Shigeru Ishiguro

Stimulation of bradykinin B2-receptors on endothelial cells induces relaxation and contraction in porcine basilar artery in vitro

The aim of the present study was to characterize the subtypes of bradykinin (BK) receptors that evoke the relaxation and contraction induced by BK and to identify the main contracting and relaxing factors in isolated porcine basilar artery by measuring changes in isometric tension and a thromboxane (TX) metabolite. Endothelial denudation completely abolished both responses. [Thi5,8, D‐Phe7]‐BK (a B2‐receptor antagonist) inhibited the BK‐induced relaxation and contraction, whereas des‐Arg9, [Leu8]‐BK (a B1‐receptor antagonist) had no effect. L‐nitro‐arginine (L‐NA, a nitric oxide synthase inhibitor) completely inhibited BK‐induced relaxation. Indomethacin (a cyclo‐oxygenase inhibitor) completely and ONO‐3708 (a TXA2/prostaglandin H2 receptor antagonist) partially inhibited BK‐induced contraction, whereas OKY‐046 (a TXA2 synthase inhibitor) and nordihydroguaiaretic acid (a lipoxygenase inhibitor) did not. In the presence of L‐NA, the contractile response to BK was inhibited by indomethacin or ONO‐3708 and was competitively antagonized by [Thi5,8, D‐Phe7]‐BK (pA2=7.50). In the presence of indomethacin, the relaxant response to BK was inhibited by L‐NA and was competitively antagonized by [Thi5,8, D‐Phe7]‐BK (pA2=7.59). TXA2 release was not induced by BK‐stimulation. These results suggest that the endothelium‐dependent relaxation and contraction to BK in the porcine basilar artery is mediated via activation of endothelial B2‐receptors. The main relaxing factor may be NO and the main contracting factor may be prostaglandin H2.

Magnesium-deficient medium enhances NO production in alveolar macrophages isolated from rats

Magnesium deficiency has been shown to increase nitric oxide (NO) levels in plasma and to aggravate endotoxin lethality. The present study was performed to examine the effects of magnesium (Mg(2+))-deficient culture medium, with and without endotoxin (LPS), on NO release and inducible NOS (iNOS) mRNA levels in alveolar macrophages isolated from rats. Decreasing the Mg(2+) concentration in the culture medium from 0.39 mM (normal-Mg(2+) medium) to 0.021 mM (Mg(2+)-deficient medium) increased NO release from alveolar macrophages for 2 h. However, LPS stimulation in Mg(2+)-deficient medium had little effect on NO release. The increased NO release in Mg(2+)-deficient medium was suppressed completely by L-NAME and aminoguanidine. Dexamethasone, pyrrolidine dithiocarbamate and curcumin strongly inhibited NO release. Verapamil, U73122, TMB-8 and W-7 had no significant effect on NO release induced by Mg(2+) deficiency. Preculture of macrophages with Mg(2+)-deficient medium for 22 h markedly increased NO release and iNOS mRNA levels for a further 2 h; these increments were suppressed completely by curcumin. These results suggest that Mg(2+) deficiency enhances NO production via iNOS by alveolar macrophages. In this experimental condition, we can not suggest that NO production from alveolar macrophage plays an essential role in the pathogenesis of enhanced endotoxin lethality in Mg-deficient rats.

Enhancement of endotoxin-induced vascular hyporeactivity to phenylephrine in the thoracic aortas of Mg-deficient rats ex vivo.

Since endotoxin lethality is enhanced by Mg deficiency in animals, we determined whether endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is enhanced in Mg-deficient rats. Normal and Mg-deficient adult male Wistar rats were injected with Escherichia coli 011: B4 lipopolysaccharide (1 or 5 mg/kg, i.p.). Six h later, rings prepared from their thoracic aortas showed severe hyporeactivity to PE. This was more pronounced in the Mg-deficient rats, and was reversed by in vitro treatment with a highly selective inducible nitric oxide (NO) synthase inhibitor, 1400 W, or a highly selective soluble guanylyl cyclase inhibitor, ODQ. However, reversal required high doses of both inhibitors in Mg-deficient rats. Endotoxemia for 6 h was associated with elevated serum interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha levels, and strong TNF receptor mRNA expression in the abdominal aortas, which were significantly greater in the Mg-deficient rats. Treatment of the thoracic aortas, isolated from control and Mg-deficient rats before endotoxic challenge, with IL-1beta or TNF-alpha for 6 h in vitro caused hyporeactivity to PE, but its severity did not differ significantly between the two groups. These results suggest that high serum IL-1beta and TNF-alpha levels, and increased TNF receptor production in the vascular tissue, contribute to vascular hyporeactivity to PE in endotoxemia, and to its enhancement in Mg-deficient rats, via NO/cGMP signaling.

Vasopressin receptor subtypes on mesenteric and cremasteric arterioles in rat.

We studied the effects of a selective vasopressin V(1A) receptor antagonist [1-(1-(4-(3-acetylaminopropoxy)benzoyl)-4-piperidyl)-3, 4-dihydro-2(1H)-quinolinone (OPC-21268)] and a selective vasopressin V(2) receptor antagonist [5-dimethylamino-1(4-(2-methylbenzoylamino)benzoyl)-2,3,4, 5-tetrahydro-1H-benzazepine (OPC-31260)] on vasopressin-induced contraction of mesenteric and cremasteric arterioles in urethane-anaesthetized rats. Vasopressin was infused intravenously for 60 min or applied topically to arterioles directly. Vasopressin infusion (50, 100 or 500 ng/kg/min) decreased the diameter of both mesenteric and cremasteric arterioles. Vasopressin (500 ng/kg/min)-induced vasoconstriction was antagonized by OPC-21268 (0. 2, 1.0 and 5.0 mg/kg, i.v.), dose-dependently, but not by OPC-31260. Topically applied vasopressin (4.6x10(-10)-4.6x10(-8) M) dose-dependently constricted both microvessels. Pre-administration of OPC-21268 (5.0 mg/kg, i.v.) completely inhibited topically applied vasopressin-induced vasoconstriction in both microvessels, and OPC-31260 partially inhibited it in cremasteric arterioles. These results suggest that vasopressin induces vasoconstriction in rat mesenteric and cremasteric arterioles mainly by stimulating vasopressin V(1A) receptors, while vasoconstriction in cremasteric arterioles is partly associated with stimulation of vasopressin V(2) receptors.

In Vitro Application of Endotoxin to Thoracic Aortas from Magnesium-Deficient Rats Enhances Vascular Hyporeactivity to Phenylephrine

Objective: Endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is well described in rat aortas, but has not been investigated in those from magnesium (Mg)-deficient rats in vitro. Methods: Segments of thoracic aorta from control and Mg-deficient rats were incubated in culture medium for 6 hours in the presence or absence of bacterial lipopolysaccharide (LPS; 0.001–10 μg/mL). Contractions to PE were measured with or without an inducible nitric oxide synthase (iNOS) inhibitor (1400W; 0.1 and 1 μM), a guanylate cyclase inhibitor (ODQ; 0.1 and 1 μM), or a potassium channel inhibitor (TEA; 1 and 10 mM). Results: LPS induced hyporeactivity in a concentration-dependent manner under relatively low concentrations (0.001–0.1 μg/mL), however, there was no significant difference at 0.1, 1 and 10 μg/mL. LPS-induced hyporeactivity was not significantly affected by endothelium-denudation. The hyporeactivity was enhanced in thoracic aortas from Mg-deficient rats by LPS (0.01, 0.1 and 1 μg/mL). LPS (1 μg/mL) induced hyporeactivity was reversed with 1400W, ODQ or TEA in both aortas in a concentration-dependent manner, however the degree of reversal was weaker in the Mg-deficient rat aorta than in the control rat one. iNOS mRNA level was increased by LPS (0.1 μg/mL) and the increment was significantly high in Mg-deficient rat thoracic aorta. Conclusions: From these results it is clearly demonstrated that LPS-induced vascular hyporeactivity to PE is enhanced in thoracic aorta from Mg-deficient rats, and it is suggested that LPS-induced NO production might contribute to the enhancement via stimulation of NO-cyclic GMP-potassium channel pathway.