Molecular characterisation of the smooth endoplasmic reticulum Ca2+-ATPase of Porcellio scaber and its expression in sternal epithelia during the moult cycle (original) (raw)
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The Journal of Experimental Biology, 2003
Recent investigations on specialized epithelia suggest a role of the smooth endoplasmic reticulum Ca 2+-ATPase (SERCA) in epithelial Ca 2+-transport (Franklin et al., 2001; Hagedorn and Ziegler, 2002). These epithelia are involved in quick mineralisation processes in which epithelial Ca 2+-transport predominantly follows a transcellular route (transport through the cells) (Hubbard, 2000; Roer, 1980; Wheatly, 1997; Ziegler, 2002), rather than a paracellular pathway in which Ca 2+ would move extracellularly across the apical cell contacts and between the epithelial cells along its electrochemical gradient (Bronner, 1991). In the transcellular pathway, Ca 2+ enters the cells passively across the plasma membrane at one side of the cells and is actively extruded on the other side (Ahearn and Franco, 1993; Ahearn and Zhuang, 1996; Roer, 1980). The most critical step in transcellular epithelial Ca 2+-transport, however, is the Ca 2+ transport within the cells, through the cytoplasm from one side to the plasma membrane on the opposite side. Sustained elevated concentrations of Ca 2+ within the cytoplasm would interfere with the multiple regulatory functions of cytosolic free Ca 2+ signals. In addition, such elevated concentrations of ionized calcium can lead to cell damage and even cell death (Berridge, 1993). How such a toxic rise is prevented during epithelial Ca 2+-transport is still unknown. Simkiss (1996) proposed a model in which organelles, e.g. the smooth endoplasmic reticulum (SER), function as a transient calcium store allowing vectorial bulk flow through epithelial cells without toxic effects. Within cells the SER generally functions as source and sink for cytosolic Ca 2+ signals, with the inositol 1,4,5 trisphosphate (IP3)-receptors and/or ryanodine-receptors releasing Ca 2+ and the SERCA pumping Ca 2+ back in a regulated fashion (Hussain and Inesi, 1999). Previous investigations on the anterior sternal epithelium (ASE) of Porcellio scaber (Hagedorn and Ziegler, 2002; Ziegler, 2002) suggest that the SER contributes to transcellular calcium transport. Like most crustaceans, P. scaber has a calcified cuticle, which is moulted regularly to allow for growth of the animal. During premoult the ASE transports Ca 2+ , originating from the posterior cuticle to form large CaCO3 deposits located within the ecdysial gap of the first four anterior sternites (Messner, 1965
Cell Calcium, 2002
It is thought that a plasma membrane Ca 2+-transport ATPase (PMCA) and a Na + /Ca 2+-exchange (NCE) mechanism are involved in epithelial Ca 2+ transport (ECT) in a variety of crustacean epithelia. The sternal epithelium of the terrestrial isopod Porcellio scaber was used as a model for the analysis of Ca 2+-extrusion mechanisms in the hypodermal epithelium. Using RT-PCR, we amplified a cDNA fragment of 1173 bp that encodes a protein sequence possessing 72% identity to the PMCA from Drosophila melanogaster and a cDNA fragment of 791 bp encoding a protein sequence with 50% identity to the NCE from Loligo opalescens. Semiquantitative RT-PCR revealed that the expression of both mRNAs increases from the non-Ca 2+-transporting condition to the stages of CaCO 3 deposit formation and degradation. During Ca 2+-transporting stages, the expression of PMCA and NCE was larger in the anterior sternal epithelium (ASE) than in the posterior sternal epithelium (PSE). The results demonstrate for the first time the expression of a PMCA and a NCE in the hypodermal epithelium of a crustacean and indicate a contribution of these transport mechanisms in ECT.
Journal of Comparative Physiology B, 2008
We have previously reported on calcium transport mechanisms in American lobster, Homarus americanus, using 45 Ca 2+ coupled with vesicle preparations of hepatopancreatic endoplasmic reticulum. The active transport of calcium across membranes bordering calcium-sequestering stores such as sarcoplasmic or endoplasmic reticulum is catalyzed by membrane-spanning proteins, the sarco-endoplasmic Ca 2+-ATPases (SERCAs). In the study described here we used advanced bioinformatics and molecular techniques to clone SERCA from the economically important Caribbean spiny lobster, Panulirus argus. We report the complete cloning of a fulllength SERCA from P. argus antenna cDNA (GenBank accession number AY702617). This cDNA has a 1020-amino acid residue open reading frame which is 90% identical to published sequences of other crustacean SERCA proteins. Our data support the hypothesis that one crustacean and three vertebrate genes controlling calcium transport were derived from a common ancestral gene.
Journal of Experimental Biology, 2004
SUMMARY The formation and resorption of CaCO3 by epithelial cell layers require epithelial transport of protons. We used the anterior sternal epithelium of the terrestrial isopod Porcellio scaber as a model to study the expression pattern and immunolocalization of a V-type H+-ATPase during the mineralization and demineralization of intermittent CaCO3 deposits. Semiquantitative RT-PCR revealed that the expression of the V-type H+-ATPase increases from non Ca2+-transporting control stages to the stages of CaCO3deposit formation and resorption. In the Ca2+-transporting stages the expression was larger in the anterior than in the posterior sternal epithelium, which is not involved in deposit formation and transports just moderate amounts of CaCO3. Immunocytochemistry of the B-subunit of the V-type H+-ATPase in the anterior sternal epithelium reveals an increase in the abundance of the protein within the basolateral membrane, from undetectable to strong signals in the control stage to th...
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2018
Crustacean growth is characterized by molting, whereby the old exoskeleton is shed and replaced by a new and larger version. The cellular events that lead to molting are driven by steroid hormones (ecdysteroids) secreted by paired endocrine glands (Y-organs). Between molts, ecdysteroid production is suppressed by a polypeptide moltinhibiting hormone (MIH) released from neurosecretory cells in the eyestalks. Although a decrease in the MIH titer precedes the upsurge in ecdysteroidogenesis, it is hypothesized that a positive regulatory signal is also required for full activation of Y-organs. Existing data point to an intracellular Ca 2+ signal. Ca 2+ signaling is dependent on a tightly regulated Ca 2+ gradient, achieved through membrane transport proteins. One such protein, the sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA), pumps Ca 2+ from cytosol to the lumen of the ER. We have recently cloned from Y-organs of the blue crab (Callinectes sapidus) a cDNA encoding a putative Cas-SERCA protein. In studies reported here, quantitative PCR (QPCR) was used to quantify Cas-SERCA transcript abundance in Y-organs during a molting cycle, and radioimmunoassay was used to quantify ecdysteroids in hemolymph. The abundance of the Cas-SERCA transcript in Y-organs increased gradually during pre-molt. Similarly, the level of ecdysteroids in hemolymph increased during pre-molt. The results are consistent with the hypothesis that Cas-SERCA functions to maintain Ca 2+ homeostasis in Y-organs. Cas-SERCA transcript abundance also changed in several non-ecdysteroidogenic tissues during a molting cycle. The pattern of change differed among tissues suggesting a functional role for SERCA in each. (Guerini et al., 2005). Ca 2+ signals are widely used across cell types to trigger a broad range of functions, including fertilization, apoptosis, muscle contraction, and neurotransmitter release (Berridge et al., 2000; Kahl and Means, 2003; Clapham, 2007). To preserve the functional integrity of the Ca 2+ signal, and to prevent cellular toxicity, the intracellular Ca 2+ concentration is tightly regulated. The Ca 2+ concentration in cytosol is controlled mainly by proteins intrinsic to the plasma membrane and to the membranes of organelles. Two main families of proteins are involved, Ca 2+ pumps (ATPases), and Ca 2+ exchangers. The family of Ca 2+ pumps includes a plasma membrane Ca 2+ ATPase (PMCA) and a sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA). The former transports Ca 2+ out of the cell; the latter transports Ca 2+ into the sarco/endoplasmic reticulum (Carafoli et al., 2001; Guerini et al., 2005). Na + /Ca 2+ exchangers (NCX) are bidirectional transporters that mediate the exchange of Ca 2+ for Na + using the energy of the Na + gradient generated by Na + /K + ATPase (Philipson and
Calcium homeostasis in crustaceans: subcellular Ca dynamics
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2002
The molting cycle of crustaceans, associated with renewal and remineralization of the cuticle, has emerged as a model system to study regulation of genes that code for Ca -transporting proteins, common to all eukaryotic cells. This article 2q reviews state-of-the-art knowledge about how crustacean transporting epithelia (gills, hepatopancreas and antennal gland) effect mass transcellular movement of Ca while preventing cytotoxicity. The current model proposed is based on in 2q vitro research on the intermolt stage with extrapolation to other molting stages. Plasma membrane proteins involved in apical and basolateral Ca movement (NCX, PMCA) are contrasted between aquatic species of different osmotic origin 2q and among transporting epithelia of an individual species. Their roles are assessed in the context of epithelial Ca flux 2q derived from organismic approaches. Exchange with extracellular environments is integrated with Ca sequestration 2q mechanisms across endomembranes of the ERySR and mitochondria. Finally, the review postulates how new Ca 2q imaging techniques will allow spatial and temporal resolution of Ca concentration in subcellular domains. ᮊ 2002 2q Elsevier Science Inc. All rights reserved.
Calcium regulation in crustaceans during the molt cycle: a review and update
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2004
Epithelial cells of the gut, gills, antennal glands and integument regulate calcium concentrations in crustaceans during the molt cycle. A cellular calcium transport model has been proposed suggesting the presence of calcium pumps, cation antiporters and calcium channels in transporting epithelial membranes that regulate the movements of this cation across the cell layer. Basolateral calcium transport during postmolt appears mainly regulated by the low affinity NCX antiporter, while calcium regulating 'housekeeping' activities of these cells in intermolt are controlled by the high affinity calcium ATPase (PMCA). A model is proposed for the involvement of the epithelial ER in the massive transepithelial calcium fluxes that occur during premolt and postmolt. This model involves the endoplasmic reticulum SERCA and RyR proteins and proposed cytoplasmic unstirred layers adjacent to apical and basolateral plasma membranes where calcium activities may largely exceed those in the bulk cytoplasmic phase. A result of the proposed transepithelial calcium transport model is that large quantities of calcium can be moved through these cells by these processes without affecting the low, and carefully controlled, bulk cytoplasmic calcium activities.
Journal of Comparative Physiology B, 2008
Early studies on the outer mantle epithelium (OME) cells of the freshwater bivalve Anodonta cygnea (Linnaeus, 1758) revealed high ionic calcium concentrations by electrophysiological methods and subsequently a high tendency to reach an intracellular toxic condition. This toxicity could be neutralized by speciWc mechanisms in the cytosol of OME cells of A. cygnea. The present immunocytochemistry studies of OME cells by light and transmission electron microscopy (TEM) clearly showed a positive reaction of an antibody directed against the human plasma membrane Ca 2+ -ATPase 1 (PMCA-1) in the cytoplasm of OME cells. Also, western blot analysis of diVerent fractions of OME cells with anti human PMCA-1 and C28R2 antibodies conWrmed the presence of a PMCA-like protein with an unusual topographical localization and a molecular weight of only 70-80 kDa. These results lead us to speculate that this PMCA-like protein is distributed either in the plasma membrane or in the entire cytosol, where it eventually regulates intracellular calcium levels. Interestingly, the antibody reactions showed seasonal variations, being highest in OME samples prepared during summer when A. cygnea live under natural acidosis and absent in samples taken in winter conditions, which is in accordance with the seasonal variation of shell calciWcation rates. During winter, PMCA-1 antibody reaction was also detected in OME cells of animals kept only under experimentally induced acidosis conditions. Therefore, we assume that a functional role for this PMCA-like protein in the intracellular calcium regulation of OME cells during the mineralization of the shells of A. cygnea can be speculated.
Calcium transport and homeostasis in gill cells of a freshwater crab Dilocarcinus pagei
Journal of Comparative Physiology B, 2010
Crustaceans present a very interesting model system to study the process of calcification and calcium (Ca 2? ) transport because of molting-related events and the deposition of CaCO 3 in the new exoskeleton. Dilocarcinus pagei, a freshwater crab endemic to Brazil, was studied to understand Ca 2? transport in whole gill cells using a fluorescent probe. Cells were dissociated, all of the gill cell types were loaded with fluo-3 and intracellular Ca 2? change was monitored by adding Ca as CaCl 2 (0, 0.1, 0.25, 0.50, 1.0 and 5 mM), with a series of different inhibitors. For control gill cells, Ca 2? transport followed Michaelis-Menten kinetics with K m = 0.42 ± 0.04 mM and V max = 0.50 ± 0.02 lM (Ca 2? change 9 initial intracellular Ca -1 9 180 s -1 ; N = 14, r 2 = 0.99). Verapamil (a Ca 2? channel inhibitor) and amiloride (a Na ? /Ca 2? exchanger [NCX] inhibitor) completely reduced intracellular Ca 2? transport, while nifedipine, another Ca 2? channel inhibitor, did not. Vanadate, a plasma membrane Ca 2? -ATPase inhibitor (PMCA), increased intracellular Ca 2? in gill cells through a decrease in the efflux of Ca 2? . Ouabain increased intracellular Ca 2? , similar to the effect of KB-R, a specific NCX inhibitor for Ca 2? in the influx mode. Alterations in extracellular [Na] in the saline did not affect intracellular Ca 2? transport.
BMC Genomics, 2007
Background: Freshwater fish absorb Ca 2+ predominantly from ambient water, and more than 97% of Ca 2+ uptake is achieved by active transport through gill mitochondrion-rich (MR) cells. In the current model for Ca 2+ uptake in gill MR cells, Ca 2+ passively enters the cytosol via the epithelium Ca 2+ channel (ECaC), and then is extruded into the plasma through the basolateral Na + / Ca 2+ exchanger (NCX) and plasma membrane Ca 2+ -ATPase (PMCA). However, no convincing molecular or cellular evidence has been available to support the role of specific PMCA and/or NCX isoforms in this model. Zebrafish (Danio rerio) is a good model for analyzing isoforms of a gene because of the plentiful genomic databases and expression sequence tag (EST) data.