Pharmacological and functional biochemical properties of D-Ala2-D-Nle5-enkephalin-Arg-Phe.
Tyr-D-Ala-Gly-Phe-D-Nle-Arg-Phe (DADN) a synthetic analogue of the endogenous Met-enkephalin-Arg-Phe (Tyr-Gly-Gly-Phe-Met-Arg-Phe; MERF), was investigated in radioligand binding assays, [(35)S]GTPgammaS stimulation experiments as well as in in vivo algesiometric tests. Binding properties of [(3)H]DADN were measured in crude membrane fractions of rat spinal cord tissues and in homogenates of Chinese hamster ovary (CHO) cells selectively expressing delta-, kappa-or micro-opioid receptors. The highest affinity for [(3)H]DADN binding was observed in membranes from CHO cells transfected with micro-opioid receptors confirming the micro-selectivity of the peptide. Unlabeled DADN was also investigated in functional biochemical experiments by measuring opioid receptor-mediated G-protein activation in rat brain membrane fractions. The peptide stimulated the activity of the regulatory G-proteins in a concentration dependent manner, and the stimulation was efficiently inhibited in the presence of micro-receptor specific antagonist ligands further supporting the selectivity profile of DADN. Intrathecally administered DADN produced a dose-related, naloxone-reversible antinociception in rat hot water tail-flick tests. Among the selective opioid antagonists tested, the delta-selective naltrindole (NTI) and the kappa-specific norbinaltorphimine (norBNI) showed only slight blocking effects compared with naloxone. The results obtained in the in vitro agonist-stimulated [(35)S]GTPgammaS binding assays are in good agreement with the opioid agonist effect seen in the in vivo pain test.
Tóth
Fanni
F
Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary.
Horváth
Gyöngyi
G
Szikszay
Margit
M
Farkas
Judit
J
Tóth
Géza
G
Borsodi
Anna
A
Benyhe
Sándor
S
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004745:Enkephalins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006358:Hot Temperature; D020128:Inhibitory Concentration 50; D051381:Rats; D017208:Rats, Wistar; D011957:Receptors, Opioid / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism; D013312:Stress, Physiological / Q000139:chemically induced / Q000503:physiopathology; D013623:Tail / Q000187:drug effects / Q000502:physiology; D013997:Time Factors; D014867:Water / Q000494:pharmacology
C498603:D-Ala2-D-Nle5-enkephalin-Arg-Phe; D004745:Enkephalins; D011957:Receptors, Opioid; D014867:Water; D016244:Guanosine 5'-O-(3-Thiotriphosphate)