No abstract

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...

Background-Although dysfunction of VE-cadherin-mediated adherence junctions in vascular endothelial cells (ECs) is thought to be one of the initial steps of atherosclerosis, little is known regarding how VE-cadherin is disrupted during atherogenic development. This study focused on the role of calpain, an intracellular cysteine protease, in the proteolytic disorganization of VE-cadherin and subsequent progression of atherosclerosis. Methods and Results-Increased expression of m-calpain was observed in aortic ECs in atherosclerotic lesions in humans and low-density lipoprotein receptor-deficient (ldlr Ϫ/Ϫ ) mice. Furthermore, proteolytic disorganization of VE-cadherin was shown in aortic ECs in ldlr Ϫ/Ϫ and apolipoprotein E-deficient (apoE

No abstract

Mitochondrlal free Ca 2+ may regulate mitochondrial ATP production during cardiac exercise. Here, using laser scanning confocal microscopy of adult rabbit cardiac myocytes co-loaded with Flue-3 to measure free Ca 2+ and tetramethylrhodamine methylester to identify mitochondria, we measured ! 2+ cytosolic and mitochondrla Ca transients during the contractile cycle. In resting cells, cytosolle and mitochondrlal Fiu,r~.3 signals were similar. During electrical pacing, transients of Flue-3 fluorescence occurred in both the cytosolic and mitochondrlal compartments. Both the mitochondrlal~ and the cytosollc transients were potentiated by isoproterenol. These experiments show directly that mitochondrlal free Ca 2+ rises and falls during excitation-contraction coupling in cardiac myocytes an6 that changes of mitochondrlal Ca z+ are kinetically competent to regulate mitochondrlal metabolism on a beat-to-beat basis.

No abstract

Background and Purpose-Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. Methods-The impact of rt-PA-and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. Results-Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion.The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1␤, TNF-␣, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 g/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. Conclusions-Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs. (Stroke. 2011; 42:1097-1104.)

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...