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Vasoactive intestinal peptide (VIP) concentrations were shown to be regulated by adrenal steroids. Therefore, we investigated whether adrenal steroids affect VIP mRNA levels, which would suggest an effect on VIP mRNA expression. Adrenalectomy performed on adult male rats resulted in a significant decrease in VIP mRNA in the hypothalamus (from 10.6 +/- 0.3 to 3.5 +/- 0.2 arbitrary units). In situ hybridization experiments revealed that a major site of VIP mRNA expression in the hypothalamus is the suprachiasmatic nucleus. Indeed, adrenalectomy resulted in an approximate decrease by half in VIP transcripts in this nucleus. However, this decrease was not reversed by replacement treatment with corticosterone or the glucocorticoid agonist, RU28362. Thus, VIP mRNA may be regulated by indirect mechanisms, influenced by the adrenal gland.

Vasoactive intestinal polypeptide (VIP) is a regulatory neuropeptide/neurotransmitter of 28 amino acids involved in a wide variety of physiological functions. Using synthetic oligodeoxynucleotide probes related to the rat VIP-cDNA, we have isolated and characterized the gene encoding the rat pre-pro VIP/PHI-27 and compared it to the human VIP gene. The rat VIP gene spanned 7400 base pairs, and contained 7 exons interrupted by 6 introns. 100% identity was found between the gene exons and the cDNA sequence. Differences in sizes of introns 2, 4 and 5 (shorter in the rat gene) are the reason for the shorter rat gene compared with the human gene of 8837 base pairs. Comparison of the genes in the two species showed a high homology in the exon sequences, 80-90% in exons 2, 4, 5, 6 and 30-50% in exons 1 and 7. In addition, the exon-intron junctions shared high identity between the genes. The rat untranslated exon 1 had little homology (30%) with human exon 1 and was 13 base pairs shorter. Interestingly, the 160 base pairs at the 5'-flanking region upstream of the cap-site share more than 75% identity between the two genes, including the exact position of TATA-boxes in positions -28, -145, -155, a cAMP-responsive element in position -80 and a CAAT sequence in position -127. The conservation of the 5'-flanking region of the VIP gene in parallel with the conservation of its coding exons emphasize the importance of these sequences during evolution.

To identify the VIP biosynthetic pathways, we have isolated the human VIP gene, using synthetic oligodeoxynucleotides. These specific hybridization probes were constructed according to the neuroblastoma VIP-cDNA sequence and contained up to 39 bases. The gene structure was deduced by direct chemical nucleotide sequencing. Six exons were thus far discovered; among them two short exons, one encoding VIP and the second encoding PHM-27 (a peptide having a N-terminal histidine and C-terminal methionine amide, closely related in sequence and activity to VIP). As a model system for VIP gene expression, we used a human buccal tumor producing elevated amounts of VIP. In these cells, a major transcript of the VIP-gene was identified as a long RNA containing intron sequences. The occurrence of elevated quantities of a high molecular weight, intron containing, gene transcript which is not processed directly into mature RNA suggests that VIP gene expression may be regulated at the RNA processing level.

Vasoactive intestinal peptide (VIP) gene transcripts were demonstrated by RNA blot hybridization using VIP-specific RNA hybridization probes. High levels of expression were observed as early as in 16-day-old embryos. In aging rats, the VIP-mRNA levels were reduced significantly (in the cerebral cortex) as compared to 21-day-old rats. Our results suggest a role for the VIP gene protein products during embryonal development. During aging processes the decrease in VIP gene transcripts may be a consequence of either a reduction in the transcriptional activity of VIP neurons or death of VIP-producing cells.

Vasoactive intestinal peptide (VIP) has been proposed as an inducer of PRL release. Moreover, immunocytochemical studies suggested an increase in the VIP content in the hypothalamus of lactating rats. We investigated whether this increase is regulated at the level of gene transcription. A sensitive RNA detection assay that uses in vitro transcribed RNA probes corresponding to specific exons of the VIP gene was devised to study VIP gene expression. Using this method, an approximately 2000-base long RNA containing the coding sequences for VIP was detected in rat hypothalamus. This RNA also contains the coding sequences for the VIP-related peptide PHM/I (peptide histidine methionine amide or isoleucine amide). An identical VIP-encoding RNA was previously identified in the rat cerebral cortex. A 2-fold increase was observed in hypothalamic VIP mRNA during lactation. In addition, the levels of this mRNA increased in the hypothalamus at the time of sexual maturation. Taken together, our results suggest a physiological regulation of VIP gene expression associated with its potential role as a neuroendocrine hormone.

The human gene coding for vasoactive intestinal peptide was recently isolated and shown to contain seven exons. We now demonstrate that an intron-containing precursor RNA can be the major vasoactive intestinal peptide-related RNA in the cell, which is in contrast to most known genes. By RNA blot analysis using a variety of genomic and cDNA-related probes we show that in a human tumor producing vasoactive intestinal peptide, most of the RNA encoding the peptide is of the precursor type. Similar precursor transcripts were found in total rat brain RNA as well. A proof of the identity of the intron-containing RNA, cDNA clones corresponding to this RNA sequence have been isolated.

In view of the potential biological importance of VIP, we have begun to examine the regulation of its biosynthesis. For this purpose we have, as a first step, searched for an enriched source of VIP biosynthesis. By a combination of chromatographic procedures and radioimmunoassays we discovered an as yet unknown source for VIP production, namely a human buccal tumor, containing 0.67 +/- 0.05 ng VIP/micrograms protein which is greater than the richest source in brain (the cerebral cortex). Thus, we decided to use the tumor tissue for VIP-mRNA purification and characterization. To identify VIP-mRNA we are using as hybridization probes, synthetic oligodeoxynucleotides with relatively unambiguous nucleotide sequence complementary to the predicted VIP-mRNA sequence. These probes are synthesized, using the deoxynucleoside phosphoramidite approach, to a length of 17 bases each, and contain all the possible DNA sequences according to the genetic code. These specific probes are then radioactively labelled using the reaction catalyzed by the enzyme polynucleotide kinase and afterwards hybridized to mRNA, which had been resolved on denaturing agarose gels. Employing this approach, we identified a single putative VIP-mRNA band which was then partially purified by sucrose gradient centrifugation. Upon in vitro translation in a rabbit reticulocyte lysate cell free system, this mRNA was found to code for VIP immunoreactive proteins. In conclusion, our studies suggest the existence of high molecular weight precursors to VIP cross-reactive with anti-VIP antibodies, that are coded for by a partially purified mRNA containing VIP sequences.

To understand the regulation of the production of peptide hormones, it is vital to elucidate their biosynthetic pathways. We chose to study a major regulatory peptide, vasoactive intestinal peptide (VIP), a peptide possessing both neurotransmitter and neurohormone actions. To identify the specific peptide mRNA we are using, as hybridization probes, radiolabeled synthetic oligodeoxynucleotides with sequence complementary to the predicted peptide mRNA sequence. Employing this approach, we identified and partially purified a approximately 1600-base long mRNA containing VIP related sequences which can be translated in vitro into VIP-immunoreactive polypeptides. Such mRNA was detected in normal VIP producing tissue (rat brain), as well as in a tumor producing VIP (human buccal tumor). This mRNA differs in size from a known VIP-mRNA identified in human neuro-blastoma cells, suggesting the possibility of different VIP-mRNAs in different cell types.

The participation of gonadal steroid hormones in regulation of the vasoactive intestinal peptide (VIP) gene expression in the hypothalamus was studied using a quantitative densitometric hybridization assay. In the female rat the levels of VIP mRNA were found to be significantly decreased following ovariectomy (4.41 +/- 0.7 arbitrary units of absorbance vs. 8.52 +/- 0.18). This decrease was largely reversed after three days of treatment with estradiol dibenzoate. In contrast to the female rats, no significant change in VIP mRNA levels was observed in the male rats, following orchidectomy. These results suggest a sexual dimorphism with regard to the steroid regulation of hypothalamic VIP gene expression in the rat.

Vasoactive intestinal peptide (VIP) is a major regulatory peptide in the nervous system, playing a role in normal brain activity. VIP levels change dramatically during postnatal rat brain development, raising the question of how these changes are regulated. To study VIP-gene expression, a sensitive RNA detection assay which uses in vitro-transcribed RNA hybridization probes, corresponding to 4 exons of the VIP-gene, was adapted. Results show that the major VIP-mRNA was 2000-2100 bases long in the rat. The amounts of this RNA varied markedly with development. In the frontal cortex of the rat brain, the 2000-2100-base mRNA increased by at least 5-fold from birth to 3-4 days, showing a maximal content at 14-16 days. VIP-mRNA synthesis therefore apparently precedes peptide synthesis by several days, as VIP in the rat cortex begins to increase only at about 7 days of age. Similarly, in the parietal cortex, VIP-mRNA was detected by 3 days of age. However, the increase in the mRNA content from 3 to 14 days of age was greater than in the frontal cortex, while almost no VIP-mRNA was detected in the newborn rat parietal cortex. In contrast, the hypothalamus and intestine contained significant quantities of VIP-mRNA at birth, the hypothalamic levels in newborns being much higher than anticipated from the peptide levels. In the hippocampus, the major peak in VIP-mRNA content occurred at 8 days of age. Taken together, these results indicate local controls of VIP-gene expression and a developmentally associated role for VIP-gene products. As the VIP-mRNA levels did not always parallel the peptide levels, regulation at the post-transcriptional stage may be essential for normal VIP function.

Vasoactive intestinal peptide (VIP) is a major neuropeptide involved in multiple functions such as vasodilation, smooth-muscle relaxation, sweat secretion, gastrointestinal peristalsis, pancreatic function, and brain activity. In view of the multiple roles associated with VIP, it is important to understand its gene regulation. We have recently isolated the human VIP gene and determined its structure. By in situ hybridization techniques we have now localized this gene to the long arm of chromosome 6, 6q24, a chromosomal region that has been shown previously to contain the coding sequences for the nuclear protooncogene c-myb. Genes found in close proximity to each other on the chromosome are often functionally related and, as VIP is primarily expressed in the nervous system, we investigated the possible correlation of c-myb to VIP in neuronal tissue. A sharp peak of c-myb mRNA was observed in the hippocampus of 3-day-old rats, preceding the peak of VIP mRNA that occurs in this brain area at 8 days of age. Thus, the protooncogene c-myb may be associated with events in brain development occurring prior to the appearance of elevated concentrations of VIP.