Corticotropin-releasing Factor 2 Receptor Localization in Skeletal Muscle
Research Division, Procter & Gamble Pharmaceuticals, Mason, Ohio (SS,JSL,RTH,RJI), and Department of Pediatrics and Medicine, McMaster University, Hamilton, Ontario, Canada (MT)
Correspondence to: Dr. Robert J. Isfort, Research Division, Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 MasonMontgomery Road, Mason, OH 45040-9317. E-mail: isfort.rj{at}pg.com
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(J Histochem Cytochem 52:967977, 2004)
Key Words: corticotropin-releasing factor receptor skeletal muscle neural tissues
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently we have discovered that activation of the CRF2R modulates skeletal muscle mass under physiological and pathological conditions (Hinkle et al. 2003). We have also demonstrated that the CRF2R protein expressed in skeletal muscle is active and positively coupled to G
s (Hinkle et al. 2003
). Therefore, the CRF2Rs expressed in skeletal muscle are biologically active and appear to have a role in the maintenance of skeletal muscle mass. Although CRF2R protein localization has been demonstrated in several tissues, the location of the CRF2R in skeletal muscle has not been investigated. We have undertaken experiments designed to localize CRF2R expression in skeletal muscle.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunocytochemical analysis was performed on 7-µm frozen sections by indirect labeling, as follows. Cryostat sections were attached, the slides were warmed to room temperature, sections were encircled with a PAP pen, and were hydrated in PBS. Sections were permeabilized in PBS/0.1% Triton X-100 for 30 min, aspirated, and covered with blocking buffer of 10% normal donkey serum (NDS) in BSA buffer [2% bovine serum albumin (BSA)/0.1 M phosphate buffer, pH 7.4] for 1 hr at room temperature (RT). Primary antibodies were diluted in BSA buffer, and sections were incubated directly, after aspiration of blocking solution, for 1 hr at RT. Slides were washed three times for 10 min in PBS, then incubated with fluorescein-conjugated donkey secondary antibodies (Jackson Immunoresearch Labs; West Grove, PA) at 5 µg/ml in BSA buffer with 0.5 µM diamidinophenylindole dihydrochloride (DAPI; Molecular Probes, Eugene, OR) for 1 hr at RT. Sections were washed twice for 10 min in PBS with a final wash in 0.1 M Tris buffer, pH 8.5. Sections were coverslipped under PPD glycerol (10% 1 M Tris, 90% glycerol, pH 8.5, containing 1 mg/ml p-phenylenediamine), sealed with nailpolish, and stored at 20C. Tissue sections were examined with a Nikon Microphot FXA (Melville, NY). Images were captured with a Spot II digital camera (Diagnostic Instruments; Sterling Heights, MI) and processed using Metamorph 4.6 imaging software (Universal Imaging; Downingtown, PA).
Co-localization analyses were performed with the following landmark antibodies and filter sets appropriate for fluorescein and Cy-3: -smooth muscle actin (A2547; Sigma Chemical, St Louis, MO); ß-tubulin (T4026; Sigma Chemical; neurofilament (34-1000; Zymed Laboratories, San Francisco, CA); PGP9.5 (UC RA 95101; Accurate Chemical and Scientific, Westbury, NY); nestin (rat-401; Developmental Studies Hybridoma Bank, Iowa City, IA); and rhodamine-labeled
-bungarotoxin (B13421; Molecular Probes).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Do CRF2Rs present on blood vessels or in the endo/perimysium function to modulate skeletal muscle mass? CRF2Rs present on blood vessels are known to have an important role in the control of blood pressure and vasodilatation (Coste et al. 2000). It is unclear how vasodilatation would function to regulate skeletal muscle mass other than to release fluid content in muscle. We have previously demonstrated that the increase in muscle mass observed after CRF2R activation is mediated by increased myocyte mass and by myocyte cross-sectional and myocyte force production (Hinkle et al. 2003
). Therefore, CRF2R activation does more than increase fluid content in muscle. It is possible that vasodilatation leads to increased exposure of myocytes to serum anabolic growth factors, such as IGF-1, a known skeletal muscle anabolic hormone. CRF2R present in the endo/perimysium may have a role in regulating myocyte mass and contractile apparatus protein levels by the production, by fibroblasts or other connective tissue cells present in the endo/perimysium, of anabolic growth factors. This hypothesis does have some support from previously reported experimental evidence demonstrating that activation of CRFR in fibroblast modulates prostaglandin synthesis (Fleisher-Berkovich and Danon 1995
). The results we report here are not sufficient to validate any of these possible mechanisms. However, the data we present are important because they provide a set of observations that provide insights necessary for the design of future experiments to evaluate the validity of these potential mechanisms of CRF2R action.
In conclusion, CRF2R protein has been localized to neural structures, myotendinous junction, blood vessels, and endo/perimysial space in skeletal muscle. CRF2R expression was not observed on myocytes, even though treatment of skeletal muscle in vivo resulted in increased myocyte mass. The mechanism underlying the effects of CRF2R activation on regulating skeletal muscle mass is presently unknown.
![]() |
Footnotes |
---|
Received for publication February 10, 2004; accepted February 24, 2004
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bale TL, Contarino A, Smith GW, Chan R, Gold LH, Sawchenko PE, Koob GF, et al. (2000) Mice deficient for corticotropin-releasing hormone receptor 2 display anxiety-like behavior and are hypersensitive to stress. Nature Genetics 24:410414[CrossRef][Medline]
Bale TL, Giordano FJ, Hickey RP, Huang Y, Nath AK, Peterson KL, Vale WW, et al. (2002) Corticotropin-releasing factor receptor 2 is a tonic suppressor of vascularization. Proc Natl Acad Sci USA 99:77347739
Blumer R, Wasicky R, Hotzenecker W, Lukas JR (2001) Presence and structure of innervated myotendinous cylinders in rabbit extraocular muscle. Exp Eye Res 73:787796[CrossRef][Medline]
Bray JJ, Harris AJ (1975) Dissociation between nerve-muscle transmission and nerve trophic effects on rat diaphragm using type D botulinum toxin. J Physiol 253:5377[Abstract]
Carlsson L, Li Z, Paulin D, Thornell LE (1999) Nestin is expressed during development and in myotendinous and neuromuscular junctions in wild type and desmin knockout mice. Exp Cell Res 251:213223[CrossRef][Medline]
Chalmers DT, Lovenberg TW, Gridoriadis DE, Behan DP, De Souza EB (1996) Corticotropin-releasing factor receptors: from molecular biology to drug design. Trends Pharmacol Sci 17:166172.[CrossRef][Medline]
Coste SC, Kesterson RA, Heldwein KA, Stevens SL, Heard AD, Hollis JH, Murray SE, et al. (2000) Abnormal adaptations to stress and impaired cardiovascular function in mice lacking corticotropin-releasing hormone receptor 2. Nature Genet 24:403409[CrossRef][Medline]
Dautzenberg FM, Hauger RL (2002) The CRF peptide family and their receptors: yet more partners discovered. Trends Pharmacol Sci 23:7177[CrossRef][Medline]
Davis HL (1983) Trophic action of nerve extract on denervated skeletal muscle in vivo: dose dependency, species specificity, and timing of treatment. Exp Neurol 80:383394[CrossRef][Medline]
De Souza EB (1995) Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders. Psychoneuroendocrinology 20:789819[CrossRef][Medline]
Dieterich KD, Lehnert H, De Souza EB (1997) Corticotropin-releasing factor receptors: an overview. Exp Clin Endocrinol Diabetes 10:6582
Fernandez HL, Ross GS, Nadelhaft I (1999) Neurogenic calcitonin gene-related peptide: a neurotrophic factor in the maintenance of acetylcholinesterase molecular forms in adult skeletal muscle. Brain Res 844:8397[CrossRef][Medline]
Fleisher-Berkovich S, Danon A (1995) Effect of corticotropin-releasing factor on prostaglandin synthesis in endothelial cells and fibroblasts. Endocrinology 136:40684072[Abstract]
Garzon J, Hollt V, Schulz R, Herz A (1985) Excitatory neuropeptides activate opioid mechanisms in the guinea pig ileum. Neuropeptides 5:583586[Medline]
Grill HJ, Markison S, Ginsberg A, Kaplan JM (2000) Long-term effects on feeding and body weight after stimulation of forebrain or hindbrain CRH receptors with urocortin. Brain Res 867:1928[CrossRef][Medline]
Heldwein KA, Duncan JE, Stenzel P, Rittenberg MB, Stenzel-Poore MP (1997) Endotoxin regulates corticotropin-releasing hormone receptor 2 in heart and skeletal muscle. Mol Cell Endocrinol 131:167172[CrossRef][Medline]
Helgren ME, Squinto SP, Davis HL, Parry DJ, Boulton TG, Heck CS, Zhu Y, et al. (1994) Trophic effect of ciliary neurotrophic factor on denervated skeletal muscle. Cell 76:493504[Medline]
Heymann-Monnikes I, Tache Y, Trauner M, Weiner H, Garrick T (1991) CRF microinjected into the dorsal vagal complex inhibits TRH analog- and kainic acid-stimulated gastric contractility in rats. Brain Res 554:139144[CrossRef][Medline]
Hinkle RT, Donnelly E, Cody DB, Samuelsson S, Lange JS, Bauer MB, Tarnopolsky M, et al. (2003) Activation of the CRF2 receptor modulates skeletal muscle mass under physiological and pathological conditions. Am J Physiol 285:E889898
Hsu SY, Hsueh AJ (2001) Human stresscopin and stresscopin-related peptide are selective ligands for the type 2 corticotropin-releasing hormone receptor. Nature Med 7:605611[CrossRef][Medline]
Hunt CC (1990) The mammalian muscle spindle: peripheral mechanisms. Physiol Rev 70:643
Kawabuchi M, Zhou CJ, Wang S, Nakamura K, Liu WT, Hirata K (2001) The spatiotemporal relationship among Schwann cells, axons and postsynaptic acetylcholine receptor regions during muscle reinnervation in aged rats. Anat Rec 264:183202[CrossRef][Medline]
Kihara N, Fujimura M, Yamamoto I, Itoh E, Inui A, Fujimiya M (2001) Effects of central and peripheral urocortin on fed and fasted gastroduodenal motor activity in conscious rats. Am J Physiol 280:G406419
Kishimoto T, Pearse RN, Lin CR, Rosenfeld MG (1995) A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle. Proc Natl Acad Sci USA 92:11081112[Abstract]
Kishimoto T, Radulovic J, Radulovic M, Lin CR, Schrick C, Hooshmand F, Hermanson O, et al. (2000) Deletion of Crhr2 reveals an anxiolytic role for corticotropin-releasing hormone receptor-2. Nature Genet 24:415419[CrossRef][Medline]
Krikler DM, Rode J, Davies MJ, Woolf N, Moss E (1992) Atrial myxoma: a tumour in search of its origins. Br Heart J 67:8991[Abstract]
Lewis K, Li C, Perrin MH, Blount A, Kunitake K, Donaldson C, Vaughan J, et al. (2001) Identification of urocortin III, an additional member of the corticotropin-releasing factor (CRF) family with high affinity for the CRF2 receptor. Proc Natl Acad Sci USA 98:75707575
Lovenberg TW, Chalmers DT, Liu C, De Souza EB (1995) CRF2 alpha and CRF2 beta receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues. Endocrinology 136:41394142[Abstract]
Markelonis GJ, Kemerer VF, Oh TH (1980) Sciatin: purification and characterization of a myotrophic protein from chicken sciatic nerves. J Biol Chem 255:89678970
Marques MJ, Neto HS (1997) Ciliary neurotrophic factor stimulates in vivo myotube formation in mice. Neurosci Lett 234:4346[CrossRef][Medline]
Martinez V, Wang L, Rivier JE, Vale W, Tache Y (2002) Differential actions of peripheral corticotropin-releasing factor (CRF), urocortin II, and urocortin III on gastric emptying and colonic transit in mice: role of CRF receptor subtypes 1 and 2. J Pharmacol Exp Ther 301:611617
McAlexander MA, Undem BJ (1997) Enhancement of tachykinin-induced contractions of guinea pig .isolated bronchus by corticotropin-releasing factor. Neuropeptides 31:293299[Medline]
McCarthy JR, Heinrichs SC, Grigoriadis DE (1999) Recent advances with the CRF1 receptor: design of small molecule inhibitors, receptor subtypes and clinical indications. Curr Pharmacol Des 5:289315[Medline]
Mense S, Meyer H (1985) Different types of slowly conducting afferent units in the cat skeletal muscle and tendon. J Physiology 363:403417[Abstract]
Miampamba M, Maillot C, Million M, Tache Y (2002) Peripheral CRF activates myenteric neurons in the proximal colon through CRF(1) receptor in conscious rats. Am J Physiol 282:G857865
Nahirney PC, Ovalle WK (1993) Distribution of dystrophin and neurofilament protein in muscle spindles of normal and Mdx dystrophic mice: an immunocytochemical study. Anat Rec 235:501510[Medline]
Nora EH, Munzenmaier DH, Hansen-Smith FM, Lombard JH, Greene AS (1998) Localization of the ANG II type 2 receptor in the microcirculation of skeletal muscle. Am J Physiol 275:H13951403[Medline]
Radulovic J, Ruhmann A, Liepold T, Spiess J (1999) Modulation of learning and anxiety by corticotropin-releasing factor (CRF) and stress: differential role of CRF receptors 1 and 2. J Neurosci 19:50165025
Ralphs JR, Benjamin M, Thornett A (1991) Cell and matrix biology of the suprapatella in the rat: a structural and immunocytochemical study of fibrocartilage in a tendon subject to compression. Anat Rec 231:167177[Medline]
Reyes TM, Lewis K, Perrin MH, Kunitake KS, Vaughan J, Arias CA, Hogenesch JB, et al. (2001) Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors. Proc Natl Acad Sci USA 98:28432848
Smith GW, Aubry JM, Dellu F, Contarino A, Bilezikjian LM, Gold LH, Chen R, et al. (1998) Corticotropin releasing factor receptor 1-deficient mice display decreased anxiety, impaired stress response, and aberrant neuroendocrine development. Neuron 20:10931102[Medline]
Stacey MJ (1969) Free nerve endings in skeletal muscle of the cat. J Anat 105:231254[Medline]
Swett JE, Schoultz TW (1975) Mechanical transduction in the Golgi tendon organ. A hypothesis. Arch Ital Biol 113:374382[Medline]
Tamaoki J, Sakai N, Kobayashi K, Kanemura T, Shibasaki T, Takizawa T (1998) Corticotropin-releasing factor potentiates the contractile response of rabbit airway smooth muscle to electrical field stimulation but not to acetylcholine. Am Rev Respir Dis 140:13311335
Timpl P, Spanagel R, Sillaber I, Kresse A, Reul JM, Stalla GK, Blanquet V, et al. (1998) Impaired stress response and reduced anxiety in mice lacking a functional corticotropin-releasing hormone receptor 1. Nature Genet 19:16216[CrossRef][Medline]
Vaittinen S, Lukka R, Sahlgren C, Rantanen J, Hurme T, Lendahl U, Eriksson JE, et al. (1999) Specific and innervation-regulated expression of the intermediate filament protein nestin at neuromuscular and myotendinous junctions in skeletal muscle. Am J Pathol 154:591600
Wang L, Martinez V, Vale W, Tache Y (2000) Fos induction in selective hypothalamic neuroendocrine and medullary nuclei by intravenous injection of urocortin and corticotropin-releasing factor in rats. Brain Res 855:4757[CrossRef][Medline]
Yarnitzky T, Min L, Volk T (1997) The drosophila neuregulin homolog vein mediates inductive interactios between myotubes and their epidermal attachment cells. Genes Dev 11:26912700