Adrenergic-agonist-induced Ca2+ fluxes in rat parotid cells are not Na+-dependent.
Ontology highlight
ABSTRACT:
SUBMITTER: Helman J
PROVIDER: S-EPMC1152620 | biostudies-other | 1985 Sep
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Pancreatic islets are complex micro-organs consisting of at least three different cell types: glucagon-secreting ?-, insulin-producing ?- and somatostatin-releasing ?-cells1. Somatostatin is a powerful paracrine inhibitor of insulin and glucagon secretion2. In diabetes, increased somatostatinergic signalling leads to defective counter-regulatory glucagon secretion3. This increases the risk of severe hypoglycaemia, a dangerous complication of insulin therapy4. The regulation of somatostatin secretion involves both intrinsic and paracrine mechanisms5 but their relative contributions and whether they interact remains unclear. Here we show that dapagliflozin-sensitive glucose- and insulin-dependent sodium uptake stimulates somatostatin secretion by elevating the cytoplasmic Na+ concentration ([Na+]i) and promoting intracellular Ca2+-induced Ca2+ release (CICR). This mechanism also becomes activated when [Na+]i is elevated following the inhibition of the plasmalemmal Na+-K+ pump by reductions of the extracellular K+ concentration emulating those produced by exogenous insulin in vivo 6. Islets from some donors with type-2 diabetes hypersecrete somatostatin, leading to suppression of glucagon secretion that can be alleviated by a somatostatin receptor antagonist. Our data highlight the role of Na+ as an intracellular second messenger, illustrate the significance of the intraislet paracrine network and provide a mechanistic framework for pharmacological correction of the hormone secretion defects associated with diabetes that selectively target the ?-cells.
| S-EPMC6986923 | biostudies-literature
Project description:Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of α₁-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the α1A-AR subtype, but not the α1B- and α1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La(3+), implying the participation of store-operated Ca(2+) channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La(3+), suggesting the involvement of extracellular Ca(2+) entry. Thus, α1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca(2+)/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca(2+) entry.
| S-EPMC4964398 | biostudies-literature
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.
| S-EPMC2978561 | biostudies-literature
Project description:Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.
| S-EPMC3029690 | biostudies-literature
Project description:beta-Adrenergic stimulation of rat parotid cells by isoprenaline (isoproterenol) results in 2-3-fold increases in [3H]mannose incorporation into N-linked oligosaccharides. This occurs without perceptible lag and is linear with time for 60 min after agonist addition. Concomitantly, isoprenaline markedly increases cellular cyclic AMP. Examination of individual proteins by sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis reveals that glycosylation changes are primarily associated with four secretory proteins, of approx. Mr 17000, 32000, 38000 and 220000. Beta-Adrenoreceptor activation additionally elicits a slight increase in parotid protein synthesis. The greatest increase in [14C]leucine incorporation is that into another secretory protein (Mr approx. 24000). Exposure of cells to dibutyryl cyclic AMP yields results comparable with those after isoprenaline treatment. Forskolin, which increases parotid-cell cyclic AMP, also causes similar effects. Conversely, dibutyryl cyclic GMP shows no such response. The data are consistent with the notion that beta-adrenergic stimulation of N-linked protein glycosylation in rat parotid cells is mediated by cyclic AMP.
| S-EPMC1144138 | biostudies-other
Project description:The contribution of intracellular stores to axonal Ca2+ overload during chemical ischemia in vitro was examined by confocal microscopy. Ca2+ accumulation was measured by fluo-4 dextran (low-affinity dye, KD approximately 4 microM) or by Oregon Green 488 BAPTA-1 dextran (highaffinity dye, KD approximately 450 nM). Axonal Na+ was measured using CoroNa Green. Ischemia in CSF containing 2 mM Ca2+ caused an approximately 3.5-fold increase in fluo-4 emission after 30 min, indicating a large axonal Ca2+ rise well into the micromolar range. Axonal Na+ accumulation was enhanced by veratridine and reduced, but not abolished, by TTX. Ischemia in Ca2+-free (plus BAPTA) perfusate resulted in a smaller but consistent Ca2+ increase monitored by Oregon Green 488 BAPTA-1, indicating release from intracellular sources. This release was eliminated in large part when Na+ influx was reduced by replacement with N-methyl-D-glucamine (NMDG+; even in depolarizing high K+ perfusate), Li+, or by the application of TTX and significantly increased by veratridine. Intracellular release also was reduced significantly by neomycin or 1-(6-[(17beta-methoxyestra-1,3,5 [10]-trien-17-yl) amino] hexyl)-1H-pyrrole-2,5-dione (U73122 [GenBank]) (phospholipase C inhibitors), heparin [inositol trisphosphate (IP3) receptor blocker], or 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP37157; mitochondrial Na+/Ca2+ exchange inhibitor) as well as ryanodine. Combining CGP37157 with U73122 [GenBank] or heparin decreased the response more than either agent alone and significantly improved electrophysiological recovery. Our conclusion is that intra-axonal Ca2+ release during ischemia in rat optic nerve is mainly dependent on Na+ influx. This Na+ accumulation stimulates three distinct intra-axonal sources of Ca2+: (1) the mitochondrial Na+/Ca2+ exchanger driven in the Na+ import/Ca2+ export mode, (2) positive modulation of ryanodine receptors, and (3) promotion of IP3 generation by phospholipase C.
| S-EPMC6725557 | biostudies-literature
Project description:Glucose output from perfused livers of 48 h-starved rats was stimulated by phenylephrine (2 microM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone or fructose were used as gluconeogenic precursors. Phenylephrine-induced increases in glucose output were immediately preceded by a transient efflux of Ca2+ and a sustained increase in oxygen uptake. Phenylephrine decreased the perfusate [lactate]/[pyruvate] ratio when sorbitol or glycerol was present, but increased the ratio when alanine, dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in the perfusate [beta-hydroxybutyrate]/[acetoacetate] ratio and increased total ketone-body output by 40-50% with all substrates. The oxidation of [1-14C]octanoate or 2-oxo[1-14C]glutarate to 14CO2 was increased by up to 200% by phenylephrine. All responses to phenylephrine infusion were diminished after depletion of the hepatic alpha-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+ re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol and glycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75% and 66% respectively. Data presented suggest that the mobilization of an intracellular pool of Ca2+ is involved in the activation of gluconeogenesis by alpha-adrenergic agonists in perfused rat liver. alpha-Adrenergic activation of gluconeogenesis is apparently accompanied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. An enhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrial compartment may also be involved in the stimulation of glucose output from the relatively reduced substrates glycerol and sorbitol and may arise principally from an increased flux through the malate-aspartate shuttle.
| S-EPMC1153129 | biostudies-other
Project description:In cardiac mitochondria, matrix free Ca(2+) ([Ca(2+)]m) is primarily regulated by Ca(2+) uptake and release via the Ca(2+) uniporter (CU) and Na(+)/Ca(2+) exchanger (NCE) as well as by Ca(2+) buffering. Although experimental and computational studies on the CU and NCE dynamics exist, it is not well understood how matrix Ca(2+) buffering affects these dynamics under various Ca(2+) uptake and release conditions, and whether this influences the stoichiometry of the NCE. To elucidate the role of matrix Ca(2+) buffering on the uptake and release of Ca(2+), we monitored Ca(2+) dynamics in isolated mitochondria by measuring both the extra-matrix free [Ca(2+)] ([Ca(2+)]e) and [Ca(2+)]m. A detailed protocol was developed and freshly isolated mitochondria from guinea pig hearts were exposed to five different [CaCl2] followed by ruthenium red and six different [NaCl]. By using the fluorescent probe indo-1, [Ca(2+)]e and [Ca(2+)]m were spectrofluorometrically quantified, and the stoichiometry of the NCE was determined. In addition, we measured NADH, membrane potential, matrix volume and matrix pH to monitor Ca(2+)-induced changes in mitochondrial bioenergetics. Our [Ca(2+)]e and [Ca(2+)]m measurements demonstrate that Ca(2+) uptake and release do not show reciprocal Ca(2+) dynamics in the extra-matrix and matrix compartments. This salient finding is likely caused by a dynamic Ca(2+) buffering system in the matrix compartment. The Na(+)- induced Ca(2+) release demonstrates an electrogenic exchange via the NCE by excluding an electroneutral exchange. Mitochondrial bioenergetics were only transiently affected by Ca(2+) uptake in the presence of large amounts of CaCl2, but not by Na(+)- induced Ca(2+) release.
| S-EPMC4065551 | biostudies-literature
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.
| S-EPMC5840080 | biostudies-literature
Project description:The effect of ionophore A23187 on cellular Ca2+ fluxes, glycogenolysis and respiration was examined in perfused liver. At low extracellular Ca2+ concentrations (less than 4 microM), A23187 induced the mobilization of intracellular Ca2+ and stimulated the rate of glycogenolysis and respiration. As the extracellular Ca2+ concentration was elevated, biphasic cellular Ca2+ fluxes were observed, with Ca2+ uptake preceding Ca2+ efflux. Under these conditions, both the glycogenolytic response and the respiratory response also became biphasic, allowing the differentiation between the effects of extracellular and intracellular Ca2+. Under all conditions examined the rate of Ca2+ efflux induced by A23187 was much slower than the rate of phenylephrine-induced Ca2+ efflux, although the net amounts of Ca2+ effluxed were similar for both agents. The effect of A23187 on phenylephrine-induced Ca2+ fluxes, glycogenolysis and respiration is dependent on the extracellular Ca2+ concentration. At concentrations of less than 50 microM-Ca2+, A23187 only partially inhibited alpha-agonist action, whereas at 1.3 mM-Ca2+ almost total inhibition was observed. The action of A23187 at the cellular level is complex, dependent on the experimental conditions used, and shows both differences from and similarities to the hepatic action of alpha-adrenergic agonists.
| S-EPMC1152261 | biostudies-other