Ca2+-driven intestinal HCO(3)(-) secretion and CaCO3 precipitation in the European flounder in vivo: influences on acid-base regulation and blood gas transport

Abstract

Marine teleost fish continuously ingest seawater to prevent dehydration and their intestines absorb fluid by mechanisms linked to three separate driving forces: 1) cotransport of NaCl from the gut fluid; 2) bicarbonate (HCO(3)(-)) secretion and Cl(-) absorption via Cl(-)/HCO(3)(-) exchange fueled by metabolic CO(2); and 3) alkaline precipitation of Ca(2+) as insoluble CaCO(3), which aids H(2)O absorption). The latter two processes involve high rates of epithelial HCO(3)(-) secretion stimulated by intestinal Ca(2+) and can drive a major portion of water absorption. At higher salinities and ambient Ca(2+) concentrations the osmoregulatory role of intestinal HCO(3)(-) secretion is amplified, but this has repercussions for other physiological processes, in particular, respiratory gas transport (as it is fueled by metabolic CO(2)) and acid-base regulation (as intestinal cells must export H(+) into the blood to balance apical HCO(3)(-) secretion). The flounder intestine was perfused in vivo with salines containing 10, 40, or 90 mM Ca(2+). Increasing the luminal Ca(2+) concentration caused a large elevation in intestinal HCO(3)(-) production and excretion. Additionally, blood pH decreased (-0.13 pH units) and plasma partial pressure of CO(2) (Pco(2)) levels were elevated (+1.16 mmHg) at the highest Ca perfusate level after 3 days of perfusion. Increasing the perfusate [Ca(2+)] also produced proportional increases in net acid excretion via the gills. When the net intestinal flux of all ions across the intestine was calculated, there was a greater absorption of anions than cations. This missing cation flux was assumed to be protons, which vary with an almost 1:1 relationship with net acid excretion via the gill. This study illustrates the intimate link between intestinal HCO(3)(-) production and osmoregulation with acid-base balance and respiratory gas exchange and the specific controlling role of ingested Ca(2+) independent of any other ion or overall osmolality in marine teleost fish.

Fig. 1.

Net production and excretion of bicarbonate equivalents (HCO₃⁻ + 2CO32−; μeq·kg⁻¹·h⁻¹) by the intestine of the flounder perfused with salines containing varying concentrations of Ca²⁺. J, flux. Bars represent the total amount of bicarbonate equivalents excreted (via the intestinal fluid + rectal fluid + precipitates). ^a,b,cSignificant difference (P < 0.05; ANOVA followed by Holm-Sidak post hoc test). Values are means ± SE; n = 8, 7, and 8 for the control, 40 mM, and 90 mM Ca²⁺ treatments, respectively.

Fig. 2.

Changes in venous blood pH (A), total CO₂ (TCO₂; mM) (B), and the partial pressure of CO₂ (Pco₂; mmHg) (C) over 3 days when flounder intestines were perfused with varying concentrations of Ca²⁺. Black circles represent control (n = 8), white circles; 40 mM Ca²⁺ (n = 8); and black triangles, 90 mM Ca²⁺ (n = 8–7) treatments. A: *significant difference in blood pH on day 3 for fish perfused with 90 mM Ca²⁺ (P < 0.01; 90 vs. 10 and 40 mM Ca²⁺); #significant difference in blood pH on day 2 (P < 0.05, 10 vs. 90 and 40 mM Ca²⁺); $significant difference in blood pH for fish perfused with 40 mM Ca²⁺ between days 1 and 2 vs. day 3 (P < 0.05). C: *significant difference in blood Pco₂ on day 3 for fish perfused with 90 mM Ca²⁺ (P < 0.01, 90 vs. 10 and 40 mM Ca²⁺). All statistical analyses were by ANOVA followed by Holm-Sidak post hoc test. Values are means ± SE.

Fig. 3.

Fluxes of titratable alkalinity (J_TAlk, gray bars), total ammonia (J_Tamm, white bars), and net acidic equivalents (J_H+^net, black bars) between flounder and surrounding water. Negative values indicate fluxes that are equivalent to branchial acid excretion. Bars labeled with different letters indicate a significant difference (P < 0.05; ANOVA followed by Holm-Sidak post hoc test). Values are means ± SE; n = 8, 7, and 8 for control, 40 mM, and 90 mM Ca²⁺ treatments, respectively.

Fig. 4.

Relationship between the net acid excretion via the gills (μeq·kg⁻¹·h⁻¹) and the net flux of the missing cation, presumed to be H⁺ (μeq·kg⁻¹·h⁻¹), absorbed by the intestine of the flounder perfused with salines containing varying concentrations of Ca²⁺ (n = 8, 7, and 8 for control, 40 mM, and 90 mM Ca²⁺ treatments, respectively). Note that the linear regression crosses the x-axis at a value for gill net acid excretion of about 118 μeq·kg⁻¹·h⁻¹. This would represent the metabolic acid that is produced normally by metabolism in tissues other than the intestine and is typical of gill net acid excretion in many studies on fish.