The novel nicotinic receptor antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), inhibits nicotine-evoked [(3)H]norepinephrine overflow from rat hippocampal slices

Abstract

Smoking is a significant health concern and strongly correlated with clinical depression. Depression is associated with decreased extracellular NE concentrations in brain. Smokers may be self-medicating and alleviating their depression through nicotine stimulated norepinephrine (NE) release. Several antidepressants inhibit NE transporter (NET) function, thereby augmenting extracellular NE concentrations. Antidepressants, such as bupropion, also inhibit nicotinic receptor (nAChR) function. The current study determined if a recently discovered novel nAChR antagonist, N,N'-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), inhibits nicotine-evoked NE release from superfused rat hippocampal slices. Previous studies determined that bPiDDB potently (IC(50)=2 nM) inhibits nicotine-evoked striatal [(3)H]dopamine (DA) release in vitro, nicotine-evoked DA release in nucleus accumbens in vivo, and nicotine self-administration in rats. In the current study, nicotine stimulated [(3)H]NE release from rat hippocampal slices (EC(50)=50 microM). bPiDDB inhibited (IC(50)=430 nM; I(max)=90%) [(3)H]NE release evoked by 30 microM nicotine. For comparison, the nonselective nAChR antagonist, mecamylamine, and the alpha7 antagonist, methyllycaconitine, also inhibited nicotine-evoked [(3)H]NE release (IC(50)=31 and 275 nM, respectively; I(max)=91% and 72%, respectively). Inhibition by bPiDDB and mecamylamine was not overcome by increasing nicotine concentrations; Schild regression slope was different from unity, consistent with allosteric inhibition. Thus, bPiDDB was 200-fold more potent inhibiting nAChRs mediating nicotine-evoked [(3)H]DA release from striatum than those mediating nicotine-evoked [(3)H]NE release from hippocampus.

Fig. 1. The chemical structure of N,N′-dodecane-1,12-diyl-bis-3-picolinium dibromide (bPiDDB)

Fig. 2. Time course and concentration dependence of nicotine-evoked fractional [³H]NE release (top) and nicotine-evoked [³H]NE overflow (bottom) from superfused rat hippocampal slices

Hippocampal slices were superfused in the absence or presence of a single concentration (1-300 μM) of nicotine for 30 min. Arrow indicates the time point at which nicotine was added to the superfusion buffer. Each experiment included a buffer control condition in which one slice was superfused with buffer only and fractional [³H]NE release (top) and [³H]NE overflow (bottom) determined. Fractional release data are expressed as a percentage of basal (mean ± SEM), n = 19 rats. Basal fractional release was 0.44 ± 0.004 as percentage of tissue-[³H] content. Fractional release data were used to calculate [³H]NE overflow data, which are expressed as mean ± S.E.M. total [³H]NE overflow as a percentage of tissue-[³H] content. n = 19. The concentration-response curve for nicotine was generated using nonlinear regression. * indicates difference from buffer control, p < 0.05.

Fig. 3. Time course and concentration dependence of mecamylamine inhibition of nicotine-evoked fractional [³H]NE release (top) and nicotine-evoked [³H]NE overflow (bottom) from superfused rat hippocampal slices

Hippocampal slices were superfused in the absence or presence of a single concentration of mecamylamine (MEC; 1 nM – 1 μM) for 30 min, and then superfused for an additional 30 min with nicotine (30 μM) added to the buffer. Arrow indicates the time point at which nicotine was added to the superfusion buffer. Fractional release data are expressed as a percentage of basal (mean ± SEM), n = 12 rats. Basal fractional release was 0.46 ± 0.012 as percentage of tissue-[³H] content. Fractional release data were used to calculate [³H]NE overflow data, which are expressed as mean ± S.E.M. total [³H]NE overflow as a percentage of tissue-[³H] content. n = 12. Control [³H]NE overflow represents response to 30 μM nicotine in the absence of mecamylamine. The mecamylamine concentration-response curve was generated using nonlinear regression. * indicates difference from control, p < 0.05.

Fig. 4. Time course and concentration dependence of MLA inhibition of nicotine-evoked fractional [³H]NE release (top) and nicotine-evoked [³H]NE overflow (bottom) from superfused rat hippocampal slices

Hippocampal slices were superfused in the absence or presence of a single concentration of MLA (0.1–10 μM) for 30 min, and then superfused for an additional 30 min with nicotine (30 μM) added to the buffer. Arrow indicates the time point at which nicotine was added to the superfusion buffer. Fractional release data are expressed as a percentage of basal (mean ± SEM), n = 6 rats. Basal fractional release was 0.40 ± 0.006 as a percentage of tissue-[³H] content. Time course data for MLA-induced inhibition of nicotine-evoked fractional [³H]NE release were used to generate the [³H]NE overflow data, expressed as mean ± S.E.M. total [³H]NE overflow as a percentage of tissue-[³H] content. n = 6. Control [³H]NE overflow represents response to 30 μM nicotine in the absence of MLA. The MLA concentration-response curve was generated using nonlinear regression. * indicates difference from control, p < 0.05.

Fig. 5. Time course and concentration dependence of bPiDDB inhibition of nicotine-evoked fractional [³H]NE release (top) and nicotine-evoked [³H]NE overflow (bottom) from superfused rat hippocampal slices

Hippocampal slices were superfused in the absence or presence of a single concentration of bPiDDB (1 nM – 10 μM) for 30 min, and then superfused for an additional 30 min with nicotine (30 μM) added to the buffer. Time course data for bPiDDB-induced inhibition of nicotine-evoked fractional [³H]NE release were used to generate [³H]NE overflow data. Arrow indicates the time point at which nicotine was added to the superfusion buffer. Fractional release data are expressed as a percentage of basal (mean ± SEM), n = 6 rats. Basal fractional release was 0.33 ± 0.006 as a percentage of tissue-[³H] content. [³H]NE overflow data are expressed as mean ± S.E.M. total [³H]NE overflow as a percentage of tissue-[³H] content. n = 6. Control [³H]NE overflow represents response to 30 μM nicotine in the absence of bPiDDB. The bPiDDB concentration-response curve was generated using nonlinear regression. * indicates difference from control, p < 0.05.

Fig. 6. Schild analysis for mecamylamine inhibition of nicotine-evoked [³H]NE overflow from superfused rat hippocampal slices

After collection of the third basal sample, slices were superfused with buffer in the absence and presence of mecamylamine (MEC; 1 nM-10 μM; between-groups factor) for 30 min before the addition of nicotine (1 – 300 μM; within subjects factor) to the buffer, and superfusion continued for an additional 30 min. Control is the concentration-response for nicotine in the absence of mecamylamine, and the nicotine concentration response was determined contemporaneously for each concentration of mecamylamine. Concentration-response curves were generated using nonlinear regression. Curves illustrated for the 0.1 and 1 μM mecamylamine are superimposed. Data are presented as mean ± S.E.M. total [³H]NE overflow during the 30-min exposure to nicotine in the absence or presence of mecamylamine; n = 4-5 rats/mecamylamine concentration; control, n = 16 rats.

Fig. 7. Schild analysis for bPiDDB inhibition of nicotine-evoked [³H]NE overflow from superfused rat hippocampal slices

After collection of the third basal sample, slices were superfused with buffer in the absence and presence of bPiDDB (0.01-1.0 μM; between-groups factor) for 30 min before the addition of nicotine (1 – 300 μM; within subjects factor) to the buffer, and superfusion continued for an additional 30 min. Control is the concentration-response for nicotine in the absence of bPiDDB, and the nicotine concentration response was determined contemporaneously for each concentration of bPiDDB. Concentration-response curves were generated using nonlinear regression. Data are presented as mean ± S.E.M. total [³H]NE overflow during the 30-min exposure to nicotine in the absence or presence of mecamylamine; n = 5-7 rats/bPiDDB concentration; control, n = 12 rats. Inset shows the Schild regression in which the log of dr – 1 was plotted as a function of log of bPiDDB concentration and data were fit by linear regression.