Corticotropin-releasing factor immunostaining in the rat spinal cord and medulla oblongata: An unexpected form of cross-reactivity with substance P |
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| Authors: | F. Berkenbosch J. Schipper F.J.H. Tilders |
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| Affiliation: | 1. Department of Pharmacology, Medical Faculty, Free University Amsterdam, The Netherlands;2. Duphar B.V., Weesp, The Netherlands;1. IVF-clinic Altra Vita, 4A, Nagornaya st., Moscow, 117186, Russia;2. Faculty of Moscow State University, Russia;1. Sorbonne University, Inserm, CNRS, Institut du Cerveau, Pitié-Salpêtrière Hospital, F-75013 Paris, France;2. IHU ICAN (ICAN OMICS Lipidomics) Foundation for Innovation in Cardiometabolism and Nutrition, Pitié-Salpêtrière Hospital, F-75013 Paris, France;3. CNRS UMR 7221, Sorbonne University, Muséum National d’Histoire Naturelle, F-75005 Paris France;1. Department of Cellular and Molecular Pharmacology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, Hiroshima, 734-8553, Japan;2. Department of Dental Anesthesiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, Hiroshima, 734-8553, Japan;3. School of Dentistry, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, Hiroshima, 734-8553, Japan;4. Faculty of Odonto-Stomatology, University of Health Sciences, #73, Monivong Blvd., Sangkat Sras Chak, Khan Daun Penh, Phnom Penh, 12201, Cambodia;5. Faculty of Dental Medicine, Universitas Airlangga, Jl. Mayjen Prof. Dr. Moestopo No. 47, Surabaya, East Java, 60132, Indonesia;6. Department of Neurochemistry and Environmental Health Sciences, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan;7. Faculty of Odonto-Stomatology, Hong Bang International University, 215 Dien Bien Phu Street, Ward 15, Binh Thanh District, Ho Chi Minh City, Viet Nam;8. Department of Pharmacology, Graduate School of Dentistry, Osaka University, Suita, Osaka, 565-0871, Japan;9. Department of Pharmacology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, Hiroshima, 734-8553, Japan;10. Department of Advanced Prosthodontics, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, Hiroshima, 734-8553, Japan;11. Molecular Research Center for Children''s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Suita, Osaka, 565-0871, Japan;12. Department of Physiological Sciences, School of Pharmaceutical Sciences, Wakayama Medical University, Wakayama, Wakayama, 640-8156, Japan;13. Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka, 565-0871, Japan;14. Global Center for Medical Engineering and Informatics, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan |
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| Abstract: | ![]()
By use of two antisera (alpha-CRFA, alpha-CRFB) raised against conjugates of o-CRF and bovine thyroglobulin, cryostat sections of formaldehyde-fixed gelatin models containing o-CRF can be stained. The staining intensity was quantitated by use of an automated microfluorimeter and was shown to be dependent on the concentration of o-CRF (1-300 microM) added to the gel. Determination of the CRF staining intensity after incorporation of o-CRF-related peptides and fragments indicated that both antisera reacted with the C-terminal region of o-CRF. They showed poor cross reactivity with r-CRF fixed in the gel. In the same models, r-CRF could be immunostained efficiently by use of an antiserum (alpha-CRFC) raised to a conjugate of r-CRF and thyroglobulin. This antiserum reacted with the N-terminal and midportion parts but not with the C-terminal fragment of o-CRF fixed in the gels. By use of both o-CRF antisera nerve fibers can be stained in the rat hypothalamus (median eminence) and in the medulla oblongata (spinal trigeminal tract and nucleus) and spinal cord (dorsal horn). Immunoinhibition experiments showed that o-CRF caused a concentration-dependent quenching (0.001-1 microM) of the immunostaining of o-CRF-containing models, rat median eminence and medulla oblongata preparations. alpha-CRFC also stained CRF immunoreactive (CRFi) fibers in the rat hypothalamus with an equal distribution to that found with the o-CRF antisera. However, no immunostaining was found in the spinal trigeminal nucleus and tract and in the dorsal horn, indicating that these fibers store different CRF-related products from those found in the hypothalamus. The CRFi in the medulla oblongata and spinal cord induced by alpha-CRFA was completely abolished 1 week after treatment of adult rats with capsaicin, a substance known to deplete Substance P (SP) from those areas. Gels incorporated with SP showed a concentration-dependent increase (range 10-1000 microM) in immunostaining with both o-CRF sera but not with the r-CRF antiserum. In addition, incubation of o-CRF sera with SP caused a concentration-dependent quenching (range 10-100 microM) of immunostaining in SP-containing models. SP at a concentration of 100 microM was also effective in quenching the CRFi in the dorsal horn and spinal trigeminal area. Quenching was also obtained with the C-terminal part of o-CRF (range 0.002-0.1 microM), which indicates that both CRF antisera contain an immunoglobulin which recognizes determinants on CRF as well as on SP.(ABSTRACT TRUNCATED AT 400 WORDS) |
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| Keywords: | Immunocytochemistry Medulla oblongata Spinal cord Hypothalamo-infundibular system Corticotropin-releasing factor Substance P |
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