Inhibition of the angiotensin-converting enzyme N-terminal catalytic domain prevents endogenous opioid degradation in brain tissue
Filip Hanak, Jessica L. Swanson, Krzysztof Felczak, Prakashkumar Dobariya, Ursula C.H. Girdwood, Kenneth E. Bernstein, Swati S. More, Patrick E. Rothwell
Filip Hanak, Jessica L. Swanson, Krzysztof Felczak, Prakashkumar Dobariya, Ursula C.H. Girdwood, Kenneth E. Bernstein, Swati S. More, Patrick E. Rothwell
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Research Letter Neuroscience Vascular biology

Inhibition of the angiotensin-converting enzyme N-terminal catalytic domain prevents endogenous opioid degradation in brain tissue

Abstract

Authors

Filip Hanak, Jessica L. Swanson, Krzysztof Felczak, Prakashkumar Dobariya, Ursula C.H. Girdwood, Kenneth E. Bernstein, Swati S. More, Patrick E. Rothwell

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Figure 1

Met-enkephalin-Arg-Phe (MERF) degradation by the angiotensin-converting enzyme (ACE) N-terminal catalytic domain.

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Met-enkephalin-Arg-Phe (MERF) degradation by the angiotensin-converting ...
(A) Illustration of ACE catalytic domains. (B) Experimental design: Exogenous MERF was applied to brain slices and cleaved by ACE to produce Met-enkephalin. (C) Genetic mutations inactivating the ACE N-terminal domain (NKO) or C-terminal domain (CKO). (D) MERF conversion to Met-enkephalin in NKO mice (n = 47) and wild-type (WT) littermates (n = 32). ***P < 0.001 by 1-way ANOVA, genotype main effect. (E) MERF conversion to Met-enkephalin in CKO mice (n = 45) and WT littermates (n = 32). (F) Pharmacological inhibition of ACE N- and C-terminal domains with RXP407 and RXPA380, respectively. (G) Effects of RXP407 on MERF conversion to Met-enkephalin (n = 8–16/dose). *P < 0.05 by Dunnett’s post hoc test after significant 1-way ANOVA dose main effect (P < 0.05). (H) Effects of RXPA380 on MERF conversion to Met-enkephalin (n = 13–16/dose). (I) Experimental design: Stimulation of brain slices with 50 mM KCl to release endogenous MERF. (J) Effects of RXP407 on MERF extracellular concentration (n = 15–16/dose). **P < 0.005 by Dunnett’s post hoc test after significant 1-way ANOVA dose main effect (P < 0.01). (K) Whole-cell patch-clamp recording from nucleus accumbens Drd1-expressing medium spiny neuron (D1-MSN). (L) Bath application of RXP407 (10 μM, n = 15) caused long-term depression of evoked excitatory postsynaptic current (EPSC) amplitude, which was significantly greater than vehicle (n = 10). **P < 0.01 by 2-way ANOVA, treatment × time interaction. (M) Effects of RXP407 on EPSC amplitude and paired-pulse ratio. *P < 0.05, ****P < 0.0001 by 1-sample t test versus reference value (100%). Graphs display mean ± SEM, with individual data points from female and male mice shown as open and closed circles, respectively.