Subcellular Distribution of Opioid Peptides in Rat Hypothalamus and Pituitary

Homogenates of rat anterior lobe (AL) and neurointermediate lobe (NIL) pituitary and rat hypothalamus were subjected to subcellular fractionation and density gradient centrifugation. The subcellular distribution of immunoreactive dynorophin A (ir-Dyn A) in NIL was found to be similar to that of ir-arginine vasopressin (ir-AVP). ir-Dyn A migrated as a discrete band on sucrose density gradients, which corresponded in sedimentation rate to that of ir-AVP, suggesting that these two peptides are stored within organelles of similar size and density. Two other products of prodynorphin, ir-alpha-neoendorphin (ir-alpha-nEND) and ir-Dyn A-(1-8) also comigrated with ir-AVP. ir-[Leu5]-enkephalin (ir-LE), which may be a product of prodynorphin or proenkephalin, was also found to migrate in this region of the gradient. When a homogenate of rat hypothalamus was prepared using a method that has been developed for synaptosome isolation, ir-Dyn A was found to comigrate with Na+/K+-activated adenosine triphosphatase (Na/K-ATPase), a synaptosomal marker enzyme. Using a more concentrated homogenate ir-Dyn A was found to migrate to a less dense region where peptide-containing synaptic vesicles have previously been localized. When a synaptosomal preparation was lysed in hypotonic solution a shift was seen in the migration rate of ir-Dyn A to this region of the gradient (containing putative synaptic vesicles). Thus the bulk of hypothalamic dynorphin appears to be present within synaptosome-like structures which, upon lysis, release a less dense, smaller subcellular organelle corresponding in sedimentation characteristics to other types of peptide-containing synaptic vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-endorphin concentrations in the hypothalamus, pituitary and plasma of streptozotocin-diabetic rats with and without insulin substitution therapy

The concentrations of beta-endorphin like immunoreactivity (beta-END) in the hypothalamus, pituitary and plasma were studied in rats of either sex, one month after induction of diabetes by single iv injection of streptozotocin. As controls, both normal and undernourished rats, weight-matched with diabetic rats, were used. Diabetic male and female rats had a marked depletion of beta-END stores in the hypothalamus and neurointermediate lobe (NIL) but not in the anterior pituitary. Depletion of beta-END was reversed to normal by insulin replacement therapy. Severe undernourishment was not as effective as diabetes to reduce beta-END stores in the hypothalamus and NIL. A significant reduction of beta-END was observed only in the NIL of undernourished female rats. Plasma beta-END and beta-lipotropin (beta-LPH) concentrations were not significantly altered in diabetic rats. These results indicate that the lack of insulin may affect beta-END synthesis in the hypothalamus and NIL.     

Hypothalamo-posterior pituitary system in Brattleboro rats: immunoreactive levels of leucine-enkephalin, dynorphin (1-17), dynorphin (1-8) and alpha-neo-endorphin

The levels of immunoreactive leucine-enkephalin, alpha-neo-endorphin, dynorphin (1-17) and dynorphin (1-8) have been determined in the hypothalamus and posterior pituitary from male and female Brattleboro rats homozygous (unable to produce vasopressin) and heterozygous (producing vasopressin) for diabetes insipidus, and from male and female Long Evans rats. In the hypothalamus we found no significant differences in the levels of these peptides while there were great differences in extracts from the posterior pituitary: female homozygous animals have greatly reduced levels in all four peptides compared to the heterozygous controls. In male homozygous animals the differences in the dynorphin (1-17) and leucine-enkephalin levels were small whereas the concentrations of alpha-neo-endorphin and dynorphin (1-8) showed a significant decrease compared to the male heterozygous controls. The results indicate a reduction in opioid peptides linked to the vasopressin deficiency in a partially sex dependent manner.     

The effect of streptozotocin-induced diabetes on pituitary and hypothalamic endorphin equivalents

This study examines the effect of experimentally induced diabetes mellitus in rats on tissue concentrations of opioid peptides in the neurointermediate lobe (NIL), anterior pituitary (AP) and hypothalamus. Diabetic animals were found to have a marked increase in endorphin equivalents, measured by opiate receptor binding assay, in the NIL whereas no change was observed in beta endorphin-like immunoreactivity (beta ELI) or ACTH measured by RIA. These results may indicate the presence of a feedback mechanism and suggest the possibility that opioid peptides may be physiologically important in the maintenance of glucose homeostasis.     

Presence of dynorphin-like immunoreactivity in rat pituitary gland and hypothalamus

Using a highly specific and sensitive radioimmunoassay for dynorphin(1-13), dynorphin-like immunoreactivity (dynorphin-LI) was detected in rat pituitary and hypothalamus. Gel chromatographic studies on Sephadex G-50 revealed three components of dynorphin-LI with molecular weights of approximately 7500-9500 (big dynorphin), 3500-5500 (intermediate dynorphin) and 1600-1900 (small dynorphin), the latter of which eluted at the same position as authentic dynorphin contamination in porcine ACTH extracts (Sigma). Dynorphin-LI in rat anterior pituitary existed mainly as big dynorphin, whereas dynorphin-LI in rat intermediate-posterior pituitary and hypothalamus eluted mainly at the position of authentic small dynorphin.     

Neurotransmitters and estrogen interact to affect beta-endorphin levels in castrated female rats

The possibility of an interaction between neurotransmitter systems and estrogen in affecting levels of immunoreactive beta-endorphin (IR-BE) in the anterior pituitary (AP), the neurointermediate lobe of the pituitary (NIL) and the hypothalamus was investigated in ovariectomized (OVX) female rats. Chronic administration of the dopamine antagonist, haloperidol (HALO), had no effect on IR-BE levels in the AP. By contrast, the content of IR-BE in the NIL was increased and the content of IR-BE in the hypothalamus was decreased by HALO. Chronic treatment with estradiol benzoate (EB) produced a decrease in IR-BE in all three tissues. The effect of EB on IR-BE levels in the AP and NIL was reversed by administration of HALO, while EB and HALO appeared to act independently on the hypothalamus. Gel chromatography indicated that alterations in IR-BE in the AP corresponded to similar changes in beta-endorphin (BE) and beta-lipotropin (LPH) and that BE alone comprised the immunoreactivity detected in the NIL and hypothalamus regardless of treatment. Chronic treatment with the alpha-adrenergic agonist, clonidine (CLON), increased, whereas treatment with EB decreased, IR-BE levels in the AP, NIL and hypothalamus. EB attenuated the effect of CLON on IR-BE levels in the AP and hypothalamus. Chronic treatment with CLON appeared to promote the formation of BE in the AP, whereas the proportions of BE and LPH were similar in the AP of controls and animals treated with EB or EB and CLON. BE alone was detected in the NIL and hypothalamus of treated and control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

The isolation of purified neurosecretory vesicles from bovine neurohypophysis using isoosmolar density gradients

Two different density gradients are described for the isolation of highly purified fractions of neurosecretory vesicles in isoosmotic solutions (300 mosm/kg) from bovine neurohypophyses. The techniques involve differential centrifugation of neural lobe homogenates followed by density gradient centrifugation on metrizamide-sucrose or Percoll-sucrose gradients. The purified fraction contained 44 and 65 μg vasopressin/mg protein, respectively. Neurosecretory vesicles thus isolated were only slightly contaminated with other subcellular organelles, e.g., mitochondria and lysosomes. These vesicles were highly stable in isotonic sucrose solutions (pH 7.5 and 5.5) even at 37°C for at least 2 h, retaining more than 90% of their hormonal content.     

Gonadal steroids and anterior lobe dynorphin in the male rat

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.     

Expression of corticosterone-binding globulin in the rat hypothalamus.

We observed coexistence of corticosteroid-binding globulin (CBG) with vasopressin (VP) and oxytocin (OT) in magnocellular neurons in rat hypothalamus by combined immunoperoxidase staining and immunofluorescence. A portion of the supraoptic and of the paraventricular neurons showed double immunostaining of CBG with either VP or with OT. CBG staining was intensified by pretreating animals with colchicine to block axonal transport. CBG was also observed in widespread axonal projections throughout the lateral hypothalamus, the median eminence and the posterior pituitary lobe. Single ependymal cells and some of the endocrine cells in the anterior lobe contained specific CBG immunoreactivity. IN SITU hybridization of semithin sections with a synthetic oligonucleotide probe to CBG mRNA provided staining of magnocellular hypothalamic neurons, but not ependymal cells or anterior lobe cells. Western blots of CBG extracted by affinity chromatography from hypothalamus homogenates showed a band at approximately 50 kDa. Our observations indicate the intrinsic expression of CBG in peptidergic hypothalamus neurons in rat. The multiple locations of CBG-expressing neurons indicate multiple functional properties, probably exceeding the role of a mere steroid transporter. CBG is likely to be subject to axonal transport and secretion in a neuropeptide-like fashion, perhaps involved in neuroendocrine regulation, which may include stress responses.     

CNS tissue levels of dynorphin-A immunoreactivity and the anorexia associated with sodium chloride imbibition in the rat

Forced imbibition of increasing concentrations of sodium chloride (NaCl) in rats reduced daytime 2-deoxy-D-glucose (2-DG) induced feeding in a concentration dependent manner. Pituitary neurointermediate lobe (NIL) levels of immunoreactive (ir)-dynorphin-A 1-17 and 1-8 were also decreased by the NaCl regimen in a concentration dependent manner. However, there was no significant association between the reduction of NIL dynorphin levels and the suppression of 2-DG induced feeding on a within-animal basis. NaCl imbibition did not affect levels of either ir-dynorphin-A 1-17 or 1-8 in the hypothalamus, cerebral cortex, hippocampus, medulla/pons or anterior pituitary. Neither the acute changes following 2-DG administration, nor the comparison of ir-dynorphin-A 1-8/1-17 ratios appeared useful for the assessment of dynorphin-A turnover. Thus, the present results did not support the hypothesis that anorexia of NaCl treated animals results from the depletion of dynorphin-A.     

Trypsin Activation, Partial Characterization, and Distribution of Kallikrein-Like and Thrombin-Like Proteases in the Neurointermediate Lobe of the Rat Pituitary

This study examined whether the neurointermediate lobe (NIL) of the rat pituitary contains latent kallikrein- and thrombin-like proteases activated by trypsin. Partial characterization of such proteases was attempted. Also examined were the distribution of proteolytic activity within the NIL and levels in both male and female lobes. NIL homogenates were assayed for proteolytic activity at pH 8.0 before and after incubation with trypsin (10 micrograms/ml). Trypsin caused a 10-fold activation of kallikrein-like activity and a 40-fold activation of thrombin-like activity in NIL homogenates. The kallikrein-like activity was separated into two components using diethylaminoethyl-Sephadex. The predominant kallikrein-like protease was a potent kininogenase closely related or identical to glandular kallikrein and was almost exclusively localized to the intermediate lobe. The second kallikrein-like protease (kallikrein A) was a weak kininogenase sensitive to inhibition by both soybean trypsin inhibitor and aprotinin and was similarly concentrated in both the neural lobe and the intermediate lobe. The thrombin-like protease was sensitive to inhibition by hirudin (a specific thrombin inhibitor), clotted fibrinogen, and was slightly more concentrated in the neural lobe than in the intermediate lobe. NILs from female rats contained approximately 40% less kallikrein activity than NILs from male rats but did not differ in their content of thrombin-like activity.     

Effects of dehydration on pro-dynorphin derived peptides in the neuro-intermediate lobe of the rat pituitary

Dehydration significantly reduced the concentration of immunoreactive dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, beta-neo-endorphin, and leu-enkephalin in the rat pituitary posterior-intermediate lobe. A statistically significant increase in immunoreactive dynorphin A(1-8), alpha-neo-endorphin and leu-enkephalin was observed in the hypothalamus. Comparison of the molar ratios of dynorphin A(1-17): dynorphin A(1-8) and alpha-neo-endorphin: beta-neo-endorphin showed an altered profile of stored pro-dynorphin cleavage products in the posterior-intermediate lobe of the pituitary of dehydrated rats.     

Cocaine influences beta-endorphin levels and release

Immunoreactive beta-endorphin (IR-BE) was measured in the plasma, anterior pituitary (AP), neurointermediate lobe of the pituitary (NIL) and hypothalamus of male rats treated chronically (once daily for ten days) with cocaine. Cocaine produced a consistent elevation in the concentration of IR-BE in the plasma, the AP and the NIL at doses of 2.5 - 20 mg/kg/ip. The release of IR-BE from the AP and the NIL was determined in vitro and was found to be increased by treatment with cocaine. Chronic administration of cocaine did not affect the concentration of IR-BE in the hypothalamus. Chromatographic analysis revealed that cocaine produced a slight decrease in the amount of beta-endorphin relative to beta-lipotropin in the AP. Beta-endorphin was the major form of IR-BE released by the AP and the sole constituent and secretory product of the NIL. These data indicate that chronic administration of cocaine stimulates the endogenous opiate system, elevating the levels of IR-BE in the pituitary and promoting beta-endorphin release.     

Secretin in the rat hypothalamo-pituitary system: Localization,identification and characterization

Secretin-like immunoreactivity (SLI) has been identified and characterized in the pituitary of the rat. The concentration in the neurointermediate lobe is about 45 fold higher than the concentration of SLI observed in the anterior lobe. Transections of the pituitary stalk of the rat caused a significant depletion of SLI in the neurointermediate lobe without affecting the content in the anterior lobe. In view of the relatively high concentration of SLI reported to occur in the hypothalamus, it appears that there may be a secretinergic pathway between the brain and the neurointermediate lobe of the pituitary.     

Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels

Summary In situ hybridization histochemistry and indirect immunofluorescence histochemistry were used to study changes in the expression of vasopressin (VP), oxytocin (OXY), tyrosine hydroxylase (TH), galanin (GAL), dynorphin (DYN) and cholecystokinin (CCK) in hypothalamic magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei of rats. After prolonged administration of 2% sodium chloride as drinking water (salt-loading), the treatment increased the levels of VP, OXY, TH, GAL, DYN and CCK mRNA in the PVN and SON. The increase in CCK mRNA was, however, proportionally higher in the PVN than in the SON. Within cell bodies of the PVN and SON of salt-loaded rats, a depletion of VP- and OXY-like immunoreactivity (LI) and an increase in TH-LI were seen. In salt-loaded/colchicine-treated rats, a marked decrease in GAL- and DYN-LI, but no specific changes in CCK-LI were observed. Within nerve fibers of the posterior pituitary of salt-loaded rats, a marked depletion of VP-, GAL- and DYN-LI was found. Less pronounced depletion was observed in OXY- and CCK-LI, and no specific changes in TH-LI were seen. The results show that high plasma osmolality induces increased mRNA levels for VP, OXY, TH, GAL, DYN and CCK, presumably indicating increased synthesis, an increased export from cell somata of VP, OXY, GAL and DYN, and a decrease in levels of these peptides in the posterior pituitary, suggesting increased release. The catecholamine-synthesizing enzyme TH, however, which has a cytoplasmic localization and is not released from nerve endings, remains high in the cell bodies and nerve endings during this state of increased activity.     

Estrogen influences the effect of immobilization stress on immunoreactive beta-endorphin levels in the female rat pituitary

Immunoreactive beta-endorphin (IR-BE) levels in the plasma, anterior pituitary (AP), the neurointermediate lobe of the pituitary (NIL), and the hypothalamus were determined in castrated female rats and castrated female rats treated with estradiol benzoate (estrogen), after exposure to acute (once for 45 min) or chronic (45 min each day for 15 consecutive days) immobilization stress. Acute and chronic stress increased plasma levels of IR-BE to the same extent in castrated female rats and castrated female rats treated with estrogen. In castrated female rats, acute stress produced an increase in the concentration of IR-BE in the AP, which was attenuated by the administration of estrogen. Although IR-BE in the NIL was not influenced by acute stress in castrated animals, exposure to acute stress resulted in an elevation in IR-BE levels in the NIL of rats given estrogen. Chronic stress did not affect the concentration of IR-BE in the AP of castrated females or castrated females treated with estrogen. Chronic stress did, however, increase the concentration of IR-BE in the NIL of castrated animals. This affect of stress on IR-BE levels in the NIL was potentiated by estrogen administration. IR-BE levels in the hypothalamus were reduced by estrogen and were not affected by acute or chronic stress, regardless of the gonadal steroid environment. As determined by column chromatography, administration of estrogen, as well as subjection to chronic stress, promoted the processing of the proopiomelanocortin precursor to form beta-lipotropin rather than beta-endorphin in the AP. By these methods, the only immunoreactivity detected in the NIL and the hypothalamus was beta-endorphin. These data indicate that IR-BE levels in the plasma, the AP, and the NIL of female rats are affected by immobilization stress and that estrogen modulates the effects of acute immobilization stress on IR-BE levels in the AP and the NIL and the effects of chronic immobilization stress on the levels of IR-BE in the NIL.     

Neurotensin-like immunoreactivity in the pituitary and hypothalamus of bony fishes

Using an antiserum raised against synthetic neurotensin (NT), the distribution of immunoreactivity in the pituitary and hypothalamus has been examined by immunocytochemistry at light and electron microscope level in a number of species of bony fishes. In most species immunoreactive perikarya were found in the preoptic region of the hypothalamus, with fibres throughout the tuberal hypothalamus and neurohypophysis (neural lobe and median eminence). In the neurohypophysis of teleosts NT-like immunoreactivity was seen in a dense band of fibres bordering the ACTH cells of the rostral pars distalis: absorption controls showed that this was due to the presence of an NT(8-13)-like or xenopsin-like sequence, which, according to electron microscopic observations, was contained in small dense cored vesicles. The antiserum also stained the pituitary ACTH cells of some species, apparently due to cross-reaction with the 17-19 sequence of ACTH. These results suggest that an NT-like peptide may have a role in control of the adenohypophysis in fishes.     

Potential involvement of galanin in the regulation of fluid homeostasis in the rat

A physiological role for galanin, a 29-amino acid neuropeptide, has not been established. However, anatomical studies have demonstrated the presence of galanin in brain regions associated with the control of water balance in the rat, most notably in the paraventricular nucleus (PVN) of the hypothalamus and the neurointermediate lobe of the pituitary gland (NIL). In the PVN, galanin coexists with arginine vasopressin (AVP) in magnocellular neurons. The present study demonstrates that homozygous Brattleboro rats, which lack AVP, produce galanin. Galanin concentrations in the median eminence (ME) of the homozygous Brattleboro rat do not differ from the galanin concentrations in the ME of either heterozygous Brattleboro or Sprague-Dawley rats. However, galanin concentrations in the NIL of the homozygous Brattleboro rat were reduced by 75%. Similarly, dehydration induced by salt-loading reduced galanin concentrations in the NIL and produced transient changes in the ME. These data demonstrate that galanin concentrations are influenced by changes in fluid homeostasis and suggest that galanin may be an important component in the regulation of neurohypophyseal function and AVP secretion.     

Effect of neonatal treatment with monosodium glutamate on vasopressin release during foot shock stress in the rat

Several lines of evidence indicate that beta-endorphin inhibits the release of vasopressin during foot shock-induced stress in the rat. This study was to evaluate the relative importance of the hypothalamic versus the pituitary pool of beta-endorphin. Neonatal treatment with monosodium glutamate (MSG) reduced drastically the content of beta-endorphin-like immunoreactivity (beta-EI) of hypothalamus but not the beta-EI concentration in the pituitary; the content of vasopressin in the hypothalamus and the pituitary was not altered by MSG treatment. MSG treatment had no effect on the plasma vasopressin response to inescapable electric foot shock stress, when compared to controls. Naloxone enhanced vasopressin release during stress both in MSG-treated rats and in controls. These results suggest that hypothalamic beta-endorphin is not involved in the control of vasopressin release during foot shock-induced stress in the rat.