Extensive biochemical and pharmacological studies of the first systemically-active, mixed inhibitors of enkephalin-degrading enzymes

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Selective effects of [D-Ser2(O-t-butyl),Leu5]enkephalyl-Thr6 and [D-Ser2(O-t-butyl),Leu5]enkephalyl-Thr6 (O-t-butyl), two new enkephalin analogues, on neurotransmitter release and adenylate cyclase in rat brain slices

The selectivity and potency of two new enkephalin-derived δ-opioid receptor agonists, DSTBULET ([D-Ser2(O-t-butyl), Leu5]enkephalyl-Thr6) and BUBU ([D-Ser2(O-t-butyl),Leu5]enkephalyl-Thr6 (O-t-butyl)) were determined with functional tests in vitro of μ-, δ-, and κ-opioid receptor activation in the rat brain. Both peptides concentration dependently (1 nM-1 μM) inhibited the release of radiolabeled acetylcholine (ACh) from striatal slices (pD2 7.6-7.9), an effect exclusively mediated by δ-opioid receptor activation. Fentanyl isothiocyanate (FIT), an irreversible δ-antagonist, completely blocked the inhibitory effects of DSTBULET and BUBU. Up to a concentration of 1 μM, the peptides did not affect striatal [3H]dopamine (DA) release nor cortical [3H]noradrenaline (NA) release, processes which are known to be inhibited by opioids activating κ and μ-receptors, respectively. Furthermore, both DSTBULET and BUBU caused a strong inhibition (pD2 8.2-8.3) of D-1 dopamine receptor-stimulated cyclic AMP efflux from striatal slices, an effect known to be mediated by μ- and/or δ-opioid receptor activation. However, the peptides were without effect when D-1 and D-2 dopamine receptors were stimulated simultaneously, a situation in which only μ-agonists are able to inhibit the resulting cAMP efflux. In conclusion, DSTBULET and BUBU appear to display a high selectivity and potency toward functional δ-opioid receptors in the brain

Evidence of a role for NK1 and CGRP receptors in mediating neurogenic vasodilatation in the mouse ear

1. The aims of this study were to develop a technique to measure blood flow in the mouse ear and to investigate the nature of the vasodilator mediator(s) involved in the response to capsaicin. 2. The response to capsaicin, applied topically, was investigated in anaesthetized CD1 or Sv129+C57BL/6 wild-type (+/+) or NK(1) receptor knockout mice (−/−). Blood flow was assessed by laser Doppler flowmetry and oedema formation by (125)I-albumin accumulation. 3. Capsaicin induced significant increases in blood flow (0.2–200 μg in 20 μl) and oedema (2–200 μg in 20 μl). The oedema response was absent in NK(1)−/− mice and NK(1)+/+mice treated with the selective NK(1) receptor antagonist SR140333 (480 nmol kg(−1)) as expected. Furthermore, the capsaicin-evoked increase in blood flow was significantly potentiated in the knockout mice (203% of wild-type response, P<0.05) and wild-type mice treated with SR140333 (201%, P<0.05). 4. The CGRP receptor antagonist CGRP(8–37) (400 nmol kg(−1)) had no effect on capsaicin-induced blood flow in NK(1)+/+mice but abolished the increased blood flow to capsaicin in NK(1)−/−, and NK(1)+/+wild-type mice pre-treated with SR140333. 5. The results indicate that neurogenic vasodilatation can be measured in the mouse ear. The capsaicin-induced increased blood flow involves activation of, and possible interactions between, both NK(1) and CGRP(1) receptors