Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1994 Mar;111(3):703–710. doi: 10.1111/j.1476-5381.1994.tb14794.x

Demonstration of the neurotransmitter role of calcitonin gene-related peptides (CGRP) by immunoblockade with anti-CGRP monoclonal antibodies.

K K Tan 1, M J Brown 1, J Longmore 1, C Plumpton 1, R G Hill 1
PMCID: PMC1910088  PMID: 7912623

Abstract

1. Monoclonal antibodies (MAbs) against rat alpha-calcitonin gene-related peptide (alpha CGRP) were produced. Those which bound CGRP in a radioimmunoassay and inhibited the binding of 2-[125I]-iodohistidyl10-CGRP in a receptor binding assay were selected for immunoblockade experiments. 2. The effect of MAbs on CGRP inhibition of electrically stimulated contractions of the rat isolated vas deferens was characterized. Four out of 11 MAbs tested shifted the concentration-response curve of CGRP to the right compared with vehicle or irrelevant MAb control. MAb C4.19 produced equipotent blockade of rat alpha CGRP and rat beta CGRP and was chosen for further studies. MAb C4.19 had no pharmacologically significant effect on the concentration-response relationship of isoprenaline, rat beta-endorphin or somatostatin. 3. We demonstrated that the pharmacological response to CGRP in the presence of MAb C4.19 could be predicted when the dissociation constant and concentration of binding sites of the antibody were known. Comparison of experimental and computer simulated data showed good agreement for EC50 and maximum effect of CGRP in the presence of MAb C4.19. 4. Capsaicin at 1 microM inhibited the electrically stimulated contractions by 60.8% (95% confidence interval 51.8% to 69.9%). This effect was significantly attenuated by MAb C4.19 to 26.0% (95% confidence interval 15.2% to 36.8%; P < 0.003). 5. The immunoblockade of exogenous and endogenous CGRP described here, together with complementary evidence from other studies, strongly suggest that CGRP has a major neurotransmitter role at the neuroeffector junction of the rat vas deferens.

Full text

PDF
703

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Al-Kazwini S. J., Craig R. K., Marshall I. Postjunctional inhibition of contractor responses in the mouse vas deferens by rat and human calcitonin gene-related peptides (CGRP). Br J Pharmacol. 1986 May;88(1):173–180. doi: 10.1111/j.1476-5381.1986.tb09484.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amara S. G., Arriza J. L., Leff S. E., Swanson L. W., Evans R. M., Rosenfeld M. G. Expression in brain of a messenger RNA encoding a novel neuropeptide homologous to calcitonin gene-related peptide. Science. 1985 Sep 13;229(4718):1094–1097. doi: 10.1126/science.2994212. [DOI] [PubMed] [Google Scholar]
  3. Bos E. S., van der Doelen A. A., van Rooy N., Schuurs A. H. 3,3',5,5' - Tetramethylbenzidine as an Ames test negative chromogen for horse-radish peroxidase in enzyme-immunoassay. J Immunoassay. 1981;2(3-4):187–204. doi: 10.1080/15321818108056977. [DOI] [PubMed] [Google Scholar]
  4. Carpenter J. R. A method for presenting and comparing dose-response curves. J Pharmacol Methods. 1986 Jul;15(4):283–303. doi: 10.1016/0160-5402(86)90009-4. [DOI] [PubMed] [Google Scholar]
  5. Dennis T., Fournier A., Cadieux A., Pomerleau F., Jolicoeur F. B., St Pierre S., Quirion R. hCGRP8-37, a calcitonin gene-related peptide antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery. J Pharmacol Exp Ther. 1990 Jul;254(1):123–128. [PubMed] [Google Scholar]
  6. Galfrè G., Milstein C. Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol. 1981;73(Pt B):3–46. doi: 10.1016/0076-6879(81)73054-4. [DOI] [PubMed] [Google Scholar]
  7. Goto K., Kimura S., Saito A. Inhibitory effect of calcitonin gene-related peptide on excitation and contraction of smooth muscles of the rat vas deferens. J Pharmacol Exp Ther. 1987 May;241(2):635–641. [PubMed] [Google Scholar]
  8. Holzer P. Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol Rev. 1991 Jun;43(2):143–201. [PubMed] [Google Scholar]
  9. Long W. J., McGuire W., Palombo A., Emini E. A. Enhancing the establishment efficiency of hybridoma cells. Use of irradiated human diploid fibroblast feeder layers. J Immunol Methods. 1986 Jan 22;86(1):89–93. doi: 10.1016/0022-1759(86)90269-3. [DOI] [PubMed] [Google Scholar]
  10. Maggi C. A., Bevan S., Walpole C. S., Rang H. P., Giuliani S. A comparison of capsazepine and ruthenium red as capsaicin antagonists in the rat isolated urinary bladder and vas deferens. Br J Pharmacol. 1993 Mar;108(3):801–805. doi: 10.1111/j.1476-5381.1993.tb12881.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Maggi C. A., Chiba T., Giuliani S. Human alpha-calcitonin gene-related peptide-(8-37) as an antagonist of exogenous and endogenous calcitonin gene-related peptide. Eur J Pharmacol. 1991 Jan 3;192(1):85–88. doi: 10.1016/0014-2999(91)90072-x. [DOI] [PubMed] [Google Scholar]
  12. Maggi C. A., Meli A. The sensory-efferent function of capsaicin-sensitive sensory neurons. Gen Pharmacol. 1988;19(1):1–43. doi: 10.1016/0306-3623(88)90002-x. [DOI] [PubMed] [Google Scholar]
  13. Maggi C. A., Santicioli P., Patacchini R., Geppetti P., Giuliani S., Astolfi G. M., Baldi E., Parlani M., Theodorsson E., Fusco B. Regional differences in the motor response to capsaicin in the guinea-pig urinary bladder: relative role of pre- and postjunctional factors related to neuropeptide-containing sensory nerves. Neuroscience. 1988 Nov;27(2):675–688. doi: 10.1016/0306-4522(88)90297-7. [DOI] [PubMed] [Google Scholar]
  14. Maggi C. A., Santicioli P., Theodorsson-Norheim E., Meli A. Immunoblockade of response to capsaicin in the rat vas deferens: evidence for the involvement of endogenous calcitonin gene-related peptide. Neurosci Lett. 1987 Jul 9;78(1):63–68. doi: 10.1016/0304-3940(87)90562-3. [DOI] [PubMed] [Google Scholar]
  15. Mimeault M., Fournier A., Dumont Y., St-Pierre S., Quirion R. Comparative affinities and antagonistic potencies of various human calcitonin gene-related peptide fragments on calcitonin gene-related peptide receptors in brain and periphery. J Pharmacol Exp Ther. 1991 Sep;258(3):1084–1090. [PubMed] [Google Scholar]
  16. Moritoki H., Iwamoto T., Kanaya J., Ishida Y., Ando K., Kitagawa K. Capsaicin enhances the non-adrenergic twitch response of rat vas deferens. Br J Pharmacol. 1987 Oct;92(2):469–475. doi: 10.1111/j.1476-5381.1987.tb11344.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mulderry P. K., Ghatei M. A., Spokes R. A., Jones P. M., Pierson A. M., Hamid Q. A., Kanse S., Amara S. G., Burrin J. M., Legon S. Differential expression of alpha-CGRP and beta-CGRP by primary sensory neurons and enteric autonomic neurons of the rat. Neuroscience. 1988 Apr;25(1):195–205. doi: 10.1016/0306-4522(88)90018-8. [DOI] [PubMed] [Google Scholar]
  18. Nakamuta H., Fukuda Y., Koida M., Fujii N., Otaka A., Funakoshi S., Yajima H., Mitsuyasu N., Orlowski R. C. Binding sites of calcitonin gene-related peptide (CGRP): abundant occurrence in visceral organs. Jpn J Pharmacol. 1986 Oct;42(2):175–180. doi: 10.1254/jjp.42.175. [DOI] [PubMed] [Google Scholar]
  19. Noguchi K., Senba E., Morita Y., Sato M., Tohyama M. Co-expression of alpha-CGRP and beta-CGRP mRNAs in the rat dorsal root ganglion cells. Neurosci Lett. 1990 Jan 1;108(1-2):1–5. doi: 10.1016/0304-3940(90)90696-7. [DOI] [PubMed] [Google Scholar]
  20. Rosenfeld M. G., Mermod J. J., Amara S. G., Swanson L. W., Sawchenko P. E., Rivier J., Vale W. W., Evans R. M. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature. 1983 Jul 14;304(5922):129–135. doi: 10.1038/304129a0. [DOI] [PubMed] [Google Scholar]
  21. Saito A., Tomobe Y., Goto K. Effect of capsaicin on smooth muscles of rat vas deferens: involvement of calcitonin gene-related peptide? J Pharmacol Exp Ther. 1987 Aug;242(2):666–672. [PubMed] [Google Scholar]
  22. Santicioli P., Maggi C. A., Geppetti P., Del Bianco E., Theodorsson E., Meli A. Release of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) from organs of the genitourinary tract in rats. Neurosci Lett. 1988 Oct 5;92(2):197–201. doi: 10.1016/0304-3940(88)90060-2. [DOI] [PubMed] [Google Scholar]
  23. Shaw N. E., Foulkes R., Andrew D. P., Brown D. T., Hughes B. The effect of monoclonal antibodies to calcitonin gene-related peptide (CGRP) on CGRP-induced vasodilatation in pig coronary artery rings. Br J Pharmacol. 1992 May;106(1):196–198. doi: 10.1111/j.1476-5381.1992.tb14314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Steenbergh P. H., Höppener J. W., Zandberg J., Lips C. J., Jansz H. S. A second human calcitonin/CGRP gene. FEBS Lett. 1985 Apr 22;183(2):403–407. doi: 10.1016/0014-5793(85)80820-6. [DOI] [PubMed] [Google Scholar]
  25. Sternini C., Anderson K. Calcitonin gene-related peptide-containing neurons supplying the rat digestive system: differential distribution and expression pattern. Somatosens Mot Res. 1992;9(1):45–59. doi: 10.3109/08990229209144762. [DOI] [PubMed] [Google Scholar]
  26. Van Heyningen V., Brock D. J., Van Heyningen S. A simple method for ranking the affinities of monoclonal antibodies. J Immunol Methods. 1983 Aug 26;62(2):147–153. doi: 10.1016/0022-1759(83)90000-5. [DOI] [PubMed] [Google Scholar]
  27. Van Oers J. W., Tilders F. J. Antibodies in passive immunization studies: characteristics and consequences. Endocrinology. 1991 Jan;128(1):496–503. doi: 10.1210/endo-128-1-496. [DOI] [PubMed] [Google Scholar]
  28. Vizi E. S., Horváth T., Somogyi G. T. Evidence that somatostatin releases endogenous substance(s) responsible for its presynaptic inhibitory effect on rat vas deferens and guinea pig ileum. Neuroendocrinology. 1984 Aug;39(2):142–148. doi: 10.1159/000123970. [DOI] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES