Über die Verteilung einiger Hydrolasen in der Rattenniere

Zusammenfassung Die Verteilung der sauren Phosphatase (SPB=nach Barka und Anderson; SPG=nach Gomori), -Glucuronidase (-Glu), Arylsulfatase (AS), -N-Acetylglucosaminidase (NAG), saure 5-Nucleotidase (s5-Nucl), unspezifische Esterase (UE) und der alkalischen Phosphatase (AP) wurden in den Nieren männlicher und weiblicher Ratten mit Hilfe unterschiedlicher Methoden (freischwimmende Gefiermikrotomschnitte, Kryostatschnitte, gefriergetrocknete Schnitte, Azofarbstoffmethode, Metallsalzmethode, Indigogenmethode) und nach verschiedenen Modifikationen (u.a. der Substratkonzentration, des pH, der Temperatur und Inkubationsdauer) untersucht. Ein optimaler Nachweis dieser Enzyme gelingt mit der Inkubation freischwimmender Schnitte nach Standard-Fixierung (2 Std. in Formol-Calcium und anschließend für 18–22 Std. in Formol-Calcium mit 0,88 M Saccharose bei 4°C) von Nierenscheiben (für die SPB und UE jedoch nach Holtscher Fixierung). Ferner sind bei einigen Enzymen für ihre bestmögliche Darstellung folgende Bedingungen einzuhalten: AP und UE Inkubation bei 4° C, End-pH für SPB 5,5, SPG 5,0, UE 6,5, UE als Kuppler Fast Garnet GBC Salt. Für alle Hydrolasen bestehen in der Niere Geschlechtsunterschiede. Bei Weibchen kommt außerdem eine erhöhte Aktivität im Östrus vor. In den S1-Segmenten der juxtamedullären Nephrone reagieren die SPB, s5-Nucl und AP bei Männchen und die SPG bei Weibchen kräftiger als in den S1-Segmenten der übrigen Rinde. In den Markstrahlen sind in den S2-Segmenten weiblicher Ratten die Aktivitäten der UE und s5-Nucl stärker als bei Männchen. Höhere Enzymaktivität weisen die in den Markstrahlen gelegenen S3-Abschnitte für SPG und AP bei Männchen und für NAG und UE bei Weibchen auf. In der Markinnenzone haben die Sammelrohre bei Männchen eine starke -Glu und bei Weibchen eine stärkere NAG-Aktivität. In den cortical gelegenen distalen Tubuli ist die SPB-Reaktion bei Männchen intensiver.

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Distribution of some hydrolases in the rat kidney

Summary Most of the available histochemical methods and techniques (azodye, metal salt and indigogenic methods, cryostat, free-floating and lyophilized section techniques) and different modifications of these methods (different substrate concentrations, pH, temperature, incubation time e.g.) were applied to study the distribution of acid phosphatase (AcPB=after Barka and Anderson; AcPG=after Gomori), -glucuronidase (-Glu), aryl sulfatase (AS), -N-acetylglucosaminidase (NAG), acid 5-nucleotidase (a5-Nucl), non-specific esterase (NE) and alkaline phosphatase (AlP) in the kidneys of rats of both sexes. The optimal conditions for the demonstration of these enzymes were established. As most important proved: the incubation of free-floating sections cut from standard-fixed (2 h in formol-calcium continued for another 18–22 h in the same fixative plus 0.88 M sucrose at 4° C) kidney slices — only for AcPB and NE material fixed after Holt had to be used; the incubation for AlP and NE at 4° C; final pH of the incubation medium for AcPB 5.5, AcPG 5.0 and NE 6.5; the use of Fast Garnet GBC Salt as coupler in the NE azo-dye reaction. Sex differences and for the female rats an increased activity during oestrus were established for all hydrolases studied. In particular the following results were obtained: AcPB, a5-Nucl and AlP are more intensive in male and AcPG in female S1 segments of the juxtamedullary nephrons in relation to the nephrons of the other parts of the cortex. In the medullary rays the NE and the a5-Nucl show a higher activity in the S2 segments of the female rats than in the male ones. The S3 segments of the medullary rays of female rats demonstrate a more intensive activity for NAG and NE. This is true for AcPG and AlP in male rats. In the inner medulla a stronger -Glu activity in male rats and a stronger NAG activity in female rats is observed. The AcPB activity of the cortical distal tubules is higher in male rats.

     

Quantitative distribution of lysosomal hydrolases in the rat nephron.

The activities of N-acetyl-beta, D-glucosaminidase (NAG, EC 3.2.1.30), beta, D-galactosidase (beta-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and beta-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

Extracellular alkaline phosphatase activity in mineralizing matrices of cartilage and bone: ultrastructural localization using a cerium-based method

Summary The ultrastructural localization of alkaline phosphatase (AlP) activity has been demonstrated in epiphyseal growth cartilage and metaphyseal bone of rats. Epiphyso-metaphyseal specimens were decalcified with EDTA and treated with MgCl₂ to regenerate the enzymatic activity before incubation in a medium containing beta-glycerophosphate, MgCl₂ and CeCl₃. AlP activity was present on the outer surface of the plasmamembrane of maturing and hypertrophic chondrocytes and of osteoblasts. Moreover, the reaction product was present in chondrocyte lacunae, in matrix vesicles, and in cartilage matrix, as well as among uncalcified collagen fibrils of osteoid tissue in bone. The intensity of reaction was the lowest, or completely lacking, where the degree of matrix calcification was the highest. These results suggest that alkaline phosphatase is transported from the cells into the cartilage and bone matrix by its association with matrix vesicles and plasmamembrane components, and that its activity in cartilage and bone matrix is inhibited as it is incorporated in the mineral substance.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneously demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3-7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH 7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histoenzymological studies on the distribution of certain phosphatases in the retinae of owlet (Athene brama) and house sparrow (Passer domesticus)

A comparative study on the distribution of alkaline phosphatase (AlP), acid phosphatase (AcP) and 5'-nucleotidase (5-N) amongst the different constituents of retinae of owlet and house sparrow revealed some interesting aspects of the localization of such phosphatases in both the cases. The outer segment of photoreceptors, where light strikes first, are positive for all the phosphatases. Further, areas composed of synapses, reveal activity of the three enzymes. Another interesting aspect is related to the total absence of the activity of AlP and 5-N in the ganglion cells of both the animals. Other sites of phosphatases in various layers have been also identified. The possible metabolic roles of various phosphatases at different sites have been discussed.     

Protective effect of lupeol and lupeol linoleate in hypercholesterolemia associated renal damage

The association between hypercholesterolemia and kidney damage has been well known for last few decades. The oxidative stress and inflammatory responses are involved in renal injury, which is upregulated in hypercholesterolemic condition. The present study is aimed to evaluate the possible effect of lupeol and its ester derivative, lupeol linoleate in renal damage associated with hypercholesterolemic rats. Hypercholesterolemia was induced in male Wistar rats by feeding them with a high cholesterol diet (HCD) comprising normal rat chow supplemented with 4% cholesterol and 1% cholic acid for 30 days. Lupeol and lupeol linoleate were supplemented (50 mg/kg body wt/day) to HCD fed rats during the last 15 days. Increased levels of renal total cholesterol, triglycerides and phospholipids, along with altered serum biochemical parameters of tissue injury indices and elevated activities of renal marker enzymes (lactate dehydrogenase and alkaline phosphatase) were noted in HCD fed rats. Elevated lipid peroxidation levels coupled with decreased antioxidant status (enzymatic and non enzymatic antioxidants) were observed in hypercholesterolemic rats, which indicate the onset of oxidative changes in the renal tissue. Renal lysosomal acid hydrolase activities (ACP, beta-Glu, beta-Gal, NAG and Cat-D) and acute phase proteins like C-Reactive protein and fibrinogen were significantly increased in HCD fed rats, which further indicates the heightening of inflammation. In addition, histopathological findings also confirmed the renal damage in hypercholesterolemic condition. Lupeol and lupeol linoleate effectively reverted the above abnormalities and was comparable with that of the control. These observations highlight the protective effect of lupeol and its ester derivative in ameliorating the renal injury associated with hypercholesterolemia.     

Quantitative distribution of lysosomal hydrolases in the rat nephron

Summary The activities of N-acetyl-,d-glucosaminidase (NAG, EC 3.2.1.30),,d-galactosidase (-gal, EC 3.2.1.23) and acid phosphatase (ac-Pase, EC 3.1.3.2) were measured in the glomeruli, five segments of the proximal and four segments of the distal tubule of normal male Wistar rats. The activities of NAG and-gal are 3- to 5-fold higher in the first part of the proximal tubule than in other segments and very low in glomeruli. We propose that the distribution of these two glycosidases reflects the contribution of the different tubular segments to the reabsorption of glycoproteins. The maximal activity of ac-Pase was found in the straight part of the proximal tubule. It was only 1.5-fold higher than in the distal tubule. Moreover, the activity in glomeruli is rather high. We conclude that ac-Pase is not primarily involved in the handling of reabsorbed molecules.This research was supported by the Swiss National Science Foundation, grant 3.904-077     

Sexual dimorphism of growth plate prehypertrophic and hypertrophic chondrocytes in response to testosterone requires metabolism to dihydrotestosterone (DHT) by steroid 5-alpha reductase type 1

Rat costochondral growth plate chondrocytes exhibit sex-specific and cell maturation dependent responses to testosterone. Only male cells respond to testosterone, although testosterone receptors are present in both male and female cells, suggesting other mechanisms are involved. We examined the hypothesis that the sex-specific response of rat costochondral cartilage cells to testosterone requires further metabolism of the hormone to dihydrotestosterone (DHT). Resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) chondrocytes from male and female Sabra strain rats exhibited sex-specific responses to testosterone and DHT: only male cells were responsive. Testosterone and DHT treatment for 24 h caused a comparable dose-dependent increase in [3H]-thymidine incorporation in quiescent preconfluent cultures of male GC cells, and a comparable increase in alkaline phosphatase specific activity in confluent cultures. RC cells responded in a differential manner to testosterone and DHT. Testosterone decreased DNA synthesis in male RC cells but DHT had no effect and alkaline phosphatase specific activity of male RC cells was unaffected by either hormone. Inhibition of steroid 5alpha-reductase activity with finasteride (1, 5, or 10 microg/ml), reduced the response of male GC cells to testosterone in a dose-dependent manner, indicating that metabolism to DHT was required. RT-PCR showed that both male and female cells expressed mRNAs for steroid 5alpha-reductase type 1 but lacked mRNAs for the type 2 form of the enzyme. Male cells also exhibited 5alpha-reductase activity but activity of this enzyme was undetectable in female cells. These observations show that sex-specific responses of rat growth zone chondrocytes to testosterone requires the further metabolism of the hormone to DHT and that the effect of DHT in the male growth plate is maturation-state dependent. Failure of female chondrocytes to respond to testosterone may reflect differences in testosterone metabolism, since these cells possess greater ability to aromatize the hormone to estradiol.     

Immature rat epididymal epithelial cells grown in static primary monolayer culture on permeable supports

Summary N-acetylglucosaminidase (NAG), acid phosphatase (ACP) and alkaline phosphatase (AKP) were localised histochemically in fixed cells from the 37-day-old rat epididymis grown in static monolayer culture for 2–8 days. ACP and NAG were cytosolic enzymes found in perinuclear positions, whereas staining of AKP was consistent with a membranous position. These enzymes were also examined in frozen tissue sections of the epididymis, from rats of the equivalent age, where NAG had intense activity in both supra- and infra-nuclear cytoplasm and ACP was more active apically. For the first time AKP was localised along basolateral membranes of the epithelium and in the lumen of the mid-caput region. The monolayer in culture was of principal cells only and they maintained their polarity and ultrastructural characteristics, but the height of the cells was reduced compared to that obtained in situ.     

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.     

Norepinephrine levels in the vomeronasal system and their responses to male urine in young and aged female rats

Norepinephrine (NE) levels in brain areas of the vomeronasal system in young (4-5 months) and aged (25-26 months) ovariectomized Sprague-Dawley rats, which were implanted with a 17 beta-estradiol silastic capsule and then exposed to male rat urine, were investigated. The unilateral vomeronasal organ was removed in all rats one week before exposure to urine stimulation. NE levels in the medial nucleus of the amygdala (MA), medial preoptic area (MPOA), ventromedial nucleus of hypothalamus (VMH) and bed nucleus of stria terminalis (BST) were measured. NE concentrations in these brain areas of the surgical side served as the control. Urine collected from young adult male rats was poured into the female's cage at 12:00h and the animals were sacrificed before and 1, 2, or 3 hours after the male urine was given. The NE basal levels in the MA and MPOA of young rats decreased significantly from 13:00h to 15:00h, and those in young rat VMH declined markedly from 13:00h to 14:00h compared to those at 12:00h. No marked alterations in NE basal levels in young rat BST were found. In contrast, no obvious changes in the NE concentrations were observed in these brain areas of old rats. Continuous exposure to male urine did not affect the NE levels in any of these brain areas of young and aged rats. We concluded that (1) the time-dependent fluctuation of the NE basal levels in some brain areas of the vomeronasal system in female rats is age-related, and (2) the NE in all these nuclei of the vomeronasal system is not involved in pheromone-induced effects.     

大鼠尿中酶类参考值检测

目的调查正常大鼠尿液样本中酶类参考值范围。方法采集95只正常SD大鼠的4 h尿液,用生化仪检测尿液中的N-乙酰基-β-D氨基葡萄糖酶（NAG）、碱性磷酸酶（ALP）、谷氨酰氨基转移酶（γ-GT）和乳酸脱氢酶（LDH）的值,参考范围以95%百分位上界表示。结果参考值范围为,雌性：NAG〈5.3 U/L,ALP〈110.8 U/L,γ-GT〈753.0 U/L,LDH〈32.6 U/L;雄性：NAG〈27.3 U/L,ALP〈329.0 U/L,γ-GT〈769.8 U/L,LDH〈40.0 U/L。以/g Cre为单位的参考值范围,雌性：NAG〈28.9 U/g,ALP〈723.5 U/g,γ-GT〈1763.2 U/g,LDH〈113.1 U/g;雄性：NAG〈84.3 U/g,ALP〈680.2 U/g,γ-GT〈2522.3 U/g,LDH〈121.8 U/g。结论尿中酶类参考值范围为药物肾毒性评价提供了背景数据。     

Influence of gender on tamoxifen-induced biochemical changes in serum of rats

Tamoxifen, the widely prescribed drug in the prevention and therapy of breast cancer, may cause side effects which may be influenced by gender. The present study was undertaken to investigate the impact of gender on tamoxifen-induced toxic and biochemical changes following oral administration of tamoxifen at high dose level of 20 mg/kg once daily for a 2-week period in both male and female rats. The results showed marked increases in serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in female rats. In contrast, treatment with tamoxifen in male animals significantly decreased the activity of ALT, with a tendency for a decrease in serum AST levels. In female rats, a significant reduction in the serum activity of acid phosphatase (ACP) was noted, compared with a non-significant decrease in males. Non-significant changes in serum levels of alkaline phosphatase (ALP) were seen in both sexes. Tamoxifen lowered serum contents of total lipid and total cholesterol in both male and female rats. Serum levels of triglycerides were reduced in female rats as compared to a non-significant decrease in male animals. The serum albumin concentration was decreased in both male and female rats, while total protein was decreased only in female animals. Tamoxifen markedly increased serum levels of creatinine in female rats, compared with a non-significant rise in males. Total serum contents of calcium were similarly reduced in both males and females. This is the first study which points to gender-related differences in tamoxifen-induced toxic and metabolic changes in rats. The results indicated that females are more susceptible than males to tamoxifen toxicity, probably due to the ability of tamoxifen to antagonize the action of estrogen in females.     

Determination of urinary and serum beta-glucuronidase and alkaline phosphatase in various renal disease and kidney rejection transplanted patients

Beta-glucuronidase (beta-Glu) and alkaline phosphatase (ALP) were evaluated in serum and urine in 50 subjects classified into five equal groups. Group I was control healthy subjects, while groups II, III, IV, and V were patients with nephritic syndrome, pyelonephritis, kidney rejection, and end-stage renal disease, respectively. Urinary beta-Glu was significantly elevated in all four groups; while serum enzyme showed no change. On the other hand, serum ALP showed a significant elevation in all abnormal groups. Accordingly, urinary beta-Glu and serum ALP could be used as diagnostic markers for various renal diseases.     

Effect of flutamide on 5 alpha-reductases, 5 beta-reductases, and 3-hydroxysteroid dehydrogenases in rat liver

Flutamide (0.5 mM) decreased in vitro the activity of NADH-5 alpha-reductase (substrate testosterone) in liver homogenate of male and female rats, whereas no change of activity of NADPH-5 alpha-reductase was observed. NADH- and NADPH-5 beta-reductase activity increased only in liver of female, but not of male rats. NAD+-3 beta-hydroxysteroid dehydrogenase and NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydro-testosterone) in liver homogenate from female rats were inhibited by flutamide (0.5 mM), whereas the activity of NADP+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 alpha-dihydrotestosterone) and of NAD+-3 alpha-hydroxysteroid dehydrogenase (substrate 5 beta-dihydrotestosterone) increased in presence of flutamide. The activity of NADH- and NADPH-5 alpha-reductase decreased after flutamide administration to female rats at a dose of 5 mg per day for 7 days.     

Alpha-1-C-octyl-1-deoxynojirimycin as a pharmacological chaperone for Gaucher disease

The most common lysosomal storage disorder, Gaucher disease, is caused by inefficient folding and trafficking of certain variants of lysosomal beta-glucosidase (beta-Glu, also known as beta-glucocerebrosidase). Recently, Sawker et al. reported that the addition of subinhibitory concentrations (10 microM) of the pharmacological chaperone N-nonyl-1-deoxynojirimycin (NN-DNJ) (10) to Gaucher patient-derived cells leads to a 2-fold increase in activity of mutant (N370S) enzyme [Proc. Natl. Acad. Sci. U.S.A.2002, 99, 15428]. However, we found that the addition of NN-DNJ at 10 microM lowered the lysosomal alpha-glucosidase (alpha-Glu) activity by 50% throughout the assay period in spite of the excellent chaperoning activity in N370S fibroblasts. Hence, we prepared a series of DNJ derivatives with an alkyl chain at the C-1alpha position and evaluated their in vitro inhibitory activity and potential as pharmacological chaperones for Gaucher cell lines. Among them, alpha-1-C-octyl-DNJ (CO-DNJ) (15) showed 460-fold stronger in vitro inhibitory activity than DNJ toward beta-Glu, while NN-DNJ enhanced in vitro inhibitory activity by 360-fold. Treatment with CO-DNJ (20 microM) for 4 days maximally increased intracellular beta-Glu activity by 1.7-fold in Gaucher N370 cell line (GM0037) and by 2.0-fold in another N370 cell line (GM00852). The addition of 20 microM CO-DNJ to the N370S (GM00372) culture medium for 10 days led to 1.9-fold increase in the beta-Glu activity without affecting the intracellular alpha-Glu activity for 10 days. Only CO-DNJ showed a weak beta-Glu chaperoning activity in the L444P type 2 variant, with 1.2-fold increase at 5-20 microM, and furthermore maximally increased the alpha-Glu activity by 1.3-fold at 20 microM. These experimental results suggest that CO-DNJ is a significant pharmacological chaperone for N370S Gaucher variants, minimizing the potential for undesirable side effects such as alpha-Glu inhibition.     

Heterogeneity of glomerular barrier function in early adriamycin nephrosis of MWF rats

Adriamycin (ADR), selectively toxic to glomerular epithelial cells, was administered (5 mg/kg BW, i.v.) to MWF/Ztm rats to study its early effects on glomerular barrier function with respect to albumin and high molecular weight (HMW) proteins. After 7 days of ADR incubation (glomerular filtration rate remains unchanged), protein excretion was significantly increased in treated rats. The proteinuria was due to a nonselective glomerular lesions resulting in an increase in both, but not a changed ratio of HMW proteins to albumin. However, this ADR-induced proteinuria seen in the final urine was not confirmed by free-flow micropuncture studies of superficial glomeruli. The albumin and HMW protein concentrations in samples taken from Bowman's capsular space of ADR-treated rats did not significantly differ from control samples. These data suggest that cortical nephrons are less sensitive to ADR than juxtamedullary nephrons.     

Gender modulates activation of renin-angiotensin and endothelin systems in hypertension and heart failure.

Sexual dimorphism may occur during the development of hypertension and congestive heart failure (CHF). Male and female spontaneous hypertension heart failure (SHHF) rats with established hypertension, but before CHF (age 5-8 mo) and during cardiac decompensation leading to CHF (age 18-20 mo in male rats and 22-24 mo in female rats), were studied. At 5-8 mo, male SHHF rats showed early activation of the renin-angiotensin system (RAS), as indicated by increased plasma renin activity (PRA) and higher serum angiotensin-converting enzyme activity compared with female rats. The increase in PRA in female rats was delayed compared with males rats, but it reached comparable levels just before CHF. Urinary endothelin excretion was significantly greater in 5- to 8-mo-old female rats compared with age-matched male rats. Urinary endothelin excretion increased in both male and female rats as CHF developed. Plasma atrial natriuretic peptide (ANP) was comparable at both time points, and both genders showed similar, marked increases as CHF developed. In conclusion, male rats show early activation of the RAS, whereas female rats show early activation of the endothelin vasopressor system. During cardiac decompensation, generalized activation of the RAS, endothelin, and ANP systems occurs and is similar in male and female SHHF rats.     

Effect of DL-alpha-lipoic acid on cyclophosphamide induced lysosomal changes in oxidative cardiotoxicity

Cyclophosphamide (CP), one of the widely prescribed antineoplastic drugs can cause fatal cardiotoxicity. The present study is aimed at evaluating the cardioprotective role of lipoic acid in CP induced toxicity. Male albino rats of Wistar strain were divided into four groups and treated as follows: Group I served as control, Group II received a single dose of CP (200 mg/kg b.wt., i.p.), Group III received lipoic acid (25 mg/kg b.wt., orally) for 10 days, and Group IV received CP immediately followed by lipoic acid for 10 days. In CP administered rats, the levels of protein carbonyl and 8-hydroxy-2-deoxyguanosine were increased significantly (P<0.001) indicating oxidative changes in the heart tissue. The activities of lysosomal acid hydrolases, beta-Glu, beta-Gal, NAG, Cat-D and ACP increased significantly (P<0.001) in the serum as well as in the heart tissue after CP administration. An increase in hydroxyproline was observed in CP induced rats. Lipoic acid effectively reverted these abnormal biochemical changes to near normalcy. These observations highlight the protective role of lipoic acid in CP induced cardiotoxicity.