Colocalization of prostaglandin F(2alpha) receptor FP and prostaglandin F synthase-I in the spinal cord

Abstract

Prostaglandin F(2alpha) is synthesized by prostaglandin F synthase, which exists in two types, prostaglandin F synthase I (PGFS I) and prostaglandin F synthase II (PGFS II). Prostaglandin F(2alpha) binds to its specific receptor, FP. Our previous immunohistochemical study showed the distinct localization of prostaglandin F synthases in rat spinal cord. PGFS I exists in neuronal somata and dendrites in the gray substance, and PGFS II exists in ependymal cells and tanycytes surrounding the central canal. Both enzymes are also present in endothelial cells of blood vessels in the white and gray substances of the spinal cord. In this study, we found that FP localizes in neuronal somata and dendrites but not in ependymal cells, tanycytes, or endothelial cells. Immunohistochemical analysis of serial sections showed the colocalization of FP and PGFS I. FP immunoreactivity was intense in spinal laminae I and II of the dorsal horn, a connection site of pain transmission, and was similar to that of PGFS I in neuronal elements. These findings suggest that prostaglandin F(2alpha) synthesized in the neuronal somata and dendrites exert an autocrine action there.

Fig. 1.

Western blot analysis of FP in rat spinal cord. Rat uterus (lane 1) and spinal cord (lane 2) microsomal fractions centrifuged at 100,000 g (40 μg of protein) were used as the protein source and applied on 10–20% SDS-polyacrylamide gel. These were analyzed using anti-FP IgG (A) or antigen-absorbed antibody (B). The analyses were performed under the same condition as described in the text. The positive band in the spinal cord using anti-FP IgG migrated to a position of ∼60 kDa, which is visible in a same position in the uterus, used as a positive control.

Fig. 2.

Immunohistochemistry of FP and PGFS I in a transverse section of rat spinal cord. In the observation with Nomarski differential interference microscope of single immunostaining, the coloration of the DAB reaction indicates the localization of FP and PGFS I. Bar = 500 μm.

Fig. 3.

Immunohistochemistry of FP in rat spinal cord. A: Transverse section immunostained for FP. B: Lamina X surrounding the central canal. C: The dorsal gray column at higher magnification. D: Immunoreactivity in lamina IX of the ventral gray column. E: A section treated with antigen-absorbed anti-FP IgG as a negative control. F: Higher magnification of lamina IX of a part of the image shown in E. The immunoreactive neuronal somata (large arrowheads) and dendrites (arrows) appear. Small arrowheads and the asterisk indicate blood vessels and the central canal, respectively. Bars = 500 μm in A and E, 200 μm in C, and 100 μm in B, D, and F.

Fig. 4.

Immunohistochemistry of FP in C57BL/6 mouse and FP-deficient mouse spinal cords. The specificity of the anti-FP IgG was confirmed using C57BL/6 mouse (A–C) and FP-deficient mouse (D–F). A and D: Transverse section immunostained for FP. B and E: Immunoreactivity in lamina IX of the ventral gray column. C and F: The dorsal gray column at higher magnification. Bars = 100 μm in A and D, 20 μm in B and E, and 50 μm in C and F.

Fig. 5.

Immunohistochemistry of FP and PGFS I in serial sections of lamina IX. Single immunostaining of FP or PGFS I in serial sections developed an image after the DAB reaction. The same neuronal somata (arrows) and dendrites with transverse and vertical sections (arrowheads) were immunoreactive for FP and PGFS I. Bar = 50 μm.

Fig. 6.

Multiple immunofluorescent labeling of FP, MAP2, tomato lectin, and vimentin. A and C: Lamina IX of the ventral gray column. B: The dorsal gray column. D: Lamina X surrounding the central canal. An optical section using CLSM shows that FP (red) and MAP2 (green in A and B) colocalized in the neuronal somata and dendrites (yellow in A and B). The merged image shows that FP colocalized with MAP2 (violet) but not with tomato lectin (green) in C. In lamina X, FP (red) did not colocalize with vimentin (green in D). Bars = 100 μm in A, B, and D and 20 μm in C.