Prolylcarboxypeptidase (PRCP) as a new target for obesity treatment

Recently, we serendipitously discovered that mice with the deficiency of the enzyme prolylcarboxypeptidase (PRCP) have elevated α-melanocyte-stimulating hormone (α-MSH) levels which lead to decreased food intake and weight loss. This suggests that PRCP is an endogenous inactivator of α-MSH and an appetite stimulant. Since a modest weight loss can have the most profound influence on reducing cardiovascular risk factors, the inhibitors of PRCP would be emerging as a possible alternative for pharmacotherapy in high-risk patients with obesity and obesity-related disorders. The discovery of a new biological activity of PRCP in the PRCP-deficient mice and studies of α-MSH function indicate the importance and complexity of the hypothalamic pro-opiomelanocortin (POMC) system in altering food intake. Identifying a role for PRCP in regulating α-MSH in the brain may be a critical step in enhancing our understanding of how the brain controls food intake and body weight. In light of recent findings, the potential role of PRCP in regulating fuel homeostasis is critically evaluated. Further studies of the role of PRCP in obesity are much needed

Effect of Continuous Phorbol Ester Treatment on Muscarinic Receptor-Mediated Calmodulin Redistribution in SK-N-SH Neuroblastoma Cells

Stimulation of muscarinic receptors by carbachol and activation of protein kinase C elicits the translocation of calmodulin (CaM) from membranes to cytosol in the human neuroblastoma cell line SK-N-SH. Our previous studies have suggested a role for protein kinase C in the regulation of CaM redistribution. To explore further the role of protein kinase C in carbachol-induced calmodulin translocation, we treated cells for 17 h with 12- O -tetradecanoylphorbol 13-acetate (TPA) to down-regulate protein kinase C isozymes or 72 h to differentiate the cells. Treatment of SK-N-SH cells for 17 h with 70 n M TPA nearly abolished the effect of carbachol on CaM redistribution. After 72 h of TPA, however, the cells appeared differentiated, and the ability of carbachol to increase cytosolic CaM levels was restored. In untreated control cells, the carbachol-mediated increase in cytosolic CaM content was mimicked by TPA and blocked by pretreatment with the selective protein kinase C inhibitor Ro 31-8220 at 10 µ M . In the 72-h TPA-treated cells, however, the ability of TPA to increase cytosolic CaM levels was significantly reduced, and the action of carbachol was no longer blocked by Ro 31-8220. The effect of prolonged TPA treatment on select protein kinase C isozymes was examined by immunoblotting. Treatment of cells for either 17 or 72 h abolished the Α-isozyme in the cytosol and reduced (17 h) or abolished (72 h) the content in the membranes. In both 17- and 72-h TPA-treated cells, the ε-isozyme was nearly abolished in the cytosol and slightly reduced in the membranes. Some protein kinase C activity may have been maintained during TPA treatment because the basal level of phosphorylation of the protein kinase C substrate myristoylated alanine-rich C kinase substrate was enhanced in cells treated for either 17 or 72 h with TPA. The potential dissociation of carbachol and protein kinase C in eliciting increases in cytosolic CaM content was a function of prolonged TPA treatment and not differentiation per se because carbachol-mediated increases in cytosolic CaM levels were inhibited by Ro 31-8220 in retinoic acid-differentiated SK-N-SH cells. This study demonstrates that continuous TPA treatment, although initially down-regulating the protein kinase C-mediated effect of carbachol on CaM redistribution, uncouples carbachol and protein kinase C at longer times.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65656/1/j.1471-4159.1997.68010040.x.pd

Cytosolic Calmodulin Is Increased in SK-N-SH Human Neuroblastoma Cells Due to Release of Calcium from Intracellular Stores

Muscarinic receptor stimulation elicits a redistribution of calmodulin (CaM) from the membrane fraction to cytosol in the human neuroblastoma cell line SK-N-SH. Increasing the intracellular Ca 2+ concentration with ionomycin also elevates cytosolic CaM. The aim of this study was to investigate the roles of extracellular and intracellular Ca 2+ pools in the muscarinic receptor-mediated increases in cytosolic CaM in SK-N-SH cells. Stimulus-mediated changes in intracellular Ca 2+ were monitored in fura-2-loaded cells, and CaM was measured by radioimmunoassay in the 100,000- g cytosol and membrane fractions. The influx of extracellular Ca 2+ normally seen with carbachol treatment in SK-N-SH cells was eliminated by pretreatment with the nonspecific Ca 2+ channel blocker Ni 2+ . Blocking the influx of extracellular Ca 2+ had no effect on carbachol-mediated increases in cytosolic CaM (168 18% of control values for carbachol treatment alone vs. 163 28% for Ni 2+ and carbachol) or decreases in membrane CaM. Similarly, removal of extracellular Ca 2+ from the medium did not affect carbachol-mediated increases in cytosolic CaM (168 26% of control). On the other hand, prevention of the carbachol-mediated increase of intracellular free Ca 2+ by pretreatment with the cell-permeant Ca 2+ chelator BAPTA/AM did attenuate the carbachol-mediated increase in cytosolic CaM (221 37% of control without BAPTA/AM vs. 136 13% with BAPTA/AM). The effect of direct entry of extracellular Ca 2+ into the cell by K + depolarization was assessed. Incubation of SK-N-SH cells with 60 m M K + elicited an immediate and persistent increase in intracellular free Ca 2+ concentration, but there was no corresponding alteration in CaM localization. On the contrary, in cells where intracellular Ca 2+ was directly elevated by thapsigargin treatment, cytosolic CaM was elevated for at least 30 min while particulate CaM was decreased. In addition, treatment with ionomycin in the absence of extracellular Ca 2+ , which releases Ca 2+ from intracellular stores, induced an increase in cytosolic CaM (203 30% of control). The mechanism for the CaM release may involve activation of the isozyme of protein kinase C, which was translocated from cytosol to membranes much more profoundly by thapsigargin than by K + depolarization. These data demonstrate that release of Ca 2+ from the intracellular store is important for the carbachol-mediated redistribution of CaM in human neuroblastoma SK-N-SH cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66213/1/j.1471-4159.1998.70010139.x.pd

Recombinant prolylcarboxypeptidase activates plasma prekallikrein

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106127/1/jth03969.pd

Scupa Or Fxii Stimulate Erk1/2 Or Akt Through Upar And Beta 1 Integrins

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106134/1/jth00092.pd

Mapping the interaction between uPAR and high molecular weight kininogen

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106070/1/jth03972.pd

A monoclonal antibody against kininogen reduces inflammation in the HLA-B27 transgenic rat

The human leukocyte antigen B27 (HLA-B27) transgenic rat is a model of human inflammatory bowel disease, rheumatoid arthritis and psoriasis. Studies of chronic inflammation in other rat models have demonstrated activation of the kallikrein–kinin system as well as modulation by a plasma kallikrein inhibitor initiated before the onset of clinicopathologic changes or a deficiency in high-molecular-mass kininogen. Here we study the effects of monoclonal antibody C11C1, an antibody against high-molecular-mass kininogen that inhibits the binding of high-molecular-mass kininogen to leukocytes and endothelial cells in the HLA-B27 rat, which was administered after the onset of the inflammatory changes. Thrice-weekly intraperitoneal injections of monoclonal antibody C11C1 or isotype IgG(1 )were given to male 23-week-old rats for 16 days. Stool character as a measure of intestinal inflammation, and the rear limbs for clinical signs of arthritis (tarsal joint swelling and erythema) were scored daily. The animals were killed and the histology sections were assigned a numerical score for colonic inflammation, synovitis, and cartilage damage. Administration of monoclonal C11C1 rapidly decreased the clinical scores of pre-existing inflammatory bowel disease (P < 0.005) and arthritis (P < 0.001). Histological analyses confirmed significant reductions in colonic lesions (P = 0.004) and synovitis (P = 0.009). Decreased concentrations of plasma prekallikrein and high-molecular-mass kininogen were found, providing evidence of activation of the kallikrein–kinin system. The levels of these biomarkers were reversed by monoclonal antibody C11C1, which may have therapeutic potential in human inflammatory bowel disease and arthritis

Kinin B1 Receptor Enhances the Oxidative Stress in a Rat Model of Insulin Resistance: Outcome in Hypertension, Allodynia and Metabolic Complications

BACKGROUND: Kinin B(1) receptor (B(1)R) is induced by the oxidative stress in models of diabetes mellitus. This study aims at determining whether B(1)R activation could perpetuate the oxidative stress which leads to diabetic complications. METHODS AND FINDINGS: Young Sprague-Dawley rats were fed with 10% D-Glucose or tap water (controls) for 8-12 weeks. A selective B(1)R antagonist (SSR240612) was administered acutely (3-30 mg/kg) or daily for a period of 7 days (10 mg/kg) and the impact was measured on systolic blood pressure, allodynia, protein and/or mRNA B(1)R expression, aortic superoxide anion (O(2)(*-)) production and expression of superoxide dismutase (MnSOD) and catalase. SSR240612 reduced dose-dependently (3-30 mg/kg) high blood pressure in 12-week glucose-fed rats, but had no effect in controls. Eight-week glucose-fed rats exhibited insulin resistance (HOMA index), hypertension, tactile and cold allodynia and significant increases of plasma levels of glucose and insulin. This was associated with higher aortic levels of O(2)(*-), NADPH oxidase activity, MnSOD and catalase expression. All these abnormalities including B(1)R overexpression (spinal cord, aorta, liver and gastrocnemius muscle) were normalized by the prolonged treatment with SSR240612. The production of O(2)(*-) in the aorta of glucose-fed rats was also measured in the presence and absence of inhibitors (10-100 microM) of NADPH oxidase (apocynin), xanthine oxidase (allopurinol) or nitric oxide synthase (L-NAME) with and without Sar[D-Phe(8)]des-Arg(9)-BK (20 microM; B(1)R agonist). Data show that the greater aortic O(2)(*-) production induced by the B(1)R agonist was blocked only by apocynin. CONCLUSIONS: Activation of kinin B(1)R increased O(2)(*-) through the activation of NADPH oxidase in the vasculature. Prolonged blockade of B(1)R restored cardiovascular, sensory and metabolic abnormalities by reducing oxidative stress and B(1)R gene expression in this model