R-isovaline does not activate GABA_B receptors in cultured hippocampal neurons.

<p><b>a</b>) Current at a holding potential of -80 mV in a high extracellular K⁺ solution at baseline (open bars) and on application of R-baclofen (n = 7), R-baclofen + CGP 52432 (n = 7) and R-isovaline at low (1 μM; n = 3) and high (50 μM; n = 8) concentrations (shaded bars). <b>b</b>) Example shows R-baclofen-mediated current <b>c</b>) Example shows lack of effect of low concentration of R-isovaline in a cell that responded to R-baclofen both before and after R-isovaline application. <b>d</b>) Example shows the effect of R-baclofen after 5 min pretreatment with 50 μM R-isovaline. <b>e</b>) Example shows the effects of two applications of R-baclofen after prolonged incubation (> 1 hour) with R-isovaline. <b>f</b>) Graph shows the magnitude of the current evoked by 5 μM R-baclofen in control extracellular solution (n = 8), after 5 minutes pretreatment with R-isovaline (n = 4) or after > 1 hour pretreatment with R-isovaline (n = 8). Data represent mean ± SEM. ** = P < 0.01.</p

GABA, R-baclofen and R-isovaline do not evoke currents in untransfected AtT-20 cells.

<p><b>a)</b> Baseline subtracted IV curve for GABA (n = 4), R-baclofen (n = 4) and R-isovaline (n = 5). <b>b</b>) Example currents at the maximum hyperpolarising step (-112.5 mV, voltage step depicted above currents) at baseline (black) and on application (grey) of GABA, R-baclofen or R-isovaline. <b>c</b>) Summary graph showing currents recorded at -112.5 mV at baseline (open bars) and on application of GABA, R-baclofen, or R-isovaline (shaded bars). Data are represented as mean ± SEM.</p

GABA and R-baclofen, but not isovaline, evoke inwardly rectifying currents in transfected AtT-20 cells.

<p><b>a)</b> Baseline subtracted IV curve for GABA and GABA + CGP 52432 (n = 7). Inset shows example currents at baseline (black), in the presence of GABA (dark grey) or in the presence of GABA + CGP 52432 (light grey). <b>b</b>) Baseline subtracted IV curve for R-baclofen (n = 6). Inset shows example currents at baseline (black) or in the presence of R-baclofen (dark grey). <b>c</b>) Baseline subtracted IV curves for R-isovaline at three concentration (50 μM n = 6; 250 μM n = 8; 1 mM n = 4). <b>d</b>) Example currents at baseline (black) and on application of three concentrations of R-isovaline (dark grey). <b>e</b>) Baseline subtracted IV curve for S-isovaline (n = 4). Inset shows example currents at baseline (black) or in the presence of S-isovaline (dark grey). <b>f</b>) Summary graph showing the currents recorded at -112.5 mV at baseline (open bars) or on application of GABA, R-baclofen, R-isovaline or S-isovaline (shaded bars). Data are represented as mean ± SEM. *** = p < 0.001.</p

R-isovaline does not modulate GABA-evoked currents in transfected AtT20 cells.

<p><b>a</b>) Baseline subtracted IV curves for GABA (1 μM; n = 3) and co-application of R-isovaline with GABA (1 μM n = 3; 10 nM n = 6). <b>b</b>) Graph shows that R-isovaline does not alter the current evoked by a high concentration of GABA. Inset shows example currents at baseline (black), on application of GABA (1 μM; dark grey) and on co-application of 1 μM GABA with 250 μM R-isovaline (light grey). <b>c</b>) Graph shows that R-isovaline does not alter the current when co-applied with a low concentration of GABA. Inset shows example currents at baseline (black), on application of 1 μM GABA (dark grey), and on co-application of 10 nM GABA with 250 μM R-isovaline (light grey). Data are expressed as mean ± SEM. * = p < 0.05, ** = p < 0.01.</p

The GABA_B1 subunit expresses at the membrane in transfected AtT-20 cells.

<p>AtT-20 cells were transfected with both subunits for the GABA_B receptor along with GFP (shown in green). Immunohistochemistry was performed for the GABA_B1 subunit (red) and cell nuclei were stained with DAPI (blue). <b>a)</b> Magnified image of an isolated cell demonstrating GABA_B1 subunit protein at the cell membrane in a GFP positive cell. <b>b)</b> Negative control (no primary antibody for GABA_B1). A merged image is shown at the bottom of each column. Scale bars are 5 μm.</p

A Novel Leu-Enkephalin Prodrug Produces Pain-Relieving and Antidepressant Effects

Persistent pain is a significant healthcare problem with limited treatment options. The high incidence of comorbid chronic pain and depression significantly reduces life quality and complicates the treatment of both conditions. Antidepressants are less effective for pain and depression than for depression alone and they induce severe side effects. Opioids are highly efficacious analgesics, but rapid development of tolerance, dependence, and debilitating side effects limit their efficacy and safe use. Leucine-enkephalin (Leu-ENK), the endogenous delta opioid receptor agonist, controls pain and mood and produces potent analgesia with reduced adverse effects compared to conventional opioids. High proteolytic instability, however, makes Leu-ENK ineffective after systemic administration and limits its clinical usefulness. KK-103, a Leu-ENK prodrug, was developed to overcome these limitations of Leu-ENK via markedly increased plasma stability in mice. We showed rapid and substantially increased systemic adsorption and blood plasma exposure of KK-103 compared to Leu-ENK. We also observed brain uptake of radiolabeled KK-103 after systemic administration, indicating a central effect of KK-103. We then established KK-103’s prolonged antinociceptive efficacy in the ramped hot plate and formalin test. In both models, KK-103 produced a comparable dose to the maximum antinociceptive-effect relationship. The pain-alleviating effect of KK-103 primarily resulted from activating the delta opioid receptor after the likely conversion of KK-103 to Leu-ENK in vivo. Finally, KK-103 produced an antidepressant-like activity comparable to the antidepressant desipramine, but with minimal gastrointestinal inhibition and no incidence of sedation