Neurons that express the AMPA receptor GluR2/3 subunits in suprachiasmatic nuclei of Syrian hamsters colocalize either vasoactive intestinal peptide, peptide histidine isoleucine or gastrin-releasing peptide.

In mammals, many circadian rhythms are driven by a clock located inside the suprachiasmatic nucleus of the hypothalamus. They are synchronized to environmental light-dark cycles by information coming directly from the retina via glutamatergic afferents. In rodents, retinal fibres make direct synaptic contacts with neurons synthesizing vasoactive intestinal peptide and gastrin-releasing peptide. These two neuropeptides, administered alone or combined with the peptide histidine isoleucine, phase-shift the clock in the same way that light does. Using ICC and light and electron microscopy, our study demonstrates that subunits 2 and 3 of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-type glutamatergic receptors are colocalized in neurons expressing one or other of these three neuropeptides. Double-labelled neurons were located in the ventral and lateral ventral parts and near the symmetrical plane of the intermediate and caudal thirds of the nucleus. In light microscopy, brown and granular blue stainings of chromogens revealing both antigens were easily identifiable and spatially separated in perikarya. In electron microscopy, almost all the cells observed in these zones expressed the receptor subunits. A few labelled dendritic profiles, some of them post-synaptic, were observed; axon terminals were always unlabelled. Colocalization with vasoactive intestinal peptide and gastrin-releasing peptide was confirmed by the immunogold technique in perikarya and some dendrites. The present study suggests that peptidergic neurons expressing the AMPA receptors are involved in photic entrainment of the clock by the retina without excluding some glutamatergic information coming from other hypothalamic nuclei.

Enhancement of glucose-induced insulin secretion in transgenic mice overexpressing human VIP gene in pancreatic beta cells.

Using transgenic mice technology, it has now become possible to test directly whether VIP and PHM-27 can enhance glucose-induced insulin secretion and reduce blood glucose in vivo. By microinjecting the entire human VIP gene ligated to the rat insulin II promoter, we have established a mouse model that overproduces VIP and PHM-27 in pancreatic beta cells. VIP was secreted from transgenic islets in a glucose-dependent manner. Analyses of these VIP-transgenic mice indicated that the transgene efficiently enhances glucose-induced insulin secretion and significantly reduces blood glucose as compared with control mice. The transgene also ameliorated glucose intolerance of 70% depancreatized mice. The present results suggest that somatic cell gene therapy directed to diabetic islets by human VIP/PHM-27 gene introduction may provide a means to improve the secretory function of the diabetic islets.

Vasoactive intestinal polypeptide/peptide histidine isoleucine messenger RNA in the rat retina: adult distribution and developmental expression.

In the adult nervous system, vasoactive intestinal polypeptide acts as a neurotransmitter or neuromodulator, and during development, it may also act as a neurotrophic factor. In the adult mammalian retina, this peptide is contained in a population of wide-field amacrine cells. Using in situ hybridization histochemistry, we examined the distribution and developmental expression of vasoactive intestinal polypeptide/peptide histidine isoleucine messenger RNA in the rat retina. Retinas collected from birth to adulthood were hybridized with an RNA probe as whole mounts, and then cut either perpendicular or parallel to the vitreal surface. Adult retinas were used in double labeling experiments for the visualization of both the hybridization signal and vasoactive intestinal polypeptide immunoreactivity in the same tissue section. In adult retinas, vasoactive intestinal polypeptide/peptide histidine isoleucine messenger RNA is localized to amacrine cells positioned in the proximal inner nuclear layer, and rarely to displaced amacrine cells in the inner plexiform layer and ganglion cell layer. The neurons expressing this messenger RNA are sparsely distributed, with a non-random distribution and densities of about 190 cells/mm2. An estimate of their total number gives about 12,350 cells/retina. The double labeling experiments showed that the hybridization signal is specifically confined to neurons displaying vasoactive intestinal polypeptide immunoreactivity. Vasoactive intestinal polypeptide/peptide histidine isoleucine messenger RNA is first detected at postnatal day 5 in cells located in the proximal part of the neuroblastic layer. A greater number of these neurons is present in the inner nuclear layer at postnatal day 10, and a few labeled neurons are also detected in the inner plexiform layer and in the ganglion cell layer. At this time, vasoactive intestinal polypeptide/peptide histidine isoleucine messenger RNA-containing amacrines in the inner nuclear layer are non-randomly distributed on the retinal surface, as in adult retinas. At postnatal day 15 (eye opening), there is a peak in both the density and the estimated number of labeled neurons, and their pattern of distribution in the retinal layers is similar to that in the adult. The present study shows that in the adult rat retina vasoactive intestinal polypeptide and peptide histidine isoleucine are synthesized in a sparsely distributed amacrine cell population, extending previous immunohistochemical findings. The appearance of vasoactive intestinal polypeptide peptide histidine isoleucine messenger RNA during the first postnatal week is consistent with the reported appearance of other transmitter-identified amacrine cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)

Gene expression and transcript size of the prepro-peptide VIP/PHM-27 in normal human tissue.

The transcript size of VIP/PHM-27 mRNA (vasoactive intestinal peptide/peptide histidine methionine) and the relative distribution of VIP/PHM-27 gene expression in 10 normal human tissues was examined. After mRNA extraction from tissue, VIP/PHM-27 transcript size and relative abundance of mRNA was determined by Northern blot analysis and densitometry of the autoradiograms. VIP/PHM-27 mRNA was detectable in brain, pancreas, colon, ileum and striated muscle while no hybridization signal was observed in liver, kidney, lung, heart, prostate and placental tissue. VIP/PHM-27 transcript in human brain and gut was a single band of 1.7 kb; by contrast, a 7.0-kb transcript was detected in striated skeletal muscle. The highest relative levels of mRNA were observed in brain and pancreas.

In situ hybridization reveals VIP precursor mRNA-containing neurons in monkey and rat neocortex.

The cDNAs encoding monkey vasoactive intestinal polypeptide (VIP) and PHM-27 and rat VIP and PHI-27 were cloned and used to generate antisense RNA probes. Using in situ hybridization, neurons expressing the VIP/PHM or VIP/PHI precursor mRNAs were localized in monkey and rat somatic sensory and visual cortex. In both neocortical areas of both species, labeled cells were observed in all 6 layers as well as the subcortical white matter.

Synthesis, cloning and expression in Escherichia coli of artificial genes coding for biologically active elongated precursors of the vasoactive intestinal polypeptide.

Synthetic genes coding for elongated precursors of the vasoactive intestinal polypeptide (VIP) were synthesized and cloned in a highly efficient Escherichia coli expression vector. The synthetic genes code for VIP with its methionine (at position 17) replaced by leucine and elongated at the C-terminus by Gly (vasoactive intestinal polypeptide-Gly, i.e. VIPa) or by Gly-Lys-Arg (vasoactive intestinal polypeptide-Gly-Lys-Arg, i.e. VIPb). The synthetic genes fused to the N-terminal part of the E. coli beta-galactosidase gene were expressed to yield high amounts of fusion proteins reaching upon induction at least 60% of the total cellular protein. The fusion proteins of 314 and 316 amino acids carrying in their C-terminal portion either the 29 or 31 amino acids long VIP precursor polypeptide were shown to be immunoreactive with VIP antisera and were further purified and cleaved by CNBr. The resulting purified peptide precursors (VIPa and VIPb) were recognized by VIP receptors in rat liver plasma membranes and by antibodies to porcine VIP in a radioimmunoassay. Both precursors activated adenylate cyclase in rat liver membranes and stimulated pancreatic secretion in the cat. The affinity and potency of the cloned precursors is close to that of VIP purified from porcine intestine, suggesting that the elongated VIP precursors may act even without being converted into the C-terminal amide form of the peptide. The elongated VIP precursors expressed in E. coli may provide a cheap, large-scale source of experimental material for studies on VIP actions.