Immunolocalization of betaine aldehyde dehydrogenase in porcine kidney.

Polyclonal anti-BADH serum was raised in rabbits against native BADH purified from porcine kidney. The antiserum cross-reacted strongly with BADH purified from kidney, Amaranthus palmierii, and Pseudomona aeuroginosa (1:1000), and weakly with Amaranthus hypochondriacus L (1:100). Antibodies bound to purified native kidney BADH in immunoblots showed a major band of an apparent molecular mass of 340 kDa and a subunit with an apparent molecular mass of 52 kDa. Data on activity assays showed higher activity in cortex sections (81.3 nmol/min/mg protein) than in medulla sections (21.3 nmol/min/mg protein). Immunolocalization of BADH in kidney tissue sections showed that BADH is found in cortex and medulla. In inner medulla, the enzyme was mainly localized in cells surrounding the tubules. Western blot analysis on extracts from the cortex and medulla sections showed higher concentration of BADH protein in cortex than in medulla. These results were in accordance with immunolocalization and activity analysis.     

Characterisation of neutral endopeptidase 3.4.24.11 (NEP) in the kidney: comparison between normotensive, genetically hypertensive and experimentally hypertensive rats.

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY), DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280 microM), (3) IC50 of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH 8.0), (5) heat inactivation profiles (half-life 20 min at 65 degrees C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.     

Characterisation of Neutral Endopeptidase 3.4.24.11 (NEP) in the Kidney: Comparison Between Normotensive,Genetically Hypertensive and Experimentally Hypertensive Rats

Abstract

Neutral endopeptidase 3.4.24.11 (NEP) has been identified as the major atrial natriuretic factor (ANF) degrading enzyme in rat kidney, therefore, suggesting a possible role for this enzyme in blood volume and pressure regulation. Various experimentally induced and genetically hypertensive rat models have been used to test NEP inhibitors. The presence of different isoforms of NEP in the various hypertensive rat models would have relevance when searching for novel NEP inhibitors. Therefore, we compared the properties of NEP in kidney cortex homogenates in order to test for possible differences in the following hypertensive rat models and their appropriate controls: spontaneously hypertensive rats (SHR), Wistar Kyoto strain (WKY). DOCA-salt hypertensive rats, and Sprague Dawley control rats (SD). No relevant differences were found when comparing the following parameters,: (1) specific activity (mean: 204 U/mg protein), (2) Michaelis constant (mean: 280μM), (3) IC₅₀ of thiorphan (mean: 6.5 nM) and phosphoramidon (mean: 54 nM), (4) pH profiles (optimum at pH8.0), (5) heat inactivation profiles (half-life 20min at 65°C), (6) immunotitration of kidney cortex homogenates, (7) molecular weight as determined by gel filtration (92,000 Dalton) and (8) affinity chromatography with concanavalin A. Without evidence for the presence of different NEP isoforms, it is unlikely that divergent findings in DOCA-salt rats and SHR using a given NEP inhibitor are due to isoforms of NEP.     

Hepatocyte nuclear factors as possible C-reactive protein transcriptional inducer in the liver and white adipose tissue of rats with experimental chronic renal failure

Little is known about the effects of coffee that are not related to the presence of caffeine. The aim of the study was to analyse changes in kidney function and nucleotide metabolism related to high intake of decaffeinated coffee. Mice consumed decaffeinated coffee extract for two weeks. Activities of AMP deaminase, ecto5′-nucleotidase, adenosine deaminase, purine nucleoside phosphorylase were measured in kidney cortex and medulla by analysis of conversion of substrates into products using HPLC. Concentration of nucleotides in kidney cortex, kidney medulla and serum were estimated by HPLC. Activity of ecto5′-nucleotidase increased from 0.032 ± 0.006 to 0.049 ± 0.014 nmol/mg tissue/min in kidney cortex of mice administered high-dose decaffeinated coffee (HDC) together with increase in cortex adenosine concentration and decrease in plasma creatinine concentration. HDC leads to increased activity of ecto5′-nucleotidase in kidney cortex that translates to increase in concentration of adenosine. Surprisingly this caused improved kidney excretion function.     

Estrone sulfatase activity in the human brain and estrone sulfate levels in the normal menstrual cycle

When the plasma concentrations of estrone sulfate (E1S) were measured in five menstrual cycles, the highest concentrations were found on the day of LH peak (14.25 nmol/l +/- 2.94 [SE]). Peak levels of E1S were 20 times higher than the highest E2 levels measured (0.769 +/- 0.276 nmol/l). To determine whether E1S can be metabolized by adult and fetal tissues we examined estrone (E1) sulfatase activity in brain and other tissues. E1 Sulfatase activity was present in all tissues studied including adult endometrium, fat and skin. When the rate of sulfatase activity was measured in homogenates of fetal hypothalamus, frontal cortex and pituitary (n = 4), the hypothalamic activity (306.0 +/- 39.1 [SE] pmol/min/mg protein) was significantly higher than that of the frontal cortex (127.4 +/- 19.4, P less than 0.002) or pituitary (193.7 +/- 43.3, P less than 0.03). This was not apparent in the adult (n = 2) where the enzyme activity was similar in the hypothalamus (413.9 +/- 27.3) and frontal cortex (446.3 +/- 82.2) and lower in the pituitary (98.2 +/- 19.2). The Km for E1 sulfatase in the fetal frontal cortex was 28.9 microM. The high E1 sulfatase activity in estrogen responsive target tissues, particularly fetal hypothalamus, accompanied by a large circulating reservoir of E1S, suggest that this enzyme could possibly have a regulatory role in controlling the level of intracellular estrogens and in modulating their intracellular function.     

Topology of membrane exposure in the renal cortex slice. Studies of glutathione and maltose cleavage

We measured glycine release from ([2-3H]glycine)-labelled GSH and glucose formation from maltose incubated with rat kidney whole cortex homogenate, thin cortex slices or collagenase-treated tubule fragments. Liberation of glycine was inhibited (74-83%) by serine borate (20 mM), indicating a gamma-glutamyltransferase-dependent hydrolysis of GSH. In whole cortex homogenate, the GSH cleavage activity was 17.4 +/- 0.6 nmol GSH degraded/mg protein per min (mean +/- S.D.); cleavage activity by intact slices was 3.5 +/- 0.7 (P less than 0.001 relative to whole cortex homogenate) and in tubule fragments 9.4 +/- 0.8 (P less than 0.001). Homogenizing the tissue preparation increased cleavage rate in slices about 4-fold (12.4 +/- 2.9; P less than 0.005 relative to intact slice) but did not change the rate in tubule fragments (9.8 +/- 0.5). Maltose cleavage activity in whole cortex homogenate was 512 +/- 22 nmol glucose formed/mg protein per min, in slices 162 +/- 12, and in tubules 884 +/- 48. These findings imply that substrate in the incubation medium has a limited access to the luminal membrane of cortex slices but not of tubule fragments. They further imply that basolateral membrane is preferentially exposed in the slice preparation.     

Loss of ecto-5'nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation

The endothelial cell surface expression of ecto-5'-nucleotidase (E5'N, CD73) is thought to be essential for the extracellular formation of cytoprotective, anti-thrombotic and immunosuppressive adenosine. Decreased E5'N activity may play a role in xenograft acute vascular rejection, preventing accommodation and tolerance mechanisms. We investigated the extent of changes in E5'N activity and other enzymes of purine metabolism in porcine hearts or endothelial cells when exposed to human blood or plasma and studied the role of humoral immunity in this context. Pig hearts, wild type (WT, n = 6) and transgenic (T, n = 5) for human decay accelerating factor (hDAF), were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were exposed for 3 h to autologous porcine plasma (PP), normal (NHP) or heat inactivated human plasma (HHP), with and without C1-inhibitor. Enzyme activities were measured in heart or endothelial cell homogenates with an HPLC based procedure. The baseline activity of E5'N in WT and T porcine hearts were 6.60 +/- 0.33 nmol/min/mg protein and 8.54 +/- 2.10 nmol/min/mg protein respectively (P < 0.01). Ex vivo perfusion of pig hearts with fresh human blood for 4 h resulted in a decrease in E5'N activity to 4.01 +/- 0.32 and 4.52 +/- 0.52 nmol/min/mg protein (P < 0.001) in WT and T hearts respectively, despite attenuation of hyperacute rejection in transgenic pigs. The initial PAEC activity of E5'N was 9.10 +/- 1.40 nmol/min/mg protein. Activity decreased to 6.76 +/- 0.57 and 4.58 +/- 0.47 nmol/min/mg protein (P < 0.01) after 3 h exposure of HHP and NHP respectively (P < 0.05), whereas it remained unchanged at 9.62 +/- 0.88 nmol/min/mg protein when incubated with PP controls. C1-inhibitor partially preserved E5'N activity, similar to the effect of HHP. Adenosine deaminase, adenosine kinase and AMP deaminase (other enzymes of purine metabolism) showed a downward trend in activity, but none were statistically significant. We demonstrate a specific decrease in E5'N activity in pig hearts following exposure to human blood which impairs adenosine production resulting in a loss of a cytoprotective phenotype, contributing to xenograft rejection. This effect is triggered by human humoral immune responses, and complement contributes but does not fully mediate E5'N depletion.     

Spectrophotometric assay of renal ouabain-resistant Na(+)-ATPase and its regulation by leptin and dietary-induced obesity

Apart from Na(+),K(+)-ATPase, a second sodium pump, Na(+)-stimulated, K(+)-independent ATPase (Na(+)-ATPase) is expressed in proximal convoluted tubule of the mammalian kidney. The aim of this study was to develop a method of Na(+)-ATPase assay based on the method previously used by us to measure Na(+),K(+)-ATPase activity. The ATPase activity was assayed as the amount of inorganic phosphate liberated from ATP by isolated microsomal fraction. Na(+)-ATPase activity was calculated as the difference between the activities measured in the presence and in the absence of 50 mM NaCl. Na(+)-ATPase activity was detected in the renal cortex (3.5 +/- 0.2 mumol phosphate/h per mg protein), but not in the renal medulla. Na(+)-ATPase was not inhibited by ouabain or an H(+),K(+)-ATPase inhibitor, Sch 28080, but was almost completely blocked by 2 mM furosemide. Leptin administered intraperitoneally (1 mg/kg) decreased the Na(+),K(+)-ATPase activity in the renal medulla at 0.5 and 1 h by 22.1% and 27.1%, respectively, but had no effect on Na(+)-ATPase in the renal cortex. Chronic hyperleptinemia induced by repeated subcutaneous leptin injections (0.25 mg/kg twice daily for 7 days) increased cortical Na(+),K(+)-ATPase, medullary Na(+),K(+)-ATPase and cortical Na(+)-ATPase by 32.4%, 84.2% and 62.9%, respectively. In rats with dietary-induced obesity, the Na(+),K(+)- ATPase activity was higher in the renal cortex and medulla by 19.7% and 34.3%, respectively, but Na(+)-ATPase was not different from control. These data indicate that both renal Na(+)-dependent ATPases are separately regulated and that up-regulation of Na(+)-ATPase may contribute to Na(+) retention and arterial hypertension induced by chronic hyperleptinemia.     

NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes

Sodium-hydrogen exchanger regulatory factor isoform-1 (NHERF-1) and NHERF-2 are two structurally related PDZ-domain-containing protein adapters that effectively transduce cyclic AMP (cAMP) signals that inhibit NHE3, the sodium-hydrogen exchanger isoform present at the apical surface of kidney and gut epithelia. The mouse renal proximal tubule expresses both NHERF isoforms, suggesting their redundant functions as regulators of renal electrolyte metabolism. To define the role of NHERF-1 in the physiological control of NHE3, we analyzed NHE3 activity in isolated brush border membrane (BBM) preparations from renal proximal tubules of wild-type (WT) and NHERF-1 (-/-) mice. Basal Na(+)-H(+) exchange was indistinguishable in BBMs from WT and NHERF-1 (-/-) mice (0.96+/-0.08 and 0.95+/-0.10 nmol/mg protein/10 s, respectively). Activation of membrane bound cAMP-dependent protein kinase (PKA) by cAMP inhibited NHE3 activity in WT BBMs (0.55+/-0.07 nmol/mg protein/10 s or 40+/-9%, P<0.01) but had no discernible effect on Na(+)-H(+) exchange in the NHERF-1 (-/-) BBM (0.97+/-0.07 nmol/mg protein/10 s; P=not significant). This was associated with a significant decrease in cAMP-stimulated phosphorylation of NHE3 immunoprecipitated from solubilized NHERF-1 (-/-) BBMs. As the protein levels for NHE3, NHERF-2, PKA and ezrin were not changed in the NHERF-1 (-/-) BBMs, the data suggest a unique role for NHERF-1 in cAMP-mediated inhibition of NHE3 activity in the renal proximal tubule of the mouse.     

17-oxidoreduction of 17beta estradiol, estrone and their 3-sulfates by kidney slices from guinea pig and human.

Slices of whole kidney and kidney cortex from the female guinea pig catalyzed a marked reduction of estrone 3-sulfate (E13S) and estrone (E1) to 17beta-estradiol 3-sulfate (E23S) and 17beta-estradiol (E2), respectively, as well as the reverse (dehydrogenation) reactions. Slices of medulla did not appear active in E23S-E13S interconversion but did possess the ability to interconvert E2 and E1, besides possessing considerable sulfatase activity. The use of [3H-55S]E13S and [3H-55S]E23S as substrates, together with a demonstrated lack of estrogen sulfate synthesis by the tissue slices, provided ample evidence that the intact sulfates were involved in direct oxidoreduction. Slices of human kidney cortex catalyzed the reduction of E13S to a very limited extent. Slices of whole kidney and of cortex from guinea pig formed small amounts of estrogen glucuronide(s).     

Renal neuraminidase. Characterization in normal rat kidney and measurement in experimentally induced nephrotic syndrome.

Several lines of evidence suggest that increased neuraminidase activity may be responsible for the loss of glomerular N-acetylneuraminic acid (AcNeu) observed in various glomerular diseases. However, virtually no information is available on the activity of neuraminidase in glomeruli or the potential role of this enzyme in glomerular pathophysiology. Utilizing 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (4MU-AcNeu) as substrate, we defined optimal assay conditions and characterized neuraminidase activity in glomeruli and, for comparison, in other renal fractions and liver. Neuraminidase activity in glomeruli, cortex and tubules was maximal at pH 4.4. The Km for 4MU-AcNeu was estimated to be 195 microM for glomeruli and 226 microM for cortex. Glomerular neuraminidase was inhibited by AcNeu (90% at 25 mM) and high concentrations of Triton X-100 (26% at 0.5%), but unaffected by CaCl2, EDTA or N-ethylmaleimide (each 1 mM). Neuraminidase activity (nmol/h per mg of protein; mean +/- S.E.M.) in normal rat kidney was: cortex, 14.47 +/- 0.76; medulla, 7.85 +/- 0.64; papilla, 2.64 +/- 0.11; tubules, 13.79 +/- 0.70; glomeruli, 5.57 +/- 0.28. In comparison, neuraminidase activity in rat liver was 2.58 +/- 0.14. Puromycin aminonucleoside (PAN)-induced nephrotic syndrome is a model of glomerular disease in which the loss of glomerular AcNeu is well documented. In two separate studies, we observed no change in the specific activity of neuraminidase in either glomeruli or cortex isolated from rats treated with PAN (15 mg/100 g, intraperitoneally) and killed at either the onset or the peak of proteinuria. Results were similar whether neuraminidase activity was expressed per mg of protein or per microgram of DNA.     

Angiotensin-(1-7) reverts the stimulatory effect of angiotensin II on the proximal tubule Na(+)-ATPase activity via a A779-sensitive receptor.

Recently, we demonstrated that the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubules is reversed, in a dose-dependent manner, by Ang-(1-7) [Biochim. Biophys. Acta 1467 (2000) 189]. In the present paper, we characterized the receptor involved in this phenomenon. The preincubation of the Na(+)-ATPase with 10(-8) M Ang II increases the enzyme activity from 7.50+/-0.02 (control) to 12.40+/-1.50 nmol Pi mg(-1) min(-1) (p<0.05). Addition of 10(-9) M Ang-(1-7) completely reverts this effect returning the ATPase activity to the control level. This effect seems to be specific to Ang-(1-7) since Ang III (10(-12)-10(-8) M) does not modify the stimulation of the renal proximal tubule Na(+)-ATPase activity by Ang II. Saralasin abolishes the Ang-(1-7) effect in a dose-dependent manner being the maximal effect obtained at 10(-11) M. The increase in A779 concentration (from 10(-12) to 10(-7) M), a specific Ang-(1-7) antagonist, also abolishes the Ang-(1-7) effect. On the other hand, PD123319 (10(-8)-10(-6) M), an AT(2) antagonist receptor, and losartan (10(-12)-10(-7) M), an AT(1) antagonist receptor, does not modify the effect of Ang-(1-7). Taken together, these data indicate that Ang-(1-7) reverts the stimulatory effect of Ang II on the Na(+)-ATPase activity in proximal tubule through a A779-sensitive receptor.     

Presence of ectonucleotidases in cultured chromaffin cells: hydrolysis of extracellular adenine nucleotides

The granular ATP released from chromaffin cells during the secretory response can be hydrolyzed by ectonucleotidases that are present in the plasma membrane of these cells. The ecto-ATPase activity showed a Km for ATP of 250 +/- 18 microM and a VMAX value of 167 +/- 25 nmol/10(6) cells x min (1.67 mumol/mg protein x min) for cultured chromaffin cells, while the ecto-ADPase activity showed a Km value for ADP of 375 +/- 40 microM and a VMAX of 125 +/- 20 nmol/10(6) cells x min (1.25 mumol/mg protein x min). The ecto 5'-nucleotidase activity of cultured chromaffin cells was more specific for the purine nucleotides, AMP and IMP, than for the pirimidine nucleotides, CMP and TMP. The Km for AMP was 55 +/- 5 microM and the VMAX value was 4.3 +/- 0.8 nmol/10(6) cells x min (43 nmol/mg protein x min). The nonhydrolyzable analogs of ADP and ATP, alpha, beta-methylene-adenosine 5'-diphosphate and adenylyl-(beta, gamma-methylene)-diphosphonate were good inhibitors of ecto 5'-nucleotidase activity, the KI values being 73.3 +/- 3.5 nM and 193 +/- 29 nM, respectively. The phosphatidylinositol-specific phospholipase C released the ecto-5'-nucleotidase from the chromaffin cells in culture, thus suggesting an anchorage through phosphatidylinositol to plasma membranes. The presence of ectonucleotidases in chromaffin cells may permit the recycling of the extracellular ATP exocytotically released from these neural cells.     

Adrenal, kidney, and heart angiotensins in female murine ren-2 transfected hypertensive rats

We analyzed by high-performance liquid chromatography and radioimmunoassay angiotensin I (Ang I), Ang II, Ang-(1–7), and metabolites in the adrenal, kidney and heart of normotensive female Sprague–Dawley (SD) and transgenic hypertensive [TGR(mRen-2)27] rats carrying the murine Ren-2^d renin gene. The monogenetic model of hypertensive rats had significant increases in adrenal Ang II; whereas in the kidney Ang II was unchanged, but Ang I and Ang-(1–7) were significantly lower. Cardiac Ang I, Ang II, and Ang-(2–10) were significantly reduced in transgenic rats, while Ang-(2–7) was increased. In SD and transgenic rats kidney and adrenal angiotensins increased primarily during estrus or proestrus. In female transgenic rats the increased adrenal Ang II and the sustained renal Ang II may contribute to the established phase of hypertension.     

Seasonal changes in angiotensin converting enzyme activity in male and female frogs (Rana esculenta)

Gonad, lung, kidney and serum angiotensin converting enzyme (ACE) activities were determined by specific substrate hydrolysis in male and female Rana esculenta over 1 year. Ovary ACE activity showed the highest values among the different tissues, with a significant peak (223+/-52 nmol min(-1) mg protein(-1)) in late winter-early spring. Testis ACE activity followed a significant seasonal cycle, increasing from September to peak in April (2.5+/-0.8 nmol min(-1) mg protein(-1)) and then decreased in the post-reproductive period. Lung and kidney ACE activities were not correlated with the annual reproductive cycle phases. In serum a peak of activity was present in the post-reproductive period both in male and female frogs. The present data show a correlation between ACE and the annual reproductive cycle of R. esculenta.     

Chronic estradiol-17β exposure increases superoxide production in the rostral ventrolateral medulla and causes hypertension: reversal by resveratrol

Women are exposed to estrogen in several forms, such as oral contraceptive pills and hormone replacement therapy. Although estrogen was believed to be cardioprotective, lately, its beneficial effects are being questioned. Recent studies indicate that oxidative stress in the rostral ventrolateral medulla (RVLM) may play a role in the development of hypertension. Therefore, we hypothesized that chronic exposure to low levels of estradiol-17β (E(2)) leads to hypertension in adult-cycling female Sprague Dawley (SD) rats potentially through generation of superoxide in the RVLM. To test this hypothesis, young adult (3 or 4 mo old) female SD rats were either sham-implanted or implanted (subcutaneously) with slow-release E(2) pellets (20 ng/day) for 90 days. A group of control and E(2)-treated animals were fed lab chow or chow containing resveratrol (0.84 g/kg of chow), an antioxidant. Rats were implanted with telemeters to continuously monitor blood pressure (BP) and heart rate (HR). At the end of treatment, the RVLM was isolated for measurements of superoxide. E(2) treatment significantly increased mean arterial pressure (mmHg) and HR (beats/min) compared with sham rats (119.6 ± 0.8 vs. 105.1 ± 0.7 mmHg and 371.7 ± 1.5 vs. 354.4 ± 1.3 beats/min, respectively; P < 0.0001). Diastolic and systolic BP were significantly increased in E(2)-treated rats compared with control animals. Superoxide levels in the RVLM increased significantly in the E(2)-treated group (0.833 ± 0.11 nmol/min·mg) compared with control (0.532 ± 0.04 nmol/min·mg; P < 0.05). Treatment with resveratrol reversed the E(2)-induced increases in BP and superoxide levels in the RVLM. In conclusion, these findings support the hypothesis that chronic exposure to low levels of E(2) induces hypertension and increases superoxide levels in the RVLM and that this effect can be reversed by resveratrol treatment.     

Sodium-dependent phosphate transport inhibited by parathyroid hormone and cyclic AMP stimulation in an opossum kidney cell line

In the present study we investigated the characteristics of the transport of inorganic phosphate (Pi) in an opossum kidney cell line endowed with parathyroid hormone (PTH) receptors. In confluent epithelial cell culture, a Na-dependent Pi transport (NaPiT) was identified. Preincubation for 1 h with bovine (b)PTH(1-34) at 10(-7) M inhibited the NaPiT from 2.76 +/- 0.11 to 1.08 +/- 0.10 nmol/mg protein X 2 min-1 (p less than 0.001). This inhibition was already expressed 5 min after exposure to 10(-7) M bPTH. It was associated with a 4-fold increase in cellular cyclic AMP. The NaPiT was significantly inhibited at 10(-9) M bPTH, a hormonal concentration which stimulated the cellular cyclic AMP by only 30%. Kinetic analysis of the NaPiT inhibition by 10(-7) M bPTH revealed a decrease in Vmax (from 4.14 +/- 0.32 to 2.41 +/- 0.14 nmol/mg protein X 2 min-1) with no change in Km (0.093 +/- 0.016 versus 0.094 +/- 0.012 mM). The effect of bPTH on NaPiT was not associated with a change in the Na-dependent glucose methylglucopyranoside transport also present in the opossum kidney cell line. The inhibitory influence of bPTH on NaPiT was not affected by blockage of new protein synthesis by cycloheximide. Stimulation of cyclic AMP production by 10(-5) M forskolin, 10 micrograms/ml cholera toxin, 10(-5) M prostaglandin E2 or addition of 10(-5) M dibutyryl cyclic AMP mimicked the PTH-induced reduction in NaPiT. In conclusion, the present study indicates that the opossum epithelial cell line is endowed with a Na-dependent Pi transport system which is selectively inhibited by PTH and agents which increase cyclic AMP production.     

Mass spectrometry for the molecular imaging of angiotensin metabolism in kidney

To better understand the tissue distribution and activity of enzymes involved in angiotensin II (Ang II) processing, we developed a novel molecular imaging method using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Mouse kidney sections (12 μm) were incubated with 10-1,000 μmol/l Ang II for 5-15 min at 37°C. The formed peptides Ang III and Ang-(1-7) were identified by MALDI-TOF/TOF. A third metabolite, Ang-(1-4), was generated from further degradation of Ang-(1-7). Enzymatic processing of Ang II was dose and time dependent and absent in heat-treated kidney sections. Distinct spatial distribution patterns (pseudocolor images) were observed for the peptides. Ang III was localized in renal medulla, whereas Ang-(1-7)/Ang-(1-4) was present in cortex. Regional specific peptide formation was confirmed using microdissected cortical and medullary biopsies. In vitro studies with recombinant enzymes confirmed activity of peptidases known to generate Ang III or Ang-(1-7) from Ang II: aminopeptidase A (APA), Ang-converting enzyme 2 (ACE2), prolyl carboxypeptidase (PCP), and prolyl endopeptidase (PEP). Renal medullary Ang III generation was blocked by APA inhibitor glutamate phosphonate. The ACE2 inhibitor MLN-4760 and PCP/PEP inhibitor Z-pro-prolinal reduced cortical Ang-(1-7) formation. Our results establish the power of MALDI imaging as a highly specific and information-rich analytical technique that will further aid our understanding of the role and site of Ang II processing in cardiovascular and renal pathologies.     

17 alpha-oxidoreductase activity in kidneys of male and female mice of various ages

Male and female mice at 0-120 days of age were used. Homogenates of kidneys were incubated with [14C]4-androstene-3,17-dione, and 17 alpha-oxidoreductase activity per g tissue was examined. The activities of 17 alpha-oxidoreductase in the kidneys of both sexes increased markedly with age during sexual development by up to 150-fold and reached the maximum values (2700 and 1500 nmol/g/h in male and female kidneys, respectively) at 60 days of age. In the adult male mouse kidney, the activity in isolated cortex fractions was 14 times as high as the activity in isolated medulla fractions; in the medulla fractions renal tubules from the cortex accounted for 3-15% of the total tissue. Furthermore, histochemical examination showed the activity present only in the cortex, at which much higher levels in the tubules than in the glomerulus. Activity at 35-120 days of age was significantly higher in male kidneys than in female kidneys. The difference appears to be induced by testicular androgens during sexual development, since neonatal castration in males resulted in decreases of activity to levels similar to those in female kidneys. However, castration at 60 days of age showed no significant effect on the activity. The present results show that the activity per g tissue of 17 alpha-oxidoreductase in the mouse kidney increases markedly with age, and that the activity is largely confined to the renal tubules of the cortex.     

Changes in ovarian NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase activity in pregnant and pseudopregnant rabbits.

The specific activity of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (PGDH) was found to increase in the ovaries of pregnant and pseudopregnant rabbits. The mean specific activity of cytosolic ovarian PGDH in 14- to 28-day pregnant rabbits was 24.3 +/- 8.1 nmol NADH formed/min/mg protein (n = 16) using PGE1 as substrate whereas in nonpregnant rabbits the specific activity was 1.5 +/- 0.8 nmol NADH formed/min/mg protein (n = 8). The reaction was dependent on NAD+; NADP+ did not support the reaction. In grouping the PGDH activities from pregnant rabbits into second (14-18 days) and third (2-28 days) trimester periods, no significant difference between values was found (26.1 +/- 8.9 vs 23.4 +/- 8.1 nmol NADH formed/min/mg protein, respectively). Western blot analysis of the ovarian cytosol using an antibody which was made to the purified lung PGDH of pregnant rabbits recognized an ovarian protein of identical molecular mass (30 kDa). Ovarian PGDH activities were also examined in rabbits treated with pregnant mare's serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG) to induce a state of superovulatory/pseudopregnancy and only on day 11 following hCG treatment was an increase in PGDH specific activity observed. On day 11, the specific activity was 14.8 +/- 4.3 nmol NADH formed/min/mg protein whereas values on days 10 and 12 were only 1.1 +/- 1.1 and 1.0 +/- 0.8, respectively. PGDH activities on days 3, 7 and 16 were also low.(ABSTRACT TRUNCATED AT 250 WORDS)