Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neurogranin, a peptide capable of binding the calcium-poor form of calmodulin, was tested in vitro for its ability to modulate a typical calmodulin target. The target employed was the calcium/calmodulin-dependent form of nitric oxide synthase, which is produced by several different types of neurons. Neurogranin for the study was purified from perchloric acid-soluble calf brain proteins by a combination of calmodulin-Sepharose affinity chromatography and reverse-phase HPLC. The protocol yielded highly purified neurogranin that was active in assays using purified nitric oxide synthase. The titration of the enzyme activity with neurogranin demonstrated a concentration-dependent effect of the peptide on enzyme activation. Subsequent analysis of the ability of increased calcium concentrations to activate the enzyme was performed in the presence of different amounts of neurogranin. The effect of neurogranin on the calcium-dependent activation of the enzyme was to depress enzyme activity in the range of 0.2 to approximately 1 microM calcium. Treatment of the neurogranin peptide with protein kinase C eliminated its inhibition on nitric oxide synthase activation. Treatment of the protein kinase C-phosphorylated peptide with calcineurin did not restore the ability of neurogranin to inhibit enzyme activity, whereas treatment with alkaline phosphatase did restore this ability. These results suggest that neurogranin may serve as a member of a unique class of endogenous calmodulin inhibitor that functions to regulate the activation of calmodulin-requiring targets in neurons.
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PMID:The dendritic peptide neurogranin can regulate a calmodulin-dependent target. 752 68

Endogenous alkaline phosphatase activity has been localized histochemically on the surface of enteric neurons of the guinea-pig small intestine by both light and electron microscopy. The enzyme activity was associated with some myenteric neurons that had Dogiel type I morphology, and the histochemical reaction products typically formed a honeycomb-like structure on labelled cell bodies. No Dogiel type II neurons in the myenteric plexus or submucous neurons showed alkaline phosphatase reactivity. Nerve fibres reactive for alkaline phosphatase were present in the myenteric plexus and ran in bundles in the circular muscle and deep muscular plexus. In addition, reactive varicose axons supplied the submucous plexus and non-ganglionated plexus of the mucosa. The results of interruption of the enteric neuronal pathways demonstrated that alkaline phosphatase-reactive myenteric neurons project anally to other myenteric ganglia, to the circular muscle and to the submucous plexus. Sequential enzyme histochemistry showed that virtually all alkaline phosphatase-reactive neurons also contained nitric oxide synthase, revealed by NADPH-diaphorase reactivity. It was estimated that 14-18% of all myenteric neurons showed alkaline phosphatase reactivity. About one-third of nitric oxide synthase-containing myenteric neurons, however, did not contain alkaline phosphatase activity. At the ultrastructural level, alkaline phosphatase activity was associated specifically with the plasma membranes of nerve cell bodies, axons and dendrites of some myenteric neurons. Reactive nerve fibres made close appositions with non-reactive submucous neurons and, within myenteric ganglia, predominantly with other alkaline phosphatase-reactive neurons. In addition to its presence in neurons, alkaline phosphatase reactivity was also present in some endothelial cells in blood vessels in the submucosa and in capillary pericytes. It is concluded, on the basis of the projections and neurochemistry, that in the guinea-pig small intestine alkaline phosphatase activity is associated with nitric oxide synthase-containing neurons which include inhibitory motor neurons to the circular muscle, and anally-directed interneurons to other myenteric and submucous neurons.
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PMID:Characterization of alkaline phosphatase-reactive neurons in the guinea-pig small intestine. 753 94

The NADPH-diaphorase (NADPH-d) reaction is frequently used to visualize the diaphorase activity of nitric oxide synthase (NOS). However, this tetrazolium salt procedure can be of limited specificity at sites where non-specific alkaline phosphatase (alP) and NADHd activity co-exist. This is shown in the present paper using methods of catalytic histochemistry for these three enzymes and levamisole as alP inhibitor for certain mouse tissues. In the urothelium, portio, vaginal and endometrial epithelium as well as in some smooth muscle cells alP hydrolyzes NADPH to NADH which in turn serves as substrate for NADHd leading to false-positive formazan production. To exclude this possibility, it is recommended always to include levamisole in the incubation medium if the NADPHd activity of NOS has to be investigated.
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PMID:Nonspecific alkaline phosphatase activity can be responsible for staining of NADPH-diaphorase activity in certain non-neural cells. 755 70

NADPH diaphorase is a histochemical activity which, in formaldehyde-fixed tissue, is rather specific for nitric oxide synthase. Recently, it was shown that NADPH diaphorase activity is inhibited by ethylenediaminetetraacetic acid (EDTA) in neurons but not in the choroid plexus epithelium. The present study, while confirming these results, demonstrates that the apparent sensitivity of NADPH diaphorase for EDTA reflects only the dependence of malic enzyme, which is used as the source of reduced cofactor, on Mg2+ or Mn2+ ions. Furthermore, evidence is provided that the apparent EDTA-insensitive NADPH diaphorase activity in the choroid plexus reflects the activity of alkaline phosphatase in conjunction with NADH diaphorase. Apart from these pitfalls, the use of the indirect, malic enzyme based method for NADPH diaphorase was found to cause much higher background staining compared to the direct method using NADPH, and is therefore proposed to be abandoned.
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PMID:NADPH diaphorase is not inhibited by ethylenediaminetetraacetic acid and is not specific for nitric oxide synthase in the choroid plexus of rat and mouse. 773 45

Human breast (MCF-7, HBL 100, T47D, BT20, HS578T), colon (HT29, CACO2, SW620, SW480, COLO320DM) and small cell lung cancer (NCI-N417, OH3, SW2) cell lines were transplanted subcutaneously into severe combined immunodeficient (SCID) mice. When sizeable tumours developed, the mice were sacrificed and the following enzyme activities were detected histochemically: presumed nitric oxide synthase-associated diaphorase (NOSaD), beta-D-glucuronidase (beta-Gluc) and non-specific alkaline phosphatase (alP). Except for HT29 and MCF-7 presumed NOSaD activity was not detected in the tumour itself or in the neo-vasculature of the tumours. beta-Gluc activity was found in all tumour cells (except N417 and COLO 320), in the necrotic parts of the tumours and in stromal cells of the tumour bed. AlP activity was present in all tumours including their necrotic areas. However, the activities of beta-Gluc and alP varied considerably even within one tumour, ranging from very weak to very strong. Principally the results show that the human/SCID mouse tumour model is well suited to test modern applications of tumour therapy involving the enzymes NOSaD, beta-Gluc and alP. In particular, antibody directed enzyme prodrug therapy concepts and activation of prodrugs by enzymes released from tumour cells into the necrotic areas of the tumour can be evaluated in this in vivo model.
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PMID:Histochemistry of therapeutically relevant enzymes in human tumours transplanted into severe combined immunodeficient (SCID) mice: nitric oxide synthase-associated diaphorase, beta-D-glucuronidase and non-specific alkaline phosphatase. 896 Mar 2

The effect of nitric oxide (NO) on osteoblastic differentiation was examined in cultured mouse osteoblasts. Interleukin-1beta and tumor necrosis factor-alpha expressed inducible NO synthase gene with little effect on constitutive NO synthase gene. These cytokines increased NO production, which was inhibited by L-NMMA pretreatment, and decreased alkaline phosphatase (AIPase) activity, which was not restored by L-NMMA. Furthermore, NO donors, sodium nitroprusside and NONOate dose-dependently elevated AIPase activity and expression of osteocalcin gene. These results suggest that NO directly facilitates osteoblastic differentiation and the cytokine-induced inhibition of AIPase activity is mediated via mechanism other than NO.
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PMID:Direct action of nitric oxide on osteoblastic differentiation. 923 37

Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) have been implicated in the pathogenesis of osteoporosis. These proinflammatory cytokines induce both cyclooxygenase (COX) and nitric oxide synthase (NOS) with the release of prostaglandin (PG) and NO, respectively. The present study was undertaken to examine the interaction between COX and NOS pathways and their role in the regulation of osteoblastic function in MC3T3-E1 cells. Addition of IL-1 alpha and TNF-alpha induced a marked increase in the production of both NO and PGE2. Reverse transcription-polymerase chain reaction analysis showed that the increase in NO production was preceded by the expression of inducible NOS mRNA. The temporal profile of PGE2 production revealed a biphasic pattern: the first small peak at 3 h was caused by de novo synthesis of PGE2 through inducible COX (COX-2) mRNA, while the subsequent progressive accumulation of PGE2 was mediated through the activation of COX pathway by NO since (1) aminoguanidine (AG), a selective inhibitor of inducible NOS, significantly suppressed the PGE2 production by IL-1 alpha and TNF-alpha, (2) NOC-18, an NO donor, reversed this suppression, and (3) NOC-18 increased PGE2 production by itself. The increase in NO production in response to IL-1 alpha and TNF-alpha was further stimulated by aspirin and inhibited by exogenous addition of PGE2, suggesting that PGE2 produced by the cytokines, in turn, negatively modulates NO production. IL-1 alpha and TNF-alpha inhibited alkaline phosphatase (ALP) activity, which was significantly reversed by AG. NOC-18 not only suppressed ALP activity by itself but also blocked the effect of AG, suggesting the role of NO in the inhibition of ALP activity. PGE2 decreased ALP activity, and the inhibitory effect of NOC-18 was attenuated in the presence of aspirin, suggesting the involvement of PGE2 in the negative modulation of ALP activity by NO. These results suggest that NO produced in response to proinflammatory cytokines participates in the modulation of ALP activity via the activation of COX pathway. The interaction between NO and the COX pathways may play an important role in the regulation of osteoblastic functions under physiologic as well as pathologic conditions.
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PMID:Interaction between nitric oxide synthase and cyclooxygenase pathways in osteoblastic MC3T3-E1 cells. 938 83

The aim of this study was to assess the involvement in decidual proliferation of nitric oxide (NO), a regulator of many cellular processes, that is synthesized from L-arginine by NO synthase. The investigation was conducted on pseudopregnant (PG) rats in which the decidual cell reaction, the basis for the decidualization process, was surgically induced by uterine trauma on PG Day 4. Groups of animals (n = 5) were pretreated with either 2 doses/day of N(G)-nitro-L-arginine methyl ester (L-NAME) that inhibits NO synthase, or twice daily doses of L-NAME plus L-arginine combined. Drug application times coincided with 3 hr after lights on or 3 hr before lights off. The two treatment regimens (PG Days 1-4 or 5-8) respectively preceded or followed decidual induction. Animals were sacrificed at mid-light on PG Day 9, the day of maximal growth response to the deciduogenic stimulus. Parallel, time-dependent increases in both NO synthase activity and decidual growth occurred mainly in the endometrium. L-NAME produced reductions in endometrial and myometrial growth that were reversed by the combined L-NAME plus L-arginine treatments. These inhibitory effects by L-NAME were caused by only the pretraumal (PG Days 1-4) administration. Hormonally, circulating progesterone levels were similarly affected by this early treatment and may also contribute to the reduced decidual sensitivity. In contrast, serum estradiol, along with the zinc metalloenzymes, alkaline phosphatase and the matrix metalloproteinases--prominent decidualization biomarkers--were all unaffected by either the pre- or post-decidual induction dosings. The study demonstrates that inducible NO synthase/endogenous NO may physiologically participate in uterine metabolism during the decidual cell reaction. Moreover, by virtue of L-NAME inhibition of the decidual response, it appears that NO synthase/NO may influence decidual growth either by directly increasing uterine sensitivity to the deciduogenic stimulus or by indirectly affecting endometrial vascularity and subsequent availability of decidual metabolites.
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PMID:Antiproliferative effects of inducible nitric oxide synthase inhibition on decidualization in pseudopregnant rats. 957 51

Previous studies have shown evidence of constitutive and cytokine-inducible nitric oxide (NO) synthase activity in cultured osteoblast-like cells from various species. Although cytokine-induced NO production has been found to inhibit osteoblast growth, the role of constitutive NO production in regulating osteoblast function is less clear and the isoforms of nitric oxide synthase (NOS) that are expressed by human osteoblasts have not been determined. Here, we investigated NOS expression in cultured human osteoblast-like cells and studied the effects of constitutive and cytokine-induced NO on osteoblast growth and differentiation. Low levels of NO were produced constitutively by osteoblast-like cells as reflected by analysis of medium nitrite concentrations, and evidence of ecNOS mRNA, protein, and bioactivity was found in primary osteoblasts (hOBs), TE85, and MG63 osteosarcoma cells. None of the osteoblast-like cells expressed nNOS, however, and iNOS was produced only by hOB cells after stimulation with the cytokines IL-1beta, TNF-alpha, and IFN-gamma. The NOS inhibitor, L-NMMA, did not affect growth or alkaline phosphatase activity in unstimulated osteoblasts. Incubation of hOB cells with cytokines inhibited growth and stimulated alkaline phosphatase activity and these effects were abrogated by L-NMMA. Cytokines also inhibited growth of TE85 cells and MG63 cells, but these effects appeared to be NO independent because they were not influenced by L-NMMA. Our experiments show that human osteoblasts constitutively produce NO through the ecNOS pathway, but demonstrate that this does not appear to exert an appreciable effect on osteoblast growth or differentiation under basal conditions. In contrast, IL-1beta, TNF-alpha, and IFN-gamma exerted growth-inhibiting and differentiation-inducing effects on osteoblasts that were partly NO dependent, indicating that NO may act predominantly as a modulator of cytokine-induced effects on osteoblast function.
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PMID:Expression and functional role of nitric oxide synthase isoforms in human osteoblast-like cells. 1007 9

We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+-dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells.
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PMID:Morphological and biochemical investigation of nitric oxide synthase and related enzymes in the rat and pig urothelium. 1033 Apr 50


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