A. Samora

In vivo Effects of Serotonergic Agents on AIpha-MeIanocyte-StimuIating Hormone Secretion

We used an in vivo pharmacological approach to investigate the potential influence of serotonin (5-HT) on peptide release from the intermediate lobe (IL) of the rat pituitary. Plasma levels of alpha-melanocyte-stimulating hormone (alpha-MSH) as well as the ultrastructural appearance of IL cells were used as indicators of IL secretory activity. Plasma beta-endorphin levels were also measured to assess the effectiveness of 5-HT-acting drugs. Intraperitoneal administration of 5-hydroxy-L-tryptophan, the synthetic precursor of 5-HT, dramatically elevated the content of 5-HT in the neurointermediate lobe but did not alter plasma titers of alpha-MSH. Treatment with the 5-HT reuptake blocker fluoxetine elevated plasma levels of beta-endorphin but not alpha-MSH. Administration of the 5-HT1/5-HT2 agonist MK-212 produced an elevation in both plasma alpha-MSH and beta-endorphin levels. Quantitative morphometry of IL cells at the ultrastructural level revealed that MK-212 treatment selectively causes depletion of electron-lucent but not electron-dense secretory granules from the cytoplasm of IL cells. Pretreatment with the selective D2 dopamine agonist apomorphine blocked MK-212-induced release of alpha-MSH but not beta-endorphin. Our results show that manipulation of 5-HT synthesis/reuptake does not affect release of alpha-MSH, but that direct activation of 5-HT receptors with the nonselective agonist MK-212 stimulates alpha-MSH release. The ability of apomorphine to block MK-212-induced release of alpha-MSH suggests a direct antagonism between dopaminergic and serotonergic regulation of alpha-MSH release.

Organization of tyrosine hydroxylase-immunoreactive neurons in the di- and mesencephalon of the American bullfrog (Rana catesbeiana) during metamorphosis

SummaryWe examined the immunocytochemical distribution of tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis, in the di-and mesencephalon of developing bullfrog tadpoles. Special attention was given to catecholaminergic innervation of the median eminence and pituitary. In premetamorphic tadpoles, tyrosine hydroxylase-immunoreactive neurons were visualized in the suprachiasmatic and infundibular hypothalamus, the ventral thalamus, and midbrain tegmentum by Taylor-Kollros stage V. The number of labeled neurons in all these areas increased as metamorphosis progressed. By mid-prometamorphosis, labeled neurons appeared in the preoptic recess organ as well as in the posterior thalamic nucleus. The majority of cells in the preoptic recess organ, as well as occasional neurons in the suprachiasmatic nucleus, exhibited labeled processes which projected through the ependymal lining of the preoptic recess to contact cerebrospinal fluid. The modified CSF-contacting neurons of the nucleus of the periventricular organ were devoid of specific staining. By late prometamorphosis, labeled fibers from the suprachiasmatic nucleus were observed projecting caudally to enter the hypothalamo-hypophysial-tract en route to innervating the median eminence and pituitary. Labeled fibers arising from the dorsal infundibular nucleus projected ventrolaterally to contribute to catecholaminergic innervation of the median eminence and pituitary. Immunoperoxidase staining of tyrosine hydroxylase-immunoreactive fibers and terminal arborizations in the median eminence were restricted to non-ependymal layers, while labeled fibers in the pituitary were observed in the pars intermedia and pars nervosa. Staining of tyrosine hydroxylase-immunoreactive fibers in the median eminence and pituitary was sparse or absent in premetamorphic tadpoles, but became increasingly more intense as metamorphosis progressed.

Benzodiazepine suppression of corticotropin-releasing factor (CRF)-induced beta-endorphin release from rat neurointermediate pituitary

Dopamine and gamma-aminobutyric acid (GABA) inhibit POMC peptide release from the pituitary intermediate lobe, via interaction with D2 or GABA-A/benzodiazepine receptors. Here, we examined the effects of an antianxiety triazolobenzodiazepine, adinazolam, on corticotropin-releasing factor (CRF)-stimulated POMC peptide secretion from the rat neurointermediate pituitary. Neurointermediate lobes (NILS) were incubated with CRF (10(-7) M), then adinazolam (10(-8) or (10(-9) M) was added, with CRF remaining in the medium. Aliquots were removed at 15-min intervals and frozen for radioimmunoassay of beta-endorphin. Adinazolam alone did not significantly affect secretion as compared to controls or CRF alone. Adinazolam incubated with CRF led to significant inhibition of beta-endorphin secretion, as compared to CRF alone. In addition, adinazolam was as effective as dopamine or the CRF antagonist, alpha-helical CRF, in preventing CRF-induced beta-endorphin release. Adinazolam appears to act directly on the pituitary to suppress hormone release induced by a stress-related hypothalamic peptide.

Interaction of corticotropin-releasing factor (CRF) and alpha-helical CRF on rat neurointermediate lobes: In vitro studies

Neurointermediate lobes (NILS) of the pituitary glands of adult male Sprague-Dawley rats were incubated in media in the presence of corticotropin-releasing factor (CRF), a stimulator of proopiomelanocortin (POMC) peptide release. Alpha-helical CRF, a peptide known to inhibit CRF induced POMC peptide release from the anterior pituitary, was incubated with NILS for a period of 90 min, to study its potential ability to modulate peptide release from the intermediate lobe. The alpha-helical peptide reduced beta-endorphin release from NILS, as measured by radioimmunoassay (RIA), when added for the entire incubation, or when added 30 min after start of the incubation period, with CRF present. Alpha-helical CRF alone reduced beta-endorphin release, as compared to control or CRF-treated lobes. Ultrastructural examination of intermediate lobes fixed at the end of incubations revealed a reduction in the numbers of Golgi-associated dense granules, an indicator of new peptide synthesis, in intermediate lobe tissue treated with alpha-helical CRF alone, both peptides together, or with CRF followed by alpha-helical peptide. The in vitro studies demonstrate the effectiveness of the antagonist peptide on intermediate lobe peptide secretion, thereby extending its effects to both POMC-secreting areas of the pituitary gland.

Stress-induced peptide release from rat intermediate pituitary : an ultrastructural analysis

SummaryWe tested the hypothesis that acute restraint stress results in ultrastructural evidence for enhanced release of alpha-melanocyte-stimulating hormone (α-MSH) and β-endorphin from the intermediate lobe (IL) of the rat pituitary. Measurements of plasma α-MSH-and β-endorphin-immunoreactivity (ir) were used to confirm ultrastructural findings. Plasma α-MSH-ir was elevated after 20 and 30 min of restraint while plasma β-endorphin-ir peaked 10 min after the onset of restraint. Ultrastructural analysis revealed a decrease in the content of secretory granules within IL cells of stressed rats. Analysis of Golgi-related immature secretory granules in IL cells indicated that new peptide synthesis was not enhanced after 30 min of restraint. These results confirm previous studies showing and elevation of plasma β-endorphin and α-MSH-ir during acute restraint. Furthermore, these results indicate that quantitative analysis at the ultrastructural level can be used to assess peptide release from IL secretory cells during stress.

Effects of the enkephalin analog (D-Met2, Pro5)-enkephalinamide on α-melanocyte-stimulating hormone secretion

We used the met-enkephalin analog (D-Met2,Pro5)-enkephalinamide (DMPEA) to investigate enkephalinergic control of alpha-melanocyte-stimulating hormone (alpha-MSH) secretion. Systemic (s.c.) administration of DMPEA elevated plasma titers of alpha-MSH in a dose- and time-related manner. Pretreatment with the opiate antagonist naltrexone had no effect on basal plasma levels of alpha-MSH but blocked DMPEA-induced alpha-MSH release. Treatment with a dose of naltrexone sufficient to block DMPEA-induced secretion of alpha-MSH had no effect on stress-induced secretion of alpha-MSH. Although pretreatment with the dopamine receptor agonist apomorphine prevented DMPEA-induced alpha-MSH secretion, DMPEA had no effect on the synthetic activity of tuberohypophysial dopamine neurons as gauged by measuring the accumulation of 3,4-dihydroxyphenylalanine in the neurointermediate lobe (NIL) following administration of NSD-1015. In vitro treatment of isolated NILs with DMPEA resulted in a significant increase in alpha-MSH release. Naltrexone completely blocked the stimulatory effects of DMPEA on alpha-MSH release in vitro. Our results indicate that DMPEA stimulates alpha-MSH secretion by acting directly through opiate receptors at the level of the NIL.

Degeneration and regeneration of nerve terminals in the rat pituitary pars intermedia after 6-hydroxydopamine treatment.

6-hydroxydopamine (6-OHDA), a catecholamine neurotoxin, has been shown previously to induce degenerative changes in nerve terminals innervating proopiomelanocortin (POMC) cells of the pituitary intermediate lobe. The present study provides evidence for regeneration of nerve fibers to the pituitary within 4 weeks of drug treatment. Adult male Sprague-Dawley rats were treated with 6-hydroxydopamine (150 mg/kg) on Days 1 and 3 and then perfused for light or electron microscopy (EM) of the pituitary intermediate lobes at 1, 2, 3, or 4 weeks after the first injection. At 1, 2, and 3 weeks after drug treatment, the number of normal-appearing nerve terminals was significantly lower than that in controls, while at 4 weeks after 6-OHDA, the number of normal nerve terminals did not differ significantly from that of control rats. Tyrosine hydroxylase-immunoreactive fibers appeared more intensely stained at 1 and 2 weeks after 6-OHDA treatment, suggesting continued and perhaps enhanced synthesis of the enzyme by hypothalamic cell bodies to the remaining terminals. Serotonin immunoreactivity after 5-hydroxytryptophan pretreatment was not detectable at 1 week after drug treatment, but was clearly visible in fibers innervating the intermediate lobe at 3 weeks, indicating a return of uptake ability and conversion of the precursor to serotonin by the regenerating terminals. Chemical denervation of the intermediate lobe followed by regeneration of nerve fibers will be useful for examination of regulatory mechanisms for the POMC secretory cells.