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Gene Expression and Enzyme Activities of the Sodium Pump During Sea Urchin Development: Implications for Indices of Physiological State.

ADAM G. MARSH [*]

PATRICK K.K. LEONG [+]

DONAL T. MANAHAN [++]

Abstract. The sodium pump consumes a large portion of the metabolic, energy (40%) in sea urchin larvae. Understanding the developmental regulation of ion pumps is important for assessing the physiological state of embryos and larvae. We sequenced a partial cDNA clone (1769 bp) from the sea urchin Strongylocentrotus purpuratus and found it to contain the C-terminal portion of an open reading frame coding for 195 amino acids that exhibited high sequence similarity (89%) to invertebrate [alpha]-subunits of the [Na.sup.+], [K.sup.+]- ATPase sodium pump. Northern blots using the 3' untranslated region of this cDNA specifically recognized a 4.6-kbp transcript under high stringency. During embryonic development, a rapid increase in levels of this mRNA transcript during gastrulation (25 h postfertilization) was paralleled by a concomitant increase in the total enzymatic activity of [Na.sup.+], [K.sup.+] -ATPase. Expression of this subunit during gastrulation increased to a maximum at 36 h, followed by a rapid decline to trace levels by 60 h. The rate of removal of the transcript from the total RNA pool after 36 h closely followed a first-order exponential decay model ([r.sup.2]= 0.988), equivalent to a degradation rate of 7.8% [h.sup.-1]. By 83 h, transcription of the [alpha]-subunit gene was low, yet sodium pump activity remained high. Molecular assays for the expression of this gene would underestimate sodium pump activities for assessing physiological state because of the temporal separation between maximal gene expression in a gastrula and maximal enzyme activities in the later larval stage. This finding illustrates the difficulty of using molecular probes for assessing the physiological state of invertebrate larvae.

Introduction

Maintaining [Na.sup.+] and [K.sup.+] ion gradients is one of the most energetically demanding processes of an organism's maintenance physiology. In general, animal cells routinely expend 20%-30% of their total metabolic energy on the activity of a single protein complex, the sodium pump ([Na.sup.+], [K.sup.+]-ATPase; Siems et al., 1982, 1992), and for adult marine invertebrates, the sodium pump can potentially account for 30%-70% of tissue metabolism (Baker and Connelly, 1966; Lucu and Pavicic, 1995). The ion gradients established by the sodium pump are critical for maintaining a cell's osmotic balance and resting membrane potential, as well as providing the electrochemical gradient necessary for the uptake of other ions, sugars, amino acids, and neurotransmitters via [Na.sup.+] coupled co-transporters (Blanco and Mercer, 1998).

The requirements for ion regulation change rapidly during embryonic development. The increase in cell number during early embryogenesis and the consequent increase in cellular-membrane surface area necessitates the production of more sodium pumps to regulate intracellular ion flux. The in vivo physiological activity of [Na.sup.+],[K.sup.+]-ATPase has been characterized during early development in the sea urchins Strongylocentrotus purpuratus and Lytechinus pictus (Leong and Manahan, 1997). Using [[blank].sup.86][Rb.sup.+] as a radioactive tracer for [K.sup.+]ion transport, Leong and Manahan (1997) described the ontogenetic changes in activity of [Na.sub.+], [K.sub.+] -ATPase in living embryos. They found a large increase in activity--from nondetectable levels prior to fertilization to a high level accounting for 40% of total metabolic energy consumption at the pluteus larval stage (72 h postfertilization). By the same radiotracer techniques, the metabolic energy demand of [Na.sub.+], [K.sub.+]-ATPase activit y in the Antarctic sea urchin Sterechinus neumayeri was found to be as high as 80% of total metabolism at the pluteus larval stage at --l.5[degrees]C (Leong and Manahan, 1999). In the sea urchin Hemicentrotus pulcherrimus, the total protein activity and gene expression of [Na.sup.+], [K.sup.+] -ATPase increases rapidly during gastrulation (Mitsunaga-Nakatsubo et al., 1992a, b). Overall, the physiological importance of [Na.sup.+], [K.sup.+] -ATPase activity during embryogenesis in sea urchins has significant implications for metabolic energy consumption during development.

This universal importance of [Na.sup.+],[K.sup.+] -ATPase in animals suggests that measurements of this enzyme could be a useful indicator of physiological state. For larval stages in which direct enzyme assays are limited by the small amount of protein in an individual, measurements of gene expression might provide the sensitivity necessary to assay small amounts of tissue. Functional [Na.sup.+], [K.sup.=] -ATPase pumps are a heterodimer ([alpha], [beta] subunits; Jorgensen and Skou, 1969), with the [alpha]-subunit possessing the ATP binding site and catalytic activity (Kyte, 1971). In this study, we describe the timing between transcription of the [alpha]-subunit and the appearance of functional sodium pumps during the development of Strongylocentrotus purpuratus. We also describe the ontogenetic changes in expression of the [alpha]-subunit to determine the developmental timing between increases in enzyme activity and the potential for using these measures as an index of physiological state in embryos and larvae.

Materials and Methods

Embryo cultures

Adult Strongylocentrotus purpuratus were induced to release gametes (injections of 0.5 M KC1), and fertilized eggs were divided into six 20-liter culture containers at a concentration of about 20 individuals per milliliter of filtered seawater (0.2 [micro]m). Culture temperatures were maintained at 15[degrees]C during development. Embryos were maintained in suspension by paddles connected to slow stirring motors ([sim]30 rpm). For the gene expression analysis, time-course samples were collected throughout development at the following times from an egg to a 4-arm pluteus larva: 0, 6, 8, 10, 12, 14, 16, 18, 20, 25, 31, 36, 42, 48, 60, 72, and 83 h postfertilization (n = 17). For each sample, about 100,000 embryos were removed by sieving (80-[micro]m mesh) and pelleted by centrifugation (1000 X g) into 50-ml screw-cap tubes. Embryos were immediately dissolved in an acidguanidinium buffer (4 M guanidinium isothiocyanate, 25 mM Na-citrate, 0.2% Sarkosyl and 215 mM ([beta]-mercapto-ethanol; pH 5.2; Chomzinsky and Sacchi, 1987) and frozen at -800C.

cDNA clone: sequencing and analysis

An expressed sequence tag (EST) library from activated coelomocytes of adult S. purpuratus was prepared by Smith et al. (1996), and a sequence fragment of one cDNA clone (#020) was found to have a high nucleotide similarity to the bovine [alpha]-subunit of [Na.sup.+], [K.sup.+] -ATPase. We sequenced this clone (provided by C. L. Smith and E. H. Davidson) by random transposon insertion in a modified pBluescript (Stratagene) plasmid (pMOB; Strathmann et al., 1991). The introduced transposon elements contained defined priming sites for subsequent manual sequencing of double-stranded plasmid templates using standard dideoxy termination reactions with 355-labeled dATP (Sequenase Reaction Kit, USB). Sequencing gels were visualized by autoradiography on X-ray film (Kodak, XAR 5). Nucleotide sequences were entered and edited using the software package MacVector 5.0 (Mac OS; Oxford Molecular Group), and contiguous overlaps between fragments were identified using the software package AssemblyLign 2.0 (Mac OS; Oxford M olecular Group). Both strands of the open reading frame (ORF) were sequenced by overlapping subclones so that most of the contiguous ORF sequence was assembled from three independent sequencing reactions. For phylogenetic comparisons, nucelotide and putative amino acid sequences from other animal species were structurally analyzed and aligned using the OMIGA 2.0 software package (Oxford Molecular Ltd.). Identity and similarity scores for the deduced amino acid alignments were calculated from the FASTA routine available in the GCG Wisconsin Package 8.0 (UNIX OS).

mRNA analysis: isolation and quantification

Total RNA was extracted from each sample by an acid guanidinium-phenol method (after Chomzinsky and Sacchi, 1987) and further purified by sequential precipitations in lithium chloride (4 M LiCl), sodium acetate (3 M NaOAc, pH 4.2) and ethanol (70% EtOH). After each precipitation, the RNA pellets were washed in 70% EtOH and dried under vacuum; before proceeding with the next precipitation, the pellets were resuspended in RNase-free TEN buffer (10 mM Tris pH 8.0, 1 mM EDTA and 10 mM NaCl). The final RNA precipitates were resuspended in RNase-free water and quantified by their optical density at 260 nm. From each developmental time point, 10 [micro]g of total RNA was size-separated by formaldehyde gel electrophoresis and blotted overnight via capillary transfer onto nylon membranes. RNA on the nylon membranes was UV cross-linked (Stratalinker), and the membranes were stored dry at room temperature. A cDNA probe was generated from the 3'-untranslated region (UTR) of clone #020. The terminal 1185 bp were PCR ampl ified (5'- TGG GAT TGA AGG AGT CAG -3' and T7 oligonucleotide primers) and gel purified for further use in standard Northern hybridizations (see general methods in Ausubel et al., 1992). Membranes were prehybridized for several hours in 40% formamide, 25 mM [Na.sub.3][PO.sub.4] (pH 7.2), 5X SSC, 0.1% SDS, 5X Denhardt's, and 50 j[micro]g/ml yeast RNA at 45[degrees]C in a hybridization oven. The 3'-UTR PCR probe (1185 bp) was radiolabeled by random priming (Promega) with [[alpha].sup.-32]P-dCTP (3000 Ci [mmol.sup.-1]), added to the hybridization tube with a fresh 10-ml aliquot of hybridization buffer (as above), and incubated overnight at 500C. The blots were initially washed with 0.1X SSC, 1.0% SDS, and 0.5% [Na.sub.4][P.sub.2][O.sub.7] at 45[degrees]C for 1 h. Additional washes at higher temperatures (max. 55[degrees]C) were performed as necessary to further reduce the background signal. Autoradiograms (Kodak Biomax X-ray film) were digitized on a high-resolution scanner (1200 dpi), and grain densities for th e signal bands were quantified using the image analysis routines in the software program PhotoShop 4.0 (Win95 OS; Adobe).

[Na.sup.+],[K.sup.+]-ATPase enzyme activity

Total enzyme activity of [Na.sup.+],[K.sup.+]-ATPase was measured at short intervals between 20 and 50 h postfertilization, the period during which enzyme activity increases rapidly during development in S. purpuratus (Leong and Manahan, 1997). Ouabain-sensitive [Na.sup.+],[K.sup.+]-ATPase activity (details in Leong and Manahan, 1997) was determined in all samples on the same day with one set of standards to minimize the between-sample assay error. Total [Na.sup.+],[K.sup.+]-ATPase activity was measured as the rate of hydrolysis of ATP (Esmann, 1988). Briefly, embryo tissues were thawed, sonicated, and resuspended in histidine buffer (10% sucrose, 5 mM EDTA and 5 mM histidine, pH 7.7) at a final protein concentration of 0.5 to 1.0mg [ml.sup.-1]. In the present study, the total [Na.sup.+],[K.sup.+]-ATPase activity of the sea urchin embryos was measured as the difference in ATPase activity in the presence and absence of 2 mM ouabain at 25[degrees]C. A detailed consideration of the inclusion of detergents in th e [Na.sup.+],[K.sup.+]-ATPase assay is presented in Leong and Manahan (1997). In summary, neither deoxycholate (a common detergent used in [Na.sup.+],[K.sup.+]-ATPase assays) nor alamethicin (a membrane-permeabilizing agent) had any effect on the total [Na.sup.+],[K.sup.+]-ATPase activity in homogenates of S. purpuratus embryos, suggesting that inside-out and right-side-out vesicles are not a significant problem in assaying [Na.sup.+],[K.sup.+]-ATPase activity in sea urchin embryos (Leong and Manahan, 1997). The protein content of the samples was determined by the Bradford assay with the modifications of Jaeckle and Manahan (1989).

Results

A partial Strongylocentrotus purpuratus cDNA clone (#020; Smith et al., 1996) was characterized in this study and found to contain 1769 bp with the terminal portion of an ORF coding for 195 amino acids (588 bp with the stop codon). The remaining sequence (1181 bp) comprised a putative 3' UTR domain. The clone's ORF was compared to other [alpha]-subunits of [Na.sup.+],[K.sup.+]-ATPase, and the S. purpuratus nucelotide sequence ranged from 64% to 70% identity to these terminal ORF domains (Table 1). The deduced amino acid sequences of these organisms were aligned to the putative amino acid sequence of the S. purpuratus clone and evidenced a high degree of sequence conservation in this terminal domain (Table 2). When compared to the S. purpuratus sequence, the derived amino acid sequence was 68%-73% identical and 89%-90% similar (Table 1).

The terminal region of the ORF of known [alpha]- [Na.sup.+],[K.sup.+]-ATPase is believed to contain several transmembrane domains; there is some debate over the exact number of these domains and the extra- vs. intracellular orientation of some of the intervening regions in [alpha]- [Na.sup.+],[K.sup.+]-ATPase (Shull and Greeb, 1988; Takeyasu et al., 1990; Blanco and Mercer, 1998). The hydropathy of the S. purpuratus sequence was estimated with Kyte-Doolittle scoring using a grouping of 11 amino acid residues (Fig. 1) and suggests a high probability of four transmembrane domains in the terminal portion of this ORE. Overlaying these domains on a structure detailed by Takeyasu et al. (1990) indicates that the region between the seventh and eighth transmembrane domains could have an extracellular localization. In the absence of crystallographic data, it is generally believed that most [alpha]-subunits of transmembrane ATPases (both [Na.sup.+] and [Ca.sup.++]) are structurally similar, with 10 transmembrane domai ns and a large extracellular loop between transmembrane domains 7 and 8 (Canfield and Levenson, 1993; Blanco and Mercer, 1998).

Northern blots using the 3' UTR of clone #020 specifically recognized a 4.5 to 4.7 kb transcript under high stringency (Fig. 2). In another sea urchin, Hemicentrotus pulcherrimus, the full-length [alpha]-[Na.sup.+],[K.sup.+]-ATPase cDNA has been cloned and has an mRNA transcript size of 4.6 kb (Mitsunaga-Nakatsubo et al., 1992a). The [alpha]-[Na.sup.+],[K.sup.+]-ATPase gene is differentially expressed during development in S. purpuratus (Fig. 2). The level of mRNA transcript is low during early cleavage, then rises rapidly around gastrulation (at 25-36 h postfertilization; Fig. 3). After gastrulation, mRNA returns to a low level comparable to that initially found in the egg (Fig. 3). The rapid disappearance of the [alpha]-[Na.sup.+],[K.sup.+]-ATPase transcript from the total RNA pool after gastrulation closely followed a first-order exponential decay model [F(X) = 98.512 [e.sup.(-0.128x)]; [r.sup.2] = 0.988; Fig. 3]. The decay constant of the regression is equivalent to a degradation rate of 7.8% [h.sup.-1] o f the transcript. At 83 h, [alpha]-subunit transcripts were barely detectable under the conditions we used for Northern blots of total RNA.

The rapid increase in [alpha]-[Na.sup.+], [K.sup.+] -ATPase mRNA transcripts during gastrulation in S. purpuratus was paralleled by a concomitant increase in the total activity of the sodium pump (Fig. 4). Activity levels were very low during early development in S. purpuratus and then increased after 20 h to a maximum level at the pluteus larval stage (Leong and Manahan, 1997). The rapid increase in activity between 20 and 40 h of development (Fig. 4) can be described by the exponential function [f(x) = 1.167(1 + [e.sup.[[(x-[x.sub.o]/4,57]).sup.-1]; [r.sup.2] = 0.9664; maximum activity of 1.17 [micro]mol [P.sub.i][h.sup.-1] [mg.sup.-1] protein]. The present study resolves the increase in enzyme activity at a finer time scale (cf.Leong and Manahan, 1997) and reveals the close coordination between [alpha]-subunit gene transcription and the assembly of functional sodium pumps in sea urchin embryos between fertilization and gastrulation.

Discussion

It has long been a general goal of physiological ecologists to identify a sensitive biochemical indicator of an animal's physiological state or metabolic activity--for example, the ratio of RNA to DNA (Westerman and Holt, 1994) or the glycolytic enzyme activities (Childress and Somero, 1990). For developmental stages with low biochemical contents, such assays are often not possible. Molecular biological techniques have the necessary sensitivity and potentially offer an alternative for assessing physiological state in larvae and small zooplankton. Because the sodium pump consumes such a large portion of cellular energy metabolism (e.g., 40% in sea urchin larvae, Leong and Manahan 1997), it would seem to be a good candidate for such an assay, with the potential to provide sensitive information regarding rates of energy utilization in a single larva.

Several lines of evidence strongly support the conclusion that the partial cDNA clone (#020) in Strongylocentrotus purpuratus is the [alpha]-subunit of the sodium pump: (1) the putative amino acid sequences show a high similarity to those of other animals; (2) the 3'-UTR probe recognizes a 4.6-kb transcript, which is the full-length transcript size in other invertebrate species; (3) the ontogenetic increase in expression during gastrulation is similar to the expression pattern in another sea urchin (Mitsunaga-Nakatsubo et al., 1992b); (4) total [Na.sup.+],[K.sup.+]-ATPase enzyme activities show a concomitant increase as mRNA transcripts of clone #020 accumulate during gastrulation.

In the sea urchin Hemicentrotus pulcherrimus, the expression of the [alpha]-[Na.sup.+],[K.sup.+]-ATPase gene increases rapidly during gastrulation (Mitsunaga-Nakatsubo et al., 1992b). In S. purpuratus, the expression of the [alpha]-[Na.sup.+],[K.sup.+]-ATPase gene evidences a similar pattern of ontogenetic regulation, with a sharp rise during gastrulation followed by a subsequent decline to much lower levels. In conjunction with the total [Na.sup.+],[K.sup.+]-ATPase enzyme activity that is present during development (this study, Fig. 4; see also Leong and Manahan, 1997), temporal changes in both mRNA transcripts and protein activity indicate that the enzyme activity is low during early cleavage. At the point when an embryo approaches gastrulation, [alpha]-subunit gene transcription and subsequent mRNA translation increase greatly, producing a large increase in sodium pumps (Fig. 3), presumably as a necessary component of the physiological function of proliferating cells.

Once these pumps have been synthesized, mRNA transcripts for the [alpha]-[Na.sup.+],[K.sup.+]-ATPase are rapidly lost. The decrease in mRNA levels over time fits a first-order exponential decay model (7.8% [h.sup.-1]) so that by 83 h, transcription of the [alpha]-subunit gene was barely detectable (Fig. 2). At gastrulation, S. purpuratus appears to have synthesized most of the necessary sodium pumps. Total enzymatic activities show little increase after 50 h, further supporting this observation that the number of [Na.sup.+],[K.sup.+]-ATPase ion pumps is set by the rapid transcription during gastrulation, and that once these transcripts are degraded, an early larva's sodium pump complement remains unchanged until further growth occurs, usually after feeding is initiated.

In vertebrates, the [alpha]-subunit [Na.sup.+],[K.sup.+]-ATPase has several isoforms (Rossier et al., 1987) that differ in many aspects, including sensitivity to proteases and cross-linking agents (Sweadner, 1979), electrophoretic mobility (Peterson et al., 1982), and affinity for ouabain (Lytton et al., 1985). In brine shrimp (Artemia sauna), the [alpha]-[Na.sup.+],[K.sup.+]-ATPase is present in two isoforms that are differentially expressed during early development (Peterson et al., 1982). In the sea urchin Hemicentrotus pulcherrimus, two [alpha]-subunit isoforms are expressed during embryogenesis (Yamazaki et al. 1997). However, these two isoforms are encoded by a single gene and have identical sequences except for the 5' leader sequences (Yamazaki et al., 1997). If S. purpuratus, like H. pulcherrimus, has a similar isoform complement, then the cDNA probe we used for the present study (from the 3'-UTR) should hybridize to other [alpha]-subunit isoforms expressed during early development. Regardless of the mechanism, the disparity at 83 h postfertilization between the transcript measurements and the complement of active sodium pumps indicates the difficulty in isolating a single molecular factor to be used as an index for physiological rate processes.

The observation that [Na.sup.+],[K.sup.+]-ATPase gene transcription and translation events are limited to a brief developmental period is intriguing. The sodium pump is considered to be a "housekeeping" protein. Consequently, for such an important physiological process, we would have expected the expression of a subunit gene to be constitutive and at a low level so that there would always be some subunit synthesis to replace any turnover in functional pump proteins. Such a continual level of replacement might have offered a sensitive assay for assessing the physiological state of individual larvae by providing a molecular index of the activity of one of the most energy-demanding cellular processes. However, this is not the case. The [alpha]-subunit expression is developmentally regulated so that gene expression is initiated rapidly at about 20 h, peaks at about 36 h, and is subsequently "turned-off." Such a temporal pattern of regulation highlights the difficulty of using molecular probes as simple indices o f physiological state. Similar difficulties in the interpretation of physiological activity and expression have been found for other specific housekeeping genes (e.g., Weinstein et al., 1992; Yang and Somero, 1996). For the multiple enzymes in metabolic pathways, the control mechanisms at the level of genes and proteins are even more complex (Hochachka et al., 1998).

Ontogenetic changes in the metabolic rates of embryos have important consequences for subsequent survival because of the finite quantity of energy reserves in an egg. During development, metabolic rates increase in embryos as their cell numbers increase (Marsh et al., 1999), and the activity of the sodium pump can consume a large fraction of total metabolism in some sea urchin embryos and larvae (Leong and Manahan, 1997, 1999). Understanding ontogenetic changes in sodium pump activities is important for assessing the metabolic energy costs of development. In the pluteus larval stage of S. purpuratus (at 83 h postfertilization), the in vivo sodium pump activity consumes 40% of total metabolism, with a potential reserve activity that could increase to a maximum of 77% of metabolism (Leong and Manahan, 1997). However, gene expression is barely detectable at this point in larval development (Fig. 3). Consequently, molecular assays for expression of this gene would not be informative for assessing sodium pump act ivity as an index of a larva's physiological state. It is likely that during development and growth many physiological processes have functional rates of protein activity that are not strictly paralleled in time by the expression of their genetic components. A knowledge of the temporal relationship between gene and enzyme activities is critical to developing a molecular genetic index of physiological state in larval forms.

Acknowledgments

We thank E. Davidson and C. Smith for providing the clone that we have characterized. D. Pace and M. Moore assisted with the culture sampling. This project was supported by California Sea Grant #R/MP-75C, and NSF #9420803.

(*.) Present address: College of Marine Studies, University of Delaware, Lewes, DE 19958.

(+.) Present address: Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, CA 90033.

(++.) To whom correspondence should be addressed. E-mail: manahan@usc.edu

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Author:MARSH, ADAM G.; LEONG, PATRICK K.K.; MANAHAN, DONAL T.
Publication:The Biological Bulletin
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Date:Oct 1, 2000
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