Prominent expression of phosphodiesterase 5 in striated muscle of the rat urethra and levator ani

Abstract

Purpose: We investigated phosphodiesterase 5 distribution and activity in the urethra.

Materials and methods: Rat tissues were examined for phosphodiesterase 5 and alpha-smooth muscle actin expression. Urethral phosphodiesterase 5 activity was examined by tissue bath in the presence of sildenafil (Pfizer, New York, New York).

Results: Anti-alpha-smooth muscle actin antibody (Abcam) stained all known smooth muscles in all tested tissues and revealed a few smooth muscle fibers in the levator ani muscle. Anti-phosphodiesterase 5 antibody (Abcam) stained smooth muscle in the penis and bladder but not striated leg muscle. However, it stained predominantly striated muscle in the urethra and the levator ani muscle. In the urethra the amount of phosphodiesterase 5 in striated muscle was 6 times that in smooth muscle. In urethral striated muscle phosphodiesterase 5 expression was localized to Z-band striations. Smooth and striated muscle intermingling was clearly visible on the inner and outer rims of the circularly arranged striated muscle layer. Relaxation of precontracted urethral tissues by sodium nitroprusside (Sigma-Aldrich) was enhanced by sildenafil, indicating phosphodiesterase 5 activity, which was primarily located in the striated muscle according to phosphodiesterase 5 staining.

Conclusions: Despite its presumed smooth muscle specificity phosphodiesterase 5 was predominantly expressed in the striated muscle of the urethra and in the levator ani muscle. Results are consistent with earlier studies in which these striated muscles were developmentally related to smooth muscle. They also suggest that these striated muscles are possibly regulated by phosphodiesterase 5.

Figure 1

Localization of PDE5 expression. SMA staining (red) was used to identify smooth muscles (sm) in the indicated tissues. The specificity of the anti-SMA was confirmed by the staining of all of the known smooth muscles including the bladder smooth muscle (bsm), the cavernous smooth muscle (csm), and the vascular smooth muscle of blood vessels (bv). The presence of smooth muscle in the levator ani smooth muscle specimens has been reported previously (see Discussion). PDE5 staining (green) of smooth muscles is clearly visible in the bladder and penis but is less in the urethra and leg blood vessels. PDE5 staining of the striated muscle (st) is prominent in the urethra and levator ani muscle. DAPI staining (blue) serves to locate cellular nuclei. Optical magnification is indicated on the right lower corner of each picture.

Figure 2

Differential distribution of PDE5 and smooth muscle actin in the urethra. SMA staining (red) mainly occurred in the longitudinal smooth muscle (lsm) and the circular smooth muscle (csm). Lesser SMA staining was found with blood vessels (bv) and presumed smooth muscle (sm?) located outside of the striated muscle (st) layer. PDE5 staining (green) was found mainly in the striated muscle. Note that the two blood vessels are also devoid of PDE5 staining. DAPI staining (blue) serves to locate cellular nuclei. Optical magnification is indicated on the right lower corner of each picture. Boxed areas in the 40x pictures are shown in the 200x pictures.

Figure 3

Differential expression of PDE5 in the urethral smooth and striated muscles. The pixel numbers of the PDE5 (green)-stained areas within the striated muscle (ST) and the smooth muscle (SM) compartments (A) of each urethra sample were counted. The results are compiled and shown in B. The number of pixels shown on the y-axis is the average number of 12 urethra samples from 12 different female rats. * indicates p<0.01.

Figure 4

Detailed structures of the urethral musculature. At 1000x magnification, PDE5 staining (green) revealed the characteristic striations of striated muscles, as demonstrated clearly in A. The same picture (A) also shows the SMA (red)-stained fibers are sandwiched between the much larger striated fibers. Pictures B and C show the intermingling striated and smooth muscle fibers on the outer and inner rims, respectively, of the circular striated muscle layer. DAPI staining (blue) serves to locate cellular nuclei. Note that the intermingled fiber centrally located in B can also be seen at the 200x magnification in Fig. 2 (labeled by “sm?”). In this case, the absence of DAPI staining allows better visualization of the green color within the red color.

Figure 5

Identification of PDE5 activity. Urethral samples shaped as rings were used in tissue bath experiments. They were treated with sildenafil (n=7) or its solvent (n=6, as controls), contracted with phenylephrine, and then relaxed with sodium nitroprusside (SNP) at the indicated concentrations. * indicates p<0.05.