Neurointermediate Lobe Research Articles

Involvement of protein kinase C and protein tyrosine kinase in thyrotropin-releasing hormone-induced stimulation of alpha-melanocyte-stimulating hormone secretion in frog melanotrope cells.

We have previously shown that the stimulatory effect of TRH on alpha-MSH secretion from the frog pars intermedia is associated with Ca2+ influx through voltage-dependent Ca2+ channels, activation of a phospholipase C and mobilization of intracellular Ca2+ stores. The aim of the present study was to investigate the contribution of protein kinase C (PKC), adenylyl cyclase (AC), Ca2+/calmodulin-dependent protein kinase II (CAM KII), phospholipase A2, and protein tyrosine kinase (PTK) in TRH-induced alpha-MSH release. Incubation of frog neurointermediate lobes (NILs) with phorbol 12-myristate-13-acetate (24 h), which causes desensitization of PKC, or with the PKC inhibitor NPC-15437, reduced by approximately 50% of the effect of TRH on alpha-MSH release. In most melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca2+ concentration ([Ca2+]i). Preincubation with phorbol 12-myristate-13-acetate or NPC-15437 suppressed the plateau phase of the Ca2+ response. Incubation of NILs with TRH (10(-6) M; 20 min) had no effect on cAMP production. In addition, the AC inhibitor SQ 22,536 did not affect the secretory response of NILs to TRH. These data indicate that the phospholipase C/PKC pathway, but not the AC/protein kinase A pathway, is involved in TRH-induced alpha-MSH release. The calmodulin inhibitor W-7 and the CAM KII inhibitor KN-93 did not significantly reduce the response to TRH. Similarly, the phospholipase A2 inhibitors quinacrine and 7-7'-DEA did not impair the effect of TRH on alpha-MSH secretion. The PTK inhibitors ST638 and Tyr-A23 had no effect on TRH-induced [Ca2+]i increase but inhibited in a dose-dependent manner TRH-evoked alpha-MSH release (ED50 = 1.22x10(-5) M and ED50 = 1.47x10(-5) M, respectively). Taken together, these data indicate that, in frog melanotrope cells, PKC and PTK are involved in TRH-induced alpha-MSH secretion. Activation of PKC is responsible for the sustained phase of the increase in [Ca2+]i, whereas activation of PTK does not affect Ca2+ mobilization.

Oxidative Stress and a Murine Superoxide Dismutase-1 Mutation Promoting Amyotrophic Lateral Sclerosis Alter Neurosecretion in the Hypothalamo-Neurohypophyseal Axis

In this study, we examined the effects of oxidative stress on a nitric oxide (NO)-regulated neuroendocrine function, the release of arginine vasopressin (AVP) by the hypothalamo-neurohypophyseal axis. Treatment of mouse-isolated hypothalami and neurointermediate lobes () with H₂O₂ increased AVP release. This effect was inhibited by copper-zinc superoxide dismutase-1 (SOD1) analogs. By measuring cGMP accumulation as an indicator of biologically active NO, we found that H₂O₂ treatment decreased cGMP formation in both hypothalami and . We have previously shown that NO inhibits AVP release by a cGMP-independent mechanism. Given that H₂O₂ stimulated AVP release, while it reduced cGMP production, our findings strongly suggest that oxidative damage affects neurosecretion by reducing NO availability. To test whether such a mechanism may operate under pathological conditions with pronounced oxidative stress, we compared neurosecretion in wild-type and transgenic mice carrying a mutated form of SOD1 associated with human familial amyotrophic lateral sclerosis. Reminiscent of the data obtained from H₂O₂-treated tissues, hypothalami and from SOD1 mutants displayed decreased cGMP accumulation and increased AVP release, compared with tissues from wild-type littermates. Since neuronal NO synthase expression was not modified, we conclude that the perturbed free radical metabolism associated with the SOD1 mutation is likely to trap NO, and thereby alter neurosecretion, a mechanism that can be exacerbated in specific physiopathological conditions.

The distribution and gene expression of adrenomedullin in the rat brain: peptide/mRNA and precursor/active peptide relationships

The highest immunoreactive adrenomedullin (AM) concentrations were found in the pituitary gland, hypothalamus and brainstem. Rat preproadrenomedullin mRNA levels were highest in the pituitary, followed by the hypothalamus. The peptide to mRNA ratios are higher in the neurointermediate lobe, brainstem and hypothalamus. Immunocytochemical study showed discrete localization of AM immunostaining in various brain regions including the cerebral cortex, hypothalamus and brainstem and in the pituitary gland. By gel filtration chromatography, the precursor peaks were low in the pituitary, hypothalamus and brainstem but high in other brain regions.

Neuroendocrine and Behavioral Effects of CRH Blockade and Stress in Male Rats

Our previous data have shown that restraint (RT), a mild nonpainful stressor, acutely impairs nonsocial and social behavior in male rats. Corticotropin-releasing hormone (CRH) is a regulator of these behavioral responses. To evaluate whether CRH mediates the neuroendocrine and behavioral alterations present 24 h after restraint stress, we administered the CRH antagonist α-helical CRH 9–41 (α-hCRH) intracerebroventricularly to male rats and we compared its effects with those of saline. Twenty-four hours after treatment, nonsocial behaviors were significantly decreased by α-hCRH, this effect being independent of RT. Among social behaviors, only introductory activity showed significant differences as a result of both RT and α-hCRH. The concentrations of ACTH in the plasma and those of β-endorphin in the anterior and neurointermediate lobes of the pituitary were affected by α-hCRH treatment. The effect on ACTH was simply related to the administration of the α-hCRH, while for β-endorphin, significant interactions between α-hCRH and RT were found. On the whole, these results point to the role played by CRH in the control of neuronal mechanisms involved in the stress-induced effects.

Endothelin-1 and the release of neurohypophysial hormones under dehydration or haemorrhage

Rats, euhydrated or dehydrated for 2 days, were given intracerebroventricularly (i.c.v.) endothelin-l (ET-1), once daily in a dose of 10 ng. In euhydrated rats treatment with ET-1 was followed by a decrease in the content of vasopressin (VP) and oxytocin (OT) in the neurointermediate lobe (NL); simultaneously, the plasma concentrations of VP and OT increased. In animals deprived of water for 48 h, plasma concentrations of both VP and OT were increased whereas their content in the NL decreased; if ET-1 was administered during dehydration, however, these effects were markedly attenuated. Under urethane anaesthesia, neither the plasma concentration nor the NL content of both VP and OT were affected by i.c.v. injection of 10 ng ET-1. On haemorrhage (1 ml per 100 g body weight) the plasma levels of both VP and OT were increased. The OT content in the NL was then decreased but no changes in the VP content in the NL were seen on haemorrhage. In haemorrhaged rats prior treatment with ET-1 decreased the VP content in the NL and increased the VP concentration in the plasma when compared with rats haemorrhaged but not treated with ET-1. Simultaneously, the decrease in the neurohypophysial OT content as brought about by bleeding was inhibited and the plasma OT level was distinctly diminished when compared with animals haemorrhaged but not injected with ET-1. These results suggest that ET-1 may have a regulatory role in neurohypophysial function, the regulatory events being possibly modified by afferentation of osmoreceptor origin and also by changes of the central input from arterial pressoreceptors as well as from atrial stretch receptors.

L1 and laminin : their expression during rathypophysis ontogenesis and in adult neurohemal areas

L1 is a murine multidomain glycoprotein implicated in cell aggregation, fasciculation,neurite outgrowth and synaptogenesis. Laminin, a trimeric polypeptide, is implicated in neuronalsurvival, growth cone guidance, neurite outgrowth and cell differentiation. Laminin can alsointeract with the cell adhesion molecule L1. Their expressions were investigated from embryonicday 15 (E15) to adult in the rat hypophysis, and in adult neurohemal zones.Detected in the neural lobe from E17, the L1 immunoreactivity increased during prenataldevelopment and persisted in adulthood mainly related to the neuropeptidergic fibers. Pituicyteswere only labelled on the plasmalemma apposed to axons. In the intermediate lobe, L1 appearedat birth on folliculo-stellate cells extensions, constituting a network which densified duringpostnatal development. L1 is also expressed in all neurohemal areas on neuronal profiles.Laminin was clearly detectable in the hypophysis at E15 before the first blood vesselspenetrate the Rathke pouch. At E20, all the basal membranes of the blood vessels were stained. Inthe intermediate lobe, a spotted laminin immunoreactivity was detected at E21. At this stage, weobserved the staining of intercellular spaces and the intracellular labelling of melanotrophs,concerning reticulum or vesicles. The staining of melanotrophs seemed to maintain duringadulthood. In contrast with blood vessels of the adult cerebral tissue, adult capillaries of the neurallobe and the others neuro-hemal zones were intensely labelled with the anti-laminin antibody.These results suggest that neurite outgrowth and neurite guidance could be promoted byL1 and laminin in the neurointermediate lobe. The intercellular tunnels could also be an importantguidance cue for migrating cells in the intermediate lobe. These data also demonstrate thatmelanotrophic cells, secreting the laminin, have a role in the ontogenesis of the gland. Finally, wesuggest that L1 and laminin can collaborate to reinforce neurons-capillaries interactions inneurohemal zones.

Tissue-specific processing of cocaine- and amphetamine-regulated transcript peptides in the rat.

Cocaine- and amphetamine-regulated transcript (CART) is a recently discovered hypothalamic peptide regulated by leptin and with a potent appetite-suppressing activity. In the rat, the CART gene encodes a peptide of 116 amino acid residues (or a splice variant 13 residues longer). The predicted signal sequence is 27 amino acid residues, resulting in a prohormone of 89 residues. The CART prohormone contains several potential posttranslational processing sites in the form of mono- and dibasic sequences. In the present study we have purified CART peptides from extracts of adrenal gland, hypothalamus, nucleus accumbens, and pituitary gland (anterior and neurointermediate lobe) of the rat and determined the peptide structures by using microsequencing and mass spectrometry. In none of the tissues examined the long splice variant was found. From the adrenal gland, the CART(1-89) and CART(10-89) peptides were isolated, in contrast to the hypothalamus and nucleus accumbens, from which the shorter form peptides CART(42-89) and CART(49-89) were purified. From the anterior lobe of the pituitary gland, CART(42-89) was isolated, in contrast to the neurointermediate lobe, which contains only CART(49-89). This tissue-specific processing indicates that CART peptides may have different biological functions in the periphery and in the central nervous system.

Dynorphin A processing enzyme: tissue distribution, isolation, and characterization.

Limited proteolysis of the dynorphin precursor (prodynorphin) at dibasic and monobasic processing sites results in the generation of bioactive dynorphins. In the brain and neurointermediate lobe of the pituitary, prodynorphin is processed to produce alpha and beta neo endorphins, dynorphins (Dyn) A-17 and Dyn A-8, Dyn B-13, and leucine-enkephalin. The formation of Dyn A-8 from Dyn A-17 requires a monobasic cleavage between Ile and Arg. We have identified an enzymatic activity capable of processing at this monobasic site in the rat brain and neurointermediate lobe of the bovine pituitary; this enzyme is designated "dynorphin A-17 processing enzyme." In the rat brain and neurointermediate lobe, a majority of the Dyn A processing enzyme activity is membrane-associated and can be released by treatment with 1% Triton X-100. This enzyme has been purified to apparent homogeneity from the membrane extract of the neurointermediate lobe using preparative iso-electrofocussing in a granulated gel pH 3.5 to 10, FPLC using anion exchange chromatography, and non-denaturing electrophoresis. The Dyn A processing enzyme exhibits a pI of about 5.8 and a molecular mass of about 65 kDa under reducing conditions. The Dyn A processing enzyme is a metalloprotease and has a neutral pH optimum. It exhibits substantial sensitivity to metal chelating agents and thiol agents suggesting that this enzyme is a thiol-sensitive metalloprotease. Specific inhibitors of other metallopeptidases such as enkephalinase [EC 3.4.24.11], the enkephalin generating neutral endopeptidase [EC 3.4.24.15], or NRD convertase do not inhibit the Dyn A processing enzyme activity. In contrast, specific inhibitors of angiotensin converting enzyme inhibit the activity. The purified enzyme is able to process a number of neuropeptides at both monobasic and dibasic sites. These characteristics are consistent with a role for the Dyn A processing enzyme in the processing of Dyn A-17 and other neuropeptides in the brain.

Detection of melanocortin-3 receptor mRNA in immature rat pituitary: functional relation to gamma3-MSH-induced changes in intracellular Ca2+ concentration?

We have previously shown that gamma3-MSH stimulates DNA replication in lactotrophs, somatotrophs and thyrotrophs of early postnatal rat pituitary in culture. Since the melanocortin-3 (MC-3) receptor is the only known receptor displaying high affinity for gamma3-MSH, the present study explored whether mRNA of the latter receptor is present in the pituitary and whether the receptor is functional. RT-PCR of RNA extracts from 14-day-old rat pituitary revealed the presence of MC-3 receptor mRNA in both the anterior and the neurointermediate lobe. The identity of the amplified products was confirmed by sequence analysis. Dispersed cells of 14-day-old female rats (24 h in culture) were exposed to gamma3-MSH, and changes in intracellular free calcium levels ([Ca2+]i) were assessed by means of fluo-3 video imaging. Gamma3-MSH evoked a rapid and maintained oscillating [Ca2+]i increase in 5%, 10% and 15% of the cells at a dose of 0.1, 1 and 10 nM, respectively. The MC-3/MC-4 receptor antagonist Ac-Nle4-c[Asp5,(D-Nal(2)7,Lys10]alpha-MSH-(4-10)-NH2 (SHU 9119) blocked the effect of gamma3-MSH in about 50% of the responsive cells. The present data suggest that the MC-3 receptor is expressed in the rat pituitary but that this receptor mediates only half of the effects of its putative ligand, gamma3-MSH, on [Ca2+]i. Part of the effect of gamma3-MSH may be mediated by a MC-3 receptor in a functional state different from the one studied previously in transfected cell lines or by a hitherto unidentified MC receptor.

Serotonergic innervation of the pituitary pars intermedia of xenopus laevis.

At this point three brain centres are thought to be involved in the regulation of the melanotrope cells of the pituitary pars intermedia of Xenopus laevis: the magnocellular nucleus, the suprachiasmatic nucleus and the locus coeruleus. This study aims to investigate the existence of a fourth, serotonergic, centre controlling the melanotrope cells. In-vitro superfusion studies show that serotonin has a dose-dependent stimulatory effect on peptide release (1.6 x basal level at 10(-6) M serotonin) from single melanotrope cells. Retrograde neuronal tract tracing experiments, with the membrane probe FAST Dil applied to the pars intermedia, reveals retrogradely labelled neurones in the magnocellular nucleus, the suprachiasmatic nucleus, the locus coeruleus and the raphe nucleus. Of these brain centres, after immunocytochemistry only the raphe nucleus revealed serotonin-immunoreactive cell bodies. In addition, serotonin-immunoreactive cell bodies were found in the nucleus of the paraventricular organ, the posteroventral tegmental nucleus and the reticular istmic nucleus. In the pituitary, the pars nervosa, pars intermedia and pars distalis all reveal serotonin-immunoreactive nerve fibres. With immunocytochemical double-labelling for tyrosine hydroxylase and serotonin no colocalization of serotonin and tyrosine hydroxylase was observed in cell bodies in the brain, and in the pituitary hardly any colocalization was found in the nerve fibres. However, after in-vitro loading of neurointermediate lobes with serotonin, tyrosine hydroxylase and serotonin appear to coexist in a fibre network in the pars intermedia. On the basis of these data we propose that the melanotrope cells in the Xenopus pars intermedia are innervated by a 5-HT network originating in the raphe nucleus; this network represents the first identified stimulatory input to the pars intermedia of this species.

The effect of streptozotocin-diabetes on β-endorphin level and proopiomelanocortin gene expression in the rat pituitary

Streptozotocin-induced diabetes for 4 weeks resulted in a decrease in proopiomelanocortin (POMC) mRNA in both the anterior lobe (AL) and the neuro-intermediate lobe (NIL) of the rat pituitary. The β-endorphin levels decreased in the NIL but not in the AL. It is concluded that the synthesis of POMC in the pituitary is inhibited in diabetic rats and that there is a decrease in β-endorphin release from the anterior pituitary.

Effect of pre- and postnatal nicotine exposure on vasopressinergic system in rats

Timed pregnant Sprague–Dawley rats were infused subcutaneously either with nicotine (NIC, 6 mg kg −1 day −1; n=17) or saline (control, n=15) on the 3rd day of gestation, via Alzet osmotic pumps, for 28 days. After the parturition, the pups of both, control and NIC infused dams, were each randomly divided into 2 groups and placed to be nursed as following: (1) control dams nursing pups born to control mother (control group); (2) control dams nursing pups born to NIC-infused mother (prenatal NIC group); (3) NIC-infused dams nursing pups born to control mother (postnatal NIC group); (4) NIC-infused dams nursing pups born to NIC-infused mother (pre- and postnatal NIC group). Vasopressin (VP) was measured by RIA in plasma, neurointermediate lobe (NIL) and hypothalamus (HT) in the pups of both sexes, at the following age: 0 (within 24 h after birth); 1, 2, 3, 4 and 6 weeks. At the age of 3, 4 and 6 weeks, the isolated NILs were individually superfused and VP was measured as a basal release and the response to a 10-min 56 mM potassium stimulation. A marked suppression in the activity of VP-ergic system was observed in both sexes of offspring exposed to NIC prenatally, being first detectable at the age of 3 weeks, when the HT-NIL system becomes fully developed. However, the significant changes were observed at the age of 6 weeks: decreased serum VP concentration, lower VP contents in the HT and NIL, and suppressed VP release, basal and stimulated, from the isolated NIL. Postnatal exposure to nicotine was ineffective.

Raloxifene analog LY 117018 effects on central and peripheral β-endorphin

Raloxifene is a selective estrogen receptor modulator with a benzothiophene structure, that exerts an estrogen-like action on some target tissues and an anti-estrogenic action on the uterus and breasts. A limited number of data are available on the effect of raloxifene on neuroendocrine function. Since beta-endorphin (β-EP) is considered a marker of neuroendocrine function, the aim of the present study was to evaluate the effects of a 14 day treatment with a raloxifene analog, LY 117018, on β-EP content in the hypothalamus, hippocampus, anterior and neuro-intermediate pituitary lobe, and in the plasma of fertile and ovariectomized (ovx) rats. The effect of LY 117018 in ovx rats was compared to that of 17β-estradiol. β-EP contents were measured by a specific radioimmunoassay.While ovariectomy determined a significant decrease in β-EP levels in the anterior and neurointermediate pituitary lobe and plasma (p < 0.01), no changes of β-EP content in the hypothalamus and hippocampus were found. The administration of 17β-estradiol or LY 117018 in ovx rats significantly increased β-EP concentration in the anterior and neurointermediate pituitary lobe, in the hypothalamus and plasma (p < 0.01), though they did not significantly modify hippocampal β-EP content. When LY 117018 was administered together with 17β-estradiol in ovx animals, a clear anti-estrogenic effect in all organs and in plasma was observed, resulting in significantly lower β-EP content with respect to the group treated with 17β-estradiol alone (p < 0.01).The chronic administration of LY 117018 in fertile rats significantly decreased β-EP content in the anterior pituitary, hippocampus and plasma (p < 0.01), while it increased β-EP hypothalamic content and did not change β-EP content in the neurointermediate lobe.In conclusion, raloxifene analog LY 117018 has an estrogen-like action on neuroendocrine opiatergic pathways when administered alone in ovx rats, while it exerts an anti-estrogen effect infertile or in ovx rats treated with 17β-estradiol.

Expression of proopiomelanocortin and prohormone convertase-1 and -2 in the late gestation fetal sheep pituitary.

The biological activity of fetal plasma immunoreactive ACTH has been reported to increase during the final weeks of gestation in fetal sheep, indicative of enhanced processing of POMC to ACTH. The present study was aimed at examining the expression and localization of the prohormone convertases, PC1 and PC2, in the pituitary of fetal sheep during the final weeks of gestation. Pituitaries were obtained from fetal sheep during the final 50 days gestation (dGA) at 100-107 dGA (n = 6), 117-121 dGA (n = 6), 126-130 dGA (n = 7), and 144-147 dGA (n = 8; term = approximately 148 dGA). Pituitaries were cryosectioned and subjected to dual labeling in situ hybridization using 35S-labeled PC1 and/or PC2 complementary RNA probes with a digoxigenin-labeled POMC complementary RNA to localize and quantify PC1 and PC2 messenger RNA (mRNA) in POMC-hybridizing cells. Immunocytochemistry was also performed to assess coexpression of PC1 and PC2 with ACTH in the fetal pituitary. PC1 mRNA was heterogeneously distributed in the anterior pituitary (AP) at all gestational ages examined, with hybridization signals observed over POMC-expressing cells (corticotropes) as well as over noncorticotrope phenotypes. The inferior region of the AP contained an approximately 3-fold greater (P < 0.01) percentage of POMC cells containing PC1 transcripts compared with the superior region of the AP. The proportion of POMC cells containing PC1 was significantly higher (P < 0.01) in the 100-107 dGA and 144-147 dGA groups than in the 117-121 dGA and 126-130 dGA groups in both inferior and superior AP. The intensity of the PC1 hybridization signal over POMC-expressing cells was also about 2- to 4-fold greater (P < 0.01) in the inferior compared with the superior region of the fetal AP; the intensity of the PC1 hybridization signal associated with POMC cells remained constant within the AP region and did not change over the gestational ages examined. Hybridization for PC1 was highly variable over regions of AP not hybridizing for POMC, probably due to differences in the level of mRNA for PC1 between phenotypes. Similar to POMC cells, the average hybridization signal for PC1 over non-POMC-hybridizing regions was about 2-fold greater in the inferior vs. superior AP. A weak PC2 hybridization signal was observed over a small number of unidentified phenotypes in the fetal AP at all ages examined; no POMC cells were found to contain PC2 hybridization signal. In the neurointermediate lobe, POMC, PC1, and PC2 were ubiquitously expressed at all ages. Levels of PC1 and PC2 mRNA in the fetal neurointermediate lobe did not change over the period of gestation examined. Immunocytochemical analysis of PC1 and PC2 with ACTH confirmed the pattern of expression and the extent of coexpression observed with in situ hybridization methods. We conclude that both PC1 and PC2 are likely to contribute to POMC processing in the fetal pituitary during the final weeks of gestation.

Expression of Proopiomelanocortin and Prohormone Convertase-1 and -2 in the Late Gestation Fetal Sheep Pituitary

The biological activity of fetal plasma immunoreactive ACTH has been reported to increase during the final weeks of gestation in fetal sheep, indicative of enhanced processing of POMC to ACTH. The present study was aimed at examining the expression and localization of the prohormone convertases, PC1 and PC2, in the pituitary of fetal sheep during the final weeks of gestation. Pituitaries were obtained from fetal sheep during the final 50 days gestation (dGA) at 100–107 dGA (n = 6), 117–121 dGA (n = 6), 126–130 dGA (n = 7), and 144–147 dGA (n = 8; term= ∼148 dGA). Pituitaries were cryosectioned and subjected to dual labeling in situ hybridization using 35S-labeled PC1 and/or PC2 complementary RNA probes with a digoxigenin-labeled POMC complementary RNA to localize and quantify PC1 and PC2 messenger RNA (mRNA) in POMC-hybridizing cells. Immunocytochemistry was also performed to assess coexpression of PC1 and PC2 with ACTH in the fetal pituitary. PC1 mRNA was heterogeneously distributed in the anterior pituitary (AP) at all gestational ages examined, with hybridization signals observed over POMC-expressing cells (corticotropes) as well as over noncorticotrope phenotypes. The inferior region of the AP contained an approximately 3-fold greater (P < 0.01) percentage of POMC cells containing PC1 transcripts compared with the superior region of the AP. The proportion of POMC cells containing PC1 was significantly higher (P < 0.01) in the 100–107 dGA and 144–147 dGA groups than in the 117–121 dGA and 126–130 dGA groups in both inferior and superior AP. The intensity of the PC1 hybridization signal over POMC-expressing cells was also about 2- to 4-fold greater (P < 0.01) in the inferior compared with the superior region of the fetal AP; the intensity of the PC1 hybridization signal associated with POMC cells remained constant within the AP region and did not change over the gestational ages examined. Hybridization for PC1 was highly variable over regions of AP not hybridizing for POMC, probably due to differences in the level of mRNA for PC1 between phenotypes. Similar to POMC cells, the average hybridization signal for PC1 over non-POMC-hybridizing regions was about 2-fold greater in the inferior vs. superior AP. A weak PC2 hybridization signal was observed over a small number of unidentified phenotypes in the fetal AP at all ages examined; no POMC cells were found to contain PC2 hybridization signal. In the neurointermediate lobe, POMC, PC1, and PC2 were ubiquitously expressed at all ages. Levels of PC1 and PC2 mRNA in the fetal neurointermediate lobe did not change over the period of gestation examined. Immunocytochemical analysis of PC1 and PC2 with ACTH confirmed the pattern of expression and the extent of coexpression observed with in situ hybridization methods. We conclude that both PC1 and PC2 are likely to contribute to POMC processing in the fetal pituitary during the final weeks of gestation.

Derivatization of progesterone to a neurally active steroid by pituitary neurointermediate lobe

The melanotrophs of the neurointermediate lobe and peptidergic terminals of the neural lobe are regulated by gamma-aminobutyric acid (GABA) via GABA-A receptors and therefore, may be important sites for the modulatory actions of neurally active steroids. These steroid compounds might be produced peripherally, synthesized de novo in the pituitary, or derivatized from circulating steroids, each pathway having different physiological implications. In the present study, we show that neurointermediate lobe tissue can derivatize progesterone to the neurally active steroid 3α-hydroxy-5α-pregnan-20-one. The neurointermediate lobe was found to be four times as active as anterior pituitary and mediobasal hypothalamus in conversion of progesterone to 3α-hydroxy-5α-pregnan-20-one; mediobasal hypothalamus was relatively more active in the production of the intermediate 5α-pregnan-3,20-dione. The identity of the compounds was confirmed by the method of serial isotopic dilution. We observed rates of synthesis in the neurointermediate lobe consistent with the production of physiologically relevant quantities of 3α-hydroxy-5α-pregnan-20-one from concentrations of progesterone which can occur naturally. In support of these findings, we demonstrate the presence of 3α-hydroxysteroid oxidoreductase in neurointermediate lobe by immunocytochemistry.

The effect of neurointermediate lobe denervation on hypothalamic neuroendocrine dopaminergic neurons

The contribution of tuberohypophyseal and periventricular-hypophyseal dopaminergic neurons to the regulation of the secretion of prolactin (PRL) has yet to be clarified. In this study, we used pituitary stalk compression to disrupt hypothalamic neural input to the neurointermediate lobe (NIL). Neurointermediate lobe denervation (NIL-D) selectively disrupts the axons of tuberohypophyseal and periventricular-hypophyseal dopaminergic neurons, while leaving tuberoinfundibular dopaminergic neurons and the vascular supply of the pituitary gland intact. NIL-D was performed in ovariectomized (OVX) rats. The concentration of DA and 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence (ME) and various regions of the pituitary gland of OVX and OVX+NIL-D rats were measured by HPLC-EC. The concentration of PRL, α-melanocyte stimulating hormone (α-MSH), and luteinizing hormone (LH) in serum were determined by radioimmunoassay. Successful NIL-D was confirmed by increased water intake. One week after NIL-D, serum PRL and α-MSH were elevated, but there was no change in the concentration of LH in serum. The concentration of DA was increased in the median eminence (ME), decreased in the outer zone of the anterior lobe (AL-OZ), as well as the intermediate (IL) and neural lobes (NL), and remained unchanged in the inner zone of the anterior lobe (AL-IZ). The concentration of DOPAC was increased in the ME and NL, decreased in the IL, and remained unchanged in both the AL-IZ and AL-OZ. These data confirm that pituitary stalk compression denervates the NIL. Moreover, decreases in the concentration of DA in the IL and AL-OZ, coupled with elevation of serum PRL and α-MSH indicate that DA from the NIL contributes to the increased inhibition of the secretion of PRL and α-MSH in OVX rats.

Immunohistochemical distribution of oestrogen receptor and luteinizing hormone B subunit in the ovine pituitary gland during foetal development.

The presence of oestrogen receptor in the developing hypothalamo-hypophyseal system is an essential prerequisite for the development of sex-steroid feedback on gonadotrophin secretion. We have used dual immunocytochemistry to examine the ontogeny and regional distribution of oestrogen receptor and LHbeta subunit in the ovine pituitary gland during foetal development. At day 65 gestation (term= 145 days) oestrogen receptor and LH/ immunopositive cells are found in a small region at the base of the anterior pituitary gland, and also in a band immediately adjacent to the neurointermediate lobe. By day 100 gestation there was a significant increase in the number of immunopositive LHbeta cells accounting for around 12% of the total cell population, and these were widely distributed throughout the anterior pituitary gland. There was also a significant increase in the proportion of gonadotrophs which contain oestrogen receptor compared with day 65. By day 130 gestation the percentage of LH containing cells had declined to around 7% of the total population, but the proportion which also contained oestrogen receptor remained the same. There were no differences in the numbers or distribution of cells containing LH or oestrogen receptors between male and female foetuses, at any age. These data describing a parallel change in the number of oestrogen receptors and LHbeta containing cells in the pituitary gland throughout gestation suggest that the development of pituitary sex-steroid feedback is not solely dependent on changes in the numbers of oestrogen receptor containing cells alone.

Pharmacological and functional characterization of muscarinic receptors in the frog pars intermedia.

The secretion of alphaMSH from the intermediate lobe of the frog pituitary is regulated by multiple factors, including classical neurotransmitters and neuropeptides. In particular, acetylcholine (ACh), acting via muscarinic receptors, stimulates alphaMSH release from frog neurointermediate lobes (NILs) in vitro. The aim of the present study was to characterize the type of receptor and the transduction pathways involved in the mechanism of action of ACh on frog melanotrope cells. The nonselective muscarinic receptor agonists muscarine and carbachol both stimulated alphaMSH release from perifused frog NILs, whereas the M1-selective muscarinic agonist McN-A-343 was virtually devoid of effect. Both the M1>M3 antagonist pirenzepine and the M3>M1 antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide inhibited muscarine-induced alphaMSH release. Administration of a brief pulse of muscarine in the vicinity of cultured melanotrope cells provoked a 4-fold increase in the cytosolic calcium concentration ([Ca2+]i). Suppression of Ca2+ in the culture medium or addition of 3 mM Ni2+ abrogated the stimulatory effect of muscarine on [Ca2+]i and alphaMSH release. In contrast, omega-conotoxin GVIA and nifedipine did not significantly reduce the stimulatory effect of muscarine on [Ca2+]i and alphaMSH secretion. Exposure of NILs to muscarine provoked an increase in inositol phosphate formation, and this effect was dependent on extracellular Ca2+. The inhibitor of polyphosphoinositide turnover neomycin significantly attenuated the muscarine-evoked alphaMSH release. Similarly, pretreatment of frog NILs with phorbol ester markedly reduced the secretory response to muscarine. In contrast, the stimulatory effect of muscarine on alphaMSH release was not affected by the phospholipase A2 inhibitor dimethyl eicosadienoic acid or by the tyrosine kinase inhibitors lavendustin A, genistein, and tyrphostin 25. Muscarine at a high concentration (10(-4) M) only produced a 40% increase in cAMP formation. Preincubation of frog NILs with pertussis toxin did not significantly affect the muscarine-induced stimulation of alphaMSH release. These results indicate that frog melanotrope cells express a muscarinic receptor subtype pharmacologically related to the mammalian M3 receptor. Activation of this receptor causes calcium influx through Ni2+-sensitive Ca2+ channels and subsequent activation of the phopholipase C/protein kinase C transduction pathway.

Alterations in brain and pituitary beta-endorphin content in genetically epilepsy-prone rats.

We measured beta-endorphin concentrations in the anterior and neurointermediate lobes of the pituitary gland and in microdissected brain regions of moderate-seizure genetically epilepsy-prone rats (GEPR-3), severe-seizure GEPR-9s and Sprague-Dawley non-epileptic control rats. Plasma concentrations of beta-endorphin and beta-melanocyte-stimulating hormone (alpha-MSH) were also measured as indicators of pituitary POMC-peptide secretion. Concentrations of beta-endorphin in the anterior lobe of GEPR-3s were 53% higher compared to controls and 57% higher compared to GEPR-9s. There were no differences in neurointermediate lobe beta-endorphin concentrations between control and either GEPR strain. Plasma beta-endorphin concentrations were significantly lower in GEPR-9s than controls. Plasma levels of alpha-MSH did not differ between control and GEPRs. In the hypothalamus of GEPR-9s beta-endorphin concentrations in the arcuate nucleus were significantly greater than in GEPR-3s. Concentrations of beta-endorphin in the central amygdala of GEPR-9s were two- to threefold greater than in control or GEPR-3s. Beta-Endorphin concentrations in the central gray of GEPR-3s were 58% lower than control or GEPR-9s. These data suggest that anterior lobe beta-endorphin secretion is reduced in GEPR-9s. Furthermore, brain endorphinergic pathways appear to be differentially altered in GEPR-3s and GEPR-9s. Alterations in pituitary beta-endorphin secretion and brain endorphinergic systems may contribute to seizure susceptibility in GEPRs and to differences in seizure severity between GEPR-3s and GEPR-9s.