Interactive roles of CD73 and tissue nonspecific alkaline phosphatase in the renal vascular metabolism of 5′-AMP

CD73 metabolizes extracellular 5′-AMP to adenosine; yet recent experiments in brain tissue suggest that CD73 is not required for the metabolism of 5′-AMP to adenosine because of tissue nonspecific alkaline phosphatase (TNAP), which like CD73 is a GPI-anchored ecto-enyzme with 5′-nucleotidase activity. Because adenosine importantly regulates renovascular function, we investigated whether both TNAP and CD73 are involved in the renovascular metabolism of 5′-AMP. To test this, we examined in isolated, perfused mouse kidneys the metabolism of 5′-AMP (applied to the lumen of the renal vasculature via intrarenal artery administration) to adenosine by measuring renal venous levels of 5′-AMP, adenosine, and inosine (adenosine metabolite) by mass spectrometry. In one study, we compared 5′-AMP metabolism in naive CD73+/+ (wild-type, n = 16) vs. CD73−/− (knockout, n = 16) kidneys; and in a second study, we compared 5′-AMP metabolism in CD73+/+ ( n = 9) vs. CD73−/− ( n = 8) kidneys pretreated with levamisole (1 mmol/l; TNAP inhibitor). In naive kidneys, 5′-AMP increased renal venous 5′-AMP, adenosine, and inosine, and these responses were similar in CD73+/+ vs. CD73−/− kidneys. Levamisole per se did not inhibit renovascular 5′-AMP metabolism; however, in the presence of levamisole, 5′-AMP increased renal venous 5′-AMP threefold more in CD73−/− vs. CD73+/+ kidneys and knockout of CD73 inhibited 5′-induced adenosine and inosine by 81 and 86%, respectively. TNAP mRNA, protein, and activity were similar in CD73+/+ vs. CD73−/− kidneys. In conclusion, CD73 and TNAP play interactive roles to metabolize luminally applied 5′-AMP in the renal vasculature such that inhibition of both is required to inhibit the production of adenosine.

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Analysis of Localization of Mutated Tissue-Nonspecific Alkaline Phosphatase Proteins Associated with Neonatal Hypophosphatasia Using Green Fluorescent Protein Chimeras1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.83.11.5267 ◽

1998 ◽

Vol 83 (11) ◽

pp. 3936-3942

Author(s):

Guiming Cai ◽

Toshimi Michigami ◽

Takehisa Yamamoto ◽

Natsuo Yasui ◽

Kenichi Satomura ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Enzymatic Activity ◽

Fluorescent Protein ◽

Clinical Severity ◽

Wild Type ◽

Neonatal Patient ◽

Tissue Nonspecific Alkaline Phosphatase ◽

Green Fluorescent

Hypophosphatasia is associated with a defect of the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The onset and clinical severity are usually correlated in hypophosphatasia; patients with perinatal hypophosphatasia die approximately at the time of birth. In contrast, we describe a male neonatal patient with hypophosphatasia who had no respiratory problems and survived. He was compound heterozygous for the conversion of Phe to Leu at codon 310 (F310L) and the deletion of a nucleotide T at 1735 (delT1735), causing the frame shift with the result of the addition of 80 amino acids at the C-terminal of the protein. Because the C-terminal portion of TNSALP is known to be important for TNSALP to bind to the plasma membrane, the localization of wild-type and mutated TNSALP proteins was analyzed using green fluorescent protein chimeras. The expression vectors containing the complementary DNA of fusion proteins consisting of signal peptide, green fluorescent protein, and wild-type or mutated TNSALP, caused by delT1735 or F310L mutation, were introduced transiently or stably in Saos-2 cells. The delT1735 mutant failed to localize at the cell surface membrane, whereas the wild-type and the F310L mutants were located in the plasma membrane and cytoplasm. The assay for enzymatic activity of TNSALP revealed that the delT1735 mutant lost the activity and that the F310L mutant exhibited an enzymatic activity level that was 72% of the normal level. The F310L mutation was also detected in another neonatal patient with relatively mild (nonlethal) hypophosphatasia (reported in J Clin Endocrinol Metab, 81:4458–4461, 1996), suggesting that residual ALP activity of the F310L mutant contributes to the less severe phenotype. The patient is unique, with respect to a discrepancy between onset and clinical severity in hypophosphatasia.

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Abstract 37: Deletion of Notch3 Gene Impairs Contractility of Renal Resistance Vessels

Hypertension ◽

10.1161/hyp.60.suppl_1.a37 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Frank Helle ◽

Panagiotis Kavvadas ◽

Michael Hultström ◽

Bjarne Iversen ◽

Christos Chatziantoniou

Keyword(s):

Vascular Reactivity ◽

Channel Blocker ◽

Chelating Agent ◽

Dependent Manner ◽

Ang Ii ◽

Wild Type ◽

Renal Vasculature ◽

Resistance Vessels ◽

And Function ◽

Renal Vascular

Background: Notch3 plays an important role in the differentiation and development of vascular smooth muscle cells. In previous studies we showed that mice lacking Notch3 display deficient renal autoregulation. Objective: Our aim was to study the mechanisms involved in the Notch3-mediated control of renal vascular response. Methods and Results: To this end, renal afferent arterioles were isolated from Notch3-/- and wild type littermates and stimulated with ANG II. Contractions and intracellular Ca2+ concentrations were blunted in Notch3-/- vessels in a dose-dependent manner (diameter decrease: 15±3 vs. 38±5%, p<0.01; change of fura 2 ratio: 0.18±0.02 vs. 0.39±0.05, p<0.001, at 10-7Ang II for Notch3-/- and wt vessels, respectively). Differential transcriptomic analysis of 47 genes known to participate in vasoreactivity indicated an important downregultion of the cacna1h gene expressing the α1H subunit of the T-type Ca2+ channel in the renal vessels of Notch3-/- mice. This finding was confirmed by real-time qPCR and western blotting. In subsequent experiments, addition of EGTA (Ca2+ chelating agent), nifedipine (L-type channel blocker) or mibefradil (T-type channel blocker) blunted as expected the response to ANG II in wild-type vessels. In sharp contrast, these agents did not affect vessel responsiveness in Notch3-/- indicating dysfunctional extracellular Ca2+-entry. Abolishing stored Ca2+ with thapsigargine, reduced Ca2+ responses equally in both strains, signifying intact Ca2+-mobilization in Notch3-/- vessels. In contrast to renal resistance vessels, pre-capillary muscle arterioles of Notch3-/- mice reacted normally to ANG II, suggesting a focal role of Notch3 in the renal vasculature. Conclusions: Notch3-/- mice display deficient renal vascular reactivity because of blunted expression and function of calcium channels. Consequently, intact Notch3 expression is necessary for proper regulation of renal vascular tone in the kidney, whereas dysfunction of Notch3 can have important physiopathological consequences by impairing regulation of renal hemodynamics

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Chronic recurrent multifocal osteomyelitis in children with hypophosphatasia explained by anti-inflammatory nucleotidase activity of tissue nonspecific alkaline phosphatase in mesenchymal and hematopoietic cells

Bone Abstracts ◽

10.1530/boneabs.7.lb5 ◽

2019 ◽

Author(s):

Laurence Bessueille ◽

Anne Briolay ◽

Juna Como ◽

Cylia Mansouri ◽

Marie Gleizes ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Hematopoietic Cells ◽

Chronic Recurrent Multifocal Osteomyelitis ◽

Nucleotidase Activity ◽

Multifocal Osteomyelitis ◽

Tissue Nonspecific Alkaline Phosphatase ◽

Anti Inflammatory

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Osteoblast tissue-nonspecific alkaline phosphatase antagonizes and regulates PC-1

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.2000.279.4.r1365 ◽

2000 ◽

Vol 279 (4) ◽

pp. R1365-R1377 ◽

Cited By ~ 105

Author(s):

K. A. Johnson ◽

L. Hessle ◽

S. Vaingankar ◽

C. Wennberg ◽

S. Mauro ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Specific Activity ◽

Bone Matrix ◽

Pericellular Matrix ◽

Wild Type ◽

Matrix Mineralization ◽

Inorganic Pyrophosphate ◽

Nucleotide Pyrophosphatase ◽

Tissue Nonspecific Alkaline Phosphatase ◽

Calvarial Osteoblast

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent 45Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP−/− mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PPi), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PPi. Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PPi and inhibited 45Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated 45Ca precipitation. Furthermore, the PPi content of MV fractions was greater in cultured TNAP−/− than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP−/− mice. Thus TNAP attenuates PC-1/NTPPPH-induced PPigeneration that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.

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Properties of Repressible Alkaline Phosphatase from Wild Type and a Wall-less Mutant of Neurospora crassa

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)42374-0 ◽

1974 ◽

Vol 249 (15) ◽

pp. 4679-4688

Author(s):

Earl G. Burton ◽

Robert L. Metzenberg

Keyword(s):

Alkaline Phosphatase ◽

Neurospora Crassa ◽

Wild Type

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Conversion of secretory proteins into membrane proteins by fusing with a glycosylphosphatidylinositol anchor signal of alkaline phosphatase

Biochemical Journal ◽

10.1042/bj3010577 ◽

1994 ◽

Vol 301 (2) ◽

pp. 577-583 ◽

Cited By ~ 8

Author(s):

K Oda ◽

J Cheng ◽

T Saku ◽

N Takami ◽

M Sohda ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Endoplasmic Reticulum ◽

Aspartic Acid ◽

Chimeric Protein ◽

Attachment Site ◽

In Vitro Translation ◽

Placental Alkaline Phosphatase ◽

Wild Type ◽

Aspartic Acid Residue

Placental alkaline phosphatase (PLAP) is initially synthesized as a precursor (proPLAP) with a C-terminal extension. We constructed a recombinant cDNA which encodes a chimeric protein (alpha GL-PLAP) comprising rat alpha 2u-globulin (alpha GL) and the C-terminal extension of PLAP. Two molecular species (25 kDa and 22 kDa) were expressed in the COS-1 cell transfected with the cDNA for alpha GL-PLAP. Only the 22 kDa form was labelled with both [3H]stearic acid and [3H]ethanolamine. Upon digestion with phosphatidylinositol-specific phospholipase C the 22 kDa form was released into the medium, indicating that this form is anchored on the cell surface via glycosylphosphatidylinositol (GPI). A specific IgG raised against a C-terminal nonapeptide of proPLAP precipitated the 25 kDa form but not the 22 kDa form, suggesting that the 25 kDa form is a precursor retaining the C-terminal propeptide. When a mutant alpha GL-PLAP, in which the aspartic acid residue is replaced with tryptophan at a putative cleavage/attachment site, was expressed in COS-1 cells, the 25 kDa precursor was the only form found inside the cell and retained in the endoplasmic reticulum, as judged by immunofluorescence microscopy. In vitro translation programmed with mRNAs coding for the wild-type and mutant forms of alpha GL-PLAP demonstrated that the C-terminal propeptide was cleaved from the wild-type chimeric protein, but not from the mutant one. This gave rise to the 22 kDa form attached with a GPI anchor, suggesting that GPI is covalently linked to the aspartic acid residue (Asp159) of alpha GL-PLAP. Taken together, these results indicate that the C-terminal propeptide of PLAP functions as a signal to render alpha GL a GPI-linked membrane protein in vitro and in vivo in cultured cells, and that the chimeric protein constructed in this study may be useful for elucidating the mechanism underlying the cleavage of the propeptide and attachment of GPI, which occur in the endoplasmic reticulum.

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