Opiate receptor subtype regulation of CRF-41 release from rat hypothalamus in vitro

We have previously shown that the release of corticotrophin-releasing factor 41 (CRF-41) induced by a variety of neurotransmitters and depolarizing agents from the rat hypothalamus in vitro is inhibited by morphine. In order to further characterize the opiate receptors mediating this inhibitory action, we have now investigated the effects of a variety of opioid compounds with relatively high selectivity for mu-, kappa- and delta-opiate receptors on K(+)-stimulated CRF-41 release. The selective mu-opioid receptor agonist 202-250 inhibited K(+)-evoked CRF-41 release in a dose-dependent manner with a maximum inhibition of approximately 60% at 10(-5) M (p less than 0.01), as did the kappa-selective agonists PD-117,302 and U-50,488, with a similar plateau in response of approximately 40% inhibition at 10(-6) M (p less than 0.05). The effects of these agonists were specifically reversed by the mu- and kappa-receptor antagonists naloxone and MR2266, respectively, while the specific delta-receptor antagonist ICI 154,129 was ineffective. Both naloxone and MR2266 slightly but significantly increased the basal release of CRF-41. The delta-agonist D-Pen2,5-enkephalin was without significant effect in the same dose range. These data suggest that both mu- and kappa-receptors, but not delta-receptors, mediate the inhibitory effect of opiates on stimulated CRF-41 release from the rat hypothalamus.     

Localization and actions of cholecystokinin in the rat pituitary neurointermediate lobe

Rat pituitary neural lobe contained high concentrations of cholecystokinin-like immunoreactivity (CCK-LI). Section of the pituitary stalk resulted in loss of CCK-LI, and both lactation and replacement of drinking water with 2% saline resulted in marked depletion of CCK-LI. Rats with congenital diabetes insipidus (Brattleboro strain) had a 73% reduction in CCK-LI below the levels of hooded Long-Evans controls, where as levels in the brain were unchanged. Release of CCK-LI, labeled dopamine, and gamma-amino butyric acid in response to potassium depolarization was studied. There was a low fractional release of CCK-LI. Addition of sulfated CCK-8 (CCK-8s) to the medium enhanced the calcium-dependent potassium-stimulated release of dopamine, but basal release was unaffected. gamma-Amino butyric acid release was only poorly calcium dependent and not effected by extracellular CCK-8s. Vasopressin and oxytocin release were stimulated by electrical stimulation of the pituitary stalk, and were unaffected by the addition of CCK-8s to the medium. In vivo, however, the injection of 5 micrograms CCK-8s into the third ventricle resulted in increased plasma vasopressin concentrations.     

Estrogen lowers immunoreactive beta-endorphin in the neurointermediate lobe of the rat pituitary

The effects of ovariectomy and sex steroids on tissue levels of immunoreactive beta-endorphin (ir-beta EP) were examined. Adult female Sprague-Dawley rats were sham operated, ovariectomized (OVX), adrenalectomized (ADRX), or ADRX-OVX. Animals were given estradiol (E2) or vehicle (oil) by six daily im injections, and tissue levels of immunoreactive beta-endorphin (ir-beta EP) determined. In OVX animals, neurointermediate lobe (NIL) content of ir-beta EP was double that in controls; plasma ir-beta EP was modestly but significantly raised, but levels in the anterior pituitary (AP) remained unaltered. The effects of ovariectomy on NIL and plasma ir-beta EP were reversed by E2 in a dose-related manner. ADRX-OVX animals showed elevations of AP and NIL ir-beta EP to levels double those in SHAM, and plasma levels 5 times higher. E2 administration to ADRX-OVX rats normalized ir-beta EP in NIL, but not that in AP nor plasma. These findings suggest that the ovary may exert a specific tonic influence on NIL ir-beta EP in the rat, and that this inhibition appears to be mediated, at least in part, through ovarian estrogen.     

Inhibition by prostaglandin E2 of the release of vasopressin and beta-endorphin from rat pituitary neurointermediate lobe or medial basal hypothalamus in vitro

The present study was performed to examine the effect of the cyclo-oxygenase inhibitor, indomethacin, and that of various prostaglandins on the release of vasopressin and beta-endorphin-like immunoreactivity (beta-EI) from the rat neurointermediate lobe of the hypophysis, which was superfused in vitro. Indomethacin (2.8 and 28 mumol/l) changed neither basal secretion of vasopressin nor that evoked by electrical stimulation, whereas the resting release of beta-EI was enhanced by indomethacin (28 mumol/l). Prostaglandin (PG) E2 did not influence resting release of vasopressin but markedly inhibited (by about 50%) electrically induced release of vasopressin (least effective concentration: 300 nmol/l) as well as spontaneous secretion of beta-EI (least effective concentration: 100 nmol/l) in the presence of indomethacin (28 mumol/l). Prostaglandin F2 alpha (5 mumol/l) also inhibited the evoked release of vasopressin, whereas PGD2 (5 mumol/l) did not. Prostaglandin F2 alpha (5 mumol/l), D2 and I2 (1.5 mumol/l each) produced no effects on beta-EI release. As observed in the neurohypophysis, PGE2 inhibited the electrically induced release of vasopressin from the medial basal hypothalamus in vitro. We conclude that prostaglandins (especially PGE2) can inhibit (1) the stimulated release of vasopressin when acting on vasopressin-containing nerve terminals of either neurosecretory system (neurohypophysis, median eminence region), and (2) the secretion of beta-EI and, as can be inferred, alpha-MSH, by a direct action on intermediate lobe cells.     

On the presence of a MSH-release inhibiting system in the rat neurointermediate lobe.

Rat neurointermediate lobes were superfused in vitro and showed a stable secretion rate of MSH after 30 min. MSH secretion from lobes of untreated rats was reversibly inhibited during superfusion with medium containing 45 mM K+. This inhibition could not be induced using lobes from median eminence lesioned or reserpinized rats. Superfusion with 45 mM K+ also induced release of dopamine from lobes preincubated with the labeled transmitter. It is concluded that the MSH-release inhibiting system present in the rat neurointermediate lobe may be identical to the dopaminergic arcuate-intermediate lobe system.     

Biosynthesis of pro-opiomelanocortin-derived peptides in the mouse neurointermediate lobe

This report concerns biosynthetic studies conducted with neurointermediate lobes of the mouse pituitary gland. High performance liquid chromatography was used to resolve newly synthesized peptides after in-vitro incubation of lobes with radioactive amino acids. Among the newly synthesized peptides identified were alpha-MSH, des-N alpha-acetyl-alpha-MSH, two forms of corticotrophin-like intermediate lobe peptide and beta-endorphin. The biosynthesis of a glycosylated gamma 3-MSH-like peptide was also demonstrated. While no newly synthesized beta-MSH could be identified, a peptide designated gamma-lipotrophin was found. Pulse-chase analysis revealed that the major biosynthetic pathway leading to the production of alpha-MSH involved the acetylation of the des-acetyl form of this peptide. Furthermore, it was evident that newly synthesized beta-endorphin was largely converted to modified forms of this peptide; most of the terminal product was probably N-acetylated endorphin.     

Central GABAergic innervation of neurointermediate pituitary lobe: biochemical and immunocytochemical study in the rat.

Activity of glutamic acid decarboxylase GluDCase, the biosynthetic enzyme of gamma-aminobutyric acid (GABA) was measured in low-speed homogenate supernatant of the neural and intermediate (neurointermediate) lobe (28--30 pmol of CO2 per microgram of protein per hr) and of the anterior lobe (2--4 pmol of CO2 per microgram of protein per hr). In the neurointermediate lobe, stalk transection reduced the GluDCase activity by more than 95%. By using an antiserum to rat brain GluDCase and the unlabeled antibody--peroxidase method of Sternberger, GluDCase immunoreactivity was localized in many terminals within the neurointermediate lobe of the hypophysis. In pars intermedia, immunoreactive terminals occurred in apposition to secretory cells and to glial cells and were near nonimmunoreactive axonal profiles; in pars neuralis they were apposed to pituicytes and to unlabeled axons including the neurosecretory terminals and were along fenestrated portal capillaries. GluDCase immunoreactive axons terminals exhibited diverse morphological features and would not have been identified as a distinct population without the GluDCase antiserum. No GluDCase-immunoreactivity was found in the anterior pituitary lobe. Stalk transection abolished GluDCase immunoreactivity in the neurointermediate lobe. These data provide biochemical and morphological evidence for a central GABAergic innervation of neural and intermediate lobes of the hypophysis.     

Characterization of pro-opiocortin-converting activity in purified secretory granules from rat pituitary neurointermediate lobe.

Lysates of secretory granules from rat pituitary neurointermediate lobes were incubated with [3H]arginine- or [3H]phenylalanine-labeled toad pro-opiocortin. The processed products formed were identified by immunoprecipitation with adrenocorticotropin (ACTH) and endorphin antisera and by migration behavior on acid/urea/polyacrylamide gels. Pro-opiocortin was cleaved by the proteolytic activity in the secretory granule fraction to approximately 21,000 Mr ACTH, approximately 13,000 Mr ACTH, alpha-melanotropin, 16,000 Mr NH2-terminal glycopeptide, beta-lipotropin, and an endorphin-related peptide. Characterization of this pro-opiocortin-converting activity shows that it (i) is present in membrane and soluble fractions of the granule lysates, (ii) has a pH optimum of 5.0, (iii) appears to cleave at pairs of basic amino acid residues in the precursor, and (iv) is inhibited by leupeptin, pepstatin A, and p-chloromercuribenzoate but not diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, or EDTA. These inhibitor studies suggest that the converting-enzyme activity is due to an acid thiol, arginyl protease, distinct from any known cathepsin B-like activity.     

Hyperprolactinemia exerts a negative effect on the beta-endorphin content of the rat neurointermediate pituitary lobe

Implantation of the PRL, ACTH, beta-endorphin (beta-EP), and beta-lipotropin (beta-LPH)-secreting transplantable rat pituitary tumor 7315a resulted in a suppression of the PRL and the ACTH content of the anterior pituitary gland and also of the beta-EP/beta-LPH content of the neurointermediate (NI) lobe. Treatment with bromocriptine further diminished the anterior lobe PRL content, whereas haloperidol partially inhibited this tumor-mediated diminution. The administration of these drugs did not influence the suppressed ACTH content of the anterior pituitary lobe. The diminished beta-EP/beta-LPH content of the NI lobe of tumor-bearing rats became completely normal after treatment with haloperidol, whereas bromocriptine administration further diminished the NI lobe beta-EP/beta-LPH content. There was a close correlation between the anterior pituitary lobe PRL content and the beta-EP/beta-LPH content of the NI lobe in all four groups of rats taken together (including nontumor-bearing controls, control tumor rats, and tumor rats treated with bromocriptine or haloperidol; P less than 0.01). Implantation of the pure PRL-secreting pituitary tumor 7315b resulted in hyperprolactinemia and a suppression of the PRL content of the anterior lobe and the beta-EP/beta-LPH content of the NI lobe, without affecting the ACTH content of the anterior pituitary lobe. There was a negative correlation between the level of the circulating PRL concentration and the beta-EP/beta-LPH content of the NI lobe. These results suggest a possible relationship between the synthesis of PRL by the anterior pituitary lactotroph and of the hormones of the NI lobe. The level of the circulating PRL concentration may play, directly or indirectly, a role in the regulation of both systems.     

Opiate receptor: autoradiographic localization in rat brain.

Opiate receptor sites in rat brain can be labeled in vivo by [3H]diprenorphine, a potent opiate antagoinst. Using techniques to minimize diffusion in fresh, frozen, unfixed brain, we have localized [3H]diprenorphine by autoradiography to visualize the distribution of opiate receptors. Silver grains indicative of the binding of labeled [3H]diprenorphine are discretely localized in numerous areas of the brain with very high densities in the locus coeruleus, the substantia gelatinosa of the spinal cord, and in clusters within the caudate-putamen, amygdala, and parts of the periventricular gray matter.     

gamma-Aminobutyric acid inhibits beta-endorphin secretion from the anterior pituitary but not the neurointermediate lobe in the rat

We have evaluated the role of gamma-aminobutyric acid (GABA) in the neuroendocrine control of beta-endorphin (beta-EP) secretion in the rat. Plasma beta-EP and beta-lipotropin (beta-LPH) levels and beta-EP-like immunoreactivity (beta-EPLI) in the anterior pituitary (AP) and neurointermediate lobe (NIL) were determined after administration of GABA antagonist or agonist drugs in male rats under resting conditions or after potent physical stresses. Bicuculline (0.1-0.8 mg/kg BW ip), a GABA receptor antagonist, induced a dose-related rise in plasma beta-EP and beta-LPH levels and a concomitant decrease in beta-EPLI concentrations in the AP but not in the NIL. Muscimol, a potent GABA-mimetic drug, did not alter baseline plasma beta-EP and beta-LPH levels, whether given systemically (1.0-2.0 mg/kg BW ip) or intracerebroventricularly (500 ng/kg BW), but prevented the effect of bicuculline on plasma and AP-beta-EP and beta-LPH concentrations. Administration of foot shock or restraint stress induced a clear-cut activation of the AP-related beta-EP secretion, an effect that was prevented by pretreatment with muscimol. Together, these data show that GABA-ergic mechanisms, probably operating at a central nervous system level, exert an inhibitory action on resting and stimulated beta-EP and beta-LPH secretion. Since no alterations in beta-EP concentrations in the NIL occurred after manipulations with GABA-ergic drugs or stress, and these were detected only in the AP, an interaction between GABA-ergic neurons and CRF neurons is the most likely explanation for the reported findings.     

Thyrotrophin-releasing hormone stimulates polyphosphoinositide metabolism in the frog neurointermediate lobe

Previous studies have demonstrated that TRH is a potent stimulator of alpha-MSH secretion from frog pituitary melanotrophs. In order to determine the intracellular events responsible for TRH-evoked alpha-MSH release, we have investigated the effect of TRH on polyphosphoinositide breakdown in frog pars intermedia. Neurointermediate lobes were labelled to isotopic equilibrium with myo-[3H]inositol. TRH stimulated the rate of incorporation of [3H]inositol into the phospholipid fraction. The effect of TRH was concentration-dependent; half-maximal stimulation of alpha-MSH release and inositol incorporation occurred at 12 and 28 nmol TRH/l respectively. In prelabelled neurointermediate lobes, lithium (10 mmol/l) enhanced the radioactivity in inositol monophosphate, bisphosphate (IP2) and trisphosphate (IP3). LiCl (10 mmol/l) induced a 38% inhibition of alpha-MSH release from perifused neurointermediate lobes but did not impair TRH-induced alpha-MSH secretion. In the presence of LiCl, TRH (1 mumol/l) induced a transient increase of the radioactivity in IP3, which was evident by 30 s and maximal by 1 min (+100%). TRH treatment also increased the radioactivity in IP2, which reached a plateau after 5 min (+100%). The increase in radioactivity in IP3 induced by TRH was closely paralleled by a rapid loss of [3H]phosphatidylinositol bisphosphate (PIP2), which was maximal by 1 min (-70%). These results indicate that, in frog pars intermedia, TRH-evoked alpha-MSH secretion is coupled to breakdown of PIP2. The data suggest that, in amphibian melanotrophs, as previously shown in GH3 tumour cells and in rat pituitary mammotrophs, TRH causes rapid stimulation of polyphosphoinositide-hydrolysing phospholipase C.     

Opiate receptor ontogeny in the rat medial preoptic area is androgen-dependent

The relationship of opiate receptors in the medial preoptic area of the hypothalamus (MPOA) to the gonadal steroid hormone environment during development was assessed using regional densitometric analysis of [3H]naloxone binding in autoradiographs prepared using brain sections from 5-day-old male and female rats treated postnatally either with tamoxifen (0.5 mg/kg), flutamide (20 mg/kg), dihydrotestosterone (DHT; 12.5 mg/kg), or sesame oil vehicle. Tamoxifen, a specific estrogen receptor antagonist, did not alter MPOA binding density in either males or females. Flutamide, a specific androgen receptor antagonist, and DHT, a nonaromatizable androgenic compound, altered the MPOA binding density in males and females, respectively. No treatment altered the binding density in several other brain regions. The results suggest that androgen, not estrogen, regulates the differentiation of MPOA opiate receptors. Since the neuronal development in the region is thought to be mediated by estrogen, both hormones probably act concurrently to affect the ontogeny of different parameters in the same brain region.     

Effects of ethanol treatment and withdrawal on biosynthesis and processing of proopiomelanocortin by the rat neurointermediate lobe

Male Sprague Dawley rats were chronically pair-fed with liquid diets containing 6.5% (vol/vol) ethanol, or equicaloric sucrose. After 21 days the ethanol-containing diet was discontinued and both groups were fed the sucrose diet. Groups of animals were killed on day 22 (0 day of ethanol withdrawal) and 1, 3, 8, and 15 days after ethanol withdrawal and the neurointermediate lobes (NILs) were removed and incubated with [3H]phenylalanine for 3 h. Chronic ethanol treatment induced an increase in the biosynthesis and release of beta-endorphin-like peptides by the rat NIL. After ethanol withdrawal the beta-endorphin-like immunoreactivity content in the NIL and the in vitro release of immunoreactive beta-endorphin (beta EP) by the NIL were significantly lower than in the controls on the first day, whereas no significant difference was found on days 3, 8, and 15 after ethanol withdrawal. The in vitro incorporation of [3H]phenylalanine into POMC, beta-lipotropin and beta EP was found to be higher in the ethanol-treated animals than in the controls on days 0, 1, and 3 after ethanol withdrawal, with no significant difference on days 8 and 15 after ethanol withdrawal. Furthermore, in both the ethanol-treated animals and their pair-fed controls the rate of incorporation of [3H]phenylalanine into total proteins, POMC, beta-lipotropin, and beta EP was significantly higher on days 8 and 15 after ethanol withdrawal than on the day of ethanol withdrawal (day 0), suggesting the implication of a nutritional factor. HPLC analysis of the beta EP peptides indicated that the percentage of acetylated forms of beta EP was higher in the NIL of the alcohol-treated animals, especially on days 8 and 15 after ethanol withdrawal. This observation suggests that though the rates of biosynthesis and release of beta EP-related peptides have returned to normal at 15 days after ethanol treatment, the activity of the enzyme responsible for the acetylation of beta EP remained elevated.     

Differential orexigenic effects of hexarelin and its analogs in the rat hypothalamus: indication for multiple growth hormone secretagogue receptor subtypes

We have previously reported that hexarelin and some of its analogs, including EP 50885, stimulated GH secretion and feeding after systemic administration in the rat, whereas EP 40904 selectively stimulated food intake and EP 40737 only GH release. The precise mechanism of growth hormone-releasing peptides (GHRPs) actions is still unclear, but the integrity of the arcuate nucleus of the hypothalamus (ARC) appears crucial for their endocrine effects. To better characterize the site(s) and mechanisms(s) of the orexigenic action of GHRPs, we have investigated their effects after infusion into the arcuate, paraventricular, ventromedial and medial preoptic areas of the hypothalamus. Food intake was measured for 60 min following injection of the test compound (2 microg/rat). Hexarelin, EP 40904 and EP 50885 had significant orexigenic effects after injection into the ARC. A specific NPY antagonist significantly inhibited the effect of hexarelin, whereas a GHRH antagonist was ineffective. In the paraventricular nucleus, only EP 50885 stimulated feeding, whereas all peptides were ineffective in the ventromedial nucleus and medial preoptic area. Taken altogether, these results demonstrate that GHRPs are endowed with site-specific orexigenic actions and that endogenous NPY, but not GHRH, mediates these effects. The additional orexigenic action of EP 50885 in the paraventricular nucleus suggests the existence of a GHRP receptor subtype different from the already cloned one.     

The effects of cholera toxin in the release of beta-endorphin from the dispersed cells of the rat neurointermediate lobe

The intermediate lobe cells of pituitary gland synthesize and secrete bioactive peptides derived from proopiomelanocortin. In the present study, we investigated the effects of cholera toxin on the release of beta-endorphin (beta-Ep) from dispersed-intermediate lobe cells of rats. Cholera toxin added into culture medium, enhanced the intracellular accumulation of adenosine 3', 5'-monophosphate (cAMP) and the release of beta-endorphin like immunoreactive substance (beta-END-LIS). A positive dose-response relationship existed between the concentration of cholera toxin and the release of beta-END-LIS or the accumulation of cAMP. Maximal response was obtained with approximately 3 X 10(-10) M (in beta-END-LIS release) and 1 X 10(-9) M (in cAMP accumulation) concentration of cholera toxin. Incubation with cholera toxin (3 X 10(-8) M) resulted in a significant rise of cAMP accumulation after 20-30 min, and a 2-2.5 fold increase of beta-END-LIS release occurred after 60 min in comparison with nontreated cells. cAMP analog and phosphodiesterase inhibitor also increased the beta-END-LIS release). These results suggested the close relationship between cAMP accumulation and its biological effect (i.e. beta-END-LIS release).     

Diethylstilbesterol- and pregnancy-induced changes in rat neurointermediate lobe oxytocin, arginine vasopressin, methionine enkephalin and dynorphin.

The neurointermediate lobe of the pituitary (NIL) contains the opioid peptides methionine enkephalin (MENK) and dynorphin 1-8 (DYN) in addition to oxytocin (OT) and vasopressin (AVP). If the opioids have a functional role, such as feedback control on OT or AVP release, the content or release of the opioids might be expected to change under conditions in which OT or AVP change. This expectation was examined by studying the synthesis, storage and release of the 4 peptides under conditions in which OT and AVP dynamics are known to be altered. Diestrus, diethylstilbesterol(DES)-treated, and day 22 pregnant rats were decapitated, the hypothalamo-neurohypophysial system (HNS) excised and either superfused in oxygenated Krebs buffer at 37 degrees C or stored at -80 degrees C for measurement of paraventricular and supraoptic nuclei mRNA content by in situ hybridization analysis. Peptide content of superfusates and NIL homogenates were determined by specific RIAs. Compared with diestrus animals, DES treatment increased NIL OT but decreased MENK. In term pregnant rats, NIL OT, AVP, and DYN were increased over diestrus values, while MENK was again decreased. Release of the peptides from the isolated HNS paralleled changes in NIL content. The hypothalamic mRNA for OT was increased in DES-treated and pregnant rats while MENK mRNA was decreased. AVP and DYN mRNA was increased in pregnant animals. Although the NIL was found to contain much more immunoreactive OT and AVP than MENK or DYN, under basal conditions the release of MENK was equal to or greater than the release of OT, while the release of DYN approached that of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aromatic L-amino acid decarboxylase activity in the rat median eminence, neurointermediate lobe and anterior lobe of the pituitary. Physiological and pharmacological implications for pituitary regulation

Recent evidence demonstrating a direct effect of 5-hydroxytryptamine (5-HT) upon anterior pituitary (AP) hormone secretion has made the question of determining the location of possible sites that could supply 5-HT to the AP an important one. It has been assumed, based on indirect evidence, that aromatic L-amino acid decarboxylase (L-AAD), the enzyme responsible for the conversion of L-5-hydroxytryptophan (5-HTP) to 5-HT as well as L-3,4-dihydroxyphenylalanine (L-dopa) to dopamine (DA), is ubiquitously distributed in most tissues of the body including the AP. The present study examined the ability of the AP and two neural areas anatomically connected to the AP, the neurointermediate lobe (NIL) of the pituitary and the median eminence (ME), to decarboxylate 5-HTP to 5-HT or L-dopa to DA following either the in vitro incubation of the various tissues with 5-HTP or L-dopa or the in vivo administration of 5-HTP to rats treated previously with saline or a peripheral decarboxylase inhibitor, MK 486. The in vivo effects of 5-HTP, alone, or following MK 486 pretreatment were also examined on 5-HT synthesis and metabolism in AP tissues which were transplanted 5 days previously under the renal capsule and were, thus, isolated from central influences that might be regulating 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) concentrations in the animal's own AP. In addition, the direct radioisotopic measurement of L-AAD activity in the ME, NIL, and AP was also analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)