The histochemical demonstration of brush border endopeptidase.

A histochemical method for the demonstration of a brush border endopeptidase is described based on results of biochemical and histochemical experiments. The substrate of choice is Glut-Ala-Ala-Ala-MNA which displays a very good localization ability and suitable kinetic properties. Km estimated in rat kidney homogenate amounts to 2.35 X 10(-4) M. pH optimum of this endopeptidase associated with the brush border membrane is in the alkaline range. The activity is dependent on the buffer used. In phosphate and cacodylate buffers of pH 7.2 about 30% lower activity in rat kidney and about 25% lower activity in rat small intestine than in Tris-HCl buffer of the same pH was found. The most suitable diazonium salt for the detection "in situ" is Fast Blue B. It inhibits the endopeptidase activity of rat kidney by about 85% at pH 7.2 AND BY ABOUT 55% AT PH 6.0. The best results are obtained in cryostat sections adherent to semipermeable membranes treated with chloroform-acetone before the incubation. A microdensitometric evaluation of the reaction product is possible and results are in good agreement with those of the biochemical determination. When Suc-Ala-Ala-Ala-INA is used as substrate hexazonium-p-rosaniline is the most suitable coupling agent although it inhibits more than Fast Blue B. The reaction using acylated trialanyl naphthylamides as substrates runs in two steps. Endopeptidase sets free Ala-NA which is attacked by aminopeptidase M. Aminopeptidase M is not reaction rate or localization limiting factor because its activity in the brush border is very high and the enzyme is anchored to the cell membrane very closely to endopeptidase. In homogenates of rat kidney and jejunal mucosa the endopeptidase activity was inhibted by EDTA (2X10(-3) M) by 75% in the kidney and by 68% in the jejunum, by DFP (10(-3) M) by 41% in the kidney and by 35% in the intestine, by Mn2+ (5X10(-3) M) by 25% in the kidney and by 30% in the intestine. No inhibition was exerted by E 600. In sections the results were similar. 1,10-phenanthroline (10(-2) M) caused a substantial inhibition. Endopeptidase activity was detected in the brush border of cells of proximal convuluted tubules of the kidney and in the brush border of differentiated enterocytes of the small intestine. In the same species enterocytes display a lower activity than kidney tubular cells. There are species differences in the distribution pattern of endopeptidase in the kidney. In the rabbit and man the positive reaction occurs in the whole cortex. It is distributed unevenly, however. In the rat the tubules of the inner cortex display a very high activity. In the outer cortex straight portions react strongly. In the rabbit kidney cells of the parietal layer of Bowman's capsule display a weak reaction as well. No sex differences were found in the distribution pattern of endopeptidase in the rat kidney. In the intestine of all species examined a proximo-distal gradient was found...     

Indigogenic methods for glycosidases

Summary 4-Cl-5-Br-3-indolyl--D-fucoside (IbF) was tested in histochemical and bio-chemical experiments. Sections from differently treated parts of the small intestine of suckling and adult rats, of jejunum of adult hamsters, guinea pigs, cooks, monkeys, of human jejunal biopsies, of kidney of suckling and adult rats, adult monkeys, guinea pigs and hamsters, and of some other rat organs were used in histochemical experiments. Neutral and acid -D-galactosidases prepared from homogenates of the small intestine of suckling rats by chromatography on Sephadex G 200 were used in biochemical experiments.The recommended medium for histochemical studies consists of 0.1 M citrate phosphate buffer pH 6 (brush border enzyme) and pH 4 (lysosomal enzyme), 3.6·10^–4M IbF and 3.1·10^–3 M potassium ferri- and ferrocyanide.IbF is split quite efficiently by the brush border -D-galactosidase (=lactase) and the recommended histochemical method with IbF at pH 6 is the method of choice in studies concerned with the localization of intestinal lactase. In unfixed cold microtome sections the brush border activity can be easily detected within 15–240 minutes, depending on the lactase activity of the studied sample. In this study this activity was shown to be present in the brush border of enterocytes in the upper part of crypts reaching its maximum in differentiated enterocytes covering the sides and tops of villi in the small intestine of suckling (the highest activity) and adult rats (3–4x lower activity according to the time of appearance of the staining), and of human, monkey and hamster jejunum. In the cock jejunum traces of brush border staining were seen only after 24 hours of incubation. In enterocytes of patients with celiac sprue the brush border activity was very much reduced in dependence on the stage of the disease. Brush border staining along with a diffuse cytoplasmic reaction was found in the proximal convoluted tubules of the monkey kidney. The question of localization of enzyme activity splitting IbF in the monkey kidney deserves further investigation.IbF is also split by isolated intestinal acid -D-galactosidase and histochemically a positive reaction was found in lysosomes of many cells displaying a high activity of -D-galactosidase when IbF was used in the recommended medium of pH 4. The use of aldehyde fixation (glutaraldehyde is to be preferred) is a prerequisite for the assessment of this lysosomal localization. The lysosomal activity splitting IbF is not firmly bound structurally and escapes from cold microtome sections prepared from unfixed tissue samples into the incubation solution.The recommended method is also very suitable for processing zymograms and immunoprecipitation lines of lactase with antisera obtained by Ouchterlony's technic and by immunoelectrophoresis.     

Indigogenic methods for glycosidases

Summary In the histochemical detection of -D-glucosidase the indigogenic method of Pearson et al. was tested and evaluated. 4-Cl-5-Br-3-indolyl--D-glucoside was used as the substrate.Intestinal -D-glucosidase is firmly bound to the structure. About 60% of activity survives 2 hours fixation in cold 4% formaldehyde and some activity can be demonstrated even in paraffin sections after a shortened embedding.The localization obtained with the original method is not correct. Due to a slow oxidation of indoxyl in the acid pH range and to hydrogen peroxide formation indigo is deposited in sites with an active peroxidase or pseudoperoxidase. The addition of horse-radish peroxidase improves the localization but does not entirely prevent diffusion artifacts. A striking improvement of the localization can be achieved by a mixture of ferri-ferrocyanide. 3.1 · 10^–3 M concentration of this oxidation catalyst which is still very effective causes only a 26% inhibition of the enzyme activity as revealed by biochemical assays in homogenates of rat intestine.A new medium was devised consisting of 0.1 M citrate phosphate buffer pH 5.5, 8 · 10^–4 M 4-Cl-5-Br-3-indolyl--D-glucoside and 3.1 · 10^–3 M ferri-ferrocyanide mixture. With this medium a very clear brush border localization of the enzyme activity (activities) in enterocytes of the rat and human intestine was demonstrated. This activity is present in differentiated enterocytes covering the villi. The highest activity resides in the jejunum. Enzyme activity is considerably inhibited by galactonolactone (5 · 10^–3 M) and gluconolactone (4 · 10^–4 M). It is completely inhibited by florizin (5 · 10^–3 M). Cellobiose (8 · 10^–2 M) caused 65%, lactose (8 · 10^–2 M) 48% and glucose (8 · 10^–2 M) 35% inhibition (data were obtained by cytospectrophotometry). In patients with celiac sprue the activity is very much decreased. Its restitution after a gluten-free diet proceeds roughly parallel to that of lactase. The relationship of the demonstrated activity (activities) to florizin hydrolase and lactase is discussed.In the kidney the reaction is very weak and is confined to the cells of proximal convoluted tubules (diffuse staining with some enhancement at the luminal surface of the brush border). The method is also very useful for processing zymograms.     

Detection of different adenosine triphosphatases in human placental brush border membranes

The microvillous membrane of human placental syncytiotrophoblast cells contains a high ATPase activity. The purpose of this study was to characterize this activity and to investigate the presence of vacuolar type H⁺ ATPase in this membrane. Intact brush border membrane vesicles strongly hydrolyzed ATP, reflecting the presence of ATPase on the external side of the membrane. The ATPase activity was entirely Mg²⁺ dependent and increased with pH. At pH 7.5, Vmax was 31.0 ± 1.7 mol/mg/20 min and Km 0.18 ± 0.03 mm ATP. Hydrolysis of ATP was not influenced by the presence of bicarbonate or alkaline phosphatase inhibitors, but at pH 8 it decreased by half following addition of 100 m dicyclohexylcarbodiimide (DCCD). At pH 7.5, 1 mm N-ethylmaleimide (NEM) depressed this activity by less than 5%. Opening the membrane vesicles with 0.1% desoxycholate (DOC) or Triton-X neither revealed any additional ATPase activity nor altered the low sensitivity to NEM. Treatment of these membranes with 1% cholate decreased the ATPase activity by more than 70% and did not enhance the sensitivity of ATP hydrolysis to NEM. 10^–7 m Bafilomycin, which reduced by 56 ± 9% the ATPase activity in dog kidney brash border membranes treated with 0.1% DOC, had no effect on placental brush border membranes subjected to the same procedure. Finally, neither immunocytochemical staining using monoclonal antibody to the Mr 31000 subunit of V-type H⁺ ATPase, nor electron microscopic examination detected the presence of H⁺ ATPase in placental membranes.In conclusion, the placental brush border membrane is the site of a strong ecto ATPase activity which is partially DCCD sensitive. However, our results did not detect the presence of any vacuolar type H⁺ ATPase activity in these membranes.This study was supported by the MRC grant N MA 9565 and by an extramural grant from Baxter Health Care Corporation. The authors are indepted to Dr. Patrick Vinay for this help in providing Bafilomycin and dog kidney membranes, as well as for his insightful discussion.     

Immunocytochemical localization of γ-glutamyl-transferase on isolated renal cortical tubular fragments

Summary The localization of -Glutamyltransferase (-GT, E.C.2.3.2.2.) was studied on isolated tubular fragments from rat kidney cortex immunocytochemically. Monospecific antibodies raised in the goat against rat kidney -GT were used. Antigoat immunoglobulin from the rabbit conjugated with ferritin was used for visualisation of the antibody binding sites. The enzyme was found to be localized at the brush border membrane of proximal tubules, the luminal membrane of distal tubules and collecting duct segments.The enzyme could further be localized on the antiluminal or basolateral cell membranes of proximal and distal tubular fragments, whereas no such localization was verified for collecting duct segments. The role of this basolateral -GT localization in context with the kidney's ability to extract over 83% of the renal arterial glutathione (GSH) input during a single passage is discussed.     

Somatic embryogenesis from immature embryos in meadowfoam (Limnanthes alba)

Developing embryos from immature seeds were excised and cultured. Optimal proliferation of differentiated secondary embryos occurred on Murashige-Skoog media containing 7% sucrose, 0.1 M 2,4-D, and 0.1–1.0 M zeatin. Higher levels of auxins inhibited embryo proliferation. Secondary embryos were subcultured to produce more embryos. The results indicate the feasibility of clonal propagation of meadowfoam.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-Benzyladenine     

Subcellular localization of enterokinase (Enteropeptidase EC 3.4.21.9) in rat small intestine

The subcellular localization of enterokinase is controversial. In this study, enterokinase was extracted from a soluble fraction and a brush border fraction of rat small intestine by differential centrifugation. The soluble fraction contained 41% of the initial enterokinase activity while the brush border fraction contained only 4.6% of the initial activity. In contrast, alkaline phosphatase monitored as a brush border marker, yielded 26.3 in the brush border fraction and only 6% in the soluble fraction. Further separation of the soluble fraction on a Sepharose 4B column revealed three peaks of enterokinase activity. One small peak (3%) of a bound enzyme (M_r, 2·10^?6) and two larger peaks of free enzyme (M_r, 3·10⁵ and 9·10⁵). In contrast, alkaline phosphatase major fraction was in a high molecular weight peak of bound enzyme. When the brush border fraction was chromatographed only a single peak of bound enterokinase and alkaline phosphatase were found. In the lower part of the small intestine, no brush border-bound enterokinase was found, while the peak of alkaline phosphatase was the same as in the upper intestine. These data suggest that enterokinase activity in the rat intestine is mainly in a free form localized in the mucin and soluble fraction and to a negligible extent in the brush border.     

Localization of nucleotide pyrophosphatase in the rat kidney

Summary Hydrolysis of NAD by a nucleotide pyrophosphatase of renal membrane fractions has been reported previously. The aim of the present study was to localize this enzyme in the rat kidney. Nucleotide pyrophosphatase was assayed in glomeruli, in three parts of the proximal tubule and in four parts of the distal tubule dissected form freezedried sections. Nucleotide pyrophosphatase activity, expressed in mol·min^–1·mg protein^–1, ranged between 9.8 and 32.3 in the proximal tubular segments and between 1.1 and 2.7 in the distal tubular segments. It was 3.4 in the glomeruli. The enrichement of the activity during the purification of brush border vesicles was measured. A tenfold higher specific activity was found in the brush border vesicles as compared to the renal cortical homogenates. Thus, most of the renal nucleotide pyrophosphatase appears to be localized in the luminal membrane of the proximal tubule. A permeabilization of the membrane did not increase the activity of brush border vesicles. This indicates that all catalytic sites are accessible at the outer surface of the membrane.Supported by the Swiss National Foundation, grant nr. 3.813.084     

Variations in the distribution pattern of acid and alkaline phosphatase activity in the kidney of some Selachians

Summary Notable species differences in the distribution pattern of acid phosphatase activity are described in the brush border cells in the convoluted tubules (Hauptstück) of Selachian kidney. In representatives of Ord. Rajiformes (Myliobatis aquila L., Raja clavata L., and Torpedo marmorata Risso) a typical droplet staining of acid phosphatase occured throughout the cells. The staining pattern highly resembles the lysosomal activity of the enzyme in the proximal convoluted tubules of the rat kidney. In kidneys of Mustelus laevis Risso and Scyllium stellare Risso, which both belong to the order Squaliformes, however, a dense uniform and rather finely granulated staining occured in the apical zone of the cells. All acid phosphatase positive structures are believed to be lysosomes and their derivates. Topographical coincidence between material stained for acid phosphatase and PAS positive structures is also described.Marked species differences in the brush border alkaline phosphatase activity also described here, correspond to the development of the brush border as revealed by the PAS staining method.Supported by Yugoslav Academy of Sciences and Arts and by grant from the Institute of Biology, University of Zagreb.     

Establishment of regional differences in brush border enzymatic activities during the development of fetal mouse small intestine

Summary In order to study the establishment of regional differences in brush border enzymic activities during the development of fetal mouse small intestine we have followed (1) the differentiation of microvilli by morphometry, and (2) the developmental pattern of three brush border enzymes (lactase, glucoamylase and alkaline phosphatase). From day 16 to day 19 of gestation, the height of duodenal microvilli increases 2.4 times on the absorptive cells located near the tip of the villi. During the same period in the upper half of the duodenal villi, the number of microvilli per square m rises by a factor of 2.4 and the microvillous surface area increases by a factor of 5.2. The differentiation of ileal microvilli follows a similar pattern but they are always shorter and less numerous than those of the duodenum. Lactase activity appears at 18 days of gestation; the other two brush border enzymes are first detected at 16 days of gestation. Afterwards all three enzyme activities increase rapidly and a decreasing gradient of activity is established from the proximal to the distal segment of the small intestine. Hence, the structural development of the microvilli and the appearance of brush border enzyme activities occur simultaneously and a proximo-distal gradient is already established at 16 days of gestation.Supported by MRC of Canada research grant MA-6069Mr. D. Malka was supported by a studentship from the F.C.A.C.Dr. D. Ménard is a chercheur boursier du Conseil de la Recherche en Santé du Québec     

Regulation of cellular Gsα levels and basal adenylyl cyclase activity by expression of the β2-adrenoceptor in neuroblastoma cell lines

Mouse neuroblastoma x rat glioma hybrid NG108-15 and mouse neuroblastoma x embryonic hamster brain NCB20 cells were transfected with a construct containing a human 2 adrenoceptor cDNA under the control of the actin promoter. Clones were selected on the basis of resistance to geneticin sulphate and those expressing a range of levels of the receptor expanded for further study. Membranes from a clone of NG108-15 cells expressing high levels of the receptor (N22) but not one expressing only low levels of the receptor (N17) exhibited a markedly elevated adenylyl cyclase activity when measured in the presence of Mg²⁺ compared to wild type cells. This was not due to elevated levels of the adenylyl cyclase catalytic moiety however as there was no difference in these membranes when the adenylyl cyclase activity was measured in the presence of Mn²⁺. The elevated basal activity was partially reversed by addition of the -adrenoceptor antagonist propranolol. Agonist activation of N22 but not N17 cells led to a large selective down-regulation of cellular G_s levels which was independent of the generation of cyclic AMP. Isoprenaline stimulation of adenylyl cyclase activity and of the specific high affinity binding of [³H] forskolin was achieved with substantially greater potency (some 30 fold) in N22 cell membranes than in N17. By contrast agonist activation of the endogenously expressed IP prostanoid receptor caused stimulation of adenylyl cyclase and stimulation of high affinity [³H] forskolin binding which was equipotent in each of N22, N17 and wild type NG108-15 cells. Agonist activation of the IP prostanoid receptor caused an equivalent degree of G_s down-regulation in each cell type. Expression of an epitope tagged variant of G_s in NG108-15 cells resulted in prostanoid agonist-induced down-regulation of this polypeptide in a manner indistinguishable from that of wild type G_s. Isolation of clones of NCB20 cells expressing high levels of the 2 adrenoceptor also resulted in a specific agonist-induced down-regulation of G_s.     

l-Alanine uptake in brush border membrane vesicles from the gill of a marine bivalve

Summary Brush border membrane vesicles were prepared from mussel gills using differential and sucrose density gradient centrifugation. These vesicles contained both the maximal Na⁺-dependent alanine transport activity found in the gradient and the maximal activities of -glutamyl transpeptidase and alkaline phosphatase. Electron micrographs showed closed vesicles of approximately 0.1–0.5 m diameter. Transport experiments using these vesicles demonstrated a transient 18-fold overshoot in intravesicular alanine concentration in the presence of an inwardly directed Na⁺ gradient, but not under Na⁺ equilibrium conditions. A reduced overshoot (10-fold) was seen with an inwardly directed K⁺ gradient. Further studies revealed a broad cation selectivity, with preference for Na⁺, which was characteristic of alanine transport but not glucose transport in these membranes. The apparent amino acid specificity of the uptake pathway(s) was similar to that of intact gills and supported the idea of at least four separate pathways for amino acid transport in mussel gill brush border membranes. The apparent Michaelis constant for alanine uptake was approximately 7m, consistent with values forK _t determined with intact tissue.     

A Creatine Transporter Is Operative at the Brush Border Level of the Rat Jejunal Enterocyte

Although ergogenic effects and health benefits have been reported for creatine used as nutritional supplement, to date little is known about the mechanism of creatine absorption in the small intestine. Thus the current study was undertaken to elucidate the mechanism of creatine intake in rat jejunum with the use of well-purified brush border membrane vesicles, isolated from jejunal enterocyte. Creatine uptake was found markedly stimulated by inwardly directed Na⁺ and Cl^– gradients, potential-sensitive, strongly reduced by the substitution of Na⁺ and Cl^– with various cations and anions and positively affected by intravesicular K⁺. Moreover, creatine uptake is: 1) significantly inhibited by creatine stuctural analogs, 2) abolished by low concentrations of 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA), 3) saturable as a function of creatine concentration with an apparent Michaelis-Menten constant of 24.08±0.80 M and a maximal velocity of 391.30±6.19 pmoles mg protein^–1 30 s^–1. The transport is electrogenic since at least two Na⁺ and one Cl^– are required to transport one creatine molecule. Western blot analysis showed the same amount of creatine transport protein in the jejunal apical membrane when compared to ileum. Thus, these data demonstrate the existence of a Na⁺- and Cl^–-dependent, membrane potential-sensitive, electrogenic carrier-mediated mechanism for creatine absorption in rat jejunal apical membrane vesicles, which is biochemically and pharmacologically similar to those observed in other tissues. However, in other cell types the stimulatory effect of intravesicular K⁺ was never detected.     

Indigogenic methods for glycosidases

Summary The indigogenic method for -D-galactosidase of Pearson et al. (1963) with 4-Cl-5-Br-3-indolyl--D-galactoside was tested and evaluated.The acid -D-galactosidase is not firmly associated with structures and escapes from cryostat sections prepared in the usual manner into incubation solutions. This leakage cannot be prevented by a short postfixation of these sections in cold acetone or Baker's formol-calcium chloride. The leakage is negligible from frozen sections prepared from tissue blocks fixed 12–24 h in cold Baker's solution or in 3% buffered glutaraldehyde (the latter fixation is preferred). Even if this fixation causes about 70–80% inactivation of acid -D-galactosidase it is a prerequisite for studies concerned with its localization. The brush border -D-galactosidase of enterocytes is more firmly structurally bound. Since its activity against 4-Cl-5-Br-3-indolyl--D-galactoside cannot be proved after overnight fixation in cold aldehyde fixatives its demonstration is to be performed in sections prepared from specimens fixed in cold Baker's solution for 2 h at the most, or in cold microtome sections.The localization obtained with the original method is not correct. The addition of horseradish peroxidase did not result in any improvement of the localization because the employed samples of this peroxidase contained a concomitant -D-galactosidase activity.A striking improvement of the localization was achieved by a mixture of ferri- and ferrocyanide which causes a 40–75% inhibition of acid -D-galactosidase when used in concentrations of 1 · 10^–3 M to 1 · 10^–2 M.A new medium was devised consisting of 0,1 M citrate phosphate buffer pH 3,5–5,5, 8 · 10^–4M 4-Cl-5-Br-3-indolyL--D-galactoside, and 3,1 · 10^–3M potassium ferri- and ferrocyanide. This medium enabled to achieve a very good correlation with biochemical studies and to localize acid and neutral -D-galactosidases in situ.The acid enzyme was demonstrated first of all in lysosomes of many cells. Its activity is inhibited by galactonolactone, lactose and p-chloromercuribenzoate. The nature of the diffuse extralysosomal staining cannot be decided at present. The distribution pattern of this enzyme in many animal organs is given.The neutral -D-galactosidase (lactase) was localized by the improved method in the brush border of differentiated rat, human and monkey enterocytes and is inhibited by galactonolactone, lactose, gluconolactone, and cellobiose. In patients with celiac sprue this activity is very much reduced or absent. It is restituted after a gluten-free diet.Our revised method proved also very useful in processing zymograms and immunoprecipitation lines of -D-galactosidase(s) with homologous antisera obtained by Ouchterlony's technic and by immunoelectrophoresis.     

In vitro induction of nitric oxide by fructose-1,6-diphosphate in the cardiovascular system of rats

Nitric oxide (NO) functions as a cellular messenger in a number of organs and cell systems in the cardiovascular system (CVS); it is a significant determinant of basal vascular tone and regulates myocardial contractility and platelet aggregation. The present study focused upon understanding the in vitro effects of fructose-1,6-diphosphate (FDP) on the rat cellular NO pathway. The iNOS activity was measured by monitoring the formation of (³H)-citrulline in 50,000 g soluble fractions of crude homogenates of endothelial (ET) and smooth muscle cells (SMC) from the arteries of rats, and macrophages (MAC) and lymphocytes (LYM) from rat blood. FDP in concentrations of 10-1000 M stimulated rat cellular iNOS activity in a concentration-dependent manner. FDP-stimulated rat cellular iNOS was found to be completely reversed by 5 M concentration of NG-monomethyl-L-arginine (L- NMMA), the potent mammalian NOS inhibitor. These studies demonstrated that FDP may induce the formation of NO by stimulating rat cardiovascular iNOS activity.     

Mechanisms of Cl− uptake through brush border membranes isolated from the posterior intestine of the freshwater trout,Oncorhynchus mykiss,R.

Summary This paper presents a study of the mechanisms of Cl^– transport through the brush border membranes of the posterior part of the intestine in the freshwater trout, Oncorhynchus mykiss. The mechanisms for Cl^– transport in the posterior intestine are distinct from those in the middle intestine; an inwardly directed pH gradient stimulates Cl^– uptake by bursh border membrane vesicles, indicating a Cl^–/OH^– exchange. A pH-regulated Cl^– conductance is present, which is not activated at normal intracellular pH. Cl^– uptake is stimulated by an outwardly directed HCO ₃ ^– gradient revealing the presence of a Cl^–/HCO ₃ ^– exchange but, conversely, Cl^– is not exchanged against SO ₄ ^2- . In addition, carbonic anhydrase activities have been detected in both the intracellular and extracellular leaflets of the bursh border membranes which favour the establishment of a bicarbonate gradient. A model of Cl^– transport mechanisms through the brush-border membranes of the posterior intestine of the freshwater trout is proposed.Abbreviations BBM brush border membrane - CA carbonic anhydrase - EGTA ethylene-bis(oxyethylenenitrilo)tetra-acetic acid - FW fresh water - Hepes N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulphonic acid - Mes 2-(N-morpholino)ethane sulphonic acid - SITS 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid - TEA triethanolamine - TMA tetramethylammonium - TRIS tris(hydroxymethyl)aminomethane     

The effects of nitric oxide synthesis on the Na+,K+-ATPase activity in guinea pig kidney exposed to lipopolysaccharides

Endotoxins (lipopolysaccharides; LPS) are known to cause multiple organ failure, including renal dysfunction. LPS triggers the synthesis and release of cytokines and the vasodilatör nitric oxide (NO). A major contributor to the increase in NO production is LPS-stimulated expression of inducible nitric oxide synthase (iNOS). This occurs in vasculature and most organs including the kidney. During endotoxemia, NO and superoxide react spontaneously to form the potent and versatile oxidant peroxynitrite (ONOO^–) and the formation of 3-nitrotyrosine (nTyr)-protein adducts is a reliable biomarker of ONOO^– generation. Therefore, the present study was aimed at investigating the role of endogenous nitric oxide in regulating Na⁺,K⁺-ATPase activity in the kidney, and at investigating the possible contribution of reactive nitrogen species (RNS) by measuring of iNOS activity. In addition, the present study was aimed at investigating the relationship between nTyr formation with iNOS and Na⁺,K⁺-ATPase activities. Previously in our study, nTyr was not detectable in kidney of normal control animals but was detected markedly in LPS exposed animals. In this study, kidney Na⁺,K⁺-ATPase activity were maximally inhibited 6 h after LPS injection (P:0.000) and LPS treatment significantly increased iNOS activity of kidney (P:0.000). The regression analysis revealed a very close correlation between Na⁺,K⁺-ATPase activity and nTyr levels of LPS treated animals (r = –0.868, P = 0.001). Na⁺,K⁺-ATPase activity were also negatively correlated with iNOS activity (r = –0.877, P = 0.001) in inflamed kidney. These data suggest that NO and ONOO^– contribute to the development of oxidant injury. Furthermore, the source of NO may be iNOS. iNOS are expressed by the kidney, and their activity may increase following LPS administration. In addition, NO and ONOO^– formation inhibited Na⁺,K⁺-ATPase activity. This results also have strongly suggested that bacterial LPS disturbs activity of membrane Na⁺,K⁺-ATPase that may be an important component leading to the pathological consequences such as renal dysfunction in which the production of RNS are increased as in the case of LPS challenge. (Mol Cell Biochem 271: 107–112, 2005)     

Beneficial effects and mechanism of action of Momordica charantia juice in the treatment of streptozotocin-induced diabetes mellitus in rat

This study investigated the beneficial effects and mechanism of action of the juice of Momordica charantia in streptozotocin (STZ)-induced diabetes mellitus in rats. Diabetes mellitus was associated with significant (p < 0.01) time course reductions in body weight, plasma insulin and the number of insulin positive cells per islet and significant (p < 0.01) time course elevation in blood glucose and osmolarity and systolic blood pressure compared to age-matched healthy controls. Oral intake of M. charantia juice by STZ-induced diabetic rats partially reversed all the diabetes-induced effects measured. Daily oral administration of M. charantia juice to STZ-induced diabetic rates significantly (p < 0.01) reduced the Na⁺- and K⁺ -dependent absorptions of glucose by the brush border membrane vesicles of the jejunum compared to the responses obtained in STZ-induced diabetic rat. Either insulin (100 MM) or the fruit juice lyophilised extract (5 g · ml^–1) can stimulate ¹⁴C-D-glucose uptake in L6 myotubes. These effects were completely blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. High concentrations (10–200 g · ml^-1) of M. charantia juice extract inhibited ¹⁴C-D-glucose uptake in L6 myotubes compared to the control response. The effect of M. charantia treatment was also investigated on myelinated fibre abnormalities in the tibial nerve of STZ-induced diabetic and control rats. The results show that diabetes was associated with significant (p < 0.05) reduction in the mean cross-sectional myelinated nerve fibres, axonal area, myelin area and maximal fibre area compared to end controls. Treatment of STZ-induced diabetic rats with M. charantia juice normalised the structural abnormalities of peripheral nerves. The results indicate that M. charantia can exert marked beneficial effects in diabetic rats, and moreover, it can regulate glucose uptake into jejunum membrane brush border vesicles and stimulate glucose uptake into skeletal muscle cells similar to the response obtained with insulin. (Mol Cell Biochem 261: 63–70, 2004)