Activation of glycerophosphocholine phosphodiesterase in rat forebrain by Ca2+.
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ABSTRACT:
SUBMITTER: Spanner S
PROVIDER: S-EPMC1154040 | biostudies-other | 1982 Dec
REPOSITORIES: biostudies-other
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Project description:Glycerophosphocholine (GPC) is an abundant osmoprotective renal medullary organic osmolyte. We previously found that its synthesis from phosphatidylcholine is catalyzed by tonicity-regulated activity of the phospholipase B, neuropathy target esterase. We also found that its degradation is catalyzed by glycerophosphocholine phosphodiesterase (GPC-PDE) activity and that elevating osmolality from 300 to 500 mosmol/kg by adding NaCl or urea, inhibits GPC-PDE activity, which contributes to the resultant increase of GPC. In the present studies we identify GDPD5 (glycerophosphodiester phosphodiesterase domain containing 5) as a GPC-PDE that is rapidly inhibited by high NaCl or urea. Recombinant GDPD5 colocalizes with neuropathy target esterase in the perinuclear region of HEK293 cells, and immuno-precipitated recombinant GDPD5 degrades GPC in vitro. The in vitro activity is reduced when the cells from which the GDPD5 is immuno-precipitated have been exposed to high NaCl or urea. In addition, high NaCl decreases mRNA abundance of GDPD5 via an increase of its degradation rate, although high urea does not. At 300 mosmol/kg siRNA knockdown of GDPD5 increases GPC in mouse inner medullary collecting duct-3 cells, and over expression of recombinant GDPD5 increases cellular GPC-PDE activity, accompanied by decreased GPC. We conclude that GDPD5 is a GPC-PDE that contributes to osmotic regulation of cellular GPC.
| S-EPMC2504799 | biostudies-literature
Project description:Glycerophosphocholine (GPC) is high in cells of the renal inner medulla where high interstitial NaCl and urea power concentration of the urine. GPC protects inner medullary cells against the perturbing effects of high NaCl and urea by stabilizing intracellular macromolecules. Degradation of GPC is catalyzed by the glycerophosphocholine phosphodiesterase activity of glycerophosphodiester phosphodiesterase domain containing 5 (GDPD5). We previously found that inhibitory posttranslational modification (PTM) of GDPD5 contributes to high NaCl- and urea-induced increase of GPC. The purpose of the present studies was to identify the PTM(s). We find at least three such PTMs in HEK293 cells: (i) Formation of a disulfide bond between C25 and C571. High NaCl and high urea increase reactive oxygen species (ROS). The ROS increase disulfide bonding between GDPD5-C25 and -C571, which inhibits GDPD5 activity, as supported by the findings that the antioxidant N-acetylcysteine prevents high NaCl- and urea-induced inhibition of GDPD5; GDPD5-C25S/C571S mutation or over expression of peroxiredoxin increases GDPD5 activity; H2O2 inhibits activity of wild type GDPD5, but not of GDPD5-C25S/C571S; and peroxiredoxin is relatively low in the renal inner medulla where GPC is high. (ii) Dephosphorylation of GDPD5-T587. GDPD5 threonine 587 is constitutively phosphorylated. High NaCl and high urea dephosphorylate GDPD5-T587. Mutation of GDPD5-T587 to alanine, which cannot be phosphorylated, decreases GPC-PDE activity of GDPD5. (iii) Alteration at an unknown site mediated by CDK1. Inhibition of CDK1 protein kinase reduces GDE-PDE activity of GDPD5 without altering phosphorylation at T587, and CDK1/5 inhibitor reduces activity of GDPD5- C25S/C571S-T587A.
| S-EPMC3645535 | biostudies-literature
Project description:The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I(Ca)) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. IBMX (100 microM), a broad-spectrum PDE inhibitor, increased basal I(Ca) by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 microM). However, MIMX (1 microM), a PDE1 inhibitor, EHNA (10 microM), a PDE2 inhibitor, cilostamide (0.1 microM), a PDE3 inhibitor, or Ro20-1724 (0.1 microM), a PDE4 inhibitor, had no effect on basal I(Ca) and little stimulatory effects on [cAMP]i (20-30%). Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I(Ca) or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40-50%), only cilostamide (0.1 microM)+ Ro20-1724 (0.1 microM) produced a significant stimulation of I(Ca) (50%). Addition of EHNA (10 microM) to this mix increased I(Ca) to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 microM) or isoprenaline (1 microM). When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I(Ca) by (approximately 40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in I(Ca). Maximal effects on I(Ca) were approximately 8% for MIMX (3 microM), approximately 20% for EHNA (1-3 microM), approximately 30% for cilostamide (0.3-1 microM) and approximately 50% for Ro20-1724 (0.1 microM). Our results demonstrate that PDE1-4 subtypes regulate I(Ca) in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal I(Ca), all four PDE subtypes determine the response of I(Ca) to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3>PDE2>PDE1.
| S-EPMC1565993 | biostudies-literature
Project description:The phosphatidylinositol phosphodiesterase of rat brain shows little activity under conditions likely to pertain in vivo (neutral pH and micromolar Ca(2+) concentrations). A short incubation of a brain supernatant with trypsin, or a longer pre-incubation of the supernatant alone, produce new forms of the enzyme, which are active under such conditions. A possible role of receptor-linked proteinases in initiating phosphatidylinositol catabolism is discussed.
| S-EPMC1158640 | biostudies-other
Project description:alpha1-Adrenoceptor stimulation (alpha1ARS) modulates cardiac muscle contraction under physiological conditions by means of changes in Ca2+ current through L-type channels (ICa,L) and Ca2+ sensitivity of the myofilaments. However, the cellular mechanisms of alpha1ARS are not fully clarified. In this study, we investigated the role of Ca2+/calmodulin-dependent PK II (CaMKII) in the regulation of ICa,L during alpha1ARS in isolated adult rat ventricular myocytes by using the perforated patch-clamp technique. CaMKII inhibition with 0.5 microM KN-93 abolished the potentiation in ICa,L observed during alpha1ARS by 10 microM phenylephrine. In the presence of PKC inhibitor (10 microM chelerythrine), the potentiation of ICa,L by phenylephrine also disappeared. In Western immunoblotting analysis, phenylephrine (> or =1 microM) increased the amount of autophosphorylated CaMKII (active CaMKII) significantly, and this increase was abolished by CaMKII inhibition or PKC inhibition. Also, we investigated changes in the subcellular localization of active CaMKII by using immunofluorescence microscopy and immunoelectron microscopy. Before alpha1ARS, active CaMKII was exclusively located just beneath the plasmalemma. However, after alpha1ARS, active CaMKII was localized close to transverse tubules, where most of L-type Ca2+ channels are located. From these results, we propose that CaMKII, which exists near transverse tubules, is activated and phosphorylated by alpha1ARS and that CaMKII activation directly potentiates ICa,L in rat ventricular myocytes.
| S-EPMC1166620 | biostudies-literature
Project description:p-Nitrophenylphosphocholine phosphodiesterase activity was purified 5000-fold from mouse brain by treatment of membranes with Bacillus cereus phospholipase C preparation and sequential chromatographies on concanavalin A-Sepharose and CM-Sephadex columns. The phosphodiesterase (Zn(2+)-requiring) showed Km and Vmax. values of 5.5 microM and 4.2 mumol/min per mg respectively in the hydrolysis of p-nitrophenylphosphocholine, and possessed an optimum pH of 10.5 and a molecular mass of approx. 74 kDa. The purified enzyme was found to convert glycerophosphocholine into glycerol and phosphocholine, with Km and Vmax. of 48 microM and 5 mumol/min per mg respectively. In the hydrolysis of glycerophosphocholine the enzyme also exhibited a Zn2+ requirement and optimal pH at 10.5. Additionally, the p-nitrophenylphosphocholine phosphodiesterase activity was competitively inhibited by glycerophosphocholine, with a Ki value of 50 microM. These observations, together with chromatographic behaviour and heat-denaturation analyses, indicate that both p-nitrophenylphosphocholine phosphodiesterase and glycerophosphocholine cholinephosphodiesterase activities reside in the same protein.
| S-EPMC1132917 | biostudies-other
Project description:Hippocalcin is a Ca(2+)-binding protein that belongs to a family of neuronal Ca(2+)sensors and is a key mediator of many cellular functions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remain illusive. Here we studied whether glutamate receptor activation induced by locally applied or synaptically released glutamate can be decoded by hippocalcin translocation. Local AMPA receptor activation resulted in fast hippocalcin-YFP translocation to specific sites within a dendritic tree mainly due to AMPA receptor-dependent depolarization and following Ca(2+)influx via voltage-operated calcium channels. Short local NMDA receptor activation induced fast hippocalcin-YFP translocation in a dendritic shaft at the application site due to direct Ca(2+)influx via NMDA receptor channels. Intrinsic network bursting produced hippocalcin-YFP translocation to a set of dendritic spines when they were subjected to several successive synaptic vesicle releases during a given burst whereas no translocation to spines was observed in response to a single synaptic vesicle release and to back-propagating action potentials. The translocation to spines required Ca(2+)influx via synaptic NMDA receptors in which Mg(2+) block is relieved by postsynaptic depolarization. This synaptic translocation was restricted to spine heads and even closely (within 1-2 microm) located spines on the same dendritic branch signalled independently. Thus, we conclude that hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site- and time-specific translocation to its targets. Hippocalcin also possesses an ability to produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity.
| S-EPMC3069492 | biostudies-literature
Project description:The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca(2+) currents (I(Ca-N)) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca(2+) currents (I(Ca)), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on I(Ca). This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca(2+) channel blocker, suggesting the involvement of N-type Ca(2+) channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited I(Ca-N) in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca(2+) channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca(2+) channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.
| S-EPMC4255100 | biostudies-literature
Project description:RATIONALE:Baseline contractility of mouse hearts is modulated in a phosphatidylinositol 3-kinase-?-dependent manner by type 4 phosphodiesterases (PDE4), which regulate cAMP levels within microdomains containing the sarcoplasmic reticulum (SR) calcium ATPase type 2a (SERCA2a). OBJECTIVE:The goal of this study was to determine whether PDE4D regulates basal cardiac contractility. METHODS AND RESULTS:At 10 to 12 weeks of age, baseline cardiac contractility in PDE4D-deficient (PDE4D(-/-)) mice was elevated mice in vivo and in Langendorff perfused hearts, whereas isolated PDE4D(-/-) cardiomyocytes showed increased whole-cell Ca2+ transient amplitudes and SR Ca2+content but unchanged L-type calcium current, compared with littermate controls (WT). The protein kinase A inhibitor R(p)-adenosine-3',5' cyclic monophosphorothioate (R(p)-cAMP) lowered whole-cell Ca2+ transient amplitudes and SR Ca2+ content in PDE4D(-/-) cardiomyocytes to WT levels. The PDE4 inhibitor rolipram had no effect on cardiac contractility, whole-cell Ca2+ transients, or SR Ca2+ content in PDE4D(-/-) preparations but increased these parameters in WT myocardium to levels indistinguishable from those in PDE4D(-/-). The functional changes in PDE4D(-/-) myocardium were associated with increased PLN phosphorylation but not cardiac ryanodine receptor phosphorylation. Rolipram increased PLN phosphorylation in WT cardiomyocytes to levels indistinguishable from those in PDE4D(-/-) cardiomyocytes. In murine and failing human hearts, PDE4D coimmunoprecipitated with SERCA2a but not with cardiac ryanodine receptor. CONCLUSIONS:PDE4D regulates basal cAMP levels in SR microdomains containing SERCA2a-PLN, but not L-type Ca2+ channels or ryanodine receptor. Because whole-cell Ca2+ transient amplitudes are reduced in failing human myocardium, these observations may have therapeutic implications for patients with heart failure.
| S-EPMC4138878 | biostudies-literature
Project description:Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca2+ from internal stores in round spermatids and pachytene spermatocytes. In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca2+ stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca2+ using fluorescent Ca2+ probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca2+ stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca2+ release from intracellular Ca2+ stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis.
| S-EPMC5305069 | biostudies-literature