: densitometric analysis of the gel photograph. Representative experiment from at least three. Lower panel: mRNA for iNOS and GAPDH.

Figure 7

Effects of YS 49 and other related chemicals on iNOS protein expression in LPS+INF-γ stimulated RAW 264.7 macrophages and RAVSMC. (a) Exposure of RAW 264.7 macrophages to LPS (10 ng ml⁻¹)+INF-γ (10 u ml⁻¹) for 18 h resulted in the expression of a 130 kDa protein. This expression was significantly inhibited by treatment of the cells with YS 49, tetrandrine, higenamine or dexamethasone. Lane 1, control; Lane 2, LPS+INF-γ; Lane 3, YS 49 (10 μM) with LPS+INF-γ; Lane 4, YS 49 (30 μM) with LPS+INF-γ; Lane 5, YS 49 (100 μM) with LPS+INF-γ; Lane 6, dexamethasone (0.1 μM) with LPS+INF-γ; Lane 7, higenamine (10 μM) with LPS+INF-γ; Lane 8, tetrandrine (10 μM) with LPS+INF-γ; Lane 9, iNOS protein. (b) In RAVSMC, exposure to LPS (300 ng ml⁻¹)+INF-γ (10 u ml⁻¹) for 18 h also led to the expression of the 130 kDa protein, which was inhibited concentration-dependently by YS 49. Lane 1, control; Lane 2, LPS+INF-γ; Lane 3, YS 49 (10 μM) with LPS+INF-γ; Lane 4, YS 49 (30 μM) with LPS+INF-γ; Lane 5, YS 49 (100 μM) with LPS+INF-γ. This immunoblot is representative of three separate experiments.

Figure 8

Effects of YS 49 on the survival rate of LPS-treated mice. Each group consisted of 20 animals. Saline was injected (i.p.) in control groups and LPS (20 mg kg⁻¹, i.p.) in LPS-treated groups. Two different doses of YS 49 (10 and 20 mg kg⁻¹) were injected (i.p.) 30 min before the LPS-injection. Survival was monitored every 6 h up to 48 h. ^**Represents significantly different at P<0.01.

Figure 9

Effects of YS 49 on NOx production in rat plasma 8 h after injection of LPS (10 mg kg⁻¹, i.p.). Blood was collected by cardiac puncture using a 21 G injection needle. Samples were centrifuged and serum fraction was analysed for its content of nitrite/nitrate. As control, saline and YS 49 were injected (i.p.) in separate animals. Data represent mean±s.e.mean of three separate experiments. ^*Significantly different at P<0.05.