Steve F. Perry

Acid–base balance and CO2 excretion in fish: Unanswered questions and emerging models

Carbon dioxide (CO(2)) excretion and acid-base regulation in fish are linked, as in other animals, though the reversible reactions of CO(2) and the acid-base equivalents H(+) and HCO(3)(-): CO(2)+H(2)O<-->H(+)+HCO(3)(-). These relationships offer two potential routes through which acid-base disturbances may be regulated. Respiratory compensation involves manipulation of ventilation so as to retain CO(2) or enhance CO(2) loss, with the concomitant readjustment of the CO(2) reaction equilibrium and the resultant changes in H(+) levels. In metabolic compensation, rates of direct H(+) and HCO(3)(-) exchange with the environment are manipulated to achieve the required regulation of pH; in this case, hydration of CO(2) yields the necessary H(+) and HCO(3)(-) for exchange. Because ventilation in fish is keyed primarily to the demands of extracting O(2) from a medium of low O(2) content, the capacity to utilize respiratory compensation of acid-base disturbances is limited and metabolic compensation across the gill is the primary mechanism for re-establishing pH balance. The contribution of branchial acid-base exchanges to pH compensation is widely recognized, but the molecular mechanisms underlying these exchanges remain unclear. The relatively recent application of molecular approaches to this question is generating data, sometimes conflicting, from which models of branchial acid-base exchange are gradually emerging. The critical importance of the gill in acid-base compensation in fish, however, has made it easy to overlook other potential contributors. Recently, attention has been focused on the role of the kidney and particularly the molecular mechanisms responsible for HCO(3)(-) reabsorption. It is becoming apparent that, at least in freshwater fish, the responses of the kidney are both flexible and essential to complement the role of the gill in metabolic compensation. Finally, while respiratory compensation in fish is usually discounted, the few studies that have thoroughly characterized ventilatory responses during acid-base disturbances in fish suggest that breathing may, in fact, be adjusted in response to pH imbalances. How this is accomplished and the role it plays in re-establishing acid-base balance are questions that remain to be answered.

Effects of cortisol on gill chloride cell morphology and ionic uptake in the freshwater trout,Salmo gairdneri

SummaryDaily intramuscular injection of cortisol (4 mg kg−1 body weight) in rainbow trout,Salmo gairdneri, for 10 days caused significant increases in the number and individual apical surface area of gill chloride cells per mm2 of filament epithelium. Concomitantly, whole body influxes of sodium (Na+) and chloride (Cl−) increased. Acute (3 h) intra-arterial infusion of cortisol did not affect whole body Na+ or Cl− influx. A significant correlation was observed between both Na+ and Cl− influxes and the fractional apical surface area of filament chloride cells in control, sham (saline-injected) and experimental (cortisol-injected) fish. The chloride cells displayed similar ultrastructural modifications in trout undergoing cortisol treatment as in trout transferred to ion-deficient water. These findings suggest the existence of structure/function relationships in which branchial chloride cell morphology is an important determinant of Na+ and Cl− transport capacity. We conclude that chronic cortisol treatment enhances whole body Na+ and Cl− influxes by promoting proliferation of branchial chloride cells. The results of correlation analysis indicate that the chloride cell is an important site of NaCl uptake in freshwater rainbow trout.

Environmental Effects on Fish Gill Morphology

Fish gill morphology and function are intricately related. Adjustments in gill morphology during adaptation of fish to environmental changes are often instrumental in conserving physiological functions. But optimization of one particular gill function by an environmentally induced morphological modification may be detrimental to other gill functions. Moreover, changes in gill morphology in response to a particular environment may not always be beneficial and indeed may contribute to the death of fish in polluted or acidified waters. In this paper we discuss both adaptive and nonadaptive changes in gill morphology when fish encounter changing environments. Emphasis is placed on recent findings concerning environmental oxygen, ionic composition, and xenobiotics. An assimilation of the results of these studies reveal a common response to widely different environmental disturbances (e.g., hyperoxia, hypercapnia, acidification, low [ NaCl], low [ Ca²⁺ ], freshwater-to-seawater transfer), namely branchial chloride cell proliferation. We suggest therefore that the chloride cell is multifunctional and plays a role in Ca²⁺ uptake, acid-base balance, NaCl uptake in freshwater fish, and NaCl excretion in seawater fish.

Environmental effects on fish gill structure and function

Aquatic habitats are remarkably diverse with respect to the chemical and physical properties of the water. For example, salinity may vary between full-strength seawater and near distilled water while pH may differ by as much as 6.0 pH units. Furthermore, aquatic environments, especially the freshwater ecosystems, are typically unstable and characterized by marked natural fluctuations of temperature, pH, oxygen, carbon dioxide, and dissolved ions. The ability of fish to inhabit these diverse and oscillating environments arises from a variety of adaptive physiological mechanisms. Owing to the location of the gill between the external and internal environments and its crucial role in gas transfer, acid-base balance, and ionic regulation, adaptive changes in branchial function are especially important. In many instances, the compensatory adjustments of gill function originate from profound morphological changes. Generally, the morphological adjustments to environmental changes are considered to be adaptive, although in certain instances gill function may actually be impaired. Moreover, owing to the multi-functional nature of the gill, morphological amelioration of a particular physiological function might compromise another.

Fish gill water boundary layer: a site of linkage between carbon dioxide and ammonia excretion

SummaryCarbon dioxide excreted across fish gills is hydrated catalytically to form HCO3− and H+ ions in water near the gill surface. We tested the possibility that CO2 excretion is functionally linked to ammonia excretion through chemical reactions in the gill-water boundary layer. A bloodperfused trout head preparation was utilized in which the convective and diffusive components of branchial gas transfer were controlled. Pre-incubation of blood perfusate with the carbonic anhydrase inhibitor, acetazolamide, reduced both carbon dioxide and ammonia excretion in the blood-perfused preparation. Increasing the buffering capacity of inspired ventilatory water significantly reduced ammonia excretion, but carbon dioxide excretion was unaffected. Each of these experimental treatments significantly reduced the acidification of ventilatory water flowing over the gills. It is proposed that the catalysed conversion of excreted CO2 to form HCO3− and H+ ions provides a continual supply of H+ ions need for the removal of NH3 as NH4+. We suggest, therefore, that acidification of boundary layer water by CO2 enhances blood-to-water NH3 diffusion gradients and facilitates ammonia excretion.

Evidence for a morphological component in acid-base regulation during environmental hypercapnia in the brown bullhead (Ictalurus nebulosus)

SummaryExposure of adult brown bullheads Ictalurus nebulosus (120–450 g) to environmental hypercapnia (2% carbon dioxide in air) and subsequent recovery caused transient changes in whole body net sodium flux (JnetNa+) and net chloride flux (JnetCl-) resulting largely from changes in whole body sodium influx (JinNa+) and chloride influx (JinCl-). Scanning electron microscopy (SEM) revealed that the fractional area of chloride cells (CCs) on the interlamellar regions was reduced by 95% during environmental hypercapnia. During post-hypercapnic recovery, gill filament CC fractional area increased. The changes in JinCl-during and after environmental hypercapnia were closely associated with the changes in CC fractional area while the changes in JinNa+did not correspond to the changes in CC fractional area. Transmission electron microscopy (TEM) supported the SEM observations of CC surface area changes and demonstrated that these changes were caused by covering/uncovering by adjacent pavement cells (PVCs). Lamellar and filament PVC microvilli density increased during hypercapnia while there was a subsequent reduction in the post-hypercapnic period. These data suggest that an important mechanism of acid-base regulation during hypercapnic acidosis is modification of the chloride cell-associated Cl-/HCO3-exchange mechanism. We suggest that bullheads vary availability, and thus functional activity, of this transporter via reversible morphological alterations of the gill epithelium. The increase in density of PVC microvilli may be associated with sodium uptake and/or acidic equivalent excretion during acidosis.

Carbonic anhydrase and acid–base regulation in fish

SUMMARY Carbonic anhydrase (CA) is the zinc metalloenzyme that catalyses the reversible reactions of CO2 with water. CA plays a crucial role in systemic acid–base regulation in fish by providing acid–base equivalents for exchange with the environment. Unlike air-breathing vertebrates, which frequently utilize alterations of breathing (respiratory compensation) to regulate acid–base status, acid–base balance in fish relies almost entirely upon the direct exchange of acid–base equivalents with the environment (metabolic compensation). The gill is the critical site of metabolic compensation, with the kidney playing a supporting role. At the gill, cytosolic CA catalyses the hydration of CO2 to H+ and HCO3– for export to the water. In the kidney, cytosolic and membrane-bound CA isoforms have been implicated in HCO3– reabsorption and urine acidification. In this review, the CA isoforms that have been identified to date in fish will be discussed together with their tissue localizations and roles in systemic acid–base regulation.

Gill Morphology and Acid-Base Regulation in Freshwater Fishes

This review examines the recent advances in our understanding of the mechanisms of ion transport and acid-base regulation in the freshwater fish gill. The application of a combination of morphological, immunocytochemical and biochemical techniques has yielded considerable insight into the field. An important mechanism for regulation of Cl- uptake/base excretion is by morphological modification of the gill epithelium. During acidosis, the chloride cell associated Cl-/HCO3- exchanger is effectively removed from the apical epithelium because of a covering by adjacent pavement cells; this mechanism reduces base excretion and contributes to the compensation of the acidosis. In addition, acidosis induces changes in both the surface structure and ultrastructure of pavement cells. Evidence is accumulating to support the hypothesis that Na+ uptake/H+ excretion is accomplished by the pavement cell. Further, specific localization of a V-type H+-ATPase on the pavement cell epithelium and an increased expression during acidosis provides support for the model originally proposed, that this exchange is accomplished by an electrochemically coupled H+-ATPase/Na+ channel mechanism.

Hydrogen sulfide as an oxygen sensor in trout gill chemoreceptors.

O2 chemoreceptors elicit cardiorespiratory reflexes in all vertebrates, but consensus on O2-sensing signal transduction mechanism(s) is lacking. We recently proposed that hydrogen sulfide (H2S) metabolism is involved in O2 sensing in vascular smooth muscle. Here, we examined the possibility that H2S is an O2 sensor in trout chemoreceptors where the first pair of gills is a primary site of aquatic O2 sensing and the homolog of the mammalian carotid body. Intrabuccal injection of H2S in unanesthetized trout produced a dose-dependent bradycardia and increased ventilatory frequency and amplitude similar to the hypoxic response. Removal of the first, but not second, pair of gills significantly inhibited H2S-mediated bradycardia, consistent with the loss of aquatic chemoreceptors. mRNA for H2S-synthesizing enzymes, cystathionine beta-synthase and cystathionine gamma-lyase, was present in branchial tissue. Homogenized gills produced H2S enzymatically, and H2S production was inhibited by O2, whereas mitochondrial H2S consumption was O2 dependent. Ambient hypoxia did not affect plasma H2S in unanesthetized trout, but produced a PO2-dependent increase in a sulfide moiety suggestive of increased H2S production. In isolated zebrafish neuroepithelial cells, the putative chemoreceptive cells of fish, both hypoxia and H2S, produced a similar approximately 10-mV depolarization. These studies are consistent with H2S involvement in O2 sensing/signal transduction pathway(s) in chemoreceptive cells, as previously demonstrated in vascular smooth muscle. This novel mechanism, whereby H2S concentration ([H2S]) is governed by the balance between constitutive production and oxidation, tightly couples tissue [H2S] to PO2 and may provide an exquisitely sensitive, yet simple, O2 sensor in a variety of tissues.

Nitrogen excretion in developing zebrafish (Danio rerio): a role for Rh proteins and urea transporters

Injection of antisense oligonucleotide morpholinos to elicit selective gene knockdown of ammonia (Rhag, Rhbg, and Rhcg1) or urea transporters (UT) was used as a tool to assess the relative importance of each transporter to nitrogen excretion in developing zebrafish (Danio rerio). Knockdown of UT caused urea excretion to decrease by approximately 90%, whereas each of the Rh protein knockdowns resulted in an approximately 50% reduction in ammonia excretion. Contrary to what has been hypothesized previously for adult fish, each of the Rh proteins appeared to have a similar effect on total ammonia excretion, and thus all are required to facilitate normal ammonia excretion in the zebrafish larva. As demonstrated in other teleosts, zebrafish embryos utilized urea to a much greater extent than adults and were effectively ureotelic until hatching. At that point, ammonia excretion rapidly increased and appeared to be triggered by a large increase in the mRNA expression of Rhag, Rhbg, and Rhcg1. Unlike the situation in the adult pufferfish (35), the various transporters are not specifically localized to the gills of the developing zebrafish, but each protein has a unique expression pattern along the skin, gills, and yolk sac. This disparate pattern of expression would appear to preclude interaction between the Rh proteins in zebrafish embryos. However, this may be a developmental feature of the delayed maturation of the gills, because as the embryos matured, expression of the transporters in and around the gills increased.