Intrathecally injected Ile-Pro-Ile, an inhibitor of membrane ectoenzyme dipeptidyl peptidase IV, is antihyperalgesic in rats by switching the enzyme from hydrolase to synthase functional mode to generate endomorphin 2.

We have found recently that membrane-bound dipeptidyl peptidase IV (DPP-IV) generated extracellularly immunoreactive endomorphin-2 from Tyr-Pro precursor in a depolarisation-sensitive manner in rat isolated L4,5 dorsal root ganglia when the enzyme was switched to synthase mode by the hydrolase inhibitor Ile-Pro-Ile. Presently, we induced hyperalgesia in rats by injecting carrageenan into the right hindpaw and measured the reduction in nociceptive threshold (hyperalgesia) to pressure (Randall-Selitto test). The hyperalgesia, peaking at 180 min after injection, was fully reversed by intrathecal administration of 30 nmol/rat Ile-Pro-Ile. The antihyperalgesic action was antagonized by s.c. naloxone (1 mg/kg) and intrathecally injected specific antiserum to endomorphin-2 indicating that the opioid receptor-mediated effect was produced by an endogenously generated endomorphin-2-like immunoreactive substance. Intrathecal Ile-Pro-Ile was ineffective as an analgesic in the acute nociceptive test such as the rat tail-flick, whereas endomorphin-2 (EC(50)=13.3 nmol/rat), endomorphin-1 (6.8 nmol/rat), morphine (0.11 nmol/rat) and DAMGO (0.0059 nmol/rat) exerted opioid receptor-mediated analgesia given by the same route. We concluded that carrageenan-induced C-fiber barrage (wind-up) may create ideal conditions for the de novo synthesis of endomorphin-2 in rat spinal cord dorsal horns if the DPP-IV enzyme is switched to the synthase functional mode by Ile-Pro-Ile.

Development of peptide receptor binding assays: methods to avoid false negatives.

Selection of appropriate ligand receptor binding assay conditions is critical for peptides, where the possibility of obtaining false negative results is pertinent due to their inherent adsorption and instability characteristics, as well as high response-sensitivity to operational conditions. The aim of this study was thus to develop a cost-effective multivariate screening method for determination of the influence of different factors on the outcome of such studies, using (125)I-labelled vasoactive intestinal peptide binding on lung homogenate as a model. The study was divided into two parts: investigation of filtration for bound-unbound ligand separation, and screening of sample incubation variables. Experimental designs were used (including Plackett-Burman) to evaluate adsorption, total binding, non-specific binding, specific binding and (non-)specific/total binding ratio. Several significant factors were identified. For filtration, a combination of polyethylenimine and BSA filter pretreatment was best, whereas albumin-containing washing solvent negatively influenced the amount of specific bound radioligand. For sample incubation, significant effects on one or more of the studied responses were observed for several factors. Bacitracin protease inhibitor also decreased adsorption. We report here multivariate experimental designs for screening of peptide (radio)ligand receptor binding assay conditions. This approach efficiently minimizes the risk on false negative results due to inappropriate operational conditions.