Neuropeptide Y (NPY) Receptor Assays

Alex Daniels1, Wallace Harrington1

1 Glaxo Wellcome Research and Development Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 4.11
DOI:  10.1002/0471141755.ph0411s01
Online Posting Date:  May, 2001
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Neuropeptide Y (NPY) is a very abundant neuropeptide that has been implicated in the regulation of a wide variety of physiological functions such as vascular tone, food ingestion, mood, and hormone secretion. In the kidney, NPY is a potent vasoconstrictor, localized predominantly in adrenergic nerve terminals supplying the afferent arteriole and the juxtaglomerular apparatus. Therefore, isolated rat kidney provides a convenient model for examining the vasoconstriction produced by NPY and its analogs. This unit describes a general method for the preparation, handling, and perfusion of the isolated rat kidney, along with measurement of the tissue response upon exposure to NPY (or its analogs) and NPY antagonists. Neuropeptide Y (NPY) is a very abundant neuropeptide that has been implicated in the regulation of a wide variety of physiolog.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • 10× perfusion solution (see recipe)
  • Carbogen gas (95% O 2/5% CO 2) with regulator capable of delivering gas at 3 to 4 lb/in.2
  • Male Sprague‐Dawley rat (200 to 400 g)
  • Ketamine/acepromazine anesthetic (see recipe), prewarmed to 37°C
  • 100 U/ml heparin (sodium salt; Elkins‐Sinn) in saline (0.9% NaCl)
  • NPY (Peninsula Labs) or other peptide test compound (pressor substance; see recipe for peptide preparation)
  • Surgical instruments: scalpel, fine scissors, forceps
  • Pressure transducer with a range of 0 to 200 ± 1 mmHg (e.g., Statham P23 ID, Spectramed)
  • Heated circulating water bath, adjustable to within 0.1°C
  • Heated isolated organ chamber (e.g., Radnoti Glass)
  • Syringe pump (Model 355, Sage Instruments, or equivalent)
  • Peristaltic pump capable of maintaining constant flow within 0.1 ml (e.g., Minipuls 2, Gilson)
  • Recording instrument able to accurately record perfusion pressure changes within 1 mmHg (Polygraph model 7D and Polygraph DC driver amplifier, Grass)
  • Stainless steel 20‐G blunt‐tipped needle
  • Tygon perfusion tubing (R3603, 1/16‐in. i.d., 3/16‐in. o.d., 1/16‐in. wall)
  • Rubber injection ports: short latex‐free male adapter plug (Abbott Labs)
  • Fine (2‐0 or 3‐0) silk thread
  • Mercury sphygmomanometer (Baum Manometer)
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Literature Cited

Literature Cited
   Allen, J.M., Raine, A.E.G., Ledingham, J.G.G., and Bloom, S.R. 1985. Neuropeptide Y: A novel renal peptide with vasoconstrictor and natriuretic activity. Clin. Sci. 68:373‐377.
   Ballesta, J., Polak, J.M., Allen, J.M., and Bloom, S.R. 1984. The nerves of the juxtaglomerular apparatus of man and other mammals contain the potent peptide NPY. Histochemistry 80:483‐485.
   Daniels, A.J., Matthews, J.E., Slepetis, R.J., Jansen, M., Viveros, O.H., Tadepalli, A., Harrington, W., Heyer, D., Landavazo, A., Leban, J.J., and Spaltenstein, A. 1995. High‐affinity neuropeptide Y receptor antagonists. Proc. Natl. Acad. Sci. U.S.A. 92:9067‐9071.
   Daniels, A.J., Heyer, D., and Spaltenstein, A. 1997. Peptide antagonists of neuropeptide Y: Design, structure and pharmacological characterization. In Neuropeptide Y and Drug Development (L. Grundemar and S.R. Bloom, eds.) pp. 127‐155. Academic Press, London.
   Doods, H.N., Wienen, W., Entzeroth, M., Rudolf, K., Eberlein, W., Engel, W., and Wieland, H.A. 1995. Pharmacological characterization of the selective nonpeptide neuropeptide Y Y1 receptor. J. Pharmacol. Exp. Ther. 275:136‐142.
   Fuhlendorff, J., Gether, U., Aakerlund, L., Langeland‐Johansen, N., Thoegersen, H., Melberg, S.G., Olsen, U.B., Thastrup, O., and Schwartz, T.W. 1990. [Leu31,Pro34] neuropeptide Y: A specific Y1 receptor agonist. Proc. Natl. Acad. Sci. U.S.A. 87:182‐186.
   Gu, J., Polak, J.M., Allen, J.M., Huang, W.M., Sheppard, M.N., Tatemoto, K., and Bloom, S.R. 1984. High concentration of a novel peptide, Neuropeptide Y, in the innervation of mouse and rat heart. J. Histochem. Cytochem. 32:467‐472.
   Hackental, E., Aktories, K., Jakobs, K.H., and Lang, R.E. 1987. Neuropeptide Y inhibits renin release by a pertussis toxin–sensitive mechanism. Am. J. Physiol. 252:F543‐F550.
   Mohy El‐Din, M.M. and Malik, K.U. 1988. Neuropeptide Y stimulates renal prostaglandin synthesis in the isolated rat kidney: Contribution of Ca and calmodulin. J. Pharmacol. Exp. Ther. 246:479‐484.
   Tadepalli, A.S., Harrington, W.W., Hashim, M.A., Matthews, J., Leban, J.J., Spaltenstein, A., and Daniels, A.J. 1996. Hemodynamic characterization of a novel neuropeptide Y receptor antagonist. Cardiovasc. Pharmacol. 27:712‐718.
   Tatemoto, K. 1982. Neuropeptide Y: Complete amino acid sequence of the brain peptide. Proc. Natl. Acad. Sci. U.S.A. 79:5485‐5489.
   Wahlestedt, C. and Reis, D.J. 1993. Neuropeptide Y–related peptides and their receptors: Are the receptors potential therapeutic drug targets? Annu. Rev. Pharmacol. Toxicol. 32:309‐352.
   Zukowska‐Grojec, Z. and Wahlestedt, C. 1993. Origin and actions of neuropeptide Y in the cardiovascular system. In The Biology of Neuropeptide Y and Related Peptides (W.F. Colmers and C. Wahlestedt, eds.) pp. 315‐388. Humana Press, Totowa, N.J..
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