Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and alpha 1b-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca2+]i) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+]i which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.

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The human retinoblastoma susceptibility gene, a member of the tumor suppressor gene family, is located on chromosome 13q14.12-13q14.2 and consists of 27 exons that are distributed over 180 kb. This study shows that intron 17, the largest in size, consisting of nearly 72,000 bp, contains an open reading frame encoding a novel G protein-coupled receptor in the reverse orientation relative to the transcription of the retinoblastoma susceptibility gene. Correction of a frameshift mutation revealed that this novel G protein-coupled receptor is the human homolog of a chicken T-cell-specific receptor cDNA. This is an additional description of an actively transcribed protein-encoding gene positioned within an intron of another gene, suggesting that introns can have important structural functions.A common feature of eukaryotic genes is the presence of intervening sequences or introns, which must be excised from each RNA transcript to convert the precursor to an mRNA molecule that can code for the synthesis of a continuous protein.Introns range in size from <100 to >10,000 nucleotides and differ significantly from coding regions in exons, in that most of the nucleotide sequence of an intron can be altered without greatly affecting gene function. In addition, introns seem to have accumulated repetitive sequences and mutations rapidly during evolution, leading to the suggestion that they have no function at all and are largely genetic "junk." An exception to the view that introns do not encode functionally important sequences occurs in several genes specifying proteins involved in RNA processing and translation. Such genes often encode in their introns small nucleolar RNAs (snoRNAs), suggesting that cotranscription of snoRNAs with mRNAs for ribosomal or nucleolar proteins may provide a regulatory mechanism to coordinate accumulation of the different components required for ribosome biogenesis (Kiss et al. 1995;Tycowski et al. 1996). Although the majority of eukaryotic genes contain introns, to date such sequences have not been found to add to the level of genetic complexity by containing other protein-encoding genes. Only a few genes are known that contain additional open reading frames (ORFs) in their introns, such as the neurofibromatosis type 1 gene (Cawthorn et al. 1990;O'Connell et al. 1990;Viskochil et al. 1991). We show here that an actively transcribed gene encoding a T-cell specific, G protein-coupled receptor is located within intron 17 of the human retinoblastoma (RB) susceptibility gene.The human RB gene is located on chromosome 13q14.12-13q14.2 and consists of 27 exons that are distributed over 180 kb (Toguchida et al. 1993). The gene belongs to a family of tumor suppressor genes, and defects in this locus are responsible for retinoblastoma, a malignant tumor arising in the eyes of young children. Normally a 4.7-kb mRNA transcript is produced that encodes a 928 amino acid nuclear phosphoprotein. Of the 26 introns, intron 17 is the largest in size, consisting of nearly 72,000 bp.In the process of using PCR with ...

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beta cells in the human fetal pancreas are immature in that they release little or no insulin in response to nutrients, such as glucose. The aim of this study was to examine further the immaturity of these cells, specifically regarding the storage and release of the precursor of insulin, proinsulin. Explants of human fetal pancreas were cultured in vitro for 3 weeks. Levels of proinsulin remained relatively constant throughout at 0.04 +/- 0.002 (S.E.M.) pmol/mg per day with a molar ratio of proinsulin to insulin of 2.2 +/- 0.11%. This low ratio was slightly greater than that observed in culture medium conditioned by adult human islets (0.3 +/- 0.1%), but similar to that found in acid-ethanol extracts of cultured explants (1.4 +/- 0.3%). Passaging of human fetal pancreas for 3 months in diabetic nude mice, which should have caused some maturation of the fetal beta cell, did not change the proportion of proinsulin present. Culture of explants in the presence of 12-O-tetra-decanoylphorbol-13-acetate resulted in some inhibition of proinsulin release, but much less than that for insulin, so that the molar ratio increased to 15.4 +/- 1.6% from the control 3.5 +/- 0.3%. Static stimulation of cultured explants with 10 mmol Ca2+/l, 10 mmol theophylline/l, and these two agents together caused 15-, 4- and 10-fold enhancement respectively of proinsulin release; glucose, leucine, arginine and KCl had no effect. In contrast, all these agents caused significant insulin release, the last four to a much smaller extent (less than or equal to three fold) than the first three (10-, 19- and 65-fold respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

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