Electrical activity in endocrine pituitary cells in situ: a support for a multiple-function coding.

The anterior pituitary is an endocrine gland that controls basic body functions. Pituitary cell functioning depends on membrane excitability, which induces cytosolic calcium rises. Here, we reported the first identification of small-amplitude voltage fluctuations that controlled spike firing in endocrine cells recorded in situ.

A single subunit (GB2) is required for G-protein activation by the heterodimeric GABA(B) receptor.

Although G-protein-coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the gamma-aminobutyric acid (GABA) type B receptor (GABA(B)R) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone.

Characterization of the methylation-sensitive promoter of the imprinted ZAC gene supports its role in transient neonatal diabetes mellitus.

ZAC is a recently isolated zinc finger protein that induces apoptosis and cell cycle arrest. The corresponding gene is imprinted maternally through an unknown mechanism and maps to 6q24-q25, within the minimal interval harboring the gene responsible for transient neonatal diabetes mellitus (TNDM) and a tumor suppressor gene involved in breast cancer. Because of its functional properties, imprinting status, and expression pattern in mammary cell lines and tumors, ZAC is the best candidate so far for both disease conditions.

PAC1 null females display decreased fertility.

We report here that PAC1 KO females display decreased fertility, whereas male fertility was normal. ICC on pituitary section showed that FSH, LH, and prolactin synthesis were not affected in KO mice. Moreover, the pituitary-gonadal axis responded properly to an acute fasting test in KO mice.

Rhythmic bursts of calcium transients in acute anterior pituitary slices.

Endocrine cells isolated from the anterior pituitary fire intracellular Ca2+ ([Ca2+]i) transients due to voltage-gated Ca2+ entry. However, the patterns of [Ca2+]i transients within the glandular parenchyma of the anterior pituitary are unknown. Here we describe, using real-time confocal laser microscopy, several spontaneous patterns of calcium signaling in acute pituitary slices prepared from male as well as cycling and lactating female rats.

5-HT4 receptors: cloning and expression of new splice variants.

On the basis of differences in the potencies and intrinsic activity of 5-HT4 receptor agonists in different biological models it has been suggested that there is heterogeneity among 5-HT4 receptors. Here, we report the molecular cloning of several 5-HT4 receptor splice variants in mouse, rat, and human brain. Our data suggest that the differences in efficacy of 5-HT4 ligands on 5-HT4 receptor-mediated responses in several tissues is due to differences in coupling efficiency rather than to the presence of different 5-HT4 receptor isoforms.

5-HT4 receptors: gene, transduction and effects on olfactory memory.

In this paper we discuss 1) the primary structures, pharmacology, and brain distribution of cloned 5-HT4 receptors; 2) the chromosomal localization of the h5-HT4 receptor; 3) whether benzamides are full or partial agonists because of a species or a coupling difference; 4) the intrinsic activity of 5-HT4 receptors and inverse agonism of GR125487 in COS-7 cells but not in colliculi neurons; 5) the modulation of 5-HT4 receptor binding and activity; and 6) the long-term blockade of K+ channels by 5-HT4 agonists and its effect on olfactory memory. We conclude that 1) the cloning of 5-HT4 receptors in different species using RT-PCR from different tissues reveals the presence of several splice variants for 5-HT4 receptors differing in the C-terminal part, downstream from the amino acid L358; 2) the pharmacological properties of 5-HT4 receptors are dependent on the cellular context in which they are expressed; and 3) 5-HT4 agonists can be added to the list of compounds having pro-cognitive properties.

PACAP-38 protects cerebellar granule cells from apoptosis.

Pituitary adenylate cyclase-activating polypeptides (PACAP-27 and -38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. PACAP receptors are expressed in different brain regions including the cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP-38 on apoptosis induced by potassium deprivation.

Identification of residues responsible for the selective binding of peptide antagonists and agonists in the V2 vasopressin receptor.

To improve our understanding of the functional architecture of G protein-coupled receptors, we have taken advantage of differences among mammalian species in ligand binding to search for the rat versus human selectivity determinants of the V2 vasopressin receptor and of its peptide ligands. Our data indicate that residue 2 of species-selective peptide antagonists such as d(CH2)5-[D-Ile2,Ile4, Tyr-NH29]arginine vasopressin controls their rat versus human selectivity. For species-selective agonists such as desmopressin, residues 1 and 8 modulate the binding selectivity.

Synthesis and biological activity of chimeric structures derived from the cytotoxic natural compounds dolastatin 10 and dolastatin 15.

The natural cytotoxic compounds dolastatins 10 and 15 exhibit great similarities in structure and in their biological activity profiles. Two compounds (1 and 2) formed by interchanging the dolaisoleuine residue of dolastatin 10 and the MeVal-Pro dipeptide of dolastatin 15 were synthesized in order to evaluate the possible equivalence of these units. These compounds can be considered as chimeras of dolastatins 10 and 15 formed by the N-terminal part of the former and the C-terminal part of the latter and vice versa.

A cluster of basic residues in the carboxyl-terminal tail of the short metabotropic glutamate receptor 1 variants impairs their coupling to phospholipase C.

Among phospholipase C-coupled metabotropic glutamate receptors (mGluRs), some have a surprisingly long carboxyl-terminal intracellular domain (mGluR1a, -5a, and -5b), and others have a short one (mGluR1b, -1c, and -1d). All mGluR1 sequences are identical up to 46 residues following the 7th transmembrane domain, followed by 313, 20, 11, and 26 specific residues in mGluR1a, mGluR1b, mGluR1c, and mGluR1d, respectively. Several functional differences have been described between the long isoforms (mGluR1a, -5a, and -5b) and the short ones (mGluR1b, -1c, and -1d).

Cloning and expression of human 5-HT4S receptors. Effect of receptor density on their coupling to adenylyl cyclase.

We have isolated a cDNA encoding the 5-HT4S receptor by RT-PCR on poly (A)+ RNA from both human heart and brain. The sequence homology with the rat and mouse 5-HT4 receptors was high: 93.8% of identity in the amino acid sequence.

Properties of a new radioiodinated antagonist for human vasopressin V2 and V1a receptors.

A vasopressin receptor antagonist, [1-(beta-mercapto-beta,beta-pentamethylenepropionic acid), 2-o-ethyl-D-tyrosine, 4-valine, 9-tyrosylamide] arginine vasopressin (d(CH2)5[o-ethyl-D-Tyr2,Val4,Tyr-NH9(2)]AVP), has been prepared. This antagonist is a potent antiantidiuretic, antivasopressor and antioxytocic peptide with pA2 values of 7.69-7.

Cloning, expression and pharmacology of the mouse 5-HT(4L) receptor.

Since most of our knowledge on pharmacological properties of brain 5-HT4 receptors have been discussed for mouse colliculi neurons, we cloned the corresponding receptor using the RT-PCR approach. As expected, the homology with the already cloned rat 5-HT(4L) receptor was high, revealing only 16 differences at the amino-acid level. One of the differences, proline75 in mouse, alanine75 in the already published rat sequences was not confirmed.

Alternative splicing in the N-terminal extracellular domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor modulates receptor selectivity and relative potencies of PACAP-27 and PACAP-38 in phospholipase C activation.

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides of the vasoactive intestinal peptide/secretin/glucagon family. We previously described alternative splicing of the region encoding the third intracellular loop of the PACAP receptor generating six isoforms with differential signal transduction properties (Spengler, D., Waeber, C.

Molecular and functional characterization of V1b vasopressin receptor in rat adrenal medulla.

In rat adrenal medulla, PCR experiments reveal the expression of messenger RNA encoding the gene for the V1b vasopressin receptor. Complementary DNA amplified sequences corresponded to the cloned rat pituitary V1b vasopressin receptor. Video microscopy experiments performed on fura-2-loaded adrenal medullary or adrenal glomerulosa cell primary cultures showed that vasopressin dose dependently mobilized intracellular calcium, suggesting that functional vasopressin receptors are expressed in these tissues.

Comparative effects of PACAP and VIP on pancreatic endocrine secretions and vascular resistance in rat.

1. The effects of pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP) and secretin on pancreatic endocrine secretions and vascular resistance were investigated and compared in the isolated perfused pancreas of the rat. The PACAP/VIP receptor types involved have been characterized.

[Large and small G proteins in vesicular transport].

The movement of proteins between compartments of the exocytic and endocytic pathways of eukaryotic cells is mediated by carrier vesicles. They bud from a donor compartment and are targetted to and fuse with the acceptor compartment. GTPases, proteins which bind and hydrolyze GTP, play key roles in the regulation of this vesicular protein transport.

The binding site of neuropeptide vasopressin V1a receptor. Evidence for a major localization within transmembrane regions.

To identify receptor functional domains underlying binding of the neurohypophysial hormones vasopressin (AVP) and oxytocin (OT), we have constructed a three-dimensional (3D) model of the V1a vasopressin receptor subtype and docked the endogenous ligand AVP. To verify and to refine the 3D model, residues likely to be involved in agonist binding were selected for site-directed mutagenesis. Our experimental results suggest that AVP, which is characterized by a cyclic structure, could be completely buried into a 15-20-A deep cleft defined by the transmembrane helices of the receptor and interact with amino acids located within this region.

Vasopressin stimulates steroid secretion in human adrenal glands: comparison with angiotensin-II effect.

Autoradiographic experiments using iodinated vasopressin analog revealed the presence of specific vasopressin-binding sites in the human adrenal cortex (zona glomerulosa and zona fasciculata). These receptors exhibited a good affinity for arginine vasopressin (3.3 nM), with classical V1a pharmacology and densities of 65 and 135 fmol/mg protein-enriched membranes from zona glomerulosa and fasciculata, respectively.

The metabotropic glutamate receptors: structure and functions.

Glutamate is the main excitatory neurotransmitter in the brain. For many years it has been considered to act only on ligand-gated receptor channels--termed NMDA, AMPA and kainate receptors--involved in the fast excitatory synaptic transmission. Recently, glutamate has been shown to regulate ion channels and enzymes producing second messengers via specific receptors coupled to G-proteins.

Mutation of valine residue unique to alpha subunit of Gs abolishes activation.

We recently characterized a decapeptide sequence (residues 367-376) that is important for the membrane association of the activated alpha subunit of Gs. We report here that when this sequence is replaced by the cognate sequence of Gi1 alpha subunit, the chimeric protein (Gsis alpha) still interacts with the membrane but cannot be activated, regardless of the mode of activation. Construction of various chimeras demonstrates that the single replacement of valine 367 by threonine, the cognate residue of Gi1 alpha subunit, fully reproduces the loss of activation.

Ca(2+)-dependent protein kinase C isoforms in rat pituitary hyperplasia: effect of in vivo treatment with quinagolide.

Ca(2+)-dependent protein kinase C (PKC) activity, diacylglycerol levels and PKC alpha, beta I, beta II and gamma expression were analyzed in the pituitary of female rats treated with estradiol alone (2 months) or in combination with quinagolide in the second month. Polymerase chain reaction (PCR) and Western blot analysis revealed the presence of PKC alpha, beta I and beta II isoenzymes in the rat pituitary gland but not of PKC gamma isoenzymes. Increases in pituitary weight and plasma prolactin levels induced by estradiol were associated with an increase in diacylglycerol pituitary content (1.