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Query: UNIPROT:P06889 (Mol)
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In the present paper, P1 and P2 purinergic receptors and their control of signal transduction pathways were investigated in NCB-20 cells. ATP elicited an increase in [Ca2+]i. The purinergic receptor subtype involved was identified by comparing the actions of a range of nucleotides. UTP was the most potent agonist in elevating [Ca2+]i, with an EC50 value of 6.2 +/- 0.5 microM. UTP, ATP (EC50, 17.3 +/- 1.5 microM), adenosine-5'-O-(3-thio)triphosphate (23 +/- 3 microM), and ITP (55 +/- 4 microM) exerted similar maximal effects. Other nucleotides tested, including beta, gamma-methylene-ATP and 2-methylthio-ATP, which are considered prototypic agonists for P2x and P2y receptors, respectively, were ineffective; in general, modifications in the ribose-triphosphate chain and substitution on the 2-position of the purines reduced the efficacy of nucleotides. This pharmacological characterization indicated that a putative P2u receptor mediates the [Ca2+]i elevation elicited by nucleotides in NCB-20 cells. The increase in [Ca2+]i originates from intracellular Ca2+ stores; blockade of Ca2+ entry does not affect the rise in [Ca2+]i. In contrast, pretreatment with the Ca(2+)-ATPase inhibitor thapsigargin or with bradykinin, a hormone that releases Ca2+ from inositol trisphosphate-sensitive stores, does preclude the increase in [Ca2+]i induced by ATP. ATP and UTP also transiently inhibit cAMP accumulation in the intact cell, presumably via a Ca(2+)-mediated mechanism. The finding of a P2u receptor in NCB-20 cells adds to a growing perception that P2 receptors are widely distributed. Besides the P2u receptor, NCB-20 cells express adenosine A2 receptors, coupled to stimulation of cAMP accumulation. The presence of both P1 and P2 purinergic receptors permits a sequential modulation of distinct second messenger levels associated with a common stimulus, ATP.
Mol Pharmacol 1992 Apr
PMID:Purinergic receptor regulation of signal transduction in NCB-20 cells. 131 45

Previous studies showed that apolipoprotein-E (apoE) mRNA is regulated in rat adrenal gland by treatments that alter adrenal gland cholesterol content and steroidogenesis. In the present study cell types expressing apoE mRNA were determined by in situ hybridizations using an [alpha-35S]UTP-labeled RNA probe. Autoradiographic grains were counted to compare apoE expression in adrenal glands from control and experimentally treated animals. In control adrenal gland, zona (z.) fasciculata and z. reticularis exhibited the highest level of apoE mRNA expression, with lower levels in z. glomerulosa and medulla. Dexamethasone (DEX) treatment selectively increased apoE mRNA 3-fold in outer z. fasciculata, but not in other adrenal zones. ApoE mRNA expression appeared to be lower in adrenal glands from 4-aminopyrazolopyrimidine-treated rats, in that differences among adrenal gland zones were abolished. DEX treatment increased adrenal gland cholesteryl ester and oil red O staining in z. fasciculata cells in which the apoE mRNA concentration was increased as well as in other cortical cells in which apoE mRNA was unchanged. Aminoglutethimide administration led to a large increase in oil red O staining throughout the cortex, including z. fasciculata, without affecting apoE mRNA expression. These data suggest that adrenal gland apoE mRNA expression is not closely coupled to cellular cholesterol concentrations. Increased apoE mRNA expression in z. fasciculata of DEX-treated animals suggests an inverse relationship between apoE mRNA concentration and the level of steroidogenesis. This result is consistent with the proposal that apoE may play a role in regulating the utilization of cholesterol for steroid production.
Mol Endocrinol 1992 Feb
PMID:Differential regulation of apolipoprotein-E messenger RNA in zona fasciculata cells of rat adrenal gland determined by in situ hybridization. 137 19

The ability of deoxycytidine kinase (dCK) to phosphorylate 2'-deoxycytidine (dCyd) and its analogs in the presence of eight nucleoside triphosphates (NTPs), simulating the cellular milieu, was investigated. Using highly purified dCK from MOLT-4 T lymphoblasts, Km and Vmax values were determined for the phosphorylation of dCyd in the presence of cellular concentrations of the eight endogenous NTPs. The results demonstrated that the efficiency of dCyd phosphorylation was greatest in the presence of all eight nucleotides, relative to ATP alone, according to relative Vmax/Km values. UTP was a better phosphate donor than ATP but was less efficient than the NTP mixture. The greater efficacy of the NTP mixture, compared with ATP alone, was due in large part to the presence of UTP, although the results suggested that the presence of other nucleotide(s) also enhanced dCyd phosphorylation. Previous results demonstrated that dCTP was a potent competitive or noncompetitive (with respect to dCyd) inhibitor of dCK, with a Ki value of approximately 1 microM. In contrast, the results presented here demonstrated that, in the presence of either the NTP mixture or UTP, inhibition of dCK was uncompetitive with respect to dCyd, with a Ki value of approximately 60 microM. Furthermore, the results demonstrated that the clinically relevant nucleoside analogs 1-beta-D-arabinofuranosylcytosine, 2',2'-difluoro-2'-deoxycytidine (dFdC), and 9-beta-D-arabinofuranosyl-2-fluoroadenine also preferred UTP or the NTP mixture, compared with ATP alone, as a phosphate donor. Of the three nucleoside analogs tested, dFdC was the most efficient dCK substrate. These data indicate that the preferred phosphate donor for dCK is UTP or a combination of UTP and another nucleotide. Furthermore, the dCTP concentration in intact cells, which is typically 10-20 microM, is not sufficient to cause substantial inhibition of dCK, due to the presence of UTP. Strategies to increase cellular dCK activity should focus on optimizing UTP concentrations.
Mol Pharmacol 1992 Sep
PMID:Nucleotide specificity of human deoxycytidine kinase. 140 3

The earliest time of onset of embryonic genome activation in golden hamsters was investigated. The inhibition of transcription by alpha-amanitin (11 micrograms/ml) in cultured embryos resulted in a total arrest of development of early 2-cell embryos (26 hr post-egg activation); under similar conditions, immediate cleavage divisions of 1-, late 2-, 4-, and 8-cell embryos were not affected. Electrophoretic analysis of [35S]methionine-labeled embryonic proteins showed that alpha-amanitin treatment apparently inhibited transcription-dependent protein synthesis in early 2-cell and, to some extent, in late 2-cell when compared to 4-cell embryos. Analysis of total RNA synthesis, using [alpha 32P]-UTP or [32P]-orthophosphate, showed that there was a high proportion of radioactivity associated with the macromolecular fraction (RNA) at the early and late 2-cell stages and at the 4-cell stage compared to that at the 1-cell stage. These results indicate that the de novo synthesis of RNA, encoded by the embryonic genome, occurs at the 2-cell stage and that the second and subsequent cleavage divisions of hamster preimplantation embryos are dependent on new transcriptional activity. This initial activity of the embryonic genome in hamsters is coincident with several characteristic features of in vitro development such as a block to development, synthesis of major proteins, change in energy substrate preference, phosphate-inhibition of development and a requirement for amino acids.
Mol Reprod Dev 1992 Jul
PMID:Golden hamster embryonic genome activation occurs at the two-cell stage: correlation with major developmental changes. 149 72

We have investigated the characteristics of the receptor for ATP on neuronal cells and the involvement of phospholipase C and phospholipase D in the effector mechanisms, using PC12 rat phaeochromocytoma cells in culture. We show that the cells respond, with generation of total inositol phosphates, to ATP and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) but not to 2-methylthioadenosine5'-triphosphate (2MeSATP), beta,gamma-methylene ATP, or adenosine 5'-O-(2-thiodiphosphate) (ADP beta S). The largest response to ATP gamma S was mainly independent of extracellular calcium, had an EC50 of 7.93 +/- 0.76 microM, and was competitively inhibited by the nonspecific antagonist suramin. The pyrimidine nucleotide UTP also elicited a response in these cells. Measurement of [3H]inositol triphosphate showed a rapid rise to maximum (10-15 sec) in response to both ATP gamma S and UTP but no response to 2MeSATP. Cells prelabeled with 32Pi and stimulated in the presence of 50 mM butanol responded to ATP gamma S, ATP, and UTP with enhanced formation of [32P]phosphatidylbutanol as well as [32P]phosphatidic acid, indicating that agonist-stimulated phosphatidic acid occurs by both phospholipase D and phospholipase C activity. The stimulation of phospholipase D was inhibited by the presence of a protein kinase C inhibitor, Ro 31-8220. The dose-response curve for the stimulation by ATP gamma S of phospholipase C was shifted to the right by the presence of UTP, indicating that both compounds act on the same receptors. The data provide the first evidence for the existence of a nucleotide receptor on neuronal cells (insensitive to both purines and pyrimidines) and show that this receptor is linked to both phospholipase C and phospholipase D.
Mol Pharmacol 1992 Mar
PMID:Neuronal "nucleotide" receptor linked to phospholipase C and phospholipase D? Stimulation of PC12 cells by ATP analogues and UTP. 154 77

The five small nuclear RNAs (snRNAs) involved in splicing occur on the loops of amphibian lampbrush chromosomes and in hundreds to thousands of extrachromosomal granules called B snurposomes. To assess the role of these snRNAs during transcription and to explore possible relationships between the loops and B snurposomes, we injected single-stranded antisense oligodeoxynucleotides (oligos) against U1 and U2 snRNA into toad and newt oocytes. As shown before, antisense U1 and U2 oligos caused truncation of U1 and complete destruction of U2 snRNAs, respectively. However, injection of any oligo, regardless of sequence, brought on dramatic cytological changes, including shortening of the chromosomes and retraction of the lateral loops, with concomitant shutdown of polymerase II transcription, as well as disappearance of some or all of the B snurposomes. When injected oocytes were incubated for 12 h or longer in physiological saline, these changes were reversible; that is, the chromosomes lengthened, transcription (detected by 3H-UTP incorporation) resumed on newly extended lateral loops, and B snurposomes reappeared. In situ hybridization showed that loops and B snurposomes had negligible amounts of U2 snRNA after recovery from injection of the anti-U2 oligo, whereas these structures had normal levels of U2 snRNA after recovery from a control oligo. Thus, the morphological integrity of B snurposomes and lampbrush chromosome loops is not dependent on the presence of U2 snRNA. Because transcription occurs in the absence of U2 snRNA, we conclude that splicing is not required for transcription on lampbrush chromosome loops.
Mol Biol Cell 1992 Mar
PMID:Transcription on lampbrush chromosome loops in the absence of U2 snRNA. 162 29

The mechanism underlying the formation of easily releasable myofilaments, from myofibrils treated with an ATP-containing relaxing solution, was examined in this investigation. The proportion of releasable myofilaments purified from myofibrils of cardiac, fast- and slow-twitch muscles increased as the [ATP] was raised from 0 to 8.5 mM. The protein composition of the easily releasable myofilaments did not differ with increasing ATP concentrations as observed by 5-15% linear gradient SDS-PAGE. There is a nucleotide specificity to the release of myofilaments in the order of ATP greater than GTP much greater than UTP greater than CTP. Experiments with AMP-PNP and inorganic phosphate (Pi) showed that ATP hydrolysis and the build up of Pi are not requirements in the formation of the easily releasable myofilaments. The release of myofilaments was found to be insensitive to variations in pH from 6.5 to 7.5. The ATP stimulation of myofilaments release is ubiquitin-independent, since incubation of purified myofibrils with ubiquitin (1-100 micrograms/ml) at both 20 and 37 degrees C did not change the amount released. Modifying the free sulfhydryl group content by treatment of myofibrils with NEM (0.01-1 mM) or silver nitrate (0.1-10 mM) decreased the proportion of myofilaments that were releasable. Exclusion of 1 mM DTT from the preparation of myofibrils had similar results. These results indicate that the formation of easily releasable myofilaments can be mediated by metabolically related parameters such as the adenosine nucleotides and the reduction-oxidation status of the myofibrillar proteins of striated muscle.
Mol Cell Biochem 1991 May 15
PMID:Regulation of ATP-stimulated releasable myofilaments from cardiac and skeletal muscle myofibrils. 164 79

A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP greater than GTP greater than ITP greater than CTP) supported this enzymic activity, which was stimulated by Mg2+ but not by Ca2+. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 Mr protein. Maximal phosphorylation of the 55,000 Mr protein occurred in the presence of Mg(2+)-ATP. Addition of cAMP, cGMP, Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 Mr protein. However, in the presence of Ca2+/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg(2+)-NTPase activity. These results indicate that a protein with Mr 55,000 may be involved in the regulation the Mg(2+)-NTPase activity associated with rat cardiac nuclei.
Mol Cell Biochem 1991 Apr 10
PMID:Regulation of rat cardiac nuclei-associated Mg(2+)-NTPase by phosphorylation. 165 81

This study was designed to investigate whether growth hormone (GH) influences the expression of its own receptor in chondrocytes. To investigate this possibility GH-receptor mRNA was measured in cultured rat epiphyseal chondrocytes in the absence or presence of GH under various experimental conditions. Chondrocytes were isolated enzymatically from epiphyseal growth plates of the proximal tibia of 20-day-old male rats and cultured in monolayer in Ham's F-12 medium supplemented with 10% calf serum and 1% of a serum substitute. The cells were seeded at various densities (100,000-1,000,000 cells per flask) and cultured for 14 days. Subsequently, the calf serum-containing medium and the cells cultured for various periods of time (0-24 h) before total nucleic acid preparation. GH-receptor mRNA was measured with a solution hybridization technique using [35S]UTP-labeled RNA growth hormone receptor cloned from rat liver cDNA. Human GH (hGH; 50 ng/ml) increased GH-receptor mRNA after 3 h and maximal levels were seen 12 h after GH addition. This effect of hGH was time and dose dependent with a significant effect of hGH at a concentration of 0.5 ng/ml and a maximal effect at 50 ng/ml. The hGH-stimulated increase of GH-receptor mRNA was completely blocked by actinomycin-C1 (1.0-0.1 micrograms/ml), while cycloheximide (10 micrograms/ml) only slightly counteracted the hGH effect. Rat and human GH were equally potent, and ovine prolactin was effective at 500 ng/ml but not 5 and 50 ng/ml. A high dose of insulin-like growth factor-I (IGF-I; 1 microgram/ml) caused a small stimulatory effect and addition of 10% calf serum caused a marked increase in GH-receptor mRNA. The level of GH receptor mRNA after 14 days of culture was inversely proportional to the cell density at the start of culture. These results show that GH specifically regulates mRNA levels for its own receptor in rat epiphyseal chondrocytes by interacting with somatogenic binding sites. These findings also suggest a transcription-dependent regulatory system between the GH-receptor and the GH-receptor gene.
Mol Cell Endocrinol 1990 May 07
PMID:Growth hormone regulation of the growth hormone receptor mRNA in cultured rat epiphyseal chondrocytes. 169 5

Expression of the Escherichia coli pyrE gene is regulated by transcription attenuation in the intercistronic orfE-pyrE region and modulated by the distance between the transcribing RNA polymerase and the leading ribosome as a function of the supply of UTP and GTP. In this communication we show that pyrE expression is hyper-repressed in vivo following addition of uracil in strains carrying the nusAcs10 mutation. This phenotype, previously seen in rpsL1204 strains whose ribosomes are pseudodependent on streptomycin and work at suboptimal elongation rate, indicates that RNA polymerase escapes from the ribosomes in the pyrE attenuator region in the nusA mutant. In vitro transcription studies revealed that the build-up of the full-length attenuated orfE transcript occurred more slowly in the presence of the NusA protein than in its absence. Moreover, the NusA protein enhanced several transcription pauses through the orfE gene. These effects were more pronounced when low concentrations of either UTP or GTP were used than at low concentrations of either CTP or ATP. The results indicate that the NusA protein is required for proper regulation of pyrE gene expression and is involved, together with the NTP pools, in maintaining the coupling between transcription and translation in the pyrE attenuator region by inhibiting RNA chain elongation.
Mol Microbiol 1991 Feb
PMID:Role of transcription pausing in the control of the pyrE attenuator in Escherichia coli. 171 Mar 13


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