Academic literature on the topic 'Acid-base regulation'

Since the topic of the role of the kidneys in the regulation of acid-base balance was last reviewed from a teaching perspective (Koeppen BM. Renal regulation of acid-base balance. Adv Physiol Educ 20: 132–141, 1998), our understanding of the specific membrane transporters involved in H+, HCO3−, and NH4+ transport, and especially how these transporters are regulated in response to systemic acid-base disorders, has advanced considerably. In this review, these new aspects of renal function are presented, as are the broader and more general concepts related to the role of the kidneys in maintaining the acid-base balance. It is intended that this review will assist those who teach this aspect of human physiology to first-year health profession students.

Three sets of in vivo experiments were conducted to investigate several aspects of acid-base regulation in fishes. There are two possible ways that involve the gills of fishes in which the acid-base regulation of the extracellular fluid can be adjusted. First, CO₂ excretion can be adjusted by altering gill water flow to increase or decrease the PCO₂ tensions in the blood. The second mechanism would involve the exchange of ions across the gill epithelium to change the concentrations of H⁺, HCO₃⁻ or NH₄⁺ in the blood. The first two sets of experiments were, respectively, designed to investigate these two possibilites. The third set of experiments investigated the role that plasma catecholamines might play in regulating the pH of the extracellular fluid as well as the intracellular compartment of the red blood cell. Experimental manipulation of ventilation in rainbow trout in steady state showed that gill water flow affected CO₂ excretion only at levels lower than about 100ml/min. Carbon dioxide excretion was retarded and blood PCO₂ pressures increased at these levels of gill ventilation. Increasing gill water flow above control levels effected neither O₂ or CO₂ exchange across the gill. Dogfish, subjected to environmental hyperoxia and various levels of hypercapnia, showed the best correlation between gill ventilation and plasma pH. There was a very weak correlation with plasma PCO₂ tension and plasma HCO₃⁻ concentrations did not affect ventilation at all. Gill ventilation increased exponentially as plasma pH declined. Experiments that involved the fresh water trout and the sea water conger eel showed that water salinity had a direct effect on the acid-base regulation of the plasma. Recovery of plasma pH in both species, after an initial decline in response to exposure to environmental hypercapnia, was dependent on water salinity. The recovery was effected by an increase in plasma HCO₃⁻ concentration. There was also an associated decrease in plasma Cl⁻ concentration in both species, indicating the possible involvement of a Cl⁻/ HCO₃⁻ exchange process. When carp were exposed to environmental hypercapnia, a reduction in the active uptake of water Cl⁻, while maintaining normal efflux rates, caused the reduction of the plasma concentration of this ion. Therefore, it seems that the modulation of this active Cl⁻/ HCO₃⁻ exchange process effected the HCO₃⁻ accumulation in the carp, and probably also in the trout and conger in fresh water. Consistent with the data from the above carp experiment, further analyses of the electrochemical gradients for Cl⁻ in trout exposed to environmental hypercapnia at the three salinities showed that active exchange processes must have accumulated the plasma HCO₃⁻ by the proposed Cl⁻/ HCO₃⁻ mechanism. These analyses also showed that the trout gill was about 2.5 times more permeable to Na⁺ than to Cl⁻ in steady state control conditions. Furthermore, Na⁺ is maintained out of electrochemical equilibrium more than Cl⁻ by a factor of about 1.5 - 2.0. This latter calculation was based on the comparison between the measured plasma concentrations of these ions and the expected concentrations based on a distribution according to the existing electrochemical gradents Catecholamines are released in trout immediately after acid infusion. This release is proportional to the change in plasma pH relative to control values and functions to maintain the oxygen carrying capacity of the blood which would otherwise be compromised due to the Root shift. This data supports existing data showing that some of the effects which catecholamines have on the physiology of fishes include those which enhance the regulation of the acid-base status of the extracellular and red cell compartments. This data also suggests that the release of catecholamines during burst exercise is due, at least partially, to the excess proton load from the lactacidosis.
Science, Faculty of
Zoology, Department of
Graduate

This thesis examines the branchial mechanisms utilized by freshwater fish to regulate internal acid-base status and presents a model to explain the underlying basis of the compensatory processes. Rainbow trout, Oncorhynchus mykiss, brown bullhead, Ictalurus nebulosus, and American eels, Anguilla rostrata, were examined under a variety of experimental treatments which induced both respiratory (hyperoxia, hypercapnia) and metabolic (post-hyperoxia, post-hypercapnia, HCl infusion, NaHCO$\sb3$ infusion) acid-base disturbances. Acid-base-regulation was achieved by appropriate adjustments of Na$\sp+$ and Cl$\sp-$ net fluxes across the gills which, in turn, were accomplished by variable contributions of three different branchial mechanisms; (i) morphological adjustments to the gill epithelium, (ii) changes in internal (H$\sp+,$ HCO$\sb3\sp-)$ and external (Na$\sp+,$ HCO$\sb3\sp-)$ substrate availability, and (iii) differential changes in Na$\sp+$ versus Cl$\sp-$ net fluxes through regulation of Cl$\sp-$ efflux. This thesis determined the variable contribution of each of these mechanisms to overall compensation of acid-base disturbances. In brown bullhead and trout, respiratory acidosis caused a reduction in chloride cell (CC) surface area whereas alkalosis was associated with increases in CC surface area. Increases in the density of microvilli displayed on the external surface of the PVC coupled with ultrastructural modifications during hypercapnic acidosis were associated with increases in Na$\sp+$ uptake $\rm(J\sb{in}\sp{Na+}).$ In addition to the effect that alterations in CC surface area have on the rate of Cl$\sp-$/HCO$\sb3\sp-$ exchange $\rm(J\sb{in}\sp{Cl-}),$ it was demonstrated that changes in the concentration of the internal counter-ion (HCO$\sb3\sp-)$ may alter the rates of acid-base compensation. When (HCO$\sb3\sp-$) is elevated, $\rm J\sb{max}\sp{Cl-}$ is elevated thereby increasing the capacity to excrete HCO$\sb3\sp-$ via the Cl$\sp-$/HCO$\sb3\sp-$ exchanger over and above those determined by CC morphology. This is an important mechanism to increase the rate of acid-base compensation during metabolic alkalosis. (Abstract shortened by UMI.)

The current increase in the atmospheric CO2 concentration results in two major consequences in the marine environment: an increase of the sea surface temperature (0.7 °C since pre-industrial times) and a decreased seawater pH. This decrease is being measured continuously in different parts of the world and ranges from -0.0017 to -0.04 units per year according to the location considered. Based on CO2 emissions models provided by the IPCC, it was predicted that the average open ocean pH would decrease further by 0.4 units by 2100 and 0.8 by 2300 (corresponding to about a three-fold and six-fold increase of the proton concentration). Also, saturation states of seawater for the different forms of calcium carbonate, such as calcite, magnesium calcite and aragonite which are produced by calcifying marine organisms, are decreasing and consequently the saturation horizons of these minerals are shoaling. Today, some environments are characterized by pH values lower than the average open ocean pH. These are intertidal rock pools, upwelling zones, the deep-sea and CO2 vents. In these environments, pH is either constantly low or fluctuates. Those changes are either due to biological activity, geological CO2 leakage, or water masses movements. Within these environments, it has been hypothesized that organisms could be adapted or acclimatized to low pH values such as those predicted for the near-future.

Tolerance to ocean acidification in metazoans is linked to their acid-base regulation capacities when facing environmental hypercapnia (i.e. increased CO2 concentration in the surrounding environment). The latter may result in a hypercapnia of the internal fluids and a concomitant acidosis (i.e. reduced pH of the internal fluids due to the dissociation of CO2 in this case). Organisms have two buffer systems allowing the compensation of this acidosis: the CO2-bicarbonate and the non-bicarbonate buffers. Homeostasis of the internal fluids thanks to these systems is essential for the proper functioning of enzymes and processes. As hypometabolic calcifying osmoconformers, three of the characteristics conferring a relative vulnerability to ocean acidification, echinoderms are considered “at risk” for the near-future conditions. Nonetheless, post-metamorphic (juveniles and adults) echinoderms inhabit all environments showing naturally low pH. Furthermore, sea urchins which are highly calcified (compared to sea stars or sea cucumbers) are also found in these environments. This suggests that echinoderms have strategies to adapt or acclimate to low pH environments. Recent studies indicated that while sea urchins are able to regulate their coelomic (extracellular) fluid by accumulation of bicarbonate, sea stars seem to tolerate the acidosis linked to environmental hypercapnia. However, this information was obtained on a reduced number of species and significant interspecific differences were evidenced. Some taxa have not been investigated at all. Furthermore, several aspects of the acid-base physiology were unexplored, like the buffering capacity of the extracellular fluid and the origin of carbon within these fluids.

Accordingly, the goal of this study was to characterize the acid-base physiology in post-metamorphic echinoderms of different taxa in order to understand their response to ocean acidification.

The acid-base regulation capacities within the different echinoderm taxa were compared. A method was designed to measure the total alkalinity in small volumes (500 µl) of the main extracellular fluid (the coelomic fluid). This study showed that regular euechinoids have an increased buffer capacity in their coelomic fluid compared to seawater and the other echinoderm groups. In sea urchins, bicarbonate and non-bicarbonate buffers come into play, the former playing the major role. This buffer capacity was increased in fed individuals compared to fasted ones and increased further when seawater pH was lowered.

The acid-base regulation capacities of sea urchins from different taxa were investigated. Regular euechinoids possess an increased buffer capacity of the coelomic fluid allowing them to maintain a higher pH compared to cidaroids at current seawater pH. This pattern was found for temperate, tropical and Antarctic sea urchins. Data was also obtained for irregular echinoids which also showed a particularly low extracellular pH and a buffer capacity close to seawater like cidaroids. When exposed to reduced seawater pH (8.0, 7.7, and 7.4) for 4-6 weeks, regular euechinoids showed an increasing buffer capacity of the coelomic fluid accompanied by a homeostasis of the pH. On the contrary, cidaroids showed no changes in their acid-base status whatever the seawater pH (8.0 to 7.4). The origin of coelomic fluid carbon, investigated by stable carbon isotope analysis, also differs according to taxa. The δ13CDIC of regular euechinoids evidenced a mixing between CO2 from metabolic origin and that from the surrounding seawater. This is further supported by the correlation between the seawater signal of reduced pH conditions (modified by the addition of industrial gas, changing the δ13C to more negative values) and that of the coelomic fluid. On the other hand, cidaroids exhibit a signal reflecting principally metabolic CO2 (very negative δ13C), and the δ13C did not change under varying pH conditions (i.e. did not adapt to the seawater δ13CDIC signature). For irregular echinoids, the carbon origin is unclear as some species show signals close to that of regular euechinoids whereas others are similar to cidaroids.

The impact of acid-base regulation was investigated by testing the effect of ocean acidification on the mechanical properties of the skeleton (test plates) in the sea urchin Paracentrotus lividus. Individuals from intertidal pools, CO2 vents and a one year acidification experiment (pH 8.0, 7.9 and 7.7) were compared. Only the intertidal pool individuals showed a difference of the Young’s modulus and fracture forces of their plates. Sea urchins from the tide pool with the largest pH fluctuations showed a lower stiffness and strengthened test. On the contrary, sea urchins from CO2 vents and experimental acidification did not display any differences in the several mechanical properties tested. We suggest that the different food qualities (calcified vs. uncalcified algae) in the different tide pools significantly contributed to the observed difference.

The acid-base regulation ability of sea cucumbers was assessed in two species from contrasted habitats (mangrove intertidal vs. coral reef species). These organisms underwent acidosis of the coelomic fluid when exposed to reduced seawater pH for a short time (6 to 12 days). The δ13C signal of the coelomic fluid mirrored that of the surrounding seawater in all conditions, indicating that the CO2 accumulated (cause of the acidosis) comes also from the seawater. This is still unexplained to date. However, metabolic processes such as respiration and ammonium excretion rates were not affected. No difference was evidenced between the two species.

The results obtained in this study compiled with data from the literature indicate that post-metamorphic echinoderms have contrasted acid-base physiology with most regular euechinoids compensating the coelomic fluid pH by accumulation of bicarbonate ions (and possibly ophiuroids also), cidaroids and at least one regular euechinoid (Arbacia lixula) having a naturally low coelomic fluid pH which is not affected by acidification, and sea stars and sea cucumbers which do not compensate their coelomic fluid pH when submitted to acidified seawater. In regular euechinoids, negative effects are linked to resource allocation with growth usually being reduced in favor of acid-base regulation mechanisms. Starfish and sea cucumbers appear as resilient to acidification, with very few functions being negatively impacted. In conclusion, it seems that post-metamorphic echinoderms studied so far will not be particularly at risk when facing ocean acidification levels expected by 2100. Furthermore, tolerance to ocean acidification does not seem linked to the present day ambient pH regime. Nevertheless, more studies need to be carried out on brittle stars and sea cucumbers to confirm preliminary results, as well as crinoids which have not been investigated to date. Long-term exposure experiments to estimate energy budget changes as well as more assessments of evolutionary potential in echinoderms are crucially needed./L’augmentation actuelle de la concentration en CO2 atmosphérique a deux conséquences majeures dans l’environnement marin :une augmentation de la température des eaux de surface (0.7°C depuis l’époque préindustrielle) et une diminution du pH de l’eau de mer. Cette diminution est mesurée continuellement dans différentes régions du monde et varie de -0.0017 à -0.04 unités de pH par an en fonction du site considéré. Basé sur des modèles d’émissions de CO2 du GIEC, il a été prédit que le pH moyen de l’océan diminuerait encore de 0.4 unités d’ici 2100 et 0.8 d’ici 2300 (correspondant à une augmentation de la concentration en protons d’environ 3 fois et 6 fois). De même, les états de saturation de l’eau de mer vis-à-vis des différentes formes de carbonate de calcium, telles que la calcite, la calcite magnésienne et l’aragonite produites par les organismes calcifiants, sont en train de diminuer et par conséquent, les horizons de saturation remontent vers les eaux de surface. Aujourd’hui, certains environnements sont caractérisés par des valeurs de pH plus basses que celle de l’océan. Ceux-ci sont les mares intertidales, les zones d’upwelling, l’océan profond et les évents volcaniques. Dans ces environnements, le pH est soit constamment bas ou fluctue. Ces changements sont soit dû à une activité biologique, une fuite de CO2 géologique, ou au mouvement des masses d’eau. Dans ces environnements, il a été suggéré que les organismes pourraient être adaptés ou acclimatés à des valeurs basses de pH, telles que celles prédites pour le futur proche.

La tolérance à l’acidification des océans chez les métazoaires est liée à leur capacité de régulation acide-base lorsqu’ils sont exposés à une hypercapnie environnementale (c’est-à-dire, une augmentation de la concentration en CO2 dans l’environnement entourant l’organisme). Ce phénomène peut résulter en une hypercapnie des liquides internes et une acidose concomitante (c’est-à-dire, un pH des liquides internes réduit dû à la dissociation du CO2 dans ce cas précis). Les organismes ont deux systèmes tampons leur permettant de compenser l’acidose :les tampons CO2-bicarbonate et non-bicarbonate. L’homéostasie des liquides internes grâce à ces systèmes est essentielle pour le fonctionnement correct des enzymes et processus. En tant qu’osmoconformes calcifiant hypométaboliques, trois caractéristiques menant à une certaine vulnérabilité face à l’acidification des océans, les échinodermes sont considérés « à risque » pour les conditions du futur proche. Cependant, les échinodermes post-métamorphiques (juvéniles et adultes) occupent tous les environnements montrant un pH faible naturellement. De plus, les oursins qui sont hautement calcifiés (par rapport aux étoiles de mer ou aux concombres de mer) sont également retrouvés dans ces environnements. Ceci suggère que les échinodermes ont des stratégies d’adaptation ou d’acclimatation à ces environnements à bas pH. Alors que des études récentes montrent que les oursins sont capables de réguler le pH du liquide cœlomique (extracellulaire) par l’accumulation de bicarbonates, les étoiles semblent tolérer l’acidose liée à l’hypercapnie environnementale. Néanmoins, ces informations ont été obtenues sur un petit nombre d’espèces et des différences interspécifiques significatives ont été mises en évidence. Certains taxa n’ont pas été étudié du tout. Par ailleurs, différents aspects de la physiologie acide-base sont inexplorés, tels que la capacité tampon du liquide extracellulaire et l’origine du carbone dans ces liquides.

Par conséquent, le but de cette étude était de caractériser la physiologie acide-base chez les échinodermes post-métamorphiques de différents taxa afin de comprendre leur réponse à l’acidification des océans.

Les capacités de régulation acide-base au sein des différents groupes d’échinodermes ont été comparées. Une méthode a été mise au point afin de mesurer l’alcalinité totale dans de petits volumes (500 µl) de liquide extracellulaire (le liquide cœlomique). Cette étude démontra que la capacité tampon du liquide cœlomique des euéchinoïdes réguliers est accrue comparée à celle de l’eau de mer ainsi que celle des autres groupes d’échinodermes. Dans les oursins, les tampons bicarbonate et non-bicarbonate entrent en jeux, le premier étant majoritaire. Cette capacité tampon est augmentée chez les individus nourris par rapport à ceux à jeuns et est augmentée plus encore lorsque le pH de l’eau de mer est diminué.

Les capacités de régulation acide-base ont été étudiées plus spécifiquement dans les différents groupes d’oursins. Les euéchinoïdes réguliers possèdent une capacité tampon accrue du liquide cœlomique leur permettant de maintenir un pH élevé comparé aux oursins cidaroïdes, au pH de l’eau de mer actuel. Ce patron se retrouve dans les oursins tempérés, tropicaux et antarctiques. Des données ont également été obtenues pour les oursins irréguliers qui ont également un pH extracellulaire particulièrement bas et une capacité tampon proche de celle de l’eau de mer comme les cidaroïdes. Lorsqu’ils sont exposés à un pH de l’eau de mer réduit (7.7 et 7.4 par rapport à 8.0) pour 4 à 6 semaines, les euéchinoïdes réguliers ont montré une augmentation de la capacité tampon du liquide cœlomique accompagnée d’une homéostasie du pH de ce liquide. A l’inverse, les cidaroïdes n’ont montré aucune modification de leur statut acide-base quel que soit le pH (8.0 à 7.4). L’origine du carbone du liquide cœlomique, étudié par analyse des isotopes stables du carbone, diffère également en fonction du groupe. Le δ13CDIC des euéchinoïdes réguliers met en évidence un mélange entre du CO2 d’origine métabolique et celui de l’eau environnante. Ceci est également démontré par la corrélation entre le signal de l’eau de mer dont le pH est réduit (modifié par l’ajout de CO2 industriel, changent le δ13C vers des valeurs plus négatives) et celui du liquide cœlomique. En revanche, les cidaroïdes montrent un signal reflétant principalement celui du CO2 métabolique (δ13C très négatif), et le δ13C n’est pas influencé par des conditions de pH variées (c’est-à-dire, qu’il ne s’adapte pas à la signature du δ13CDIC de l’eau de mer). Pour les oursins irréguliers, l’origine du carbone est incertaine puisque certaines espèces montrent un signal proche de celui des euéchinoïdes réguliers et d’autres similaire à celui des cidaroïdes.

L’impact de la régulation acide-base a été étudié en testant l’effet de l’acidification des océans sur les propriétés mécaniques du squelette (plaques squelettiques) de l’oursin Paracentrotus lividus. Des individus de mares intertidales, d’évents volcaniques et d’une expérience d’acidification d’un an (pH 8.0, 7.9 et 7.7) ont été comparés. Seuls les individus des mares intertidales montrèrent une différence pour le module de Young et la force des fractures des plaques. Les oursins venant de la mare intertidale montrant les plus grandes variations de pH avaient une rigidité plus faible et un squelette renforcé. A l’inverse, les oursins des évents volcaniques et de l’expérience d’acidification n’ont montrés aucune différence dans les diverses propriétés mécaniques étudiées. Nous suggérons que les variations en termes de qualité de nourriture (algues calcifiées vs. non-calcifiées) dans les différentes mares intertidales ont contribués de manière significative à la différence observée.

L’habilité des concombres de mer à réguler leur balance acide-base a été évaluée dans deux espèces d’habitats contrastés (espèce intertidale des mangroves vs. subtidale des récifs coralliens). Ces organismes ont subis une acidose du liquide cœlomique lorsqu’ils ont été exposés à un pH réduit de l’eau de mer pour une courte durée (6 à 12 jours). Le signal δ13C du liquide cœlomique reflétait celui de l’eau environnante dans toutes les conditions, indiquant que le CO2 accumulé (cause de l’acidose) venait de l’eau. Ceci est encore inexpliqué à l’heure actuelle. Cependant, les processus métaboliques tels que la respiration ou l’excrétion d’ammonium n’ont pas été affecté. Aucune différence n’a été observée entre les deux espèces.

Les résultats obtenus dans cette étude compilés avec ceux de la littérature indiquent que les échinodermes post-métamorphiques ont une physiologie acide-base contrastée avec la plupart des euéchinoïdes réguliers qui compensent le pH du liquide cœlomique par l’accumulation d’ions bicarbonates (et peut-être les ophiures aussi), les cidaroïdes et au moins un euéchinoïde régulier (Arbacia lixula) qui ont naturellement un pH du liquide cœlomique bas et qui ne sont pas affectés par l’acidification, et les étoiles de mer et les concombres de mers qui ne compensent pas le pH du liquide cœlomique lorsqu’ils sont soumis à une eau acidifiée. Chez les euéchinoïdes réguliers, des effets négatifs sont liés à un changement de l’allocation des ressources avec souvent un taux de croissance réduit en faveur des mécanismes de régulation acide-base. Les étoiles de mer et les concombres de mer apparaissent plus tolérants à l’acidification, avec peu de fonctions négativement impactées. En conclusion, il semble que les échinodermes post-métamorphiques étudiés jusqu’à présent ne seront pas particulièrement à risque lorsqu’ils seront exposés au niveau d’acidification attendu pour 2100. De plus, la tolérance à l’acidification des océans ne semble pas liée au régime de pH subit actuellement. Cependant, plus d’études doivent être menées sur les ophiures et les concombres de mer afin de confirmer les résultats préliminaires, ainsi que sur les crinoïdes qui n’ont à l’heure actuelle pas encore été étudiés. Des expériences à long terme afin d’estimer le budget énergétique des organismes ainsi que plus d’évaluations du potentiel d’évolution chez les échinodermes sont absolument nécessaires.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

As one of the most successful invasive species in the marine environment around the globe, the green crab Carcinus maenas possesses efficient regulatory mechanisms to quickly acclimate to environmental changes. The most important organs in this process are the nine pairs of gills that not only allow for osmoregulation, but have been shown to be involved in ammonia excretion and respiratory gas exchange. To date, however, little is known about the gills’ contribution to acid-base regulation that might become increasingly important in a “future ocean scenario” whereby surface ocean pH is predicted to drop by up to 0.5 units by the year 2100. The present thesis aims to characterize the green crab gills’ role in acid-base regulation and how it is linked to ammonia excretion. After exposure to hypercapnia (0.4 kPa pCO2 for 7 days), osmoregulating green crabs were capable of fully compensating for the resulting extracellular respiratory acidosis, while osmoconforming green crabs only partially buffered the accompanying drop in hemolymph pH after acclimation to 1% CO2 for 48 hours. Perfusion experiments on isolated green crab gills showed that different gills contributed to the excretion of H+ in an individual pattern and indicated that NH4+ is an important component of branchial acid excretion. Experiments on gill mRNA expression and pharmaceutical effects on isolated gills identified distinct epithelial transporters to play significant roles in branchial acid base regulation: Rhesus-like protein, basolateral bicarbonate transporter(s), cytoplasmic V-(H+)-ATPase, Na+/H+-exchanger, basolateral Na+/K+-ATPase, cytoplasmic and membrane bound carbonic anhydrase, and basolateral K+ channels. Regarding the latter, the present work provides the first sequence-based evidence for a potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel (CmHCN) capable of promoting NH4+ transport in the green crabs’ gill epithelium, and further demonstrates its direct involvement in branchial acid-base regulation. This highly conserved protein is a potentially important novel key-player in acid-base regulation in all animals. Interestingly, the observed principles linking acid-base to ammonia regulation in the decapod crustacean gill epithelium resemble many observations previously made in vertebrates. The data of the present thesis therefore provides valuable information for general acid-base regulation, while contributing substantially to our understanding of acid-base regulation in invertebrates.
February 2016

In the final step of protein C pathway, activated protein C (APC) is neutralized with a plasma inhibitor, termed protein C inhibitor (PCI). PCI was first described by Marlar and Griffin (1980) and then isolated from human plasma as a homogeneous form and characterized by the authors (1983). PCI is a single chain glycoprotein with M 57,000 and a plasma concentration of 5 ug/ml. Analysis of a cDNA nucleotide sequence has clarified that a precursor of human PCI consists of a mature protein of 387 amino acid residues (M 43,759) and a signal peptide of 19 amino acid residues. Only one cysteine residue is present in the entire protein as in α1antitrypsin (α1AT) and α1antichymotrypsin (α1ACT). Three Asn-X-Ser/Thr sequences and two Ser/Thr-X-X-Pro sequences are present as potential attachment sites of carbohydrate chains. Based on the amino acid sequence of the carboxyl-terminal peptide released from the inhibitor by APC digestion, the reactive site peptide bond of PCI was found to be Arg(354)-Ser(355). It is similar to the reactive sites of the other serine protease inhibitors which are located to their carboxyl-terminal Arg(393)-Ser (394), Met(358)-Ser(359) and Leu(358)-Ser(359) in antithrombin III, α1AT and α1ACT, respectively. The alignment of the amino acid sequence of PCI with heparin cofactor II, α1plasmin inhibitor, ovalbumin, angiotensinogen and the above noted plasma inhibitors showed that PCI is a member of serine protease inhibitor superfamily. PCI inhibits APC noncompetitively in a 1:1 stoichiometry and forms a covalent acyl-bond with a Ser residue in the active center of APC. The half life of APC in plasma approximately 30 min, which is rather slow compared with the other protease inhibitors. However, optimal concentrations of heparin, dextran sulfate and its derivatives potentiate the rate of inhibition 30-60 fold. PCI has Ki of 10-8m for APC, and can inhibit thrombin, Factor Xa, urokinase and tissue plasminogen activator as well in the presence of heparin or dextran sulfate, though the Ki for these enzymes is slightly higher. During the complex formation with APC, PCI is cleaved by the complexed APC to form a modified form with M 54,000. PCI is synthesized in several hepatoma cell lines and decreased in plasma of patients with liver cirrhosis. It is also decreased in patients with DIC or those during cardiopulmonary bypass in parallel with the decrease in protein C, suggesting that PCI participates in regulation of the protein C pathway in intravascular coagulation. Recently, we have obtained the recombinant PCI from COS-1 cells which were transfected with expression vector pSV2 containing the cDNA of PCI. The recombinant PCI had the same Mr and specific activity as the protein purified from plasma. It also had an affinity for heparin and dextran sulfate. Moreover, we have predicted a three dimentional structure of the proteolytically modified PCI with computer graphics based on its amino acid sequence homology with the modified α1AT whose structure had been elucidated with X-ray crystallography. All potential carbohydrate attachment sites were estimated to exist on the surface of the protein. Succesively we have constructed the interaction model between the intact PCI predicted from the modified form and the active center of APC which was simulated from that of trypsin. From the model, it was observed that the amino-group of Arg (354, PI site) of PCI could strongly interact with the carboxy1-group of Asp (88, SI site) of the heavy chain of APC at the base of the active center pocket of the enzyme.