Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The whole-cell patch-clamp technique was used to examine nonselective conductances in single proximal tubule cells isolated from mouse kidney. Single cells were isolated in either the presence or absence of a cocktail designed to stimulate cAMP. Patches were obtained with Na+ Ringer in the bath and Cs+ Ringer in the pipette. On initially achieving the whole-cell configuration, whole-cell currents were small. In cAMP-stimulated cells, with 5 mM ATP in the pipette solution, whole-cell currents increased with time. The activated current was linear, slightly cation-selective, did not discriminate between Na+ and K+ and was inhibited by 100 microM gadolinium. These properties are consistent with the activation of a nonselective conductance, designated G(NS). Activation of G(NS) was abolished with pipette AMP-PNP, ATP plus alkaline phosphatase or in the absence of ATP. In unstimulated cells G(NS) was activated by pipette ATP together with PKA. These data support the hypothesis that G(NS) is activated by a PKA-mediated phosphorylation event. G(NS) was also activated by a hypertonic shock. However, G(NS) does not appear to be involved in regulatory volume increase (RVI), as RVI was unaffected in the presence of the G(NS) blocker gadolinium. Instead, the ATP sensitivity of G(NS) suggests that it may be regulated by the metabolic state of the renal proximal tubule cell.
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PMID:A hypertonicity-activated nonselective conductance in single proximal tubule cells isolated from mouse kidney. 1282 Jun 64

To clarify the regulating mechanism of vascular calcification, the investigators observed the effects of three vasoactive peptides, adrenomedullin (ADM), C-type natriuretic peptide (CNP), and parathyroid hormone-related peptide (PTHrP) on calcification in rat vascular smooth muscle cells (VSMCs). Beta-glycerophosphate stimulated growth and calcification in VSMCs. Adrenomedullin and CNP lowered beta-glycerophosphate-induced increase in VSMC growth. All three vasoactive peptides attenuated the increases of 45Ca accumulation, calcium content, and alkaline phosphatase activity in calcified VSMCs. As for comparing the inhibitory effects, the strongest was PTHrP. Both ADM and PTHrP increased cyclic adenosine monophosphate (cAMP) content in calcified VSMCs, but CNP upregulated cyclic guanosine monophosphate (cGMP) content. The PKA inhibitor PKAI completely reversed the inhibition of ADM on cell growth and all inhibitory effects of PTHrP on the parameters of calcification. The PKG inhibitor H8, however, strongly antagonized all the inhibitory effects of CNP on calcification. These data suggested that beta-glycerophosphate-induced calcification in VSMCs was inhibited by ADM, CNP, and PTHrP. Adrenomedullin and PTHrP inhibited VSMC calcification partially through the cAMP/PKA pathway, whereas CNP inhibited VSMC calcification through the cGMP/PKG pathway. This study could be of help in understanding the pathogenesis of vascular calcification, and providing new target for clinical treatment of cardiovascular diseases associated with vascular calcification.
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PMID:Effects of adrenomedullin, C-type natriuretic peptide, and parathyroid hormone-related peptide on calcification in cultured rat vascular smooth muscle cells. 1282 32

Hydrophobic bile salts induce either necrosis or apoptosis depending on the severity of the injury caused by them. Since bile salt-induced apoptosis is influenced by Ca2+- and protein kinase-signaling pathways, and both necrosis and apoptosis share common initiating mechanisms, we analyzed whether these signaling cascades also influence bile salt-induced necrosis in isolated rat hepatocytes. Taurochenodeoxycholate (TCDC, 0.25-1.50 mM, 2 h) reduced, in a dose-dependent manner, the percentage of viable hepatocytes, and increased the release of the cytosolic enzyme, lactate dehydrogenase (LDH) and alanine aminotransferase (ALAT), and that of the plasma membrane enzyme, alkaline phosphatase (AP). The PKC inhibitors, H7 (100 microM) and chelerythrine (2.5 microM), both prevented significantly TCDC-induced necrosis. On the contrary, the PKA activator, dibutyryl-cAMP, exacerbated TCDC-induced cell damage in a dose-dependent manner; this effect was more likely due to cAMP-mediated PKA activation, as the PKA inhibitor, KT5720 (1 microM), counteracted this effect. Instead, the intracellular Ca2+ chelator, BAPTA/AM (20 microM), was without effect. TCDC (1 mM) increased lipid peroxidation from 0.7 +/- 0.2 to 7.5 +/- 0.9 nmol of malondialdehyde per mg of protein, p < 0.001; the addition of the free radical scavenger, diphenyl-p-phenylendiamine, completely blocked this increase and prevented significantly TCDC-induced necrosis. PKC inhibition induced only a slight attenuation of TCDC-induced lipid peroxidation. Possible mechanisms accounting for the modulatory effect of signal transduction pathways on TCDC-induced necrosis, including signaling influence on TCDC transport events and TCDC-induced oxidative stress, are discussed.
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PMID:Signaling modulation of bile salt-induced necrosis in isolated rat hepatocytes. 1549 97

CCN2/connective tissue growth factor (CCN2/CTGF) is known to promote both the proliferation and differentiation of chondrocytes, which actions are mediated by ERK and p38 MAPK, respectively. In this study, we first re-evaluated the involvement of multiple MAPKs therein and found that JNK also mediated such CCN2 signals. Thereafter, we further analyzed the roles of upstream kinases. The involvement of PKC, PI3K and PKA in the CCN2 signaling to promote the maturation, proliferation and terminal differentiation of a human chondrocytic cell line, HCS-2/8 and rabbit primary growth cartilage cells was investigated. As a result, the PKC inhibitor calphostin C repressed all of the effects of CCN2, which were represented by increased synthesis of DNA and proteoglycans and the display of alkaline phosphatase activity. In addition, evaluation of the effect of the PI3K inhibitor wortmannin disclosed the contribution of PI3K in transducing CCN2 signals to promote chondrocyte hypertrophy. This signal was known to be mediated by PKB, which was translocated into the nucleus upon CCN2 stimulation. Of note, calphostin C showed inhibitory effects on the activation of p38 MAPK, ERK and also PKB, whereas it exerted no effect on JNK activation. These results suggest that PKC is a driver of multiple signal transducing kinases that promote the proliferation and differentiation of chondrocytes. The requirement of PI3K in transmitting the signal for terminal differentiation and PKC-independent signaling pathways for the promotion of chondrocytic growth and differentiation, which was mediated by JNK, were also uncovered.
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PMID:Roles of PKC, PI3K and JNK in multiple transduction of CCN2/CTGF signals in chondrocytes. 1643 Nov 70

Different C-terminal fragments of parathyroid hormone (PTH)-(1-84) in blood participate in the regulation of calcium homeostasis by PTH-(1-84), and an antagonizing effect for the large carboxyl-terminal parathyroid hormone (C-PTH)-fragment (7-84) on calcium release has been described in vivo and in vitro. In this study the smaller C-PTH-fragment (53-84) and mid-regional PTH fragment (28-48), which represent discrete areas of activity in the PTH-(7-84) molecule, were assayed for their effects on calcium release and alkaline phosphatase (ALP) activity in a chick bone organ culture system. Neither PTH-(28-48) nor PTH-(53-84) had any effect on calcium release into the medium and both fragments stimulated ALP activity in the bone tissue, suggesting that the cAMP/PKA signalling pathway was not affected by these fragments. However they suppressed the calcium release induced by PTH-(1-34) and attenuated the down regulation of ALP activity caused by PTH-(1-34), suggesting that the effect on the cAMP/PKA signalling pathway may be indirectly. In conclusion, the study shows that the PTH-fragments (53-84) and (28-48) antagonize the PTH-(1-34) induced effects on calcium release and inhibition of ALP activity in a chick bone organ culture system.
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PMID:hPTH-fragments (53-84) and (28-48) antagonize the stimulation of calcium release and repression of alkaline phosphatase activity by hPTH-(1-34) in vitro. 1646 18

The biological activities of parathyroid hormone (PTH) on bone are quite complex as demonstrated by its catabolic and anabolic activities on the skeleton. Although there have been many reports describing genes that are regulated by PTH in osteoblast cells, the goal of this study was to utilize a well-established in vivo PTH anabolic treatment regimen to identify genes that mediate bone anabolic effects of PTH. We identified a gene we named PTH anabolic induced gene in bone (PAIGB) that has been reported as brain and acute leukemia cytoplasmic (BAALC). Therefore, using the latter nomenclature, we have discovered that BAALC is a PTH-regulated gene whose mRNA expression was selectively induced in rat tibiae nearly 100-fold (maximal) by a PTH 1-34 anabolic treatment regimen in a time-dependent manner. Although BAALC is broadly expressed, PTH did not regulate BAALC expression in other PTH receptor expressing tissues and we find that the regulation of BAALC protein by PTH in vivo is confined to mature osteoblasts. Further in vitro studies using rat UMR-106 osteoblastic cells show that PTH 1-34 rapidly induces BAALC mRNA expression maximally by 4 h while the protein was induced by 8 h. In addition to being regulated by PTH 1-34, BAALC expression can also be induced by other bone forming factors including PGE(2) and 1,25 dihydroxy vitamin D(3). We determined that BAALC is regulated by PTH predominantly through the cAMP/PKA pathway. Finally, we demonstrate in MC3T3-E1 osteoblastic cells that BAALC overexpression regulates markers of osteoblast differentiation, including downregulating alkaline phosphatase and osteocalcin expression while inducing osteopontin expression. We also demonstrate that these transcriptional responses mediated by BAALC are similar to the responses elicited by PTH 1-34. These data, showing BAALC overexpression can mimic the effect of PTH on markers of osteoblast differentiation, along with the observations that BAALC is induced selectively with a bone anabolic treatment regimen of PTH (not a catabolic treatment regimen), suggest that BAALC may be an important mediator of the PTH anabolic action on bone cell function.
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PMID:Identification of a PTH regulated gene selectively induced in vivo during PTH-mediated bone formation. 1651 68

The goals of this study were to determine (a) if melatonin enhances human adult mesenchymal stem cell (hAMSC) differentiation into osteoblasts as assessed by measuring alkaline phosphatase (ALP) enzyme activity, and (b) identify potential signal transduction pathways that mediate this process. ALP activity significantly increased in hAMSCs following a 10-day incubation in osteogenic medium, relative to hAMSCs incubated in basal growth medium alone. Melatonin (50 nm), added in combination with the osteogenic medium, significantly increased ALP activity relative to osteogenic medium alone. Co-exposure of hAMSCs to osteogenic medium supplemented with melatonin and either pertussis toxin or the melatonin receptor antagonists, luzindole or 4P-PDOT (MT2 receptor selective), inhibited the melatonin-induced increase in ALP activity, indicating the involvement of melatonin receptors, in particular, MT2 receptors. Assessment of melatonin receptor function following exposure to osteogenic medium containing either vehicle or melatonin produced dichotomous results. That is, if the differentiation of hAMSCs into an osteoblast was induced by osteogenic medium alone, then 2-[125I]-iodomelatonin binding and melatonin receptor function increased. However, examination of melatonin receptor function following chronic melatonin exposure, an exposure that resulted in a 50% enhancement in ALP activity, revealed that these receptors were desensitized. This was reflected by a complete loss in specific 2-[125I]-iodomelatonin binding as well as melatonin efficacy to inhibit forskolin-induced cAMP accumulation. Further characterization of the mechanisms underlying melatonin's effects on these differentiation processes revealed that MEK (1/2) and ERK (1/2), epidermal growth factor receptors, metalloproteinase and clathrin-mediated endocytosis were essential while PKA was not. Our results are consistent with a role for melatonin in osteoblast differentiation. If so, then, the decrease in plasma melatonin levels observed in humans during late adulthood may further enhance susceptibility to osteoporosis.
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PMID:Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade. 1663 21

Emerin is a ubiquitously expressed inner nuclear membrane protein of unknown function. Mutations in its gene give rise to X-linked Emery-Dreifuss muscular dystrophy (X-EDMD), a neuromuscular condition with an associated life-threatening cardiomyopathy. We have previously reported that emerin is phosphorylated in a cell cycle-dependent manner in human lymphoblastoid cell lines [Ellis et al. (1998) Aberrant intracellular targeting and cell cycle-dependent phosphorylation of emerin contribute to the EDMD phenotype. J. Cell Sci. 111, 781-792]. Recently, five residues in human emerin were identified as undergoing cell cycle-dependent phosphorylation using a Xenopus egg mitotic cytosol model system (Hirano et al. (2005) Dissociation of emerin from BAF is regulated through mitotic phosphorylation of emerin in a Xenopus egg cell-free system. J. Biol. Chem.280, 39 925-39 933). In the present paper, recombinant human emerin was purified from a baculovirus-Sf9 heterogeneous expression system, analyzed by protein mass spectrometry and shown to exist in at least four different phosphorylated species, each of which could be dephosphorylated by treatment with alkaline phosphatase. Further analysis identified three phosphopeptides with m/z values of 2191.9 and 2271.7 corresponding to the singly and doubly phosphorylated peptide 158-DSAYQSITHYRPVSASRSS-176, and a m/z of 2396.9 corresponding to the phosphopeptide 47-RLSPPSSSAASSYSFSDLNSTR-68. Sequence analysis confirmed that residue S49 was phosphorylated and also demonstrated that this residue was phosphorylated in interphase. Using an in vitro protein kinase A assay, we observed two phospho-emerin species, one of which was phosphorylated at residue S49. Protein kinase A is thus the first kinase that has been identified to specifically phosphorylate emerin. These results improve our understanding of the molecular mechanisms underlying X-EDMD and point towards possible signalling pathways involved in regulating emerin's functions.
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PMID:The Emery-Dreifuss muscular dystrophy associated-protein emerin is phosphorylated on serine 49 by protein kinase A. 1697 41

Many growth factors or cytokines regulate cell proliferation via different intracellular signaling pathways. The mechanisms remained quite unclear in avian primordial germ cells (PGCs). In the present study, two major protein kinases, PKA and PKC, were investigated to be involved in signal transduction of PGC proliferation. PGCs were isolated from genital ridge of 3.5-day chicken embryos and primary culture was performed with 5% fetal calf serum (FCS)-supplemented medium 199. After culture for 24 h, PGCs were subcultured on chicken embryonic fibroblast feeder (CEF) and the cells were characterized by histochemical stainings of alkaline phosphatase (ALP) and periodic acid-Schiff (PAS) reagent as well as immunocytochemical stainings of c-kit and stage-specific embryonic antigen-1 (SSEA-I). In addition, cells were challenged with adenylate cyclase activator forskolin (FRSK) and PKC activator phorbol-12-myristate-13-acetate (PMA) alone or in combinations with PKA inhibitor H(89) and PKC inhibitor H(7), respectively. Results showed that subcultured PGCs on CEF displayed positive histochemical and immunocytochemical stainings for ALP, PAS, c-kit and SSEA-I and manifested intensive proliferating activity by colony formation. Downstream activation of PKA by FRSK (10(-7) to 10(-5)M) significantly promoted the proliferation of PGCs by increasing colony number (ALP-stained) in a dose-dependant manner. PMA (10(-8)M) also increased PGC colony number (P<0.05). However, the proliferating effects elicited by FRSK or PMA could be inhibited by the respective protein kinase inhibitor H(89) or H(7). Therefore, the above results suggest that activation of intracellular protein kinases A and C by external factors may promote proliferation of cultured PGCs and PKA represents the most likely mediator of PGC proliferation in embryonic chickens.
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PMID:Activation of protein kinases A and C promoted proliferation of chicken primordial germ cells. 1705 97

Increased bone formation by PTH mainly results from activation of osteoblasts, an effect largely mediated by the cAMP-PKA pathway. Other pathways, however, are likely to be involved in this process. In this study we investigated whether PTH can activate p38 MAPK and the role of this kinase in osteoblastic cells. Bovine PTH(1-34) and forskolin markedly increased alkaline phosphatase (ALP) activity and doubled osteocalcin (Oc) expression in early differentiating MC3T3-E1 cells. These effects were associated with increase in cellular cAMP and activation of the MAP kinases ERK and p38. Activation of these MAP kinases was detectable after 1 h incubation with 10(-7) M PTH and lasted 1-2 h. Activation of p38 was mimicked by 10 microM forskolin and prevented by H89 suggesting a cAMP-PKA-dependent mechanism of p38 activation. Interestingly, PTH-induced ALP stimulation was dose-dependently inhibited by a specific p38 inhibitor with no change in the generation of cAMP and the production of osteocalcin. Similar inhibitory effect was obtained in cells stably expressing a dominant-negative p38 molecule. Finally, treatment of MC3T3-E1 cells with PTH for 3 weeks significantly enhanced matrix mineralization and this effect was markedly reduced by a selective p38 but not a specific MEK inhibitor. In conclusion, data presented in this study indicate that PTH can activate p38 in early differentiating osteoblastic cells. Activation of p38 is cAMP-PKA-dependent and mediates PTH-induced stimulation of ALP which plays a critical role for the calcification of the bone matrix.
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PMID:Evidences for a role of p38 MAP kinase in the stimulation of alkaline phosphatase and matrix mineralization induced by parathyroid hormone in osteoblastic cells. 1743 17


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