Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.27.5 (RNase)
 17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies from this laboratory have demonstrated that the 5-hydroxytryptamine (5-HT2) receptor subtype is transcriptionally regulated by 5-HT (serotonin) itself in rat myometrial smooth muscle cells. To better understand this transcriptional regulation, we have isolated and characterized the 5'-flanking region of the 5-HT2 receptor gene. Screening of a rat genomic library was accomplished using 5'-directed fragments of 5-HT2 cDNA, and a 5.2-kilobase fragment was isolated. Sequencing demonstrated that the fragment overlapped the 5'-end of the 5-HT2 cDNA by 226 base pairs. Primer extension and RNase protection analyses indicated that three transcriptional start sites, which are common to both rat brain and myometrium, appear to exist and that the 5'-untranslated region of the 5-HT2 receptor cDNA is 1120 base pairs long. Neither classical TATA boxes nor CCAAT sequences were found upstream of any of the start sites identified. Upstream of the dominant start site, however, an initiator consensus sequence, two GC boxes (SP-1 binding sites), and several AP-2 binding sites were identified. Based on this information, a 1.4-kilobase fragment beginning 64 base pairs downstream from the dominant start site was constructed by polymerase chain reaction and ligated into a pCAT vector. Transient transfection of this construct into rat myometrial smooth muscle cells displayed both constitutive and serotonin-induced promoter activity. Serotonin-inducible activity was abolished by a selective 5-HT2 receptor antagonist; however, antagonists selective for other 5-HT receptor subtypes were without effect. Conversely, a selective 5-HT2 receptor agonist completely substituted for serotonin as an inducer. Preliminary deletion experiments indicate that regulation of basal and serotonin-inducible activity likely depends upon different cis elements in the 5-HT2 receptor gene promoter.
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PMID:Isolation and characterization of the rat 5-hydroxytryptamine type 2 receptor promoter: constitutive and inducible activity in myometrial smooth muscle cells. 802 6

Although serotonin (5-hydroxytryptamine; 5-HT) is used for provocation of coronary spasm, 5-HT receptor subtypes in spastic coronary arteries remain undetermined. We demonstrated the supersensitivity of isolated coronary artery to ergonovine, 5-HT, and sumatriptan, a 5-HT1D receptor agonist, in a patient with variant angina. Furthermore, we detected gene expression of 5-HT1Dbeta and 5-HT2A receptors in spastic coronary artery using RNase protection assay. These findings suggest that the leftward shift of the dose-response curve for 5-HT, which plays an important role in the pathogenesis of coronary spasm, is mediated by activation of 5-HT1Dbeta receptor.
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PMID:5-HT1Dbeta receptor mediates the supersensitivity of isolated coronary artery to serotonin in variant angina. 944 May 99

Astrocytes are coupled via gap junctions, predominantly formed by connexin-43 proteins, into cellular networks. This coupling is important for the propagation of intercellular calcium waves and for the spatial buffering of K+. Using the scrape-loading/dye transfer technique, we studied gap junction permeability in rat astrocytes cultured from four different brain regions. The cultures were shown to display regional heterogeneity with the following ranking of the gap junction coupling strengths: hippocampus = hypothalamus > cerebral cortex = brain stem. Similar relative patterns were found in connexin-43 messenger RNA and protein levels using solution hybridization/RNase protection assay and western blots, respectively. The percentages of the propagation area of mechanically induced intercellular calcium waves for cortical, brain stem and hypothalamic astrocytes compared with hippocampal astrocytes were approximately 77, 42, and 52, respectively. Thus, the extent of calcium wave propagation was due to more than just gap junctional permeability as highly coupled hypothalamic astrocytes displayed relatively small calcium wave propagation areas. Incubation with 5-hydroxytryptamine decreased and incubation with glutamate increased the calcium wave propagation area in hippocampal (67% and 170% of the control, respectively) and in cortical astrocytes (82% and 163% of the control, respectively). Contrary to hippocampal and cortical astrocytes, the calcium wave propagation in brain stem astrocytes was increased by 5-hydroxytryptamine incubation (158% of control), while in hypothalamic astrocytes, no significant effects were seen. Similar effects from 5-hydroxytryptamine or glutamate treatments were observed on dye transfer, indicating an effect on the junctional coupling strength. These results demonstrate a strong relationship between connexin-43 messenger RNA levels, protein expression, and gap junction permeability among astroglial cells. Furthermore, our results suggest heterogeneity among astroglial cells from different brain regions in intercellular calcium signaling and in its differential modulation by neurotransmitters, probably reflecting functional requirements in various brain regions.
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PMID:Extent of intercellular calcium wave propagation is related to gap junction permeability and level of connexin-43 expression in astrocytes in primary cultures from four brain regions. 1039 48

The RNase protection assay (RPA) is an extremely sensitive procedure for detection of messenger RNA (mRNA) in complex sample mixture of total RNA. However, its usefulness has been limited by the requirement for the DNA to be cloned onto an appropriate vector. We have utilized the polymerase chain reaction (PCR) to directly incorporate a T7 RNA polymerase promoter sequence onto the cDNA for the 5-hydroxytryptamine(1B) (5-HT(1B)) receptor. Radiolabeled riboprobe was then synthesized using the PCR product as a template and used in RPA to detect mRNA for 5-HT(1B) receptor in rat brain. The internal control was the beta-Actin mRNA. Due to the simplicity of its design and the lack of need for subcloning, the DNA template synthesis by PCR facilitates the implementation of the RPA. Since the 5-HT(1B) receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters and the protocol described here permits the determination of 5-HT(1B) receptor mRNA levels in the rat cerebellum, striatum, hippocampus and frontal cortex, this protocol is helpful in understanding the involvement of 5-HT(1B) receptors in various physiological phenomena.
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PMID:Application of the polymerase chain reaction to the RNase protection assay for 5-HT(1B) receptor mRNA levels measurement in rat brain tissues. 1059 41

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.
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PMID:Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome. 2643 82

Gastrointestinal enterochromaffin cells regulate bone and gut homeostasis via serotonin (5-hydroxytryptamine [5-HT]) production. A recent report suggested that gut microbes regulate 5-HT levels; however, the precise underlying molecular mechanisms are unexplored. Here, we reveal that the cation channel Piezo1 in the gut acts as a sensor of single-stranded RNA (ssRNA) governing 5-HT production. Intestinal epithelium-specific deletion of mouse Piezo1 profoundly disturbed gut peristalsis, impeded experimental colitis, and suppressed serum 5-HT levels. Because of systemic 5-HT deficiency, conditional knockout of Piezo1 increased bone formation. Notably, fecal ssRNA was identified as a natural Piezo1 ligand, and ssRNA-stimulated 5-HT synthesis from the gut was evoked in a MyD88/TRIF-independent manner. Colonic infusion of RNase A suppressed gut motility and increased bone mass. These findings suggest gut ssRNA as a master determinant of systemic 5-HT levels, indicating the ssRNA-Piezo1 axis as a potential prophylactic target for treatment of bone and gut disorders.
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PMID:RNA Sensing by Gut Piezo1 Is Essential for Systemic Serotonin Synthesis. 3264 Jan 90