Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells

Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade ¹²⁵I-ANP in the presence of EDTA. In this study, we have characterized this degradation of ¹²⁵I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a K_m of 320 ± 60 nM and V_max of 35 ± 14 pmol of ANP degraded/10 min/10⁵ cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0–8.5. Potato carboxypeptidase inhibitor and bestatin inhibited ¹²⁵I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when ¹²⁵I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe²⁶ and tyr²⁸ were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC₅₀ of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.     

Feeding in Euchlanis dilatata lucksiana Hauer on filamentous cyanobacteria and a prochlorophyte

Ingestion and assimilation rates of Euchlanis dilatata lucksiana Hauer, isolated from Lake Loosdrecht (The Netherlands) and cultured on lake water (seston < 33 µm), were measured in the laboratory using the ¹⁴C-tracer technique. The five taxa used as tracer foods, together with 6–7 mg C l^–1 of lake seston in each case, included four species of filamentous cyanobacteria (Oscillatoria redekei, O. limnetica, Aphanizomenon flos-aquae, Anabaena PCC 7120) and a prochlorophyte (Prochlorothrix hollandica). Except Anabaena, they are all commonly encountered in eutrophic Loosdrecht lakes, including Lake Loosdrecht, and their dimensions ranged between 150 and 250 µm in length and 2 and 3.5 µm in width. The small and large Euchlanis used as experimental animals had mean lengths of 217–223 µm and 276–305 µm, respectively. Euchlanis fed on all the taxa offered as food. Clearance rates ranged from 51 to 99 µl ind^–1 d^–1 for the large animals and from 22 to 41 µl ind^–1 d^–1 for the small animals. The highest ingestion rate observed, 1.7 µg ind^–1 d^–1, was for the large animals feeding on Aphanizomenon. The daily ration for both size classes far exceeded 100% of body weight, reaching up to 690% for the small animals feeding on Aphanizomenon. The small animals generally appeared to assimilate the ingested food more efficiently (assimilation efficiencies: 37–100%) than the large animals (34–77%). Compared with an earlier study in which only lake seston (<33 µm) was used as food, the specific clearance rates of Euchlanis on the cyanobacteria and Prochlorothrix were generally somewhat lower.     

The effects of posterolateral spine length and body length on feeding rate in the rotifer,Brachionus calyciflorus

The length of the posterolateral spines in the rotifer, Brachionus calyciflorus, is variable. The effect of the length of these spines on the clearance rates of B. calyciflorus was examined under laboratory conditions using radioactively-labelled yeast (Rhodotorula) and bacteria (Aerobacter). The hypothesis that individuals with longer spines have higher clearance rates than those of similarly-sized individuals with shorter spines was rejected. The effect of body length (L) on the clearance rates (C) of short-spined and long-spined animals was also examined. Statistically significant linear regressions of the form log₁₀ C = aL + b, were derived for both types. The slopes of both regressions were similar and suggest that clearance rate in B. calyciflorus increases as a cube function of body length.     

Lipoic acid attenuates hypertension and improves insulin sensitivity,kallikrein activity and nitrite levels in high fructose-fed rats

Chronic feeding of fructose to normal rats causes impaired glucose tolerance, loss of tissue sensitivity to insulin, hyperinsulinemia and hypertension. -Lipoic acid (LA), a co-enzyme known for its potent antioxidant effects, stimulates insulin-mediated glucose uptake in clinical and experimental diabetes. The purpose of this study was to examine whether LA can mitigate fructose-induced insulin resistance and associated abnormalities. Male Wistar rats of body weights 150–170 g were divided into 4 groups containing 12 rats each. Control rats received a control diet containing starch and water ad libitum. Fructose rats received a fructose-enriched diet (>60% of total calories). Fructose + LA rats received a fructose diet and LA (35 mg/kg b.w.) intraperitoneally. Control + LA rats received a normal diet and LA (35 mg/kg b.w.) intraperitoneally. After the treatment period of 20 days, blood pressure (BP) was measured. Oral glucose-tolerance test, insulin-sensitivity index, urea and creatinine clearance tests, and plasma and urinary sodium and potassium levels were analysed. Kallikrein activity and nitrite content were assayed. Additionally, the activities of RBC-membrane Na⁺/K⁺ ATPase and Ca²⁺ ATPase enzymes were assayed. Fructose rats showed increased BP, decreased glucose tolerance, decreased insulin sensitivity and altered sodium and potassium levels and renal clearance. LA supplementation mitigated these alterations. The increase in BP was attenuated and the levels of biochemical parameters were brought close to normal. The BP-lowering effect of LA in fructose rats may be related to improvement in insulin sensitivity.Communicated by L.C.-H. Wang.     

Immunology of Apoptosis and Necrosis

A complex of reactions regulating the number of cells in organs and tissues under normal and pathologic conditions is one of the most important systems of multicellular organisms. In this system, which controls both cell proliferation and clearance, clearance has been given special attention during the last three decades. Some stages of the clearance are known (the choice of "unwanted" cells, their destruction not affecting the surrounding tissue, and, finally, removal of the corpses), and undeniable progress has been achieved in the understanding of the second stage mechanisms, whereas mechanisms of elimination per se of cells or their fragments still continue to be terra incognita. The clearance of such cells is mainly determined by different components of natural and adaptive immunity: phagocytes, complement, opsonins, antigen-presenting cells, etc. Recently specific "danger signals", such as hydrolases, DNA, heat shock proteins, and other potential immunogens released by cells during their elimination have been discovered. Entering the extracellular space, these signals induce inflammation and injury of the surrounding tissues, i.e., autoimmune reactions. Heat shock proteins, in addition to chaperon activity, act as signaling, costimulating, and antigen-carrying molecules in the interactions of dying cells and the immune system.     

Rapid simultaneous determination of metabolic clearance of multiple compounds catalyzed in vitro by recombinant human UDP-glucuronosyltransferases

The purpose of this study was to test the applicability of n-in-one (cocktail) incubations in the determination of intrinsic clearance (Cl(int)) as the slope of the linear portion of the Michaelis-Menten curve (velocity V vs. substrate concentration [S]) where substrate concentrations were low. A rapid, sensitive, and selective liquid chromatography tandem mass spectrometry (LC/MS/MS) method was developed for the analysis of samples produced by single-substrate and n-in-one (seven substrates: entacapone, 17beta-estriol, umbelliferone, 4-methylumbelliferone, tolcapone, hydroxyquinoline, and paracetamol) incubations conducted in 96-well plates with different recombinant UDP-glucuronosyltransferases (UGTs). The Cl(int) values obtained with n-in-one incubations were compared with those obtained in single-compound incubations and with V(max)/K(m) values determined by estimating the enzyme kinetic parameters V(max) and K(m) from the Michaelis-Menten curve. When substrate concentrations were well below their K(m) values, Cl(int) values determined as the slope of the linear part of the Michaelis-Menten fitting correlated well with the values determined as V(max)/K(m) ratios from the Michaelis-Menten curve. The correlation between Cl(int) values determined in single-substrate and n-in-one incubations was high as well. Together, the n-in-one incubations, the determination of Cl(int) values as the slope of the linear part of the Michaelis-Menten fitting, and LC/MS/MS as an analytical method proved to be effective approaches for increasing throughput in the first-phase screening of metabolic properties.     

Effects of fluoxetine on the activity of phagocytosis in stressed mice

We studied the effects of 1, 2, 5, 10 and 20 mg/kg of fluoxetine on the activity of phagocytosis in mice subjected to a chronic auditory stressor. Both the in vitro and in vivo activity of phagocytosis, measured using the zymosan-particle uptake method and the carbon clearance test, respectively, were reduced after 2, 4, 8 and 16 days of stress exposure. A partial recovery on the in vivo activity of phagocytosis was found on day 16th. Daily treatment with fluoxetine partially reversed the adverse effects of stress in a dose-dependent manner on both parameters but did not significantly affect the activity of phagocytosis in unstressed mice. Significant differences appeared when fluoxetine was administered at 2 mg/kg. Maximum effect was reached at 5 mg/kg.