Control and consequences of adrenergic activation of red blood cell Na+/H+ exchange on blood oxygen and carbon dioxide transport in fish.

The catecholamines, adrenaline and noradrenaline, are released into the circulation of fish during a variety of physical and environmental disturbances that share the common feature of a requirement for enhanced blood oxygen transport. Indeed, the dominant factor controlling the mobilization of catecholamines from chromaffin tissue is a depression of blood oxygen content usually coinciding with a reduction of hemoglobin-O2 (Hb-O2) binding to 50-60% saturation. The elevation of plasma catecholamine levels, under such conditions, activates a beta-adrenergic cyclic AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness AMP-dependent Na+/H+ exchanger on the red blood cell (rbc) membrane. The adrenergic responsiveness of the rbc Na+/H+ exchanger to catecholamines varies both within and between species. Such inter- and intra-specific differences may reflect, in part, the availability of cell surface beta-adrenoceptors that are functionally coupled to adenylate cyclase. The activation of rbc Na+/H+ exchange and the accompanying profound adjustments of intracellular and extracellular acid-base status, nucleoside triphosphate (NTP) levels, and cooperativity of Hb-O2 binding have important consequences on both O2 and CO2 transfer and transport in the blood that vary markedly at the sites of oxygenation (the gill) and deoxygenation (the tissues) thereby enabling simultaneous amelioration of O2 loading and unloading. At the gill, oxygen transfer is enhanced owing to increases in Hb-O2 affinity and capacity while at the tissues, oxygen delivery is facilitated by a reduction of Hb-O2 affinity. This reduction in affinity at the tissues is a consequence of the combined effects of increased cooperativity of Hb-O2 binding and a rise in venous PCO2 (PvCO2) caused by the titration of HCO3- by H+ extruded by the rbc Na+/H+ exchanger. This elevation of PvCO2 may contribute to the rise in arterial PCO2 (PaCO2) observed after adrenergic activation of rbc Na+/H+ exchange that is caused primarily by impairment of rbc CO2 excretion related to modification of the intracellular acid-base status.     

Nucleoside uptake by red blood cells from a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), is mediated by a nitrobenzylthioinosine-insensitive transport system

Red blood cells from the Pacific hagfish (Eptatretus stouti) were found to possess a facilitated diffusion nucleoside transport system insensitive to inhibition by the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). Uridine uptake by this route was saturable (apparent Km 0.14 mM; Vmax 2 mmol/l cells per h at 10 degrees C), inhibited by inosine and adenosine, and blocked both by the vasodilator dipyridamole and by the thiol-reactive agent p-chloromercuriphenylsulphonate. The properties of this carrier resemble closely those of NBMPR-insensitive nucleoside transport systems in some mammalian neoplastic cell lines and in rat red cells. The presence of this type of carrier in a primitive vertebrate suggests that such transporters have a broad biological distribution and that they pre-date or arose at an early stage of vertebrate evolution.     

Acrosome reaction in spermatozoa of the hagfish Eptatretus burgeri (Agnatha)

Fertilization of the hagfish or myxiniformes, a member of the most primitive vertebrate group and an animal of phylogenic interest, is unknown. Here, induction of an acrosome reaction for spermatozoa in the hagfish, Eptatretus burgeri, was successfully achieved by treatment of mature spermatozoa with ionomycin and excess Ca2+. The spermatozoon produced an acrosomal process that elongated from the apex of the long sperm head. The reaction bears resemblance to that of invertebrate spermatozoa rather than that of vertebrate spermatozoa. The result provides insights into the phylogenetical changes that have occurred in this sperm reaction.     

Bicarbonate permeability and immunological evidence for an anion exchanger-like protein in the red blood cells of the sea lamprey,Petromyzon marinus

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 H¹⁴CO ₃ ^- 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.     

Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

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 enzymes 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.Abbreviations Az acetazolamide - CA carbonic anhydrase - MP maximum parsimony - NJ neighbour joining - RACE rapid amplification of cDNA ends - rbc red blood cellCommunicated by L.C.-H. Wang     

Carbonic anhydrase and red blood cell anion exchange in the neotenic aquatic salamander, Necturus maculosus

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.     

Mineral content and blood parameters of dying brown trout (Salmo trutta L.) exposed to acid and aluminum in soft water.

1. Whole body mineral content and blood parameters were monitored in dying brown trout exposed to acid (pH 4.5) and aluminium (12 mumol l-1) at low external calcium concentration (20 mumol l-1). 2. Fish surviving the treatment had elevated haematocrit, low plasma sodium levels, and low whole body sodium and potassium content. 3. Dying trout had haematocrit and plasma sodium levels similar to treatment survivors, but whole body mineral content was unaffected and moisture content increased. 4. Death was probably caused by redistribution of fluid from the extracellular compartment to the intracellular compartment.     

cDNA cloning of a mannose-binding lectin-associated serine protease (MASP) gene from hagfish (Eptatretus burgeri)

Hagfish, agnathan cyclostome, is the most primitive extant vertebrate and its complement (C) system seems to be a primordial system in comparison with a well-developed C system in gnathostome vertebrates. From a phylogenic perspective of defense mechanisms, we have isolated complement C3 from the serum of hagfish (Eptatretus burgeri). In this study, we first attempted to identify a hagfish Bf or C2 as a C3 convertase by RT-PCR using degenerative primers designed on the basis of the conserved amino acid stretches among the several kinds of serine proteases. Contrary to our expectation, homology search of cloned RT-PCR product suggested that there was a partial cDNA encoding the homologue of neither Bf nor C2 but a mannose-binding lectin-associated serine protease (MASP). Analyses of a full-length cDNA clone isolated from a hagfish liver cDNA library by using the partial cDNA as a probe indicated that this cDNA encoded hagfish MASP 1. This evidence strongly suggests that the hagfish defends itself against pathogens at least by the complement system composed of lectin pathway.     

Fish red blood cell carbon dioxide transport in vitro: A comparative study

Red blood cell (rbc) carbon dioxide transport was examined in vitro in three teleosts (Oncorhynchus mykiss, Anguilla anguilla, Scophthalmus maximus) and an elasmobranch (Scyliorhinus canicula) using a radioisotopic assay that measures the net conversion of plasma HCO₃⁻ to CO₂. The experiments were designed to compare the intrinsic rates of rbc CO₂ excretion and the impact of haemoglobin oxygenation/deoxygenation among the species.Under conditions simulating in vivo levels of plasma HCO₃⁻ and natural haematocrits, the rate of whole blood CO₂ excretion varied between 14.0 μmol ml⁻¹ h⁻¹ (S. canicula) and 17.6 μmol ml⁻¹ h⁻¹ (O. mykiss). The rate of CO₂ excretion in separated plasma was significantly greater in the dogfish, S. canicula. The contribution of the rbc to overall whole blood CO₂ excretion was low in the dogfish (46 ± 6%) compared to the teleosts (trout, 71 ± 4%; turbot, 64 ± 5%; eel, 55 ± 3%).To eliminate the naturally occurring differences in haematocrit and plasma [HCO₃⁻] as inter-specific variables, the rates of whole blood CO₂ excretion were determined in blood that had been resuspended to constant [HCO₃⁻] (5 mmol⁻¹) and haematocrit (20%) in appropriate teleost and elasmobranch Ringer solutions. Under such normalized conditions, the rate of whole blood CO₂ excretion was significantly higher in the turbot (22.4 ± 1.3 μmol ml⁻¹ h⁻¹) in comparison to the other species (16.4–18.4 μmol ml⁻¹ h⁻¹) and thus revealed a greater intrinsic rate of rbc CO₂ excretion in the turbot.To study the contribution of Bohr protons, the rates of whole blood CO₂ excretion were assessed in blood subjected to rapid oxygenation during the initial phase of the 3 min assay period. Rapid oxygenation significantly enhanced the rate of CO₂ excretion in the teleosts but not in the elasmobranch. The extent of the increase provided by the rapid oxygenation of haemoglobin was a linear function of the extent of the Haldane effect, as quantified in each species from in vitro CO₂ dissociation (combining) curves. Under steady-state conditions, deoxygenated blood exhibited greater rates of CO₂ excretion than oxygenated blood in the teleosts but not in the elasmobranch. As a consequence of the Haldane effect, rbc intracellular pH was increased in the teleosts by deoxygenation but was unaltered in the elasmobranch.The results, by extrapolation, suggest that the rates of CO₂ excretion in vivo are influenced by the magnitude of the Haldane effect and the extent of haemoglobin oxygenation during gill transit in addition to the intrinsic rate at which the rbc converts plasma HCO₃⁻ to CO₂.     

Recovery from blood alkalosis in the Pacific hagfish (Eptatretus stoutii): involvement of gill V-H+-ATPase and Na+/K+-ATPase

To investigate the base secretory mechanisms in the Pacific hagfish (Eptatretus stoutii), we injected animals with NaHCO3 into the subcutaneous sinus. In the first series of experiments, hagfish were injected with 6000 micromol kg(-1) NaHCO3 (base-infused hagfish, BIH) or NaCl (controls). Blood pH increased significantly 1 h after injection in BIH (8.05+/-0.05 vs. 7.82+/-0.03 pH units), but returned to control values by t=6 h. Plasma total CO2 (TCO2) followed the same pattern. Immunolabeled sections revealed that Na+/K+-ATPase and V-H+-ATPase were usually located in the same cells. Western blotting revealed that the abundance of both proteins remained unchanged in whole gill homogenates and in a fraction enriched in cell membranes 6 h after the injections. The second experimental series was to induce long-term alkalosis by serially injecting 6000 micromol kg(-1) NaHCO3 every 6 h for 24 h. Blood pH completely recovered from the base loads within 6 h after each injection. Moreover, plasma TCO2 was not elevated 3 h after the second infusion, suggesting that HCO3(-) secreting mechanisms had been upregulated by that time. Na+/K+-ATPase and V-H+-ATPase cellular localizations did not change in the 24 h base infusion protocol. Na+/K+-ATPase abundance was similar in gill homogenates from fish from both treatments. However, Na+/K+-ATPase abundance in the membrane fraction was significantly lower in BIH, while V-H+-ATPase was greater both in whole gill and membrane fractions. Our results suggest that differential insertion of V-H+-ATPase and Na+/K+-ATPase into the basolateral membrane is involved in recovering from alkalotic stress in hagfish.     

Plasma cells in adult Atlantic hagfish,Myxine glutinosa

Summary Hagfishes, the most primitive vertebrates, are of special interest for the evolution of immune responses. Eptatretus stoutii, the Pacific hagfish, is able to mount cellular and humoral immune responses but all attempts to demonstrate in them the presence of plasma cells have failed. In the present study we demonstrate for the first time plasma cells identifiable by ultrastructural criteria in the pronephros, a primitive lymphohaemopoietic organ, of Myxine glutinosa, the Atlantic hagfish.     

Responses of the red blood cells from two high-energy-demand teleosts, yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis), to catecholamines

In fishes, catecholamines increase red blood cell intracellular pH through stimulation of a sodium/proton (Na⁺/H⁺) antiporter. This response can counteract potential reductions in blood O₂ carrying capacity (due to Bohr and Root effects) when plasma pH and intracellular pH decrease during hypoxia, hypercapnia, or following exhaustive exercise. Tuna physiology and behavior dictate exceptionally high rates of O₂ delivery to the tissues often under adverse conditions, but especially during recovery from exhaustive exercise when plasma pH may be reduced by as much as 0.4 pH units. We hypothesize that blood O₂ transport during periods of metabolic acidosis could be especially critical in tunas and the response of rbc to catecholamines elevated to an extreme. We therefore investigated the in vitro response of red blood cells from yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis) to catecholamines. Tuna red blood cells had a typical response to catecholamines, indicated by a rapid decrease in plasma pH. Amiloride reduced the response, whereas 4,4′diisothiocyanatostilbene-2,2′-disulphonic acid enhanced both the decrease in plasma pH and the increase in intracellular pH. Changes in plasma [Na⁺], [Cl⁻], and [K⁺] were consistent with the hypothesis that tuna red blood cells have a Na⁺/H⁺ antiporter similar to that described for other teleost red blood cells. Red blood cells from both tuna species were more responsive to noradrenaline than adrenaline. At identical catecholamine concentrations, the decrease in plasma pH was greater in skipjack tuna blood, the more active of the two tuna species. Based on changes in plasma pH, the response of red blood cells to catecholamines from both tuna species was less than that of rainbow trout (Oncorhynchus mykiss) red blood cells, but greater than that of cod (Gadus morhua) red blood cells. Noradrenaline had no measurable influence on the O₂ affinity of skipjack tuna blood and only slightly increased the O₂ affinity of yellowfin tuna blood. Our results, therefore, do not support our original hypothesis. The catecholamine response of red blood cells from high-energy-demand teleosts (i.e., tunas) is not enhanced compared to other teleosts. There are data on changes in cardio-respiratory function in tunas caused by acute hypoxia and modest increases in activity, but there are no data on the changes in cardio-respiratory function in tunas accompanying the large increases in metabolic rate seen during recovery from exhaustive exercise. However, we conclude that during those instances where high rates of O₂ delivery to the tissues are needed, tunas' ability to increase cardiac output, ventilation volume, blood O₂ carrying capacity, and effective respiratory (i.e., gill) surface area are probably more important than are the responses of red blood cells to catecholamines. We also use our data to investigate the extent of the Haldane effect and its relationship to blood O₂ and CO₂ transport in yellowfin tuna. Yellowfin tuna blood shows a large Haldane effect; intracellular pH increases 0.20 units during oxygenation. The largest change in intracellular pH occurs between 40–100% O₂ saturation, indicating that yellowfin tuna, like other teleosts, fully exploit the Haldane effect over the normal physiological range of blood O₂ saturation. Accepted: 27 March 1998     

Comparative studies of bile salts. Myxinol disulphate, the principal bile salt of hagfish (Myxinidae)

1. The principal bile salt, myxinol disulphate, of two hagfish species, Eptatretus stoutii and Myxine glutinosa, has been shown by chemical methods and by optical-rotatory and mass-spectral considerations to be probably the C-3,27-disulphate ester of 3beta,7alpha,16alpha,27-tetrahydroxy-5beta-cholestane. 2. Myxine liver sterols were about half ;free' and half esterified: cholesterol only was identified. 3. The chemical nature of myxinol is what might be expected for the bile alcohol of a very primitive vertebrate.     

Calculation of whole blood CO2 content

Currently used methods for calculating whole blood CO2 content from calculated plasma content, measured blood pH, hemoglobin concentration ([Hb]), and O2 saturation yield materially different results. In this study the constants of the fundamental equations relating blood CO2 content to plasma content have been reevaluated. An iterative computer technique was used to empirically derive appropriate constants from data obtained from nine healthy male subjects at rest and at several exercise work loads. A calculation was derived that fitted the data well [difference 0.02 +/- 1.19 ml/100 (SD) ml, r = 0.98] blood CCO2 = plasma CCO2 (Formula: see text) where plasma CCO2 = 2.226.s.plasma PCO2.(1 + 10pH-pK'), CCO2 is CO2 content, SO2 is O2 saturation, s is the plasma CO2 solubility coefficient, and pK' is the apparent pK [s and pK' are from the equations of Kelman (Respir. Physiol. 3: 111-115, 1967)].     

Cell surface expression of the endoplasmic reticular heat shock protein gp96 is phylogenetically conserved.

In mammals, the heat shock protein gp96 complexed to antigenic peptides elicits T cell adaptive immunity. By itself, however, gp96 can evoke responses that are characteristic of innate immunity. Interestingly, this protein, which resides in the endoplasmic reticulum, is expressed on the surface of certain mouse tumor cells. Given that heat shock proteins are highly conserved, we investigated whether the cell surface expression of gp96 is also evolutionarily conserved. Our data reveal that gp96, most likely containing the endoplasmic reticulum retention motif (KDEL), is expressed on the surface of three different Xenopus lymphoid tumor cell lines, each derived from a different spontaneously arising thymic tumor. Levels of expression differ among the tumor lines tested, with more immunogenic tumors expressing greater amounts of surface gp96. Moreover, a high level of gp96 surface expression is detectable on a subset of Xenopus normal nontransformed splenic lymphocytes (mainly surface IgM+ B cells) but not on other normal cells, including macrophages and nucleated erythrocytes. Surface expression of a gp96 protein homologue occurs also on some, but not all, T and B cell clones derived from peripheral blood cells of the channel catfish, as well as on lymphocyte-like cells, but not on erythrocytes from the hagfish, a primitive agnathan vertebrate lacking markers of an adaptive immune system. gp96 is actively directed to and retained on the plasma membrane of Xenopus lymphocytes and tumor cells and hagfish lymphocyte-like cells by a process that requires vesicular transport. This selective surface expression of gp96 on some immune cells from different vertebrate classes is consistent with an ancestral immunological role of gp96 as danger-signaling molecule.     

Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells

Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.     

The effects of endothelin-1 on the cardiorespiratory physiology of the freshwater trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias).

The aim of the present study was to evaluate the effects of endothelin-l-elicited cardiovascular events on respiratory gas transfer in the freshwater rainbow trout (Oncorhynchus mykiss) and the marine dogfish (Squalus acanthias). In both species, endothelin-1 (666 pmol kg(-1)) caused a rapid (within 4 min) reduction (ca. 30-50 mmHg) in arterial blood partial pressure of O2. The effects of endothelin-1 on arterial blood partial pressure of CO2 were not synchronised with the changes in O2 partial pressure and the responses were markedly different in trout and dogfish. In trout, arterial CO2 partial pressure was increased transiently by approximately 1.0 mmHg but the onset of the response was delayed and occurred 12 min after endothelin-1 injection. In contrast, CO2 partial pressure remained more-or-less constant in dogfish after injection of endothelin-1 and was increased only slightly (approximately 0.1 mmHg) after 60 min. Pre-treatment of trout with bovine carbonic anhydrase (5 mg ml(-1)) eliminated the increase in CO2 partial pressure that was normally observed after endothelin-1 injection. In both species, endothelin-1 injection caused a decrease in arterial blood pH that mirrored the changes in CO2 partial pressure. Endothelin-1 injection was associated with transient (trout) or persistent (dogfish) hyperventilation as indicated by pronounced increases in breathing frequency and amplitude. In trout, arterial blood pressure remained constant or was decreased slightly and was accompanied by a transient increase in systemic resistance, and a temporary reduction in cardiac output. The decrease in cardiac output was caused solely by a reduction in cardiac frequency; cardiac stroke volume was unaffected. In dogfish, arterial blood pressure was lowered by approximately 10 mmHg at 6-10 min after endothelin-1 injection but then was rapidly restored to pre-injection levels. The decrease in arterial blood pressure reflected an increase in branchial vascular resistance (as determined using in situ perfused gill preparations) that was accompanied by simultaneous decreases in systemic resistance and cardiac output. Cardiac frequency and stroke volume were reduced by endothelin-1 injection and thus both variables contributed to the changes in cardiac output. We conclude that the net consequences of endothelin-1 on arterial blood gases result from the opposing effects of reduced gill functional surface area (caused by vasoconstriction) and an increase in blood residence time within the gill (caused by decreased cardiac output.