Janet Genz

Effects of salinity on intestinal bicarbonate secretion and compensatory regulation of acid-base balance in Opsanus beta.

SUMMARY Marine teleosts have extracellular fluids less concentrated than their environment, resulting in continual water loss, which is compensated for by drinking, with intestinal water absorption driven by NaCl uptake. Absorption of Cl– occurs in part by apical Cl–/HCO3– exchange, with HCO3– provided by transepithelial transport and/or by carbonic anhydrase-mediated hydration of endogenous epithelial CO2. Hydration of CO2 also liberates H+, which is transported across the basolateral membrane. In this study, gulf toadfish (Opsanus beta) were acclimated to 9, 35 and 50 ppt. Intestinal HCO3– secretion, water and salt absorption, and the ensuing effects on acid–base balance were examined. Rectal fluid excretion greatly increased with increasing salinity from 0.17±0.05 ml kg–1 h–1 in 9 ppt to 0.70±0.19 ml kg–1 h–1 in 35 ppt and 1.46±0.22 ml kg–1 h–1 in 50 ppt. Rectal fluid composition and excretion rates allowed for estimation of drinking rates, which increased with salinity from 1.38±0.30 to 2.60±0.92 and 3.82±0.58 ml kg–1 h–1 in 9, 35 and 50 ppt, respectively. By contrast, the fraction of imbibed water absorbed decreased from 85.9±3.8% in 9 ppt to 68.8±3.2% in 35 ppt and 61.4±1.0% in 50 ppt. Despite large changes in rectal base excretion from 9.3±2.7 to 68.2±20.4 and 193.2±64.9 μmol kg–1 h–1 in 9, 35 and 50 ppt, respectively, acute or prolonged exposure to altered salinities was associated with only modest acid–base balance disturbances. Extra-intestinal, presumably branchial, net acid excretion increased with salinity (62.0±21.0, 229.7±38.5 and 403.1±32.9 μmol kg–1 h–1 at 9, 35 and 50 ppt, respectively), demonstrating a compensatory response to altered intestinal base secretion associated with osmoregulatory demand.

The involvement of H+-ATPase and carbonic anhydrase in intestinal HCO3- secretion in seawater-acclimated rainbow trout.

SUMMARY Pyloric caeca and anterior intestine epithelia from seawater-acclimated rainbow trout exhibit different electrophysiological parameters with lower transepithelial potential and higher epithelial conductance in the pyloric caeca than the anterior intestine. Both pyloric caeca and the anterior intestine secrete HCO3– at high rates in the absence of serosal HCO3–/CO2, demonstrating that endogenous CO2 is the principal source of HCO3– under resting control conditions. Apical, bafilomycin-sensitive, H+ extrusion occurs in the anterior intestine and probably acts to control luminal osmotic pressure while enhancing apical anion exchange; both processes with implications for water absorption. Cytosolic carbonic anhydrase (CAc) activity facilitates CO2 hydration to fuel apical anion exchange while membrane-associated, luminal CA activity probably facilitates the conversion of HCO3– to CO2. The significance of membrane-bound, luminal CA may be in part to reduce HCO3– gradients across the apical membrane to further enhance anion exchange and thus Cl– absorption and to facilitate the substantial CaCO3 precipitation occurring in the lumen of marine teleosts. In this way, membrane-bound, luminal CA thus promotes the absorption of osmolytes and reduction on luminal osmotic pressure, both of which will serve to enhance osmotic gradients to promote intestinal water absorption.

Concentration of MgSO4 in the intestinal lumen of Opsanus beta limits osmoregulation in response to acute hypersalinity stress

Marine teleosts constantly lose water to their surrounding environment, a problem exacerbated in fish exposed to salinity higher than normal seawater. Some fish undergo hypersaline exposures in their natural environments, such as short- and long-term increases in salinity occurring in small tidal pools and other isolated basins, lakes, or entire estuaries. Regardless of the degree of hypersalinity in the ambient water, intestinal absorption of monovalent ions drives water uptake to compensate for water loss, concentrating impermeable MgSO(4) in the lumen. This study considers the potential of luminal [MgSO(4)] to limit intestinal water absorption, and therefore osmoregulation, in hypersalinity. The overall tolerance and physiological response of toadfish (Opsanus beta) to hypersalinity exposure were examined. In vivo, fish in hypersaline waters containing artificially low [MgSO(4)] displayed significantly lower osmolality in both plasma and intestinal fluids, and increased survival at 85 parts per thousand, indicating improved osmoregulatory ability than in fish exposed to hypersalinity with ionic ratios similar to naturally occurring ratios. Intestinal sac preparations revealed that in addition to the osmotic pressure difference across the epithelium, the luminal ionic composition influenced the absorption of Na(+), Cl(-), and water. Hypersalinity exposure increased urine flow rates in fish fitted with ureteral catheters regardless of ionic composition of the ambient seawater, but it had no effect on urine osmolality or pH. Overall, concentrated MgSO(4) within the intestinal lumen, rather than renal or branchial factors, is the primary limitation for osmoregulation by toadfish in hypersaline environments.

Intestinal transport following transfer to increased salinity in an anadromous fish (Oncorhynchus mykiss).

The ability to transition from freshwater to seawater environments is an intrinsic requirement of the life history of some fish species, including the anadromous rainbow trout (Oncorhynchus mykiss). The differences between hyper- and hypoosmoregulation are developed quickly (in hours to days), and at all scales, from gene expression to organ function. In this study, intestinal ion and water transport was examined in O. mykiss following acute transfer from freshwater (FW) to 70% seawater (SW). Plasma [Mg²+] increased at 24h post-transfer but recovered by 72 h. In the intestinal fluids, total CO₂ was found to increase with SW exposure/acclimation, while [Na+] decreased after 24h of SW exposure. Overall, in vitro experiments demonstrated the importance of base secretion to epithelial water uptake, and suggested that the primary physiological adjustments occurred 24-72 h after acute SW transfer. The mRNA expression of ion transporters important for intestinal osmoregulation and maintenance of acid-base balance was also investigated. A Na+/H+ exchanger (NHE2) and anion exchanger (SLC26a6) were hypothesized to be involved in the transport of acid-base equivalents, Na+, and Cl⁻, but were not uniformly expressed across tissue samples, and expression, where present, did not change following salinity transfer. NHE1, however, was expressed in all examined tissues (gill, kidney, anterior intestine, and pyloric cecae), but exhibited no changes in expression following acute salinity transfer.

Measuring intestinal fluid transport in vitro: Gravimetric method versus non-absorbable marker

The gut sac is a long-standing, widely used in vitro preparation for studying solute and water transport, and calculation of these fluxes requires an accurate assessment of volume. This is commonly determined gravimetrically by measuring the change in mass over time. While convenient this likely under-estimates actual net water flux (Jv) due to tissue edema. We evaluated whether the popular in vivo volume marker [(14)C]-PEG 4000, offers a more representative measure of Jvin vitro. We directly compared these two methods in five teleost species (toadfish, flounder, rainbow trout, killifish and tilapia). Net fluid absorption by the toadfish intestine based on PEG was significantly higher, by almost 4-fold, compared to gravimetric measurements, compatible with the latter under-estimating Jv. Despite this, PEG proved inconsistent for all of the other species frequently resulting in calculation of net secretion, in contrast to absorption seen gravimetrically. Such poor parallelism could not be explained by the absorption of [(14)C]-PEG (typically <1%). We identified a number of factors impacting the effectiveness of PEG. One was adsorption to the surface of sample tubes. While it was possible to circumvent this using unlabelled PEG 4000, this had a deleterious effect on PEG-based Jv. We also found sequestration of PEG within the intestinal mucus. In conclusion, the short-comings associated with the accurate representation of Jv by gut sac preparations are not overcome by [(14)C]-PEG. The gravimetric method therefore remains the most reliable measure of Jv and we urge caution in the use of PEG as a volume marker.