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Aim: To identify the physiological role of the acid-base sensing enzyme, soluble adenylyl cyclase (sAC), in red blood cells (RBC) of the model teleost fish, rainbow trout., Methods: We used: (i) super-resolution microscopy to determine the subcellular location of sAC protein; (ii) live-cell imaging of RBC intracellular pH (pH i ) with specific sAC inhibition (KH7 or LRE1) to determine its role in cellular acid-base regulation; (iii) spectrophotometric measurements of haemoglobin-oxygen (Hb-O 2 ) binding in steady-state conditions; and (iv) during simulated arterial-venous transit, to determine the role of sAC in systemic O 2 transport., Results: Distinct pools of sAC protein were detected in the RBC cytoplasm, at the plasma membrane and within the nucleus. Inhibition of sAC decreased the setpoint for RBC pH i regulation by ~0.25 pH units compared to controls, and slowed the rates of RBC pH i recovery after an acid-base disturbance. RBC pH i recovery was entirely through the anion exchanger (AE) that was in part regulated by HCO 3 - -dependent sAC signaling. Inhibition of sAC decreased Hb-O 2 affinity during a respiratory acidosis compared to controls and reduced the cooperativity of O 2 binding. During in vitro simulations of arterial-venous transit, sAC inhibition decreased the amount of O 2 that is unloaded by ~11%., Conclusion: sAC represents a novel acid-base sensor in the RBCs of rainbow trout, where it participates in the modulation of RBC pH i and blood O 2 transport though the regulation of AE activity. If substantiated in other species, these findings may have broad implications for our understanding of cardiovascular physiology in vertebrates., (© 2024 The Author(s). Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Marine fishes excrete excess H + using basolateral Na + -K + -ATPase (NKA) and apical Na + /H + exchanger 3 (NHE3) in gill ionocytes. However, the mechanisms that regulate H + excretion during exposure to environmentally relevant hypercapnia (ERH) remain poorly understood. Here, we explored transcriptomic, proteomic, and cellular responses in gills of juvenile splitnose rockfish ( Sebastes diploproa ) exposed to 3 days of ERH conditions (pH ∼7.5, ∼1,600 μatm Pco 2 ). Blood pH was fully regulated at ∼7.75 despite a lack of significant changes in gill 1 ) mRNAs coding for proteins involved in blood acid-base regulation, 2 ) total NKA and NHE3 protein abundance, and 3 ) ionocyte density. However, ERH-exposed rockfish demonstrated increased NKA and NHE3 abundance on the ionocyte plasma membrane coupled with wider apical membranes and greater extension of apical microvilli. The observed gill ionocyte remodeling is consistent with enhanced H + excretion that maintains blood pH homeostasis during exposure to ERH and does not necessitate changes at the expression or translation levels. These mechanisms of phenotypic plasticity may allow fishes to regulate blood pH during environmentally relevant acid-base challenges and thus have important implications for both understanding how organisms respond to climate change and for selecting appropriate metrics to evaluate its impact on marine ecosystems. NEW & NOTEWORTHY Splitnose rockfish exposed to environmentally relevant hypercapnia utilize existing proteins (rather than generate additional machinery) to maintain homeostasis.

The inner ear of teleost fish regulates the ionic and acid-base chemistry and secretes protein matrix into the endolymph to facilitate otolith biomineralization, which is used to maintain vestibular and auditory functions. The otolith is biomineralized in a concentric ring pattern corresponding to seasonal growth, and this calcium carbonate (CaCO 3 ) polycrystal has become a vital aging and life-history tool for fishery managers, ecologists, and conservation biologists. Moreover, biomineralization patterns are sensitive to environmental variability including climate change, thereby threatening the accuracy and relevance of otolith-reliant toolkits. However, the cellular biology of the inner ear is poorly characterized, which is a hurdle for a mechanistic understanding of the underlying processes. This study provides a systematic characterization of the cell types in the inner ear of splitnose rockfish ( Sebastes diploproa ). Scanning electron microscopy revealed the apical morphologies of six inner ear cell types. In addition, immunostaining and confocal microscopy characterized the expression and subcellular localization of the proteins Na + -K + -ATPase, carbonic anhydrase, V-type H + -ATPase, Na + -K + -2Cl - -cotransporter, otolith matrix protein 1, and otolin-1 in six inner ear cell types bordering the endolymph. This fundamental cytological characterization of the rockfish inner ear epithelium illustrates the intricate physiological processes involved in otolith biomineralization and highlights how greater mechanistic understanding is necessary to predict their multistressor responses to future climate change.