Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) - PNAS

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 3416-3420, April 1995

Physiology

Ethanol inhibits luteinizing hormone-releasing hormone (LHRH) secretion by blocking the response of LHRH neuronal terminals to nitric oxide (medial basal hypothalamic explants/sodium

nitroprusside/[14C]citrulline/prazosine/cGMP)

GRISELDA CANTEROS*, VALERIA RETrORI*, ANA FRANCHI*, ANA GENARO*, ELISA CEBRAL*, ALICIA FALETTI*, MARTHA GIMENO*, AND SAMUEL M. MCCANNtt *Centro de Estudios Farmacologicos y Botanicos, Consejo Nacional de Investigaciones Cientificas y Tecnicas (CEFYBO-CONICET), Serrano 665, 1414 Buenos Aires, Argentina; and tNeuropeptide Division, Department of Physiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235

Contributed by Samuel M. McCann, December 29, 1994

ABSTRACT It has previously been shown that alcohol can suppress reproduction in humans, monkeys, and small rodents by inhibiting release of luteinizing hormone (LH). The principal action is via suppression of the release of LH-releasing hormone (LHRH) both in vivo and in vitro. The present experiments were designed to determine the mechanism by which alcohol inhibits LHRH release. Previous research has indicated that the release of LHRH is controlled by nitric oxide (NO). The proposed pathway is via norepinephrine-induced release of NO from NOergic neurons, which then activates LHRH release. In the present experiments, we further evaluated the details of this mechanism in male rats by incubating medial basal hypothalamic (MBH) explants in vitro and examining the release of NO, prostaglandin E2 (PGE2), conversion of arachidonic acid to prostanoids, and production of cGMP. The results have provided further support for our theory of LHRH control. Norepinephrine increased the release of NO as measured by conversion of [14C]arginine to [14C]citrulline, and this increase was blocked by the a1 receptor blocker prazosin. Furthermore, the release of LEIRH induced by nitroprusside (NP), a donor of NO, is related to the activation of soluble guanylate cyclase by NO since NP increased cGMP release from MBHs and cGMP also released LHRH. Ethanol had no effect on the production of NO by MBH explants or the increased release of NO induced by norepinephrine. Therefore, it does not act at that step in the pathway. Ethanol also failed to affect the increase in cGMP induced by NP. On the other hand, as might be expected from previous experiments indicating that LHRH release was brought about by PGE2, NP increased the conversion of [14C]arachidonic acid to its metabolites, particularly PGE2. Ethanol completely blocked the release of LHRHI induced by NP and the increase in PGE2 induced by NP. Therefore, the results support the theory that norepinephrine acts to stimulate NO release from NOergic neurons. This NO diffuses to the LHRH terminals where it activates guanylate cyclase, leading to an increase in cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP increases intracellular free calcium, activating phospholipase A2 to provide arachidonic acid, the substrate for conversion by the activated cyclooxygenase to PGE2, which then activates the release ofLHRH. Since alcohol inhibits the conversion of labeled arachidonic acid to PGE2, it must act either directly to inhibit cyclooxygenase or perhaps it may act by blocking the increase in intracellular free calcium induced by cGMP, which is crucial for activation of both phospholipase A2 and cyclooxygenase.

chronic administration of alcohol not only inhibits the estrous cycle but it can also delay onset of puberty (4, 5). In conscious animals, administration of alcohol via an indwelling gastric cannula in doses that produce mild intoxication inhibits pulsatile release of luteinizing hormone (LH), but not folliclestimulating hormone, within a few minutes (4). In this situation, the responsiveness of the pituitary gland to acute injection of LH-releasing hormone (LHRH) is unaffected, which shows that the mechanism of this effect is via suppression of the pulsatile LHRH release into the hypophyseal portal vessels that triggers release of LH from the gonadotropes of the adenohypophysis. The interference with estrous cycles and the delayed onset of puberty in the female rat is likely brought about by this suppression by ethanol of LHRH release (5). Secretion of LH is required to stimulate the ovary to produce ovarian steroids responsible for the estrous cycle, and the onset of puberty is consequently delayed. We have recently shown that nitric oxide (NO) controls the release of LHRH both in vivo and in vitro (6). On the basis of in vitro experiments employing incubation of medial basal hypothalamic (MBH) explants in a static incubation system, it has been determined that norepinephrine activates constitutive NO synthase (NOS) in neurons in this region (NOergic neurons) (6, 7). The NO released from these neurons diffuses to LHRH terminals, where it induces the release of LHRH, probably by activating cyclooxygenase 1. The activated cyclooxygenase then converts arachidonate into prostaglandin E2 (PGE2) (8). PGE2 activates adenylate cyclase, causing generation of cAMP, which acts via protein kinase A to evoke exocytosis of LHRH granules into the hypophyseal portal vessels for its delivery to the anterior pituitary gland (9, 10). The LHRH acts on the gonadotropes and causes a pulse of LH release. Support for this theoretical pathway stems from the ability of inhibitors of NOS, such as NG-monomethyl-Larginine, to inhibit LHRH release, whereas releasers of NO, such as sodium nitroprusside (NP), induce LHRH release both in vitro and in vivo (6-9). We have identified the site of inhibitory action of interleukin 1 (IL-1) on LHRH release in in vitro studies with MBH explants. This was made possible by the fact that NP, the releaser of NO, can directly cause the LHRH terminals to release LHRH. IL-la blocked the response of the LHRH terminals to NO (11). Because of the similarity in action of IL-1 and ethanol on LHRH, we wondered whether it also inhibits the response to NO of the LHRH terminals in the postulated pathway. The results indicate that the principal site

Alcohol suppresses reproductive function in humans, monkeys, and small rodents, such as the rat (1-4). In the rat,

Abbreviations: LHRH, luteinizing hormone-releasing hormone; MBH, medial basal hypothalamus; NOS, nitric oxide synthase; PG, prostaglandin; NP, sodium nitroprusside; PLA2, phospholipase A2. TTo whom reprint requests should be addressed.

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Physiology: Canteros et at of action of ethanol to suppress LHRH release is via a blockade of the response of the LHRH terminals to NO.

MATERIALS AND METHODS Male rats of the Wistar strain (200-250 g) from our colony were used. All rats were kept in group cages in a light (0500-1900 hr) and temperature (23-25°C)-controlled room with free access to laboratory chow and water. In Vitro Studies. After decapitation and removal of the brain, the MBH was dissected by making frontal cuts just behind the optic chiasm, extending dorsally 1.0 mm. A horizontal cut extended from this point caudally to just behind the pituitary stalk, where another frontal cut was made. Longitudinal cuts were made 1 mm lateral to the midline bilaterally. The hypothalami were preincubated in Krebs-Ringer bicarbonate-buffered medium (pH 7.4) containing 0.1% glucose (KRB) for 30 min prior to replacement with fresh medium or medium containing the substances to be tested. The incubation was continued for 30 min followed by removal of the medium and storage of samples at -20°C prior to assay for LHRH. Incubations were carried out in a Dubnoff metabolic shaker (50 cycles per min; 95% 02/5% CO2) (6). LHRH was measured by RIA using highly specific LHRH antiserum kindly provided by A. Barnea (The University of Texas Southwestern Medical Center, Dallas). The sensitivity of the assay was 0.2 pg per tube and the curve was linear up to 100 pg of LHRH. cGMP and PGE2 were also measured in media and tissue in certain experiments by RIA. For determination of NO release from the incubated MBH explants, we used a modification of the method of Bredt and Snyder (12), which measures the conversion of [14C]arginine into ['4C]citrulline since citrulline remains in the sample, whereas the equimolar amounts of NO produced are rapidly

destroyed. Metabolism of [14C]Arachidonic Acid by MBH. After preincubation of MBHs for 30 min in KRB, they were incubated for 1 hr in 2 ml of medium containing 0.25 ,tCi of [14C]arachidonic acid (New England Nuclear; 52.9 Ci/mol; 1 Ci = 37 GBq). At the end of the incubation period, tissues were removed and the remaining incubation medium was acidified to pH 3.0 with 1.0 M HCl. The arachidonic acid metabolites were extracted two times with 2 ml of ethyl acetate. Pooled ethyl acetate extracts were dried under nitrogen. The residue was suspended in chloroform/methanol (2:1, vol/vol) and applied to silica gel TLC plates. Before application of extracts, reference compounds, 6-keto-prostaglandin F (PGFia), PGF2a, thromboxane B2, and PGE2 were placed on the plates. The plates were developed in a solvent system of benzene/dioxane/ glacial acetic acid (60:30:3.0, vol/vol/vol). The position of authentic PGs was visualized by spraying the dried plates with 10% phosphomolybdic acid in ethanol, followed by heating at 110°C for 10 min; the plates were scraped off and 14C radioactivity was measured by liquid scintillation counting. Average Rf values were 0.30 for 6-keto-PGFia, 0.35 for PGF2,a, 0.47 for PGE2, 0.57 for thromboxane B2, and 0.80 for arachidonic acid. Results were expressed as percentage conversion of [14C]arachidonic acid per mg of MBH over 60 min. This method has already been used effectively with MBHs (13). The following compounds were purchased from Sigma: norepinephrine HCl, sodium NP, NG-monomethyl-L-arginine, nitroarginine methyl ester, and 8-bromo-cGMP (Na+ salt). Statistics. Data were analyzed by one-way analysis of variance and the Student-Newman-Keuls multiple comparison test for unequal replicates. Differences with P values < 0.05 were considered significant.

Proc. Natl. Acad. Sci. USA 92 (1995)

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RESULTS Indirect Measurement of NO Released by the ['4C] Citrulline Method. The initial experiments were designed to test the validity of our model of LHRH control via norepinephrine and NO (6). NOS converts arginine in the presence of oxygen to NO plus citrulline in stoichiometric amounts. To determine whether NO formed in our MBH explants was sufficient to be detected by the [14C]citrulline method, two MBHs were incubated with [14C]arachidonate and the [14C]citrulline produced was measured. Substantial NO was produced under these "basal" conditions (Fig. 1). When the enzyme was inhibited by coincubation with nitroarginine methyl ester (300 ,uM), a specific inhibitor of NOS (14), citrulline production was markedly reduced, indicating that the enzyme was active and producing significant quantities of NO in vitro (data not shown). Effect of Norepinephrine on NO Production by MBHs. According to our theory, norepinephrine should increase NO release from MBH explants (6). Indeed, norepinephrine (10-5 M) caused a highly significant increase in labeled citrulline production (Fig. 1). We had postulated that this was via activation of the NOergic neuron by a receptors. Indeed, the a, receptor blocker prazosin (10-5 M) completely blocked the response to norepinephrine, although it had no effect on basal release of NO (Fig. 1). Role of cGMP in NO-Induced LHRH Release. In our original theory, we postulated that an a receptor also exists on the LHRH terminals, which would cause an increase in intracellular free calcium required for activation of phospholipase A2 (PLA2) that would then convert membrane phospholipids into arachidonate for conversion to PGE2 via the NO-activated cyclooxygenase. Subsequently, we showed that an a receptor on the LHRH terminal was not necessary since the a receptor blocker phentolamine did not alter the response of the LHRH terminals to NO released by NP (15). Therefore, it appeared that intracellular calcium was elevated by another mechanism. We hypothesized that activation of soluble guanylate cyclase by the NO released from the NOergic neurons with consequent formation of cGMP (15) might elevate intracellular calcium, thereby activating PLA2. Therefore, we evaluated the effect of cGMP on LHRH release. Although cGMP (10-3 M) did not affect LHRH release by MBH explants, a higher concentration (10-2 M) produced a highly significant increase in release (Fig. 2). This would be consistent with the hypothesis that the release of NO activates the release of LHRH from LHRH terminals by activating guanylate cyclase to increase cGMP. Lack of Effect of Ethanol on the Production of NO by MBH Explants. Having further validated the pathway by which 80,000 m 2 60,000-

E0. 0

1640,000 0

Control

NE

NE + Praz

Praz

FIG. 1. Effect of norepinephrine (NE) (10-5 M) and prazosin (Praz) (10-4 M) on release of NO from MBH explants measured by the [14C]citrulline method. In this and subsequent figures, the height of the column represents the mean, and the vertical line represents SEM. ***, P < 0.001 vs. the other groups.

Proc. Natl. Acad Sci USA 92 (1995)

Physiology: Canteros et at

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60,000 E~~~~~~~~~~

40,0007i m1

lS1

920,000 0

Control CControl

FIG. 2.

cGMP (10-2M) cGMP (10-3M)

Effect of cGMP on LHRH release. ***, P < 0.001 vs. the

NE

NE + EtOH

FIG. 4. Increase in NO release by the citrulline method induced by norepinephrine (NE) is not altered by ethanol (EtOH). *, P < 0.05 vs. control.

other groups.

norepinephrine and NO activate LHRH release, we examined the effect of ethanol on several steps in the pathway.

To

determine whether ethanol might suppress LHRH release by

inhibiting the production of NO by the NOergic neurons, we incubated MBHs in the presence of various concentrations of

ethanol and evaluated the production of NO by the [14C]citrulline method. Several concentrations of ethanol that cause mild to severe intoxication in the rat (4) had no effect on the production

of NO by the

explants

(Fig.

3).

Furthermore,

ethanol (100 mM) had no effect on the increased NO release induced by norepinephrine

(10-5

M) (Fig. 4).

Effect of NP and Ethanol on cGMP Release from MBH. As indicatediabove, cGMP increased LHRH release from MBH explants. Therefore, NP, the releaser of NO, should have a

52

similar effect. Indeed, NP (300

significantly ,uM) ~II

increased

cGMP release and this increase was not significantly affected by ethanol at a relatively high concentration (100

,uM).

Eth-

anol by itself had no effect on levels of cGMP in the medium (Fig. 5). cGMP in the tissue at the end of the incubation was not significantly altered (data not shown).

Effect of NP and Ethanol on Conversion of ['4CJArachidonic Acid to Its Metabolites. Since norepinephrine releases

NP also produced a highly significant increase in production of PGE2. It also increased formation of 6-keto-PGFia, the metabolite of prostacyclin, and produced a borderline significant increase in the production of PGF2a, but it had no effect on the synthesis of thromboxane B2 (Fig. 6). Ethanol at the relatively high concentration of 100 mM had no effect on thromboxane B2 formation in the presence or absence of NP; however, it tended to lower basal production of PGE2, PGF2a, and 6-ketoPGFia. The former two changes were significant. Ethanol highly significantly suppressed the synthesis of PGE2, PGF2a, and 6-keto-PGFia induced by NP (Fig. 6). Effect of Ethanol on the Release of LHRH Induced by NP. As shown earlier (6, 11), NP (300 ,uM) produced a marked stimulation of LHRH release from MBH fragments. Although ethanol (100 mM) failed to suppress basal release of LHRH, it completely blocked the LHRH release induced by NP (Fig. 7). In an additional experiment, we tested the response to a lower concentration of ethanol (50 mM). It also completely blocked the LHRH release caused by NP (300 ,uM) and had no effect on basal release (Fig. 8). However, when the concentration of NP was increased to 600 ,uM, this concentration of ethanol did not suppress the already elevated release of LHRH induced by NP.

LHRH by increasing PGE2 production, we postulated that NP, the releaser of NO, would stimulate PGE2 release. Indeed, NP

DISCUSSION

highly significantly increased release of PGE2 into the medium (data not shown). Conversely, acetylsalicylic acid, an inhibitor of cyclooxygenase, blocked the release of PGE2 induced by NP (data not shown). In the uterus, we have found effects of NO on conversion of arachidonate (16) into its metabolites. Indeed, as in the uterus,

On the basis of the present experiments, we have modified our model of control of LHRH release in the MBH (Fig. 9). Norepinephrinergic neurons with cell bodies in the brainstem-e.g., locus ceruleus-project to the MBH region. There, they release norepinephrine which acts on a, receptors on NOergic neurons, thereby increasing intracellular free cal40

.105000

I 30 CD

E~

20-

0 IL.

Ethanol,

mM-I

FIG. 3. Lack of effect of ethanol at various concentrations release of NO measured by the [14C]citrulline method.

on

Control

NP

NP + EtOH EtOH

FIG. 5. Lack of effect of ethanol (EtOH) (100 mM) on cGMP release from MBH explants and its inability to alter the increased cGMP release induced by NP (300 ,uM). NP vs. NP + EtOH, P < 0.05; EtOH vs. NP or NP + EtOH, P < 0.001.

Physiology: Canteros et at

Proc. Natl. Acad. ScL USA 92 (1995)

3419

12

10 U Control

10

&NP