IntroductionGrowth hormone secretagogues (GHSs), which were developed from met-enkephalin based on conformational energy calculations, peptide chemistry, and biological activity, stimulate GH secretion via a specific receptor (1). An intracerebroventricular (ICV) injection of GHS also stimulates food intake in freely feeding rats (2). The GHS receptor (GHS-R) was cloned and found to be a member of the G protein-coupled receptor superfamily (3). The expression of GHS-R mRNA is observed by in situ hybridization or an RNase protection assay mainly in the arcuate nucleus (Arc) and ventromedial nucleus of the hypothalamus and in the pituitary (4). Ghrelin, an endogenous ligand for GHS-R, has recently been isolated from stomach extracts of rats and subsequently cloned in rats and humans (5). Ghrelin-producing cells are found in the hypothalamus as well as in the stomach (5). As expected, ICV administration of ghrelin stimulated GH secretion and food intake in rats (6, 7). Daily peripheral administration of ghrelin caused weight gain by reducing fat utilization in mice and rats (8). However, the physiological role of endogenous ghrelin in the hypothalamus is still unknown.To attenuate GHS-R expression in vivo, we attempted to create transgenic (Tg) rats with impaired GHS-R function in the hypothalamus, especially in the Arc. For this purpose, we used a construct that expresses GHS-R-specific antisense RNA under the control of the promoter for tyrosine hydroxylase (TH). TH is the rate-limiting enzyme in catecholamine biosynthesis and is a marker for the dopaminergic neurons in the hypothalamus. TH-like immunoreactivity is present in most neurons in the ventral part of the Arc that contain GH-releasing hormone (GHRH) (9). GHS-R mRNA hybridizing cells show an extensive overlap with GHRH-expressing neurons (10). These results suggest that a certain number of GHS-R-expressing neurons in the Arc also contain TH. Tg mice bearing a fusion gene containing the TH promoter and the coding region of the human GH gene have been generated, and these Tg mice showed human GH-like immunoreactivity in all the catecholaminergic neurons in the hypothalamus (11). Based on this report, an antisense GHS-R mRNA under the control of the TH promoter would be expected to suppress GHS-R expression in the Arc. Therefore, in the present study we have generated Tg rats that express an antisense GHS-R mRNA under the control of the TH promoter to determine the physiological role of the ghrelin/GHS-R system in the hypothalamus. MethodsGeneration of Tg rats. To construct the antisense GHS-R fusion gene, a synthetic 108-nucleotide DNA fragment spanning the 5′ extracellular region of GHS-R was cloned in the antisense orientation into the vector 4.5THpAL+ (kindly provided by Dona Chikaraishi), which contains 4.5 kb of the upstream region of the rat TH gene (11). The antisense orientation of the cloned fragments was verified by DNA sequencing. A 4.8-kb Growth hormone secretagogues (GHSs) stimulate GH secretion and food intake. GHS receptor (GHS-R) mRNA has b...
Using strips of human basilar arteries mounted in organ chambers to record isometric tension, we investigated vascular reactivity to thrombin and bradykinin. Both agents produced endothelium-dependent relaxation of basilar artery strips precontracted with phenylephrine but had no effect on resting tension in strips with or without endotheUum. The relaxations caused by thrombin were abolished by antithrombin III/heparin, hirudin, and MD805. Thrombin but not bradykinin caused complete tachyphylaxis toward a second exposure. Indomethacin did not inhibit the relaxations induced by thrombin or bradykinin, whereas bromophenacyl bromide and methylene blue did. Aging decreased the relaxation induced by thrombin but did not affect the concentration needed to reach 50% maximal relaxation, nor did it affect the maximal relaxation in response to bradykinin, calcium ionophore A23187, and sodium nitroprusside. Our results suggest that thrombin and bradykinin produce endothelium-dependent relaxations mediated by an endothelium-derived relaxing substance and that the relaxation caused by thrombin is mediated by a proteolytic action on endothelial cells. The decrease in relaxations in response to thrombin with increasing age might be due to a decrease in the number or sensitivity of thrombin receptors on endothelial cells. (Stroke 1990;21:1039-1043) A endothelium-derived relaxing substance, discovered by Furchgott and Zawadski 12 and usually called endothelium-derived relaxing factor (EDRF), mediates endothelium-dependent relaxation in response to a number of vasoactive substances. The relaxation decreases in pathophysiological states, such as hypertension 3 and atherosclerosis, 4 and with increasing age, as found in isolated preparations from dogs 5 and rats. 6 We do not know whether the results in experimental animals are applicable to isolated human arteries since there are few studies on this subject.In isolated human basilar arteries, thrombin and bradykinin have recently been shown to induce endothelium-dependent relaxation. 7 -8 Because both vasodilators are known to be generated during blood coagulation, it is important to consider whether vascular responsiveness to them changes with age. The aim of our study was therefore to
In order to elucidate the mechanism of the antiinflammatory action of baicalein and wogonin, flavonoids from the root of Scutellaria baicalensis, the effects of these compounds were investigated on lipopolysaccharide-induced nitric oxide production in a macrophage-derived cell line, RAW 264.7. Baicalein (5-25 microM) and wogonin (5-50 microM) inhibited lipopolysaccharide-induced nitric oxide generation in a concentration-dependent manner. The inhibitory effect of these compounds was observed only when they were added at the start of cell incubation soon after the stimulation with lipopolysaccharide. Baicalein (25 microM) and wogonin (25 microM) also inhibited protein expression of inducible nitric oxide synthase. This inhibitory effect of wogonin was stronger than that of baicalein, which agrees with the result that wogonin showed stronger inhibition of nitric oxide production than baicalein. These results suggest that baicalein and wogonin attenuate lipopolysaccharide-stimulated nitric oxide synthase induction in macrophages and thus may help to explain the antiinflammatory action of these flavonoid compounds.
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