Modulation of in vitro release of beta-endorphin from the separate lobes of the rat pituitary.

The rate of in vitro release of β-endorphin immunoreactivity from the anterior lobe of rat pituitary increased in response to hypothalamic extract and lys-vasopressin. Lys-vasopressin, at a low concentration, initiated a pronounced (5–6 fold) dose-dependent, parallel increase in the release of β-endorphin and ACTH from the anterior lobe. Corticosterone (5·10^?7 M) did not influence basal but could suppress such stimulated release. These stimulants did not, however, change the rate of release from the intermediate/posterior lobe.Chromatography of incubation media showed that β-endorphin and β-lipotropin were released in parallel from the anterior lobe but only β-endorphin from intermediate/posterior lobe tissue.These findings suggest that the β-endorphin pools in anterior and intermediate lobes differ both in their mechanism of release and in the regulation of this process.     

β-Endorphinergic system involvement in the inhibitory action of clonidine on induced sodium appetite

In the present study, we investigated the degree to which β-endorphin plays a role in the alpha 2-adrenergic/imidazoline receptor agonist attenuation of salt appetite. In order to evaluate whether the inhibitory action of clonidine (an α2-adrenergic/imidazoline receptor agonist) on induced sodium intake is mediated by the β-endorphinergic system, we used a β-endorphin deficient mouse line. β-endorphin knockout (βend(-/-)), heterozygous (βend(+/-)) and wild-type (βend(+/+)) mice were submitted to acute sodium depletion by a combined treatment of furosemide and low sodium diet and, 20h later, were administered with clonidine (0.5mg/kg). An hour later, the animals were subjected to a two-bottle choice test (water/2% NaCl). The results indicate that clonidine administration during the first stage of the test exerts an equivalent inhibition on sodium intake regardless of the genotype; however, in the final stage of the test, a reversal of the inhibitory response on induced sodium appetite becomes evident in the mice lacking β-endorphin. Moreover no differences in dipsogenic response were observed between the genotypes. Considering these results and the fact that plasma half-life of clonidine at the dose administered is approximately 3h, it is possible to speculate that the inhibitory effect of clonidine on sodium appetite may be independent of β-endorphin modulation during the first stage; however, the long-lasting inhibitory effect of clonidine may be mediated by the β-endorphinergic system. This evidence supports the existence of adrenergic and β-endorphinergic system interaction in the osmoregulatory response to achieve sodium balance.     

Localization of multiple forms of ACTH- and β-endorphin-related substances in the pituitary of the reptile, Anolis carolinensis

Immunohistochemical studies on the pituitary of Anolis carolinensis detected ACTH-like, β-endorphin-like, and 16K fragment-like immunoreactivity in distinct clusters of cells in the anterior lobe; ACTH-like, αMSH-like, β-endorphin-like, and 16K fragment-like immunoreactivity was detected in all the cells of the intermediate lobe. Crude acid extracts of both lobes, when alayzed by radioimmunoassay, gave displacement curves in ACTH and β-endorphin assays which were parallel to the appropriate synthetic standard. Only extracts of the intermediate lobe gave parallel displacement curves in an αMSH radioimmunoassay. Extracts of both lobes crossreacted with antiserum to 16K fragment, but the displacement curves were not parallel to that of mouse 16K fragment standard. The levels of immunoreactive ACTH and β-endorphin in the intermediate lobe were approximately 8-fold higher than in the anterior lobe. Fractionation of anterior lobe and intermediate lobe extracts by either gel filtration on Sephadex G-75 in 10% formic acid or sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed multiple forms of ACTH-related and β-endorphin-related substances in both lobes. In the anterior lobe the major forms of immunoreactivity were, respectively, ACTH-sized and β-endorphin-sized. In the intermediate lobe the major forms of immunoreactivity were αMSH-sized, CLIP-sized, and β-endorphin-sized. In both lobes, antisera directed against ACTH and β-endorphin detected high molecular weight material with an apparent molecular weight slightly less than that of mouse pro-ACTH/endorphin; this material probably represents the putative common precursor for ACTH and β-endorphin in this species.     

Acetylated and nonacetylated forms of beta-endorphin in rat brain and pituitary

Extracts of rat posterior intermediate pituitary and extracts of brains from normal and hypophysectomized rats were separated by gel filtration chromatography and fractions were analyzed by both a classical β-endorphin radioimmunoassay and by a radioimmunoassay specific for α-N-acetyl β-endorphin. In posterior intermediate pituitary extracts, more than 90 percent of the β-endorphin-sized immunoreactive material was α-N-acetylated. In extracts of brains from normal rats, less than 2 percent of the β-endorphin-sized immunoreactive material corresponded to α-N-acetylβ-endorphin, whereas in brains from hypophysectomized animals, no α-N-acetylβ-endorphin-like material could be detected. Immunofluorescence on normal brain sections, using either affinity purified antibodies to α-N-acetylβ-endorphin or conventional β-endorphin antibodies, showed no α-N-acetylβ-endorphin immunoreactivity in β-endorphin neurons. Only in brain sections which had been acetylated $\text{in}\text{vitro}$ prior to immunostaining could α-N-acetylβ-endorphin-like material be detected in the β-endorphin neurons. These results suggest that—in contrast to the cells in the intermediate lobe of the pituitary—the β-endorphin in brain neurons is not α-N-acetylated and that the small amount of α-N-acetyl β-endorphin which can be found in extracts of brains from normal animals is probably of pituitary origin.     

Immunocytochemical colocalization of progestin receptors and β-endorphin or enkephalin in the hypothalamus of female guinea pigs

Double-label immunocytochemistry was used to determine whether estradiol-induced progestin receptors and either β-endorphin or leucine-enkephalin are colocalized in female guinea pig brain. Ovariectomized, adult guinea pigs were implanted with capsules containing estradiol-17β to induce high levels of progestin receptors, and injected intracerebroventricularly with co chicine to improve visualization of the opiate peptides. Sections through the hypothalamus and preoptic area were processed for progestin receptor, followed by β-endorphin or leucine-enkephalin immunocytochemistry. As reported previously, high concentrations of progestin receptor-immunoreactive (PR-IR) cells were found in the preoptic area (medial and periventricular portions, medial preoptic nucleus) and hypothalamus (anterior hypothalamic and arcuate nuclei, ventrolateral area). Many β-endorphin-IR cells contained PR-IR in the arcuate nucleus and its surroundings (33%) and in the dorsomedial area of the hypothalamus (64%). Scattered enkephalin-IR cells were found in the septal nucleus, medial and lateral preoptic area, bed nucleus of the stria terminalis, and the arcuate nucleus. The ventromedial nucleus of the hypothalamus and dorsolateral magnocellular nucleus, respectively, contained moderate and heavy concentrations of enkephalin-IR cells. Although some of these areas also contained PR-IR, enkephalin-IR was colocalized consistently with PR-IR only in a small number of cells in the arcuate nucleus and ventromedial/ventrolateral area of the hypothalamus. These data, taken together with earlier observations that virtually all cells containing estradiol-induced PR-IR also contain estrogen receptor-IR, provide neuroanatomical evidence that hypothalamic actions of progesterone and estradiol may be mediated by β-endorphin and/or enkephalin.     

Lesion of central part of the dorsomedial nucleus alters vasopressin but not corticotropin releasing hormone mRNA levels in rat hypothalamic paraventricular nucleus

Functional significance of neural projections from the hypothalamic dorsomedial nucleus (DMN) to the paraventricular nucleus (PVN) was investigated using surgical lesion of the central part of the DMN. Under basal conditions, DMN lesion resulted in a decrease in magnocellular vasopressin (AVP) mRNA levels in the PVN, rise in pituitary proopiomelancortin (POMC) mRNA concentrations and elevated plasma corticosterone levels. Corticotropin-releasing hormone (CRH) mRNA levels remained unaffected. In sham operated animals, osmotic stress induced by hypertonic saline injection failed to modify AVP mRNA, but increased CRH and POMC mRNA levels and peripheral hormone release. The rise in CRH mRNA levels after osmotic stress was potentiated in DMN lesioned animals. Thus, the DMN participates in the control of hypothalamic peptide gene expression and pituitary adrenocorticotropic function.     

Potentiation of vasopressin analgesia in rats treated neonatally with monosodium glutamate

The analgesic response elicited by central administration of arginine vasopressin (AVP) appears to be dependent upon the integrity of the hypothalamic paraventricular nucleus (PVN), since lesions placed in the PVN eliminate AVP analgesia. A projection to the zona externa of the median eminence constitutes one of the VP-containing efferents of the PVN. Neonatal treatment with monosodium glutamate (MSG) destroys perikarya of the arcuate nucleus and median eminence. The present study examined whether AVP analgesia was affected in the MSG-treated rat and whether these alterations were accompanied by specific changes in VP immunoreactivity in the zona externa of the median eminence. Female rats, neonatally treated with either MSG or a saline control, were tested as adults on the tail-flick test following intracerebroventricular injections of 0, 75, 150 and 500 ng doses of AVP. After testing, selected animals were prepared for AVP and oxytocin immunocytochemistry of the median eminence. Significant potentiations in the magnitude of AVP analgesia were observed in MSG-treated rats. AVP and oxytocin immunoreactivity in the zona interna and oxytocin immunoreactivity in the zona externa of the median eminence were similar in MSG-treated and control rats. In contrast, AVP immunoreactivity in the zona externa of the median eminence was markedly reduced in the MSG-treated rat. These data suggest that VP analgesia may normally be inhibited by those medial-basal hypothalamic neurons affected by neonatal MSG treatment.     

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.     

Elimination of vasopressin analgesia following lesions placed in the rat hypothalamic paraventricular nucleus

Pain thresholds are increased following central administration of arginine vasopressin (AVP), an effect which appears not to be mediated through opioid analgesic processes. In addition to magnocellular projections to the posterior lobe of the pituitary gland and parvocellular projections to the zona externa of the median eminence, the paraventricular nucleus (PVN) of the hypothalamus contains VP parvocellular neurons which also project to extrahypothalamic structures involved in pain inhibition. The present study examined whether AVP analgesia as measured by the tail-flick test was altered in animals with lesions placed in the PVN at either 7 or 35 days after surgery. VP levels in the pons-medulla and the lumbo-sacral spinal cord were measured by radioimmunoassay, as well as VP-like immunoreactivity in the PVN and spinal cord with immunocytochemistry. Lesions placed in the PVN eliminated AVP analgesia on the tail-flick test at both 7 and 35 days after surgery, and decreased radioimmunoassayable VP by 59% in the lumbo-sacral spinal cord and 36% in the pons-medulla. The extent of the lesions ranged from complete destruction of the PVN to partial sparing of ventro-medial PVN cells with VP-like immunoreactivity. These data indicate that the PVN is a critical structure for the integrity of AVP analgesia.     

Activation of Brainstem Pro-opiomelanocortin Neurons Produces Opioidergic Analgesia,Bradycardia and Bradypnoea

Opioids are widely used medicinally as analgesics and abused for hedonic effects, actions that are each complicated by substantial risks such as cardiorespiratory depression. These drugs mimic peptides such as β-endorphin, which has a key role in endogenous analgesia. The β-endorphin in the central nervous system originates from pro-opiomelanocortin (POMC) neurons in the arcuate nucleus and nucleus of the solitary tract (NTS). Relatively little is known about the NTS_POMC neurons but their position within the sensory nucleus of the vagus led us to test the hypothesis that they play a role in modulation of cardiorespiratory and nociceptive control. The NTS_POMC neurons were targeted using viral vectors in a POMC-Cre mouse line to express either opto-genetic (channelrhodopsin-2) or chemo-genetic (Pharmacologically Selective Actuator Modules). Opto-genetic activation of the NTS_POMC neurons in the working heart brainstem preparation (n = 21) evoked a reliable, titratable and time-locked respiratory inhibition (120% increase in inter-breath interval) with a bradycardia (125±26 beats per minute) and augmented respiratory sinus arrhythmia (58% increase). Chemo-genetic activation of NTS_POMC neurons in vivo was anti-nociceptive in the tail flick assay (latency increased by 126±65%, p<0.001; n = 8). All effects of NTS_POMC activation were blocked by systemic naloxone (opioid antagonist) but not by SHU9119 (melanocortin receptor antagonist). The NTS_POMC neurons were found to project to key brainstem structures involved in cardiorespiratory control (nucleus ambiguus and ventral respiratory group) and endogenous analgesia (periaqueductal gray and midline raphe). Thus the NTS_POMC neurons may be capable of tuning behaviour by an opioidergic modulation of nociceptive, respiratory and cardiac control.     

beta-Endorphin concentration in the brain of intact and hypophysectomized rats.

The brain concentration and distribution of β-endorphin immunoreactivity in the brain have been studied in intact and hypophysectomized rats. The results obtained with different methods for killing the animals and extracting β-endorphin are compared. Different methodologies of killing the rat and extracting the brain yield concentrations of β-endorphin which vary ten fold. Consistently the highest concentrations of β-endorphin have been found in the hypothalamus, midbrain and hindbrain. After hypophysectomy major reduction of β-endorphin concentration in the brain was observed.     

Comparison of β-endorphin immunoreactivity in hypothalamic and brainstem nuclei of normotensive and age-matched hypertensive rat strains

The concentration of β-endorphin immunoreactivity was determined in 21 hypothalamic and brainstem nuclei of Sprague-Dawley (SD) and 6 and 14 week Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SH) rats. The concentrations of β-endorphin immunoreactivity were greater in the hypothalamic nuclei than in the brainstem nuclei approximately by a factor of 5. A significant strain-age interaction was observed in the β-endorphin immunoreactivity levels in the anterior hypothalamic area, paragigantocellular reticular nucleus and locus coeruleus of age-matched SH and WKY rats in that immunoreactivity levels fell in the age period studied (6–14 weeks) in WKY rats and rose in SH rats. These biochemical differences are related in time to a growth period during which there are large increases in blood pressure in the SH rat and may thus have a pathogenetic significance.     

Hypergravity and opioid-mediated pain suppression in rats

It is known that pain suppression in animals is induced by certain environmental stimulus. However, little is known about the effects of gravitational alteration on the nociceptive responses in rats. A recent study indicated that Fos protein expression was strongly induced in the vestibular-related brainstem regions of rats that were exposed to 2 G hypergravity (Gustave Dit Duflo et al., 2000). A number of studies indicate that Fos expression is induced in the brain by various kinds of stress. We showed that either long-term exposure or short-term exposure to 2 G hypergravity elevated the nociceptive threshold in the rat skin surfaces, in concomitant with Fos induction in the hypothalamus including the arcuate nucleus and paraventricular nucleus (Kumei et al., 2000). We have examined the possible involvement of beta-endorphin, an endogenous opioid, in the hypergravity-induced analgesic effects on rats and its counteraction by naloxone, an opioid receptor antagonist.     

Localization of γ-melanocyte stimulating hormone (γMSH) immunoreactivity in rat brain and pituitary

γ-Melanocyte stimulating hormone (γMSH) is a possibly biologically active material discovered in the cryptic N-terminus of the pro-opiocortin precursor by recombinant DNA analysis of bovine pituitary mRNA. Well-characterized antisera to synthetic bovine γ-3MSH (γ₃MSH) were used to localize immunoreactive sites in sections of formaldehyde-fixed rat brain and pituitary by the indirect immunoperoxidase technique. Specificity for staining was established by absorption with the synthetic antigen peptides or their fragments; staining was not blocked by absorption with synthetic replicates of other natural peptides that contain redundant amino acid sequences, with those of γMSH such as corticotropin or β-MSH. The general patterns of staining within adenohypophysis, intermediate lobe, and central nervous system closely followed the previously described patterns of β-endorphin immunoreactivity. Corticotrophs, all intermediate lobe cells and neuronal perikarya in the ventro-basal hypothalamus exhibited immunoreactivity for γ₃MSH as they do for β-endorphin. Furthermore, the general distribution of immunoreactive nerve fibers and terminals within the diencephalon and pons was quite similar to endorphin immunoreactivity patterns as well. In series of alternating sections, prepared for either γ₃MSH β-endorphin immunoreactivity, the same specific terminal fields were found to exhibit very similarly shaped varicose axons and probable terminal bouton configurations. However, the density of the innervation by fibers exhibiting immunoreactivity for the two peptides varied among the common target areas. Furthermore, the perikarya exhibiting γ₃MSH immunoreactivity were fewer in number, smaller in size, and more medially clustered than those exhibiting immunoreactivity for β-endorphin. These results demonstrate that γ₃MSH also occurs in rat brain and in pituitary cells which were already known to contain endorphin immunoreactivity. However, γ₃MSH-immunoreactive neurones may not be coexistent with all endorphin-immunoreactive neurons, and these cells project with varying intensity to common target fields. Such observations are in agreement with the proposal of different processing of a common precursor in different cells.     

Effects of ventromedial nucleus lesions on estradiol induced ovulation in the rat

Small bilateral stereotaxic lesions were made in the hypothalamic ventromedial nucleus (SVMN) to determine: (i) whether estrogen would restore early receptivity in unreceptive SVMN lesioned female rats and (ii) whether SVMN lesions would suppress estrogen induced ovulation in the rat. SVMN lesions were shown to completely suppress spontaneous early receptivity and seriously impair estrous receptivity in 5-day cyclic female Wistar rats. A loss of early receptivity in response to 10 μg estradiol benzoate (EB) was also observed in SVMN lesioned females, in comparison to unoperated, sham VMN and dorsomedial nucleus (DMN) lesioned animals. Isolated SVMN lesioned females exhibited a weak ovulatory response to 10 μg EB, but, where shown to be unreceptive prior to estrogen injection, they never ovulated. On the contrary, ovulation occured in about 50% of cases in isolated unoperated and in sham VMN and DMN lesioned females following estrogen administration. The mechanisms whereby EB brought about precocious ovulation in 5-day cyclic female rats were therefore concluded to be dependent on VMN functional integrity and thereby on the degree of early sexual receptivity in the rat.     

Demonstration of the tuberoinfundibular tract of the cat

Summary The distribution of dopaminergic nerve cells in the cat hypothalamus, particularly in the arcuate and periventricular nuclei, and the projections of their axons were studied by fluorescence and electron microscopy after electrothermic coagulation. The majority of these perikarya were located in the arcuate nucleus and the periventricular nucleus dorsocaudal to the optic chiasma. Large lesions caused a wide and diffuse depletion of dopamine fluorescence within the external layer; small lesions caused ipsilateral partial depletion of the dopamine fluorescence. Electron microscopic observations in animals with a lesioned arcuate nucleus revealed that in the external layer degenerating nerve terminals are engulfed by glial processes. In some cases nerve fibers had entirely disappeared and a heavy reactive proliferation of glial processes was observed. Persistence of the form of the median eminence in spite of the extensive degeneration of its nervous elements is considered to depend upon this glial proliferation.Dedicated to Professor W. Bargmann in honour of his 70th birthday     

Immunohistochemical localization of beta-LPH in the human hypothalamus.

In order to establish the presence of β-LPH and to clearly identify the nervous structures containing β-LPH in the human hypothalamus, an immunohistochemical localization of β-LPH was performed in this tissue. The immunohistochemical technique involved use of a specific antiserum to human β-LPH and the peroxidase-antiperoxidase complex. Immunostained neuronal cell bodies were observed in the arcuate nucleus whereas β-LPH-positive nervous fibers could be detected in a large area extending rostro-caudally from the anterior part of the paraventricular nucleus up to the mammillary bodies. Staining was completely abolished by previous immunoabsorption with β-LPH while β-endorphin and ovine γ-LPH_1–47 only partially prevented immunostaining. Although it cannot be excluded that the precursor 31K molecule, β-LPH_1–58 and/or β-endorphin are detected by the immunostaining, it is likely that β-LPH is at least partly responsible for the positive reaction.     

Serotonergic mechanism in the control of β-endorphin and acth release in male rats

The role of the serotonergic mechanism in the regulation of β-endorphin (β-EP) and adrenocorticotropin (ACTH)-like immunoreactivity in plasma was investigated. Increases in β-EP and ACTH-LI produced by quipazine maleate (QPZ), a serotonergic agonist, 1 hr after injection could be completely prevented by the serotonin (5-HT) antagonist, cinanserin (CIN), which when injected alone, decreased basal plasma concentrations of both β-EP-LI and ACTH-LI. Concurrent injections of L-5-HTP with the 5-HT reuptake inhibitor, fluoxetine, produced an additive increase in plasma β-EP-LI 1 hr after injection. Injection of the 5-HT antagonist, cyproheptadine, significantly decreased plasma β-EP-LI. Stress by immobilization for 30 min or exposing the rats to 40° ± 1°C for 30 min produced an approximate 4-fold increase in plasma β-EP-LI and ACTH-LI, which was potentiated by I.P. injections of fluoxetine. Furthermore, the stress induced increases in plasma concentrations of β-EP-LI and ACTH-LI were significantly reduced by the serotonin antagonists metergoline and cinanserin. These results suggest that 5-HT is a potent stimulator of both β-EP and ACTH release and the increase in plasma concentrations of ACTH and β-EP induced by stress are probably mediated, at least in part, by central serotonergic mechanisms.     

Craniofacial alterations following electrolytic lesions of the trigeminal motor nucleus in actively growing rats

The objective of this study was to define further the role of the trigeminal motor nucleus (TMNu) in the postnatal ontogeny of the mammalian craniofacial skeleton. To that end, 42 male Sprague-Dawley rats underwent stereotaxic surgery at 40 days of age; 21 received small electrolytic lesions to their left-side TMNu (lesioned group) while 21 had TMNu stimulation with no actual electrolytic lesion produced (sham group). Seven rats from each group were killed at 28, 56, and 84 days postoperative to analyze trigeminal motoneuron (TMNe) count, masticatory muscle weight, and osteological growth vector data. At all three time periods, lesioned animals showed significant differences 1) between the surgery and nonsurgery sides, and 2) from sham animals. However, sham animals also demonstrated significant between-side differences for medial pterygoid muscle weight (56 days), mandibular height (28 and 56 days), and mandibular length data (84 days); these data suggested that even relatively slight damage to TMNe can create morphological changes within the craniofacial complex. Snout deviation in a lesioned rat towards the opposite side from all other lesioned animals was correlated with unique damage to its pontine reticular formation; this suggested that the observed morphological alterations of the craniofacial complex may have been due not only to TMNu damage, but also to changed expressions of the masticatory central pattern generator (CPG). Morphological alterations of the craniofacial skeleton resulting from lesions to the TMNu were likely due to changed neuromuscular activity patterns of the masticatory muscles and their biomechanical effects upon bone.     

Content of beta-LPH and its fragments (including endorphins) in anterior and intermediate lobes of the bovine pituitary gland

Radioimmunoassays (RIAs) specific for β-LPH_1–47, β-endorphin, α-MSH and β-MSH have been used to identify immunoreactive components in acid extracts from anterior and intermediate lobes of bovine pituitary gland after separation by chromatography on Sephadex G-50. When components in extracts of both lobes, eluting at the same position, were measured with the β-endorphin and β-LPH_1–47 RIA systems, marked quantitative differences were seen. The main components reacting with the β-LPH_1–47 system in anterior pituitary extract co-migrated with β-LPH and γ-LPH while in the intermediate lobe, the main immunoreactive component eluted at a position slightly later than β-endorphin. When the β-endorphin RIA system was used, relatively low amounts of immunoreactive material co-migrating with β-endorphin were seen in the anterior lobe extract while a highly predominant peak eluting at a position slightly later than β-endorphin was observed in intermediate lobe extract. Some β-MSH was seen in the intermediate lobe. These date indicate that the processing of β-LPH is markedly different in the anterior and intermediate bovine pituitary lobes: β-endorphin immunoreactive material predominates in the intermediate lobe whereas β-LPH and γ-LPH predominate in the anterior lobe.