Sugar-Controlled Ca2+ Uptake and {alpha}-Amylase Secretion in Cultured Cells of Rice (Oryza sativa L.)

Sugar starvation-induced synthesis and extracellular liberationof -amylase molecules in suspension-cultured cells of rice (Oryzasativa L.) required Ca²⁺, although the level of translatable-amylase mRNA was not affected in the presence of Ca²⁺. Sugardepletion markedly stimulated Ca²⁺ uptake by rice cells andsucrose supplementation reduced it. Immunohistochemical andelectron probe microanalyzer studies indicated an apparent resemblancebetween the distribution pattern of Ca²⁺ and that of -amylasemolecules induced in the sugar-depleted cells. Ca²⁺ uptake wasreduced by sucrose, maltose, fructose, and glucose similarlyat more than 5 mM, but was unaffected by mannitol (88 mM), 6-deoxy-D-glucose(10 mM), and 3-O-methyl-D-glucose (10 mM). Furthermore, an effectiveCa²⁺ channel blocker, La³⁺ significantly inhibited the Ca²⁺uptake and the synthesis and extracellular liberation of -amylasemolecules in the absence of sucrose, while a general P-typeATPase inhibitor, vanadate greatly stimulated both in the presenceof sucrose. We concluded that, by controlling the Ca²⁺ uptake,metabolic sugars regulate the protein synthesis and posttranslationalsecretory processes of -amylase molecules in rice cells. ⁴ Invited research fellow of the Japan Society for the Promotionof Science. Present address: Plant Physiology Department, WarsawAgricultural University, Rakowiecka Str. 26/30 02-528 Warsaw,Poland.     

Effects of Carbon Dioxide on Metabolism by the Glycollate Pathway in Leaves

When solutions of [¹⁴C]glycollate, glycine, serine, glycerate,or glucose were supplied to segments of wheat leaves throughtheir cut bases in the light, most of the ¹⁴C was incorporatedinto sucrose in air but in CO₂-free air less sucrose was made.The synthesis of sucrose was decreased because metabolism ofserine was partly blocked. Sucrose synthesis from glucose andglycerate in CO₂-free air was decreased but to a smaller extent;relatively more CO₂ was evolved and serine accumulated. Theeffects of DCMU and light of different wavelengths on metabolismby leaves of L-[U-¹⁴C]serine confirmed that simultaneous photosyntheticassimilation of carbon was necessary for the conversion of serineto sucrose. Of various products of photosynthesis fed exogenouslyto the leaves -keto acids were the most effective in promotingphotosynthesis of sucrose and release of ¹⁴CO₂ from ¹⁴C-labelledserine. This suggests that in CO₂-free air the metabolism ofserine may be limited by a shortage of -keto acid acceptorsfor the amino group. In CO₂-free air added glucose stimulatedproduction of CO₂ and sucrose from D-[U-¹⁴C]- glycerate andno competitive effects were evident even though glucose is convertedrapidly to sucrose under these conditions. In addition to asupply of keto acid, photosynthesis may also provide substratesthat can be degraded and provide energy in the cytoplasm forthe conversion of glycerate to sugar and phosphates and sucrose.     

Hormonal Regulation of Source-sink Relationship: Effect of Hormones on Excised Source and Sink Organs of Cereals

The regulatory role of plant growth substances in relation tosource and sink activity was studied. Leaching of electrolytesfrom flag leaf cells increased with age. Abscisic acid, however,increased the permeability of leaf cells at 10^–4 M andlower concentrations. Plant growth regulators like auxin, gibberellinand cytokinin at 10^–5 M inhibited the leaching of electrolytesbut slightly enhanced the process at 10^–3 M. The effluxof ¹⁴C-sucrose loaded on the flag leaves was not detectablein the very early stage of panicle development but was prominentin the later stages. While auxin prevented sucrose efflux atall stages of panicle development, gibberellin and cytokininwere effective only in different degrees. All the three growthregulators enhanced the activity of -amylase in the flag leafcells but protease activity was enhanced only by gibberellin.Synthesis of polymers like starch, DNA, RNA and protein in developingrice grains was stimulated by gibberellin, cytokinin and auxinlargely in the order mentioned. (Received September 2, 1986; Accepted May 22, 1987)     

Heterologous expression of Na(+)-K(+)-ATPase in insect cells: intracellular distribution of pump subunits

TheNa⁺-K⁺-ATPase is a heterodimeric plasmamembrane protein responsible for cellular ionic homeostasis in nearlyall animal cells. It has been shown that some insect cells (e.g., HighFive cells) have no (or extremely low)Na⁺-K⁺-ATPase activity. We expressed sheepkidney Na⁺-K⁺-ATPase - and -subunitsindividually and together in High Five cells via the baculovirusexpression system. We used quantitative slot-blot analyses to determinethat the expressed Na⁺-K⁺-ATPase comprisesbetween 0.5% and 2% of the total membrane protein in these cells.Using a five-step sucrose gradient (0.8-2.0 M) to separate theendoplasmic reticulum, Golgi apparatus, and plasma membrane fractions,we observed functional Na⁺ pump molecules in each membranepool and characterized their properties. Nearly all of the expressedprotein functions normally, similar to that found in purified dogkidney enzyme preparations. Consequently, the measurements describedhere were not complicated by an abundance of nonfunctionalheterologously expressed enzyme. Specifically, ouabain-sensitive ATPaseactivity, [³H]ouabain binding, and cation dependencieswere measured for each fraction. The functional properties of theNa⁺-K⁺-ATPase were essentially unaltered afterassembly in the endoplasmic reticulum. In addition, we measuredouabain-sensitive ⁸⁶Rb⁺ uptake in whole cellsas a means to specifically evaluateNa⁺-K⁺-ATPase molecules that were properlyfolded and delivered to the plasma membrane. We could not measure anyouabain-sensitive activities when either the -subunit or -subunitwere expressed individually. Immunostaining of the separate membranefractions indicates that the -subunit, when expressed alone, isdegraded early in the protein maturation pathway (i.e., the endoplasmicreticulum) but that the -subunit is processed normally and deliveredto the plasma membrane. Thus it appears that only the -subunit hasan oligomeric requirement for maturation and trafficking to the plasma membrane. Furthermore, assembly of the - heterodimer within theendoplasmic reticulum apparently does not require a Na⁺pump-specific chaperone.

     

Nicotinic acetylcholine receptor alpha 7 regulates cAMP signal within lipid rafts

Neuronal nicotinic acetylcholine receptors (nAChRs) are made of multiple subunits with diversified functions. The nAChR ₇-subunit has a property of high Ca²⁺ permeability and may have specific functions and localization within the plasma membrane as a signal transduction molecule. In PC-12 cells, fractionation by sucrose gradient centrifugation revealed that nAChR₇ existed in low-density, cholesterol-enriched plasma membrane microdomains known as lipid rafts where flotillin also exists. In contrast, nAChR ₅- and ₂-subunits were located in high-density fractions, out of the lipid rafts. Type 6 adenylyl cyclase (AC6), a calcium-inhibitable isoform, was also found in lipid rafts and was coimmunoprecipitated with nAChR₇. Cholesterol depletion from plasma membranes with methyl--cyclodextrin redistributed nAChR₇ and AC6 diffusely within plasma membranes. Nicotine stimulation reduced forskolin-stimulated AC activity by 35%, and this inhibition was negated by either treatment with -bungarotoxin, a specific antagonist of nAChR₇, or cholesterol depletion from plasma membranes. The effect of cholesterol depletion was negated by the addition of cholesterol. These data suggest that nAChR₇ has a specific membrane localization relative to other nAChR subunits and that lipid rafts are necessary to localize nAChR₇ with AC within plasma membranes. In addition, nAChR₇ may regulate the AC activity via Ca²⁺ within lipid rafts. cholesterol; PC-12 cells     

The Site of Synthesis of Sucrose in Green Plant Cells

Glycollic acid-1-¹⁴C, glyoxylic acid-2-¹⁴C, glycine-1-¹⁴C, andglycine-2-¹⁴C were fed to illuminated excised leaves of Pisumsativum and the distribution of ¹⁴C determined in the glycine,serine, sucrose, and insoluble polyglucan formed. Carboxyl-labelledglycollic acid and glycine gave rise to randomly labelled polyglucanand sucrose although the serine formed was predominantly carboxyllabelled. By contrast glyoxylic acid and glycine labelled inthe -carbon did not give rise to randomly labelled serine, sucrose,or polyglucan.     

Sexual cell agglutination in relation to the formation of zygotes in Saccharomyces cerevisiae

The ratio of a to cells in the sexual cell aggregates was consistentlyabout one regardless of the ratio of a to cells at the initialmixing. Conjugating cells seemed to be formed exclusively inthe aggregates during mixed culture of a and cells. Large cellswith buds (L cells) and small cells without buds (S celb) wereseparated from a logarithmic culture by sucrose density gradientcentrifugation. L Cells showed higher sexual agglutinabilitythan S cells in a mating type, but such difference was not detectedin a mating type. The same tendency was observed in cells dividingsynchronously. Based on the above results, the biological significanceof sexual agglutination in the mating reaction is discussed. ¹ Present address: Department of Physiology, Japan Women's University,Bunkyo-ku, Tokyo 112, Japan. (Received September 8, 1978; )     

pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway

The influences of the gastric H⁺/K⁺ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the - and -subunits of human H⁺/K⁺-ATPase (H⁺/K⁺-, cells). The pH values of trans-Golgi network (pH_TGN) and recycling endosomes (pH_RE) were measured by transfecting H⁺/K⁺-, cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H⁺/K⁺-ATPase was present in the plasma membrane, TGN, and RE. The pH_TGN was similar in both H⁺/K⁺-, cells (pH_TGN 6.36) and vector-transfected ("mock") cells (pH_TGN 6.34); pH_RE was also similar in H⁺/K⁺-, (pH_RE 6.40) and mock cells (pH_RE 6.37). SCH28080 (inhibits H⁺/K⁺-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H⁺ v-ATPase) caused TGN to alkalinize from pH 6.4 up to a new steady-state pH_TGN of 7.0–7.5, close to pH_cytosol. Similar results were observed in RE. Thus H⁺/K⁺-ATPases that trafficked to the plasma membrane were active but had small effects to acidify the TGN and RE compared with H⁺ v-ATPase. Mathematical modeling predicted a large number of H⁺ v-ATPases (8,000) active in the TGN to balance a large, passive H⁺ leak (with P_H 10^–3 cm/s) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H⁺/K⁺-ATPases (estimated to be 4,000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane. pHluorin; H⁺ v-ATPase; trans-Golgi network; organelle pH; H⁺ permeability     

Insulin increases plasma membrane content and reduces phosphorylation of Na+-K+ pump alpha 1-subunit in HEK-293 cells

Insulin stimulates K⁺ uptake andNa⁺ efflux via the Na⁺-K⁺ pump inkidney, skeletal muscle, and brain. The mechanism of insulin action inthese tissues differs, in part, because of differences in the isoformcomplement of the catalytic -subunit of theNa⁺-K⁺ pump. To analyze specifically the effectof insulin on the ₁-isoform of the pump, we have studiedhuman embryonic kidney (HEK)-293 cells stably transfected with the ratNa⁺-K⁺ pump ₁-isoform tagged onits first exofacial loop with a hemagglutinin (HA) epitope. The plasmamembrane content of ₁-subunits was quantitated bybinding a specific HA antibody to intact cells. Insulin rapidly increased the number of ₁-subunits at the cell surface.This gain was sensitive to the phosphatidylinositol (PI) 3-kinaseinhibitor wortmannin and to the protein kinase C (PKC) inhibitorbisindolylmaleimide. Furthermore, the insulin-stimulated gain insurface -subunits correlated with an increase in the binding of anantibody that recognizes only the nonphosphorylated form of₁ (at serine-18). These results suggest that insulinregulates the Na⁺-K⁺ pump in HEK-293 cells, atleast in part, by decreasing serine phosphorylation and increasingplasma membrane content of ₁-subunits via a signalingpathway involving PI 3-kinase and PKC.

     

Taste of methyl-{alpha}-D-mannopyranoside: Effects of cross adaptation and Gymnema sylvestre

Methyl--D-mannopyranoside is a glycoside with a bitter-sweettaste. Adaptation to sucrose reduces the sweetness and adaptationto quinine sulphate reduces the bitterness of methyl--Dmannopyranoside.Application of Gymnema sylvestre reduces the sweetness of methyl--D-mannopyranosidewithout reducing its bitterness. These results, predicted byprevious studies, contradict a recent hypothesis and reportby Birch and Mylvaganam. ¹Supported by NIH Grant 2-RO1-NS07873-9     

The Ca2+-sensing receptor couples to Galpha12/13 to activate phospholipase D in Madin-Darby canine kidney cells

The Ca²⁺-sensing receptor (CaR) couples to multiple G proteins involved in distinct signaling pathways: G_i to inhibit the activity of adenylyl cyclase and activate ERK, G_q to stimulate phospholipase C and phospholipase A₂, and G to stimulate phosphatidylinositol 3-kinase. To determine whether the receptor also couples to G_12/13, we investigated the signaling pathway by which the CaR regulates phospholipase D (PLD), a known G_12/13 target. We established Madin-Darby canine kidney (MDCK) cell lines that stably overexpress the wild-type CaR (CaR^WT) or the nonfunctional mutant CaR^R796W as a negative control, prelabeled these cells with [³H]palmitic acid, and measured CaR-stimulated PLD activity as the formation of [³H]phosphatidylethanol (PEt). The formation of [³H]PEt increased in a time-dependent manner in the cells that overexpress the CaR^WT but not the CaR^R796W. Treatment of the cells with C₃ exoenzyme inhibited PLD activity, which indicates that the CaR activates the Rho family of small G proteins, targets of G_12/13. To determine which G protein(s) the CaR couples to in order to activate Rho and PLD, we pretreated the cells with pertussis toxin to inactivate G_i or coexpressed regulators of G protein-signaling (RGS) proteins to attenuate G protein signaling (RGS4 for G_i and G_q, and a p115RhoGEF construct containing the RGS domain for G_12/13). Overexpression of p115RhoGEF-RGS in the MDCK cells that overexpress CaR^WT inhibited extracellular Ca²⁺-stimulated PLD activity, but pretreatment of cells with pertussis toxin and overexpression of RGS4 were without effect. The involvement of other signaling components such as protein kinase C, ADP-ribosylation factor, and phosphatidylinositol biphosphate was excluded. These findings demonstrate that the CaR couples to G_12/13 to regulate PLD via a Rho-dependent mechanism and does so independently of G_i and G_q. This suggests that the CaR may regulate cytoskeleton via G_12/13, Rho, and PLD. calcium-sensing receptor; G proteins; RGS proteins     

Autocrine production of prostaglandin F2alpha enhances phenotypic transformation of normal rat kidney fibroblasts

We have used normal rat kidney (NRK) fibroblasts as an in vitro model system to study cell transformation. These cells obtain a transformed phenotype upon stimulation with growth-modulating factors such as retinoic acid (RA) or transforming growth factor- (TGF-). Patch-clamp experiments showed that transformation is paralleled by a profound membrane depolarization from around –70 to –20 mV. This depolarization is caused by a compound in the medium conditioned by transformed NRK cells, which enhances intracellular Ca²⁺ levels and thereby activates Ca²⁺-dependent Cl^– channels. This compound was identified as prostaglandin F₂ (PGF₂) using electrospray ionization mass spectrometry. The active concentration in the medium conditioned by transformed NRK cells as determined using an enzyme immunoassay was 19.7 ± 2.5 nM (n = 6), compared with 1.5 ± 0.1 nM (n = 3) conditioned by nontransformed NRK cells. Externally added PGF₂ was able to trigger NRK cells that had grown to density arrest to restart their proliferation. This proliferation was inhibited when the FP receptor (i.e., natural receptor for PGF₂) was blocked by AL-8810. RA-induced phenotypic transformation of NRK cells was partially (25%) suppressed by AL-8810. Our results demonstrate that PGF₂ acts as an autocrine enhancer and paracrine inducer of cell transformation and suggest that it may play a crucial role in carcinogenesis in general. membrane potential; intracellular calcium; mass spectrometry; FP receptor     

IP3 receptor blockade fails to prevent intracellular Ca2+ release by ET-1 and alpha -thrombin

The effect ofinositol 1,4,5-trisphosphate(IP₃) receptor blockade onplatelet-derived growth factor (PDGF), fibroblast growth factor (FGF),endothelin-1 (ET-1), or -thrombin receptor-mediated intracellularCa²⁺(Ca²⁺_i) release was examined using fura 2 microspectrofluorometry in single Chinese hamster ovary cells andmyoblasts. Blockade of the IP₃receptor was achieved by microinjection of heparin or monoclonalantibody (MAb) 18A10 into the IP₃type 1 receptor. Heparin completely inhibitedCa²⁺_i release after flash photolysis withcaged IP₃ and after exposure toPDGF and FGF. In contrast, heparin failed to blockCa²⁺_i release after -thrombin andET-1. After application of ligand, IP₃ levels were five- to sevenfoldhigher for -thrombin than for ET-1 or PDGF.IP₃ levels after PDGF and ET-1were comparable. Similar to heparin, MAb 18A10 blockedCa²⁺_i release after PDGF but failed toblock Ca²⁺_i release after ET-1 or-thrombin. These data suggest that the mechanisms of Ca²⁺_i release by tyrosine kinase andcertain 7-transmembrane receptors may differ. Although both receptortypes use the IP₃-signaling system, the ET-1 and -thrombin receptors may have a second,alternative mechanism for activatingCa²⁺_i release.

     

Receptor-mediated inhibition of renal Na+-K+-ATPase is associated with endocytosis of its alpha - and beta -subunits

The mechanisms involved in receptor-mediated inhibition ofNa⁺-K⁺-ATPaseremain poorly understood. In this study, we evaluate whether inhibitionof proximal tubuleNa⁺-K⁺-ATPaseactivity by dopamine is linked to its removal from the plasma membraneand internalization into defined intracellular compartments.Clathrin-coated vesicles were isolated by sucrose gradientcentrifugation and negative lectin selection, and early and lateendosomes were separated on a flotation gradient. Inhibition ofNa⁺-K⁺-ATPaseactivity by dopamine, in contrast to its inhibition by ouabain, wasaccompanied by a sequential increase in the abundance of the-subunit in clathrin-coated vesicles (1 min), early endosomes (2.5 min), and late endosomes (5 min), suggesting its stepwise translocationbetween these organelles. A similar pattern was found for the-subunit. The increased incorporation of both subunits in allcompartments was blocked by calphostin C. The results demonstrate thatthe dopamine-induced decrease inNa⁺-K⁺-ATPaseactivity in proximal tubules is associated with internalization of its- and -subunits into early and late endosomes via aclathrin-dependent pathway and that this process is protein kinase Cdependent. The presence ofNa⁺-K⁺-ATPasesubunits in endosomes suggests that these compartments may constitutenormal traffic reservoirs during pump degradation and/orsynthesis.

     

EFFECT OF MANGANESE IONS ON THE IAA-INDUCED ELONGATION OF AVENA COLEOPTILE SECTIONS INHIBITED BY SOME ORGANIC ACIDS

1.Organic acids, such as citric, -ketoglutaric, succinic, fumaricand L-malic acids, inhibit the IAA-induced growth of Avena coleoptilesections. But pyruvic acid has no effect on the growth. 2.High concentrations of MnCl₂ (for example 10^–3 m) alleviatethe inhibition due to L-malic, -ketoglutaric, succinic and fumaricacids, but not that due to D-malic, tartaric and malonic acids. 3.A mechanism of the alleviating effect of Mn⁺⁺ on the inhibitiondue to the organic acids is discussed with the reference tothe activating effect of Mn⁺⁺ on "malic" enzyme. ¹Contribution No. 6 from the Botanical Gardens. Faculty of Science,University of Tokyo, Koishikawa, Tokyo.     

Biosynthesis, processing, and secretion of {alpha}-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

N-Acetylglucosamine 1-phosphotransferase is a key enzyme requiredfor synthesis of the mannose 6-phosphate recognition markerthat is used by many newly made acid hydrolases for their transportto lysosomes. It has previously been found that lymphoid cellsfrom patients with I-cell disease and pseudo-Hurler polydystrophyhave nearly normal intracellular and intralysosomal activitiesof several lysosomal acid hydrolases, despite a deficiency ofN-acetylglucosamine 1-phosphotransferase. These results suggestthat lymphoid cells may provide an important system to investigatealternate mechanisms for targeting newly made acid hydrolasesto lysosomes. In the present study, the biosynthesis, processingand secretion of -L-fucosidase in I-cell and pseudoHurler lymphoidcells was used as a model system to study the existence of suchmechanisms. The level of intracellular -L-fucosidase proteinin exponentially growing I-cell or pseudo-Hurler lymphoid cultureswas statistically indistinguishable from the mean of 19 controlcultures. A 1.5 h [³⁵S]methionine pulse experiment showed that-L-fucosidase is initially sythesized by I-cell, pseudo-Hurlerand control cultures as an intracellular form (M_r = 58 000).Companion cultures chased with methionine from 2 to 21 h processedthe enzyme to an intracellular form (M_r = 60 000) and an extracellularform (M_r = 62 000). All enzyme forms were glycoproteins withpolypeptide chains of Mr 52 000. In control cells incubatedwith radioactive inorganic phosphate (³²Pi), <1% of the ³²Piincorporated into -L-fucosidase was associated with carbohydratechains and >99% with polypeptide chains. In I-cell diseaselymphoid cells, the ³²Pi incorporated into -L-fucosidase wasassociated solely with polypeptide chains. A qualitative analysisof phosphorylated residues identified phosphoserine in -L-fucosidasefrom control and I-cell lymphoid cells. Only -L-fucosidase fromcontrol cells contained mannose 6-phosphate. These results areconsistent with the proposal that I-cell lymphoid cells mayuse a mannose 6-phosphate-independent mechanism for routing-L-fucosidase. Additional metabolic labelling experiments demonstratedthe presence of ³²P-labelled -L-fucosidase in both cells andmedium of a control lymphoid culture, but only in cells of anI-cell lymphoid culture. In contrast, -L-fucosidase labelledwith [³⁵S]methionine was found in cells and medium of controland I-cell lymphoid cultures. Since phosphoserine was only foundto occur in intracellular, but not in extracellular -L-fucosidaseof the I-cell culture, we speculate that phosphoserine may beinvolved in intracellular retention of -L-fucosidase in I-celllymphoid cells. -L-fucosidase I-cell disease lymphoid cells phos-phorylation pseudo-Hurler polydystrophy     

Inflammatory cytokines (IL-1alpha , TNF-alpha ) and LPS modulate the Ca2+ signaling pathway in osteoblasts

Locally derived growth factors and cytokines in bone play acrucial role in the regulation of bone remodeling, i.e., bone formationand bone resorption processes. We studied the effect of interleukin(IL)-1, tumor necrosis factor (TNF)-, andEscherichia coli lipopolysaccharide(LPS) on the hormone-activatedCa²⁺ message system in theosteoblastic cell line UMR-106 and in osteoblastic cultures derivedfrom neonatal rat calvariae. In both cell preparations, IL-1,TNF-, and LPS did not alter basal intracellularCa²⁺ concentration([Ca²⁺]_i)but attenuated Ca²⁺ transientsevoked by parathyroid hormone (PTH) andPGE₂ in a dose (1-100 ng/ml)-and time (8-24 h)-dependent fashion. The cytokines modulatedhormonally induced Ca²⁺ influx(estimated by using Mn²⁺ as asurrogate for Ca²⁺) as well asCa²⁺ mobilization fromintracellular stores. The latter was linked to suppressed production ofhormonally induced inositol 1,4,5-trisphosphate. The effect ofcytokines on[Ca²⁺]_iwas abolished by the tyrosine kinase inhibitor herbimycin A (50 ng/ml).The cytokine's effect was, however, independent of nitric oxide (NO)production, since NO donors (sodium nitroprusside) as well as permeablecGMP analogs augment, rather than attenuate, hormonally inducedCa²⁺ transients in osteoblasts.Given the stimulatory role of cytokines on NO production inosteoblasts, the disparate effects of cytokines and NO on theCa²⁺ signaling pathway may servean autocrine/paracrine mechanism for modulating the effect ofcalciotropic hormones on bone metabolism.

     

Regulation of rat Na+-K+-ATPase activity by PKC is modulated by state of phosphorylation of Ser-943 by PKA

We have previously shown that the ratNa⁺-K⁺-ATPase₁-isoform is phosphorylated atSer-943 by protein kinase A (PKA) and at Ser-23 by protein kinase C(PKC), which in both cases results in inhibition of enzyme activity. Wenow present evidence that suggests that the phosphorylation of Ser-943by PKA modulates the response ofNa⁺-K⁺-ATPaseto PKC. RatNa⁺-K⁺-ATPase₁ or a mutant in which Ser-943was changed to Ala-943 was stably expressed in COS cells. Theinhibition of enzyme activity measured in response to treatment withthe phorbol ester, phorbol 12,13-dibutyrate (PDBu;10⁶ M), was significantlyreduced in the cells expressing the Ala-943 mutant compared with thatobserved in cells expressing wild-type enzyme. In contrast, for cellsexpressingNa⁺-K⁺-ATPase₁ in which Ser-943 was mutatedto Asp-943, the effect of PDBu was slightly enhanced. The PDBu-inducedinhibition was not mediated by activation of the adenosine3',5'-cyclic monophosphate/PKA system and was not achievedvia direct phosphorylation of Ser-943. Sp-5,6-DCl-cBIMPS, a specificPKA activator, increased the phosphorylation of Ser-943, and this wasassociated with an enhanced response to PDBu. Thus the effect of PKC onratNa⁺-K⁺-ATPase₁ is determined not only by theactivity of PKC but also by the state of phosphorylation of Ser-943.

     

Norepinephrine-induced calcium signaling and expression of adrenoceptors in avian tendon cells

Sympathetic efferent nerves are present in tendons, but their function within tendon is unknown. ₁-Adrenoceptors are expressed by a variety of cell types. In the presence of norepinephrine (NE), adrenoceptors activate G_q/11 signaling pathways that subsequently increase intracellular Ca²⁺ concentration ([Ca²⁺]_ic). It was hypothesized that avian tendon cells express functional adrenoceptors that respond to NE by increasing [Ca²⁺]_ic. Avian tendon cells were analyzed for mRNA expression of ₁-adrenoceptors by RT-PCR. Avian tendons expressed the _1A- and _1B-adrenoceptor subtypes. Furthermore, both tendon surface epitenon cells and internal fibroblasts infused with a Ca²⁺-sensitive dye, fura 2, and stimulated with NE responded by increasing [Ca²⁺]_ic. KMD-3213, an _1A-adrenoceptor antagonist, significantly reduced the Ca²⁺ response. Other adrenoceptor antagonists had no effect on the Ca²⁺ response. The absence of extracellular Ca²⁺ also significantly reduced the response to NE, indicating that Ca²⁺ influx contributed to the rise in [Ca²⁺]_ic. This study provides the first evidence that tendon cells express adrenoceptors and that the NE-induced Ca²⁺ response is coupled to the _1A-adrenoceptor subtype. -adrenoceptors; fibroblasts; catecholamines; tenocytes     

Na+ pump alpha 2-subunit expression modulates Ca2+ signaling

The role of the Na⁺ pump₂-subunit in Ca²⁺ signaling was examined inprimary cultured astrocytes from wild-type(₂^+/+ = WT) mouse fetuses and thosewith a null mutation in one [₂^+/ = heterozygote (Het)] or both [₂^/ = knockout (KO)] ₂ genes. Na⁺ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na⁺] ([Na⁺]_cyt) and[Ca²⁺] ([Ca²⁺]_cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na⁺ pumpswith both ₁- (80% of total ) and₂- (20% of total ) subunits. Het astrocytesexpress 50% of normal ₂; those from KO express none.Expression of ₁ is normal in both Het and KO cells.Resting [Na⁺]_cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only₂) equalized [Na⁺]_cyt at 8 mMin all three cell types. Resting[Ca²⁺]_cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca²⁺ pumps and unloads the ER, induces transient (inCa²⁺-free media) or sustained (in Ca²⁺-repletemedia) elevation of [Ca²⁺]_cyt. TheseCa²⁺ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca²⁺-free media, thereintroduction of Ca²⁺ induced significantly largertransient rises in [Ca²⁺]_cyt (due toCa²⁺ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat ₂ Na⁺ pumps andNa⁺/Ca²⁺ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of ₂ Na⁺ pump activitycan elevate local [Na⁺] and, viaNa⁺/Ca²⁺ exchange, [Ca²⁺] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca²⁺ stores and therebyamplifies Ca²⁺ signaling without elevating bulk[Na⁺]_cyt.