Guanylin activates Cl⁻secretion into the lumen of seawater eel intestine via apical Cl⁻channel under simulated in vivo conditions

Abstract

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na(+) permeability over Cl(-), as confirmed by the replacement by MgCl2 (no Cl(-) gradient) or Na2SO4 Ringer (no Na(+) gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl(-) secretion into the lumen, as the GN effect was blocked by apical Cl(-) channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na(+) > Cl(-)), and GN further enhanced Cl(-) secretion (Cl(-) > Na(+)), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

Molecular mechanisms for intestinal HCO3-secretion and its regulation by guanylin in seawater-acclimated eels

“…Considerable expression of cftr was reported in some teleost species (Marshall and Singer, 2002; Gregŏrio et al, 2013), and it was recently reported that eel RGN induced CFTR insertion into the apical membrane of toadfish enterocytes (Ruhr et al, 2018). However, mucosal DPC, which effectively abolished GN-induced Cl - secretion in the eel intestine (Ando and Takei, 2015), failed to inhibit GN-induced HCO 3 - secretion in the present study. Thus, it is unlikely that GN directly stimulates Cl - channel including CFTR for HCO 3 - secretion in the SW eel intestine.…”

“…As a second candidate for the route of HCO 3 - secretion, we considered Cl - channels such as CFTR, since GN stimulates apical Cl - channel in the SW eel intestine (Ando and Takei, 2015) and HCO 3 - is known to pass through the Cl - channels (Rubenstein, 2018). Two CFTR genes exist in the eel, but the expression of the cftr s was low and decreased after SW transfer in the eel intestine (Ando et al 2014; Wong et al, 2016).…”

“…Although eel plasma pH is ca. 7.8, all experiments were performed at pH 7.4 because electrical parameters were more stable (Ando et al, 2014) and the effects of GN and bumetanide were more reproducible at this pH (Ando and Takei, 2015). The mucosal side of the intestine was bathed with unbuffered Ringer solution (2.3 ml), where NaHCO 3 was replaced by NaCl and gassed with 100% O 2 passed through 20 mM NaOH.…”

“…To assess the ion transporter target(s) of GN that lead to increased HCO 3 - secretion, various inhibitors were applied on either mucosal or serosal side of the intestinal epithelia in the Ussing chamber before or after GN administration. The inhibitors applied on the mucosal side were bumetanide, DPC and DNDS/DIDS since, in our earlier studies, GN inhibited NKCC2 and stimulated Cl - channel in the eel (Ando et al, 2014; Ando and Takei, 2015), and since UGN inhibited Pat-1 in the toadfish (Ruhr et al, 2016). DNDS/DIDS was also applied to the serosal side to evaluate the role of basolateral AE and NBC that transport HCO 3 - into the cell.…”

“…Because homologous GN from the eel is available, this euryhaline teleost serves as a good model to analyze the mechanisms of GN action on the HCO 3 - secretion. It was shown that GN decreased NaCl absorption through inhibition of NKCC2b (Ando et al, 2014) and decreased Cl - secretion into the lumen through stimulation of DPC-sensitive anion channels in the intestine of SW-acclimated eels (Ando and Takei, 2015). In the marine toadfish, eel RGN was shown to decrease HCO 3 - secretion through inhibition of Slc26a6 in the posterior intestine, while it increased the secretion in the fish acclimated to concentrated (60 ppt) SW (Ruhr et al, 2015).…”