Figure 1.
Distribution histogram of resting membrane potentials in the diaphragm of control (solid bars) and nicotine-treated rats (striped bars).
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 (Methods). 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%.
Figure 2.
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.
A) 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, ENernst 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) Concentration-dependence and K values for inhibition of the α2 and α1 isozymes, computed from the data in panel A. C) 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 Methods. Measurements are from the same muscles as in Fig. 1 (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.
Table 1.
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.
Figure 3.
Na,K-ATPase α1 and α2 and nAChR content in diaphragm muscles of control and nicotine-treated rats.
A, B, C – whole homogenate; D, E – 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 Methods. Assays were made using diaphragm tissue from the same muscles used for RMP and activity measurements (Fig. 1 & 2C, oral nicotine).
Figure 4.
The nAChRα1 subunit and the Na,K-ATPase α1 and α2 subunits and PLM co-immunoprecipitate in rat diaphragm muscle after 21–31days of sham or oral nicotine treatment.
Skeletal muscle protein was prepared from control and nicotine treated animals and immunoprecipitated (IP) with monoclonal antibodies against the nAChRα1 subunit. Precipitates were probed by Western blot (WB) using antibodies against the nAChRα1, Na,K-ATPase α1 and α2, and PLM. A positive control (lane 1, input) confirmed the presence of each species in the control sample before IP. Each panel is a representative Western blot from 7–8 independent experiments. Protein homogenates were prepared using diaphragm tissue from the same muscles used for RMP and activity measurements (Fig. 1 & 2C, oral nicotine).
Figure 5.
Chronic nicotine treatment activates PKCα/β2 (A) and PKCδ (B) and increases PLM phosphorylation at Ser63 (D) and Ser68 (E).
Total PKCα/β2 (A), PKCδ (B), or PLM (C) 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 Thr638/641) 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) (Fig. 1 & 2C). * p<0.05. Y-axis, arbitrary units (AU).
Figure 6.
Effects of PMA on PKC and PLM phosphorylation and [3H]ouabain binding to intact rat skeletal muscle.
A) Activation of PKCα/β2 and PKCδ by PMA (phorbol-12-myristate-13-acetate, 100 nM) induces parallel increases in PLM phosphorylation at Ser63 and Ser68. A rat soleus muscle was dissected and equilibrated for 30 min in standard Krebs-Ringer solution, then incubated in K+-free Krebs–Ringer buffer containing 100 nM PMA and 2 µM [3H]ouabain for 0, 30, 60 and 120 min, followed by 4×15 min washout in ice-cold K+-free Krebs–Ringer buffer. Five independent experiments were performed and a representative Western blot is shown. B) [3H]ouabain binding site content in intact rat soleus muscle was determined directly in the same experiment and expressed per gram wet weight. Mean values ±S.E.M. are shown, * p<0.05, n = 5.