Evidence for the involvement of endogenous opioids in the inhibition of luteinizing hormone by corticotropin-releasing factor.

Experiments were carried out in castrate adult male rats to further examine whether endogenous opioids are involved in CRF-induced suppression of LH secretion. Serum LH levels in rats castrated 5 days earlier were significantly reduced by intracerebroventricular administration of homologous (rat) CRF (0.02-2 nmol) within 30 min posttreatment; the effects of 0.02 nmol CRF lasted for at least 2 h, whereas those of 0.2 and 2 nmol CRF were evident for up to 6 h. Rats that received sc infusions of the opioid receptor antagonist naloxone (9.6 mg/kg.day) for 48 h before testing with 0.2 nmol CRF showed a significant reduction of the LH response to CRF. Rats that received two acute injections of naloxone (2 mg/kg, iv, 30 min apart) also showed an attenuated response to the LH-suppressive effects of CRF. In another experimental model where the opioidergic control of LH secretion is absent or masked, the long term castrate rat, there was also a marked attenuation of the LH-suppressing effects of CRF. Transient replacement of testosterone in long term castrates reinstated the inhibitory effects of CRF on LH secretion. A third experiment, in which short term castrates were pretreated with an opioid antibody and then with CRF, resulted in a significant reduction of the CRF-induced reduction of serum LH levels. These observations indicate that opioid receptor-mediated events play an important part in the actions of CRF on LH secretion. On the basis of our previous experiments in vitro, we propose that CRF stimulates the release of hypothalamic opioid peptides, which in turn inhibits the activity of LHRH neurons and, thus, LH secretion.

Feedback inhibition of opioid peptide release in the hypothalamus of the rat.

Corticotropin-releasing factor initially stimulates the release of beta-endorphin and dynorphin from rat hypothalamic slices in vitro; with time, in the continued presence of corticotropin-releasing factor, the release of both these peptides declines. The studies described here were undertaken to test whether this decline could be due to the operation of inhibitory feedback mechanisms associated with the function of the opioidergic neurons. When the opioid receptor antagonist naloxone was added to the superfusion medium in the presence of corticotropin-releasing factor, the time-related decrease in opioid release was not observed. Potassium ions also caused an increase, followed by a decrease, in opioid peptide release, and naloxone also prevented the latter from occurring. In addition, naloxone on its own, produced a Ca2+-dependent increase in the non-stimulated release of beta-endorphin and dynorphin, and this action was resistant to tetrodotoxin. These findings suggest that opioid receptors mediate inhibitory feedback effects upon the secretory activity of beta-endorphin and dynorphin neurons in the hypothalamus.

Stimulation of hypothalamic opioid peptide release by lithium is mediated by opioid autoreceptors: evidence from a combined in vitro, ex vivo study.

The effect of both chronic and acute lithium treatment on hypothalamic opioid peptides was investigated. Acute treatment with lithium was found to stimulate the release of beta-endorphin, dynorphin and Met-enkephalin from perfused rat hypothalamic slices. Application of tetrodotoxin was found to have no effect upon the stimulation indicating it to be mediated at the nerve terminal level. The release of hypothalamic opioid peptides is known to be under the chronic control of a system of inhibitory autoreceptors. Blockade of these autoreceptors with, for example, the opioid receptor antagonist naloxone causes a release of all three opioid peptides. Simultaneous addition of naloxone and lithium was found to have no additive effect on the release of any opioid, suggesting lithium acts via an inhibition of the inhibitory autoreceptor. Preincubation with pertussis toxin prevented the lithium stimulation of dynorphin and Met-enkephalin, but not beta-endorphin, release, indicating lithium interacts with a G-protein to affect the autoreceptor controlling the release of dynorphin and Met-enkephalin. Chronic treatment with lithium in vivo (10 days) had no effect on the basal release or hypothalamic content of any of the opioids, but prevented the naloxone-stimulated release of dynorphin and Met-enkephalin in vitro. Long-term treatment with lithium would thus appear to inactivate the autoreceptor(s) controlling their release. These data demonstrate a lithium-stimulated release of hypothalamic beta-endorphin, Met-enkephalin and dynorphin, apparently mediated via an inhibition of the autoreceptors controlling their release. Chronic treatment with lithium permanently inactivated the autoreceptor(s) controlling the release of dynorphin and Met-enkephalin but not beta-endorphin. Lithium would appear to mediate its effects upon Met-enkephalin and dynorphin release via an interaction with a pertussis toxin-sensitive G-protein. The mechanisms underlying its release of beta-endorphin are at present uncertain.

Presynaptic auto- and allelo-receptor regulation of hypothalamic opioid peptide release.

Recent studies have shown that inhibitory feedback mechanisms regulate the release of the endogenous opioid peptides beta-endorphin (acting predominantly at mu opioid receptors in the brain), dynorphin (a kappa opioid receptor ligand) and [Met]enkephalin (a delta opioid receptor ligand) from the rat hypothalamus. By using specific antagonists of the various opioid receptor types, it is shown that the release of these peptides from hypothalamic slices in vitro is not only controlled by homologous (auto)-receptors, but that cross-regulation between the three neuronal opioid receptor types also occurs; thus, the delta receptor antagonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu increases the release of all three peptides, the mu receptor antagonist D-tetrahydroisoquinoline-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 increases that of beta-endorphin and dynorphin, and the kappa receptor antagonist nor-binaltorphimine increases that of dynorphin; all these effects occur in the presence of tetrodotoxin, indicating a presynaptic site of action. We propose the term "allelo-receptors" to describe this particular form of neuronal regulation in which an endogenous ligand, acting via its own specific receptor, also regulates the release of related peptides which activate different classes of opioid receptors.

Neuropharmacological analysis of the control of LH secretion in gonadectomized male and female rats: altered hypothalamic responses to inhibitory neurotransmitters in long-term castrated rats.

The control of LHRH and LH by neurotransmitters and neuromodulators such as the endogenous opioid peptides is essentially the same in intact adult male and female rats: adrenergic and dopaminergic agonists stimulate LH release and opioid agonists inhibit it. Several weeks after gonadectomy, however, the contribution of the endogenous ligands of adrenergic, dopaminergic and opioidergic receptors to the control of LHRH is altered. A detailed pharmacological analysis in long-term ovariectomized females confirmed previous reports that adrenergic and dopaminergic agonists still enhance secretion of LHRH and LH and opioid receptor agonists still suppress it. A similar investigation in long-term castrated males also confirmed previous reports that opioid agonists fail to block LH secretion. In addition, we have found that while adrenergic and dopaminergic agonists cause increases in serum concentrations of LH, adrenoreceptor and dopamine receptor antagonists do not inhibit LH release in long-term castrates. Furthermore, the opioid antagonist naloxone does not raise serum LH levels in either sex after long-term gonadectomy. These observations therefore imply reduced opioidergic, dopaminergic and adrenergic transmission, in relation to LHRH release, after long-term castration. In addition, opioid receptor activity (assessed by responsiveness to an opioid receptor agonist) of female rats is maintained, whereas that of male rats is lost, after long-term gonadectomy.

Diminished role of LHRH in the control of gonadotroph morphology and function in the long-term castrated male rat.

It was found in previous studies that the neurotransmitter control of the secretion of LHRH and LH differs between long-term castrated and ovariectomized rats. One interpretation of these data was that there was a reduced 'positive drive' in the male, and the question was raised 'how do the gonadotrophs of long-term castrated rats maintain a high level of LH secretion?'. In the present series of experiments, evidence for a reduced dependence of the gonadotrophs upon LHRH stimulation is provided. Although sensitivity to native LHRH was not completely lost in long-term castrated rats, two potent LHRH antagonists (D-pyroglu1,D-Phe2,D-Trp3,6)-LHRH and (N-acetyl-3,4-dehydro-Pro,p-fluoro-D-Phe2,D-Trp3,6)-LHRH, were found to inhibit LH secretion in short-term castrated and long-term ovariectomized rats, but not in long-term castrated rats. Neither blockade of axonal transport with colchicine nor immunoneutralization of LHRH with an antiserum against LHRH (both administered 48 h before blood sampling) produced reductions in serum concentrations of LH in long-term castrated rats, although these treatments significantly suppressed LH levels in short-term castrated animals. Chronic (6-day) infusions of the second LHRH antagonist (up to 450 micrograms/day) neither reduced LH secretion nor altered the morphology of the 'castration cells' in the pituitaries of long-term castrated rats. Chronic treatment with testosterone (15 days), however, reversed these parameters to some extent, and when the testosterone treatment was coupled with chronic infusions of the LHRH antagonist, significantly lower serum levels of LH and reductions in the size of the castration cells were observed. These data thus indicate that castration cells may function autonomously, without the need for LHRH, and that testosterone in some way restores the dependency on LHRH and/or the responsiveness to LHRH of these cells.

Peripheral but not intracerebroventricular corticotropin-releasing hormone (CRH) produces antinociception which is not opioid mediated.

Corticotropin-releasing hormone and endogenous opioid peptide systems are both activated during stress. An elevation of the pain threshold also occurs under conditions of stress. In the present study the effects of CRH antinociception were examined. Rats were treated with CRH either centrally (i.c.v.) or peripherally (intracardially; i.c.) and their tail-flick latencies were measured. Central application of CRH (0-30 micrograms) was without effect on the analgesic test, while after peripheral application (0-32 micrograms) CRH produced a dose-dependent increase in tail-flick latencies. In a subsequent experiment we examined the possible involvement of endogenous opioids in the peripheral CRH-induced antinociceptive effects. To this end, two approaches were used: animals were either acutely treated with the opioid antagonist naloxone (3 or 6 mg/kg), or they were rendered tolerant to morphine, and then tested with CRH. In both cases, CRH effects on the tail-flick latencies were not modified. Our findings suggest that: (a) CRH may modulate pain sensitivity during stress; (b) opioids do not mediate this effect; and (c) brain CRH receptors are probably not involved in these processes.

Hypothalamic opioid receptors mediate the inhibitory actions of corticotropin-releasing hormone on luteinizing hormone release: further evidence from a morphine-tolerant animal model.

Recent experiments have shown that corticotropin-releasing hormone (CRH) inhibits gonadotropin hormone-releasing hormone (GnRH) and luteinizing hormone (LH) release, and endogenous opioid peptides have been implicated in the mediation of these effects. To further test this hypothesis, the effects of CRH on LH secretion was tested in rats that were made tolerant to the alkaloid opiate agonist morphine. Male rats were gonadectomized and 5 days later implanted with 2 pellets containing 75 mg of morphine each, for 48 h and with a third morphine pellet for the following 24 h. Rats were killed on the 4th day and their serum levels of LH were found to be similar to those of placebo-treated controls, indicating that the neural systems controlling LH secretion had become tolerant to the chronic exposure to morphine. The tolerant condition was confirmed in a subgroup of morphine-treated rats since an acute injection of morphine (5 or 10 mg/kg) did not further suppress LH levels in the chronically morphinized rats. The LH-suppressive efficacy of CRH (0.2 mnol, i.c.v.) was found to be markedly reduced in these morphine-tolerant rats compared to the opiate-naive animals. This finding thus further supports the view that opioid receptors partially mediate the inhibitory actions of CRH upon the GnRH-LH system.

Prolonged inflammatory pain modifies corticotropin-releasing factor-induced opioid peptide release in the hypothalamus.

The influence of prolonged pain upon hypothalamic opioid peptide release in vitro was examined in rats subjected to Freund's adjuvant (FA)-induced unilateral inflammation of the hindlimb. Basal release of enkephalin (ENK) but not beta-endorphin (END) or dynorphin (DYN) was increased 10 days following FA treatment. Superfusion of corticotropin-releasing factor (CRF; 10(-8) M) stimulated the release of opioid peptides in control hypothalami. CRF, however, failed to modify beta-END and DYN release in hypothalami of FA-treated rats, whereas ENK release was markedly reduced. In contrast, KCl-stimulated opioid peptide release did not differ between FA and control hypothalami. These data demonstrate that prolonged inflammatory pain alters the responsiveness of hypothalamic opioid systems to CRF. It is suggested that this effect is mediated at the level of the CRF neuron or its receptor.

Corticotropin-releasing factor stimulates the release of methionine-enkephalin and dynorphin from the neostriatum and globus pallidus of the rat: in vitro and in vivo studies.

This study examined the changes in the in vitro and in vivo release of methionine-enkephalin (Met-enkephalin), and dynorphin from the rat neostriatum in response to corticotropin-releasing factor (CRF). The levels of each opioid peptide were measured in the same sample collected at each time interval by specific radioimmunoassay methods. The in vitro release experiments were conducted using neostriatal slices (250 microns) obtained from adult male Wistar rats whereas in the in vivo studies, the release of both Met-enkephalin and dynorphin were assessed in push-pull perfusates of the caudate nucleus (containing both Met-enkephalin and dynorphin cell bodies/fibres) and the globus pallidus (containing Met-enkephalin and dynorphin terminals) of chloral hydrate-anaesthetised adult male Wistar rats. In the in vitro studies, CRF (10(-12), 10(-10) and 10(-8) M) (applied in pulses of 75 min) stimulated both Met-enkephalin and dynorphin release from the neostriatal slices in a dose-related manner; in the presence of the CRF receptor antagonist, alpha-helical CRF9-41 (10(-6) M) the release of both Met-enkephalin and dynorphin in response to CRF (10(-8) M) were completely blocked. Push-pull perfusion experiments conducted in both the caudate nucleus and the globus pallidus, also demonstrated a dose-related increase in the release of both Met-enkephalin and dynorphin in response to CRF (10(-12), 10(-10) and 10(-8) M) applied in 60-min pulses. In addition, in each of the two brain sites, the release of both Met-enkephalin and dynorphin in response to CRF (10(-8) M) was completely blocked by alpha-helical CRF9-41 (10(-6) M). Both the in vitro and in vivo studies thus demonstrate that CRF can exert potent effects on Met-enkephalin and dynorphin release within the neostriatum-pallidum of the rat and that such effects are mediated via receptors specific for CRF, probably located on both the cell bodies and terminals of these opioid-containing neurones. The data obtained in this study thus substantiate the view that CRF, in addition to its regulation of pituitary opioid peptides, can communicate to opioid-containing neurones within the central nervous system and that many of the effects elicited by CRF may be ascribed to the opioid peptide released by CRF.(ABSTRACT TRUNCATED AT 400 WORDS)

Corticotropin-releasing factor (CRF) inhibits gonadotropin-releasing hormone (GnRH) release from superfused rat hypothalami in vitro.

A dose-dependent suppression of basal gonadotropin-releasing hormone (GnRH) release was observed when rat hypothalamic slices were superfused with human/rat corticotropin-releasing factor (CRF) (10(-12) to 10(-8) M). CRF was also found to significantly reduce the amount of GnRH released in response to 56 mM KCl. These in vitro results demonstrate that the CRF inhibition of GnRH secretion observed in vivo occurs within the hypothalamus and independently of the CRF activation of the pituitary-adrenal-gonadal axis.

Stimulation of hypothalamic beta-endorphin and dynorphin release by corticotropin-releasing factor (in vitro).

Corticotropin-releasing factor (CRF) at doses of 10(-12)-10(-8) M significantly stimulated the release of beta-endorphin and dynorphin from superfused rat hypothalamic slices. These effects were shown to be mediated by the CRF receptor since they were antagonized by the CRF receptor antagonist alpha-helical CRF9-41 (10(-6) M). The two opioid peptides showed different time courses of response and in the case of beta-endorphin, an attenuation of the response upon continued exposure to CRF was observed.

Hypothalamic LH-RH release after acute and chronic treatment with morphine studied in a combined in vivo/in vitro model.

Rats were treated with the opiate agonist, morphine, and the release of luteinizing hormone-releasing hormone (LH-RH) from their hypothalami was studied in vitro. Within 16-24 h after morphine treatment, basal LH-RH release rates were observed to be higher compared to those from hypothalami derived from opiate-naive rats, suggesting that dependence had occurred in the neural mechanisms underlying LH-RH release. Maintenance of tissues exposed to morphine in vivo in medium containing morphine in vitro did not alter the increased basal release of LH-RH, but because this was significantly greater than control rates, tolerance is not believed to have occurred. Addition of the opioid antagonist naloxone in vitro resulted in a 220% increase in the release of LH-RH by hypothalami exposed to morphine for 48 h in vivo, whereas it caused a 50% reduction in LH-RH release from tissues exposed to morphine for 96 h in vivo. This latter result shows parallels with our previous finding that naloxone paradoxically decreases serum LH levels of chronically morphine-treated rats. In view of recent evidence for presynaptic feedback inhibitory effects operating on opioid neurons, it is suggested that, following chronic exposure to morphine, the opioid neurons which normally inhibit LH-RH neurons are inhibited; upon treatment with naloxone, they are disinhibited and release more opioid peptides which then act to switch off LH-RH neuronal activity.

Prodynorphin gene expression in spinal cord is enhanced after traumatic injury in the rat.

Levels of mRNAs coding for prodynorphin (Pro-Dyn) and proenkephalin (Pro-Enk) as well as the levels of immunoreactive (ir)-dynorphin (Dyn) and (ir)-Met-enkephalin (Met-Enk) were measured in the spinal cord of rats, 65 h following transection or injury of the spinal cord at the T6 spinal segment. Levels of both Pro-Dyn mRNA and of ir-Dyn were significantly increased between 60 and 150%, above control levels in the whole spinal cord, whereas those of Pro-Enk mRNA and of ir-Met-Enk remained unchanged. The increase in spinal levels of Pro-Dyn mRNA were highest in the areas close to the side of transection and indicate an involvement of the Pro-Dyn opioid system in the response to spinal injury and transection.

In vivo modulation of rat hypothalamic opioid peptide content by intracerebroventricular injection of guanidinoethylmercaptosuccinic acid (GEMSA): possible physiological role of enkephalin convertase.

Twice-daily intracerebroventricular (i.c.v.) injections for three days of increasing doses of guanidino-ethyl-mercapto-succinic acid (GEMSA) produced a dose-dependent decrease in methionine-enkephalin- and leucine-enkephalin levels in rat hypothalami. GEMSA is a specific and potent inhibitor of a carboxypeptidase B-like processing enzyme, referred to as enkephalin convertase (EC). The administration of GEMSA (0.1 microgram) resulted in more than 50% reduction in the levels of these two opioid peptides. However, no changes occurred in the hypothalamic content of beta-endorphin or dynorphin1-17. Moreover, in GEMSA-treated animals, hypothalamic luteinizing hormone-releasing hormone and serum luteinizing hormone levels were increased by 75%. Serum prolactin concentrations were decreased by 60% at the same time. Subcutaneous naloxone administration resulted in a 75% elevation of serum LH concentrations in control animals whereas GEMSA-treated animals showed a blunted response, most likely due to a decreased amount of opioid-active peptides. The present study is in agreement with the putative role of EC in the processing of the multivalent opioid precursor (proenkephalin A) in the rat hypothalamus. The enzyme inhibition by GEMSA may result in a reduced enkephalinergic tone, which is then accompanied by an altered endocrine status.

Facilitation of ACTH secretion by morphine is mediated by activation of CRF releasing neurons and sympathetic neuronal pathways.

Exogenously applied opioid agonists have a stimulatory effect on adrenocorticotropic hormone (ACTH) secretion. The present experiments were designed to examine the mechanisms involved in the stimulatory effect of the mu-receptor agonist morphine on ACTH release in chronically cannulated, freely moving, non-stressed rats. Morphine (7.5 mg/kg, i.v.) treatment was followed by a significant increase in plasma levels of ACTH. Pretreatment with the peripheral ganglionic blocker chlorisondamine (3 mg/kg, i.p.) attenuated the response to morphine. The morphine stimulatory effect was also partially inhibited if the rats were pretreated with a specific antiserum to corticotropin-releasing factor (CRF). In rats given both CRF antiserum and chlorisondamine, the plasma ACTH levels remained unchanged after morphine application. These findings indicate that morphine stimulates the release of ACTH by activating both CRF-secretion and peripheral sympathetic neuronal pathways.

Nigrostriatal dopamine mediates the stimulatory effects of corticotropin releasing factor on methionine-enkephalin and dynorphin release from the rat neostriatum.

This study examined the effects of corticotropin-releasing factor (CRF) on the in vitro release of methionine-enkephalin (Met-enkephalin) and dynorphin in neostriatal slices taken from rats with unilateral 6-hydroxydopamine (6-OHDA)-induced lesions of the nigrostriatal DA pathway. In neostriatal slices from control saline-infused animals and in those from the contralateral hemisphere of 6-OHDA-lesioned animals, CRF (10(-10) M) administered as a 90-min pulse exerted potent stimulatory effects on both Met-enkephalin and dynorphin release. In the neostriatal slices of 6-OHDA-lesioned striata, both the basal release and tissue content of Met-enkephalin were significantly (P less than 0.01) higher (2-fold) than those of control animals and the contralateral hemisphere of 6-OHDA-lesioned animals; however, neither the basal release nor the tissue content of dynorphin in 6-OHDA-lesioned striata was significantly different from control striata. In response to CRF (10(-10) M) the release of both Met-enkephalin and dynorphin were significantly diminished in slices of 6-OHDA-lesioned striata. These data support previous studies suggesting that nigrostriatal DA itself may exert a tonic inhibitory action on the activity of striatal Met-enkephalin neurones; however, DA may not have the same influence on striatal dynorphin neurons. However, the results of this study demonstrate that the action of CRF on Met-enkephalin as well as dynorphin release from the rat neostriatum is DA dependent. The data suggest that CRF receptors in the rat neostriatum may be localized on nigrostriatal/nigropallidal DA terminals/collaterals.

The role of CRF in the release of ACTH by opiate agonists and antagonists in rats.

Opiate agonists as well as opiate antagonists release adrenocorticotropic hormone (ACTH) through unknown central mechanisms. We examined the role of corticotropin releasing factor (CRF) in opioid actions on ACTH release. Rats were pretreated with a specific antiserum to rat CRF (r-CRF) prior to injection of the opiate antagonist naloxone or the opiate agonists morphine (mu-receptor agonist) or MR 2034 (kappa-receptor agonist). This abolished the increase in ACTH caused by naloxone or by the kappa-agonist. The mu-agonist morphine, however, caused a significant increase in ACTH in control- or r-CRF antiserum treated rats. We conclude that (a) endogenous opioids tonically inhibit the release of CRF, (b) kappa-agonists stimulate the release of CRF, and (c) mu-agonists release ACTH by acting additionally through CRF-independent mechanisms.

Pre- and postsynaptic actions of GABA on the release of hypothalamic gonadotropin-releasing hormone (GnRH).

The role of gamma-aminobutyric acid (GABA) in the regulation of gonadotropin hormone-releasing hormone (GnRH) release was studied using rat hypothalamic slices in vitro. Perifusion with GABA (10(-8)-10(-4) M) caused a dose-dependent increase in GnRH release. The GABAA receptor agonist isoguvacine (10(-5) M) also stimulated GnRH release, whereas the GABAB agonist baclofen (10(-6) M) had no effect. The specific GABAA antagonist SR95103 (10(-6) M) caused a reduction of basal GnRH release and completely blocked that induced by GABA (10(-4) M). When nerve transmission was blocked with tetrodotoxin (TTX, 10(-6) M), GnRH release was slightly reduced but the stimulatory effects of both GABA and isoguvacine were abolished. The GABA-induced stimulation of GnRH release was also prevented when the hypothalamic slices were treated with a corticotropin releasing hormone (CRH) antagonist (alpha-helical CRF9-41, 10(-6) M) or the opioid antagonist naloxone (10(-6) M). Treatment with CRH (10(-8) M) resulted in a decrease in GnRH release and this effect was not reversed in the presence of GABA. Finally, GABA was found to stimulate the release of the opioid peptides beta-endorphin, dynorphin and met-enkephalin. These results lead us to conclude that GABA exerts two opposing effects upon GnRH neuronal activity: it acts in an inhibitory fashion at GnRH nerve terminals and in a stimulatory fashion at GnRH perikarya; the latter might occur through GABAergic inhibition of CRH release and, therefore, of opioid peptide release. Lastly, all the effects of GABA upon GnRH release appear to be mediated through GABAA receptors.