Roles of NCX and PMCA in basolateral calcium export associated with mineralization cycles and cold acclimation in crayfish.

Basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+ ATPase (PMCA) are the primary transmembrane proteins that export calcium (Ca2+) from cells. In our lab we use a nonmammalian animal model, the freshwater crayfish, to study cellular Ca2+ regulation. Two experimental conditions are employed to effect Ca2+ dyshomeostasis: (a) in the postmolt stage of the crustacean molting cycle increased unidirectional Ca2+ influx associated with cuticular mineralization is accompanied by elevated basolateral Ca2+ export (compared with intermolt Ca balance); and (b) exposure of the poikilothermic crayfish to cold acclimation (4 degrees C) causes influx of Ca2+ into cells, which is compensated by increased basolateral Ca2+ export (compared with exposure to 23 degrees C). This study compares expression of both NCX and PMCA mRNA (real-time PCR) and protein (Western) in both epithelial (kidney) and nonepithelial tissue (tail muscle) during elevated basolateral Ca2+ export. Both experimental treatments produced increases in NCX and PMCA expression (mRNA and protein) in both tissues. Mineralization produced greater upregulation of mRNA in kidney than in tail, whereas cold acclimation yielded comparable increases in both tissues. Protein expression patterns were generally confirmatory of real-time PCR data although expression changes were less pronounced. Both experimental treatments appear to increase basolateral Ca2+ export.

Allometric relationship of postmolt net ion uptake, ventilation, and circulation in the freshwater crayfish Procambarus clarkii: intraspecific scaling.

There are few intraspecific studies relating physiological parameters to body mass. This study relates scaling of ionic regulation and respiratory parameters with body mass in crayfish (Procambarus clarkii). These animals were chosen because of their direct development, spanning four orders of magnitude in body mass. Usually, these animals are hyperregulators and must maintain hemolymph electrolyte levels above those in the ambient freshwater. This is especially important in the postmolt, when ion imbalance can occur. Maintaining hemolymph ion levels above ambient involves active processes that are independently related to metabolic rate, ventilation, and circulation. Therefore, this study investigates relationships among size and ionic regulation, heart rate, and ventilation in crayfish, spanning a size range of 0.003-24 g. Postmolt net ion uptake of Ca, titratable base, Na, Cl, and NH4 increase with body mass (positive allometry) with slopes of 0.92, 0.79, 0.90, 0.84, and 0.87, respectively. Between 72% and 97% of variation in ionic regulation was related to body mass. The slopes differed from each other for Ca and titratable base but not for Na, Cl, and NH4. For heart rate and ventilation rate, different relationships were derived for animals smaller and larger than 0.01 g (between first and third instar). Animals larger than 0.01 g show a negative allometric relationship between heart rate and body size ([body mass](0.15)), while smaller animals show positive allometry with body size, but only 29% of variation in heart rate is explained by body size alone. For ventilation rates, the negative allometry with body size for animals larger than 0.01 g is present, but less than 15% of variation in ventilation rate is explained by size, while for smaller animals the size dependency disappears. Based on these results, predictions of physiological parameters such as ionic regulation based on body size are useful in crayfish, but estimates of respiratory parameters and body size should be used with caution.

Calcium balance in crustaceans: nutritional aspects of physiological regulation.

Calcium homeostasis in crustaceans is influenced by their natural molting cycle that periodically requires replacement of the calcified exoskeleton in order for growth to occur. Whole body Ca balance transitions from intermolt (zero net flux) to premolt (net efflux) and postmolt (net influx at the rate of 2 mmol kg(-1)h(-1)). As such, molting provides a convenient model to study up- and down-regulation of epithelial Ca transporting proteins (such as Ca pumps and exchangers), the genes that encode them, and the steroid hormone (ecdysone) that putatively regulates the genes. Species residing in either freshwater or in terrestrial environments are more limited in their Ca availability than are marine species. Further the advance towards terrestriality is accompanied by decreased reliance upon branchial Ca uptake and increased reliance upon digestive uptake. This review will correlate Ca handling strategies with environment in semi-terrestrial and terrestrial crabs through examining environmental sources of Ca uptake. Ca homeostasis will also be discussed at the whole animal level, cellular, subcellular and molecular levels of regulation.

Calcium homeostasis in crustaceans: subcellular Ca dynamics.

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(2+)-transporting proteins, common to all eukaryotic cells. This article reviews state-of-the-art knowledge about how crustacean transporting epithelia (gills, hepatopancreas and antennal gland) effect mass transcellular movement of Ca(2+) while preventing cytotoxicity. The current model proposed is based on in vitro research on the intermolt stage with extrapolation to other molting stages. Plasma membrane proteins involved in apical and basolateral Ca(2+) movement (NCX, PMCA) are contrasted between aquatic species of different osmotic origin and among transporting epithelia of an individual species. Their roles are assessed in the context of epithelial Ca(2+) flux derived from organismic approaches. Exchange with extracellular environments is integrated with Ca(2+) sequestration mechanisms across endomembranes of the ER/SR and mitochondria. Finally, the review postulates how new Ca(2+) imaging techniques will allow spatial and temporal resolution of Ca(2+) concentration in subcellular domains.

Cloning and characterization of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) from crayfish axial muscle. Sarco/Endoplasmic Reticulum Ca(2+)-ATPase.

The discontinuous pattern of muscle growth during the moulting cycle of a freshwater crustacean (the crayfish Procambarus clarkii) was used as a model system to examine the regulation of the expression of Sarco/Endoplasmic Reticulum Ca(2+)-ATPase (SERCA). We describe the cloning, sequencing and characterization of a novel SERCA cDNA (3856 bp) obtained from crayfish axial abdominal muscle by reverse transcription/polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). This complete sequence contains a 145 base pair (bp) noncoding region at the 5' end, a 3006 bp open reading frame coding for 1002 amino acid residues with a molecular mass of 110 kDa and 705 bp of untranslated region at the 3' end. This enzyme contains all the conserved domains found in 'P'-type ATPases, and the hydropathy profile suggests a transmembrane organization typical of other SERCAs. It exhibits 80% amino acid identity with Drosophila melanogaster SERCA, 79% identity with Artemia franciscana SERCA, 72% identity with rabbit fast-twitch muscle neonatal isoform SERCA1b, 71% identity with slow-twitch muscle isoform SERCA2 and 67% identity with SERCA3. Sequence alignment revealed that regions anchoring the cytoplasmic domain in the membrane were highly conserved and that most differences were in the NH(2) terminus, the central loop region and the COOH terminus. Northern analysis of total RNA from crayfish tissues probed with the 460 bp fragment initially isolated showed four bands (7.6, 7.0, 5.8 and 4.5 kilobases) displaying tissue-specific expression. SERCA was most abundant in muscle (axial abdominal, cardiac and stomach), where it is involved in Ca(2+) resequestration during relaxation, and in eggs, where it may be implicated in early embryogenesis. The level of SERCA mRNA expression in axial abdominal muscle varied during the moulting cycle as determined by slot-blot analysis. SERCA expression was greatest during intermoult and decreased to approximately 50% of this level during pre- and postmoult. Patterns of gene expression for SERCA and other sarcomeric proteins during the crustacean moulting cycle may be regulated by ecdysteroids and/or mechanical stimulation.

Calcium homeostasis in crustacea: the evolving role of branchial, renal, digestive and hypodermal epithelia.

Crustaceans serve as an ideal model for the study of calcium homeostasis due to their natural molting cycle. Demineralization and remineralization of the calcified cuticle is accompanied by bidirectional Ca transfer across the primary Ca transporting epithelia: gills, antennal gland (kidney), digestive system, and cuticular hypodermis. The review will demonstrate how a continuum of crustaceans can be used as a paradigm for the evolution of Ca transport mechanisms. Generally speaking, aquatic crustaceans rely primarily on branchial Ca uptake and accordingly are affected by water Ca content; terrestrial crustaceans rely on intake of dietary Ca across the digestive epithelium. Synchrony of mineralization at the cuticle vs. storage sites will be presented Physiological and behavioral adaptations have evolved to optimize Ca balance during the molting cycle in different Ca environments. Intracellular Ca regulation reveals common mechanisms of apical and basolateral membrane transport as well as intracellular sequestration. Regulation of cell Ca concentration will be discussed in intermolt and during periods of the molting cycle when transepithelial Ca flux is significantly elevated. Molecular characterization of the sarco-/endoplasmic reticular Ca pump in aquatic species reveals the presence of two isoforms that originate from a single gene. This gene is differentially expressed during the molting cycle. Gene expression may be regulated by a suite of hormones including ecdysone, calcitonin, and vitamin D. Perspectives for future research are presented.

ATP-dependent calcium uptake into basolateral vesicles from transporting epithelia of intermolt crayfish.

ATP-dependent Ca2+ uptake was determined into inside-out basolateral membrane vesicles (BLMV) from intermolt crayfish (Procambarus clarkii) Ca2+-transporting epithelia: gill, hepatopancreas (liver), and antennal gland (kidney). Extravesicular (EV) ATP (5 mM) increased 45Ca2+ uptake (free Ca2+ 5 microM) by fivefold but was abolished by pretreatment with either vanadate or the ionophore A-23187. Addition of A-23187 to Ca2+-loaded vesicles produced 70% efflux. The saturable carrier exhibited a Km for Ca2+ of 0.11-0.27 microM and maximal influx of 20-123 pmol. mg-1. min-1. The Km for ATP was 0.01-0.04 mM. The temperature coefficient ranged from 1.43 to 2.06. EGTA treatment of hepatopancreas and antennal gland vesicles decreased 45Ca2+ uptake by 50-90%; uptake was restorable by calmodulin. However, in gill, 45Ca2+ uptake was unaffected by EGTA treatment and calmodulin decreased uptake in both EGTA-treated and untreated vesicles. Addition of EV Na+ (5 mM) increased ATP-dependent Ca2+ uptake into hepatopancreas and antennal gland BLMV by 60%; in hepatopancreas BLMV, this increase was inhibitable by ouabain. However, ATP-dependent Ca2+ uptake in gill vesicles was Na+ independent. The relative role of each epithelium in whole animal Ca2+ homeostasis has been interpreted based on in vitro characteristics.

Isolation, visualization, characterization, and osmotic reactivity of crayfish BLMV.

Procedures were developed to isolate basolateral membrane vesicles (BLMV) from gill, hepatopancreas, and antennal gland of intermolt freshwater crayfish, Procambarus clarkii. Individual procedures involved a discontinuous sucrose gradient (gill), a 65% sucrose cushion (hepatopancreas), or differential centrifugation (antennal gland). BLMV were visualized, characterized (37 degrees C), and tested for osmotic reactivity with a view to using them for Ca2+ uptake studies. Mean diameters of BLMV were 159 nm (gill), 363 nm (hepatopancreas), and 226 nm (antennal gland). Enrichments of basolateral membranes and mitochondria in BLMV were, respectively, 18- and 1.7-fold for gill, 9- and 0.4-fold for hepatopancreas, and 10- and 1-fold for antennal gland. Apical contamination was negligible in BLMV. Percentages of resealing of vesicles as inside out, right side out, or leaky/sheets were 17:27:56% (gill), 14:26:60% (hepatopancreas), and 21:39:40% (antennal gland). Vesicles exhibited osmotic reactivity, as indicated by a linear relationship between vesicular 45Ca2+ uptake and osmolality. Nonspecific 45Ca2+ binding was 20% in gill, 39% in hepatopancreas, and 31% in antennal gland. Data were compared with published values for marine crustaceans.

The mechanisms of acid-base and ionoregulation in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia. II. The role of the kidney.

Plasma ionic status and renal excretion of acidic equivalents and electrolytes were continuously monitored in the freshwater rainbow trout (Salmo gairdneri) during 24 h normoxia (PIO2 = 120-150 torr; control); 72 h hyperoxia (PIO2 = 500-600 torr), and 24 h return to normoxia. Plasma [Cl-] progressively declined in approximate equivalence to the rise in [HCO-3] which compensated the respiratory acidosis of hyperoxia, while [Na+] increased only slightly. [Ca2+] and [K+] rose, [phosphate] declined, and [NH+4] was unchanged. During normoxic recovery, the [Na+], [Cl-] and [HCO-3] changes were reversed, [K+] and [Ca2+] showed further elevations, and [NH+4] increased sharply . Renal acid output increased greatly during hyperoxia with elevations in both NH+4 and titratable components, though the latter predominated due to a marked elevation of phosphate excretion. Renal efflux rates of other electrolytes were generally homeostatic for ECF composition, with increased Na+, K+, and Ca2+ effluxes, and decreased Cl- efflux. Clearance calculations indicated that net tubular reabsorption increased for Cl-, fell for Na+ and K+, and changed over to marked net secretion for phosphate, while net ammonia secretion increased. Most trends were reversed upon return to normoxia. The critical role of phosphate in urinary electrolyte balance and acid-base regulation is emphasized. The net renal excretion of acidic equivalents accounted for only 7-10% of the total compensation observed for the whole animal during hyperoxia. The kidney contributed primarily in conserving ECF HCO-3 and secondarily in balancing branchial exchanges.

The mechanisms of acid-base and ionoregulation in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia. III. Branchial exchanges.

Fluxes of both acidic equivalents (JH+net) and electrolytes across the gills were continuously monitored in the freshwater rainbow trout (Salmo gairdneri) during 24 h normoxia (PIO2 = 120-150 torr; control), 72 h hyperoxia (PIO2 = 500-600 torr), and 24 h return to normoxia. A highly negative JH+net (i.e., excretion) was responsible for over 90% of the compensation of respiratory acidosis induced by hyperoxia in the whole animal. Similarly, a highly positive JH+net (i.e., uptake) accounted for virtually all the compensation of metabolic alkalosis induced by normoxic recovery. Hyperoxia was associated with a small net gain of Na+ and large net losses of Cl- at the gills, while normoxic recovery was associated with large net losses of Na+ and net gains of Cl-, effects reflected in ECF composition. Unidirectional flux analyses with radiotracers (22Na, 36Cl) demonstrated that these net flux alterations resulted from rapid and complex changes in both influx and efflux components such that the difference between JNa+net and JCl-net was stoichiometrically equivalent to JH+net. The results support the concept that Na+ vs acidic equivalent (H+, NH+4) and Cl- vs basic equivalent (HCO-3, OH-) exchanges at the gill are dynamically adjusted in order to correct internal acid-base disturbances.

The mechanisms of acid-base and ionoregulation in the freshwater rainbow trout during environmental hyperoxia and subsequent normoxia. I. Extra- and intracellular acid-base status.

The extracellular acid-base status of the freshwater rainbow trout (Salmo gairdneri) was continuously monitored during 24 h normoxia (PIO2 = 120-150 torr; control), 72 h hyperoxia (PIO2 = 500-600 torr) and 24 h return to normoxia. Hyperoxia induced a marked respiratory acidosis (delta pHe = -0.23 unit) due to a 3-fold elevation in arterial CO2 tension which was completely compensated over 72 h by a comparable rise in plasma bicarbonate, reflecting effective removal of acidic equivalents from the ECF. Upon return to normoxia, arterial CO2 tension rapidly returned to normal against a background of high plasma bicarbonate, provoking a metabolic alkalosis which was largely compensated by 24 h. This effective restoration of acidic equivalents in the ECF occurred more rapidly than the original removal. Intracellular acid-base status was measured during normoxia and after 72 h hyperoxia using the steady state distribution of 14C-DMO. The rate of 14C-DMO excretion was 0.479 +/- 0.048 (% DMO lost per hour) during normoxia, and significantly decreased with hyperoxia. A considerable overestimate of mean whole body pHi would have resulted had this not been taken into account. Whole body and white expaxial muscle were similar with a pHe - pHi gradient of ca. 0.5 during normoxia, and underwent identical changes during hyperoxia. Intracellular pH was completely compensated by 72 h hyperoxia as intracellular bicarbonate increased 4-fold. The overall net removal of acidic equivalents from the ICFV was approximately one half that from the ECFV , but pHe regulation did not occur at the expense of pHi regulation. The ultimate restoration of both pHe and pHi during hyperoxia must have occurred via kidney or gills.