Culturing of HRTCs with 1 nM of C-peptide resulted in increase in PKCε phosphorylation, while phosphorylation of PKCs α/β increased by high glucose concentrations.

<p>Cells were cultured as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028294#s4" target="_blank">Materials and Methods</a>”. Total cell lysates were subject to Western blot analysis to determine PKCs phosphorylation. A representative Western blot image is shown in the upper panel of each graph. <b>A.</b> Phospho PKCα/β. <b>B.</b> Phospho PKCδ, <b>C.</b> Phospho PKCε. GAPDH protein was used as a loading control. Results are means ± SE for 6 independent experiments. * P<0.05 versus 5 mM glucose without C-peptide.</p

Na,K-ATPase expression regulates via PKC , ERK1/2 and ZEB activation.

<p><b>A.</b> Cells were cultured, incubated with kinase inhibitors and analyzed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028294#s4" target="_blank">Materials and Methods</a>”. Experiments were performed at least 5 times and representative Western blot or EMSA images are shown. GAPDH protein was used as a loading control for total cell lysate. <b>B.</b> Effect of siRNA- mediated silencing of ZEB on ZEB and Na,K-ATPase α₁-subunit protein expression in HRTC. GAPDH protein was used as a loading control. Experiments were performed at least 4 times and representative images are shown.</p

Schematic representations of intracellular signaling pathways for regulation of the sodium pump activity by C- peptide in human renal tubular cells.

<p>C- peptide binds specifically to a membrane structure, most likely a G-protein coupled receptor, with subsequent activation of PLC, isoforms of both classic and novel PKC, Rho A, MEK1/2 and ERK1/2. The latter elicits activation of ZEB (AREB6) and regulation of the gene expression for Na,K-ATPase α₁-subunit.</p

Contributions to the resting membrane potential (mV) from electrogenic active transport by the α1 and α2 Na, K-ATPase isozymes in the diaphragm muscle of control and chronic nicotine-exposed rats.

<p><b>A</b>) RMP of muscle fibers versus ouabain concentration. Each data point represents the mean ± SEM of 130–170 measurements from 4–6 muscles. The solid line is a nonlinear regression fit to a two-site binding model: RMP = RMP₀+A₁/(1+[I]/K₁)+A₂/(1+[I]/K₂), where RMP₀ is the RMP when both ouabain-binding sites are inhibited; K₁ and K₂ are the half maximal ouabain concentrations for ouabain binding to α1 and α2 isoforms, respectively; A₁ and A₂ (mV) are their respective contributions to the RMP and [I] is the inhibitor (ouabain) concentration. The left vertical bar indicates the electrogenic potentials contributed by the α1 (black) and α2 (grey) isoforms obtained from the fitted data. Horizontal dashed lines show the predicted RMP levels for three cases: when both α isoforms are inactive (∼−61 mV, E_Nernst alone), when only α1 is active (∼−74 mV), and when both α1 and α2 are active (∼−78 mV). Muscles were incubated with the indicated concentration of ouabain for one hour before the start of recording. <b>B</b>) Concentration-dependence and K values for inhibition of the α2 and α1 isozymes, computed from the data in panel A. <b>C</b>) Changes in RMP elicited by 1 µM and 500 µM ouabain in the diaphragm of control (filled circles) and nicotine-treated (open circles) rats. Rats received nicotine orally for 21–31 days, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#s2" target="_blank">Methods</a>. Measurements are from the same muscles as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#pone-0033719-g001" target="_blank">Fig. 1</a> (oral nicotine). Arrows indicate when ouabain was added and the horizontal bar indicates when ouabain was present in the solution. RMPs were measured 15, 30 and 45 minutes and stabilized to a new level within 30 min of each solution change. Left vertical bars denote the electrogenic potentials contributed by the α1 (black) and α2 (grey) isozymes. Measurements are from 10 (control) and 9 (nicotine-treated) animals.</p

Chronic nicotine treatment activates PKCα/β2 (A) and PKCδ (B) and increases PLM phosphorylation at Ser⁶³ (D) and Ser⁶⁸ (E).

<p>Total PKCα/β2 (<b>A</b>), PKCδ (<b>B</b>), or PLM (<b>C</b>) abundance was not affected by the nicotine treatment. Bar graphs show the mean density from 8–9 measurements. A representative Western Blot is shown above each graph. Blots were probed with specific antibodies to activated PKCα/β2 (PKCα/β2 Thr^638/641) and total PKCα/β2, activated PKCδ (PKCδ Thr⁵⁰⁵) and total PKCδ, total PLM or PLM phosphorylated at Ser⁶³ (pPLM Ser⁶³) or Ser⁶⁸ (pPLM Ser⁶⁸). Protein homogenates were prepared from the same samples used for RMP and activity measurements, obtained from diaphragm muscles of rats after 21–31 day treatment with oral nicotine or sham (control) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#pone-0033719-g001" target="_blank">Fig. 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#pone-0033719-g002" target="_blank">2C</a>). * p<0.05. Y-axis, arbitrary units (AU).</p

Mean RMPs in the diaphragm muscle of control and chronic nicotine-treated rats, and the electrogenic potentials generated by α1 and α2 Na,K-ATPase basal transport.

<p>RMPs were computed from measurements in muscles perfused sequentially with no ouabain (control solution), 1 µM ouabain, or 500 µM ouabain, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#pone-0033719-g002" target="_blank">Fig. 2C</a>.</p>**<p>p<0.01 and</p>***<p>p<0.001, compared to control. Treated rats received nicotine orally for 21–31 days prior to tissue removal. RMPs were measured 30–45 min after each solution change. n = number of fibers. Mean RMPs were obtained from a fit of the RMPs in each group to a Gaussian function, after confirming that the RMPs distributed normally (Kolmogorov-Smirnov test, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#s2" target="_blank">Methods</a>).</p

C-peptide in concentration of 1 nM stimulates ERK transocation into the nucleus and the kinase activatory phosphorylation.

<p>Cells were cultured as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028294#s4" target="_blank">Materials and Methods</a>”. Total cell lysate and nuclear extracts were subject to Western blot analysis to determine ERK1/2 abundance and phosphorylation. A representative Western blot image is shown in the upper panel of each graph. <b>A.</b> The total ERK1 expression. <b>B.</b> ERK1 nuclear abundance . <b>C.</b> Total ERK1/2 phosphorylation. <b>D.</b> Nuclear ERK1/2 phosphorylation . GAPDH and histone H3 proteins were used as loading controls for total cell lysate and nuclear extracts, respectively. Results are means ± SE for 6 independent experiments. * P<0.05 versus 5 mM glucose without C-peptide.</p

Distribution histogram of resting membrane potentials in the diaphragm of control (solid bars) and nicotine-treated rats (striped bars).

<p>Treated animals received nicotine orally in the drinking water for 21–31 days prior to tissue removal. RMPs were recorded from 622 fibers from 9 muscles (nicotine) and 676 fibers from 10 control muscles (vehicle). The solid and dashed curves are Gaussian fits to the RMP distribution for each group. The distribution of RMPs in each group was consistent with a normal distribution based on the Kolmogorov-Smirnov normality test (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#s2" target="_blank">Methods</a>). The classes on the histograms are grouped (using ORIGIN 6.1) with Bin size 4.1 mV for 12 bins, in the range from −50 mV to −97.5 mV. For ease of visualization, the gap between bars was chosen = 0, overlap is 60%.</p

Na,K-ATPase α1 and α2 and nAChR content in diaphragm muscles of control and nicotine-treated rats.

<p><b>A</b>, <b>B</b>, <b>C</b> – whole homogenate; <b>D</b>, <b>E</b> – plasma membrane fraction. Upper panels show representative immunoblots; lower panels show mean densities ± SE from 9–10 blots prepared using different muscle samples. * p<0.05. Nicotine was administered orally for 21–31 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#s2" target="_blank">Methods</a>. Assays were made using diaphragm tissue from the same muscles used for RMP and activity measurements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#pone-0033719-g001" target="_blank">Fig. 1</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033719#pone-0033719-g002" target="_blank">2C</a>, oral nicotine).</p

Effects of different C-peptide and glucose concentrations on Na,K-ATPase expression and activity in HRTC.

<p>Cells were cultured and analyzed as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028294#s4" target="_blank">Materials and Methods</a>”. <b>A.</b> C-peptide at 1 nM concentration stimulates Na,K-ATPase protein expression. A representative Western blot image is shown in the upper panel. GAPDH protein was used as a loading control. <b>B.</b> Ouabain-sensitive ⁸⁶Rb⁺ uptake in intact HRTC. <b>C.</b> Na,K-ATPase α₁-subunit abundance in basolateral membrane fractions isolated from HRTC. A representative Western blot image is shown in the upper panel. GLUT2 protein was used as a loading control. <b>D.</b> Na,K-ATPase enzymatic activity in basolateral membrane fractions isolated from HRTC. Results are means ± SE for 6 independent experiments. * P<0.05 versus 5 mM glucose without C-peptide. † P<0.05 versus 5 mM glucose with 1 nM of C-peptide.</p