Angiotensinase and vasopressinase activities in hypothalamus, plasma, and kidney after inhibition of angiotensin-converting enzyme: basis for a new working hypothesis

Reducing angiotensin II (Ang II) production via angiotensin-converting enzyme (ACE) inhibitors is a key approach for the treatment of hypertension. However, these inhibitors may also affect other enzymes, such as angiotensinases and vasopressinase, responsible for the metabolism of other peptides also involved in blood pressure control, such as Ang 2-10, Ang III, Ang IV, and vasopressin. We analyzed the activity of these enzymes in the hypothalamus, plasma, and kidney of normotensive adult male rats after inhibition of ACE with captopril. Aspartyl- (AspAP), glutamyl- (GluAP), alanyl- (AlaAP) and cystinyl-aminopeptidase (CysAP) activities were measured fluorimetrically using arylamides as substrates. Systolic blood pressure (SBP), water intake, and urine flow were also measured. Captopril reduced SBP and increased urine flow. In the hypothalamus, GluAP and AspAP increased, without significant changes in either AlaAP or CysAP. In contrast with effects in plasma, GluAP was unaffected, AspAP decreased, while AlaAP and CysAP increased. In the kidney, enzymatic activities did not change in the cortex, but decreased in the medulla. These data suggest that after ACE inhibition, the metabolism of Ang I in hypothalamus may lead mainly to Ang 2-10 formation. In plasma, the results suggest an increased formation of Ang IV together with increased vasopressinase activity. In the kidney, there is a reduction of vasopressinase activity in the medulla, suggesting a functional reduction of vasopressin in this location. The present data suggest the existence of alternative pathways in addition to ACE inhibition that might be involved in reducing BP after captopril treatment.     

Angiotensinase activity in mice fed an olive oil-supplemented diet

We evaluated the influence of a diet supplemented with olive oil (20% by weight) (OO) on the activity of glutamyl aminopeptidase (GluAP) and aspartyl aminopeptidase (AspAP), which are involved in angiotensin metabolism. Serum concentrations of total cholesterol and fatty acids were also measured. Animals fed on the OO diet gained significantly more weight than did controls from the second week until the end of the feeding period. Serum total cholesterol concentration was significantly higher in the OO group than in control mice. Total monounsaturated fatty acids increased in OO-fed animals, but total saturated fatty acids decreased. No differences between the two groups were observed for total polyunsaturated fatty acids. Serum from animals fed on the OO diet contained significantly lower proportions of myristic, pentadecanoic, palmitic, palmitoleic, vaccenic, alpha-linolenic, gamma-linolenic, and 11,14-eicosadienoic acids than did serum from control animals. In contrast, the OO group had higher levels of oleic, stearic, and gondoic acids. GluAP activity decreased significantly in the serum of OO-fed animals. In these animals soluble AspAP activity was significantly higher in the testis, and significantly lower in the lung and adrenal, in comparison to controls. Membrane-bound AspAP activity was higher in the testis and atrium, and lower in lung, in the OO group. Soluble GluAP activity was significantly lower in the testis of OO-fed animals. Membrane-bound GluAP activity did not differ between the two groups in any of the tissues analyzed. Serum AspAP and GluAP activities correlated negatively with palmitoleic and vaccenic acid respectively in the OO group. However, no significant correlations were found in the control group. These results may reflect functional changes in the renin-angiotensin system in the serum, adrenal, testis, lung and atrium after feeding with a diet enriched in olive oil.     

Effect of valsartan on angiotensin II- and vasopressin-degrading activities in the kidney of normotensive and hypertensive rats.

Valsartan, a selective antagonist of angiotensin II at the AT(1) receptor subtype, is an efficacious, orally active, blood pressure-lowering agent used in hypertensive patients. Given that aminopeptidases (APs) play a major role in the metabolism of local peptides involved in blood pressure control, studying them helped us to understand cardiovascular control. We studied the effect of valsartan on angiotensin II- (GluAP) and vasopressin- (CysAP) degrading activities in the kidney in the rat model of renovascular hypertension, Goldblatt two-kidney one-clip. GluAP and CysAP in renal cortex and medulla exhibited different responses to hypertension and valsartan treatment. In the renal cortex, GluAP decreased in clipped and non-clipped kidneys of hypertensive animals. However, while hypertension did not affect GluAP in the clipped kidney medulla, the non-clipped kidney exhibited an increase in soluble and a decrease in membrane-bound activity. Valsartan decreased soluble GluAP in the medulla of normotensive and hypertensive animals. In the renal cortex, CysAP activity was mainly downregulated following hypertension. Valsartan decreased soluble CysAP activity in sham-operated, but not in hypertensive animals. The renal medulla showed a significant valsartan-related decreased activity in clipped and non-clipped kidneys of both sham-operated and hypertensive animals. These results suggest a functional relationship between the AT(1) receptor and vasopressin-degrading activity.     

Angiotensinase activity in hypothalamus and pituitary of hypothyroid, euthyroid and hyperthyroid adult male rats.

A local renin-angiotensin system (RAS) that may be involved in their regulatory functions has been identified in hypothalamus and pituitary. Altered thyroid status induces modifications in the secretory function of hypothalamus and pituitary. However, few studies have analyzed the role of the RAS in hypothalamus and, to our knowledge, there is no data on the pituitary RAS during thyroid dysfunction. In the present study, angiotensinase activities (glutamyl, aspartyl and alanyl aminopeptidase: GluAP, AspAP and AlaAP, respectively) were studied in hypothalamus and in the anterior and posterior lobes of pituitary of euthyroid, hypothyroid and hyperthyroid adult male rats. In the anterior pituitary, compared with euthyroid and hyperthyroid rats, hypothyroid animals showed a highly significant increase of GluAP and AspAP activities; the percentage increase in GluAP was markedly higher than the percentage increase in AspAP. This suggests an increased metabolism of angiotensin (Ang) I and Ang II to des-Asp 1-Ang I and Ang III, respectively. We also observed an increase of Ang III-degrading activity (AlaAP) in the hypothalamus of hyperthyroid rats in soluble fraction. Increased Ang I and Ang II metabolism in the anterior pituitary of hypothyroid rats and increased metabolism of Ang III in the hypothalamus of hyperthyroid animals may be related to alterations in the secretory function of hypothalamus and pituitary in these thyroid dysfunctions.     

Oleate, linoleate and cholesterol differently modify aspartyl- and glutamyl-aminopeptidase activities in primary cultures of rat astrocytes

The intake of mono- and polyunsaturated fatty acids has been associated with a minor risk of cardiovascular diseases including hypertension. Changes in levels of fatty acids may also modify the cell activity and may be related with alterations in different regulatory processes. Aminopeptidases are zinc-metalloenzymes which metabolise circulating peptide hormones in several tissues. Glutamyl-aminopeptidase (GluAP) and to a lesser extent, aspartyl-aminopeptidase (AspAP), are related with angiotensin metabolism in the renin-angiotensin system. The present work was designed to study the effect of a range of concentrations (1-100 microM) of oleic and linoleic acids and cholesterol present in the culture medium on the activity of GluAP and AspAP in the culture of rat cerebral cortical astrocytes taken from 21-day-old fetuses. The results showed that oleic acid inhibits, while linoleic acid stimulates the activity of AspAP. Both fatty acids inhibit the activity of GluAP. Cholesterol stimulates the activity of both enzymes. On the basis of these results, a functional link may exit between the effects of fatty acids on hypertension and the modulation of aminopeptidase activity by these compounds in rat astrocytes, as an example of target cell type in the central nervous system.     

Influence of thyroid disorders on kidney angiotensinase activity.

Thyroid disorders affect renal function, which involves changes in local renin angiotensin system (RAS). Angiotensin peptide levels in the tissue are regulated by the activity of several aminopeptidases (AP) known as angiotensinases. The nature and consequences of the thyroid-induced RAS changes are not completely understood. We investigated the relationship between thyroid status (hyper- and hypothyroidism) and several kidney AP actions involved in RAS control. We have determined fluorometrically soluble (SOL) and membrane-bound (M-B) alanylaminopeptidase (AlaAP), glutamylaminopeptidase (GluAP) and aspartylaminopeptidase (AspAP) activity using naphthylamide derivatives as substrates. Sprague-Dawley rats were divided into three groups--control, hyperthyroid, and hypothyroid. Hyperthyroidism was induced by daily subcutaneous injection of L-thyroxin (300 microg/kg/day). Hypothyroidism was induced by continuous administration of methimazole (0.03%) in drinking water. Hypothyroid animals demonstrated a significant increase in SOL and M-B GluAP activity in renal cortex and a decrease in M-B AlaAP compared to euthyroid rats. This result may suggest higher Ang III availability. In hyperthyroid animals, M-B AlaAP and M-B AspAP activity increased significantly, which may suggest increased Ang III to Ang IV metabolism and greater formation of Ang 2-10, respectively. In contrast, no differences were observed between euthyroid and hypothyroid animals for SOL and M-B AP activity in renal medulla. However, hyperthyroid animals demonstrated a significant decrease in SOL and M-B GluAP activity compared to euthyroid rats, which may suggest a greater availability of Ang II in renal medulla. Alterations in angiotensin metabolism may, in part, account for some changes in renal function during thyroid disorders.     

Angiotensinase activities in the kidney of renovascular hypertensive rats

In spite of the well-known contribution of angiotensin II (Ang II) in the pathogenesis of Goldblatt two-kidney one clip (G2K1C) hypertension, the importance of other Ang peptides, such as Ang III, Ang IV or Ang 2-10, is scarcely understood. The functional status of these peptides depends on the action of several aminopeptidases called angiotensinases. The metabolism of Ang III to Ang IV by aminopeptidase M (AlaAP) and of Ang I to Ang 2-10 by aspartyl aminopeptidase (AspAP) was evaluated in the renal cortex and medulla of normotensive (Sham-operated) and hypertensive (G2K1C) rats, treated or not with the AT(1) receptor antagonist valsartan. The results demonstrated a highly significant increase of membrane-bound (MEMB) AlaAP in the cortex of the non-ischemic kidney of G2K1C rats compared with the kidney of normal rats and with the clipped kidney of G2K1C rats. This suggests an increased formation of Ang IV in the non-clipped kidney of G2R1C rats. Valsartan reduced MEMB AlaAP and AspAP activities in the renal cortex of normotensive and in the clipped kidney of hypertensive rats. The reduced metabolism of Ang III may prolong its half-life in valsartan-treated animals. These results suggest a role for AlaAP in renovascular hypertension. In addition, the higher AspAP activity of the renal cortex compared to medulla reflects its relative functional difference between both locations.     

Diurnal opposite variation between angiotensinase activities in photo–neuro–endocrine tissues of rats

Central and peripheral renin–angiotensin systems (RASs) act in a coordinated manner for the physiologic functions regulated by neuroendocrine events. However, whereas the diurnal rhythm of peripheral circulatory and tissue RASs is well known, the circadian behaviour of their components in central photo–neuro–endocrine structures, key elements for the control of circadian rhythms, has been barely studied. In the present study, we analysed the aspartyl- (AspAP) and glutamyl-aminopeptidase (GluAP) (aminopeptidase A) activities, the angiotensinases responsible for the metabolism of Ang I to Ang 2–10 and Ang II to Ang III, respectively, in the retina, anterior hypothalamus and pituitary at different light and dark time-points of a 12:12 h light:dark cycle (7–19 h light), using arylamide derivatives as substrates. The results demonstrated that while retina and pituitary exhibited their highest levels of AspAP activity in the light period and the lowest in the dark one, the contrary occurred in the hypothalamus – the lowest levels were observed in light conditions and the highest in darkness. The outcome for GluAP showed the highest levels in the light period and the lowest in the dark one in the three tissues analysed. In conclusion, changes in angiotensinase activities throughout the daytime may cause changes of their respective substrates and derived peptides and, consequently, in their functions. This observation may have implications for the treatment of hypertension.     

Proteolytic enzymes in human leukemic lymphoid cells. III. Aminopeptidases, angiotensin-converting enzyme, and its inhibitor in cells of different immunological phenotype

Activities of plasma membrane proteinases such as angiotensin-converting enzyme (ACE), aminopeptidases, and dipeptidyl peptidase IV (DPP-IV) were determined in lymphoid cells of various immunological phenotype which were obtained from 30 patients with lymphoproliferative diseases. The enzyme activities significantly varied depending on the immunological phenotype and stage of cell differentiation, but no correlation was found between activities of ACE, DPP-IV, and aminopeptidases in the cells of different type. The cell lysates studied contained at least two classes of aminopeptidases: metal- and sulfhydryl-dependent enzymes. A sulfhydryl-dependent aminopeptidase with activity optimum at pH 8. 5-9.0 was found for the first time and is suggested to be from a poorly studied aminopeptidase family. In addition to ACE, lysates of leukemic T- and B-cells were found to contain an inhibitor of ACE which was not previously described for these cells.     

Localization and characterization of soluble and plasma membrane aminopeptidase activities in Arabidopsis seedlings

We previously demonstrated that N,1-naphthylphthalamic acid is hydrolyzed at the root-hypocotyl transition zone and other regions of Arabidopsis thaliana seedlings, and that this reaction, like NPA-induced growth inhibition, is strongly promoted by blue light. In addition, NPA amidase activity was detected in plasma membrane-enriched fractions obtained from Arabidopsis seedlings. To further investigate this phenomenon, we tested the hypothesis that the arylamidase(s) responsible for NPA hydrolysis may also have aminopeptidase activity. The responses of Arabidopsis seedlings to various aminopeptidase substrates were tested. The hydrolysis of Tyr-, Trp-, Pro- and Gly-Pro-β-naphthylamide aminopeptidase substrates was shown to be histochemically localized at the root-hypocotyl transition zone and other regions where NPA hydrolysis also occurs. Blue light stimulated the in vivo activity of Tyr- and Pro-aminopeptidase activities, and far-red light stimulated the activity of the Trp-aminopeptidase. These same substrates also induced NPA-like growth inhibitory effects. In parallel experiments, aminopeptidase activities were detected in the supernatant and plasma membrane fractions of seedling extracts. The soluble AP activities resemble previously described neutral aminopeptidases with specificity for aromatic residues. The plasma membrane fraction hydrolyzed Tyr-, Trp-, Ala-Pro- and Pro-AP substrates, and also exhibited activity against Phe- and Leu-substrates. Many of the properties of the aminopeptidases, such as pH optima, metal requirements and responses to inhibitors, overlap with those of the previously characterized NPA amidase, suggesting that the latter may represent the combined activities of multiple aminopeptidases.     

Differential responses of midgut soluble aminopeptidases of Helicoverpa armigera to feeding on various host and non-host plant diets

Differential responses of midgut soluble aminopeptidases were studied in Helicoverpa armigera larvae fed on various host (chickpea and pigeon pea) and non-host (bitter gourd and chili) plant diets. Larval growth was significantly reduced by non-host plant diets. Although the serine proteinase activities were inhibited, aminopeptidase activities were significantly increased in the larvae fed on non-host plant diets. Results were qualitatively and quantitatively confirmed with in vivo and in vitro analyses. It was noted that aminopeptidases had given more preference to ApNA than LpNA on non-host plant diets and vice versa on host plant diets. However, optimum pH for aminopeptidase activity (around pH 7.0–8.0) and susceptibility to inhibitors was similar in the larvae fed on host as well as non-host plant diets. These results suggest that H. armigera regulates digestive enzyme levels to obtain better nourishment from the diet and avoid toxicity due to nutritional imbalance. A detailed biochemical and molecular analysis of gut aminopeptidases upon exposure of the insect to a particular diet will highlight their specific roles.     

Effects of changes in hydromineral balance on rat brain aspartyl, arginyl, and alanyl aminopeptidase activities.

In order to examine the possible relationship between the processing and inactivation roles of aminopeptidases and the disruption of water-electrolyte balance, we measured the activities of aspartyl aminopeptidase (Asp-Ap), arginyl aminopeptidase (Arg-Ap) and alanyl aminopeptidase (Ala-Ap) in certain brain areas (hypothalamus, hippocampus, thalamus and brain cortices) and in the pituitary gland in several models of hydrosaline change. The activity of hypothalamic membrane-bound Asp-Ap significantly decreased (more than 50%) following treatments which induced a hypovolemic state. Aminopeptidase M activity (membrane-bound Ala-Ap activity with low sensitivity to puromycin) was also significantly decreased by 53 % in the thalamus of rats under conditions of hypovolemia plus hyperosmolality in comparison to the control group. These results indicate that aminopeptidases in the central nervous system may be involved in the physiological regulation of hydromineral balance.     

Synthesis and structure activity relationships of novel non-peptidic metallo-aminopeptidase inhibitors

Racemic derivatives of 3-amino-2-tetralone were synthesised and evaluated for their ability to inhibit metallo-aminopeptidase activities. New compounds substituted in position 2 by methyl ketone, substituted oximes or hydroxamic acids as well as heterocyclic derivatives were evaluated against representative members of zinc-dependent aminopeptidases: leucine aminopeptidase (E.C. 3.4.11.1), aminopeptidase-N (E.C. 3.4.11.2), Aeromonas proteolytica aminopeptidase (E.C. 3.4.11.10), and the aminopeptidase activity of leukotriene A(4) hydrolase (E.C. 3.3.2.6). Several compounds showed K(i) values in the low micromolar range against the 'one-zinc' aminopeptidases, while most of them were rather poor inhibitors of the 'two-zinc' enzymes. This interesting selectivity profile may guide the design of new, specific inhibitors of target mammalian aminopeptidases with one active site zinc.     

Serum oxidized low density lipoprotein, paraoxonase 1 and lipid peroxidation levels during oral glucose tolerance test.

Increasing evidence suggests that the postprandial state is a contributing factor to the development of atherosclerosis. To evaluate the effects of acute hyperglycemia on the oxidative stress, concentrations of serum-oxidized low density lipoprotein (oxLDL), paraoxonase 1 (PON1), and thiobarbituric acid reactive substances (TBARS) were measured in subjects with normal glucose tolerance (NGT) (n=35), impaired glucose tolerance (IGT) (n=25), and diabetic glucose tolerance (DGT) (n=20). In NGT group, the 2 hours' TBARS and oxLDL levels were not statistically different when compared to baseline, and 2 hours' PON1 activities were higher when compared to baseline (p<0.01). Subjects with IGT and DGT have higher 2 hours' serum TBARS and oxLDL levels than their baseline levels (p<0.01, for each). Baseline oxLDL levels of both IGT and DGT groups were higher than NGT group (p<0.01 and p<0.01, respectively). While there were not any significant differences in 2 hours' versus baseline PON1 activities in the IGT group, the 2 hours' versus baseline PON1 activities in the DGT group were significantly lower (p<0.01). The postchallenge 2 hours' PON1 activities of both IGT and DGT groups were lower than NGT group (p<0.01 and p<0.01, respectively). Baseline oxLDL was positively correlated with 2 hours' glucose (r=0.613, p<0.01) in IGT and DGT groups. PON1 activities were correlated with HDL-cholesterol, total cholesterol, and fasting glucose (r=0.680, r=0.698 and r=0.431, respectively, for each p<0.01) in NGT. In conclusion, oxidative stress occurs at an early stage in diabetes, and protective effects of HDL against atherosclerosis may be dependent on the PON1 activities.     

Chloride-insensitive, glycine-phenylalanine-naphthylamide hydrolysis at neutral pH in human skin fibroblasts

Crude lysosomal preparations from a cultured human skin fibroblast line were found to contain significant levels of a neutral pH hydrolase activity towards glycine--phenylalanine--beta-naphthylamide (NA), a substrate normally used for the assay of lysosomal dipeptidyl aminopeptidase I. However, the activity was chloride ion insensitive, nonlatent, and inhibitable by cationic detergents and amino acids. Assays of substrate selectivity, relative substrate affinity, pH and anion and cation sensitivity indicated the activity to be distinct from dipeptidyl aminopeptidases I (chloride-dependent hydrolysis of Pro-Phe-, Gly-Phe-, Gly-Arg-, and Pro-Arg-NA's at acid pH), II (Lys-Ala-NA hydrolysis), III (Arg-Arg-NA hydrolysis), and IV (Gly-Pro-NA hydrolysis). The lysosomal preparations also contained significant activity towards several amino acid--naphthylamides, notably Arg-NA. Only dipepidyl aminopeptidase I activity showed sensitivity to chloride anions, both dipeptidyl aminopeptidases I and II showed substantial latency, and none of the activities displayed a significant metal cation dependent.     

Relationship between the changes in placental blood flow resistance assessed by Doppler technique and maternal serum placental aminopeptidases, which degrade vaso-active peptides, in pre-eclampsia.

Our study showed that there were statistically significant correlations between the systolic and diastolic ratio (S/D) of maternal uterine or umbilical artery and the levels of maternal serum aminopeptidase activities in pre-eclampsia. Kininase I was positively correlated with the S/D ratios, whereas placental leucine aminopeptidase (P-LAP) and aminopeptidase A were negatively correlated with the S/D ratios. It is known that the increased S/D ratios reflect the increased utero-placental blood flow resistance. Since our previous study showed that placental aminopeptidases degrade vasoactive peptides such as oxytocin, angiotensin and bradykinin, which the fetus actively produces, our present study suggests that the increased vascular resistance in feto-placental circulation in pre-eclampsia is partly controlled by changes in vaso-active peptides, via degradation by placental aminopeptidases.     

Characterisation of aminopeptidase activity in scab mites (Psoroptes spp.)

Soluble and membrane-bound aminopeptidase activities were demonstrated in extracts of P. cuniculi (Delafond). Leucine aminopeptidase (LAP) activity in the soluble fraction of P. cuniculi extracts displayed substrate preference for amino acid derivatives with terminal leucine and methionine over those with acidic, basic or heterocyclic groups. P. cuniculi LAP was inhibited by leucinethiol (IC(50) = 1.4 +/- 0.4 nM), bestatin (IC(50) = 3.9 +/- 1.7 microM), Arphamenine A (IC(50) = 0.37 +/- 0.03 mM) the chelating agent 1,10-phenanthroline (IC(50) = 2.3 +/- 0.5 mM), Zn(2+), Cu(2+) Ni(2+), and Co(2+), and activated by Mn(2+) and Mg(2+). The LAP activity was visualised as a single major band after electrophoresis on native gels and eluted from a size exclusion column as a single major peak representing a molecular mass range of 85-116 kDa. Degenerate oligonucleotide primers were used to amplify short fragments of genomic DNA containing nucleotide sequence coding for the cation-binding motifs of the co-catalytic Zn(2+) binding domains of dizinc leucine aminopeptidases in both P. cuniculi and P.ovis (Hering). The major soluble aminopeptidase from these mites therefore displays most of the characteristics associated with typical cytosolic leucine aminopeptidases belonging to the M17 family of metalloproteinases.     

Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.     

Expression of glutamyl aminopeptidase by osteogenic induction in rat bone marrow stromal cells

Glutamyl aminopeptidase (GluAP, EC 3.4.11.7, ENPEP) is a 130-kDa homodimeric zinc metallopeptidase which specifically cleaves the N-terminal glutamate or aspartate residue of peptidic substrates such as cholecystokinin-8 or angiotensin (Ang) II, in vitro. We used a DNA microarray hybridization (Genechip Rat Expression Array 230A, Affymetrix Inc., Santa Clara, CA, USA) to demonstrate that GluAP was upregulated in osteogenic induced rat bone marrow stromal cells (BMSCs). To compare the expression of GluAP in the osteogenic differentiation and non-osteogenic differentiation of rat BMSCs in vitro, the cells were osteogenic induced in vitro. We also performed an MTT assay, alkaline phosphatase assay, alizarin red staining, and an immunohistochemical analysis to determine the osteogenic differentiation of BMSCs. The expression of GluAP was examined by real-time polymerase chain reaction (PCR). The real-time PCR results showed that GluAP was upregulated in osteogenic differentiated BMSCs in vitro, suggesting that GluAP may be correlated with the osteogenic differentiation of BMSCs.     

Arsenic is decreased in target organs during viral infection in mice

Arsenic (As), a potentially toxic trace element, has been shown to influence viral replication and resistance to microbial infection. However, the impact of infection on the normal As status in target organs involved in the disease process has not been studied to date. In the present study, As was measured through inductively coupled plasma mass spectrometry in the plasma, liver, spleen, kidney, heart, pancreas and brain at days 1 and 3 of coxsackievirus B3 infection in female Balb/c mice. The severity of the infection was assessed from clinical signs of disease. The infection changed plasma As in a biphasic pattern with a small increase (n.s.) at day 1 that turned into a decreasing trend (13%, p<0.05) by day 3. In the liver, spleen, heart, pancreas and kidney As was unchanged at day 1 but, at day 3, it had decreased by 71% (p<0.01), 64% (p<0.01), 55% (p<0.01), 63% (p<0.01) and 73% (p<0.01), respectively. In the brain, As went unchanged. The pathophysiological interpretation of these findings requires further research.