Influence of endogenous opiates on anterior pituitary function

In general, the endogenous opioid peptides (EOP), morphine (MOR), and related drugs exert similar effects on acute release of pituitary hormones. Thus administration of opiates produces a rapid increase in release of prolactin (PRL), growth hormone (GH), adrenocorticotropin (ACTH), and antidiuretic hormone (ADH), and a decrease in release of gonadotropins and thyrotropin (TSH). Although not yet fully established, there is growing evidence that the EOP participate in the physiological regulation of pituitary hormone secretion. Thus naloxone (NAL), a specific opiate antagonist, has been shown to reduce basal serum levels of PRL and GH, and to elevate serum levels of LH and follicle stimulating hormone in male rats. Other reports have shown that NAL can inhibit the stress-induced rise in serum PRL, raise the castration-induced increase in serum LH to greater than normal castrate values, and counteract the inhibitory effects of estrogen and testosterone on LH secretion. Opiates appear to have no direct action on the pituitary, but there is evidence that they can alter activity of hypothalamic dopamine and serotonin in modulating secretion of pituitary hormones.     

The roles of the opioid peptides in controlling thyroid stimulating hormone release

We have studied the role of the opioid peptides in controlling TSH secretion. Morphine sulfate significantly decreased, while naloxone had no effect on, basal plasma TSH levels of female rats. In contrast, naloxone blocked the stress-induced fall in plasma TSH. Microinjection of β-endorphin into the third ventricle resulted in a fall in TSH while such injection of naloxone into the posterior hypothalamus increased TSH. Microinjection of β-endorphin directly into the pituitary caused a rise in plasma TSH. It is concluded that opioid peptides probably play no role in basal TSH secretion, but are involved in the stress-induced fall in TSH. Furthermore, it appears that opioid peptides have a site of action in the hypothalamus to decrease TSH and a direct pituitary action to increase TSH.     

Stimulation of prolactin secretion in the rat by alpha-neo-endorphin, beta-neo-endorphin and dynorphin

Intraventricular injections of α-neo-endorphin, β-neo-endorphin and dynorphins (dynorphin[1–13], dynorphin[1–17], dynorphin[1–8]) resulted in an increase in plasma prolactin levels in urethane-anesthetized rats. Dynorphin [1–13] was the most potent to stimulate prolactin release among these opioid peptides. Plasma prolactin responses to these stimuli were blunted by naloxone, an opiate antagonist. In $\text{in}\text{vitro}$ studies, prolactin release from perfused pituitary cells was stimulated by α-neo-endorphin, and the effect was blunted by naloxone, whereas neither β-neo-endorphin nor dynorphin[1–13] affected prolactin release. These results suggest that newly identified “big” Leu-enkephalins in the brain stimulate prolactin secretion in the rat and that α-neo-endorphin has a possible direct action on the pituitary.     

Effects of photoperiod, beta-endorphin, and naloxone on in vitro secretion of testosterone in white-footed mouse (Peromyscus leucopus) testes

The effects of the pro-opiomelanocortin-derived beta-endorphin (B-EP) and the opioid antagonist naloxone on in vitro secretion (accumulation of testosterone (T) in the medium) of T by testicular cells were assessed in adult white-footed mice (Peromyscus leucopus). Animals were housed under long days (16L:8D) to maintain testicular function or under short days (8L:16D) to induce gonadal regression. In vitro treatment with B-EP or naloxone did not affect basal secretion of T in dispersed cells from active or regressed testes. However, B-EP caused a dose-dependent reduction in secretion of T from cells stimulated maximally with human chorionic gonadotropin (hCG) or dibutyryl cyclic adenosine 3', 5'-monophosphate (dbcAMP). Conversely, naloxone enhanced maximal hCG- and dbcAMP-stimulated secretion of T in testicular incubates from both long- (1.5-fold) and short-day (3.5-fold)-exposed mice. The finding that the addition of naloxone to maximally stimulated cells increased further the secretion of T is evidence that B-EP may act to inhibit gonadotropin-stimulated secretion of T. Also, the stimulatory effect of naloxone on cells from regressed testes indicates that B-EP may be involved in suppressing production of T during the gonadally regressed state. Testicular B-EP-like immunostaining is present within the cytoplasm of interstitial cells and is not apparent in the seminiferous tubules. Together, these results support the idea that in P. leucopus endogenous opioid peptides in the testes may aid in the regulation of testicular function throughout the yearly breeding cycle.     

Endocrine actions of opioids

The widespread occurrence of opioid peptides and their receptors in brain and periphery correlates with a variety of actions elicited by opioid agonists and antagonists on hormone secretion. Opioid actions on pituitary and pancreatic peptides are summarized in Table 1. In rats opioids stimulate ACTH and corticosterone secretion while an inhibition of ACTH and cortisol levels was observed in man. In both species, naloxone, an opiate antagonist, stimulates the release of ACTH suggesting a tonic suppression by endogenous opioids. In rats, a different stimulatory pathway must be assumed through which opiates can stimulate secretion of ACTH. Both types of action are probably mediated within the hypothalamus. LH is decreased by opioid agonists in many adult species while opiate antagonists elicit stimulatory effects, both apparently by modulating LHRH release. A tonic, and in females, a cyclic opioid control appears to participate in the regulation of gonadotropin secretion. Exogenous opiates potently stimulate PRL and GH secretion in many species. Opiate antagonists did not affect PRL or GH levels indicating absence of opioid control under basal conditions, while a decrease of both hormones by antagonists was seen after stimulation in particular situations. In rats, opiate antagonists decreased basal and stress-induced secretion of PRL. Data regarding TSH are quite contradictory. Both inhibitory and stimulatory effects have been described. Oxytocin and vasopressin release were inhibited by opioids at the posterior pituitary level. There is good evidence for an opioid inhibition of suckling-induced oxytocin release. Opioids also seem to play a role in the regulation of vasopressin under some conditions of water balance. The pancreatic hormones insulin and glucagon are elevated by opioids apparently by an action at the islet cells. Somatostatin, on the contrary, was inhibited. An effect of naloxone on pancreatic hormone release was observed after meals which contain opiate active substance. Whether opioids play a physiologic role in glucose homeostasis remains to be elucidated.     

Regulation of hypophysial secretion by endogenous opiates: Humoral endorphin stimulates the release of growth hormone

Humural endorphin, a recently discovered endogenous opioid factor stimulates the release of growth hormone and, to some extent of prolactin, similarly to other endogenous (enkephalin, β-endorphin) and exogenous (morphine) opiates. This stimulatory effect is dose-dependent with peak values at 30 minutes following intraventricular injection to newborn rats. However, in contrast to the other opioid ligands, the effect of humoral endorphin is not blocked in a dose-dependent fashion by naloxone, the potent opiate antagonist. Thus, while moderate doses of naloxone partially inhibit the stimulatory effect, higher doses which completely block morphine, enkephalin and β-endorphin, are ineffective in antagonizing humoral endorphin. This peculiar interaction between naloxone and humoral endorphin resembles the effect of the opiate antagonist on spontaneous release of growth hormone and prolactin, suggesting the involvement of humoral endorphin in the physiological regulation of hypophysial secretion.     

Effect of naloxone and beta-casomorphin on the hypothalamic-pituitary-luteinizing hormone axis in vitro.

The effect of naloxone and beta-casomorphin on luteinizing hormone (LH) release from pituitary cell aggregates, obtained by three-dimensional culture, with or without mediobasal hypothalamic fragments was studied in vitro. Short-term naloxone perifusion at a concentration of 10(-5)M did not modify either basal or LHRH-stimulated LH release from the pituitary cell aggregates. In contrast, a 12-min naloxone perifusion at the same concentration caused an increase in LH release in the mediobasal hypothalamic-pituitary cell aggregate axis. This increase was rapid (12-16 min after time pulse), marked [up to 10 times (p less than 0.004) the initial base line], short (return to the base line secretion 32-40 min after the beginning of the time pulse) and dose-dependent, with a rise greater than 1000% at a concentration of 10(-4) (p less than 0.006). The same effect was observed when a second pulse was applied 48 min after the first one. LH release induced by naloxone was antagonized 56 +/- 2% (p less than 0.03) by beta-casomorphin (an exogenous opiate) at a concentration of 10(-5) M. beta-casomorphin alone did not modify LH basal secretion, but inhibited 25.1 +/- 2.4% (p less than 0.008) LH release enhanced by LHRH. These results indicate that naloxone, an opiate antagonist, markedly increases LH release via a mu-type opioid receptor mechanism at the hypothalamic level only, during short-term exposure.     

Relation of endogenous opioid peptides and morphine to neuroendocrine functions.

Morphine and the endogenous opioid peptides (EOP) exert similar effects on the neuroendocrine system. When adminstered acutely, they stimulate growth hormone (GH), prolactin (PRL), and adrenocorticotropin (ACTH) release, and inhibit release of luteinizing hormone (LH), follicle stimulating hormone (FSH),and thyrotropin (TSH). Recent studies indicate that the EOP probably have a physiological role in regulating pituitary hormone secretion. Thus injection of naloxone (opiate antagonist) alone in rats resulted in a rapid fall in serum concentrations of GH and PRL, and a rise in serum LH and FSH, suggesting that the EOP help maintain basal secretion of these hormones. Prior administration of naloxone or naltrexon inhibited stress-induced PRL release, and elevated serum LH in castrated male rats to greater than normal castrate levels. Studies on the mechanisms of action of the EOP and morphine on hormone secretion indicate that they have no direct effect on the pituitary, but act via the hypothalamus. There is no evidence that the EOP or morphine alter the action of the hypothalamic hypophysiotropic hormones on pituitary hormone secretion; they probably act via hypothalamic neurotransmitters to influence release of the hypothalamic hormones into the pituitary portal vessels. Preliminary observations indicate that they may increase serotonin and decrease dopamine metabolism in the hypothalamus, which could account for practically all of their effects on pituitary hormone secretion.     

Opioid inhibition of luteinizing hormone secretion in the postpartum lactating sow

Twelve lactating sows were used at 22.4 +/- 0.8 days postpartum to determine whether endogenous opioid peptides (EOP) are involved in the suckling-induced inhibition of luteinizing hormone (LH) secretion. Four sows each received either 1, 2, or 4 mg/kg body weight of naloxone (NAL), an opiate antagonist, in saline i.v. Blood was collected at 15-min intervals for 2 h before and 4 h after NAL treatment. All sows were then given 100 micrograms gonadotropin-releasing hormone (GnRH) in saline i.v., and blood samples were collected for an additional h. Pigs were weaned after blood sampling. At 40 h after weaning, sows were treated and blood samples collected as during suckling. Serum concentrations of LH after treatment with NAL were similar for all doses; therefore, the data were pooled across doses. During suckling, serum concentrations of LH were 0.41 +/- 0.04 ng/ml before NAL treatment, increased to 0.65 +/- 0.08 ng/ml at 30 min after NAL treatment, and remained elevated above pretreatment concentrations for 120 min (p less than 0.05). Naloxone failed to alter serum concentrations of LH after weaning. These data indicate that EOP may be involved in the suckling-induced suppression of LH secretion and that weaning may either decrease opioid inhibition of LH secretion or decrease pituitary LH responsiveness to endogenous GnRH released by NAL.     

Inhibitory effect of beta-endorphin on gonadotropin-releasing hormone and thyrotropin-releasing hormone releasing activity in cultured rat anterior pituitary cells

To study the effect of human beta-endorphin (beta h-End) on pituitary response to gonadotropin-releasing hormone (LH-RH) and thyrotropin-releasing hormone (TRH) in vitro, we used dispersed rat pituitary cells. When beta h-End (10(-7) M) was simultaneously added along with LH-RH, its stimulatory effect was blocked and naloxone (NAL, 10(-5) M) did not reverse the beta h-End inhibitory effect. NAL alone elicited an increase in LH release, but in the presence of both stimulants (LH-RH and NAL), LH secretion was lower than that observed with LH-RH alone. TRH stimulatory activity of TSH and PRL secretion was blunted by the presence of beta h-End (10(-7) M) and was not reversed by NAL (10(-5) and 10(-3) M). These data suggest that beta h-End directly blocks the LH, TSH- and PRL-secreting activity of both LH-RH and TRH at the pituitary level. This beta h-End effect is not reversed by the specific opiate receptor blocker NAL.     

The met-enkephalin analog FK 33-824 directly inhibits ACTH release by the rat pituitary gland in vitro

Systematic administration of the enkephalin analog FK 33-824 was previously shown to stimulate PRL secretion and to inhibit ACTH secretion in man. Naloxone prevented the effect on PRL release, but not on ACTH release. In this study, the direct action of this analog on hormone release by rat anterior pituitary lobes $\text{in}$$\text{vitro}$ were investigated. 1 uM FK 33-824 inhibited basal ACTH secretion by anterior pituitary glands in vitro, while 0.1 uM and 1 uM attenuated the lysine vasopressin stimulated ACTH release. Naloxone did not reverse the inhibitory action of the analog on ACTH release. β-Endorphin (0.01 - 1 uM) did not directly affect ACTH release. Basal and dopamine-induced inhibition of PRL release by anterior pituitary glands was neither influenced by FK 33-824 (0.1 and 1 uM), nor by β-endorphin (0.1 and 1 uM) with or without bacitracin. This study shows that the long-acting met-enkephalin analog FK 33-824 differentially affects PRL and ACTH secretion by the pituitary gland. It seems to stimulate PRL release at a suprapituitary site and this action probably involves u opiate receptors, because naloxone prevents these stimulatory effects. The inhibitory effect of FK 33-824 on ACTH release, however, is mediated via a direct effect at the pituitary level, which does not involve u receptors, as naloxone did not prevent this effect. In this respect, its action differs from that of β-endorphin, which does not directly affect ACTH release by the anterior pituitary gland.     

A possible dual mechanism involved in the inhibitory influence of suckling on luteinizing hormone release in the ovariectomized rat.

A study was conducted to further understand involvement of the endogenous opioid peptides in suckling-induced inhibition of LH release in ovariectomized rats. The first experiment was designed to determine the effect of an opioid antagonist, naloxone (NAL, 1.0 mg. kg-1h-1), on the increase in peripheral LH concentration 18 h after pup removal and on the decrease in LH concentration 18 h after pup return. Infusion of NAL during the 18 h after pup removal or during the 18 h after pup return neither accentuated nor attenuated serum LH concentrations. The second experiment was designed to determine the effect of NAL on peripheral LH concentrations in continuously suckled rats. Serum LH increased (p less than 0.10 and p less than 0.005, respectively) in response to 18 and 36 h of NAL infusion. The third experiment was designed to determine the effect of pup removal during NAL infusion on serum LH. Peripheral LH concentrations were not different in the rats treated with 36 h of NAL infusion whether they were suckled for the duration of the infusion or nonsuckled for the last 18 h of infusion. These results suggest that suckling may inhibit LH release through two mechanisms. The first may be an opioid-independent or enhanced opioid tone mechanism important for the initiation of the inhibitory effect of suckling on LH release, while the second may be an opioid-dependent mechanism important for the sustained inhibitory effect of suckling on LH release.     

Differential effects of naloxone on basal and stress-induced release of ACTH and prolactin in the male rat

The effect of i.v. injection of various doses of naloxone (NAL) on plasma adrenocorticotropin (ACTH) and prolactin (Prl) in conscious animals bearing an indwelling intrajugular catheter was assessed. The effects were evaluated in animals which were left undisturbed and in others subjected to either restraint or ether stress. The results revealed that the dose of 3 mg/kg of NAL significantly reduced basal Prl levels, whereas a dose of 6 mg/kg of NAL was required to block completely either ether or restraint stress-induced release of Prl. The behavior of ACTH contrasted with that of Prl. There was no effect whatsoever of the 3 mg/kg dose of NAL on either resting or stress-induced ACTH levels, whereas a 6 mg/kg or 12 mg/kg dose of NAL elevated resting ACTH levels and only partially attenuated the further elevation induced by stress in these animals. The results clearly indicate a NAL sensitive step in the control of resting and stress-induced Prl release but indicate that the control of resting and stress-induced release of ACTH is different in that the predominantly millimicron receptor blocker, NAL, can elevate ACTH at high doses and can only partially block the response to stress. In contrast to Prl where opioid peptide control is solely stimulatory, this control of ACTH secretion appears to have both stimulatory and inhibitory features.     

Comparison of analgesic and body temperature responses to intrathecal beta-endorphin and D-Ala2-D-Leu5-enkephalin

The inhibition of tail flick response to radiant heat and body temperature changes after intrathecal administration of β-endorphin (β-EP) and D-Ala²-D-Leu⁵-enkephalin (DADL) were studied in rats. Both opioid peptides caused inhibition of tail flick response. On a molar basis, β-EP was 73% as potent as DADL, but the duration of tail flick inhibition of β-EP was much longer than that of DADL. β-EP induced hyperthermia while DADL did not cause any significant change in body temperature. The tail flick inhibition induced by β-EP (1 nmole) was reversed by 2 mg/kg of naloxone, ip; however, the tail flick inhibition induced by DADL (7 nmole) was not reversed by 2 mg/kg and was incompletely reversed by a higher dose of naloxone one (6 mg/kg, ip). These studies demonstrate the existence of naloxone-resistant opioid receptors in the spinal cord which are sensitive to enkephalin. These results indicate that the opioid receptors involved in the production of opioid responses in the spinal cord are different from those in supraspinal brain areas.     

Opiatergic inhibition of pulsatile luteinizing hormone release during the menstrual cycle of rhesus macaques

The endogenous opioid peptides (EOPs) may inhibit the rate of hypothalamic gonadotropin-releasing hormone (GnRH) release and hence the frequency of pulsatile luteinizing hormone (LH) release, particularly in the luteal phase of the menstrual cycle. Our objectives were to compare the effects of an opiate antagonist, naloxone (NAL), on the patterns of LH, estradiol-17 beta (E2), and progesterone (P4) secretion during the follicular and luteal phases of the macaque menstrual cycle. Plasma levels of E2, P4, and bioactive LH were measured in serial, 15-min blood samples during 8-hr infusions of NAL (2 mg/hr) or saline, either on Days 5 or 6 of the follicular phase (FN and FS, n = 5 and 4, respectively) or on Days 8, 9, or 10 of the luteal phase (LN and LS, n = 5 each) of a menstrual cycle. The pulsatile parameters of each hormone were determined by PULSAR analysis and the correspondence of steroid pulses with those of LH were analyzed for each cycle stage in each animal. As expected, LH mean levels and pulse frequencies in LS monkeys were only about one-third of those values in FS animals. NAL had no effects on pulsatile LH, E2, or P4 release during the follicular phase. In contrast, luteal phase NAL infusions increased both LH mean levels and pulse frequencies to values which were indistinguishable from those in FS animals. LH pulse amplitudes did not differ among the four groups. Mean levels and pulse frequencies of P4 secretion in LS monkeys were about 4- and 14-fold greater than those values in FS animals. Mean levels and pulse amplitudes of P4 release in LN animals were greater than those values in all other groups. LH and E2 pulses were not closely correlated in follicular phase animals, and this pulse association was not altered by NAL. In FS monkeys, LH and P4 pulses were not correlated; however, NAL increased this LH-p4 pulse correspondence. LH and P4 pulses were closely correlated in luteal phase animals and this association was not affected by NAL. Our data suggest that the EOPs inhibit the frequency of pulsatile LH secretion in the presence of luteal phase levels of P4. During the midfollicular phase when LH pulses occur every 60 to 90 min, the opioid antagonist NAL alters neither the pulsatile pattern of LH release nor E2 secretion, but NAL may directly affect P4-secreting cells.     

Augmentation by naloxone of efflux of LRF from superfused medial basal hypothalamus

The effects of naloxone and β-endorphin (β-EP) on the efflux of luteinizing hormone releasing factor (LRF) from superfused rat medial basal hypothalamus (MBH) were determined. After an equilibration period of 2.5 hrs with Medium 199 at 37°C 0.5 ml fractions were collected. Infusion of medium containing 150 mM KCl for 10 min produced a prompt 4-fold rise in LR$\text{F}$ efflux. Injection of naloxone, but not medium alone, into the system significantly increased the effluent concentration of LRF from female (N = 6) MBH's by 177% (P < 0.01) and from male (N = 5) MBH's by 108% (P < 0.05). Administration of β-EP did not significantly alter LRF efflux. However, β-EP did nullify the LRF-stimulating effect of naloxone, when an equimolar mixture of β-EP and naloxone was injected. We conclude that naloxone-sensitive opiate receptors exert a tonic inhibitory effect on tuberoinfundibular LRF neurons. This action does not require the intermediation of brain centers outside the MBH.     

Unexpected effects of nalmefene, a new opiate antagonist, on the hypothalamic-pituitary-gonadal axis in the male rat

In order to gain additional information on the role of brain opioid peptides in the regulation of the hypothalamic-pituitary-gonadal axis, we studied the effects of nalmefene, a new opiate antagonist, on gonadotropin and testosterone secretion in male rats. The results were compared with those obtained with naloxone, a well-studied antagonist. Acute injections of either nalmefene or naloxone (2 mg/kg) produced 4-fold increases in LH and testosterone secretion. In castrated male rats treated with testosterone propionate (TP), nalmefene (10 mg/kg) reversed the androgen negative feedback on LH secretion; surprisingly, when higher doses (25 and 50 mg/kg) were injected, the compound lost its ability to antagonize the testosterone-induced inhibition of LH levels. In contrast, naloxone was able to increase LH levels in TP-treated castrated rats even at the highest dose tested (50 mg/kg). Chronic administration of these antagonists resulted in suppression of the acute release of LH and T secretion in nalmefene-treated but not in naloxone-injected animals. These data are consistent with previous observations suggesting that opioid peptides a) exert a tonic inhibitory effect on LH and testosterone production and b) participate in the negative androgen-induced feedback control of LH secretion. Our results also show that the antagonistic action of nalmefene, but not naloxone, is reversed when higher doses are used or following chronic administration.     

Involvement of the hypothalamus in opiate-stimulated prolactin secretion

Administration of opiate agonists to rats is known to elevate plasma prolactin, an effect which is antagonised by the opiate antagonist naloxone. However, this appears not to be a result of a direct action at the pituitary gland. We report here that opiate agonists stimulate prolactin secretion from isolated adenohypophysial cells when they are coincubated with hypothalamic fragments. Both morphine and Met-enkephalin stimulated prolactin secretion by 1.84 fold and 1.50 fold respectively, and this was antagonised by naloxone. These findings support the hypothesis that one site of action of opioid compounds on pituitary hormone secretion is at the level of hypothalamus.     

Studies of naloxone-induced secretion of β-endorphin immunoreactivity in dogs

The stimulating effect of naloxone on plasma β-endorphin immunoreactivity (βEI) was examined in dogs. Intravenous naloxone at 5, 1, or 0.1 mg/kg caused a significant increase in βEI while doses of 0.01 or 0.001 mg/kg had no effect. The peak plasma βEI levels occurred at 25 mins after naloxone. The neurotransmitter and antagonists metergoline, atropine, diphenhydramine and phentolamine all failed to significantly alter basal βEI secretion; further, they all failed to prevent the increase in βEI resulting from naloxone administration. Dexamethasone prevented the naloxone-induced rise in βEI. Our results suggest naloxone's effect on βEI is not mediated through several neurotransmitter systems known to affect ACTH secretion. Additionally, βEI secreted in response to naloxone appears to originate mainly from the anterior lobe of the pituitary.     

Gluten exorphin B5 stimulates prolactin secretion through opioid receptors located outside the blood-brain barrier

Gluten exorphin B5 (GE-B5) is a food-derived opioid peptide identified in digests of wheat gluten. We have recently shown that GE-B5 stimulates prolactin (PRL) secretion in rats; this effect is abolished by preadministration of the opioid receptor antagonist naloxone. However, since the structure of naloxone allows it to cross the blood-brain barrier (BBB) and antagonize opioid effects centrally as well as peripherally, it could not established, on the basis of those data, if GE-B5-induced PRL release is exerted through sites located inside or outside the BBB. In this study, we sought to determine the site of action of GE-B5 on PRL secretion, by pretreating male rats with naloxone methobromide (NMB), an opioid antagonist that does not cross the BBB. Four groups of rats were given the following treatments: 1) intravenous vehicle; 2) intravenous GE-B5 (3 mg kg(-1) body weight); 3) intraperitoneal NMB (5 mg kg(-1) body weight), followed by vehicle; 4) NMB, followed by GE-B5. Blood samples for PRL were taken at intervals for 40 minutes after vehicle or GE-B5 administration. GE-B5 stimulated PRL secretion; the effect was statistically significant at time 20. NMB preadministration completely abolished PRL response. Our experiment indicates that GE-B5 stimulates PRL secretion through opioid receptors located outside the BBB. Since opioid peptides do not exert their effect on PRL secretion directly, but via a reduced dopaminergic tone, our data suggest that GE-B5 can modify brain neurotransmitter release without crossing the BBB.