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:3.1.3.16 (calcineurin)
17,112 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The molecular mechanisms underlying regulation of fluid production by secretory epithelia such as the choroid plexus are poorly understood. Two cAMP-regulated inhibitors of protein phosphatase-1, inhibitor-1 (I1) and a dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000 (DARPP-32), are enriched in the choroid plexus. We show here that these two phosphoproteins are colocalized in choroid plexus epithelial cells. We have developed a novel method for studying the phosphorylation state of DARPP-32 and I1 in intact cells, using a phosphorylation state-specific monoclonal antibody. Several drugs and hormones that are known to alter fluid secretion and that increase cAMP levels (forskolin, isoproterenol, vasoactive intestinal peptide) or cGMP levels (atrial natriuretic peptide) or that may use additional second messenger pathways (5-HT), increase the phosphorylation of I1 and DARPP-32 in rat choroid plexus. In contrast, dopamine does not alter cAMP and cGMP levels, or I1 and DARPP-32 phosphorylation. Our results indicate that DARPP-32, known to be regulated by dopamine in a number of tissues, can be phosphorylated in response to non-dopaminergic factors, including hormones acting through non-cAMP-dependent pathways. Our results also raise the possibility that inhibition of phosphatase-1, as a result of I1 and DARPP-32 phosphorylation, might be part of a final common pathway in the action of several factors that are known or thought to alter cerebrospinal fluid production.
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PMID:Phosphorylation of DARPP-32 and protein phosphatase inhibitor-1 in rat choroid plexus: regulation by factors other than dopamine. 149 46

The effect of atrial natriuretic peptide (ANP) on angiotensin II- and histamine-induced contraction and muscle light chain phosphorylation was examined in strips of rabbit aorta smooth muscle. Preincubation of strips with 10(-7) M ANP prior to addition of either agonist inhibits both the increase in extent of myosin light chain phosphorylation and the contractile response to either 5 x 10(-8) M angiotensin II or 10(-5) M histamine without inhibiting the agonist-induced increase in the intracellular free Ca2+ concentration. Furthermore, in muscle strips precontracted with either angiotensin II or histamine, addition of ANP leads to a prompt relaxation and a prompt decrease in the extent of myosin light chain phosphorylation. These data argue that ANP uncouples the initial agonist-induced Ca2+ transient from the increase in extent of myosin light chain phosphorylation either by inhibiting the Ca2+-dependent activation of myosin light chain kinase or stimulating the activity of a phosphoprotein phosphatase capable of bringing about the rapid dephosphorylation of phosphorylated myosin light chains.
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PMID:Atrial natriuretic peptide inhibits the agonist-induced increase in extent of myosin light chain phosphorylation in aortic smooth muscle. 297 Oct 37

A phosphatase which exhibits strong activity toward phosphorylated atrial natriuretic peptide (ANP) was identified in the soluble fraction of rat brain homogenate. This ANP phosphatase has a neutral pH optimum, does not require divalent cations for activity, is inhibited by low concentrations of okadaic acid (50% inhibition at 1 nM) and preferentially dephosphorylates the alpha subunit of phosphorylase kinase. These properties are characteristic of serine/threonine protein phosphatase type 2A (PP2A). The apparent molecular mass of the ANP phosphatase (160 kDa), as estimated by gel filtration, is similar to that of the native heterotrimeric form of PP2A. In addition, phosphorylated ANP is an excellent substrate for the purified catalytic subunit of PP2A (Km = 42 microM, Vmax = 10.3 mumol x min-1 x mg-1). In contrast, protein phosphatase 2B (PP2B) has only very low ANP phosphatase activity (Km = 2.5 microM, Vmax = 0.008 mumol x min-1 x mg-1), and the catalytic subunit of protein phosphatase type 1 (PP1) as well as purified protein phosphatase type 2C (PP2C) are essentially inactive on ANP. These findings are consistent with the observation that PP2A-like activity accounts for virtually all ANP dephosphorylation in brain homogenate. While the phosphorylation of ANP in vitro by cAMP-dependent protein kinase is well documented, this is a first report on a phosphatase that efficiently can reverse this modification.
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PMID:Dephosphorylation of phosphorylated atrial natriuretic peptide by protein phosphatase 2A. 838 53

Attempts to activate partially purified preparations of the guanylyl cyclase-A (GC-A) receptor with atrial natriuretic peptide (ANP) have previously failed, leading to speculation that essential cofactors are lost during purification procedures. The receptor was modified to contain the FLAG epitope (DYKDDDDK), expressed in Sf9 cells, and purified to apparent homogeneity (4.3 mumol cyclic GMP formed/min/mg protein; 5.8 mmol 125I-ANP binding site/mg protein) by a combination of immunoaffinity, Q-Sepharose FF, and wheat germ agglutinin batch chromatography. High initial protein/detergent ratios, the presence of glycerol (40%), and the inclusion of protein phosphatase inhibitors in all buffers resulted in the purification of a receptor that continued to transduce the ANP/ATP activation signal. Both native and purified GC-A contained a single class of high affinity ANP binding sites (Kd = 60 pM) and an equivalent EC50 for ATP (0.3 mM). Positive cooperativity as a function of MnGTP was retained during purification. Thus, GC-A is capable of transducing a ligand binding signal in the absence of other proteins.
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PMID:The guanylyl cyclase-A receptor transduces an atrial natriuretic peptide/ATP activation signal in the absence of other proteins. 853 May 25

Vasodilating agents induce relaxation of mesangial cells, in part through cGMP-mediated activation of large calcium-activated potassium channels (BKCa). Normally quiescent in cell-attached patches, the response of BKCa to nitric oxide, atrial natriuretic peptide, and dibutyryl cGMP (Bt2cGMP) is characterized by a biphasic increase and then decrease ("rundown") in open probability. Using the patch-clamp method in conjunction with phosphatase inhibitors, we investigated whether the run-down phase was the result of dephosphorylation by an endogenous protein phosphatase. In cell-attached patches, cantharidic acid (500 nM), okadaic acid (100 nM), and calyculin A (100 nM), nondiscriminant inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A) at these concentrations, caused a significantly greater and sustained response of BKCa to Bt2cGMP. Within 2 min, the response of BKCa to the combination of cantharidic acid and Bt2cGMP was greater than the response to these agents added separately. Incubation of mesangial cells with okadaic acid for 20 min at a concentration (5 nM) specific for PP2A increased the basal open probability of BKCa and completely inhibited rundown after activation by Bt2cGMP. Incubation with calyculin A (10 nM), a more potent inhibitor of PP1, did not affect BKCa activity. In inside-out patches, Bt2cGMP plus MgATP caused a sustained activation of BKCa that was inhibited by exogenous PP2A but not PP1. It is concluded that either BKCa or a tightly associated regulator of BKCa is a common substrate for endogenous cGMP-activated protein kinase, which activates BKCa, and PP2A, which inactivates BKCa, in human mesangial cells.
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PMID:Regulation of large calcium-activated potassium channels by protein phosphatase 2A. 909 28

A rapidly emerging body of literature implicates a pivotal role for the Ca2+-calmodulin-dependent phosphatase calcineurin as a cellular target for a variety of Ca2+-dependent signaling pathways culminating in left ventricular hypertrophy (LVH). Most of the recent experimental support for this hypothesis is derived from in vitro studies or in vivo studies in transgenic mice expressing activated calcineurin or mutant sarcomeric proteins. The aim of the present study was to test whether calcineurin inhibitors, cyclosporin A (CsA) and FK 506, prevent pressure-overload LVH using 2 standard rat models: (1) the spontaneously hypertensive rat (SHR) and (2) aortic banding. The major new findings are 2-fold. First, in SHR, LVH (left ventricular weight to body weight ratio) was unaffected by a dose of CsA (5 mg. kg-1. d-1) that was sufficient to raise blood pressure and to inhibit calcineurin-mediated transcriptional activation in skeletal muscle. Second, in rats with aortic banding, LVH was unaffected by FK 506 (0.3 mg. kg-1. d-1) or even higher doses of CsA (10 and 20 mg. kg-1. d-1) that were sufficient to inhibit 90% of total calcineurin phosphatase activity in the hypertrophied myocardium. In the latter experiments, CsA blocked neither the elevated left ventricular end-diastolic pressures, a measure of diastolic function, nor the induction in atrial natriuretic peptide mRNA in the hypertrophic ventricles. Thus, in numerous experiments, systemic administration of potent calcineurin inhibitors did not prevent the development of LVH in 2 classic models of pressure-overload hypertrophy. These results demonstrate that pressure-overload hypertrophy can arise through calcineurin-independent pathways.
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PMID:Failure of calcineurin inhibitors to prevent pressure-overload left ventricular hypertrophy in rats. 1018 63

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.
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PMID:A constitutively "phosphorylated" guanylyl cyclase-linked atrial natriuretic peptide receptor mutant is resistant to desensitization. 1035 98

The cellular processes linking mechanical wall stretch to atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) secretion from the heart are unclear. In the present study, a paced perfused rat heart preparation was used to study the signaling mechanisms of atrial wall stretch-induced secretion of ANP and BNP. Vehicle or drugs were infused into the perfusate for 40 min and right atrial wall stretch was superimposed for 10 min after 25-min drug infusions by elevating the level of the pulmonary artery cannula tip. Lavendustin A, a potent inhibitor of protein tyrosine kinases, at the concentrations of 0.5 and 1.3 microM decreased atrial wall stretch-induced ANP secretion (53% and 68%, respectively, P < 0.001) in the perfused rat heart preparation, whereas no difference in the hemodynamic variables (heart rate, contractile force and perfusion pressure) were noted between groups. Lavendustin A also completely abolished the wall stretch-induced secretion of BNP. Several other protein kinase inhibitors including staurosporine (protein kinase C inhibitor), ML-9 (myosin light chain kinase inhibitor), KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) and H-89 (protein kinase A inhibitor) had no significant effect on atrial wall stretch-stimulated ANP secretion. In a separate series of experiments, in which the right atria were stretched for 2 h, administration of lavendustin A (1 microM) but not staurosporine (30 nM) significantly decreased sustained wall stretch-induced ANP secretion. Okadaic acid, a potent protein phosphatase A2 (PPA2) and PP1 inhibitor, at the concentration of 100 nM had no effect on basal ANP secretion but significantly accelerated the ANP secretory response to atrial wall stretch (P < 0.05). In conclusion, the findings that inhibitors of protein tyrosine kinase and protein phosphatase selectively modulated atrial wall stretch-induced ANP secretion suggest a new mechanism involving endogenous protein tyrosine activity in the regulation of natriuretic peptide exocytosis from cardiac myocytes.
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PMID:Inhibition of atrial wall stretch-induced cardiac hormone secretion by lavendustin A, a potent tyrosine kinase inhibitor. 1046 92

We recently reported that leukemia inhibitory factor (LIF) enhances Ca(2+)](i) through an increase in L-type Ca(2+) current (I(Ca,L)) in adult cardiomyocytes. The aim of this study was to investigate whether LIF activates Ca(2+)-dependent signaling molecules, such as calcineurin and calmodulin kinases II and IV (CaMKII and CaMKIV), and, if so, whether these Ca(2+)-mediated signaling events contribute to LIF-mediated cardiac hypertrophy. We first confirmed that LIF increased I(Ca,L) and [Ca(2+)](i) in primary cultured rat neonatal cardiomyocytes. Calcineurin, CaMKII, and CaMKIV activities increased at 2 minutes and peaked by 1.6-, 2.2-, and 2.2-fold, respectively, at 15 minutes. Nicardipine or verapamil fully inhibited these activities. Autophosphorylation of CaMKII was also observed to parallel the timing of CaMKII activity, and this phosphorylation was blocked by nicardipine, verapamil, or EGTA. LIF treatment led to a 3-fold increase in nuclear factor of activated T cell-luciferase activity. To confirm that inositol triphosphate (IP(3))-induced Ca(2+) release from sarcoplasmic reticulum was not involved in this process, IP(3) content and phosphorylation of phospholipase Cgamma were investigated. LIF did not increase IP(3) content or phosphorylate phospholipase Cgamma. KN62 (an inhibitor of CaMKII and CaMKIV) attenuated c-fos, brain natriuretic peptide, alpha-skeletal actin, and atrial natriuretic peptide expression. KN62 suppressed the LIF-induced increase in [(3)H]phenylalanine uptake and cell size. Cyclosporin A and FK506 slightly attenuated brain natriuretic peptide but did not affect c-fos or atrial natriuretic peptide expression. Cyclosporin A significantly reduced the LIF-induced increase in [(3)H]phenylalanine uptake. These findings indicated that LIF activated CaMKII, CaMKIV, and calcineurin through an increase in I:(Ca,L) and [Ca(2+)](i) and that CaMKII, CaMKIV, and calcineurin are critically involved in LIF-induced cardiac hypertrophy.
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PMID:Calmodulin kinases II and IV and calcineurin are involved in leukemia inhibitory factor-induced cardiac hypertrophy in rats. 1107 91

Cardiotoxicity resulting from detrimental environmental insults has been recognized for a long time. However, extensive studies of the mechanisms involved had not been undertaken until recent years. Advances in molecular biology provide powerful tools and make such studies possible. We are gathering information about cellular events, signaling pathways, and molecular mechanisms of myocardial toxicologic responses to environmental toxicants and pollutants. Severe acute toxic insults cause cardiac cell death instantly. In the early response to mild environmental stimuli, biochemical changes such as alterations in calcium homeostasis occur. These may lead to cardiac arrhythmia, which most often is reversible. Prolonged stimuli activate transcription factors such as activator protein-1 through elevation of intracellular calcium and the subsequent activation of calcineurin. Upregulation by activated transcription factors of hypertrophic genes results in heart hypertrophy, which is a short-term adaptive response to detrimental factors. However, further development of hypertrophy will lead to severe and irreversible cardiomyopathy, and eventually heart failure. From cardiac hypertrophy to heart failure, myocardial cells undergo extensive biochemical and molecular changes. Cardiac hypertrophy causes tissue hypoperfusion, which activates compensatory mechanisms such as production of angiotensin II and norepinephrine. Both further stimulate cardiac hypertrophy and, importantly, activate counterregulatory mechanisms including overexpression of atrial natriuretic peptide and b-type natriuretic peptide, and production of cytokines such as tumor necrosis factor-alpha. This counterregulation leads to myocardial remodeling as well as cell death through apoptosis and necrosis. Cell death through activation of mitochondrial factors and other pathways constitutes an important cellular mechanism of heart failure. Our current knowledge of cardiotoxicity is limited. Further extensive studies are warranted for a comprehensive understanding of this field.
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PMID:Molecular and cellular mechanisms of cardiotoxicity. 1125 Aug 3


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