The fish gill is a multipurpose organ that, in addition to providing for aquatic gas exchange, plays dominant roles in osmotic and ionic regulation, acid-base regulation, and excretion of nitrogenous wastes Thus, despite the fact that all fish groups have functional kidneys, the gill epithelium is the site of many processes that are mediated by renal epithelia in terrestrial vertebrates Indeed, many of the pathways that mediate these processes in mammalian renal epithelial are expressed in the gill, and many of the extrinsic and intrinsic modulators of these processes are also found in fish endocrine tissues and the gill itself The basic patterns of gill physiology were outlined over a half century ago, but modern immunological and molecular techniques are bringing new insights into this complicated system Nevertheless, substantial questions about the evolution of these mechanisms and control remain

https://people.clas.ufl.edu/devans/files/PRVGill.pdf

The Multifunctional Fish Gill: Dominant Site of Gas Exchange, Osmoregulation, Acid-Base Regulation, and Excretion of Nitrogenous Waste

CO2 currently accumulating in the atmosphere permeates into ocean surface layers, where it may impact on marine animals in addition to effects caused by global warming. At the same time, several countries are developing scenarios for the disposal of anthropogenic CO2 in the worlds' oceans, especially the deep sea. Elevated CO2 partial pressures (hypercapnia) will affect the physiology of water breathing animals, a phenomenon also considered in recent discussions of a role for CO2 in mass extinction events in earth history. Our current knowledge of CO2 effects ranges from effects of hypercapnia on acid-base regulation, calcification and growth to influences on respiration, energy turnover and mode of metabolism. The present paper attempts to evaluate critical processes and the thresholds beyond which these effects may become detrimental. CO2 elicits acidosis not only in the water, but also in tissues and body fluids. Despite compensatory accumulation of bicarbonate, acid-base parameters (pH, bicarbonate and CO2 levels) and ion levels reach new steady-state values, with specific, long-term effects on metabolic functions. Even though such processes may not be detrimental, they are expected to affect long-term growth and reproduction and may thus be harmful at population and species levels. Sensitivity is maximal in ommastrephid squid, which are characterized by a high metabolic rate and extremely pH-sensitive blood oxygen transport. Acute sensitivity is interpreted to be less in fish with intracellular blood pigments and higher capacities to compensate for CO2 induced acid-base disturbances than invertebrates. Virtually nothing is known about the degree to which deep-sea fishes are affected by short or long term hypercapnia. Sensitivity to CO2 is hypothesized to be related to the organizational level of an animal, its energy requirements and mode of life. Long-term effects expected at population and species levels are in line with recent considerations of a detrimental role of CO2 during mass extinctions in the earth's history. Future research is needed in this area to evaluate critical effects of the various CO2 disposal scenarios.

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Biological Impact of Elevated Ocean CO2 Concentrations: Lessons from Animal Physiology and Earth History

This review focuses on the structure and function of the branchial chloride cell in freshwater fishes. The mitochondria-rich chloride cell is believed to be the principal site of trans-epithelial Ca2+ and Cl- influxes. Though currently debated, there is accruing evidence that the pavement cell is the site of Na+ uptake via channels linked electrically to an apical membrane vacuolar H(+)-ATPase (proton pump). Chloride cells perform an integral role in acid-base regulation. During conditions of alkalosis, the surface area of exposed chloride cells is increased, which serves to enhance base equivalent excretion as the rate of Cl-/HCO3- exchange is increased. Conversely, during acidosis, the chloride cell surface area is diminished by an expansion of the adjacent pavement cells. This response reduces the number of functional Cl-/HCO3- exchangers. Under certain conditions that challenge ion regulation, chloride cells proliferate on the lamellae. This response, while optimizing the Ca2+ and Cl- transport capacity of the gill, causes a thickening of the blood-to-water diffusion barrier and thus impedes respiratory gas transfer.

THE CHLORIDE CELL:Structure and Function in the Gills of Freshwater Fishes

Compared to terrestrial animals, fish have to cope with more-challenging osmotic and ionic gradients from aquatic environments with diverse salinities, ion compositions, and pH values. Gills, a unique and highly studied organ in research on fish osmoregulation and ionoregulation, provide an excellent model to study the regulatory mechanisms of ion transport. The present review introduces and discusses some recent advances in relevant issues of teleost gill ion transport and functions of gill ionocytes. Based on accumulating evidence, a conclusive model of NaCl secretion in gills of euryhaline teleosts has been established. Interpretations of results of studies on freshwater fish gill Na+/Cl- uptake mechanisms are still being debated compared with those for NaCl secretion. Current models for Na+/Cl- uptake are proposed based on studies in traditionally used model species. Many reported inconsistencies are claimed to be due to differences among species, various experimental designs, or acclimation conditions. Having the benefit of advanced techniques in molecular/cellular biology, functional genomics, and model animals, several new notions have recently been raised concerning relevant issues of Na+/Cl- uptake pathways. Several new windows have been opened particularly in terms of molecular mechanisms of ionocyte differentiation and energy metabolite transport between gill cells during environmental challenge.

New insights into fish ion regulation and mitochondrion-rich cells

Currently rising CO 2 levels in atmosphere and marine surface waters as well as projected scenarios of CO 2 disposal in the ocean emphasize that CO 2 sensitivities need to be investigated in aquatic organisms, especially in animals which may well be the most sensitive. Moreover, to understand causes and effects, we need to identify the physiological processes that are sensitive to CO 2 beyond the current emphasis on calcification. Few animals may be acutely sensitive to moderate CO 2 increases, but subtle changes due to long-term exposure may already have started to be felt in a wide range of species. CO 2 effects identified in invertebrate fauna from habitats characterized by oscillating CO 2 levels include depressed metabolic rates and reduced ion exchange and protein synthesis rates. These result in shifts in metabolic equilibria and slowed growth. Long-term moderate hypercapnia has been observed to produce enhanced mortality with as yet unidentified cause and effect relationships. During future climate change, simultaneous shifts in temperature, CO 2 , and hypoxia levels will enhance sensitivity to environmental extremes relative to a change in just one of these variables. Some interactions between these variables result from joint effects on the same physiological mechanisms. Such interactions need to be considered in terms of future increases in atmospheric CO 2 and its uptake by the ocean as well as in terms of currently proposed mitigation scenarios. These include purposeful injection of CO 2 in the deep ocean or Fe fertilization of the surface ocean, which reduces subsurface O 2 levels. The resulting ecosystem shifts could develop progressively, rather than beyond specific thresholds, such that effects parallel CO 2 oscillations. It is unsure to what extent and how quickly species may adapt to permanently elevated CO 2 levels by microevolutionary compensatory processes.

Synergistic effects of temperature extremes, hypoxia, and increases in CO2 on marine animals : From earth history to global change : The ocean in a high-CO2 world

A spontaneously ventilating blood-perfused trout preparation and saline perfused gill preparations were utilized to investigate the role of the erythrocyte and branchial epithelium in CO 2 excretion and acid-base regulation. CO, excretion ( M CO CO2 ) in blood-perfused preparations was positively correlated with haematocrit (Hct), and was abolished completely during plasma-perfusion. Elevating HCO 3 - concentration of input blood from 10 to 25 mM significantly increased M CO CO2 . fourfold in blood-perfused preparations as a result of increased entry of H CO into the red blood cell and not into the gill epithelium. Increased HCO 3 - concentration was without effect in totally saline-perfused coho salmon (Onchorynchus kisutch). The addition of 4-acetamido-4′-wo-thiocyanatostilbene-2, 2 disulfonic acid (SITS; 10 −4 M) to input blood significantly reduced M CO , and oxygen uptake ( M g , O O2 ) in blood-perfused fish due to inhibition of erythrocytic HCO 3 -exchange. Unlike blood-perfused preparations, no saline-perfused preparation (isolated holobranchs or totally perfused rainbow trout or coho salmon) displayed measureable CO, excretion at physiological Pco and pH. Increased input PCOt in both blood-perfused and saline-perfused preparations significantly increased MCOt due to enhanced branchial diffusion of molecular CO 2 . It is concluded that the entry of HCO 3 - into the erythrocyte is the rate-limiting step in CO, excretion and that movement of HCO 3 - from plasma to gill epithelium cells in no way contributes to overall CO 3 elimination. Note: Department of Physiology and Anatomy, Massey University Palmerston North, New Zealand. Pacific Gamefish Foundation, P.O. Box 25115, Honolulu, Hawaii, U.S.A. 96825

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A Comparison of CO2 Excretion IA Spontaneously Ventilating Blood Perfused Trout Preparation and Salineperfused Gill Preparations: Contribution of the Branchial Epithelium and Red Blood Cell

1. 1. Amiloride (10−4 M) inhibited sodium uptake in rainbow trout by 78% and was associated with a pronounced acidosis and decreases in both plasma total CO2 ( T coco2)* and [HCO3−].

2. 2. 4-acetamido-4′-iso-thiocyanatostilbene-2,2′ disulphonic acid (SITS) (10−4M) in the bathing medium inhibited chloride uptake by 66% and following 6 h a significant decrease in plasma [H+] and significant increases in T COCO2 and [HCO3−] were observed.

3. 3. Inhibition of chloride uptake (50%) with external sodium bicarbonate (12 mM) resulted in a more rapid and pronounced alkalosis than did SITS inhibition.

4. 4. Hypercapnic acidosis had no significant effect on the rates of branchial sodium and chloride uptake.

5. 5. Increasing the concentration of sodium in the bathing water resulted in a less pronounced acidosis and a more rapid pH recovery during hypercapnia.

6. 6. These results are discussed with reference to the gill as an acid-base regulating structure. These findings are consistent with a gill model previously presented by Haswell, Randall & Perry (1980).

Branchial Ionic Uptake and Acid-Base Regulation in the Rainbow Trout, Salmo Gairdneri

Adult Pacific spiny dogfish (Squalus acanthias) were exposed to acute (approximately 20 min) hypercarbia while we monitored arterial blood pressure, systemic vascular resistance (RS), cardiac output (V . b) and frequency (fH) as well as ventilatory amplitude (VAMP) and frequency (fV). Separate series of experiments were conducted on control, atropinized (100 nmol kg -1 ) and branchially denervated fish to investigate putative CO2-chemoreceptive sites on the gills and their link to the autonomic nervous system and cardiorespiratory reflexes. In untreated fish, moderate hypercarbia (water CO2 partial pressure; PwCO•=6.4±0.1 mmHg) (1 mmHg= 0.133 kPa) elicited significant increases in VAMP (of approximately 92 %) and fV (of approximately 18 %) as well as decreases in fH (of approximately 64 %), V . b (approximately 29 %) and arterial blood pressure (of approximately 11 %); RS did not change significantly. Denervation of the branchial branches of cranial nerves IX and X to the pseudobranch and each gill arch eliminated all cardiorespiratory responses to hypercarbia. Prior administration of the muscarinic receptor antagonist atropine also abolished the hypercarbia-induced ventilatory responses and virtually eliminated all CO2-elicited cardiovascular adjustments. Although the atropinized dogfish displayed a hypercarbic bradycardia, the magnitude of the response was significantly attenuated (36±6 % decrease in fH in controls versus 9±2 % decrease in atropinized fish; means ± S.E.M.). Thus, the results of the present study reveal the presence of gill CO2 chemoreceptors in dogfish that are linked to numerous cardiorespiratory reflexes. In addition, because all cardiorespiratory responses to hypercarbia were abolished or attenuated by atropine, the CO 2 chemoreception process and/or one or more downstream elements probably involve cholinergic (muscarinic) neurotransmission.

Branchial CO(2) receptors and cardiorespiratory adjustments during hypercarbia in Pacific spiny dogfish (Squalus acanthias).

A spontaneously ventilating, blood-perfused trout preparation was used to examine gas exchange across the gills. The blood flow rate and input oxygen content to the branchial circulation were manipulated to assess the contributions of perfusion and diffusion limitations to oxygen transfer. Increases in the flow rate (Q), or the haematocrit (Hct) were positively correlated with increases in the oxygen uptake across the gills (M g, O g, O2 ). Manipulation of pulse pressure or frequency of the pump, with no changes to Q had no effect on M g, O g, O2 . Addition of adrenaline (1 × 10 −6 M) to the blood also did not effect M g, O g, O2 . Calculations of cardiac output from the Fick principle always yielded values which were overestimates of the actual cardiac output (Q) set by the mechanical pump. The difference between the measured oxygen uptake by the fish from the water (Vg 1, O 1, O2 ) and the amount of oxygen transferred to the blood across the gills (Vg 1, O 1, O2 ) was a reflection of gill tissue metabolism. It is concluded that trout gills, like mammalian lungs, are primarily perfusion limited for oxygen uptake under resting normoxic conditions, but decreases in diffusion limitations come into play under stress conditions, such as environmental hypoxia or exercise. Note:

Oxygen Uptake in a Spontaneously Ventilating, Blood-Perfused Trout Preparation

During the passage of blood through the fish gill, large oscillations in oxygen and carbon dioxide content occur. Although the increase in oxygen content is related to oxygen binding by red blood cells, the fall in carbon dioxide content is independent of red blood cells and their complement of carbonic anhydrase. This loss of venous carbon dioxide content is primarily the result of the movement of plasma bicarbonate into the gill epithelium, where it subsequently can be converted to molecular carbon dioxide by branchial carbonic anhydrase. The ultimate control of the bicarbonate flux and hence plasma hydrogen ion regulation is coupled to salt movements also occurring in the fish gill. This evidence in conjunction with carbonic anhydrase localization studies makes it possible to formulate a model capable of explaining acid-base regulation as well as salt transport in freshwater- or seawater-adapted fish. In light of this model the role of the “chloride cell” is discussed.

S. F. Perry

Papers

A Comparison of CO2 Excretion IA Spontaneously Ventilating Blood Perfused Trout Preparation and Salineperfused Gill Preparations: Contribution of the Branchial Epithelium and Red Blood Cell

Branchial Ionic Uptake and Acid-Base Regulation in the Rainbow Trout, Salmo Gairdneri

Branchial CO(2) receptors and cardiorespiratory adjustments during hypercarbia in Pacific spiny dogfish (Squalus acanthias).

Oxygen Uptake in a Spontaneously Ventilating, Blood-Perfused Trout Preparation

Fish gill carbonic anhydrase: acid-base regulation or salt transport?