Preferential pHi Regulation Research Articles

Preferential intracellular pH regulation is a common trait amongst fishes exposed to high environmental CO2.

Acute (<96 h) exposure to elevated environmental CO2 (hypercarbia) induces a pH disturbance in fishes that is often compensated by concurrent recovery of intracellular and extracellular pH (pHi and pHe, respectively; coupled pH regulation). However, coupled pH regulation may be limited at CO2 partial pressure (PCO2 ) tensions far below levels that some fishes naturally encounter. Previously, four hypercarbia-tolerant fishes had been shown to completely and rapidly regulate heart, brain, liver and white muscle pHi during acute exposure to >4 kPa PCO2 (preferential pHi regulation) before pHe compensation was observed. Here, we test the hypothesis that preferential pHi regulation is a widespread strategy of acid-base regulation among fish by measuring pHi regulation in 10 different fish species that are broadly phylogenetically separated, spanning six orders, eight families and 10 genera. Contrary to previous views, we show that preferential pHi regulation is the most common strategy for acid-base regulation within these fishes during exposure to severe acute hypercarbia and that this strategy is associated with increased hypercarbia tolerance. This suggests that preferential pHi regulation may confer tolerance to the respiratory acidosis associated with hypercarbia, and we propose that it is an exaptation that facilitated key evolutionary transitions in vertebrate evolution, such as the evolution of air breathing.

American alligator (Alligator mississippiensis) embryos tightly regulate intracellular pH during a severe acidosis

Crocodilian nests naturally experience high CO2(hypercarbia), which leads to increased blood Pco2and reduced blood pH (pHe) in embryos; their response to acid–base challenges is not known. During acute hypercarbia, snapping turtle embryos preferentially regulate tissue pH (pHi) against pHereductions. This is proposed to be associated with CO2tolerance in reptilian embryos and is not found in adults. In the present study, we investigated pH regulation in American alligator (Alligator mississippiensis (Daudin, 1802)) embryos exposed to 1 h of hypercarbia hypoxia (13 kPa Pco2, 9 kPa Po2). Hypercarbia hypoxia reduced pHeby 0.42 pH unit, while heart and brain pHiincreased, with no change in the pHiof other tissues. The results indicate that American alligator embryos preferentially regulate pHi, similar to snapping turtle embryos, which represents a markedly different strategy of acid–base regulation than what is observed in adult reptiles. These findings suggest that preferential pHiregulation may be a strategy of acid–base regulation used by embryonic reptiles.

Water pH limits extracellular but not intracellular pH compensation in the CO2-tolerant freshwater fish Pangasianodon hypophthalmus.

Preferentially regulating intracellular pH (pHi) confers exceptional CO2 tolerance on fish, but is often associated with reductions in extracellular pH (pHe) compensation. It is unknown whether these reductions are due to intrinsically lower capacities for pHe compensation, hypercarbia-induced reductions in water pH or other factors. To test how water pH affects capacities and strategies for pH compensation, we exposed the CO2-tolerant fish Pangasianodon hypophthalmus to 3 kPa PCO2 for 20 h at an ecologically relevant water pH of 4.5 or 5.8. Brain, heart and liver pHi was preferentially regulated in both treatments. However, blood pHe compensation was severely reduced at water pH 4.5 but not 5.8. This suggests that low water pH limits acute pHe but not pHi compensation in fishes preferentially regulating pHi Hypercarbia-induced reductions in water pH might therefore underlie the unexplained reductions to pHe compensation in fishes preferentially regulating pHi, and may increase selection for preferential pHi regulation.

Preferential intracellular pH regulation: hypotheses and perspectives

The regulation of vertebrate acid-base balance during acute episodes of elevated internal PCO2 is typically characterized by extracellular pH (pHe) regulation. Changes in pHe are associated with qualitatively similar changes in intracellular tissue pH (pHi) as the two are typically coupled, referred to as 'coupled pH regulation'. However, not all vertebrates rely on coupled pH regulation; instead, some preferentially regulate pHi against severe and maintained reductions in pHe Preferential pHi regulation has been identified in several adult fish species and an aquatic amphibian, but never in adult amniotes. Recently, common snapping turtles were observed to preferentially regulate pHi during development; the pattern of acid-base regulation in these species shifts from preferential pHi regulation in embryos to coupled pH regulation in adults. In this Commentary, we discuss the hypothesis that preferential pHi regulation may be a general strategy employed by vertebrate embryos in order to maintain acid-base homeostasis during severe acute acid-base disturbances. In adult vertebrates, the retention or loss of preferential pHi regulation may depend on selection pressures associated with the environment inhabited and/or the severity of acid-base regulatory challenges to which they are exposed. We also consider the idea that the retention of preferential pHi regulation into adulthood may have been a key event in vertebrate evolution, with implications for the invasion of freshwater habitats, the evolution of air breathing and the transition of vertebrates from water to land.

Embryonic common snapping turtles (Chelydra serpentina) preferentially regulate intracellular tissue pH during acid-base challenges.

The nests of embryonic turtles naturally experience elevated CO2 (hypercarbia), which leads to increased blood PCO2 and a respiratory acidosis, resulting in reduced blood pH [extracellular pH (pHe)]. Some fishes preferentially regulate tissue pH [intracellular pH (pHi)] against changes in pHe; this has been proposed to be associated with exceptional CO2 tolerance and has never been identified in amniotes. As embryonic turtles may be CO2 tolerant based on nesting strategy, we hypothesized that they preferentially regulate pHi, conferring tolerance to severe acute acid-base challenges. This hypothesis was tested by investigating pH regulation in common snapping turtles (Chelydra serpentina) reared in normoxia then exposed to hypercarbia (13 kPa PCO2 ) for 1 h at three developmental ages: 70% and 90% of incubation, and yearlings. Hypercarbia reduced pHe but not pHi, at all developmental ages. At 70% of incubation, pHe was depressed by 0.324 pH units while pHi of brain, white muscle and lung increased; heart, liver and kidney pHi remained unchanged. At 90% of incubation, pHe was depressed by 0.352 pH units but heart pHi increased with no change in pHi of other tissues. Yearlings exhibited a pHe reduction of 0.235 pH units but had no changes in pHi of any tissues. The results indicate common snapping turtles preferentially regulate pHi during development, but the degree of response is reduced throughout development. This is the first time preferential pHi regulation has been identified in an amniote. These findings may provide insight into the evolution of acid-base homeostasis during development of amniotes, and vertebrates in general.

Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid-base disturbances in the armoured catfish, Pterygoplichthys pardalis.

Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid-base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2-3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15-7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid-base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.

Acid–base and ion balance in fishes with bimodal respiration

The evolution of air breathing during the Devonian provided early fishes with bimodal respiration with a stable O2 supply from air. This was, however, probably associated with challenges and trade-offs in terms of acid-base balance and ionoregulation due to reduced gill:water interaction and changes in gill morphology associated with air breathing. While many aspects of acid-base and ionoregulation in air-breathing fishes are similar to water breathers, the specific cellular and molecular mechanisms involved remain largely unstudied. In general, reduced ionic permeability appears to be an important adaptation in the few bimodal fishes investigated but it is not known if this is a general characteristic. The kidney appears to play an important role in minimizing ion loss to the freshwater environment in the few species investigated, and while ion uptake across the gut is probably important, it has been largely unexplored. In general, air breathing in facultative air-breathing fishes is associated with an acid-base disturbance, resulting in an increased partial pressure of arterial CO2 and a reduction in extracellular pH (pHE ); however, several fishes appear to be capable of tightly regulating tissue intracellular pH (pHI ), despite a large sustained reduction in pHE , a trait termed preferential pHI regulation. Further studies are needed to determine whether preferential pHI regulation is a general trait among bimodal fishes and if this confers reduced sensitivity to acid-base disturbances, including those induced by hypercarbia, exhaustive exercise and hypoxia or anoxia. Additionally, elucidating the cellular and molecular mechanisms may yield insight into whether preferential pHI regulation is a trait ultimately associated with the early evolution of air breathing in vertebrates.

Metabolic changes associated with acid–base regulation during hypercarbia in the CO 2-tolerant chondrostean, white sturgeon ( Acipenser transmontanus)

CO 2 tolerance in white sturgeon is associated with the ability to tightly regulate intracellular pH (pHi) despite a large reduction in extracellular pH (pHe) termed preferential pHi regulation. How this regulatory response affects whole animal metabolic rate is unknown. Accordingly, we characterized oxygen consumption rate ( M ˙ O 2 ) and metabolically-relevant organismal and cellular responses in white sturgeon during exposure to hypercarbia. White sturgeon were able to protect intracellular pH (pHi) in liver and white muscle as early as 6 h (the earliest time period investigated) following exposure to severe (sub-lethal) hypercarbia (45 and 90 mmHg PCO 2). Sturgeon exposed to 15 and 30 mmHg PCO 2 exhibited pHe compensation and significant increases in M ˙ O 2 (up to 80% greater than control values). In contrast, severe hypercarbia (≥ 45 mmHg PCO 2) elicited an uncompensated reduction in pHe (up to ~ 1.0 pH units) and red blood cells (as great as ~ 0.5 pH units), and was accompanied by 30 and 60% reductions in M ˙ O 2 , respectively. While behavioral, respiratory and cellular responses to hypercarbia were observed, none corresponded well with the pattern or magnitude of changes in M ˙ O 2 . The findings of this research provide empirical support for the hypothesis that preferential pHi regulation is not metabolically costly, and thus may have been a strategy strongly selected for in fishes encountering short-term hypercarbia.

Intrinsic mechanical properties of the perfused armoured catfish heart with special reference to the effects of hypercapnic acidosis on maximum cardiac performance

The armoured catfish, Pterygoplichthys pardalis, is known to be extremely tolerant of environmental hypercarbia (elevated water CO(2) tensions), which occurs in their natural environment. In addition, previous studies have demonstrated that during exposure to hypercarbia, P. pardalis does not exhibit extracellular pH compensation and thus the heart and other organs must continue to function despite a severe extracellular acidosis. We used an in situ perfused heart preparation to determine the effects of an extracellular hypercapnic (elevated CO(2) in the animal) acidosis (1-7.5% CO(2)) on heart function, specifically cardiac output, power output, heart rate and stroke volume. The present study is the first to comprehensively examine cardiac function in an acidosis-tolerant teleost. When compared with control conditions, maximum cardiac performance was unaffected at levels of CO(2) as high as 5%, far exceeding the hypercapnic tolerance of other teleosts. Moreover, P. pardalis exhibited only a moderate decrease (approximately 35%) in cardiac performance when exposed to 7.5% CO(2), and full cardiac performance was restored in six out of seven hearts upon return to control conditions. Myocardial intracellular pH (pH(i)) was protected in situ, as has been found in vivo, and this protection extended to the highest level of CO(2) (7.5%) investigated. Thus, maintained heart function during a hypercapnic acidosis in P. pardalis is probably associated with preferential pH(i) regulation of the heart, but ultimately is not sufficient to prevent loss of cardiac function. Our findings suggest the need for further study to elucidate the mechanisms behind this remarkable cardiac hypercapnic tolerance.

10.2. Acid–base regulation during hypercapnia, exercise and anoxia in the armoured catfish, Liposarcus pardalis

During exposure to elevated water CO2 levels (hypercapnia), blood pH and tissue intracellular pH (pHi) in fish are initially reduced due to the rapid development of a respiratory acidosis. Blood pH recovery is associated with acid–base relevant ion transfer at the gills (i.e., Na+/H+ or Cl−/HCO3−), and generally occurs over 24–48 h during which time pHi recovery closely follows pH recovery of the blood. In the armoured catfish, Liposarcus pardalis, hypercapnia induces a large respiratory acidosis, but intracellular pH (pHi) of tissues such as the liver, heart and muscle is tightly regulated despite large reductions in plasma pH. Using an in situ perfused heart preparation, maximum cardiac performance (power output, cardiac output and stroke volume) was maintained at levels of up to 5% CO2 in L. pardalis. Maintenance of heart function under these conditions was associated with preferential pHi regulation. Following exhaustive exercise and 2 h of anoxia, there was little evidence for lactate incorporation into the skull or bony plates, but liver, heart and brain pHi were preferentially regulated despite a large uncompensated plasma acidosis, indicating that the type of acidosis does not alter the pattern of acid–base regulation in this species. This pattern of preferential pHi regulation differs markedly from that of most fish investigated to date; however, it has also been observed in two other facultative air-breathing fishes, Synbranchus marmoratus and Amia calva, and may be associated with the evolution of air-breathing.