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Aim: Although zebrafish are gaining popularity as biomedical models of cardiovascular disease, our understanding of their cardiac control mechanisms is fragmentary. Our goal was to clarify the controversial role of the ß1-adrenergic receptor (AR) in the regulation of heart rate in zebrafish., Methods: CRISPR-Cas9 was used to delete the adrb1 gene in zebrafish allowing us to generate a stable adrb1 -/- line. Larval heart rates were measured during pharmacological protocols and with exposure to hypercapnia. Expression of the five zebrafish adrb genes were measured in larval zebrafish hearts using qPCR., Results: Compared with genetically matched wild-types (adrb1 +/+ ), adrb1 -/- larvae exhibited ~20 beats min -1 lower heart rate, measured from 2 to 21 days post-fertilization (dpf). Nevertheless, adrb1 -/- larvae exhibited preserved positive chronotropic responses to pharmacological treatment with AR agonists (adrenaline, noradrenaline, isoproterenol), which were blocked by propranolol (general ß-AR antagonist). Regardless of genotype, larvae exhibited similar increases in heart rate in response to hypercapnia (1% CO 2 ) at 5 dpf, but tachycardia was blunted in adrb1 -/- larvae at 6 dpf. adrb1 gene expression was abolished in the hearts of adrb1 -/- larvae, confirming successful knockout. While gene expression of adrb2a and adrb3a was unchanged, adrb2b and adrb3b mRNA levels increased in adrb1 -/- larval hearts., Conclusion: Despite adrb1 contributing to the setting of resting heart rate in larvae, it is not strictly essential for zebrafish, as we generated a viable and breeding adrb1 -/- line. The chronotropic effects of adrenergic stimulation persist in adrb1 -/- zebrafish, likely due to the upregulation of other ß-AR subtypes., (© 2022 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Neuroepithelial cells (NECs) of the fish gill are respiratory chemoreceptors that detect changes in O2 and CO2/H(+) and are homologous to type I cells of the mammalian carotid body. In zebrafish (Danio rerio), stimulation of NECs by hypoxia or hypercapnia initiates inhibition of K(+) channels and subsequent membrane depolarisation. The goal of the present study was to further elucidate, in zebrafish NECs, the signalling pathways that underlie CO2/H(+) sensing and generate intracellular Ca(2+) ([Ca(2+)]i) signals. Breathing frequency was elevated maximally in fish exposed to 5 % CO2 (~37.5 mmHg). Measurement of [Ca(2+)]i in isolated NECs using Fura-2 imaging indicated that [Ca(2+)]i increased in response to acidic hypercapnia (5 % CO2, pH 6.6) and isocapnic acidosis (normocapnia, pH 6.6), but not to isohydric hypercapnia (5 % CO2, pH 7.6). Measurement of intracellular pH (pHi) using BCECF demonstrated a rapid decrease in pHi in response to acidic and isohydric hypercapnia, while isocapnic acidosis produced a smaller change in pHi. Intracellular acidification was reduced by the carbonic anhydrase inhibitor, acetazolamide, without affecting [Ca(2+)]i responses. Moreover, intracellular acidification using acetate (at constant extracellular pH) was without effect on [Ca(2+)]i. The acid-induced increase in [Ca(2+)]i persisted in the absence of extracellular Ca(2+) and was unaffected by Ca(2+) channel blockers (Cd(2+), Ni(2+) or nifedipine). The results of this study demonstrate that, unlike type I cells, extracellular H(+) is critical to the hypercapnia-induced increase in [Ca(2+)]i in NECs. The increase in [Ca(2+)]i occurs independently of pHi and appears to originate primarily from Ca(2+) derived from intracellular stores.

The presence of an interlamellar cell mass (ILCM) on the gills of goldfish significantly decreases the functional lamellar surface area and increases the diffusion distance for gas transfer and thus may impose a serious challenge for the transfer of respiratory gases (O₂ and CO₂). Here we tested the hypothesis that the presence of the ILCM in goldfish acclimated to 7°C impedes the uptake of O2 and excretion of CO₂. While Pa(O₂) remained unaltered, the baseline values of Pa(CO)₂ were significantly higher in goldfish at 7°C with ILCM present (5.55 ± 0.54 mmHg; mean ± SEM) than in goldfish at 25°C without the ILCM (3.98 ± 0.18 mmHg). Carbonic anhydrase (CA) injections relieved the apparent diffusion limitation imposed by the presence of the ILCM on CO₂ excretion (Pw(CO₂) levels dropped to 3.07 ± 0.32 mmHg). Interestingly, the exposure of fish to acute hypoxia evoked similar changes in Pa(O₂) at the two acclimation temperatures. Ethanol (EtOH) exposure was also used as a tool to further investigate the potential effects of the ILCM on branchial solute transfer. The results showed that the ILCM does not impede EtOH uptake in 7°C goldfish. Overall, the results of this study demonstrate that the remodelling of the goldfish gill associated with acclimation to 7°C water, while increasing Pw(CO₂) , has minimal impact on branchial O2 transfer., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The plainfin midshipman (Porichthys notatus) possesses an aglomerular kidney and like other marine teleosts, secretes base into the intestine to aid water absorption. Each of these features could potentially influence acid-base regulation during respiratory acidosis either by facilitating or constraining HCO(3)(-) accumulation, respectively. Thus, in the present study, we evaluated the capacity of P. notatus to regulate blood acid-base status during exposure to increasing levels of hypercapnia (nominally 1-5% CO(2)). Fish exhibited a well-developed ability to increase plasma HCO(3)(-) levels with values of 39.8 ± 2.8 mmol l(-1) being achieved at the most severe stage of hypercapnic exposure (arterial blood PCO(2) = 21.9 ± 1.7 mmHg). Consequently, blood pH, while lowered by 0.15 units (pH = 7.63 ± 0.06) during the final step of hypercapnia, was regulated far above values predicted by chemical buffering (predicted pH = 7.0). The accumulation of plasma HCO(3)(-) during hypercapnia was associated with marked increases in branchial net acid excretion (J (NET)H(+)) owing exclusively to increases in the titratable alkalinity component; total ammonia excretion was actually reduced during hypercapnia. The increase in J (NET)H(+) was accompanied by increases in branchial carbonic anhydrase (CA) enzymatic activity (2.8×) and CA protein levels (1.6×); branchial Na(+)/K(+)-ATPase activity was unaffected. Rectal fluids sampled from control fish contained on average HCO(3)(-) concentrations of 92.2 ± 4.8 mmol l(-1). At the highest level of hypercapnia, rectal fluid HCO(3)(-) levels were increased significantly to 141.8 ± 7.4 mmol l(-1) but returned to control levels during post-hypercapnia recovery (96.0 ± 13.2 mmol l(-1)). Thus, the impressive accumulation of plasma HCO(3)(-) to compensate for hypercapnic acidosis occurred against a backdrop of increasing intestinal HCO(3)(-) excretion. Based on in vitro measurements of intestinal base secretion in Ussing chambers, it would appear that P. notatus did not respond by minimizing base loss during hypercapnia; the increases in base flux across the intestinal epithelium in response to alterations in serosal HCO(3)(-) concentration were similar in preparations obtained from control or hypercapnic fish. Fish returned to normocapnia developed profound metabolic alkalosis owing to unusually slow clearance of the accumulated plasma HCO(3)(-). The apparent inability of P. notatus to effectively excrete HCO(3)(-) following hypercapnia may reflect its aglomerular (i.e., non-filtering) kidney coupled with the normally low rates of urine production in marine teleosts.

Fish aquaculture facilities strive to maintain abiotic conditions that optimise somatic growth. Two physiological systems that are critically linked to environmental abiotic factors are ion and acid‐base regulation. Given the detrimental effects of ionic and acid‐base imbalances coupled with the potential energetic costs of ion and acid‐base regulation, it is important to understand how changes in abiotic variables such as salinity, temperature and pH alter ionoregulatory and acid‐base physiology. The gill is the predominant site of ion and acid‐base regulation, two linked processes that are vital to the health of fishes. Most species maintain ion levels in the blood plasma within a narrow range, independent of external ion concentrations. In freshwater (FW), fishes must actively take up ions from their dilute environment to counteract passive ion losses, while in seawater (SW), ion excretion serves to counteract passive salt gain. Ion uptake (FW) and excretion (SW) are coordinated by different subtypes of ion‐transporting cells (ionocytes) expressed in the gill epithelium. In this review, we discuss the function of FW and SW ionocytes in the movement of ions across the gill epithelium and address other features of the gill that are integral to ionoregulation. We also discuss the mechanisms of acid‐base regulation by the gill, which is intimately related to ion regulation because it is coordinated by Na+‐ and Cl−‐linked fluxes of acid (H+) and base (HCO3‐) equivalents. These fundamental physiological principles are integral in understanding how fishes in aquaculture respond to relevant disturbances that may occur under culture conditions. [ABSTRACT FROM AUTHOR]

The ontogeny of carbon dioxide (C02) sensing in zebrafish (Danio rerio) has not been examined. In this study, C02-mediated increases in heart rate were used to gauge the capacity of zebrafish larvae to sense C02. C02 is thought to be detected via neuroepithelial cells (NECs), which are homologous to mammalian carotid body glomus cells. Larvae at 5 days post-fertilization (d.p.f.) exhibited tachycardia when exposed for 30 min to 0.75% C02 (-5.63 mmHg); at 7 d.p.f., tachycardia was elicited by 0.5% C02 (-3.75 mmHg). Based on pharmacological evidence using p-adrenergic receptor (P-AR) antagonists, and confirmed by Pi-AR translational gene knockdown using morpholinos, the reflex tachycardia accompanying hypercapnia was probably mediated by the interaction of catecholamines with cardiac Pi receptors. Because the cardiac response to hypercapnia was abolished by the ganglionic blocker hexamethonium, it is probable that the reflex cardio-acceleration was mediated by catecholamines derived from sympathetic adrenergic neurons. Owing to its likely role in facilitating intracellular acidification during exposure to hypercapnia, it was hypothesized that carbonic anhydrase (CA) is involved in C02 sensing, and that inhibition of CA activity would blunt the downstream responses. Indeed, the cardiac response to hypercapnia (0.75% C02) was reduced in fish at 5 d.p.f. exposed to acetazolamide, a CA inhibitor, and in fish experiencing zCAc (CA2-like a) knockdown. Successful knockdown of zCAc was confirmed by CA activity measurements, western blotting and immunocytochemistry. Co-injection of embryos with zCAc morpholino and mRNA modified at the morpholino binding site restored normal levels of CA activity and protein levels, and restored (rescued) the usual cardiac responses to hypercapnia. These data, combined with the finding that zCAc is expressed in NECs located on the skin, suggest that the afferent limb of the C02-induced cardiac reflex in zebrafish larvae is initiated by coetaneous C02-sensing neuroepithelial cells. [ABSTRACT FROM AUTHOR]

Abstract: Fish possess chemoreceptors able to sense increasing levels of ambient CO2 and initiate various cardiorespiratory reflexes including hyperventilation and bradycardia. These chemoreceptors are localized predominantly to the gills, are oriented to sense the external environment and typically are stimulated by changes in environmental molecular CO2 rather than H+ (although increasing H+ may be the proximate intracellular stimulus). In zebrafish, a subset of branchial neuroepithelial cells (NECs) act as bimodal sensors of CO2 and O2, similar to the Type I (glomus) cells of the mammalian carotid body. Like O2 sensing, the mechanisms underlying CO2 detection involve the inhibition of a background K+ current leading to membrane depolarization and subsequent elevation of intracellular Ca2+ levels. Carbonic anhydrase, by catalysing the hydration of CO2 to H+ and HCO3 −, appears to play a critical role in reducing NEC response times and increasing the magnitude of membrane depolarization accompanying hypercapnia. In larval zebrafish, CA activity is essential for the rapid development of hypercapnic bradycardia. [Copyright &y& Elsevier]

Experiments were conducted on rainbow trout to determine the impact of dietary salt on arterial blood pressure. After 4-6 wk, fish fed a salt-enriched diet exhibited a 37% elevation of dorsal aortic pressure (from 23.8 ± 1.2 to 32.6 ± 1.4 mmHg) and an 18% increase in ventral aortic pressure (from 33.0 ± 1.5 to 38.9 ± 1.3 mmHg). The hypertension presumably reflected the increase in cardiac output (from 31.0 ± 0.8 to 36.4 ± 2.2 ml·min-1·kg-1) because systemic and branchial resistances were statistically unaltered by salt feeding. The chronic hypertension was associated with a decrease in the pressor responses of the systemic vasculature to catecholamines and hypercapnia in the salt-fed fish. The reduction in α-adrenergic responsiveness of the systemic vasculature is consistent with desensitization or loss of functional α-adrenoceptors (α-ARs). In support of this idea, the salt-fed fish exhibited significantly decreased levels of α1D-AR mRNA in the dorsal aorta and the afferent (ABA) and efferent branchial arteries (EBA). In contrast, however, the results obtained from norepinephrine dose-response curves for EBA and ABA vascular rings in vitro did not provide evidence for loss of function of branchial artery α1-ARs in the salt-fed fish. Indeed, the EC50 for the EBA norepinephrine dose-response curve was significantly reduced (from 3.75 × 10-7 to 2.12 × 10-7 M) in the salt-fed fish, indicating an increase in the binding affinity of the α1-ARs. [ABSTRACT FROM AUTHOR]

We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC rnRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+] = 20-30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (~7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an ~100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells. [ABSTRACT FROM AUTHOR]

Deals with a study which cloned and characterized the B subunit of the trout H...V-ATPase to examine by northern analysis the expression and tissue distribution of the B subunit mRNA during external hypercapnia. Materials and methods; Results and discussion.

The tambaqui (Colossoma macropomum), migrates annually between whitewater and blackwater rivers of the Amazon. Unlike the whitewater, blackwater is characterized by higher levels of dissolved organic carbon (DOC), including humic acids (HA). Because humic substances impair sensory processes, the current study tested the hypothesis that O 2 and/or CO 2 chemoreception is impeded in blackwater owing to the presence of HA. Thus, the ventilatory responses of tambaqui to hypoxia or hypercapnia were assessed in well water transported from Manaus, local blackwater, and in well water containing HA either extracted from Rio Negro water or obtained commercially (Sigma Aldrich; SA). In well water, tambaqui exhibited typical hyperventilatory responses to hypoxia or hypercapnia. These responses were prevented by simultaneously exposing fish to SA HA (20 mg l−1). The negative effects of SA HA on ventilation were prevented when natural DOC (30 mg l−1; extracted from Rio Negro water after first removing the endogenous HA fraction) was added concurrently, indicating a protective effect of this non-humic acid DOC fraction. The hyperventilatory responses were unaffected during acute exposure or after acclimation of fish to Rio Negro water. HA extracted from Rio Negro water did not impair the hyperventilatory responses to hypoxia or hypercapnia. This study, while demonstrating a negative effect of SA HA derived from peat (coal) on the control of breathing in tambaqui, failed to reveal any detrimental consequences of HA (derived from the decomposition of a variety of lignin-rich plants) naturally occurring in the blackwaters of the Rio Negro. • This study tested the hypothesis that reflex ventilatory responses to would be impaired in Tambaqui in natural blackwater. • Commercial humic acid impaired the ventilatory response to hypoxia or hypercapnia. • Natural blackwater from the Rio Negro or humic acid derived from blackwater was without effect. • The negative effects of commercial humic acid were absent in the presence of DOC (minus the humic acid fraction) [ABSTRACT FROM AUTHOR]

Abstract: The neurochemical, serotonin (5-hydroxytryptamine; 5-HT) is involved in the regulation of toadfish pulsatile urea excretion as well as the teleost hypoxia response. Thus, the goal of this study was to determine whether environmental conditions that activate branchial chemoreceptors also trigger pulsatile urea excretion in toadfish, since environmental dissolved oxygen levels in a typical toadfish habitat show significant diel fluctuations, often reaching hypoxic conditions at dawn. Toadfish were fitted with arterial, venous and/or buccal catheters and were exposed to various environmental conditions, and/or injected with the O2 chemoreceptor agonist NaCN or the 5-HT2 receptor agonist α-methyl-5HT. Arterial PO2, as well as ammonia and urea excretion were monitored. Natural fluctuations in arterial PO2 levels in toadfish did not correlate with the occurrence of a urea pulse. Chronic exposure (24 h) of toadfish to hyperoxia was without effect on nitrogen excretion, however, exposure to hypoxia caused a significant reduction in the frequency of urea pulses, and exposure to hypercapnia resulted in a reduction in the percentage of nitrogen waste excreted as urea. Of toadfish exposed acutely to hypoxia, 20% pulsed within 1 h, whereas none pulsed after normoxic or hypercapnic treatments. Furthermore, 20% of fish injected intravenously with NaCN pulsed within 1 h of injection, but no fish pulsed after injection of NaCN into the buccal cavity. To test whether environmental conditions affected 5-HT2 receptors, toadfish were injected with α-methyl-5HT, which elicits urea pulses in toadfish. No significant differences in pulse size occurred among the various environmental treatments. Our findings suggest that neither the environmental conditions of hypoxia, hyperoxia or hypercapnia, nor direct branchial chemoreceptor activation by NaCN play a major role in the regulation of pulsatile urea excretion in toadfish. [Copyright &y& Elsevier]