The role of the rectum in osmoregulation and the potential effect of renoguanylin on SLC26a6 transport activity in the Gulf toadfish (Opsanus beta)

Abstract

Teleosts living in seawater continually absorb water across the intestine to compensate for branchial water loss to the environment. The present study reveals that the Gulf toadfish (Opsanus beta) rectum plays a comparable role to the posterior intestine in ion and water absorption. However, the posterior intestine appears to rely more on SLC26a6 (a HCO3 (-)/Cl(-) antiporter) and the rectum appears to rely on NKCC2 (SLC12a1) for the purposes of solute-coupled water absorption. The present study also demonstrates that the rectum responds to renoguanylin (RGN), a member of the guanylin family of peptides that alters the normal osmoregulatory processes of the distal intestine, by inhibited water absorption. RGN decreases rectal water absorption more greatly than in the posterior intestine and leads to net Na(+) and Cl(-) secretion, and a reversal of the absorptive short-circuit current (ISC). It is hypothesized that maintaining a larger fluid volume within the distal segments of intestinal tract facilitates the removal of CaCO3 precipitates and other solids from the intestine. Indeed, the expression of the components of the Cl(-)-secretory response, apical CFTR, and basolateral NKCC1 (SLC12a2), are upregulated in the rectum of the Gulf toadfish after 96 h in 60 ppt, an exposure that increases CaCO3 precipitate formation relative to 35 ppt. Moreover, the downstream intracellular effects of RGN appear to directly inhibit ion absorption by NKCC2 and anion exchange by SLC26a6. Overall, the present findings elucidate key electrophysiological differences between the posterior intestine and rectum of Gulf toadfish and the potent regulatory role renoguanylin plays in osmoregulation.

Keywords: CFTR; Cl− secretion; HCO3− secretion; intestine; marine teleost; water secretion.

Fig. 1.

Changes in short-circuit current (I_SC) (A) and transepithelial conductance (G_TE) (B) in rectal tissues as a function of renoguanylin (RGN) dose after mucosal application (log scale). The concentration of RGN was increased every 30 min when I_SC values became stable. Values are expressed as means ± SE (n = 6). Significant differences from the control (no dose) were revealed by one-way, repeated-measures ANOVAs, followed by Holm-Sidak tests (*P ≤ 0.05). The 50% effective concentration (EC₅₀) of RGN was calculated using a nonlinear regression, global curve fitting (n = 6; P ≤ 0.05). Positive and negative I_SC values indicate secretory and absorptive currents, respectively.

Fig. 2.

Relative gene expression levels of NKCC1 (A), NKCC2 (B), and CFTR (C) in various tissues of the Gulf toadfish. Gene expression is normalized to elongation factor (EF)-1α and scaled relative to the tissue with the lowest expression value (NKCC1: anterior intestine, NKCC2: gills, and CFTR: muscle), which was given a value of 1.0. NKCC1 gene expression was not detected in the esophagus and liver. Values are expressed as means ± SE (n = 8).

Fig. 3.

Relative gene expression levels of guanylin (GN), uroguanylin (UGN), guanylyl cyclase-C (GC-C), NKCC1, CFTR, and NKCC2 in the rectum of Gulf toadfish acclimated to 0 (control) and 96 h in 60 ppt seawater. The level of expression for each gene is normalized to elongation factor (EF)-1α and scaled relative to the 0-h control, which was given a value of 1.0. Values are means ± SE (n = 8). Significant differences between the 0 and 96-h groups were revealed by one-tailed Student's t-tests (*P ≤ 0.05).

Fig. 4.

Baseline (control) values of the short-circuit current (I_SC), transepithelial potential (TEP), and transepithelial conductance (G_TE) displayed by the posterior intestine and rectum of Gulf toadfish. Values are expressed as means ± SE and were pooled from various experiments in the present study (I_SC: n = 11 and 13, TEP: n = 13 and 13, and G_TE: n = 25 and 27, posterior intestine and rectum, respectively). Significant differences were revealed by Student's t-tests (*P ≤ 0.05). Positive and negative I_SC values indicate secretory and absorptive currents, respectively. Positive and negative TEP values indicate net anion secretion and absorption, respectively.

Fig. 5.

HCO₃⁻ (Bic) secretion (A and D), transepithelial potential (TEP) (B, E), and transepithelial conductance (G_TE) (C and F) displayed by the posterior intestine and rectum of Gulf toadfish before and after treatment with renoguanylin (RGN). Pretreatment values were taken from the final 30 min of the control flux, and post-treatment values were taken from the final 30 min of a 70-min treatment flux. Values are expressed as means ± SE (n = 7 and 8, 5 and 0 mM Bic groups, respectively). Significant differences: control vs. RGN revealed by paired t-tests (*P ≤ 0.05), posterior intestine vs. rectum revealed by Student's t-tests (†P ≤ 0.05), and 5 mM Bic vs. 0 mM (within tissue and within treatment) revealed by Student's t-tests (§P ≤ 0.05). The presence (5 mM Bic) and absence (0 mM Bic) of serosal HCO₃⁻ is indicated by solid and open bars, respectively. Mucosally administered RGN treatment is indicated by hatched bars.

Fig. 6.

Short-circuit current (I_SC) (A, C) and transepithelial conductance (G_TE) (B, D) displayed by the posterior intestine and rectum of Gulf toadfish before and after treatment with renoguanylin (RGN) or bumetanide (Bum). Pretreatment values were taken from the final 30 min of the control flux and post-treatment values were taken from the final 30-min of a 70-min treatment flux. Values are expressed as means ± SE (n = 6 and 6 or 7, RGN and Bum experiments, respectively). Significant differences: control vs. treatment revealed by paired t-tests (*P ≤ 0.05) and posterior intestine vs. rectum revealed by Student's t-tests (†P ≤ 0.05). Control and treatment values are indicated by solid and open bars, respectively. Positive and negative I_SC values indicate secretory and absorptive currents, respectively.

Fig. 7.

HCO₃⁻ secretion (A), transepithelial potential (TEP) (B), and transepithelial conductance (G_TE) (C) displayed by the posterior intestine and rectum of Gulf toadfish. Control values (solid bars) were taken from the final 30 min of the control flux. Bumetanide (Bum) (open bars) was then added, and its effects were measured from the final 30 min of a 70-min treatment flux. Finally, renoguanylin (RGN) (shaded bars) was added in combination with Bum, and its effects were measured from the final 30 min of a 70-min treatment flux (t_total = 170 min). Values are expressed as means ± SE. Significant differences were revealed by one-way, repeated-measures ANOVAs, followed by Holm-Sidak tests (^a,bP ≤ 0.05). Positive and negative TEP values indicate net anion secretion and absorption, respectively.

Fig. 8.

Absolute (A, B, D, E) and relative (C, F) HCO₃⁻ secretion rates displayed by the posterior intestine and rectum of Gulf toadfish when exposed in the absence (0 mmol/l) of serosal HCO₃⁻ (Bic). A and D: ethoxzolamide (Ethox) was added alone to the mucosal saline (n = 7 and 8, posterior intestine and rectum, respectively; t_total = 100 min). Values are expressed as means ± SE. Significant differences were revealed by paired t-tests (*P ≤ 0.05). B and E: RGN was initially added to the mucosal saline, after which Ethox was also added to the mucosal saline (n = 6 and 7, posterior intestine and rectum, respectively; t_total = 170 min). Significant differences were revealed by one-way, repeated-measures ANOVAs, followed by Holm-Sidak tests (^a,b,cP ≤ 0.05). C and F: combined effects of RGN and Ethox were compared with their individual treatments. Significant differences: RGN vs. RGN + Ethox revealed by paired t-tests and Ethox vs. RGN + Ethox revealed by Student's t-tests (*P ≤ 0.05). Control values were taken from the final 30 min of the control flux. Treatment values were taken from the final 30 min of a 70-min treatment flux.

Fig. 9.

Water (A), Na⁺ (B), and Cl⁻ (C) fluxes displayed by the posterior intestine (PI) and rectum (Rec) of Gulf toadfish acclimated to 35 or 60 ppt and treated with RGN. Values are expressed as means ± SE. Three-way ANOVAs, followed by one-tailed Holm-Sidak tests revealed significant differences in Na⁺, Cl⁻, and water fluxes (P ≤ 0.05; n_total = 55). Control vs. RGN (*), PI: 35 vs. 60 ppt (†), and 35 or 60 ppt: PI vs. Rec (§). Positive and negative values indicate net absorption or net secretion, respectively.

Fig. 10.

Simplified proposed effects of the guanylin peptides in the posterior intestinal and rectal epithelia of Gulf toadfish (Opsanus beta) acclimated to 35 ppt seawater. Guanylin (GN), uroguanylin (UGN), and renoguanylin (RGN) bind to a guanylyl cyclase-C (GC-C) receptor on the apical membrane of an enterocyte (A). The stimulation of GC-C leads to enhanced formation of cGMP, whose downstream effects increase the phosphorylation of PKA and PKG, both of which can activate CFTR and, perhaps, NKCC1 (B). The downstream effects of cGMP also lead to inhibited ion transport activity by NKCC2 and decreased anion exchange activity by SLC26a6 (C). The combined effects of GC-C stimulation results in the reversal of ion flux, from net ion absorption (mucosa-to-serosa) to net ion secretion (serosa-to-mucosa), which result in either inhibition of water absorption or net water secretion. NKA, Na⁺/K⁺-ATPase (maintains electrochemical gradients that enables ion transport); TJ, tight junction, stimulatory (+) and inhibitory (−) effects. Cell diagram modified from Ruhr et al. (41).