Bernadette Lutz-Bucher

Characterization of specific receptors for vasopressin in the pituitary gland

The present paper reports new findings concerning interaction of [3H]-Arginine-vasopressin with putative receptors in rat anterior pituitary gland. It shows the presence of a single type of receptor sites, with a limited binding capacity and a dissociation constant of nearly 1nM. The parent neurohormone oxytocin revealed weak affinity as compared with vasopressin [Ki = 100nM and Ki = 1nM, respectively]. None of the various peptides tested and, especially corticotropin-releasing factor CRF, competed for binding. Receptor characteristics appeared to be unaffected by lack of circulating vasopressin in Brattleboro rats presenting complete deficiency in synthesis of that peptide.

Differential screening of mutated SOD1 transgenic mice reveals early up-regulation of a fast axonal transport component in spinal cord motor neurons.

In the present study we analyze the molecular mechanisms underlying motor neuron degeneration in familial amyotrophic lateral sclerosis (FALS). For this, we used a transgenic mouse model expressing the Cu/Zn superoxide dismutase (SOD1) gene with a Gly(86) to Arg (G86R) mutation equivalent to that found in a subset of human FALS. Using an optimized suppression subtractive hybridization method, a cDNA specifically up-regulated during the asymptomatic phase in the lumbar spinal cord of G86R mice was identified by sequence analysis as the KIF3-associated protein (KAP3), a regulator of fast axonal transport. RT-PCR analysis revealed that KAP3 induction was an early event arising long before axonal degeneration. Immunohistochemical studies further revealed that KAP3 protein predominantly accumulates in large motor neurons of the ventral spinal cord. We further demonstrated that KAP3 up-regulation occurs independent of any change in the other components of the kinesin II complex. However, since the ubiquitous KIF1A motor is up-regulated, our results show an early and complex rearrangement of the fast axonal transport machinery in the course of FALS pathology.

Evidence for an inhibitory effect of nitric oxides on neuropeptide secretion from isolated neural lobe of the rat pituitary gland.

The present study aims at investigating the effect of pharmacological manipulation of nitric oxides (NOs) formation in the rat neurohypophysis on the secretion of vasopressin (AVP). We found that the NO synthase antagonist L-NAME and free-ferrous hemoglobin (an NO inactivator) produced a transient and significant enhancement of basal secretion of AVP from incubated glands. Conversely, the NO precursor L-arginine (but not its inactive counterpart D-arginine) antagonized the stimulatory influence of L-NAME on both AVP and oxytocin (OT) output. Elevation of NOs formation triggered by means of the NO donor SIN-1 likewise dampened spontaneous, as well as stimulated, AVP release. It is concluded that NOs molecules show up as potent regulators of neuropeptide secretion at the level of nerve terminals in the neurohypophysis.

Evidence for a direct inhibitory effect of morphine on the secretion of posterior pituitary hormones

The effect of morphine and naloxone on vasopressin and oxytocin release from incubated neurointermediate lobe and pars nervosa of rat pituitaries were investigated. It was shown that morphine and endorphins blocked hormonal output and that this inhibitory action was reversed by naloxone. It is concluded that opiates exert a direct inhibitory influence on vasopressin and oxytocin secretions from the neurohypophysis.

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates cyclic AMP formation as well as peptide output of cultured pituitary melanotrophs and AtT-20 corticotrophs

The present study was aimed at investigating whether PACAP stimulates accumulation of cAMP, as well as hormonal secretion of homogeneous populations of pituitary proopiomelanocortin (POMC) cells, namely melanotrophs and AtT-20 corticotrophs. PACAP was shown to enhance cAMP accumulation in a dose-dependent fashion in both cell types (with EC50 values of approx. 10(-10) M) and elicited additive increases of cAMP production with CRF in melanotrophs, but not in corticotrophs. PACAP also stimulated dose-dependently the secretion of alpha-MSH and ACTH, with EC50 concentrations of about 10(-9) M. In melanotrophs, bromocriptine significantly depressed PACAP-induced cAMP formation and blunted by more than 90% stimulated alpha-MSH release. This study shows that (1) pituitary POMC cells did respond to PACAP by enhancing cAMP accumulation and elevating hormone secretion as well; (2) the effect of PACAP was additive with CRF on cAMP production in melanotrophs, but not in corticotrophs, while there was no additivity on peptide output from both cell types; (3) activation of dopamine receptors in melanotrophs dampened both cAMP formation and peptide secretion. These findings are consistent with PACAP playing a possible hypophysiotropic role in the regulation of pituitary POMC cell activity.

Evidence for the Presence of Receptors for Pituitary Adenylate Cyclase-Activating Polypeptide in the Neurohypophysis that are Positively Coupled to Cyclic AMP Formation and Neurohypophyseal Hormone Secretion

This study examines the neural lobe of the pituitary gland for the presence of receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) and their possible involvement in the regulation of neurosecretion. The presence of PACAP receptors of type I was revealed in the neural lobe, as well as in anterior and intermediate lobes, by means of RT-PCR amplification using selective oligonucleotide pairs of primers. They appeared to be expressed in the tissues as a short form together with an isoform of heavier molecular weight. Activation of receptors in the presence of PACAP stimulated both formation of cyclic AMP (cAMP) and secretion of arginine vasopressin (AVP) in neural lobes, in a dose-related fashion, with half-maximum (EC50) values of 1.0 +/- 0.2 x 10(-9) M and 1.4 +/- 0.3 x 10(-8) M, respectively. Parallel with AVP, PACAP also stimulated oxytocin (OXT) output, with an EC50 value of 0.6 +/- 0.1 x 10(-8) M. In an attempt to localize receptors on cells (mainly astrocyte-like glials or pituicytes) and/or on nerve fibers of the gland, we used cultures of neural lobe cells and explants (in which nerve fibers undergo degeneration), as well as isolated nerve endings. In both cells and nerve terminals, PACAP enhanced accumulation of cAMP, while it triggered AVP secretion from the latter. The stimulatory effect of PACAP on both AVP and OXT release was mimicked by dbcAMP and blocked by H89, an inhibitor of cAMP-dependent protein kinase. We conclude that in the neural lobe, PACAP receptors are localized on both nerve terminals and pituicytes, which participate in the modulation of secretion of neurohypophyseal hormones in an interactive way and mainly through the cAMP signalling route.

Corticotropin-Releasing Factor-Like Immunoreactivity, Arginine Vasopressin-Like Immunoreactivity and ACTH-Releasing Bioactivity in Hypothalamic Tissue from Fetal and Neonatal Sheep

Corticotropin-releasing factor-like immunoreactivity (CRF-LI), arginine vasopressin-like immunoreactivity (AVP-LI) and ACTH-releasing bioactivity of hypothalamic tissue from 63- to 143-day-old ovine fetuses and from 7- and 150-day-old lambs have been assessed. CRF-LI and AVP-LI contents and concentrations increased steadily between 63 and 138 days of gestation, decreased at day 143 then rose again in postpartum animals. In terms of concentration, the AVP-LI/CRF-LI ratio remained close to 5 between 63 and 123 days of intrauterine life, decreased steadily in late gestation down to 1.2 at day 143 increased again in lambs. The total hypothalamic ACTH-releasing bioactivity increased 26-fold between 63 and 138 days of gestation and remained constant until day 143. The highest values were observed in 150-day-old-lambs. These data support the view that AVP and oCRF1-41 might be important hypothalamic factors involved in the regulation of ACTH release by the pituitary gland of the sheep fetus. In addition, they suggest that AVP is more important in young fetuses and lambs than in prepartum animals.

A mouse model of familial amyotrophic lateral sclerosis expressing a mutant superoxide dismutase 1 shows evidence of disordered transport in the vasopressin hypothalamo‐neurohypophysial axis

Amyotrophic lateral sclerosis (ALS) is a fatal, paralytic disorder that primarily affects motoneurons. By combining physiological and morphological approaches, we examined the effect of a murine superoxide dismutase 1 (SOD1) mutation (G86R), which induces neurological disorders resembling human familial ALS (FALS), on the arginine vasopressin (AVP) hypothalamo‐neurohypophysial axis, an unmyelinated tract poor in neurofilaments. First, we observed that G86R mice progressively consumed more water than wild‐type littermates. Furthermore, levels of plasma AVP and neurohypophysial AVP content were decreased in the SOD1 mutant mice, whereas the amount of hypothalamic AVP increased in an age‐dependent manner. However, hypothalamic AVP mRNA levels were not significantly modified in these animals. At the ultrastructural level, we found that the neurohypophysis of G86R mice had a decreased number of neurosecretory axons. Conversely, the presence of large axon swellings was more pronounced in the SOD1 mutant mice. In addition, the size of neurosecretory granules was higher in G86R than in wild‐type animals. All these findings strongly suggest that the FALS‐associated SOD1 mutation injures the hypothalamo‐neurohypophysial axis by provoking early, progressive disturbances in the axonal transport of neurosecretory products from neuronal perikarya to nerve terminals. This blockade could ultimately result in degeneration of the tract, as proposed for the myelinated, neurofilament‐enriched motor axons affected by ALS.

Indirect evidence that protein kinase C plays a critical role in signal transduction of both vasopressin and corticotropin-releasing pactor on pituitary cells in culture

The possible role of protein kinase C (PKC) in the cyclic AMP-dependent mechanism of action of corticotropin-releasing factor (CRF) on proopiomelanocortin cells of anterior and intermediate pituitary glands was examined after pretreatment of cells in culture with the PKC inhibitor retinal or the phorbol ester PMA, which depletes cell stores of the kinase. We found that these drugs not only abolished ACTH response to PMA and vasopressin, which both activate PKC, but unexpectably also dampened by 80-90% the stimulatory effect of CRF. Cell treatment with retinal failed to prevent CRF-induced accumulation of cyclic AMP. Retinal and PMA pretreatments of intermediate pituitary cells likewise inhibited alpha-MSH secretion stimulated by CRF. These data provide evidence to suggest that the mechanism of action of CRF on pituitary cells involves both cyclic AMP and PKC messenger systems.

Pituitary adenylate cyclase polypeptide (PACAP) stimulates cyclic AMP formation in pituitary fibroblasts and 3T3 tumor fibroblasts: Lack of enhancement by protein kinase C activation

A number of neuropeptides were shown to produce potent mitogenic effects on Swiss 3T3 fibroblasts by activating the phospholipase C pathway. Here we provide evidence for the activation by PACAP of the adenylate cyclase pathway in 3T3, as well as in non-tumoral pituitary fibroblasts, similarly to what was seen in pituitary endocrine cells. In these cells, PACAP triggered elevation of both intracellular and extracellular contents of cAMP and the effect was time- and dose-dependent, with half-maximal stimulations being induced with about 0.1 nM. Following activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA), PACAP-induced cAMP production was amplified in pituitary endocrine cells, but was either unchanged or dampened in 3T3 and pituitary fibroblasts, respectively. Pretreatment of cells with pertussis toxin (PT) failed to change the effect of PMA on PACAP-stimulated adenylate cyclase activity, irrespective of the cell type being used. However, PT dramatically reduced the potentiation by PMA of cAMP production enhanced by forskolin in 3T3 cells. These results provide new evidence pointing to the presence in fibroblasts of receptors for PACAP, coupled to cAMP production, which may play a role in the modulation of the mitogenic signal. They also indicate that, compared with pituitary endocrine cells, PKC activation in fibroblasts differentially affected PACAP-induced cAMP formation and that these effects were unaltered upon inhibition by PT of Gi-like proteins.