Nitric oxide and cGMP activate Ca2+-release processes in rat parotid acinar cells.
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ABSTRACT:
SUBMITTER: Looms DK
PROVIDER: S-EPMC1221715 | biostudies-other | 2001 Apr
REPOSITORIES: biostudies-other
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Project description:Intracellular Ca2+ mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (H2O2) on cytosolic Ca2+ accumulation in mouse parotid acinar cells. Intracellular Ca2+ levels were slowly elevated when 1 mM H2O2 was perfused in the presence of normal extracellular Ca2+. In a Ca2+-free medium, 1 mM H2O2 still enhanced the intracellular Ca2+ level. Ca2+ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H2O2. On the other hand, 10 mM H2O2 induced more rapid Ca2+ accumulation and facilitated Ca2+ entry from extracellular fluid. Ca2+ refill into intracellular Ca2+ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca2+ release from Ca2+ store was not affected by 1 mM H2O2 in permeabilized cells. Ca2+ efflux through plasma membrane Ca2+-ATPase (PMCA) was markedly blocked by 1 mM H2O2 in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H2O2-induced Ca2+ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H2O2 under pathological conditions may lead to cytosolic Ca2+ accumulation and that the primary mechanism of H2O2-induced Ca2+ accumulation is likely to inhibit Ca2+ efflux through PMCA rather than mobilize Ca2+ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.
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Project description:To date, studies suggest that biological signaling by nitric oxide (NO) is primarily mediated by cGMP, which is synthesized by NO-activated guanylyl cyclases and broken down by cyclic nucleotide phosphodiesterases (PDEs). Effects of cGMP occur through three main groups of cellular targets: cGMP-dependent protein kinases (PKGs), cGMP-gated cation channels, and PDEs. cGMP binding activates PKG, which phosphorylates serines and threonines on many cellular proteins, frequently resulting in changes in activity or function, subcellular localization, or regulatory features. The proteins that are so modified by PKG commonly regulate calcium homeostasis, calcium sensitivity of cellular proteins, platelet activation and adhesion, smooth muscle contraction, cardiac function, gene expression, feedback of the NO-signaling pathway, and other processes. Current therapies that have successfully targeted the NO-signaling pathway include nitrovasodilators (nitroglycerin), PDE5 inhibitors [sildenafil (Viagra and Revatio), vardenafil (Levitra), and tadalafil (Cialis and Adcirca)] for treatment of a number of vascular diseases including angina pectoris, erectile dysfunction, and pulmonary hypertension; the PDE3 inhibitors [cilostazol (Pletal) and milrinone (Primacor)] are used for treatment of intermittent claudication and acute heart failure, respectively. Potential for use of these medications in the treatment of other maladies continues to emerge.
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Project description:1. A method is described for the isolation of rat parotid acinar cells by controlled digestion of the gland with trypsin followed by collagenase. As judged by Trypan Blue exclusion, electron microscopy, water, electrolyte and ATP concentrations and release of amylase and lactate dehydrogenase, the cells are morphologically and functionally intact. 2. A method was developed for perifusion of acinar cells by embedding them in Sephadex G-10. Release of amylase was stimulated by adrenaline (0.1-10muM), isoproternol (1 or 10 MUM), phenylephrine (1 muM), carbamoylcholine (0.1 or 1 muM), dibutyryl cycle AMP (2 MM), 3-isobutyl-1-methylxanthine (1mM) and ionophore A23187. The effects of phenylephrine, carbamoylcholine and ionophore A23187 required extracellular Ca2+, whereas the effects of adrenaline and isoproterenol did not. 3. The incorporation of 45Ca into parotid cells showed a rapidly equilibrating pool (1-2 min) corresponding to 15% of total Ca2+ and a slowly equilibrating pool (greater than 3h) of probably a similar dimension. Cholinergic and alpha-adrenergic effectors and ionophore A23187 and 2,4-dinitrophenol increased the rate of incorporation of 45Ca into a slowly equilibrating pool, whereas beta-adrenergic effectors and dibutyryl cyclic AMP were inactive. 4. The efflux of 45Ca from cells into Ca2+-free medium was inhibited by phenylephrine and carbamoylcholine and accelerated by isoproterenol, adrenaline (beta-adrenergic effect), dibutyryl cyclic AMP and ionophore A23187. 5. A method was developed for the measurement of exchangeable 45Ca in mitochondria in parotid pieces. Incorporation of 45Ca into mitochondria was decreased by isoproterenol, dibutyryl cyclic AMP or 2,4-dinitrophenol, increased by adrenaline, and not changed significantly by phenylephrine or carbamoylcholine. Release of 45Ca from mitochondria in parotid pieced incubated in a Ca2+-free medium was increased by isoproterenol, adrenaline, dibutyryl cyclic AMP or 2,4-dinitrophenol and unaffected by phenylephrine or carbamoylcholine. 6. These findings are compatible with a role for Ca2+ as a mediator of amylase-secretory responses in rat parotid acinar cells, but no definite conclusions about its role can be drawn in the absence of knowledge of the molecular mechanisms involved, their location, and free Ca2+ concentration in appropriate cell compartment(s).
| S-EPMC1164270 | biostudies-other
Project description:Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous Rab27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.
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Project description:The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser(222), Ser(407)), three sites (Ser(217), Tyr(260), Ser(47)) previously found from large scale proteomic screens, and two sites (Ser(23), Ser(16)) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser(23) and Ser(16) and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca(2+) ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser(23) α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser(23) α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser(23) α1 subunit was not modified by ERK or PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser(23) and Ser(16), respectively, the latter because ouabain itself increased Ser(16) phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser(16) α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC.
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Project description:Rationale: Recently, abundant axial tubule (AT) membrane structures were identified deep inside atrial myocytes (AMs). Upon excitation, ATs rapidly activate intracellular Ca2+ release and sarcomeric contraction through extensive AT junctions, a cell-specific atrial mechanism. While AT junctions with the sarcoplasmic reticulum contain unusually large clusters of ryanodine receptor 2 (RyR2) Ca2+ release channels in mouse AMs, it remains unclear if similar protein networks and membrane structures exist across species, particularly those relevant for atrial disease modeling. Objective: To examine and quantitatively analyze the architecture of AT membrane structures and associated Ca2+ signaling proteins across species from mouse to human. Methods and Results: We developed superresolution microscopy (nanoscopy) strategies for intact live AMs based on a new custom-made photostable cholesterol dye and immunofluorescence imaging of membraneous structures and membrane proteins in fixed tissue sections from human, porcine, and rodent atria. Consistently, in mouse, rat, and rabbit AMs, intact cell-wide tubule networks continuous with the surface membrane were observed, mainly composed of ATs. Moreover, co-immunofluorescence nanoscopy showed L-type Ca2+ channel clusters adjacent to extensive junctional RyR2 clusters at ATs. However, only junctional RyR2 clusters were highly phosphorylated and may thus prime Ca2+ release at ATs, locally for rapid signal amplification. While the density of the integrated L-type Ca2+ current was similar in human and mouse AMs, the intracellular Ca2+ transient showed quantitative differences. Importantly, local intracellular Ca2+ release from AT junctions occurred through instantaneous action potential propagation via transverse tubules (TTs) from the surface membrane. Hence, sparse TTs were sufficient as electrical conduits for rapid activation of Ca2+ release through ATs. Nanoscopy of atrial tissue sections confirmed abundant ATs as the major network component of AMs, particularly in human atrial tissue sections. Conclusion: AT junctions represent a conserved, cell-specific membrane structure for rapid excitation-contraction coupling throughout a representative spectrum of species including human. Since ATs provide the major excitable membrane network component in AMs, a new model of atrial "super-hub" Ca2+ signaling may apply across biomedically relevant species, opening avenues for future investigations about atrial disease mechanisms and therapeutic targeting.
| S-EPMC6187065 | biostudies-literature
Project description:Cellular responsiveness to nitric oxide (NO) is shaped by past history of NO exposure. The mechanisms behind this plasticity were explored using rat platelets in vitro, specifically to determine the relative contributions made by desensitization of NO receptors, which couple to cGMP formation, and by phosphodiesterase-5 (PDE5), which is activated by cGMP and also hydrolyzes it. Repeated delivery of brief NO pulses (50 nM peak) at 1-min intervals resulted in a progressive loss of the associated cGMP responses, which was the combined consequence of receptor desensitization and PDE5 activation, with the former dominating. Delivery of pulses of differing amplitude showed that NO stimulated and desensitized receptors with similar potency (EC50 = 10-20 nM). PDE5 activation was highly sensitive to NO, with a single pulse peaking at 2 nM being sufficient to evoke a 50% loss of response to a subsequent near-maximal NO pulse. However, the activated state of the PDE subsided quickly after removal of NO, the half-time for recovery being 25 s. In contrast, receptor desensitization reverted much more slowly, the half-time being 16 min. Accordingly, with long (20-min) exposures, NO concentrations as low as 600 pM provoked significant desensitization. The results indicate that PDE5 activation and receptor desensitization subserve distinct short term and longer term roles as mediators of plasticity in NO-cGMP signaling. A kinetic model explicitly describing the complex interplay between NO concentration, cGMP synthesis, PDE5 activation, and the resulting cGMP accumulation successfully simulated the present and previous data.
| S-EPMC2757965 | biostudies-literature
Project description:BackgroundRegulatory functions of nitric oxide (NO*) that bypass the second messenger cGMP are incompletely understood. Here, cGMP-independent effects of NO* on gene expression were globally examined in U937 cells, a human monoblastoid line that constitutively lacks soluble guanylate cyclase. Differentiated U937 cells (>80% in G0/G1) were exposed to S-nitrosoglutathione, a NO* donor, or glutathione alone (control) for 6 h without or with dibutyryl-cAMP (Bt2cAMP), and then harvested to extract total RNA for microarray analysis. Bt2cAMP was used to block signaling attributable to NO*-induced decreases in cAMP.ResultsNO* regulated 110 transcripts that annotated disproportionately to the cell cycle and cell proliferation (47/110, 43%) and more frequently than expected contained AU-rich, post-transcriptional regulatory elements (ARE). Bt2cAMP regulated 106 genes; cell cycle gene enrichment did not reach significance. Like NO*, Bt2cAMP was associated with ARE-containing transcripts. A comparison of NO* and Bt2cAMP effects showed that NO* regulation of cell cycle genes was independent of its ability to interfere with cAMP signaling. Cell cycle genes induced by NO* annotated to G1/S (7/8) and included E2F1 and p21/Waf1/Cip1; 6 of these 7 were E2F target genes involved in G1/S transition. Repressed genes were G2/M associated (24/27); 8 of 27 were known targets of p21. E2F1 mRNA and protein were increased by NO*, as was E2F1 binding to E2F promoter elements. NO* activated p38 MAPK, stabilizing p21 mRNA (an ARE-containing transcript) and increasing p21 protein; this increased protein binding to CDE/CHR promoter sites of p21 target genes, repressing key G2/M phase genes, and increasing the proportion of cells in G2/M.ConclusionNO* coordinates a highly integrated program of cell cycle arrest that regulates a large number of genes, but does not require signaling through cGMP. In humans, antiproliferative effects of NO* may rely substantially on cGMP-independent mechanisms. Stress kinase signaling and alterations in mRNA stability appear to be major pathways by which NO* regulates the transcriptome.
| S-EPMC1312313 | biostudies-literature
Project description:Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.
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Project description:In exocrine acinar cells, agonist stimulation results in a polarized Ca2+ signal, termed the 'Ca2+ wave', that propagates from the apical pole towards the basolateral region. We attempted to detect the inositol 1,4,5-trisphosphate (InsP3)-induced Ca2+ wave in saponin-permeabilized rat parotid acinar cells using a digital imaging system. The permeabilized acinar cells were labelled with the membrane-bound Ca2+ indicator Calcium Green C18 to detect changes in Ca2+ concentration adjacent to the membrane of intracellular organelles. Application of InsP3 was made by the photolysis of InsP3 P4(5)-1-(2-nitrophenyl)ethyl ester (caged InsP3) to expose simultaneously all regions of the permeabilized acinar cells to InsP3. The increase in fluorescence ratio following the photolysis of 0.5 microM caged InsP3 started at the apical region of the acinar cells within 0.1 s and spread towards the basolateral region, indicating that Ca2+ release from intracellular Ca2+ stores was initially evoked at the apical region. Pretreatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pumps, failed to prevent the InsP3-induced Ca2+ wave, suggesting that the generation of the Ca2+ wave is not attributed to the polarized distribution of the Ca2+ pumps. The photolysis of a high concentration (10 microM) of caged InsP3 caused a homogeneous increase in the fluorescence ratio throughout the cells, indicating that all regions of intracellular Ca2+ stores similarly responded to the high concentration of InsP3. The present study is the first demonstration of the InsP3-induced Ca2+ wave in permeabilized exocrine acinar cells. The result provides fresh evidence that the apical region contains elements of intracellular Ca2+ stores particularly sensitive to InsP3 and that the Ca2+ wave results from a polarized distribution of InsP3-sensitive Ca2+ stores.
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