PubMed Health⌕ Search

Biomedical subjects

T E Webb

Publications and source records attributed to T E Webb.

At least 19 recordsLinked to original sources
PubMed2001

Mechanisms of P2 receptor-evoked DNA synthesis in thyroid FRTL-5 cells.

The expression of the P2 receptors and their functional responses were studied in rat thyroid FRTL-5 cells. RT-PCR analysis revealed transcripts for the G protein-coupled P2Y(2), P2Y(4) and P2Y(6) receptors, and for the transmitter-gated ion channel P2X(3), P2X(4) and P2X(5) subunits. In Fura-2-loaded cells, UTP, ATP, ATPgammaS or UDP increased [Ca(2+)](i), and behaved as potent full agonists, while 2-Methylthio-ATP (2-MeSATP), alpha,beta-methylene-ATP (alpha,beta-meATP) and pure ADP were weak agonists. The agonist-mediated [Ca(2+) ](i) increases were diminished in Ca(2+) -free buffer, and by pertussis toxin (PTX) or suramin treatments. ATP, UTP, UDP and ATPgammaS increased (3)H-thymidine incorporation into DNA and expression of the protooncogenes c-Fos and c-Jun, while 2-MeSATP was ineffective, and alpha,beta-meATP gave a response only at 100-microM dose. The ATP-stimulated expression of c-Fos and c-Jun was dependent on Ca(2+), and protein kinase C, but not on calmodulin or Ca(2+)/calmodulin-dependent protein kinase II. Extracellular signal-regulated kinases (ERK1 and ERK2) are also involved as the MEK inhibitor, PD98059, reduced both ATP-evoked (3)H-thymidine incorporation and c-Fos and c-Jun expression. These results indicate that multiple P2Y receptor subtypes and at least the P2X(5) subtype are functionally expressed in FRTL-5 cells, and that nucleotides acting via P2 receptors are involved in the regulation of DNA-synthesis.

Adenosine Triphosphate↗
PubMed2001

Aroma components of an oil-based grill flavoring by direct thermal desorption-gas chromatography-olfactometry and sample dilution analysis.

Grill flavorings are a convenient way for food processors to impart grill-like flavor to meat products that have not been grilled. In this study a commercially available oil-based processed grill flavoring was analyzed by direct thermal desorption (DTD)-gas chromatography-olfactometry (GCO) and DTD-GC-mass spectrometry (MS). Sample mass dilution analysis-GCO was used to indicate which compounds had the greatest impact on the overall aroma of the sample. Major aroma contributors included 1-octen-3-one, 2-methoxyphenol, and (E)-2-nonenal. Minor contributors were (E)-2-decenal and 2,4-decadienal. Other major contributors, characterized as having grill aroma notes, were unidentified. Excluding the possibility of artifact formation from the thermal degradation of fatty acid hydroperoxides, DTD functioned well as a GCO technique, but poorly as a qualitative GC-MS technique.

Adsorption↗
PubMed2001

Pharmacological characterisation of pyrimidinoceptor responses in NG108-15 cells.

In the present study, the P2Y receptor(s) mediating the effects of the pyrimidines UTP and UDP on phospholipase C activation in the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 was investigated. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) analysis detected transcripts for the P2Y(6) and P2Y(2) receptors, but not for P2Y(1) and P2Y(4.) UTP and UDP were equipotent agonists and their effects were partially additive. Suramin, reactive blue 2 and pyridoxal phosphate-6-azophenyl-2',4'disulfonic acid (PPADS) antagonised the phospholipase C response to both UTP and UDP. High micromolar concentrations of adenosine, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680), 2',3'-O-isopropylideneadenosine (iPAdo) and adenosine 3':5'-cyclic monophosphate (3',5'-cAMP) were able to antagonise the effect of UTP on phospholipase C but not that of UDP. The additivity of the UTP and UDP responses, novel P2 receptor antagonist profile and the distinguishing action of adenosine may indicate the expression of a pyrimidine selective P2Y receptor in addition to the P2Y(6) type in these cells.

Adenosine↗
PubMed2001

Assessing a crisis of faith and making a pastoral crisis intervention.

Evaluating the spiritual impact of a critical incident upon an individual is a vital aspect of restoring an individual to functioning in their relationships and work responsibilities. Being able to recognize and respond to spiritual symptoms of critical incident stress will allow crisis interventionists to address crisis recovery issues that are often central to an individual's identity and purpose for living. Spiritual symptoms of traumatic stress issue from the defining identity of an individual. With an understanding of the nature of a crisis of faith crisis interventionists can employ critical incident stress management skills to support a person in spiritual as well as psychological trauma.

Chaplaincy Service, Hospital↗
PubMed2000

Evidence that rat hepatocytes co-express functional P2Y1 and P2Y2 receptors.

Previous studies have indicated the expression of multiple P2Y receptors by rat hepatocytes although they have not been identified. Here we show by reverse transcriptase-polymerase chain reaction (RT - PCR) that rat hepatocytes express mRNA encoding all of the four cloned rat P2Y receptors (P2Y(1), P2Y(2), P2Y(4) and P2Y(6)). The effects of UTP have been examined on single aequorin-injected rat hepatocytes. The [Ca(2+)](i) transients induced by UTP were indistinguishable from those induced by ATP in the same cell. The modulatory effects of elevated intracellular cyclic AMP concentration were the same on both UTP- and ATP-induced [Ca(2+)](i) transients. UDP, an agonist at the P2Y(6) receptor, failed to induce transients in hepatocytes, indicating that functional P2Y(6) receptors coupled to increased [Ca(2+)](i) are not expressed. The transients evoked by ADP were more sensitive to inhibition by suramin than those induced by either ATP or UTP. Within an individual cell, the transients induced by ATP and UTP were inhibited by the same concentration of suramin. This sensitivity of ATP and UTP responses to suramin suggests action through P2Y(2) rather than P2Y(4) receptors. Co-application of 30 microM pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) caused a decrease in frequency and amplitude of transients induced by ADP. ATP- and UTP-induced transients also displayed a decrease in amplitude in response to addition of PPADS, but this was accompanied by an increase in frequency of transients. In conclusion the data presented here are consistent with the co-expression of P2Y(1) and P2Y(2) receptors by rat hepatocytes.

Adenosine Diphosphate↗
PubMed1999

Only pyrimidinoceptors are functionally expressed in mouse neuroblastoma cell lines.

The ability of UTP, UDP, ATP, and ADP to influence inositol phospholipid hydrolysis in neuroblastoma origin cell lines was assessed. The mouse neuroblastoma lines N1E 115, Neuro 2a, and NB4 1A3 and the rat glioma/mouse neuroblastoma hybrid line NG108-15 gave robust responses to both UTP and UDP, which were essentially equipotent. Thus a range of cell lines of mouse neuroblastoma origin express a pyrimidine-selective P2Y receptor. The NG108-15 cells were the only cell type tested at which ATP and ADP displayed activity with EC50 values of greater than 100 microM, compared with values of 0.58 and 1.25 microM for UTP and UDP, respectively. In contrast to the cell lines derived from mouse neuroblastoma, the human neuroblastoma lines SH-SY5Y and SK-N-SH did not respond to any nucleotides, although both responded well to carbachol.

Adenosine Diphosphate↗
PubMed1999

Dual coupling of heterologously-expressed rat P2Y6 nucleotide receptors to N-type Ca2+ and M-type K+ currents in rat sympathetic neurones.

1. The P2Y6 receptor is a uridine nucleotide-specific G protein-linked receptor previously reported to stimulate the phosphoinositide (PI) pathway. We have investigated its effect in neurones, by micro-injecting its cRNA into dissociated rat sympathetic neurones and recording responses of N-type Ca2+ (I(Ca(N))) and M-type K+ (I(K(M))) currents. 2. In P2Y6 cRNA-injected neurones, UDP or UTP produced a voltage-dependent inhibition of I(Ca(N)) by approximately 53% in whole-cell (disrupted-patch) mode and by 73% in perforated-patch mode; no inhibition occurred in control cells. Mean IC50 values (whole-cell) were: UDP, 5.9+/-0.3 nM; UTP, 20+/-1 nM. ATP and ADP (1 microM) had no significant effect. Pertussis toxin (PTX) substantially (approximately 60%) reduced UTP-mediated inhibition in disrupted patch mode but not in perforated-patch mode. 3. Uridine nucleotides also inhibited I(K(M)) in P2Y6 cRNA-injected cells (by up to 71% at 10 microM UTP; perforated-patch). Mean IC50 values were: UDP, 30+/-3 nM; UTP, 115+/-12 nM. ATP (10 microM) again had no effect. No significant inhibition occurred in control cells. Inhibition was PTX-resistant. 4. Thus, the P2Y6 receptor, like the P2Y2 subtype studied in this system, couples to both of these two neuronal ion channels through at least two different G proteins. However, the P2Y6 receptor displays a much higher sensitivity to its agonists than the P2Y2 receptor in this expression system and higher than previously reported using other expression methods. The very high sensitivity to both UDP and UTP suggests that it might be preferentially activated by any locally released uridine nucleotides.

Animals↗
PubMed1999

Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells.

1 In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors. 2 None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPgammaS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin. 3 Comparison of the concentration response curves to ATPgammaS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides. 4 Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1, 3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists. 5 These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors.

Adenosine↗
PubMed1999

Staying the course in stormy seas: establishing a central Critical Incident Stress Management team in the Navy Marine Corps environment.

Crisis response teams are becoming a standard operating procedure for most United States military organizations and for many military organizations outside the U.S. This paper describes the establishment of one specific Critical Incident Stress Management (CISM) team designed to provide psychological support during crisis, disasters, traumas, and other critical incidents that may be encountered in a Navy Marine Corps environment.

Crisis Intervention↗
PubMed1998

Phenotype changes of the vascular smooth muscle cell regulate P2 receptor expression as measured by quantitative RT-PCR.

Studies using selective agonists have suggested that the contractile effect of extracellular nucleotides, such as ATP and UTP, in blood vessels is mediated mainly by P2X1 receptors with a smaller contribution of P2Y receptors while the mitogenic effect is mediated by P2Y (P2Y1, P2Y2, P2Y4, and P2Y6) receptors with no effect of P2X1 receptors. This indicates a difference in P2 receptor expression between the contractile and the synthetic phenotype of the SMC. To measure the expression of mRNA for these receptors a competitive RT-PCR assay was developed that utilised synthetic RNA-competitors allowing determination of the number of mRNA copies for each receptor in the samples. In the synthetic phenotype the mitogenic P2Y1 and P2Y2 receptor transcripts were upregulated by 342- and 8-fold, respectively, while the contractile P2X1 receptor is totally downregulated and the P2Y4 and P2Y6 receptors were unchanged. This plasticity of the receptor expression may be important in the transition from the contractile to the synthetic SMC phenotype.

Adenosine Triphosphate↗
PubMed1998

P2Y2 nucleotide receptors expressed heterologously in sympathetic neurons inhibit both N-type Ca2+ and M-type K+ currents.

The P2Y2 receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y2 receptor to neuronal N-type Ca2+ channels and to M-type K+ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y2 cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y2 nuerones. Br J Pharmacol 121:849-851], UTP inhibited the Ca2+ current (ICa(N)) by up to 64%, with an IC50 of approximately 0.5 microM. We now find that UTP also inhibited the K+M current (IK(M)) by up to 61%, with an IC50 of approximately 1.5 microM. UTP had no effect on either current in neurons not injected with P2Y2 cRNA. Structure-activity relations for the inhibition of ICa(N) and IK(M) in P2Y2 cRNA-injected neurons were similar, with UTP >/= ATP > ITP >> GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment with Pertussis toxin (PTX) did not affect UTP-induced inhibition of IK(M) but reduced inhibition of ICa(N) by approximately 60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y2 receptor can couple to at least two G-proteins to inhibit both Ca2+N and K+M channels with near-equal facility. This implies that the P2Y2 receptor may induce a broad range of effector responses in the nervous system.

Animals↗
PubMed1998

Stimulation of P2Y receptors activates c-fos gene expression and inhibits DNA synthesis in cultured cardiac fibroblasts.

OBJECTIVES: The aims of this study were to determine (1) whether neonatal rat cardiac fibroblasts (CAFB) express P2Y receptors; (2) whether CAFB respond to extracellular ATP by inducing expression of c-fos mRNA; and (3) whether extracellular ATP modulates norepinephrine (NE)-stimulated cell growth in CAFB. METHODS: Expression of P2Y1 and P2Y2 receptors and induction of c-fos were examined by Northern blot analysis. CAFB growth was assessed by measuring [3H]thymidine incorporation and DNA content. P2Y receptor pharmacology was studied using various ATP analogues. RESULTS: Northern blot analysis of polyA enriched RNA confirmed that at least 2 subtypes of P2Y receptors (P2Y1 and P2Y2) are expressed in cultured CAFB. Extracellular ATP induced the expression of c-fos mRNA through a pathway that was sensitive to inhibitors of protein kinase C (PKC), but not to inhibitors of intracellular Ca2+ signaling. Extracellular ATP inhibited the NE-stimulated increases in DNA content and in [3H]thymidine incorporation into DNA. Whereas the potency order for stimulation of c-fos expression was ATP = UTP > ADP > adenosine, the potency order to inhibit the NE-induced increase of [3H]thymidine incorporation into DNA was ATP > ADP > UTP > adenosine. CONCLUSIONS: These data demonstrate that CAFB express both P2Y1 and P2Y2 receptor mRNA and that CAFB respond to P2Y receptor stimulation by induction of c-fos and inhibition of DNA synthesis. These findings suggest that the effects of ATP on [3H]thymidine incorporation into DNA and on expression of c-fos mRNA are exerted via distinct P2Y receptor subtypes.

Adenosine Triphosphate↗
PubMed1998

Regional distribution of [35S]2'-deoxy 5'-O-(1-thio) ATP binding sites and the P2Y1 messenger RNA within the chick brain.

The distribution of the P2Y1 receptor protein and transcript in the one-day-old chick brain were determined by quantitative in vitro ligand autoradiography and in situ hybridization histochemistry. We have previously used [35S]2'-deoxy 5'-O-(1-thio) ATP as a radioligand for the recombinant P2Y1 receptor transiently expressed in COS-7 cells and have also shown that such sites are present at high density (Bmax: approximately 37 pmol radioligand bound/mg protein) in chick brain membranes. Here we report the macroscopic localization of these [35S]2'-deoxy 5'-O-(1-thio) ATP binding sites within the chick brain. They were found to be widely distributed there (within the range of 0.047 +/- 0.012 to 0.309 +/- 0.035 pmol bound/mg wet tissue). The affinities of P2 agonists and antagonists at these binding sites was comparable to that found previously for the recombinant P2Y1 receptor. In parallel experiments, the regional and cellular localization of the P2Y1 receptor messenger RNA was examined by in situ hybridization. The transcript was also found to be widely distributed throughout the brain. High levels of hybridization were detected in the cortex piriformis, ectostriatum, hippocampus, cerebellum and in a range of discrete nuclei throughout the brain, including the ovoidalis, isthmo-opticus and spiriformis lateralis nuclei. Localization at cellular level indicates that this receptor transcript is expressed in neurons and also at non-neuronal sites. Furthermore, the distribution of the P2Y1 transcript and the [35S]2'-deoxy 5'-O-(1-thio) ATP binding sites matched in a number of the regions and structures mentioned above. The present study clarifies the anatomical distribution of the P2Y1 receptor within the chick brain. Its broad distribution coupled with its neuronal expression suggest an important role for this type of metabotropic nucleotide receptor within the brain.

Animals↗
PubMed1998

Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP.

1. Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. 2. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. 3. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2-methythio-ATP and alpha,beta-methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors. 4. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors. 5. 1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. 6. Use of the reverse transcriptase-polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected. 7. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]-thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. 8 These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.

Adenosine Triphosphate↗
PubMed1998

Molecular cloning and characterization of the rat P2Y4 receptor.

Degenerate PCR was used to amplify DNAs encoding members of the P2Y receptor family from rat brain RNA. A full-length sequence obtained for one novel clone (R5) contained an intronless open reading frame that encoded a polypeptide of 361 amino acids, sharing 84% sequence identity with the human P2Y4 receptor. When R5 was stably expressed in Jurkat cells, calcium fluxes resulting from stimulation of the receptor showed that UDP, ADP, 2-methylthio-ATP, and diadenosine tetraphosphate were inactive, whereas UTP and ATP were both full agonists with similar potency. At the human receptor, ATP has significantly lower potency than UTP. The R5 transcript was not detected in brain by northern hybridization. Therefore, its tissue distribution was assessed by PCR, and the mRNA was found to be widely distributed at a low abundance, being present in brain, spinal cord, and a variety of peripheral organs. Localization of the receptor transcript in adult rat brain sections by in situ hybridization indicated that it is expressed at highest levels in the pineal gland and ventricular system. It is presumed that R5 is a species orthologue of the human P2Y4 receptor but with this significant difference in agonist pharmacology.

Amino Acid Sequence↗
PubMed1997

Nucleotide receptors in the nervous system. An abundant component using diverse transduction mechanisms.

Extracellular nucleotides achieve their role as cell-to-cell communicators by acting at cell surface transmembrane receptors-the P2 receptors. Before molecular cloning led to the isolation of any P2-receptor sequence, a small number of receptor types had been proposed on the basis of pharmacological evidence. The application of molecular biology to this field of receptor research has indicated that a great underestimation of the number of receptor subtypes and of their abundance had occurred. There are now known to be seven characterized P2Y (G protein linked) receptors and the same number again of P2X receptors of the transmitter-gated ion channel type. In this review, we discuss the properties of these cloned receptors, their distribution within the nervous system, and their methods of signal transduction.

Adenosine Triphosphate↗