Oxygen consumption and acid secretion in isolated gas gland cells of the European eel Anguilla anguilla

Journal of Comparative Physiology B https://doi.org/10.1007/s00360-022-01432-x ORIGINAL PAPER Oxygen consumption and acid secretion in isolated gas gland cells of the European eel Anguilla anguilla Victoria Drechsel1,3 · Gabriel Schneebauer1,4 · Adolf M. Sandbichler1,2 · Birgit Fiechtner1,2 · Bernd Pelster1,2 Received: 9 December 2021 / Revised: 18 February 2022 / Accepted: 23 February 2022 © The Author(s) 2022 Abstract Swimbladder gas gland cells are known to produce lactic acid required for the acidification of swimbladder blood and decreasing the oxygen carrying capacity of swimbladder blood, i.e., the onset of the Root effect. Gas gland cells have also been shown to metabolize glucose via the pentose phosphate shunt, but the role of the pentose phosphate shunt for acid secretion has not yet been evaluated. Similarly, aerobic metabolism of gas gland cells has been largely neglected so far. In the present study, we therefore simultaneously assessed the role of glycolysis and of the pentose phosphate shunt for acid secretion and recorded oxygen consumption of isolated swimbladder gas gland cells of the European eel. Presence of glucose was essential for acid secretion, and at glucose concentrations of about 1.5 mmol l−1 acid secretion of gas gland cells reached a maximum, indicating that glucose concentrations in swimbladder blood should not be limiting acid production and secretion under physiological conditions. The data revealed that most of the acid was produced in the glycolytic pathway, but a significant fraction was also contributed by the pentose phosphate shunt. Addition of glucose to gas gland cells incubated in a glucosefree medium resulted in a reduction of oxygen uptake. Inhibition of mitochondrial respiration significantly reduced oxygen consumption, but a fraction of mitochondria-independent respiration remained in presence of rotenone and antimycin A. In the presence of glucose, application of either iodo-acetate inhibiting glycolysis or 6-AN inhibiting the pentose phosphate shunt did not significantly affect oxygen uptake, indicating an independent regulation of oxidative phosphorylation and of acid production. Inhibition of the muscarinic acetylcholine receptor caused a slight elevation in acid secretion, while forskolin caused a concentration-dependent reduction in acid secretion, indicating muscarinic and c-AMP-dependent control of acid secretion in gas gland cells. Keywords Swimbladder · Metabolism · Oxygen consumption · European eel · Lactate Introduction Communicated by G. Heldmaier. Victoria Drechsel and Gabriel Schneebauer have contributed equally to this study. * Bernd Pelster 1 Institut für Zoologie, Leopold-Franzens-Universität Innsbruck, Technikerstr.25, 6020 Innsbruck, Austria 2 Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria 3 Animal Physiological Ecology, Institute of Evolution and Ecology, University of Tübingen, Tübingen, Germany 4 Institute for Human Genetics, Medical University Innsbruck, Innsbruck, Austria The energy metabolism of swimbladder gas gland cells of physoclist fish is peculiar in that they typically experience hyperoxic conditions, but glucose is mainly converted to lactic acid to generate sufficient protons for the acidification of swimbladder blood (D’Aoust 1970; Deck 1970; Pelster 1995b; Pelster and Scheid 1993). Lactic acid production and secretion are considered the main sources of swimbladder blood acidification. On acidification, hemoglobin releases oxygen (Root effect), which is essential for the generation of hyperoxic oxygen partial pressures (Pelster and Randall 1998; Pelster and Weber 1991; Pelster 2001, 2021) required to fill the swimbladder with oxygen, even under conditions of elevated hydrostatic pressure. The initial increase in oxygen partial pressure in a second step is multiplied by countercurrent concentration in the rete mirabile of the 13 Vol.:(0123456789) Journal of Comparative Physiology B swimbladder, so that hyperbaric oxygen partial pressures can be generated, sufficient to explain the secretion of oxygen against hydrostatic pressures of several 10 s or even more than 100 atmospheres (Kuhn et al. 1963; Pelster 2001). Gas gland cells of the American eel and of the gulf toadfish have been shown to produce CO2 in the pentose phosphate shunt (Pelster et al. 1994; Walsh and Milligan 1993). The C O2 diffuses along the partial pressure gradient into the swimbladder lumen, contributing to the filling of the swimbladder, but also into the blood, supporting the acidification of the blood. The possible contribution and the importance of the pentose phosphate shunt to acid secretion of gas gland cells and acidification of the blood, however, have never been addressed. In addition, NADPH is produced in the pentose phosphate shunt, which may affect the redox equilibrium in the cell. Analysis of a number of different fish species revealed that mitochondria are not numerous in gas gland cells (Dorn 1961; Copeland 1969; Jasinski and Kilarski 1969; Morris and Albright 1975), but enzyme activities of the aerobic metabolism (Ewart and Driedzic 1990; Pelster and Scheid 1991; Walsh and Milligan 1993) as well as oxygen uptake have been reported. Oxygen uptake of a saline perfused swimbladder preparation of the European eel, which did not secrete any gas into the swimbladder, was measured, and it was significantly lower than the oxygen uptake of blood perfused eel swimbladder tissue secreting gas into the swimbladder lumen. The rate of gas secretion was correlated to the rate of lactate formation, but the elevated oxygen uptake indicated that aerobic metabolism is also of importance in the active swimbladder (Pelster 1995b; Pelster and Scheid 1992). The role of aerobic metabolism, however, has not been carefully analyzed. Regulation of gas gland cell metabolism, a crucial parameter influencing the rate of gas secretion (Pelster and Scheid 1992), remains enigmatic. Cholinergic nerve endings have been detected in swimbladder tissue of cod (McLean and Nilsson 1981; Nilsson 1983). Acetylcholine, respective the cholinomimetic drug carbachol, have been reported to stimulate lactate production in cod, but the effect was not pronounced and particularly low at acidic pH of 6.5, which is to be expected in active gas gland cells (Ewart and Driedzic 1990; Pelster 1995a). Activation of adenylate cyclase in turn has been shown to reduce the rate of acid secretion in primary cultured eel gas gland cells (Pelster and Pott 1996). An elevation of acid secretion, however, could not be elicited in these experiments. Aim of the present study therefore was to analyze oxygen consumption of isolated gas gland cells of the European eel and to assess the importance of lactate formation and of the pentose phosphate shunt for acid secretion of the cells. Materials and methods Animals European eels Anguilla anguilla were caught by local fishermen in Lake Constance, Bregenz, Austria, and kept in (...truncated)