Signaling Components Involved in the Hormone Induced Translocation of ENaC in Cultured Adult Human Fungiform (HBO) Taste Cells

The amiloride-sensitive epithelial Na+ channel, ENaC, is the Na+-specific salt taste receptor in rodents. Compared to rodents, human salt taste perception is amiloride-insensitive. In rodents the ENaC is composed of aβg-subunits. Whereas humans express an additional subunit, the d-ENaC subunit. ENaC in human taste cells is composed of aβg-subunits or dβg-subunits, with the latter being amiloride-insensitive. Currently, it is not known if dβg-ENaC expression and trafficking is regulated by hormones and their downstream intracellular signaling effectors. The aim of this study is to investigate if arginine vasopressin (AVP), aldosterone, and cAMP regulate d-ENaC expression and trafficking in cultured fungiform human taste cells (HBO cells). Secondly, we want to demonstrate the expression of downstream signaling effectors involved in the trafficking of d-ENaC in HBO cells. Using molecular and immunocytochemical techniques, our results demonstrate that AVP, cAMP, and aldosterone increase expression of d-ENaC mRNA and protein in HBO cells. Furthermore, AVP, cAMP and aldosterone increased trafficking of the d-ENaC subunit from the cytosolic compartment to the apical pole of the HBO cells. Our results further demonstrate that HBO cells express several components of signaling cascade involved in ENaC translocation from cytosol to apical pole in HBO cells. The components of this signaling cascade include AVPR2, PKA, CREB, SGK-1, Nedd4-2, and GILZ-1. These hormones in mice and rats upregulate ENaC. Currently, we are not sure if these hormones affect ENaC this way in humans. By studying d-ENaC with these hormones, we are able to see how human ENaC is regulated in the tongue

Cyclic-AMP regulates postnatal development of neural and behavioral responses to NaCl in rats

During postnatal development rats demonstrate an age-dependent increase in NaCl chorda tympani (CT) responses and the number of functional apical amiloride-sensitive epithelial Na+channels (ENaCs) in salt sensing fungiform (FF) taste receptor cells (TRCs). Currently, the intracellular signals that regulate the postnatal development of salt taste have not been identified. We investigated the effect of cAMP, a downstream signal for arginine vasopressin (AVP) action, on the postnatal development of NaCl responses in 19–23 day old rats. ENaC-dependent NaCl CT responses were monitored after lingual application of 8-chlorophenylthio-cAMP (8-CPT-cAMP) under open-circuit conditions and under ±60 mV lingual voltage clamp. Behavioral responses were tested using 2 bottle/24h NaCl preference tests. The effect of [deamino-Cys1, D-Arg8]-vasopressin (dDAVP, a specific V2R agonist) was investigated on ENaC subunit trafficking in rat FF TRCs and on cAMP generation in cultured adult human FF taste cells (HBO cells). Our results show that in 19–23 day old rats, the ENaC-dependent maximum NaCl CT response was a saturating sigmoidal function of 8-CPT-cAMP concentration. 8-CPT-cAMP increased the voltage-sensitivity of the NaCl CT response and the apical Na+ response conductance. Intravenous injections of dDAVP increased ENaC expression and γ-ENaC trafficking from cytosolic compartment to the apical compartment in rat FF TRCs. In HBO cells dDAVP increased intracellular cAMP and cAMP increased trafficking of γ- and δ-ENaC from cytosolic compartment to the apical compartment 10 min post-cAMP treatment. Control 19–23 day old rats were indifferent to NaCl, but showed clear preference for appetitive NaCl concentrations after 8-CPT-cAMP treatment. Relative to adult rats, 14 day old rats demonstrated significantly less V2R antibody binding in circumvallate TRCs. We conclude that an age-dependent increase in V2R expression produces an AVP-induced incremental increase in cAMP that modulates the postnatal increase in TRC ENaC and the neural and behavioral responses to NaCl

Effect of 8-CPT-cAMP on ENaC-dependent NaCl CT responses in 19–23 day old rats.

<p><b>(A)</b> Mean normalized tonic CT response under open-circuit conditions (filled squares) to NaCl (<i>r</i>_<i>o</i>(0)) at zero 8-CPT-cAMP (<i>N</i> = 12–18). Since <i>r</i>_<i>o</i>(0) = <i>r</i>_<i>aso</i>(0) + <i>r</i>_<i>aio</i>(0), the solid line through the data points is the sum of the regression curves for <i>r</i>_<i>aso</i>(0) and <i>r</i>_<i>aio</i>(0). Mean normalized tonic CT response under open-circuit conditions (open circles) to NaCl + CPC (<i>r</i>_<i>aso</i>(0)) at zero 8-CPT-cAMP (<i>N</i> = 10–13). Curve (long dashes) is the regression curve. CT response under open-circuit conditions (closed triangles) to NaCl + CPC + Bz at zero 8-CPT-cAMP (<i>N</i> = 3). Curve (short dashes) is the linear regression line. <b>(B)</b> Mean ENaC-dependent part of the CT response as a function of NaCl concentration under open circuit conditions in 19–23 day old rats before (r_aso(0); open circles) and following exposure to 20 mM CPT-cAMP (r_aso(20); filled circles). The curves are the least squares fits to the data using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171335#pone.0171335.e001" target="_blank">Eq 1</a> with <i>φ</i> = 0. The dotted line represents the pre-cAMP control curve and the solid curve represents the post 20 mM 8-CPT-cAMP curve. Each NaCl solution contained 2 mM CPC to block the Bz-insensitive part of the response. For r_aso(0), <i>N</i> = 10 (0.1 M NaCl), <i>N</i> = 13 (0.3 M NaCl) and <i>N</i> = 10 (0.5 M NaCl). For r_aso(20), <i>N</i> = 5 (0.1 M NaCl), <i>N</i> = 9 (0.3 M NaCl) and <i>N</i> = 10 (0.5 M NaCl).</p

Effect of 8-CPT-cAMP on the NaCl CT responses in 19–23 day old rats.

<p>The figure shows a representative response in a 21 day old rat, in which the CT responses were monitored under open-circuit conditions while the tongue was first rinsed with a rinse solution R (10 mM KCl) and then with 0.1, 0.3 and 0.5 M NaCl under controls conditions <b>(A)</b> and in NaCl solutions containing 2 mM CPC <b>(B).</b> After topical lingual application of 20 mM 8-CPT-cAMP for 10 min the CT responses were again monitored under open circuit conditions while the tongue was first rinsed with a rinse solution R (10 mM KCl) and then with 0.1, 0.3 and 0.5 M NaCl <b>(C)</b> and in NaCl solutions containing 2 mM CPC <b>(D)</b> or 2 mM CPC + 5 μM Bz <b>(E)</b>. <b>(F)</b> Shows the mean normalized tonic CT responses for various concentrations of NaCl, NaCl + CPC and ((NaCl)-(NaCl + CPC)). Each bar shows the mean CT response from the number of animals (<i>N</i>). For Control: 0.1–0.5 M NaCl (<i>N</i> = 12–18); Control NaCl + CPC (<i>N</i> = 10–13); post 8-CPT-cAMP 0.1–0.5 M NaCl (<i>N</i> = 9–12); NaCl + CPC (<i>N</i> = 5–9). No changes in the Bz-insensitive NaCl CT response ((NaCl)-(NaCl + CPC)) were observed (P>0.05) at any NaCl concentration (Fig 2F; grey bars). *p = 0.0001 with respect to control. <b>(G)</b> Shows the comparative changes in Bz-sensitive NaCl CT responses in 19–23 day old and adult (60+ day old) rats. The values of mean normalized ENaC-dependent NaCl tonic CT response magnitudes before and after 20 mM 8-CPT-cAMP treatment in 19–23 day old rats were taken from Fig F (filled bars). The relative values of ENaC-dependent NaCl tonic CT response magnitudes in control adult rats are shown in hatches bars (<i>N</i> = 3). *P = 0.0001 with respect to control adult rats.</p

Effect of dDAVP on intracellular cAMP formation in HBO cells.

<p>Treating HBO cells for 10 min with dDAVP (0.1 μM) increased intracellular cAMP. Forskolin (10 μM), a known activator of adenylyl cyclase was used as a control. The values represent mean ± SEM of triplicate measurements.</p

Effect of lingual application of 8-CPT-cAMP or 8-CPT-cGMP on fluid intake in adult rats.

<p>Adult (60+ day old) rat tongues were treated with either H₂O (Control; <i>N</i> = 15) or 20 mM 8-CPT-cAMP (Post-8-CPT-cAMP; <i>N</i> = 15) or 20 mM 8-CPT-cGMP (<i>N</i> = 14). The rats were given a choice between two bottles, one containing H₂O and the other containing varying NaCl concentrations (0.01–0.5 M NaCl) and their fluid intake was measured every day. No significant difference in the intake of H₂O or NaCl was observed between control and 8-CPT-cGMP treated rats at different NaCl concentrations. Therefore, the intake of H₂O and NaCl was presented as the mean intake observed in control and 8-CPT-cGMP treated rats (<i>N</i> = 30). The mean intake of NaCl at 0.15 M NaCl (grey bars) was significantly different from the intake at 0.01, 0.025, 0.05, 0.10, 0.25 and 0.5 M NaCl with P values of 0.0001, 0.0001, 0.0001, 0.1657, 0.0004, and 0.0001, respectively (<i>N</i> = 30; unpaired). In 8-CPT-cAMP treated rats the intake at 0.1 M NaCl (black bars) was significantly different from the intake at 0.01, 0.025, 0.05, 0.15, 0.25 and 0.5 M NaCl with P values of 0.0001, 0.0002, 0.0010, 0.0020, 0.0058, and 0.0001, respectively (<i>N</i> = 14; unpaired).</p

Fluid intake and NaCl preference in developing rats.

<p><b>(A)</b> Rat (21 day old) tongues were treated with either H₂O (Control; <i>N</i> = 3) or 20 mM 8-CPT-cAMP (Post-8-CPT-cAMP; <i>N</i> = 4) for 20 min. The 3 control and 4 treated rats were then transferred to separate cages and were given choice between two bottles, one containing H₂O and the other containing 0.15 M NaCl. Their fluid intake was measured every day between postnatal day 22 and 27 and expressed as g/g body weight (BW)/24h. The bars under the brackets represent the mean ± SEM values of fluid intake and NaCl preference over the 6 day period in control (<i>N</i> = 3) and 8-CPT-cAMP treated rats (<i>N</i> = 4). Horizontal line denotes NaCl preference ratio of 0.5. In 8-CPT-cAMP treated rats, there was a decrease in H₂O intake (*p = 0.0006) and an increase in NaCl intake (*p = 0.006) relative to control rats. The mean preference in control and 8-CPT-cAMP treated rats was 0.577 ± 0.02 and 0.818 ± 0.018, respectively (*p = 0.0004). <b>(B)</b> Rat (21d old) tongues were treated with either H₂O (Control; <i>N</i> = 5) or 20 mM 8-CPT-cAMP (Post-8-CPT-cAMP; <i>N</i> = 5) or 20 mM 8-CPT-cGMP (Post-8-CPT-cGMP; <i>N</i> = 5) for 20 min. The control and treated rats were then transferred to separate cages and were given choice between two bottles, one containing H₂O and the other containing 0.035 or 0.075 M NaCl. Their fluid intake and NaCl preference was measured every day between postnatal day 22 and 27. Only rats treated with 8-CPT-cAMP demonstrated a significant increase in NaCl preference. *p = 0.0001 (<i>N</i> = 5).</p

Age-dependent increase in ENaC expression.

<p><b>(A)</b> CV papillae from naïve rats at different age were collected for Q-PCR to determine the age dependent mRNA level of α-ENaC and γ-ENaC. The results showed the mRNA levels of both subunits is higher in adult rat CV papillae compared with the pups. <b>(B)</b> For Western blot studies, CV papillae were pooled from fifteen 14–18 day old rats and eight adult (60+ day old) rats. Forty μg total protein was used for the assay. Beta-actin was used as a protein loading control. <b>(C)</b> Shows the intensity of the α-ENaC band in 14–18 day old rats relative to adult rats from 3 different batches of CV tissues normalized to β-actin (p = 0.0001).</p

Spaciotemporal relationship between 8-CPT-cAMP, dDAVP and 8-CPT-cGMP treatment and δ-ENaC trafficking in HBO cells.

<p>The figure shows the overlay of the transmitted (DIC) image, DAPI labelled cell nuclei (blue) and δ-ENaC binding to HBO cells (green). <b>(Panel A)</b> Shows δ-ENaC antibody binding to a subset of HBO cells treated with 10 μM 8-CPT-cAMP for 10 min in a representative low resolution <b>(L)</b> and a high resolution <b>(H)</b> image. The δ-ENaC antibody binding was observed mainly in the apical compartment with much less binding in the cytosolic compartment of HBO cells (arrows). <b>(Panel B)</b> Shows δ-ENaC antibody binding to a subset of HBO cells treated with 10 nM dDAVP for 10 min in a representative low resolution <b>(L)</b> and a high resolution <b>(H)</b> image. Similar to HBO cells treated with 8-CPT-cAMP (Panel A), the δ-ENaC antibody binding was observed mainly in the apical compartment with much less binding in the cytosolic compartment in a subset of HBO cells (arrows). <b>(Panel C)</b> Shows δ-ENaC antibody binding to a subset of HBO cells treated with 10 μM 8-CPT-cGMP for 10 min in a representative low resolution <b>(L)</b> and a high resolution <b>(H)</b> image. Similar to control HBO cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171335#pone.0171335.g011" target="_blank">Fig 11</a>), δ-ENaC antibody binding was observed mainly in the cytosolic compartment with much less binding in the apical compartment of HBO cells (arrows).</p

Effect of AVP and dDAVP on NaCl CT responses in adult rats.

<p><b>(A)</b> In adult rats CT responses to 0.1 and 0.3 M NaCl were measured before and 20 min after tail vein injection of 1 nano moles AVP/Kg BW. The figure shows the mean normalized tonic NaCl CT responses in control and AVP-treated rats. *P = 0.0015 with respect to control. <b>(B)</b> In another set of rats, CT responses to 0.1 and 0.3 M NaCl were measured in the absence and presence of 5 μM Bz, before and 20 min after tail vein injection of 0.1 nano moles dDAVP/Kg BW. The values are mean normalized tonic NaCl CT responses from 3 rats in each group. *P = 0.02 with respect to control. The CT responses were normalized to 0.3 M NH₄Cl.</p