C. Kordon

Opiate Receptors Modulate LHRH and SRIF Release from Mediobasal Hypothalamic Neurons

In order to investigate the effect of opiates on luteinizing hormone-releasing hormone (LHRH) and somatostatin (SRIF) release, mediobasal hypothalamic (MBH) slices of male adult rats were superfused a

Vasoactive intestinal peptide (VIP): Brain distribution, subcellular localization and effect of deafferentation of the hypothalamus in male rats

Abstract We have studied the regional and subcellular distribution of vasoactive intestinal peptide (VIP) in the brain of adult male rat, using a specific radioimmunoassay. Selective deafferentation of the mediobasal hypothalamus (MBH) was also performed in order to investigate the origin of hypothalamic VIP. The highest concentrations of VIP were found in the neocortex, namely the occipital region. The brain stem, the posterior hypothalamus, and the pineal gland contained low amounts of the peptide. VIP was not detectable in the cerebellum and the neurohypophysis. After fractionation, most of the VIP was recovered from the crude mitochondrial fraction of the hypothalamus as well as the parietal cortex. However, a non-negligible portion of the activity was also found in the supernatant suggesting that the peptide is mainly located in nerve endings but also present in neuronal cell bodies and/or axons. Two weeks after complete deafferentation of the MBH, VIP concentrations of the caudal MBH (including the infundibular sulcus, the stalk, part of the ventromedial nucleus and premamillary structures) were decreased by 40%. In contrast, no change in VIP levels were observed in the rostral MBH, organum vasculosum of the lamina terminalis (OVLT), and cortex. This suggests that hypothalamic nerve endings containing the peptide derive from neuronal cell bodies located both outside and within the MBH.

Decreased Expression of the Two D2 Dopamine Receptor Isoforms in Bromocriptine-Resistant Prolactinomas

Bromocriptine or other dopamine agonists are usually effective for the treatment of prolactin-secreting adenomas. Five to 18% of prolactinomas, however, do not respond to such therapy. We have shown previously that such resistance to bromocriptine correlates with reduced binding to the D2 receptor subtype of dopamine, the major PRL inhibiting factor. In the present work, we demonstrated that reduced binding actually corresponds to decreased expression of the gene coding for the D2 receptor in the pituitary from bromocriptine-resistant patients, as shown by 4-fold lower levels of the corresponding mRNAs compared to those coding for actin. The existence of two D2 receptor isoforms, D2S and D2L generated by alternative splicing, has been described in several tissues, including the pituitary. Both are negatively coupled to adenylyl cyclase and inhibit prolactin secretion, but, in addition, the shortest one (D2S) is more efficiently coupled to phospholipase C. Consequently, we also investigated whether expression of a particular D2 receptor isoform was preferentially affected in resistant adenomas. The proportion of messengers corresponding to the short receptor isoform (D2S) was lower in resistant compared to responsive adenomas: D2S/D2L = 0.74 +/- 0.08 and 1.00 +/- 0.07, respectively. In parallel, much lower levels of D2 receptor mRNAs were found in growth hormone-secreting adenomas, with a D2S/D2L ratio comparable to those of both normal human pituitary and bromocriptine-sensitive prolactinomas (1.05 +/- 0.11). Thus, resistance to bromocriptine therapy seems to involve defects in D2 dopamine receptor expression and possibly in posttranscriptional splicing.

Characterization, regional distribution, and subcellular distribution of 125I-Tyr1-somatostatin binding sites in rat brain.

Abstract: 125I‐Tyr1‐somatostatin binds reversibly, in a saturable manner, and with high affinity to membranes from rat brain. Kinetic and saturation data measured at equilibrium lead to KDvalues of 0.4 nM for cortical membranes. The binding is not affected significantly by seven neuropeptides and drugs unrelated structurally to somatostatin (SRIF) while native SRIF, Tyr1‐SRIF, and D‐Trp8‐D‐Cys14‐SRIF displace 125I‐Tyr1‐SRIF in a dose‐dependent manner, with Ki of 0.23 nM, 0.90 nM, and 0.11 nM, respectively. Binding sites for 125I‐Tyr1‐SRIF were found in 9 out of 11 central structures; there was a significant correlation between binding capacity and endogenous SRIF levels measured by radioimmunoassay. In each of the two structures containing the most binding sites, the cortex and the preoptic area, Scatchard analysis suggests a single population of sites with apparent affinities of 0.8 nM and 1.4 nM, respectively. Subcellular fractionation of these two regions reveals that more than 60% of 125I‐Tyr1‐SRIF specific binding of the homogenate is found in the crude mitochondrial pellet (P2), which contains synaptosomes. When P2 is further fractionated on a discontinuous sucrose gradient, most of the initial P2 binding is recovered from membrane fractions. Each of nine SRIF analogs, with a single alanine substitution, displaces 125I‐Tyr1‐SRIF binding on cortical membranes in the same order of potency as on adenohypophyseal membranes (r= 0.84). The data demonstrate the presence of SRIF binding sites in the rat brain, with kinetic characteristics comparable to those found in the adenohypophysis, and they provide a biochemical basis for the multiple functions of SRIF in brain.

The use of bacitracin as an inhibitor of the degradation of thyrotropin releasing factor and luteinizing hormone releasing factor.

Bacitracin was found to be an effective inhibitor of the invitro degradation of both thyrotropin releasing factor1 (TRF) and luteinizing hormone releasing factor (LRF) by guinea pig hypothalamic and whole brain homegenates and rat hypothalamic homogenates and subcellular fractions. Bacitracin was effective in inhibiting the degradation of TRF and LRF, as determined by radioimmunoassay, where it exhibited no interference with the assays. Kinetic studies of the degradation of exogenous synthetic [3H]-TRF demonstrated non-competitive inhibition by bacitracin with Ki = 1.9 × 10−5 M, while studies on the degradation of [3H] LRF indicated competitive inhibition with Ki = 1.7 × 10−5 M. Electrophoretic and amino acid analysis revealed that bacitracin itself was not degraded during the course of the invitro incubation.

Endogenous opiates block dopamine inhibition of prolactin secretion in vitro.

OPIATES stimulate prolactin secretion in vivo; plasma prolactin levels are increased by morphine administration in normal1 and steroid-primed male rats2, as well as in immature3 and oestrogen-treated female rats4. Met-enkephalin and β-endorphin were also stimulatory1–6 as were enkephalin analogues3,7,8. Naloxone or naltrexone, specific opiate antagonists, reduce basal prolactin levels when given alone1,3,9, and block, at least partially, the effect of opiate agonists1–4,7,8. None of these drugs is active in vitro when tested alone on anterior pituitaries3 or dispersed pituitary cells2,4. However, as we have previously shown in a preliminary experiment, the inhibitory effect of dopamine on prolactin secretion in vitro was antagonised by the addition of morphine to the incubation medium10. In the present work, we have attempted to clarify further the mode of interaction of opiates with prolactin secretion. We confirmed that morphine, Met-enkephalin and β-endorphin do not affect the spontaneous release of prolactin in vitro, but found that they suppress the inhibitory effect of dopamine on prolactin release. We have also characterised the specificity and the kinetics of this interaction.

Hypothalamic and Hypophyseal Regulation of Growth Hormone Secretion

1. Regulation of pulsatile secretion of growth hormone (GH) relies on hypothalamic neuronal loops, major transmitters involved in their operation are growth hormone releasing hormone (GHRH) synthetized mostly in arcuate nucleus (ARC) neurons, and somatostatin (SRIH), synthetized both in hypothalamus periventricular (PVe) and ARC neurons. 2. Neurons synthetizing both peptides can inhibit each other in a reciprocal manner. Other neuropeptides synthetized in ARC neurons, such as galanin, or in ARC interneurons, such as neuropeptide Y (NPY), are able to modulate synthesis and release of GHRH and SRIH into the hypothalamohypophyseal portal system. 3. In addition, the hitherto uncharacterized endogenous ligand of the recently cloned growth hormone releasing peptide receptor, expressed mostly in the ARC, triggers GH release, presumably by actions on ARC interneurons. 4. Thyroid, gonadal, and adrenal steroid hormones also affect the GHRH-SRIH balance; a differential distribution of sex steroid receptors in the ARC and the PVe is likely to account for the different pattern of GH secretion in male and female animals. 5. Growth hormone itself is able to inhibit the amplitude of GH secretory episodes and to increase their frequency, by entering the brain (presumably by receptor-mediated internalization at the level of the choroid plexus) and acting subsequently on ARC neurons. 6. At the pituitary level, major neurotransmitters regulating GH cells act on receptors of the VIP/PACAP/GHRH family and of the somatostatin family, in particular, sst2 and sst3. Those are coupled to accumulation of cAMP as a second messenger. 7. In addition, patch-clamp experiments and measurement of intracellular Ca2+ indicate that GH cells present characteristic, GHRH-dependent, but self-maintained Ca2+ spikes and [Ca2+]i transients, which reflect adaptive mechanisms to constraints of episodic release. 8. Recent data on transcription factors affecting GH gene expression and somatotrope differentiation are also summarized. 9. Regulation and differentiation of somatotropes also depend upon paracrine processes within the pituitary itself and involve growth factors and several neuropeptides, for instance, vasoactive intestinal peptide, angiotensin 2, endothelin, and activin. 10. Finally, characteristic changes occur in the GH secretory pattern under discrete, pathological conditions, such as abnormal growth and dwarfism, diabetes, and acromegaly, as well as during inflammatory processes.

Stimulation of in vitro Prolactin Release by Vasoactive Intestinal Peptide

VIP stimulated prolactin secretion from incubated rat hemipituitaries. Under the same conditions, the secretion of GH, LH, FSH was not affected. The stimulation of prolactin was dose-dependent, with an apparent affinity of VIP of 10.9 +/- 3.1 nM and a maximal stimulation of 57.7 +/- 4.2%. Secretin, a structurally related peptide, was also active at higher concentrations whereas another partial analogue, glucagon, was ineffective. The effect of VIP was not blocked by alpha-flupentixol, a potent dopaminergic antagonist, at concentrations which antagonized the dopamine inhibition of prolactin secretion. Stimulation by VIP and TRH was additive. Neither Met-enkephalin nor naloxone interfered with the response to VIP. It thus seems that specific VIP receptors are present on pituitary prolactin cells. VIP, present in the mediobasal hypothalamus and detected in the hypothalamo-hypophyseal portal blood therefore is a good candidate as a physiological PRF.

Effect of vasoactive intestinal peptide (VIP) on the release of adenohypophyseal hormones from purified cells obtained by unit gravity sedimentation. Inhibition by dexamethasone of VIP-induced prolactin release.

The effect of vasoactive intestinal peptide (VIP) on the release of prolactin (PRL), gonadotropins (LH and FSH), growth hormone (GH) and corticotropin (ACTH) was studied using purified rat anterior pituitary cells obtained by means of velocity sedimentation at unit gravity. VIP, at concentrations ranging from 10(-10) to 10(-7) M, stimulated PRL secretion in a dose-dependent manner with an ED50 of 2 nM and a maximal response of 530% of control values. In contrast, similar concentrations of VIP did not affect the release of either LH, FSH, GH or ACTH from the corresponding enriched cell populations. Addition of dexamethasone (10(-9) M) to both preincubation and incubation medium of PRL cells completely inhibited VIP-induced PRL release. The present results further support the hypothesis that VIP is of physiological importance in the control of PRL secretion and demonstrate that corticosteroids can modify the responsiveness of PRL cells to VIP.

Growth hormone-releasing hormone-synthesizing neurons are a subpopulation of somatostatin receptor-labelled cells in the rat arcuate nucleus : a combined in situ hybridization and receptor light-microscopic radioautographic study

Distribution of growth-hormone-releasing hormone (GHRH) cell bodies and somatostatin binding sites were compared in the mediobasal hypothalamus of the rat. GHRH-synthesizing neurons were visualized by in situ hybridization, using as 35S-labelled synthetic oligonucleotide (45 mere), and 125I-Tyr0-DTrp8-somatostatin (125I-SRIH) binding sites by light-microscopic radioautography on adjacent 20-microns-thick frozen mirror sections. GHRH mRNA hybridizing cells were detected mostly in the ventrolateral portion of the arcuate nucleus (ARC) and around the perimeter of the ventromedial nucleus (VMN). Comparison with the distribution of pericellular 125I-SRIH binding sites allowed to differentiate three types of cells: (1) GHRH perikarya not associated with pericellular 125I-SRIH binding sites around the perimeter of the VMN, (2) 125I-SRIH-labelled cells, not associated with GHRH perikarya in the periventricular zone along the dorsal part of the third ventricle, and (3) in the ventrolateral portion of the ARC, GHRH mRNA-labelled neurons had the same distribution as 125I-SRIH-labelled cells. Furthermore, on adjacent sections, the number of both labelled cells were correlated (r = 0.68; p less than 0.001). In this last population, the extent of colocalization of 125I-SRIH binding sites on GHRH mRNA-labelled neurons was further investigated in adjacent 5-microns-thick sections. The proportions of cells GHRH mRNA and 125I-SRIH allowed to differentiate three subdivisions of the arcuate: the periventricular (PV), ventrobasal (VB) and lateral portions. In the PV-ARC, 27% of GHRH-synthesizing cells were coidentified as 125I-labelled while only 6% of 125I-labelled cells contained GHRH mRNA. In the VB-ARC the proportion of double-labelled cells was equivalent (31 and 26%, respectively for GHRH mRNA and 125I-SRIH).(ABSTRACT TRUNCATED AT 250 WORDS)