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In this study, myosin, the major component of myofibrillar protein in the skeletal muscle, was characterized and its expression was monitored during growth in juvenile walleye Sander vitreus. First, the coding region of myosin heavy chain (MyHC) from the fast skeletal muscle of walleye was amplified by long-distance PCR using a full-length cDNA. Phylogenetic analysis was used to determine the evolutionary relationship of this S. vitreus myosin sequence to other vertebrate myosin sequences. Next, it was established that the myosin isoform was most prevalent in the white muscle, compared with the red and cardiac muscle. Myosin expression was monitored over a series of experiments designed to influence growth. Specifically, change in MyHC mRNA was monitored after acute changes in feeding. Fish exposed to a one-week fasting period showed significant decreases in MyHC mRNA levels by the end of the fast. The effect of feeding was also examined more closely over a 24 h period after feeding, but results showed no significant change in myosin expression levels through this time period. Finally, fish with higher growth rates had higher MyHC mRNA and protein expression levels. This study indicates that MyHC mRNA expression is sensitive to the factors that may influence growth in juvenile S. vitreus.

The objective of the current study was to examine whether sublethal (moderate) levels of dissolved ammonia may be beneficial to growth in juvenile walleye Sander vitreus (recent evidence in juvenile rainbow trout Oncorhynchus mykiss has shown significant increases in protein synthesis in the presence of moderately elevated concentrations of dissolved ammonia). Moderately elevated dissolved ammonia concentrations between 100 and 300 micromol l(-1) suppressed routine aerobic metabolic activity by 20% during acute trials (2 h), while promoting specific growth rate (>50%) and elevating whole body soluble protein content by 20% in the early stages (14-42 days) in chronic ammonia exposure experiments. Juvenile S. vitreus held at ammonia concentrations between 107.6 +/- 5.5 and 225.5 +/- 4.7 micromol l(-1) (mean +/-s.e.) grew significantly faster than control fish and significantly reduced plasma cortisol levels (<3 microg dl(-1)). Results from this study suggest that chronic exposure to moderate amounts of dissolved ammonia significantly increase growth rates in juvenile S. vitreus by increasing nitrogen accessible for supplementary protein deposition leading to somatic development.

In this study, field biotelemetry and laboratory physiology approaches were coupled to allow understanding of the behavioral and physiological responses of fish to winter hypoxia. The biotelemetry study compared dissolved oxygen levels measured throughout the winter period with continually tracked locations of nine adult largemouth bass obtained from a whole-lake submerged telemetry array. Fish habitat usage was compared with habitat availability to assess whether fish were selecting for specific dissolved oxygen concentrations. The laboratory study examined behavioral and physiological responses to progressive hypoxia in juvenile largemouth bass acclimated to winter temperatures. Results from the dissolved oxygen measurements made during the biotelemetry study showed high variance in under-ice dissolved oxygen levels. Avoidance of water with dissolved oxygen <2.0 mg/L by telemetered fish was demonstrated, but significant use of water with intermediate dissolved oxygen levels was also found. Results from the lab experiments showed marked changes in behavior (i.e., yawning and vertical movement) at <2.0 mg/L of dissolved oxygen but no change in tissue lactate, an indicator of anaerobic metabolism. Combined results of the biotelemetry and laboratory studies demonstrate that a dissolved oxygen content of 2.0 mg/L may be a critical threshold that induces behavioral responses by largemouth bass during the winter. In addition, the use by fish of areas with intermediate levels of dissolved oxygen suggests that there are multiple environmental factors influencing winter behavior.

Carbonic anhydrase related proteins (CA-RP) are members of the alpha-CA gene family that have lost the ability to catalyze the hydration/dehydration of carbon dioxide. Nonetheless, these proteins are very highly conserved within mammals, suggesting they have an important biological function. To date, however, there has been no effort to examine the presence and role of CA-RP proteins in non-mammalian vertebrates. This study therefore examined whether CA-RPs are present in a representative teleost, the rainbow trout (Oncorhynchus mykiss). A CA-RP-like 846 bp coding region was amplified from whole body rainbow trout mRNA, and was shown to be expressed primarily in the brain. Phylogenetic analysis grouped this isozyme, and several other non-mammalian CA-RPs identified in Genbank, closely with mammalian CA-RP VIII. Analyses of the molecular structure revealed that mutations that led to the loss of catalytic activity in mammalian CA-RP VIII were also present in the active site pocket of the rainbow trout CA-RP protein, suggesting a similar lack of catalytic activity. Unlike mammalian CA-RP VIII, however, the N-terminal regions of non-mammalian CA-RPs do not contain an acidic motif. This suggests that these proteins do not function as a regulator of transcription, as has been proposed for mammals.

Guidelines for ammonia toxicity in fish are often determined using static exposure tests with immature fish over a 96-h period. These results may not be relevant to aquaculture, hauling or angling tournament scenarios where mature fish can be exposed to ammonia for shorter durations, often following additional stressors such as handling. The current study sought to quantify (1) the impact of ambient ammonia on the ability of largemouth bass to recover from exercise, (2) the behavioural response of largemouth bass to elevated ambient ammonia and (3) the concentration of ammonia that can accumulate in a live-release vessel at an angling tournament. After approximately 3 h, total ammonia (T(amm)) concentrations in a live-release vessel at an angling tournament were almost 200 muM. Exposure of fish to 1000 microM T(amm) (a value approximately 80% below the criteria maximum concentration for largemouth bass) caused significant reductions in ventilation rates, and increases in erratic swimming and irregular ventilation. Exposure to 100 microM T(amm) impaired the ability of largemouth bass to recover from exercise relative to fish recovering in fresh water. Therefore, sub-lethal ambient ammonia concentrations cause physiological disturbances that can impair the recovery of largemouth bass from exercise.

This study was intended to discover whether forcing largemouth bass (Micropterus salmoides) to swim at 0.5 body lengths/second following exercise would expedite recovery relative to fish recovered in static water. Exercise resulted in a suite of physiological disturbances for largemouth bass that included a depletion of anaerobic energy stores, an accumulation of lactate, and increased cardiac output. At 1 h following exercise, exhaustively exercised largemouth bass forced to swim exhibited expedited recovery relative to fish in static water, evidenced by lower concentrations of lactate in white muscle, elevated concentrations of phosphocreatine in white muscle, and reduced concentrations of glucose in plasma. By 4 h postexercise, largemouth bass forced to swim during recovery exhibited signs of physiological disturbance that were absent in fish recovered in static water. These signs of disturbance included a loss of osmotically active particles from plasma, elevated lactate in plasma, reductions of phospocreatine in white muscle, and increased cardiac output. These results are discussed in relation to the body of work with salmonid fishes showing physiological benefits to recovering fish in flowing water.

Carbonic anhydrase is a ubiquitous metalloenzyme that catalyzes the reversible hydration/dehydration of carbon dioxide. To date, 16 different CA isozymes have been identified in mammals, and several novel isozymes have also been identified in non-mammalian vertebrates. These isozymes are involved in many physiological processes; however, one of the most important roles is facilitating the transport and subsequent excretion of carbon dioxide. As such, CA isozymes are found at virtually every step of the process, including the metabolic site of CO(2) production (muscle), the circulating red blood cells, and the primary respiratory surface (gills/lungs). This review will examine the structural characteristics that are integral to CAs participation in respiration, as well as highlight the specific roles and tissues that the different CA isozymes are involved in.

Carbonic anhydrase (CA) is a multi-functional enzyme that catalyzes the hydration/dehydration of carbon dioxide. In the red blood cell (rbc), CA is necessary to facilitate the transport of carbon dioxide out of the body. Results from earlier biochemical studies indicate that ancient vertebrates, such as agnathans, possess a low activity rbc CA isozyme, whereas more recently evolved vertebrates, such as teleost fish, possess a high activity isozyme. At present, however, the changes in the molecular structure that have resulted in this large increase in catalytic efficiency are unknown. The objective of the current study was therefore to determine the molecular structure of rbc CA in lampreys and compare it to that of teleosts in an effort to ascertain how this important enzyme became more efficient over evolutionary time. Isolation and sequencing of cytoplasmic CA from rbc and gill showed only a single isozyme of 789 bp (262 amino acids). This isozyme was also found in brain and kidney, with no evidence of additional cytoplasmic CA isozymes in other tissues. Phylogenetic analysis grouped this isozyme closely to vertebrate CA VII, which is ancestral to the rbc isozymes in other vertebrates. Interestingly, active site analysis revealed a structure similar to high activity isozymes. A comparative kinetic analysis of CA from rbc lysates and CA fusion proteins showed that the traditional method of determining the turnover number may not be appropriate for all vertebrate CAs. In contrast to previous evidence, lamprey CA was found to be a high activity isozyme. These results suggest that the critical functional characteristics of rbc CA have been highly conserved throughout vertebrate evolution.

It is well established that the gills of teleost fish contain substantial levels of cytoplasmic carbonic anhydrase (CA), but it is unclear which CA isozyme(s) might be responsible for this activity. The objective of the current study was to determine if branchial CA activity in rainbow trout was the result of a general cytoplasmic CA isozyme, with kinetic properties, tissue distribution and physiological functions distinct from those of the red blood cell (rbc)-specific CA isozyme. Isolation and sequencing of a second trout cytoplasmic CA yielded a 780 bp coding region that was 76% identical with the trout rbc CA (TCAb), although the active sites differed by only 1 amino acid. Interestingly, phylogenetic analyses did not group these two isozymes closely together, suggesting that more fish species may have multiple cytoplasmic CA isozymes. In contrast to TCAb, the second cytoplasmic CA isozyme had a wide tissue distribution with high expression in the gills and brain, and lower expression in many tissues, including the red blood cells. Thus, unlike TCAb, the second isozyme lacks tissue specificity and may be expressed in the cytoplasm of all cells. For this reason, it is referred to hereafter as TCAc (trout cytoplasmic CA). The inhibitor properties of both cytoplasmic isozymes were similar (Ki acetazolamide 1.21+/-0.18 nmol l(-1) and 1.34+/-0.10 nmol l(-1) for TCAc and TCAb, respectively). However, the turnover of TCAb was over three times greater than that of TCAc (30.3+/-5.83 vs 8.90+/-1.95 e4 s(-1), respectively), indicating that the rbc-specific CA isoform was significantly faster than the general cytoplasmic isoform. Induction of anaemia revealed differential expression of the two isozymes in the red blood cell; whereas TCAc mRNA expression was unaffected, TCAb mRNA expression was significantly increased by 30- to 60-fold in anaemic trout.

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzyme's kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.

In order to gain insight into the early evolution of carbonic-anhydrase (CA) isozymes in vertebrates, the main objective of the present study was to determine whether the hearts of an ancient vertebrate species, Petromyzon marinus, possess a membrane-bound CA isozyme. Since a significant amount of CA activity appeared to be strongly associated with the heart membrane fraction after differential centrifugation and washing, further experiments were conducted to examine the inhibitor properties of the CA from the membrane fraction in comparison with lamprey cytoplasmic CA from the red blood cell (rbc) fraction. These experiments showed that the inhibitor properties of the CA from the heart membranes were significantly different from those of the cytoplasmic CA from lamprey rbcs. A final series of experiments showed that the membrane-bound CA in the lamprey heart is not anchored via a glycosylphosphatidylinositol (GPI) linkage. Taken together, the results of these studies indicate that a membrane-bound CA does appear to be present in the hearts of lamprey, but the properties of the membrane-bound CA isozyme in these ancient vertebrates appear to differ from those in more recently evolved groups.

The different isozymes of carbonic anhydrase (CA) have been the subject of intensive study in mammals, but there is still much to be learned about the early evolution of this enzyme in vertebrates. Erythrocyte CA plays an essential role in the respiratory processes of most vertebrates and is probably the most well studied CA isozyme. The available evidence indicates that there has been a progressive increase in the efficiency of erythrocyte CA during the early evolution of vertebrates. There also appears to be a substantial increase in erythrocyte CA activity during development in some species. At the present time, however, the selective pressures that may be influencing the properties of erythrocyte CA during vertebrate evolution and development have not been clearly determined. When the available molecular sequence information is examined, it is evident that the erythrocyte CAs of early vertebrates have active sites that are more similar to those of mammalian CA VII and II, rather than CA I. We can now also begin to examine the phylogenetic relationships between the different rbc CAs in vertebrates, but more CA sequence information is clearly required from different groups of vertebrates before we have a complete picture of the molecular evolution of erythrocyte CA.

Cellular aging in nucleated erythrocytes from lower vertebrates is accompanied by losses in mitochondria but it remains unclear (i) how these losses accrue (ii) if these changes alter energetics and (iii) whether such changes increase the propensity for apoptosis. We addressed these questions using trout erythrocytes that were separated into age classes using inherent differences in buoyant density. The oldest cells showed a profound decline in mtDNA transcripts, due to reductions in both transcription (90% decline in total RNA) and mtDNA copy number (35%). No alterations in the ratio of 16S rRNA to COX I mRNA were detected, nor was there an accumulation of unprocessed mtDNA transcripts. While older cells had reduced basal respiration, there were no changes in mitochondrial enzymes stoichiometries, tissue ATP levels or dinitrophenol-induced (maximal) respiration rates. Apoptosis could not be induced in either whole blood, young or old erythrocytes by pro-oxidants, mitochondrial inhibitors or staurosporine. In contrast, cyclosporin A (CsA) caused caspase 3 activation, DNA laddering and LDH leakage, but only in young cells. Both CsA and a combination of azide, oligomycin and dinitrophenol cause mitochondrial depolarization and caspase 9 activation, but only CsA induced caspase 3 and apoptosis. Caspase inhibitor studies support the conclusion that mitochondrial changes may accompany CsA-induced cell death, but are not essential in its progression. While pifithrin failed to induce cell death, it enhanced the effects of CsA, implicating a role for p53. Collectively, these studies suggest that the mitochondrial changes with aging do not compromise cellular function, although trout erythrocytes can initiate apoptosis by non-mitochondrial pathways.

This study investigates the evolutionary history of vertebrate red blood cell carbonic anhydrase (CA) by characterizing the isozyme properties and nucleotide sequence of an ancient fish, the longnose gar ( Lepisosteus osseus). The inhibitor sensitivities of gar rbc CA closely resembled those for mammalian CA II, as well as those for CAs from more recently evolved fishes. The kinetic properties of gar rbc CA were not closely aligned with either mammalian CA I and CA II, but fit well into an emerging phylogenetic pattern for early vertebrates. Gar rbc CA cDNA was also amplified from mRNA using 5' and 3'-RACE and the open reading frame consisted of 786 bp. This sequence shares approximately 65% identity with the nucleotide and amino acid sequences of both mammalian CA I and CA II. When the amino acid sequences within the active site are compared, gar rbc CA differs from mammalian CA I, CA II and CA VII by 9, 4 and 3 of the 36 amino acids, respectively. Phylogenetic analyses suggest that gar rbc CA diverged before the amniotic CAs (CA I, CA II and CA III), but after CA V and CA VII.

This main purpose of this study was to examine the subcellular distribution and isozyme characteristics of branchial carbonic anhydrase (CA) in Chaenocephalus aceratus, an Antarctic icefish that lacks erythrocytes. The Antarctic fish, Notothenia coriiceps, which possesses erythrocytes, was also studied for comparative purposes. The gills of both species were found to have measurable activity of CA. N. coriiceps also had normal levels of blood CA activity. In contrast, the icefish, C. aceratus, lacked blood CA activity, but was found to possess an endogenous plasma CA inhibitor. The large majority of branchial CA in the gills of these species was located in the cytoplasmic fraction whereas less than 3% was associated with the membrane fraction. In both species, CA from the cytoplasmic gill fraction and membrane fraction differed markedly in terms of their sensitivity to the plasma CA inhibitor from C. aceratus. In addition, treatment with the cleaving enzyme phosphatidylinositol-specific phospholipase C indicated that CA from the branchial membrane fraction of both species is anchored to the membrane via a phosphatidylinositol-glycan linkage. Taken together, these results provide evidence for a CA IV-like isozyme in the gills of Antarctic fish. At present, the functional significance of this membrane-bound CA is unknown, but the relative amount of this isozyme appeared to be greater in the gills of C aceratus, the species that lacked erythrocytes.

The purpose of the present study was to examine the distribution of the anion exchange protein in the sea lamprey. Southern blots showed that genomic DNA of juvenile lampreys possesses several regions that are similar to segments of the AE gene from other vertebrates. However, physiological experiments examining rapid anion fluxes across the red blood cell (rbc) membrane and molecular experiments examining mRNA transcript levels both indicated that the anion exchange protein is absent in sea lamprey rbcs. In contrast, lamprey kidney, skeletal muscle, liver and heart tissue all appeared to possess mRNA transcripts for an AE protein. Further evidence for the presence of an AE protein in kidney tissue was obtained from Western blots. In order to evaluate the impact of the apparent rbc anion exchange limitations, the bicarbonate permeability of lamprey rbcs was also evaluated using mass spectrometry. The bicarbonate permeability of the lamprey rbc membrane was an order of magnitude lower than that of trout rbcs. Taken together, these results indicate that the gene for the AE protein is indeed present in lampreys, but it is not expressed in the rbc. Moreover, the process of CO2 transport in lamprey probably does not involve bicarbonate transport across the rbc membrane.

Teleost fishes, living in fresh water, engage in active ion uptake to maintain ion homeostasis. Current models for NaCl uptake involve Na(+) uptake via an apical amiloride-sensitive epithelial Na(+) channel (ENaC), energized by an apical vacuolar-type proton pump (V-ATPase) or alternatively by an amiloride-sensitive Na(+)/H(+) exchange (NHE) protein, and apical Cl(-) uptake mediated by an electroneutral, SITS-sensitive Cl(-)/HCO(3-) anion-exchange protein. Using non-homologous antibodies, we have determined the cellular distributions of these ion-transport proteins to test the predicted models. Na(+)/K(+)-ATPase was used as a cellular marker for differentiating branchial epithelium mitochondria-rich (MR) cells from pavement cells. In both the freshwater tilapia (Oreochromis mossambicus) and rainbow trout (Oncorhynchus mykiss), V-ATPase and ENaC-like immunoreactivity co-localized to pavement cells, although apical labelling was also found in MR cells in the trout. In the freshwater tilapia, apical anion-exchanger-like immunoreactivity is found in the MR cells. Thus, a freshwater-type MR chloride cell exists in teleost fishes. The NHE-like immunoreactivity is associated with the accessory cell type and with a small population of pavement cells in tilapia.

The effects of cell age on protein synthesis were examined in the nucleated red blood cells of rainbow trout (Oncorhynchus mykiss). Total DNA content was unaffected by cell age, whereas total RNA content in young red blood cells was roughly ten times as high as that in old red blood cells. The mRNA levels for haemoglobin, carbonic anhydrase and the chloride/bicarbonate (Cl(-)/HCO(3)(-)) exchanger were also approximately tenfold higher in young red blood cells. Although young red blood cells synthesized roughly five times more protein under steady-state conditions, total protein concentration was not affected by cell age. Despite large reductions in mRNA levels with red blood cell ageing, the concentrations and/or activities of the respiratory proteins were largely preserved. In contrast, the ability to mount a heat shock response was greatly reduced in older red blood cells. Young red blood cells produced 13 times more heat shock protein 70 mRNA following heat shock and four times more 70 kDa protein after recovery. They also transcribed much more heat shock cognate 71 and heat shock factor mRNA than did older red blood cells under steady-state conditions.

The effects of cell age on metabolism in the nucleated red blood cells of rainbow trout (Oncorhynchus mykiss) were examined. Red blood cells were separated according to age using fixed-angle centrifugation. The mean erythrocyte haemoglobin concentration in old red blood cells was found to be 120 % of that in young red blood cells. In young red blood cells, the activities of the mitochondrial enzymes citrate synthase and cytochrome oxidase were 135-200 %, respectively, of those measured in old red blood cells. The activity of the glycolytic enzyme lactate dehydrogenase in young red blood cells was 170 % of that in old red blood cells, whereas the activity of the glycolytic enzyme pyruvate kinase was not significantly affected by cell age. In addition, young red blood cells consumed over twice as much O(2) and devoted 50 % more O(2) to protein synthesis and the activity of Na(+)/K(+)-ATPase than old red blood cells. Red blood cell age did not significantly affect the rate of lactate production. This study shows that ageing in rainbow trout nucleated red blood cells is accompanied by a significant decline in aerobic energy production and the processes it supports, as well as a corresponding increase in the glycolytic contribution to metabolism.

Carbonic anhydrase (CA) activity in blood and other tissues and red blood cell (rbc) anion exchange were measured in the mud puppy, Necturus maculosus, in order to gain insight into the strategy for CO2 transport used by these neotenic salamanders and to further explore evolutionary relationships between rbc CA activity and anion exchange in nonmammalian vertebrates. CA activity was detectable in all of the tissues examined, but CA activity in blood was much lower than that in most vertebrates. There was no indication, however, that additional CA had been incorporated into the membrane fraction of other tissues to compensate for this low blood CA activity. In further contrast to most other animals, low levels of CA activity were also detectable in mud puppy plasma. Preliminary characterization of the rbc CA indicated that the Type II, fast-turnover enzyme was indeed present, but that there are a very low number of active sites in mud puppy rbc's. Further experiments showed that the rbc's were highly permeable to anions and that the relative rate of anion flux could be inhibited by 4, 4-diisothiocyanostilbene-2,2-disulphonic acid. Thus, the process of CO2 transport in the blood of mud puppies probably involves components of the Jacobs-Stewart cycle, as in most other vertebrates.

The physiological and biochemical signals that induce stress protein (HSP) synthesis remain conjectural. In this study, we used the nucleated red blood cells from rainbow trout, Oncorhynchus mykiss, to address the interaction between energy status and HSP gene expression. Heat shock (25 degrees C) did not significantly affect ATP levels but resulted in an increase in HSP70 mRNA. Hypoxia alone did not induce HSP transcription in these cells despite a significant depression in ATP. Inhibition of oxidative phosphorylation with azide, in the absence of thermal stress, decreased ATP by 56% and increased lactate production by 62% but did not induce HSP gene transcription. Inhibition of oxidative phosphorylation and glycolysis with azide and iodoacetic acid respectively, decreased ATP by 79% and prevented lactate production, but did not induce either HSP70 or HSP30 gene transcription in these cells. This study demonstrates that a reduction in the cellular energy status will not induce stress protein gene transcription in rainbow trout red blood cells and may, in fact, limit induction during extreme metabolic inhibition.

The purpose of this study was to examine the subcellular distribution and isoenzyme characteristics of carbonic anhydrase from the gills and respiratory air bladder of bowfin Amia calva, a primitive air-breathing fish. Separation of subcellular fractions by differential centrifugation revealed that the vast majority of carbonic anhydrase from the gills of bowfin originated from the cytoplasmic fraction. Washing of the gill microsomal pellet also indicated that the carbonic anhydrase originally associated with this pellet was largely due to contamination from the cytoplasmic fraction. Experiments with a carbonic anhydrase inhibitor, sulphanilamide, and the plasma carbonic anhydrase inhibitor from this species confirmed that the bowfin gill probably contains only one carbonic anhydrase isoenzyme which had properties resembling those of CA II. In contrast to the situation in the gills, a relatively large percentage (27%) of the total air bladder carbonic anhydrase was associated with the microsomal fraction. Washing of the air bladder microsomal pellet removed little of the carbonic anhydrase activity, indicating that most of the carbonic anhydrase in the microsomal fraction was associated with the membranes. Like the mammalian pulmonary CA IV isoenzyme, microsomal carbonic anhydrase from the bowfin air bladder was less sensitive to the bowfin plasma carbonic anhydrase inhibitor, sodium dodecylsulphate (SDS) and sulphanilamide than was cytoplasmic carbonic anhydrase from the air bladder. Microsomal carbonic anhydrase from the bowfin air bladder also resembled CA IV in that it appears to be anchored to the membrane via a phosphatidylinositol-glycan linkage which could be cleaved by phosphatidylinositol-specific phospholipase C. Taken together, these results suggest that a membrane-bound carbonic anhydrase isoenzyme resembling mammalian CA IV in terms of inhibition characteristics and membrane attachment is present in the air-breathing organ of one of the most primitive air-breathing vertebrates.

This study examines how the different red blood cell (rbc) characteristics in two lower vertebrates, the phylogenetically primitive hagfish and a more recent teleost, the rainbow trout, influence their strategies for blood CO2 transport. Deoxygenation of the blood resulted in a significant increase in rbc CO2 content in hagfish, but there were no significant changes in the CO2 content of plasma or whole blood under these conditions. In contrast, deoxygenation increased the CO2 content of the rbc, plasma and whole blood in the trout. These results demonstrate that the Haldane effect is much less important for CO2 transport in the hagfish as compared to the trout. The relative importance of the rbc and plasma in blood CO2 transport were roughly similar in hagfish and trout and were very different from that previously documented in another primitive vertebrate, the lamprey. In trout, however, the role of the rbc in CO2 carriage was increased upon the addition of the beta-adrenergic agonist isoproterenol (10(-5) M) to the blood. Taken together, these results and those recently collected for lampreys demonstrate that changes in rbc characteristics during vertebrate evolution have probably resulted in several important transitions in the strategy for blood CO2 transport.

Physiological and immuno-blotting experiments were used to determine whether the red blood cell membrane of a primitive vertebrate, the sea lamprey Petromyzon marinus, contained a counterpart similar to the vertebrate anion exchange protein known as AE1 or band 3. Results of the physiological experiments which measured CO2 production after adding H14CO3- to the extracellular saline, indicated significant transmembrane bicarbonate movement in lamprey blood which unlike that in most vertebrates, was insensitive to inhibition by 4,4' diisothiocyanatostilbene-2,2' disulfonic acid. The present study also showed that lamprey red blood cells possess acetazolamide-sensitive carbonic anhydrase which is an important component of CO2 production by vertebrate red blood cells. Polyclonal immunoglobulins against a 12 amino acid domain in the C-terminus of the mouse AE1 recognized a trout red blood cell membrane protein with a relative molecular mass of 97 kDa, but failed to immunoreact with any membrane proteins from the red blood cells of lamprey. Antibodies against trout AE1 immunoreacted with trout red blood cell membrane proteins of approximately 97 kDa, 200 kDa and > 200 kDa. Interestingly, only a 200-kDa membrane protein from the red blood cells of the primitive lamprey immunoreacted with the trout anti-AE1 immunoglobulin proteins. Therefore, lamprey red blood cells appear to possess an AE1-like protein that may be physiologically different than that in most other vertebrates.

The purpose of this study was to examine the relative importance of some of the factors potentially limiting carbon dioxide (CO2) removal from the white muscle in the rainbow trout following exhaustive exercise. Inhibition of red blood cell carbonic anhydrase (CA) activity by acetazolamide (Az) infusion following exercise caused a large increase in both arterial and muscle PCO2 indicating retention of CO2 in the animal. Despite the increase in muscle PCO2, there was no significant decrease in muscle pH in Az-treated fish, probably due to the relatively high buffer capacity of white muscle. The presence of CA in the plasma had a transient, significant effect on blood PCO2 and pH, but no significant effect on muscle PCO2 or pH. Thus, CA treatment transiently enhances CO2 removal from the animal following exercise, but the extracellular catalysis of the CO2 reactions in the plasma does not appear to have a large impact on CO2 removal from the tissues.

Inhibition of red cell carbonic anhydrase (CA) activity resulted in the rapid development of a respiratory acidosis (0.25 pH depression within 15 min post-injection) in the blood of trout. In the lamprey, however, the onset of the respiratory acidosis was delayed and its magnitude was less (0.18 pH depression at 6 h post-injection). Erythrocyte pH of both species decreased by about 0.12 units by 1 h after CA inhibition. These data, combined with the lack of rapid anion (Cl-/HCO3-) exchange in the red cells of agnathans but not in other lower vertebrates, support the hypotheses that (1) the majority of total CO2 in lamprey is transported within the erythrocyte, and (2) the limiting step in the evolution of a functioning Jacobs-Stewart cycle, and thus the evolution of the common mechanism of systemic CO2 transport in vertebrate blood, was the incorporation of the band-3 anion exchange protein into the membrane of the red cell.

Arterial and venous cannulations were used to examine the characteristics of CO2 transport in pre and post branchial blood both at rest and during recovery from exercise. As in previous studies, exercise caused a marked decrease in the extracellular pH (pHe) in both arterial and venous blood. Except for a transient increase in venous blood immediately following exercise, plasma total CO2 ([CCO 2]pl) and whole blood total CO2 ([CCO 2]wb) decreased in both arterial and venous blood during recovery. Exercise also resulted in an increase in red blood cell total CO2 concentration ([CCO 2]i) and in the partial pressure of CO2 (PCO 2) in both arterial and venous blood. Activation of the adrenergic mechanism at the level of the red blood cell likely contributed to the increases observed in ([CCO 2]i) following exercise. At rest, the majority of the total [CCO 2] carried in arterial and venous blood could be attributed to the plasma, with 2 and 9% carried in the red blood cells, respectively. However, exercise resulted in an increase in the percentage of C02 carried within the red blood cell to 13.5 and 20% in arterial and venous blood, respectively. The total CO2 difference between pre and post branchial blood also increased following exercise suggesting an increase in CO2 excretion.

The separated plasma of gray seals (Halichoerus grypus) has an unusually high non-bicarbonate buffering capacity (beta NB = d[HCO3-]/dpH = -21 Slykes; Slyke = mmol l-1 pH unit-1). To examine whether this represents a trait of mammalian carnivora, or is related instead to deep diving, we examined blood from dog and killer whale, respectively. The beta NB of separated plasma of dog was typically low (-4 to -8 Slykes), as in other mammals, whereas that of the whale (-13.5 Slykes), while significantly higher than dog, was not nearly as high as in seal. As one would theoretically predict of a blood system with exceptionally high extracellular buffering and functional anion exchange across the erythrocyte membrane, there was little difference in the buffer slope between true plasma and whole blood of seal. This appears to be a novel pattern of CO2 carriage in mammals. Erythrocyte ion concentrations were similar to other mammalian carnivora, with intracellular sodium concentration being high and potassium low. The red cell water content was unaffected by changes in oxygen saturation and tended to decrease when extracellular pH decreased below 7.4. Apart from the comparatively elevated Bohr factor, -0.518 as a function of extracellular pH and -0.550 as a function of red blood cell pH, the blood oxygen binding properties were similar to terrestrial mammals of similar size.

Exhaustive exercise in cannulated sea lampreys, Petromyzon marinus, resulted in a marked extracellular acidosis in the arterial blood which had both a respiratory and a metabolic component. Blood CO2 tension (PCO2) returned to control levels within an hour after exercise, but the metabolic acidosis had a somewhat longer time course and the extracellular pH (pHe) did not fully recover until the 4 h recovery sample. The magnitude and duration of the changes in both the plasma lactate concentration and the concentration of metabolic protons were very similar and the maximal proton deficit after exercise was, therefore, only 1.5 mequiv l-1. In contrast to the changes in pHe, there were no significant changes in the erythrocyte pH (pHi) following the exercise period. The regulation of pHi was apparently not adrenergically mediated, however, since addition of catecholamines to lamprey blood in vitro had no significant effect on pHi. In addition, the period of exhaustive exercise in vivo was not associated with any significant changes in the mean cellular hemoglobin concentration. The total carbon dioxide concentration in the arterial whole blood and true plasma were both significantly reduced after exercise, but the total carbon dioxide concentration within the erythrocytes was transiently increased. Finally, there was a marked decrease in the arterial PO2 immediately after exercise, which was associated with a significant reduction in the amount of oxygen bound to hemoglobin; however, within 30 min, these values had both returned to normal. The maintenance of pHi presumably contributes to the regulation of oxygen transport in lampreys and it may be particularly important during the brief period immediately after exercise when oxygen transport is clearly compromised. Although several studies have provided evidence that chloride/bicarbonate exchange limitations may exist in agnathan blood in vitro, the present results demonstrate that the characteristics of carbon dioxide transport and acid-base regulation after exercise in P. marinus are not markedly different from those in other lower vertebrates.

CO2 transport properties of blood were examined in the lamprey Petromyzon marinus and the hagfish Myxine glutinosa. In order to evaluate possible chloride/bicarbonate exchange limitations, experiments were conducted under control conditions and in the presence of an ionophore to permit equilibrium distribution of chloride, bicarbonate, and protons across the erythrocyte membrane. The ionophore, tri-n-propyl tin chloride, markedly altered the CO2 transport properties and apparent nonbicarbonate buffering characteristics of the blood of Petromyzon marinus. In addition, the distributions of protons, bicarbonate and chloride ions across the erythrocyte membrane were very different from each other under control conditions, but became very similar in the presence of the anionic ionophore. The CO2 transport properties of the blood of Myxine glutinosa were not significantly different in the presence of the ionophore. Small but significant changes were observed, however, in erythrocyte pH, chloride concentration and water content in the presence of tri-n-propyl tin chloride. These results demonstrate that chloride/bicarbonate exchange limitations and possibly active transport of protons contribute to the unique CO2 transport properties in the blood of the lamprey, Petromyzon marinus. In the hagfish, Myxine glutinosa, the importance of anion exchange limitations or active proton transport with regard to the CO2 carrying properties of the blood are clearly much less than in the lamprey under the in vitro conditions of this study.

The CO2 transport properties of the blood of the hagfish, Myxine glutinosa are markedly different from those previously demonstrated for the lamprey, Petromyzon marinus. As in most other vertebrates, the majority of the CO2 in Myxine blood is transported in the form of plasma bicarbonate. Erythrocyte bicarbonate does have some access to the plasma in the blood of Myxine although the documented bicarbonate movements were not sensitive to the chloride/bicarbonate exchange inhibitor, 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS). The transmembrane pH gradient in hagfish erythrocytes is also quite different from the gradient previously demonstrated in other agnathans. As in other agnathans, significant differences do occur between the distribution ratios for protons, chloride and bicarbonate ions across the erythrocyte membrane, although the magnitude of these differences is smaller in Myxine. Finally, the absence of pH-dependent changes in erythrocyte water content appears to be a common feature of agnathan erythrocytes.

The effects of different pharmacological agents and incubation media on the intracellular pH and water content of Amphiuma erythrocytes were investigated in vitro. Adrenaline had no significant effect on the intracellular pH or cell water content. DIDS caused an intracellular alkalinization that could be abolished by amiloride, ouabain or removal of sodium from the incubation medium. In addition, amiloride and DIDS both caused a decrease in cell water content. The data indicate that sodium/proton and chloride/bicarbonate exchangers are present on the membrane of Amphiuma erythrocytes and these exchangers are active under steady-state conditions.

In vivo experiments were carried out to determine the effect of forced activity on circulating catecholamine levels, haematocrit, and the pH and water content of erythrocytes in the toad, Bufo marinus. In addition, the effect of the beta-adrenergic agonist isoproterenol on erythrocyte pH and water content was examined in vitro. Forced activity caused a significant decrease in both whole blood and erythrocyte pH, while haematocrit and circulating adrenaline and noradrenaline levels increased. Erythrocyte water content did not change following forced activity. Addition of isoproterenol to toad blood in vitro had no effect on either erythrocyte pH or water content. The apparent absence of beta-adrenergic effects on erythrocyte pH and water content in the toad is in sharp contrast to the response of teleost fish erythrocytes to beta-adrenergic stimulation. The significance of these differences is discussed.

The aquatic urodeles Ambystoma tigrinum and Necturus maculosus responded to hypercapnia quite differently. A. tigrinum, after 2-h exposure to 22 Torr partial pressure of CO2 (PCO2), decreased arterial pH (pHa) from 7.85 to 7.32 and increased arterial pressure of CO2 (PaCO2) to 26 Torr. Plasma [HCO-3] [( HCO-3]pl) remained constant at about 17 mM. Prolonged exposure (24 h) led to a 26% extracellular compensation as pHa rose to 7.46 while [HCO-3]pl increased to 24 mM. Plasma [K+] increased and [Cl-] decreased while [Na+] remained unchanged. Recovery in normocapnic water reversed these changes. N. maculosus did not display similar compensatory changes. Two-hour exposure to 17 Torr PCO2 resulted in a decline of pHa from 7.66 to 7.24, which was not compensated (pHa = 7.19) after 24 h. There were no significant changes in plasma [Na+], [K+], [Cl-], or [HCO-3]. The pHa decline reversed after recovery in normocapnic water, however. The fact that compensation for hypercapnic in A. tigrinum was accompanied by changes in Cl- and K+ concentrations may indicate the participation of epithelial transport mechanisms involving these ions in acid-base balance.

Ureters were cannulated in specimens of Bufo marinus (L.) in order to partition the regulatory contributions of the kidney and skin. The in vivo roles of the kidney, skin and internal calcareous deposits in the response of these animals to chronic hypercapnia were then evaluated. There was no compensatory adjustment by the skin and only a minimal regulatory response by the kidney. Major adjustments which have been attributed to combined skin and urinary tract in previous studies must therefore come from the urinary bladder. Removal of the bladder as a regulatory site in these animals completely eliminated the compensatory elevation of HCO3- in the extracellular fluid. Mobilization of internal calcareous deposits as a source of HCO3- was found to contribute 50% of the compensatory response of these animals during hypercapnia.

1. Immediately following burst swimming, the oxygen consumption of rainbow trout increased by 71%, carbon dioxide excretion by 104% and the respiratory exchange ratio by 17%. 80 min after burst swimming all of these parameters had returned to levels which were not significantly different from control values. 2. Infusion of adrenaline into resting fish had no significant effect on oxygen consumption or carbon dioxide excretion and therefore there was no significant change in the respiratory exchange ratio. 3. This infusion of adrenaline caused a significant elevation in the red blood cell pH which was still present 80 min later. 4. The present results contrast with those of van den Thillart, Randall & Lin (1983), who demonstrated carbon dioxide retention after burst swimming. While it is possible that catecholamines may inhibit bicarbonate flux through the red blood cell, our experiments indicate that this inhibition would not result in detectable changes in carbon dioxide excretion or, therefore, in the respiratory exchange ratio.

beta-Adrenergic stimulation of salmonid red cells results in a rapid decrease (within 5 min) in the nucleotide triphosphate:haemoglobin ratio (NTP:Hb), which is thereafter maintained at a constant level, presumably through increased ATP turnover via matched aerobic metabolism and energy-consuming processes. Addition of the beta-adrenergic agonist isoproterenol to rainbow trout red cells in vitro leads to a rise in intracellular pH (pHi), a corresponding decrease in extracellular pH (pHe) and an increase in red cell oxygen consumption (MO2). Moreover, the extent to which red cell pHi is maintained constant in the face of an acute extracellular acidosis in vitro or in vivo is proportional to the adrenergically stimulated increase in red cell MO2. In the absence of oxygen, these red cells remain capable of pH regulation, but cannot maintain NTP:Hb constant. As a result, membrane and metabolic functions become uncoupled in the stimulated deoxygenated cells.