Our second goal was to see whether the RGN response was mediated primarily by PKG or PKA.

Schauer Yoshio Takei 2 Department of Marine Bioscience, The Atmosphere and Ocean Research Institute, The University of Tokyo, Kashiwa, Chiba, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yoshio Takei Martin Grosell 1 Department of Marine Biology and Ecology, The Rosenstiel School of Marine and Atmospheric Science, The University of Miami, Miami, FL 33149, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site.

Most notably, they reverse normal net ion-absorbing mechanisms that are vital to water absorption, an important process for seawater teleost survival.

The role of guanylin-peptide stimulation of the intestine remains unclear, but it is hypothesized to facilitate the removal of solids from the intestine by providing fluid to enable their removal by peristalsis.

The present study used one member of this group of peptides RGN to provide evidence for the prominent role that protein kinase A (PKA) plays in mediating the effects of guanylin-peptide stimulation in the posterior intestine of the Gulf toadfish ( Opsanus beta ).

Protein kinase G was found to not mediate the intracellular effects of RGN, despite previous evidence showing that GC-C activation leads to higher cyclic guanosine monophosphate formation.

RGN reversed the absorptive short-circuit current and increased conductance in the Gulf toadfish intestine.

These effects are correlated to increased trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel to the apical membrane, which is negated by PKA inhibition.

Moreover, RGN decreased HCO 3 secretion, likely by limiting apical HCO 3 Cl exchange (possibly by reducing SLC26a6 activity), a reduction that was enhanced by PKA inhibition.

RGN seems to alter PKA activity in the posterior intestine to recruit CFTR to the apical membrane and reduce HCO 3 secretion.

INTRODUCTION Seawater presents a major osmoregulatory challenge for teleost fish, because of the direct interaction of their thin respiratory epithelia the gills with the environment, which causes unavoidable branchial fluid loss ( Evans et al., 2005 ).

To compensate, the seawater teleost constantly drinks water and absorbs it across the intestine.

Imbibed seawater undergoes rapid desalination by the esophagus, so, by the time it enters the intestine, its osmolality is roughly that of blood plasma.

In the intestine, precipitation of Ca 2 and Mg 2 by HCO 3 to form CaCO 3 and MgCO 3 precipitates and titration of HCO 3 by H facilitate further decreases in intestinal fluid osmolality by up 130 mOsmol kg 1 ( Grosell et al., 2009a, b; Wilson et al., 2002 ).

The combined effect of these processes, in addition to other ion-absorbing pathways, renders the fluid osmolality in the intestine similar to that of the extracellular fluids and allows for efficient water absorption that is coupled to Na and Cl absorption ( Skadhauge, 1969, 1974 ).

This response is also isolated to the mid and posterior intestine of the Japanese eel ( Anguilla japonica ) after stimulation by guanylin ( Ando and Takei, 2015; Ando et al., 2014; Yuge and Takei, 2007 ).

It is hypothesized that the inhibition of fluid absorption is required to facilitate the removal of solids, such as undigested food, fecal matter or carbonate precipitates.

A handful of studies on the intestinal physiology of seawater teleosts have demonstrated a reversal of ion absorption.

A more recent study on the killifish ( Fundulus heteroclitus ) similarly revealed that a combination of ionomycin, dibutyryl-cAMP and IBMX (a phosphodiesterase inhibitor) stimulates fluid secretion in the posterior intestine ( Marshall et al., 2002 ).

It is interesting that intestinal tissues from multiple species of seawater teleosts respond to the above-mentioned hormones, activators and inhibitors by decreasing ion-absorbing mechanisms that, in some cases, result in fluid secretion.

The inhibition of ion transport and water absorption is widespread across seawater teleost species and might serve a beneficial purpose for life in seawater.

In both mammalian and fish enterocytes, stimulation of the apical guanylin peptide receptor guanylyl cyclase-C (GC-C) leads to increased formation of cGMP ( Chao et al., 1994; Kuhn et al., 1994; Schulz et al., 1990; Yuge and Takei, 2007 ), which activates protein kinase G (PKG) and increases cAMP formation leading to protein kinase A (PKA) activation ( Arshad and Visweswariah, 2012, 2013 ).

Stimulation of GC-C leads to apical CFTR activation, inhibition of Na H -exchangers (NHEs) in mammals, inhibition of NKCC2 in fish and a decrease in HCO 3 secretion in fish ( Chao et al., 1994; Ruhr et al., 2014, 2016 ).

In the present study, we investigated the intracellular effects of RGN in Gulf toadfish enterocytes.

Our first goal was to determine whether RGN affected CFTR abundance in the apical membrane.

We have previously shown that CFTR is likely present in the posterior intestine of the Gulf toadfish, but that it is mostly sub-apical.

The present study corroborates these findings by measuring the I SC and transepithelial conductance ( G TE ) of the tissues.

Ppt Bioteknologi Kelas 9