HCO (3)(-) secretion and CaCO3 precipitation play major roles in intestinal water absorption in marine teleost fish in vivo

Abstract

The intestine of marine teleosts must effectively absorb fluid from ingested seawater to avoid dehydration. This fluid transport has been almost exclusively characterized as driven by NaCl absorption. However, an additional feature of the osmoregulatory role of the intestine is substantial net HCO(3)(-) secretion. This is suggested to drive additional fluid absorption directly (via Cl(-)/HCO(3)(-) exchange) and indirectly by precipitating ingested Ca(2+) as CaCO(3), thus creating the osmotic gradient for additional fluid absorption. The present study tested this hypothesis by perfusing the intestine of the European flounder in vivo with varying [Ca(2+)]: 10 (control), 40, and 90 mM. Fractional fluid absorption increased from 47% (control) to 73% (90 mM Ca(2+)), where almost all secreted HCO(3)(-) was excreted as CaCO(3). This additional fluid absorption could not be explained by NaCl cotransport. Instead, a significant positive relationship between Na(+)-independent fluid absorption and total HCO(3)(-) secretion was consistent with the predicted roles for anion exchange and CaCO(3) precipitation. Further analysis suggested that Na(+)-independent fluid absorption could be accounted for by net Cl(-) and H(+) absorption (from Cl(-)/HCO(3)(-) exchange and CO(2) hydration, respectively). There was no evidence to suggest that CaCO(3) alone was responsible for driving fluid absorption. However, by preventing the accumulation of luminal Ca(2+) it played a vital role by dynamically maintaining a favorable osmotic gradient all along the intestine, which permits substantially higher rates of solute-linked fluid absorption. To overcome the resulting hyperosmotic and highly acidic absorbate, it is proposed that plasma HCO(3)(-) buffers the absorbed H(+) (from HCO(3)(-) production), and consequently reduces the osmolarity of the absorbed fluid entering the body.

Fig. 1.

Net production and excretion of bicarbonate equivalents (μeq·kg⁻¹·h⁻¹) by the intestine of the European flounder perfused with salines containing varying concentrations of Ca²⁺ ([Ca²⁺]). The white bars represent the amount of bicarbonate equivalents recovered in the fluid (intestinal fluid+rectal fluid) and black bars show the amount incorporated into precipitates. J, flux. Data are means ± SE, and values labeled with different letters indicate a significant difference between treatments; n = 8, 7, and 8 for the control, 40-mM, and 90-mM treatments, respectively.

Fig. 2.

Influence of luminal [Ca²⁺] on the rate (ml·kg⁻¹·h⁻¹) of intestinal fluid absorption (white bars) by the European flounder in relation to perfusion rate (black bars). Corresponding fractional absorption (%) is shown in parentheses. Data are means ± SE, and values labeled with different letters indicate a significant difference between treatments; n = 8, 7, and 8 for the control, 40-mM, and 90-mM treatments, respectively.

Fig. 3.

Mean net fluxes of cations and anions (μeq·kg⁻¹·h⁻¹) by the intestine of the European flounder following perfusion with salines containing varying [Ca²⁺]; n = 8, 7, and 7 for the control, 40-mM, and 90-mM treatments, respectively. Values for SO₄²⁻ are not shown since net fluxes were not significantly different from zero (0.7 ± 5.5, 0.0 ± 2.6, and −9.2 ± 12.8 μeq·kg⁻¹·h⁻¹, for each respective treatment).

Fig. 4.

Relationship between intestinal HCO₃⁻ secretion (μeq·kg⁻¹·h⁻¹) and the predicted net flux of the missing ion (J_ion?; μeq·kg⁻¹·h⁻¹) in the fluid being absorbed by the intestine of the flounder perfused with salines containing varying [Ca²⁺]; n = 8, 7, and 7 for the control, 40-mM, and 90-mM treatments, respectively. (F_1,20 = 167.24, P < 0.001).

Fig. 5.

Relationship between the calculated, Na⁺-independent fluid transport (ml·kg⁻¹·h⁻¹) and the total (fluid+precipitates) rate of HCO₃⁻ secretion (μeq·kg⁻¹·h⁻¹) by the intestine of the European flounder following perfusion with varying [Ca²⁺], n = 8, 7, and 7 for the control, 40-mM, and 90-mM treatments, respectively. (F_1,20 = 29.44, P < 0.001).

Fig. 6.

Predicted relative contributions of NaCl cotransport (gray bars) and Na⁺-independent pathway(s) (black bars) to overall measured fluid absorption (ml·kg⁻¹·h⁻¹) by the intestine of the European flounder following perfusion with varying [Ca²⁺]. Alongside is the predicted rate of fluid absorption (white bars) associated with (Na⁺-independent) Cl⁻ absorption and the calculated H⁺ flux (thus providing an indication of solute absorption exclusively associated with Cl⁻/HCO₃⁻ exchange and CO₂ hydration, respectively), assuming the associated water movement would be isoosmotic. J_v, net flux of fluid across the intestine. Data are means ± SE, and values labeled with different letters indicate a significant difference between treatments; n = 8, 7, and 7 for the control, 40-mM, and 90-mM treatments, respectively.

Fig. 7.

Relationship between the rate of (Na⁺-independent) Cl⁻ absorption (μeq·kg⁻¹·h⁻¹) and total (fluid+precipitates) HCO₃⁻ secretion (μeq·kg⁻¹·h⁻¹) (A), the net absorption of J_ion? (μeq·kg⁻¹·h⁻¹) (B), and the rate of Na⁺-independent fluid absorption (ml·kg⁻¹·h⁻¹) (C) by the intestine of the flounder perfused with salines containing varying [Ca²⁺]; n = 8, 7, and 7 for the control, 40-mM, and 90-mM treatments, respectively.

Fig. 8.

Simple model illustrating the epithelial ion transport pathways and consequent reactions generating the driving forces for fluid absorption by the intestine of the European flounder as demonstrated and proposed by the present study (adapted from Ref. 20). Apical NaCl cotransporters (NaCl and NKCC) provide the primary driving force for fluid transport, fueled by the inward Na⁺ gradient created by basolateral Na⁺-K⁺-ATPase. Operating alongside is Cl⁻/HCO₃⁻ exchange, with the vast majority of HCO₃⁻ being supplied from intracellular CO₂ hydration, catalyzed by carbonic anhydrase (CA), with preferential basolateral export of H⁺. However, the fate of secreted HCO₃⁻ will be determined by luminal chemistry. Under normocalcaemic control conditions (10 mM Ca²⁺), the availability of Ca²⁺ for precipitation will rapidly become limited; thus to maintain the osmotic gradient for fluid absorption apical H⁺ secretion becomes important, consuming luminal HCO₃⁻, and yielding CO₂, which can potentially recycle back into the cell. Higher luminal [Ca²⁺] will increase CaCO₃ precipitation rates (as shown in 40 mM and 90 mM Ca²⁺ treatments), ultimately consuming 2HCO₃⁻ ions (as the resulting H⁺ will combine with HCO₃⁻ to produce CO₂). The osmotic gradient generated by luminal CaCO₃, in conjunction with elevated Cl⁻ and H⁺ absorption, provides the driving force for Na⁺-independent fluid absorption. The overall absorbed fluid is distinctly hyperosmotic and acidic. In the subepithelial compartment, it is predicted that H⁺ in the absorbate will be buffered by extracellular HCO₃⁻, producing CO₂. This effectively both neutralizes and removes the osmotic influence of H⁺, consequently reducing the osmotic pressure of the fluid entering the blood. The CO₂ generated enters the venous blood for excretion at the gills or alternatively could recycle back into the cell to support additional HCO₃⁻ production. In response to elevated luminal Ca²⁺, higher rates of intestinal HCO₃⁻ production and CaCO₃ formation will incur additional H⁺, which not only leads to a reduction in blood pH and an increase in Pco₂ (respiratory acidosis), but was predicted to produce a hypoosmotic absorbate, which significantly reduced the overall osmotic pressure of the body fluids.