Abstract
The amounts of dynorphin-(1–8) [dyn-(1–8)] and its seven hydrolysis products, Y, YG, YGG, YGGF, YGGFL, YGGFLR and YGGFLRR, were estimated after incubating dyn-(1–8) with a membrane fraction from either guinea-pig ileum or striatum for various times at 37°C. The major hydrolysis products during the initial 5-min incubation were YGGFLR and Y, which indicates that dipeptidyl carboxypeptidase and aminopeptidase activities were mainly involved in the hydrolysis. After 60 min of incubation, dyn-(1–8) was completely hydrolyzed in both membrane preparations. When the ileal and the striatal preparations were incubated for 60 min in the presence of both captopril, a dipeptidyl carboxypeptidase inhibitor, and amastatin, an aminopeptidase inhibitor, 63.8 and 49.3% of dyn-(1–8), respectively, were hydrolyzed. The YGG fragment was the major hydrolysis product in both preparations. When the ileal and the striatal membrane fractions were incubated with dyn-(1–8) in the presence of three peptidase inhibitors, captopril, amastatin and phosphoramidon (an inhibitor of endopeptidase-24.11), approximately 95% of the opioid octapeptide remained intact in both cases. This shows that dyn-(1–8) was almost exclusively hydrolyzed by three enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive dipeptidyl carboxypeptidase I and phosphoramidon-sensitive endopeptidase-24.11, in both ileal and striatal membranes. Additionally, the Ke (equilibrium dissociation constant) values of selective antagonists against dyn-(1–8) and its initial main hydrolysis product YGGFLR in two isolated preparations pretreated with the three peptidase inhibitors indicate that the latter acts onmu receptors in guinea pig ileum but deltareceptors in mouse vas deferens and the former acts on kappareceptors in both preparations. It is indicated, therefore, that in the absence of peptidase inhibitors endogenously released dyn-(1–8) acts either through dyn-(1–8) itself on kappa receptors or through YGGFLR on mu or delta receptors depending on both the three peptidase activities and the three receptor type densities at the target synaptic membrane.
Footnotes
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Send reprint requests to: Dr. Tetsuo Oka, Department of Pharmacology, School of Medicine, Tokai University, Isehara 259-11, Japan.
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↵1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
- Abbreviations:
- dyn-(1–8) and -(1–17)
- dynorphin-(1–8) and -(1–17)
- AsA
- amastatin-sensitive aminopeptidase(s)
- PsE
- phosphoramidon-sensitive endopeptidase-24.11 (“enkephalinase”, EC3.4.24.11)
- CsD
- captopril-sensitive dipeptidyl carboxypeptidase I (angiotensin 1 converting enzyme, kininase II, EC 3.4.15.1)
- met-enk
- [Met5]-enkephalin
- met-enk-RGL
- [Met5]-enkephalin-Arg6-Gly7-Leu8
- PI
- peptidase inhibitor
- DAMGO
- [d-Ala2,N-Me-Phe4,Gly-ol]-enkephalin
- DPDPE
- [d-Pen2,5]-enkephalin
- CTOP
- d-Phe-Cys-Tyr-d-Trp-Orn-Thr-Pen-Thr-NH2
- nor-BNI
- nor-binaltorphimine HCl tetrahydrate
- ICI-174
- 864,N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (Aib = α-aminoisobutyric acid)
- HPLC-ECD
- high-performance liquid chromatography combined with electrochemical detection
- The standard one-letter
- instead of three-letter, codes for amino acids were employed for the hydrolysis products of dyn-(1–8)
- Received January 28, 1998.
- Accepted April 10, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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