Inhibitory pathways and the inhibition of luteinizing hormone-releasing hormone release by alcohol

Abstract

In this research we examined the mechanisms by which ethanol (EtOH) inhibits luteinizing hormone-releasing hormone (LHRH) release from incubated medial basal hypothalamic explants. EtOH (100 mM) stimulated the release of two inhibitory neurotransmitters: gamma-aminobutyric acid (GABA) and beta-endorphin. EtOH also inhibited NO production, indicative of a suppression of nitric oxide synthase (NOS) activity. This inhibition was reversed by naltroxone (10(-8) M), a micro-opioid receptor blocker, indicating that the inhibition of NOS by EtOH is mediated by beta-endorphin. EtOH also blocked N-methyl-d-aspartic acid-induced LHRH release, but the blockade could not be reversed by either the GABA receptor blocker, bicuculline (10(-5) M), naltroxone (10(-8) M), or both inhibitors added together. However, increasing the concentration of naltrexone (10(-6) M) but not bicuculline (10(-4) M) reversed the inhibition. When we lowered the concentration of EtOH (50 mM), the EtOH-induced blockade of LHRH release could be reversed by either bicuculline (10(-5) M), naltroxone (10(-8) M), or the combination of the two blockers. Therefore, GABA is partially responsible for the blockade of N-methyl-d-aspartic acid-induced LHRH release. The block by GABA was exerted by inhibiting the activation of cyclooxygenase by NO, because it was reversed by prostaglandin E(2), the product of activation of cyclooxygenase. Because the inhibition caused by the higher concentration of EtOH could not be reduced by bicuculline (10(-4) M) but was blocked by naltroxone (10(-6) M), the action of alcohol can be accounted for by stimulation of beta-endorphin neurons that inhibit LHRH release by inhibition of activation of NOS and stimulation of GABA release.

Figure 1

The effect of EtOH (100 mM) on GABA release from MBH incubated in vitro. In this and subsequent figures, the height of the column represents the mean and the vertical line represents 1 SEM. Significantly different from control, *, P < 0.05. n = 7 from each column (Student-Neuman-Keuls test).

Figure 2

The effect of EtOH (100 mM), GABA (10 mM), and NP (600 μM) on β-endorphin release from MBH incubated in vitro. **, P < 0.01 as compared with Control column. n = 7 for each column (Dunnet test). EtOH stimulated, GABA did not modify, and NP inhibited β-endorphin release.

Figure 3

The effect of NMDA (20 mM), EtOH (100 mM), bicuculline (Bic) (10 μM), naltrexone (Nalt) (10nM), and their combinations on NOS activity measured by the ¹⁴C-citrulline method. Because activation of the enzyme yields equimolar release of NO and citrulline, the results are plotted as NO release. **, P < 0.01, as compared versus control column; +, P < 0.05 as compared versus NMDA column and NMDA+EtOH+Nalt+Bic column. n = 9 for each column (Student-Neuman-Keul test). NMDA increased NOS activity, EtOH blocked it, and Nalt+Bic reverted this inhibition.

Figure 4

The effect of NMDA (20 mM), EtOH (100 mM), bicuculline (Bic) (10 μM), naltrexone (Nalt) (10 nM), and their combinations on LHRH release from MBH incubated in vitro. ***, P < 0.001 versus all columns. n = 9 for each column (Student-Neuman-Keuls test). NMDA stimulated highly significant LHRH release. EtOH significantly blocked this stimulation, and neither Bic, Nalt, nor a combination of both would revert this inhibition.

Figure 5

Effect of EtOH (50 mM) on NMDA (20 mM)-induced LHRH. The same combination of drugs were used as in Fig. 4. In contrast to the results shown in Fig. 4, bicuculline (Bic) alone, naltrexone (Nalt) alone, or their combination all not only reversed the EtOH block of LHRH, but actually significantly increased the values of above those in the NMDA-stimulated controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6

Effect of EtOH on NMDA-stimulated LHRH release of EtOH (100 mM) on NMDA-induced stimulation of LHRH release. The increased concentration of bicuculline (Bic) (10⁻⁴ M) did not reverse the EtOH-induced inhibition; however, naltrexone (Nalt) (10⁻⁶ M) completely reversed it. *, P < 0.05; **, P < 0.01; a, P < 0.05 vs control.

Figure 7

Effect of NMDA (20 mM), EtOH (100 mM), AA (50 μM), 8-bromo-cGMP (nonhydrolizable analogue of cGMP) (1 mM), and their combinations on LHRH release from MBH in vitro. ***, P < 0.001 versus all columns. n = 5 per column (Student-Newman-Keuls test). NMDA highly significantly stimulated LHRH release that was blocked by EtOH, and AA or 8-bromo-cGMP failed to reverse this inhibition.

Figure 8

Effect of NMDA (20 mM) EtOH (100 mM), PGE₂ (100 nM), and their combinations on LHRH release from MBH incubated in vitro. ***, P < 0.001 versus control and NMDA+EtOH columns. ++, P < 0.01 versus NMDA column. n = 6 for each column (Student-Neuman-Keuls test). NMDA highly significantly stimulated LHRH release that was blocked by EtOH, and the addition of PGE₂ reversed this inhibition and further significantly increased LHRH release as compared with NMDA alone.

Figure 9

Diagrammatic representation of the postulated mechanism of action of EtOH to suppress NMDA-stimulated LHRH release. For explanation, see Discussion. β-end, β-endorphin; μR, μ-opioid receptor; GABA-n, GABA neuron; NO-n, NO-ergic neuron; NE-n, noradrenergic neuron; α_1r, α₁ adrenergic receptor; NMDA-r, NMDA receptor; Glut-n, glutamergic neuron; LHRH-n, LHRH neuronal terminal; lipids, membrane phospholipids; PLA₂, phospholipase A₂; GC, guanylate cyclase; AC, adenylate cyclase. Solid arrow indicates stimulation. Dashed arrow indicates inhibition.