Different red blood cell characteristics in a primitive agnathan (M. glutinosa) and a more recent teleost (O. mykiss) influence their strategies for blood CO2 transport

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.     

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.     

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     

Characterization of erythrocyte carbonic anhydrase in an ancient fish,the longnose gar ( Lepisosteus osseus)

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.     

Comparative physiology and molecular evolution of carbonic anhydrase in the erythrocytes of early vertebrates

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.     

Analysis of abnormalities of capillary CO2 exchange in vivo

Capillary CO2 exchange in vivo is affected by several interdependent reactions and transport processes. A mathematical model that includes all the significant chemical and transport events that are presumed to occur during capillary gas exchange has been used to investigate the effect of inhibition of 1) erythrocyte HCO(3-)-Cl- exchange, 2) lung carbonic anhydrase (CA) activity with access to plasma, and 3) erythrocyte CA activity on overall pulmonary CO2 excretion (VCO2) during rest and moderate exercise. Any decrement in VCO2 due to inhibition of HCO(3-)-Cl- exchange and/or CA activity, should result in compensatory alterations in cardiac output and/or an increase in the mixed venous blood-to-alveolar PCO2 gradient [(delta PCO2)V-A] to restore steady-state VCO2. Our computations show that complete inhibition of erythrocyte anion exchange would require a compensatory increment in cardiac output of approximately 30-40% or an increase in (delta PCO2)V-A from 6 to 8.3 Torr at rest and from 12 to 15.6 Torr during moderate exercise, if lung CA activity is intact. In the absence of availability of lung CA activity to plasma, the necessary (delta PCO2)V-A is 10.5 Torr at rest and 19.5 Torr during moderate exercise. Complete inhibition of lung and erythrocyte CA activity is predicted to require (delta PCO2)V-A of 39.1 Torr at rest and 74.2 Torr during moderate exercise. These results suggest that HCO(3-)-Cl- exchange might not be vital to maintenance of CO2 transfer and perhaps has a more important role in minimizing the changes in plasma pH associated with microvascular gas exchange in vivo.     

Chloride--bicarbonate exchange in red blood cells: physiology of transport and chemical modification of binding sites

About 80% of the CO2 formed by metabolism is transported from tissues to lungs as bicarbonate ions in the water phases of red cells and plasma. The catalysed hydration of CO2 to bicarbonate takes place in the erythrocytes but most of the bicarbonate thus formed must be exchanged with extracellular chloride to make full use of the carbon dioxide transporting capacity of the blood. The anion transport capacity of the red cell membrane is among the largest ionic transport capacities of any biological membrane. Exchange diffusion of chloride and bicarbonate is nevertheless a rate-limiting step for the transfer of CO2 from tissues to lungs. Measurements of chloride and bicarbonate self-exchange form the basis for calculations that demonstrate that the ionic exchange processes cannot run to complete equilibration at capillary transit times less than 0.5 s. The anion exchange diffusion is mediated by a large transmembrane protein constituting almost 30% of the total membrane protein. The kinetics of exchange diffusion must depend on conformational changes of the protein molecule, associated with the binding and subsequent translocation of the transported anion. We have characterized the nature of anion-binding sites facing the extracellular medium by acid-base titration of the transport function and modification of the transport protein in situ with group-specific amino acid reagents. Anion binding and translocation depend on the integrity and the degree of protonation of two sets of exofacial groups with apparent pK values of 12 and 5, respectively. From the chemical reactivities towards amino acid reagents it appears that the groups whose pK = 12 are guanidino groups of arginyl residues, while the groups whose pK = 5 are likely to be carboxylates of glutamic or aspartic acid. Our studies suggest that the characteristics of anion recognition sites in water-soluble proteins and in the integral transport proteins are closely related.     

海洋浮游藻类无机碳利用机理的研究

为了认识海洋浮游藻类在碳充足和碳受限条件下对水体中溶解无机碳(DIC)的利用方式与可能机理,对13种海洋浮游藻类在不同pH和CO2浓度及不同DIC条件下细胞外碳酸酐酶(CA)的活性进行了分析测定.结果显示:13种藻中,只有Amphidinium carterae和Prorocentrum minimum在碳充足条件下具细胞外CA活性.Melosira sp.、Phaeodactylum tricornutum、Skeletonema costatum、Thalassiosira rotula、Emiliania huxleyi和Pleurochrysis carterae则在碳受限条件下才具细胞外CA活性.Chaetoceros compressus、Glenodinium foliaceum、Coccolithus pelagicus、 Gephrocapsa oceanica和Heterosigma akashiwo即使在碳受限条件下也未检测到细胞外CA活性.应用封闭系统中pH漂移技术和阴离子交换抑制剂4′4′-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS)等的研究表明,Coc. pelagicus和G. oceanica可通过阴离子交换机制进行HCO-3的直接利用.H. akashiwo没有潜在的HCO-3直接利用或细胞外CA催化的HCO-3利用.     

Characterization of carbonic anhydrase and anion exchange in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish

The purpose of this study was to investigate the characteristics of carbonic anhydrase (CA) and the Cl⁻/HCO₃⁻ exchanger (Band 3; AE1) in the erythrocytes of bowfin (Amia calva), a primitive air-breathing fish, in order to further understand the strategies of blood CO₂ transport in lower vertebrates and gain insights into the evolution of the vertebrate erythrocyte proteins, CA and Band 3. A significant amount of CA activity was measured in the erythrocytes of bowfin (70 mmol CO₂ min⁻¹ ml⁻¹), although it appeared to be lower than that in the erythrocytes of teleost fish. The turnover number (K_cat) of bowfin erythrocyte CA was intermediate between that of the slow type I CA isozyme in agnathans and elasmobranchs and the fast type II CA in the erythrocytes of the more recent teleost fishes, but the inhibition properties of bowfin erythrocyte CA were similar to the fast mammalian CA isozyme, CA II. In contrast to previous findings, a plasma CA inhibitor was found to be present in the blood of bowfin. Bowfin erythrocytes were also found to possess a high rate of Cl⁻/HCO₃⁻ exchange (6 nmol HCO₃⁻ s⁻¹ cm⁻²) that was sensitive to DIDS. Visualization of erythrocyte membrane proteins by SDS-PAGE revealed a major band in the 100 kDa range for the trout, which would be consistent with the anion exchanger. In contrast, the closest major band for the membranes of bowfin erythrocytes was around the 140 kDa range. Taken together, these results suggest that the strategy for blood CO₂ transport in bowfin is probably similar to that in most other vertebrates despite several unique characteristics of erythrocyte CA and Band 3 in these primitive fish.     

Band 3 protein function in teleost fish erythrocytes: effect of oxygenation-deoxygenation

During vertebrate evolution, structural changes in red blood cells (RBC) and hemoglobin (Hb), have probably resulted in the importance of blood carbon dioxide transport. The chloride/bicarbonate exchange across the RBC membrane, which is an integral part of the blood CO(2) transport process in vertebrates, has been examined on two different species of teleost fish, Euthynnus alletteratus and Thunnus thynnus, at several oxygenation states of erythrocyte HOS (high-oxygenation state, about 90 % of saturation) and LOS (low-oxygenation state, about 15 % of saturation). The results were compared with those observed in human RBC under the same experimental conditions and with the chicken (Gallus gallus) erythrocytes, which have particular modifications at the N-terminus of the band 3 protein (B3). In fish the kinetic measurements have shown a different anion transport in several oxygenation states of erythrocytes, indicating that also at lower levels of vertebrate evolution there exists a modulation of the anionic flow affected by oxygen. The functional correlation of anion transport to changes of parts of the hemoglobin sequence responsible for alterations in the interactions with the cytoplasmic domain of band 3 protein (cdb3) allowed us to suggest a hypothesis about fish physiology. The highest values of kinetic measurements observed in fish have been attributed to the metabolic need of the RBC in response to the removal of CO(2) that in teleosts is also of endogenous origin.     

Evidence for a membrane-bound carbonic anhydrase in the heart of an ancient vertebrate,the sea lamprey (<Emphasis Type="Italic">Petromyzon marinus</Emphasis>)

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.Abbreviations Az acetazolamide - CA carbonic anhydrase - GPI glycosylphosphatidylinositol - PI-PLC phosphatidylinositol specific phospholipase C - Rbc red blood cell     

The importance of a single amino acid substitution in reduced red blood cell carbonic anhydrase function of early-diverging fish

In most vertebrates, red blood cell carbonic anhydrase (RBC CA) plays a critical role in carbon dioxide (CO₂) transport and excretion across epithelial tissues. Many early-diverging fishes (e.g., hagfish and chondrichthyans) are unique in possessing plasma-accessible membrane-bound CA-IV in the gills, allowing some CO₂ excretion to occur without involvement from the RBCs. However, implications of this on RBC CA function are unclear. Through homology cloning techniques, we identified the putative protein sequences for RBC CA from nine early-diverging species. In all cases, these sequences contained a modification of the proton shuttle residue His-64, and activity measurements from three early-diverging fish demonstrated significantly reduced CA activity. Site-directed mutagenesis was used to restore the His-64 proton shuttle, which significantly increased RBC CA activity, clearly illustrating the functional significance of His-64 in fish red blood cell CA activity. Bayesian analyses of 55 vertebrate cytoplasmic CA isozymes suggested that independent evolutionary events led to the modification of His-64 and thus reduced CA activity in hagfish and chondrichthyans. Additionally, in early-diverging fish that possess branchial CA-IV, there is an absence of His-64 in RBC CAs and the absence of the Root effect [where a reduction in pH reduces hemoglobin’s capacity to bind with oxygen (O₂)]. Taken together, these data indicate that low-activity RBC CA may be present in all fish with branchial CA-IV, and that the high-activity RBC CA seen in most teleosts may have evolved in conjunction with enhanced hemoglobin pH sensitivity.

     

Molecular mapping of human band 3 anion transport regions using synthetic peptides

Band 3 is a ubiquitous membrane transport protein found in Golgi, mitochondrial, nuclear, and cell membranes. It is the most heavily used anion transport system in the body because it is responsible for CO2 exchange in all tissues and organs and for acid-base balance. The anion transport regions are mapped along the band 3 molecule using synthetic peptides (pep) from extracellular regions of band 3 and/or suspected anion transport regions. Assays include anion transport/inhibition and immunoblotting with anti-idiotypic antibodies to a transport inhibitor. Results indicate that anion binding/transport regions of band 3 reside within residues 549-594, (588-594 being the most active) and 804-839 (822-839 being the most active), and 869-883. Pep-COOH (residues 812-827), which is part of senescent cell antigen, is an anion binding site with most of the activity localized to residues 813-818 (the six amino acids on the amino side of pep-COOH). The stilbene disulfonate inhibitors of transport bind to peptide 812-830, and possibly peptides 788-805 and 800-818, as determined with anti-idiotypic antibodies. Residues 538-554, which have been reported to be a transport segment of band 3, do not bind sulfate. Band 3 external loops containing residues 539-553 and 812-830, and internal segments containing residues 588-594 and 869-883, are in close spacial proximity in the membrane. The contribution of lysine and/or arginine to anion transport is examined by synthesizing peptides in which glycines or arginines are substituted for lysines or arginines. Lysines can contribute to anion binding but are not required.     

Effects of membrane partitioning and other physical chemical properties on the apparent potency of "membrane active" compounds evaluated using red blood cell lysis assays

The membrane-destabilizing properties of Amphotericin B and Zwittergent were used as benchmark compounds for examining in detail their membrane-altering effects in a series of human red blood cell lysis assays. The procedures included examining dose responses and the effects of different cell concentrations on potency in rbc lysis assays. In order to enhance detection of subtle membrane effects, we also used a range of NaCl concentrations to osmotically stress the rbc's. Using the benchmark compounds, a set of conditions was developed for examination of subtle membrane effects that may be applied to series of compounds with suspected membrane-perturbation activity. A group of experiments was defined that allow detection of the most important membrane-modifying behaviors among a diverse group of compounds. From an initial screen of bacterial growth inhibition over 150 compounds were examined for membrane-altering properties using the limited experimental protocols developed from the benchmark compounds. Several dose-response patterns were observed as useful for classifying compounds based on their tendency to alter membrane integrity and to partition into the lipids of membranes, as well as their propensity to form aggregates or precipitates. The methods may prove generally useful for distinguishing compounds whose primary activity is membrane destabilization from more interesting and useful pharmacological mechanisms of action.     

Anion exchange in the giant erythrocytes of African lungfish

Carbon dioxide transport in African lungfish Protopterus aethiopicus blood conformed to the typical vertebrate scheme, implying a crucial and rate-limiting role of erythrocyte Cl^–/HCO₃^– exchange. The rate coefficient for unidirectional Cl^– efflux via the anion exchanger ( k , s⁻¹) increased with temperature in African lungfish, but values were well below those reported in other species. The erythrocytes of African lungfish were, however, very large (mean cellular volume = 6940 µm³), and the ratio of cell water volume to membrane surface area was high ( V _w A _m⁻¹ = 1·89). Hence, the apparent Cl^– permeability ( P _Cl =  kV _w A _m⁻¹, µm s⁻¹) was close to that in other vertebrates. The plot of ln P _Cl against the inverse absolute temperature was left-shifted in the tropical African lungfish compared to the temperate rainbow trout Oncorhynchus mykiss , which supports the idea that P _Cl is similar among animals when compared at their preferred temperatures. Also, Q ₁₀ for anion exchange calculated from P _Cl values in African lungfish was 2·0, supporting the idea that the temperature sensitivity of erythrocyte anion exchange matches the temperature sensitivity of CO₂ production and transport in ectothermic vertebrates.     

Role of tubular secretion and carbonic anhydrase in vertebrate renal sulfate excretion

The renal proximal tubule of vertebrates performs an essential role in controlling plasma SO(4)(2-) concentration ([SO(4)(2-)]). Although net tubular SO(4)(2-) reabsorption is the predominate control process in terrestrial vertebrates, a facilitated secretory flux is also present. In contrast, marine teleosts obtain excess SO(4)(2-) from drinking, and increased plasma [SO(4)(2-)] is prevented predominately through net tubular secretion. Tubular SO(4)(2-) secretion is accomplished by at least two electroneutral anion exchange processes in series. Movement of SO(4)(2-) into the cell across the basolateral membrane is pH dependent, suggesting SO(4)(2-)/OH(-) exchange. Luminal HCO(3)(-) and Cl(-) can facilitate SO(4)(2-) movement out of the cell across the brush-border membrane. The molecular identities of the anion exchangers are unknown but are probably homologues of SO(4)(2-) transporters in the mammalian SLC26 gene family. In all species tested, glucocorticoids increase renal SO(4)(2-) excretion. Whereas glucocorticoids downregulate SO(4)(2-) reabsorptive mechanisms in terrestrial vertebrates, they may also stimulate a mediated secretory flux. In the marine teleost, cortisol increases the level of SO(4)(2-)/HCO(3)(-) exchange at the brush-border membrane, tubular carbonic anhydrase (CA) activity, CAII protein, and a proportion of tubular SO(4)(2-) secretion that is CA dependent. CA activity is required for about one-half of this net SO(4)(2-) secretion but is also required for about one-half of the net reabsorption in bird proximal epithelium. A CA-SO(4)(2-)/anion exchanger metabolon arrangement is proposed that may speed both the secretory and reabsorptive processes.     

Uroplakins do not restrict CO2 transport through urothelium

Lipid bilayers and biological membranes are freely permeable to CO(2), and yet partial CO(2) pressure in the urine is 3-4-fold higher than in blood. We hypothesized that the responsible permeability barrier to CO(2) resides in the umbrella cell apical membrane of the bladder with its dense array of uroplakin complexes. We found that disrupting the uroplakin layer of the urothelium resulted in water and urea permeabilities (P) that were 7- to 8-fold higher than in wild type mice with intact urothelium. However, these interventions had no impact on bladder P(CO2) (～1.6 × 10(-4) cm/s). To test whether the observed permeability barrier to CO(2) was due to an unstirred layer effect or due to kinetics of CO(2) hydration, we first measured the carbonic anhydrase (CA) activity of the bladder epithelium. Finding none, we reduced the experimental system to an epithelial monolayer, Madin-Darby canine kidney cells. With CA present inside and outside the cells, we showed that P(CO2) was unstirred layer limited (～7 × 10(-3) cm/s). However, in the total absence of CA activity P(CO2) decreased 14-fold (～ 5.1 × 10(-4) cm/s), indicating that now CO(2) transport is limited by the kinetics of CO(2) hydration. Expression of aquaporin-1 did not alter P(CO2) (and thus the limiting transport step), which confirmed the conclusion that in the urinary bladder, low P(CO2) is due to the lack of CA. The observed dependence of P(CO2) on CA activity suggests that the tightness of biological membranes to CO(2) may uniquely be regulated via CA expression.     

Comparative radioimmunological investigation of prolactin from various species of vertebrates

Antibodies to ovine prolactin were used in a radioimmunoassay for prolactin in pituitary extracts from various species of vertebrates. Representative species were chosen from five different classes. The degree of cross-reaction was highest in mammals (62.5%) and lowest in bony fishes (16.2%). Birds, reptiles, and amphibians were intermediary (27·0, 31·9, and 25·0% respectively). In most cases, the immunological relatedness of the prolactins agreed with biological similarities. Prolactin from a few animals (mud puppy, mouse and snake) however, displayed very low immunological reactions. These discrepancies are discussed in relation to different molecular sites for immunological and biological activities.     

Influence of proton availability on intracapillary CO2-HCO3(-)-H+ reactions in isolated rat lungs.

Transcapillary CO2 exchange entails a transient perfusate CO2-HCO3(-)-H+ disequilibrium, leading to net loading or unloading of blood HCO3-. Perfusate reequilibration may or may not reach completion during the time of capillary transit, depending on the rate of intracapillary CO2-HCO3(-)-H+ reactions. Failure to reestablish equilibrium within the "open" capillary system leads to continued reequilibration in the "closed" postcapillary vasculature with resultant shifts in postcapillary perfusate PCO2, pH, and [HCO3-]. In the present study, we determined the effects of perfusate nonbicarbonate buffer capacity (beta) on intracapillary CO2-HCO3(-)-H+ reactions in isolated saline-perfused rat lungs. Effects of beta on the rate of transcapillary CO2 excretion (VCO2) and the magnitude of the postcapillary perfusate pH disequilibrium were measured as a function of luminal vascular carbonic anhydrase (CA) activity. The data indicate that beta markedly influenced the kinetics and dynamics of intravascular CO2-HCO3(-)-H+ reactions. beta affected VCO2 and the relative enhancement of VCO2 by luminal vascular CA. The data emphasize the inadequacies of using traditional "equilibrium" models of the CO2-HCO3(-)-H+ system to investigate capillary CO2 transport and exchange, even in organs (e.g., lungs) that contain significant luminal vascular CA activity.