Reserpine inhibits rat anterior pituitary hormone secretion in vitro: Effects of prolactin and ACTH and ultrastructural observations

We measured [³H]prolactin ([³H]Prl) synthesis and secretion in female rat anterior hemipituitary glands incubated in vitro, and immunoassayable Prl secretion from dispersed anterior pituitary cells in a perfused column. Anterior pituitary glands which were incubated in 9 μM reserpine showed a marked inhibition of [³H]Prl secretion but no change in hormone synthesis, thus causing [³H]Prl accumulation within the gland. The same concentration of reserpine produced a similar effect in pituitary glands taken from rats depleted of dopamine with a-methyl-p-tyrosine. Reserpine inhibited Prl secretion from dispersed anterior pituitary cells with a gradual onset and prolonged duration. Thyrotropin-releasing hormone (TRH), but not dibutyryl cyclic AMP (dbcAMP), the calcium ionophore A23187 or excess Ca²⁺, stimulated both [³H]Prl and Prl secretion in the presence of reserpine. In contrast, neither basal nor vasopressin-stimulated ACTH (bio- and immunoassayable) secretion was inhibited by 9 μM reserpine. Ultrastructurally, pituitary glands incubated in reserpine had an increased content of Prl secretory granules. Reserpine thus selectively inhibited Prl secretion, secondarily causing accumulation of both measurable hormone and Prl secretory granules within the pituitary gland. We hypothesize that reserpine interrupted calcium-dependent mechanisms in the stimulus-secretion coupling process to inhibit Prl release.     

Sub-chronic exposure to opiates in the rat: effects on brain levels of substance P and calcitonin gene-related peptide during dependence and withdrawal.

Substance P (SP) and calcitonin gene-related peptide (CGRP) are putative transmitters in the central and peripheral (sensory) nervous systems. In this study, we examined the effects of dependence on and withdrawal from morphine and methadone on brain SP and CGRP content. Female Long Evans rats (70-100 g) were provided with plain drinking water or solutions containing opiate. No choice of drinking fluid was allowed. The maintenance level of each opiate (0.8 and 0.4 mg/ml for morphine and methadone, respectively) was continued for 4 days. Following an injection with naloxone (10 mg/kg i.p.) or saline, animals were decapitated 0, 20, or 60 min later and regional brain peptide content was measured by specific radioimmunoassays. SP and CGRP content in opiate-maintained and naive animals were similar following saline injection. However, following naloxone injection in morphine-maintained animals, SP content was elevated in the hypothalamus and midbrain at 20 min, but by 60 min was no longer distinguishable from basal (0 min) level. CGRP content was increased in the medulla oblongata and followed a comparable time course but, unlike SP, was not altered in the hypothalamus or midbrain. No alterations were observed in methadone-maintained animals. These results correlated with the peak of the behavioral morphine withdrawal syndrome and were consistent with the comparatively milder abstinence encountered in methadone medication.     

The role of opioid peptides in memory and learning

Evidence is discussed which points to the existence of a physiologic amnestic mechanism mediated by beta-endorphin and perhaps by other opioid peptides as well. This mechanism is triggered by various forms of training and by either painful or painless stimulation. It may operate through the inhibition of central dopaminergic and beta-adrenergic systems that modulate the memory consolidation process. This amnestic mechanism is unrelated to the regulation of pain perception, and operates at opioid peptide levels several orders of magnitude below those that are needed to cause analgesia or other effects. In addition, shuttle avoidance and habituation learning seem to be dependent on a state induced by the release of beta-endorphin. It is possible that this may be related to the amnestic properties of this substance. Therefore, it appears that the endogenous opioid peptides may exert their primary function in the modulation of memory processes.     

Thyrotropin-releasing hormone (TRH) content of rat striatum: Modification by drugs and lesions

Two hours after injection, D-amphetamine sulfate (10 mg/kg, i.p.) lowered thyrotropin-releasing hormone (TRH) levels in rat striatum by 50%, but produced no significant changes in the TRH contents of hypothalamus, septum, brain stem or preoptic area. The effect peaked 2 h after amphetamine injection and declined slowly thereafter. The amphetamine-induced decrease in striatal TRH could be blocked by pretreatment with haloperidol or alpha-methyltyrosine, or by production of a 6-hydroxy-dopamine lesion in the ipsilateral substantia nigra. Amphetamine did not act by inhibiting protein synthesis in as much as cycloheximide did not similarly decrease striatal TRH. Kainic acid injected into the striatum lowered TRH by 30% after 5 days. In contrast, partial deafferentiation of the striatum (by cerebral hemitransection at mid-hypothalamic level) increased striatal TRH 2-3-fold, while lesions of the dorsal raphe did not significantly change striatal TRH. Thus TRH levels in rat striatum are closely regulated by dopaminergic and other neurotransmitter systems.     

Immunological blockade of endogenous thyrotropin-releasing hormone produces hypothermia in rats

Administration of anti-serum to thyrotropin-releasing hormone (TRH) into the lateral cerebral ventricle of rats produces a dose-dependent hypothermia. Neutralization of anti-TRH serum with excess TRH abolishes this hypothermic effect. These results suggest a thermoregulatory role for endogenous TRH in the rat central nervous system.     

Endogenous opioid systems: Physiological role in the self-limitation of seizures

Immediately following a seizure, the severity of subsequent seizures is significantly reduced. The involvement of endogenous opioid systems as a physiological regulator of this postseizure inhibition was studied in rats using repeated maximal electroshock (MES) seizures. Both the opiate antagonist (−)-naloxone and morphine tolerance abolished the progressive seizure protection associated with repeated MES. We propose that endogenous opioids, activated by a prior seizure, provide a central homeostatic inhibitory mechanism which may be responsible for the initiation of a postictal refractory state in the epileptic.     

Systemic morphine blocks the seizures induced by intracerebroventricular (i.c.v.) injections of opiates and opioid peptides

Intracerebroventricular (i.c.v.) injections of the endorphins and of morphine in rats produce highly characteristic, naloxone sensitive, electrographic seizures. In contrast, systemic injections of morphine have been shown to exert a marked anticonvulsant effect. The present study demonstrates that systemic morphine pretreatment can prevent the occurrence of electrographic seizures induced by i.c.v. morphine, Leu-enkephalin and β-endorphin and that the anti-epileptic effect of morphine can be reversed by naloxone. Male albino rats, previously prepared for chronic i.c.v. injections and EEG recording, were pretreated with 0–100 mg/kg of intraperitoneal (i.p.) morphine. Thirty five minutes later morphine (520 nmol), Leu-enkephalin (80 nmol) or β-endorphin (5 nmol) were injected i.c.v. Pretreatment with i.p. morphine blocked the occurrence of seizures induced by morphine and both endogenous opioids. Lower doses of systemic morphine (50 mg/kg) were necessary to block i.c.v. morphine seizures than the dose (100 mg/kg) necessary to block seizures induced by i.c.v. Leu-enkephalin and β-endorphin. Naloxone (1 mg/kg) administered 25 min following 50 mg/kg of i.p. morphine and preceding the injections of i.c.v. morphine reversed the antiepileptic effect of systemic morphine. These results demonstrate the possible existence of two opiate sensitive systems, one with excitatory-epileptogenic effects and the other possessing inhibitory-antiepileptic properties. The possible relationship between these findings and the known heterogeneity of opiate receptors and opiate actions is discussed.     

Neuroendocrinological study of opioid and serotoninergic systems in migraine patients

PRL response to morphine in a group of migraine subjects was studied in the baseline condition and after neuroendocrinological inhibition and stimulation tests. No significant differences were found in comparison with control subjects. This demonstrated the integrity of the opioid system and serotoninergic pathways in migraine subjects.

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Sommario In un gruppo di soggetti emicranici è stata studiata la risposta in prolattina alla morfina in condizioni basali e con tests neuroendocrinologici di stimolazione ed inibizione. Non si sono riscontrate significative differenze nei confronti dei soggetti di controllo. Si è cosi documentata l'integrità del sistema degli oppiati endogeni e della via serotoninergica nei pazienti emicranici.

     

Morphine exerts testosterone-like effects in the hypothalamus of the castrated male rat

Previous research has indicated that endogenous opioids participate in the regulation of activity in the hypothalamic-pituitary-luteinizing hormone (LH) axis and mediate the negative feedback control exerted by testosterone. If this assumption is correct, then two predictions can be made. First, the effects of testosterone should be competitively inhibited by narcotic antagonists; and, second, opiates should mimic the acute and chronic effects of testosterone in the castrated male rat. The results of the present investigations support both of these predictions. We found that naloxone competitively antagonized the depressive effects of testosterone on serum LH in the castrated rat and, conversely, that testosterone competitively antagonized the LH-releasing properties of naloxone. In addition, morphine and testosterone both depressed serum LH levels in a dose-dependent fashion in the acutely castrated animal. Moreover, morphine was just as effective as testosterone in reversing the castration-induced fall in hypothalamic-LH-releasing hormone (LH-RH), which occurs in the chronically castrated male rat. On the other hand, morphine failed to reverse the long-term changes in pituitary LH content and increase in serum LH, which is consistent with prior observations that morphine affects only the hypothalamic aspect of the hypothalamic-pituitary-LH axis in the male rat. These results, thus, support the concept that an as yet unidentified opioid-containing neuronal system regulates activity in the hypothalamic-pituitary-LH axis and mediates the effects of testosterone on this axis.     

Opioid peptides and opiates differ in receptor selectivity.

(1) The binding properties of opioid receptors in whole rat cerebrum have been studied with tritium-labelled dihydromorphine, naltrexone and Leu-enkephalin as radioindicators and various opioid peptides and opiates as competitors. (2) Dihydromorphine shows high affinity binding to sites which are competitively blocked by various opiate agonists and antagonists (DHM sites). (3) Naltrexone binds to additional sites which also strongly bind other narcotic antagonists (NAL sites). (4) The morphinomimetic enkephalins show high selectivity for DHM sites while ACTH fragments and somatostatin show less selectivity. (5) Leu-enkephalin appears to bind to separate sites (EKN sites) with similar affinity to that for DHM sites. (6) Both dihydromorphine and naltrexone show higher affinity for DHM sites than for EKN sites. (7) In conclusion, at least three kinds of opioid binding sites are observable. These differences in receptor populations may relate to functional differences.     

Changes in anterior pituitary hormone secretion and hypothalamic catecholamine metabolism during morphine withdrawal in the female rat

Studies were undertaken to evaluate the acute responses of hypothalamic noradrenergic and dopaminergic neurons and anterior pituitary hormones to naloxone (NAL)-precipitated morphine (MOR) withdrawal in the rat. Ovariectomized female rats were rendered MOR-dependent and injected with NAL (1 mg/kg b.w., s.c.). During precipitated MOR withdrawal, a decline in norepinephrine (NE) concentrations was preceded by an increase in the level of its metabolite normetanephrine (NME) in the medial basal hypothalamus (MBH) as well as the preoptic area-anterior hypothalamus (POA-AH). Both dopamine (DA) and its major acid metabolite, dihydroxyphenylacetic acid (DOPAC), showed increased concentrations in these two hypothalamic regions within 30 min of NAL administration. Elevated luteinizing hormone (LH) and beta-endorphin secretion was evident within 5 min of NAL injection to MOR-dependent rats, while serum prolactin (PRL) increased 15 min into MOR withdrawal. Both growth hormone (GH) and thyroid-stimulating hormone (TSH) were depressed over the course of MOR withdrawal. Although a cause and effect relationship cannot be established, during NAL-precipitated MOR withdrawal, a heightened hypothalamic monoaminergic neuronal activity is accompanied by a differential response of anterior pituitary hormones. The observed responses, which are similar to those seen during acute stress, indicate that MOR withdrawal may activate the same mechanisms which mediate the neuroendocrine response to stress.     

Cyclosporine Effects on in vitro Responsiveness of Anterior Pituitary Hormone Release to Dopamine and Thyrotropin-Releasing Hormone in Young Female Rats

Endocrine side effects of the immunosuppressive drug cyclosporine (CyA) include changes in anterior pituitary hormone secretion. The aim of the present study was to examine the effects of CyA on the responsiveness of in situ and ectopic anterior pituitary prolactin (PRL), growth hormone (GH) and luteinizing hormone (LH) release response to dopamine (DA) and thyrotropin-releasing hormone (TRH) treatment in young female rats, and to evaluate the possible PRL participation in these effects. Thirty day old rats were rendered hyperprolactinemic by transplanting an anterior pituitary gland of a littermate donor, under the kidney capsule, and were then injected with CyA or vehicle for 2 or 8 days. Sham-operated rats were used as controls and treated in the same way. CyA treatment prevented the increase in plasma PRL levels which occurred in controls after pituitary grafting. In vitro basal PRL release of in situ pituitaries from either sham-operated and/or pituitary-grafted animals was decreased by CyA treatment at any point studied. Basal in vitro secretion of GH was only decreased in the in situ pituitaries from grafted animals after 2 days of CyA therapy. The presence of an ectopic pituitary lead to an increase in the in vitro basal LH secretion from in situ pituitaries as compared to those from sham-operated rats. Basal LH release rates were not changed by CyA treatment, although the LH release in vitro did increase in the in situ pituitaries from sham-operated animals treated with the drug for 2 days. DA addition to the incubation media decreased the in vitro release of PRL, GH and LH from the in situ pituitaries of sham-operated and pituitary-grafted animals treated with vehicle. In CyA treated animals, DA decreased in vitro PRL release from the in situ pituitaries of animals, independently of the presence or absence of an ectopic pituitary. Reductions of the in vitro GH and LH release after DA treatment were higher in the in situ pituitaries from grafted animals on day 8 of CyA or vehicle treatment. TRH increased the in vitro release of the three hormones with differential effects related to the length of the treatment with CyA and/or the presence of an ectopic pituitary. In vitro release of PRL and GH by ectopic pituitaries was inhibited by previous treatment with CyA and this effect was decreased proportional to the duration of the treatment with the drug, while LH secretion was not modified. Addition of DA to the incubation media resulted in a marked reduction of in vitro PRL and GH release, but only at day 8 of vehicle treatment on GH release did DA addition to media further decrease the release of both hormones from ectopic pituitaries from animals treated for 2 or 8 days with the drug, whereas LH secretion was not modified. TRH addition to the incubation media of ectopic pituitaries surprisingly reduced PRL and GH secretion on day 8 of CyA treatment or after surgery. The results of these studies suggest that CyA can act directly at the hypophyseal level modifying pituitary responsiveness to external stimuli. CyA seems to exert its main effects on lactotroph activity while its effects on somatotrophs and gonadotrophs are less.     

Localization of immunoreactive thyrotropin releasing hormone in the lower brainstem of the rat

The distribution of thyrotropin releasing hormone (TRH) in individual nuclei of the rat brainstem was examined by radioimmunoassay. TRH was detectable in 36 of 40 brainstem nuclei investigated and the localization of TRH in the brainstem was unlike other known brainstem neuropeptides. By far the highest concentration of TRH in the brainstem (1.2 ng/mg protein) is present in the nucleus of the solitary tract. The concentration of TRH was relatively high in the motor nuclei of the IIIrd, Vth, VIIth, Xth and XIIth cranial nerves, and less high in the area postrema, nucleus gracilis, locus coeruleus, lateral reticular nucleus (A1-catecholamine cell group), dorsal raphe and central gray matter. Cerebellum and pontine nuclei contained very low levels (<0.03ng/mg protein) of TRH.     

Effect of amitriptyline on the thyrotropin response to thyrotropin-releasing hormone in endogenous depression

Patients with endogenous depression whose depressive episodes were clinically resolved after electroconvulsive therapy were divided into two groups: one in which patients remained well (n = 16) and another in which patients relapsed within 6 months (n = 11). Treatment with amitriptyline for 3 weeks did not affect the median thyrotropin (thyroid-stimulating hormone; TSH) response to thyrotropin-releasing hormone (TRH) in recovered patients, whereas that in relapsed patients was significantly enhanced. The data suggest that amitriptyline affects the TSH response to TRH differently in stably recovered and relapsed patients. If this effect is maintained beyond the 3-week period studied, treatment with amitriptyline will invalidate the predictive value of the TRH test.     

Role of central nervous system neurotransmitters in mediating the effects of morphine on growth hormone-and prolactin-secretion in the rat

Unanesthetized adult male rats with indwelling right atrial cannulae were used in the majority of experiments. Morphine (MOR, 3.0 mg/kg) caused a large but transient increase in both GH and PRL levels, which could be prevented with naloxone. Disruption of central noradrenergic function with diethyldithiocarbamate (400 mg/kg) or phenoxybenzamine (15 mg/kg) abolished the GH-releasing effect of MOR, without interfering with the PRL secretory response. Depletion of brain serotonin stores with p-chlorophenylalanine (300 mg/kg) or 5,7-dihydroxytryptamine or administration of serotonin receptor blocker, cyproheptadine (2.5 mg/kg), did not diminish the GH respnse to MOR but it inhibited, or in the case of 5,7-DHT treatment abolished the activation of PRL secretion. Additionally, metergoline (0.1 and 1.0 mg/kg), another serotonin receptor blocker, caused an inhibition of the GH-releasing action of MOR; however, this inhibition was reversed by pretreatment with spiroperidol (0.1 mg/kg). Metergoline also markedly diminished the MOR-induced elevation of PRL. Inhibition of catecholamine synthesis with alpha-methyl-p-tyrosine (alpha-MT, 250 mg/kg) blunted the effect of MOR on GH; however, dopamine receptor blockers, spiroperidol (0.01 and 0.1 mg/kg) or (+)butaclamol (0.3 and 1.3 mg/kg), were without any influence. alpha-MT or spiroperidol did not alter the effect of MOR on PRL secretion, but the higher dose of (+)butaclamol suppressed it. It is concluded that the GH-releasing action of MOR requires unimpaired functioning of the central noradrenergic system, while the serotonergic and dopaminergic systems appear to play no significant role in it. In contrast, serotonergic systems seem to be essential for the activation of PRL secretion, whereas the noradrenergic system is not involved. It remains uncertain whether morphine activtes PRL secretion also through inhibition of dopaminergic activity. We favor the view that the dopaminergic component participates in the PRL activation by MOR, but that its contribution to the overall effect is rather small.     

Activating endogenous opioid systems by electroconvulsive shock or footshock stress inhibits recurrent kindled seizures in rats

Electroconvulsive shock (ECS) significantly decreased the behavioral manifestations of seizures elicited by amygdaloid stimulation in kindled rats. This anticonvulsant effect was significantly reduced by the opiate antagonist, naloxone, and by the development of morphine tolerance. A form of footshock stress known to cause opioid-mediated analgesia had a similar anticonvulsant effect, whereas another form causing non-opioid analgesia did not. These results suggest that the anticonvulsant effects of ECS and stress are mediated by the release of endogenous opioids.     

Stress-induced analgesia in frogs: evidence for the involvement of an opioid system

Immobilization of frogs for 1 h induces analgesia which is blocked but not reversed by low doses of naloxone. After 9 days of daily immobilization for 1 h, the treatment fails to cause analgesia thus indicating that tolerance has developed. Animals tolerant to i immobilization-induced analgesia do not show cross-tolerance to the analgesic action of morphine. The development of tolerance to this form of stress-induced analgesia and the ability of naloxone to prevent its occurrence indicate the involvement of opioid pathways. The lack of cross tolerance to morphine suggests that μ receptors are not involved.     

Effect of TRH on TSH and prolactin levels in affective disorders

The thyrotropin-releasing hormone (TRH) test was studied in 32 patients with acute major depressive disorder, 16 patients with recurrent unipolar (n = 8) or bipolar (n = 8) affective disorder in remission, and 22 healthy control subjects. Twenty-six of the 32 acutely ill patients were also studied when in remission. Outcome in these patients was correlated to serum levels of triiodothyronine (T3), 3,3',5'triiodothyronine (reverse T3), thyroxine (T4), thyroid-stimulating hormone (TSH), prolactin (PRL), melatonin, dexamethasone suppression test (DST) results, and clinical symptoms assessed by the Comprehensive Psychopathological Rating Scale (CPRS). The TSH response to TRH (delta TSH) was decreased in the acutely ill patients, but no difference was found between patients in remission and controls. The delta TSH was correlated to TSH but not to T3 and T4 levels in both acutely ill and control subjects. In the acutely ill group, delta TSH did not distinguish between patients with normal and abnormal DST results. Thus, abnormalities in the hypothalamic-pituitary-thyroid (HPT) axis are not correlated to abnormalities in the hypothalamic-pituitary-adrenal (HPA) axis. Moreover, delta TSH did not differentiate between melancholic (DSM-III) and nonmelancholic patients or between patients with primary and secondary depression. No correlation was found between delta TSH and CPRS scores. Patients with observable agitation greater than 0.25 points (item range 0-3) had higher levels of delta TSH than patients with lower levels. No significant correlation was found between delta TSH and seven specific symptom clusters on the CPRS. However, there was a possible relation between low delta TSH and violent suicide attempts or suicide. PRL levels did not distinguish acutely ill patients from controls. Finally, there was no significant regression between delta TSH and melatonin levels. The decrease in delta TSH seen in the acutely ill patients was too small to be of diagnostic value as a laboratory measure differentiating acutely ill and healthy subjects. The mechanism underlying the HPT alterations in acute major depressive disorder may be a desensitization of the TRH receptor in the thyrotrophs secondary to an increased endogenous TRH stimulation.