Role of nitric oxide in control of prolactin release by

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1 Proc. Natl. Acad. Sci. USA Vol. 9, pp , January 1995 Physiology Role of nitric oxide in control of prolactin release by the adenohypophysis (sodium nitroprusside/hemoglobin/ng-monomethyl-l-arginine/nitroarginine methyl ester/nitric oxide synthase) B. H. DUVILANSKI*, C. ZAMBRUNO*, A. SEILICOVICH*, D. PISERA*, M. LASAGA*, M. DEL C. DIAZ*, N. BELOVAt, V. RETTORIt, AND S. M. MCCANNO *Centro de Investigaciones en Reproducci6n, Facultad de Medicina, Piso 1, Universidad de Buenos Aires, 111 Buenos Aires, Argentina; tdepartment of Physiology, Medical Academy-Sofia, 1431 Sofia, Bulgaria; *Neuropeptide Division, Department of Physiology, The University of Texas Southwestern Medical Center, 533 Harry Hines Boulevard, Dallas, TX Contributed by S. M. McCann, September, 1994 ABSTRACT Nitric oxide synthase-containing cells were visualized in the anterior pituitary gland by immunocytochemistry. Consequently, we began an evaluation of the possible role of NO in the control of anterior pituitary function. Prolactin is normally under inhibitory hypothalamic control, and in vitro the gland secretes large quantities of the hormone. When hemipituitaries were incubated for 3 min in the presence of sodium nitroprusside, a releaser of NO, prolactin release was inhibited. This suppression was completely blocked by the scavenger of NO, hemoglobin. Analogs of arginine, such as NG-monomethyl-L-arginine (NMMA, where NG is the terminal guanidino nitrogen) and nitroarginine methyl ester, inhibit NO synthase. Incubation of hemipituitaries with either of these compounds significantly increased prolactin release. Since in other tissues most of the actions of NO are mediated by activation of soluble guanylate cyclase with the formation of cyclic GMP, we evaluated the effects of cyclic GMP on prolactin release. Cyclic GMP (1 mm) produced an "4%o reduction in prolactin release. Prolactin release in vivo and in vitro can be stimulated by several peptides, which include vasoactive intestinal polypeptide and substance P. Consequently, we evaluated the possible role of NO in these stimulations by incubating the glands in the presence of either of these peptides alone or in combination with NMMA. In the case of vasoactive intestinal polypeptide, the significant stimulation of prolactin release was augmented by NMMA to give an additive effect. In the case of substance P, there was a smaller but significant release of prolactin that was not significantly augmented by NMMA. We conclude that NO has little effect on the stimulatory action of these two peptides on prolactin release. Dopamine (.1,uM), an inhibitor of prolactin release, reduced prolactin release, and this inhibitory action was significantly blocked by either hemoglobin ( jig/ml) or NMMA and was completely blocked by 1 mm nitroarginine methyl ester. Atrial natriuretic factor at 1,uM also reduced prolactin release, and its action was completely blocked by NMMA. In contrast to these results with prolactin, luteinizing hormone (LH) was measured in the same medium in which the effect of nitroprusside was tested on prolactin release, there was no effect of nitroprusside, hemoglobin, or the combination of nitroprusside and hemoglobin on luteinizing hormone release. Therefore, in contrast to its inhibitory action on prolactin release NO had no effect on luteinizing hormone release. Immunocytochemical studies by others have shown that NO synthase is present in the folliculostellate cells and also the gonadotrophs of the pituitary gland. We conclude that NO produced by either of these cell types may diffuse to the lactotropes, where it can inhibit prolactin release. NO appears to play little role in the The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. 17 prolactin-releasing action of vasoactive intestinal polypeptide and substance P, but mediates the prolactin-inhibiting activity of dopamine and atrial natriuretic factor. Nitric oxide (NO) is recognized as a neurotransmitter in the central and peripheral nervous system (1-3). Immunocytochemical studies have demonstrated a wide distribution of NO synthase (NOS) in neurons (4) of the central nervous system. NO plays an important role in controlling release of hypothalamic peptides via neurons that contain constitutive NOS. Glutamic acid and norepinephrine (5, 6) activate NOergic neurons, which stimulate the release of luteinizing hormone (LH)-releasing hormone (LH-RH). NOergic neurons also control the release of corticotropin-releasing hormone (7), vasopressin (8), prolactin-releasing factors (9), growth hormone-releasing hormone (1), and somatostatin (11). A number of cells in the anterior pituitary gland also contain NOS as determined by immunocytochemical studies (4, 1). Therefore, it was of interest to determine the role of NO in the control of release of pituitary hormones. In these experiments we have investigated the role of NO in control of the release of prolactin and its possible role in mediating the action of two prolactin-releasing peptides: vasoactive intestinal polypeptide (VIP) and substance P (SP). We also determined the role of NO in dopaminergic and atrial natriuretic factor (ANF)- induced inhibition of prolactin release. The results indicate that NO exercises an inhibitory control on prolactin release, that it has little effect on the stimulatory effects of VIP and SP, but that it mediates the inhibitory actions of dopamine and ANF on prolactin release. MATERIALS AND METHODS Animals and Drugs. Male Wistar rats (-5 g) were used. The animals were maintained under controlled conditions of light (1-hr light/dark cycles) and temperature (- 5 C). Food and water are available ad libitum. The animals were killed by decapitation. The anterior lobes were dissected free of the posterior lobe and were cut longitudinally into halves. One hemipituitary per tube was preincubated in 1 ml of Krebs-Ringer bicarbonate buffer (ph 7.4) containing 1 mm glucose, 1 AM bacitracin,.1% ascorbic acid, and.1% bovine serum albumin (KRB buffer) in an atmosphere of 95% /5% CO with constant shaking at 6 cycles per min at 37 C. After preincubation for 6 min, the tissue was incubated for 3, 6, or 1 min with 1 ml of fresh Abbreviations: NMMA, NG-monomethyl-L-arginine, where NG is the terminal guanidino N; NAME, nitroarginine methyl ester; NOS, nitric oxide synthase; VIP, vasoactive intestinal polypeptide; SP, substance P; NP, nitroprusside; ANF, atrial natriuretic factor; LH, luteinizing hormone; LH-RH, LH-releasing hormone. To whom reprint requests should be addressed.

2 Physiology: Duvilanksi et al KRB buffer containing sodium nitroprusside (NP), hemoglobin (Hb) or NG-monomethyl-L-arginine (NMMA, where NG is the terminal guanidino nitrogen) (Sigma). Other hemipituitaries were incubated similarly with various concentrations of VIP, SP, ANF (Peninsula Laboratories), dopamine hydrochloride, or 8-monobutyryl cyclic guanosine monophosphate (cgmp) (Sigma). The reagents were added to the incubation medium alone or in combination as described in Results. Controls were incubated in medium alone. At the end of the incubation, the media were aspirated and frozen. Prolactin and LH were measured by double-antibody radioimmunoassay, with kits supplied by the National Institutes of Diabetes and Digestive and Kidney Diseases (Bethesda, MD). The results were expressed in terms of rat prolactin (RP-3) standard and rat LH (RP-3) standard. The tissue was homogenized, and proteins were determined by the method of Lowry et al. (13). Data were expressed as means ± SE and were evaluated by analysis of variance followed by Tukey or Dunnett's test. Differences between means were considered to be significant when P <.5. The experiments were performed at least twice. Results of individual experiments were presented. RESULTS Effect of NP on Prolactin Release. In the first experiment the hemipituitaries were incubated for 6 min with 3 1.M NP, a compound that spontaneously liberates NO. NP significantly lowered prolactin release (Fig. 1). When the glands were incubated with Hb (,Lg/ml), which scavenges NO, "basal" release was slightly but significantly increased (P <.5). Hb completely reversed the inhibitory action of NP on prolactin release. Next the glands were incubated for 3 min with various concentrations of NP (5-5,uM). A similar reduction in prolactin release was induced by each of the three concentrations (5, 1, and 5,uM) after 3 min of incubation (Fig. ). When the incubation was continued for an additional 3 min, the effect of 5,uM NP had dissipated, but NP at the two higher concentrations had a similar highly significant inhibitory effect on prolactin release (Fig. ). Effect of NMMA and Nitroarginine Methyl Ester (NAME) on Prolactin Release. The arginine analog NMMA is a competitive inhibitor of NOS. Therefore, its effect on prolactin release was evaluated at a concentration previously found to inhibit NOS effectively in the hypothalamus (5-7). Incubation of the tissue for 1 min with 3,uM NMMA induced a significant elevation of prolactin release (Fig. 3). Another 3. v. '4-4 *= 1. cu '. Proc Nati Acad Sci USA 9 (1995) 171 Control NP, FlM FIG.. Effect of NP on prolactin release from hemipituitaries when the glands were incubated for 3 (v) or 6 (o) min. *, P <.5; **, P <.1 versus control. inhibitor of NOS, NAME (1-3), was also evaluated. It also caused a highly significant increase in prolactin release (Fig. 3). Effect of cgmp on Prolactin Release. Since cgmp mediates many of the actions of NO (1-3), it was of interest to evaluate its effect on prolactin release. Incubation of the pituitaries for 3 or 6 min with cgmp (1 mm) had no effect on prolactin release (results not shown). At a higher concentration (1 mm) previously found to be effective on hypothalamic explants (V.R., unpublished data), cgmp significantly inhibited prolactin release (Fig. 4). Effect of NMMA on Prolactin Release Induced by VIP and SP. To explore a possible role of NO in the prolactin-releasing action of VIP and SP, pituitaries were incubated for 6 min in the presence or absence of the peptides with or without NMMA. Although NMMA increased prolactin release slightly, in this case the increase was not significant (P <.1) (Fig. 5). Each peptide alone was also effective (Fig. 5). When.1,uM VIP was incubated together with 3,uM NMMA, NMMA significantly augmented the effect of VIP. In the case of 1 1.M SP, its stimulatory effect was not modified by coincubation of the peptide with NMMA (Fig. 5). Addition of NMMA significantly augmented the effect of VIP. Role of NO on Dopamine and ANF-Induced Inhibition of Prolactin Release. One micromolar dopamine had the expected effect to suppress prolactin release, and this inhibition of prolactin release was markedly but not completely reversed by Hb. In each of two experiments of which only one result is c 1. '4-i. 3. i 1. C Control NP NP + Hb Hb FIG. 1. Effect of 3,uM NP on prolactin release from hemipituitaries incubated in vitro. Hb was at jig/ml. In this and subsequent figures, height of the column equals the mean and vertical lines represent one standard error of the mean. There were 8-1 glands per group. *, P <.1 versus the other columns.. Control NMMA NAME FIG. 3. Effect of the NOS inhibitors NMMA and NAME, both at 3 gm, on prolactin release. Each inhibitor produced a highly significant increase in prolactin release. ***, P <.1 versus control.

3 17 Physiology: Duvilanksi et al. Pror- Natt Acad Sci USA 9 (1995) r T-- ** _F_ 3. t5. o t 'I 1. U Control cgmp FIG. 4. Suppression of prolactin release by 1 mm monobutyryl cgmp. **, P <.1 versus control. illustrated (Fig. 6), 3,uM NMMA, the inhibitor of NOS, also had a highly significant although partial effect in reversing the inhibition of prolactin release induced by.1,um dopamine (Fig. 7A). The effect was not significant with the higher concentration of dopamine (results not shown). In contrast to the partial effect of 3,uM NMMA, 1 mm NAME, another inhibitor of NOS, completely blocked the action of.1,.m dopamine without significantly altering "basal" prolactin release (Fig. 7B). ANF also inhibited prolactin release at a concentration of 1,uM but not.1,um (data not shown). This inhibition was reversed completely by 3,.M NMMA (Fig. 8). Lack of Effect of NO on LH Release. To compare the action of NO on LH release with that of prolactin, the effect of Hb at,ug/ml, 3,uM NP, and 3,uM NMMA on LH release from the same hemipituitaries as those incubated in Fig. 1 was determined (Fig. 9). In contrast to the actions of NO on prolactin release (Fig. 1), there was no significant effect of any of the treatments on LH release. DISSSION The results of this research clearly establish an inhibitory effect of NO on prolactin release by anterior pituitaries incubated in vitro since NP, which releases NO, inhibited prolactin release in concentrations that are effective in the hypothalamus (5-7). The 5,uM concentration is near the minimal effective dose, since its action was only demonstrable upon incubation for 3 min and was dissipated by 6 mmin. The inhibitory effect of NP was abolished by Hb, a scavenger of NO. Since the inhibitor of 4 ' f>> 1x1x \\\1 Control DA DA+Hb Hb FIG. 6. Effect of 1,uM dopamine (DA) and Hb at,ug/ml on prolactin release. ***, P <.1 versus control. Hb significantly (P <.1) blocked the inhibition of prolactin release induced by DA. NOS, NMMA, caused an increase in prolactin release only when the incubation was continued for 1 min, it appears that NO suppresses the release of prolactin from the incubated pituitaries. Similarly, Hb slightly increased "basal" release of prolactin. Since Hb would remove NO formed during the incubation, this result also suggests that NO suppressed prolactin release from the incubated glands. The results indicate that the stimulation of prolactin release by SP is not mediated by NO because it was not altered by an effective concentration of the inhibitor of NOS, NMMA. The 3. v. *> 1. ) Eb U *S. 1. Control DA NAME DA + NAME. Contl VIP VIP+NMMA SP SP+NMMA NMMA FIG. 5. Effect of 3,LM NMMA on the prolactin release induced by.1,um VIP or 1,uM SP. *, P <.1 versus control; ***, P <.5 versus VIP and P <.1 versus control. FIG. 7. (A) Effect of DA and NMMA on prolactin release. ***, P <.1 versus control. The inhibition of prolactin release induced by.1 t,m DA was highly significantly reduced (P <.1) by 3 t,m NMMA. (B) Effect of.1,um DA and 1 mm NAME on prolactin release. **, P <.1 versus other groups.

4 ~ ~1 Control ANF ANF + NMMA NMMA FIG. 8. Effect of 1 ILM ANF and 3 I.M NMMA on prolactin release. **, P <.1 versus the other columns. augmentation of the stimulation of prolactin release by VIP in the presence of NMMA is consistent with the possibility that removal of NO release by NMMA augments its effect. Therefore, its effect could be related at least in part to decreased NO release. VIP releases prolactin by activating adenylate cyclase, which increases camp production (14), whereas SP acts by the phosphatidylinositol cycle (14). The difference in their mechanism of action may account for the different effect of NMMA on the prolactin-releasing action of the two peptides. In contrast to the prolactin-releasing action of these peptides, ANF inhibited the release of prolactin from pituitaries incubated in vitro with a minimal effective dose of 1 ALM. This effect was reversed by NMMA, suggesting that NO plays a role in this inhibition. The inhibitory action of dopamine (14) was partially reversed not only by Hb, which would scavenge the NO released, but also by 3 nm NMMA, which would block NOS and diminish NO formation; however, NMMA blocked the inhibitory action of the lower (.1,uM) but not the higher (1 ALM) concentration of dopamine, which suggested that the concentration of NMMA was too low to block NO synthesis completely. The presumed complete blockade of NOS by the higher concentration of NAME (1 mm) completely inhibited the action of.1,lm dopamine which indicates that NO release is the major factor in the inhibitory response. Dopamine and ANF act on their receptors on NOScontaining pituitary cells to increase the intracellular concentration of Ca+, which after combination with calmodulin activates NOS to produce NO. The accepted mechanism of action of NO in the brain and vascular system is by activation of soluble guanylate cyclase with the generation of cgmp, 4-' -E Physiology: Duvilanksi et al 5: -. Control NP NP + Hb Hb FIG. 9. Effect of 3,uM NP and Hb at jlg/ml on LH release. Alone or combined, NP and Hb had no effect on LH release from hemipituitaries used in Fig Proc NatL Acad Sci USA 9 (1995) 173 which then mediates the actions (1-3). The fact that cgmp at a high concentration (1 mm) suppressed prolactin release is consistent with this concept. Although the concentration of cgmp required is much higher than that shown earlier to be effective with cyclic AMP in responsive tissues, it was the same as that required to induce LH-RH release from hypothalamic explants (V.R., unpublished data). Therefore, it is likely that the action of NO to suppress prolactin release may be mediated by activation of guanylate cyclase and generation of cgmp. Since increased intracellular Ca+ concentration is required for prolactin release (14), cgmp may decrease intracellular Ca+ concentrations in the lactotrophs (15), thereby decreasing prolactin release. Another possible mechanism may be by altering the activity of other Fe+-containing enzymes, such as cyclooxygenase (16). In the paper of Bredt et al. (4) demonstrating NOergic neurons in various hypothalamic areas, there was heavy NOS immunostaining in the neural lobe of the pituitary gland, presumably reflecting high concentrations of the enzyme in the terminals of the neurons of the supraopticohypophyseal tract. Although it was not mentioned, the anterior lobe was also present in the section and increased magnification revealed the obvious presence of cells containing NOS in the anterior lobe of the pituitary, which was the main reason that we initiated this research. Ceccatelli et al. (1) further studied the immunocytochemical distribution of NOS in the anterior pituitary and reported the presence of NOS and its mrna in folliculostellate cells and gonadotrophs of the pituitary gland. They did not mention finding NOS in lactotrophs; however, since those constitute a small proportion of cells in the gland, it may be that the enzyme is present there as well. If the enzyme is not present in the lactotrophs, then the action of NO on prolactin release must be a paracrine effect by diffusion of the dissolved gas from gonadotrophs or folliculostellate cells to the lactrophs. Previous studies have shown paracrine actions of the gonadotrophs on the lactotrophs (17). Therefore, it is conceivable that this mechanism may operate here. Ceccatelli et al. (1) reported that NO inhibited the response of the gonadotrophs to LH-RH but had no effect on basal LH release. Our results are in agreement with theirs and indicate that NO has precious little effect on basal LH release from hemipituitaries; however, the paracrine actions of gonadotropes on prolactin secretion may be brought about by NO. From these studies, it is apparent that NO has important effects on the secretion of pituitary hormones not only by altering release of releasing hormones from the hypothamus but also by actions on the gland itself. In the case of prolactin, we have reported that injection of NMMA into the third brain ventricle had no effect on basal pulsatile prolactin release, but that it completely blocked the stimulation of prolactin release induced by injection into the third ventricle of interleukin-1 a subunit (IL-la) (9). We believe that this represents an action on the hypothalamus to suppress the secretion of prolactinreleasing factors induced IL-la. Since oxytocin is an important prolactin-releasing factor (18) and the cell bodies of the oxytocinergic neurons are in juxtaposition to the NOergic neurons (4), oxytocin is a good candidate to be responsible for the prolactin-releasing activity of IL-la. Alternatively, the action of IL-1 could have been due to removal of lactotroph inhibition by prolactin-inhibiting factors, such as dopamine (14). Therefore, the actions of NO in controlling prolactin release are complex by effects mediated both by the hypothalamus and also by the pituitary gland itself. The actions of NO within the hypothalamus stimulate, whereas the action at the pituitary level inhibits prolactin secretion. This work was supported by grants from Consejo Nacional de Investigaciones Cientificas y Tecnicas and from Universidad de Buenos Aires and by National Institutes of Health Grant DK173 (to S.M.M.).

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