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T E Webb

Publications and source records attributed to T E Webb.

At least 37 records · Page 2Linked to original sources
PubMed1997

Distribution of [35S]dATP alpha S binding sites in the adult rat neuraxis.

Highly abundant, saturable and specific binding sites for [35S]2'-deoxyadenosine 5'-O-(1-thio) triphosphate ([35S]dATP alpha S, Kd: 9 +/- 2 nM; Bmax: 39 +/- 8 pmol/mg protein) are present in adult rat brain membranes and have characteristics consistent with those expected for a P2Y1 receptor. The anatomical distribution of these binding sites in the brain and spinal cord was examined using in vitro autoradiography. The [35S]dATP alpha S binding sites showed a widespread distribution throughout the brain and spinal cord. They could be displaced by a large excess (100 microM) of 2-methylthioATP (2MeS-ATP) but not by uridine-5'-triphosphate (UTP) or alpha,beta-methyleneATP (alpha,beta-meATP). Within the cortical regions labelling was of equal medium density. However, discrete structures and nuclei within the olfactory bulb, subcortical telencephalon, hippocampal complex, thalamic regions and mesencephalon displayed a variety of densities. Within the spinal cord, gray matter was labelled at a greater density than the funiculi. The present study clarifies the anatomical distribution of P2Y1 and closely related receptors within the central nervous system of rat and extends the evidence that those receptors are abundant and widely distributed within the neuraxis.

Animals↗
PubMed1997

Inhibition by heterologously-expressed P2Y2 nucleotide receptors of N-type calcium currents in rat sympathetic neurones.

The P2Y2 nucleotide receptor has previously been shown to stimulate phosphoinositide breakdown. We now show that, when P2Y2 receptors are heterologously expressed by cRNA injection into dissociated rat sympathetic neurones, activation of these receptors by uridine 5'-triphosphate (UTP) or adenosine 5'-triphosphate (ATP) inhibits the N-type voltage-gated calcium current by approximately 65%, with an IC50 of 0.5 microM. Thus, the same molecular species of nucleotide receptor can link to two different effector pathways.

Animals↗
PubMed1996

Molecular cloning of a novel P2 purinoceptor from human erythroleukemia cells.

Screening of a human erythroleukemia cell cDNA library with radiolabeled chicken P2Y3 cDNA at low stringency revealed a cDNA clone encoding a novel G protein-coupled receptor with homology to P2 purinoceptors. This receptor, designated P2Y7, has 352 amino acids and shares 23-30% amino acid identity with the P2Y1-P2Y6 purinoceptors. The P2Y7 cDNA was transiently expressed in COS-7 cells: binding studies thereon showed a very high affinity for ATP (37 +/- 6 nM), much less for UTP and ADP (approximately 1300 nM), and a novel rank order of affinities in the binding series studied of 8 nucleotides and suramin. The P2Y7 receptor sequence appears to denote a different subfamily from that of all the other known P2Y purinoceptors, with only a few of their characteristic sequence motifs shared. The P2Y7 receptor mRNA is abundantly present in the human heart and the skeletal muscle, moderately in the brain and liver, but not in the other tissues tested. The P2Y7 receptor mRNA was also abundantly present in the rat heart and cultured neonatal rat cardiomyocytes. The P2Y7 receptor is functionally coupled to phospholipase C in COS-7 cells transiently expressing this receptor. The P2Y7 gene was shown to be localized to human chromosome 14. We have thus cloned a unique member of the P2Y purinoceptor family which probably plays a role in the regulation of cardiac muscle contraction.

Amino Acid Sequence↗
PubMed1996

Identification of 6H1 as a P2Y purinoceptor: P2Y5.

We have determined the identity of the orphan G-protein coupled receptor cDNA, 6H1, present in activated chicken T cells, as a subtype of P2Y purinoceptor. This identification is based on first on the degree of sequence identity shared with recently cloned members of the P2Y receptor family and second on the pharmacological profile. Upon transient expression in COS-7 cells the 6H1 receptor bound the radiolabel [35S]dATP alpha S specifically and with high affinity (Kd, 10 nM). This specific binding could be competitively displaced by a range of ligands active at P2 purinoceptors, with ATP being the most active (K (i)), 116 nM). Such competition studies have established the following rank order of activity: ATP ADP 2-methylthioATP alpha, beta-methylene ATP, UTP, thus confirming 6H1 as a member of the growing family of P2Y purinoceptors. As the fifth receptor of this type to be identified we suggest that it be named P2Y5.

Amino Acid Sequence↗
PubMed1996

The diverse series of recombinant P2Y purinoceptors.

A cDNA encoding a P2Y purinoceptor was originally cloned from chick brain and the bovine and human homologues have recently been obtained. These are seven-transmembrane-domain polypetides, i.e. G protein-coupled receptors. When activated by agonists, this P2Y receptor mobilizes intracellular Ca2+ and has been shown to be coupled to inositol-1,4,5-trisphosphate formation. Its pharmacology has been established in several expression systems, using both ligand binding and functional responses: 2-methylthioATP has the highest potency of nucleotides and derivatives tested, while UTP and alpha, beta-methylene ATP are inactive. This was hence assigned as a new subtype of the pharmacologically defined P2Y receptors, P2Y1. P2Y1 receptors are exceptionally abundant in the brain. A P2U receptor reported by others can be designated P2Y2. Another P2 receptor subtype, P2Y3, now cloned as a cDNA from the brain and expressed in oocytes and in transfected cells, shows a quite different ligand potency profile to the first two. A fourth subtype is expressed primarily in certain haemopoietic cells and in cardiac muscle. A putative fifth subtype is expressed only in T lymphocytes, upon activation. Yet other P2Y subtypes are indicated by recent cloning studies. The amino acid sequences of all of these P2 receptors, while displaying some homology, are strikingly diverse: they form a separate and unusual new family in the G protein-coupled receptor main superfamily.

Animals↗
PubMed1996

Molecular biology of P2Y purinoceptors: expression in rat heart.

1. Application of molecular biology to the study of P2Y purinoceptors has led to the identification of seven such receptors. Here we briefly review their properties and investigate qualitatively the expression of four rat receptor transcripts in heart. 2. The reverse transcriptase-polymerase chain reaction was used to ascertain whether the rat P2Y1, P2Y2, P2Y4 and P2Y6 receptor transcripts were expressed in whole heart, neonatal cardiac fibroblasts, neonatal cardiac myocytes and adult cardiac myocytes. 3. All receptor sequences could be amplified from neonatal rat whole heart, with P2Y6 appearing the most abundant transcript of the four. P2Y1 is expressed at higher levels in comparison to P2Y2, P2Y4 and P2Y6 in the neonatal myocyte. In the adult myocyte P2Y1, P2Y2 and P2Y6 could be amplified but P2Y4 could not be detected. In the neonatal fibroblast, P2Y1 and P2Y6 appear to be expressed at higher levels than P2Y2 and P2Y4. 4. In summary, it is concluded that multiple P2Y receptor subtypes are expressed in heart and that the expression in myocytes changes from the neonate to the adult.

Animals↗
PubMed1996

The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase.

1. B10 cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2. The radioligand [35S]-dATP alpha S bound with high affinity (Kd = 9.8 nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg-1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]-dATP alpha S led to the rank order of affinity: dATP alpha S (Ki 3.4 nM) > 2-chloroATP (2-ClATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium (PPADS) was able to displace only 20% of [35S]-dATP alpha S binding at a concentration of 100 microM. 3. 2-ClATP (EC50 = 0.22 microM), 2-MeSATP (0.54 microM), ADP (7.9 microM) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 microM. 4. A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6-2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y-type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has been shown to increase phospholipase C activity. 5. Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2-MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y1 receptor but not of any other type resembling the known P2Y receptors, it is concluded that a P2Y1 receptor on rat brain microvascular endothelial cells can account for all of the observations. This single P2Y1 receptor, therefore, appears to couple in different native cell types to either adenylate cyclase inhibition or to phospholipase C activation.

Adenylyl Cyclase Inhibitors↗
PubMed1996

A novel G protein-coupled P2 purinoceptor (P2Y3) activated preferentially by nucleoside diphosphates.

A partial cDNA was isolated by hybridization screening of an embryonic chick brain library for P2Y purinoceptors. After extension to full length, it revealed an open reading frame that encoded a protein, P2Y3, of 328 amino acids that is nearest in sequence identity to the G protein-coupled P2 purinoceptors obtained by DNA cloning. Expression of P2Y3 in cRNA-injected Xenopus oocytes confirmed that this cDNA encodes a member of the metabotropic purinoceptor family, with a novel order for the relative activities of nucleotides. At 100 microM concentrations, ADP gave the highest activity, and UTP and UDP were also strongly active. When expressed in the human T cell line Jurkat, P2Y3 mediated transient increases in intracellular Ca2+ in response to various nucleotides. Again, an unusual agonist rank order was revealed, with uridine nucleotides being more potent than adenosine nucleotides and UDP being the most potent agonist tested (half-maximal concentration, 0.13 microM) and 10-fold more potent than UTP. 2-Methylthlo-ATP was of relatively low activity in both systems. The receptor transcript is expressed in brain, spinal cord, kidney, and lung and is highly abundant in the spleen but not in other peripheral tissues that we tested. The results indicated that P2Y3 is a previously unknown P2 purinoceptor subtype with a preference for nucleoside diphosphates.

Adenosine Diphosphate↗
PubMed1995

Characterisation of a recombinant P2Y purinoceptor.

We have previously cloned a cDNA encoding a G-protein-coupled P2 purinoceptor from chick brain and designated this as a P2Y1 purinoceptor (Webb, T.E., J. Simon, B.J. Krishek, A.N. Bateson, T.G. Smart, B.J. King, G. Bumstock and E.A. Barnard, 1993, FEBS Lett. 324, 219). Here, we describe the further characterisation of this recombinant receptor expressed in both simian kidney endothelial (COS-7) cells and Xenopus oocytes. In transfected COS-7 cell membranes, the recombinant receptor showed a high level of expression (Bmax = 7.9 +/- 2.2. pmol [35S]dATP alpha S bound/mg protein) and affinity (Kd = 6.6 +/- 0.3 nM). In these COS-7 cells, the activation of the implanted purinoceptor induced a suramin-sensitive formation of inositol 1,4,5-triphosphatic (1,4,5InsP3). Upon expression in Xenopus oocytes, ATP was the only natural nucleoside triphosphate to elicit a Ca(2+)-activated chloride current. The P2 purinoceptor antagonists suramin and Reactive Blue-2 were both able to inhibit this evoked current. Utilizing both expression systems, the binding affinity profile and the functional pharmacological profile of the agonists, the common series found was: 2-methylthioATP (2-MeSATP) > or = ATP > ADP beta S > ADP. These two agonist series and the lack of activity of adenosine, alpha, beta-methyleneATP (alpha, beta-meATP), 3'-O-(4-benzoyl) benzoyl-ATP (Bz-ATP) and UTP, together confirmed that this receptor is a specific subtype of the P2Y purinoceptors.

Adenosine↗
PubMed1995

Cloning and characterisation of a bovine P2Y receptor.

Using a chick P2Y1 receptor cDNA probe we have isolated a mammalian P2Y receptor clone from a bovine aortic endothelial cell library. The sequence has a high degree of similarity to the chick P2Y1 clone. When transfected into the Jurkat cell line, the cDNA conferred sensitivity to purinoceptor agonists. Using fura-2 loaded cells the potency order at the receptor was found to be 2-methylthioadenosine 5' triphosphate = adenosine 5' diphosphate > adenosine 5' triphosphate >> alpha,beta-methyleneadenosine 5' triphosphate and uridine 5' triphosphate. This corresponds to the agonist potency order expected for the bovine aortic endothelial cell P2Y receptor.

Adenosine Diphosphate↗
PubMed1995

Characterization of a P2Y purinoceptor in the brain.

Little has been known of the abundance in the brain of any of the G protein coupled P2 purinoceptors nor their pharmacology. Here we show that [35S]dATP alpha S is a suitable radioligand for investigating these receptors and hence that they are exceptionally abundant both in one-day-old chick (Bmax: 37 pmol agonist sites/mg protein) and adult rat brain membranes (Bmax: 39 pmol/mg protein). [35S]dATP alpha S (which is selective for P2Y over the P2X types of purinoceptor) binds with high affinity to these sites in the chick (Kd: 13.3 nM) and in the rat brain membranes (Kd: 9.1 nM). The rank order of potency of purinoceptor-active agonists and antagonists displacing [35S]dATP alpha S binding is: dATP alpha S > (3'-deoxyATP, 2-methylthioATP, ATP alpha S, ATP) > 2'-deoxyATP > 2-methylthioADP > ADP >> suramin, Reactive Blue-2 >> UTP, L-beta,gamma-methyleneATP, adenosine; this defines these binding sites as P2Y subtypes of the P2 purinoceptors. This pharmacological profile of purinergic ligands is in excellent agreement with the potency order established for the recombinant P2Y1 purinoceptor from chick brain, identifying the great majority of the brain P2 purinoceptors as identical or very similar to the native P2Y1 receptor.

Animals↗
PubMed1995

Evaluation of oncofetal protein-related mRNA transport activity as a potential early cancer marker in dogs with malignant neoplasms.

A 55-kd protein with mRNA transport activity found in fetal rat liver cells and plasma from mice, rats, and human beings with malignant neoplasms has been designated oncofetal protein 55 (OFP55). Monoclonal antibody produced to rat OFP55 cross-reacts with human OFP55. Using this monoclonal antibody in a bioassay measuring mRNA transport stimulated by OFP55, we tested the plasma from 19 dogs with a variety of malignant neoplasms, including carcinomas, sarcomas, lymphomas, and melanomas, and compared the results with plasma from 20 clinically normal dogs without evidence of neoplasia. The mean mRNA transport activity from the group of dogs with malignant neoplasms was 0.43 +/- 0.28%/mg of protein. Mean transport activity from the group of control dogs was 0.04 +/- 0.02%/mg of protein. These means were significantly different (P < 0.0001). The degree of overlap between these 2 groups in their OFP55-related mRNA transport activity was minimal, and measurement of this protein appears to have potential for the early detection of malignant neoplasms in dogs.

Animals↗
PubMed1994

Pharmacokinetics relevant to the anti-carcinogenic and anti-tumor activities of glucarate and the synergistic combination of glucarate:retinoid in the rat.

Alone and in synergistic combination with retinoids, dietary glucarate inhibits both the chemical induction and growth of rat mammary tumors. To investigate the pharmacokinetics of glucarate, [14C]glucarate was synthesized, converted to the calcium salt, and administered to rats bearing primary mammary tumors. When given by gavage, [14C]glucarate, as the calcium salt, showed a biphasic response in the blood. After peaking within 1 hr of administration at a level of 0.4 mumol/mL (normal endogenous level is approximately 0.04 mumol/mL), its plasma concentration dropped to 0.1 mumol/mL at 3 hr. In the second phase, there was a semilog increase to 0.6 mumol/mL at 15 hr, followed by a slow rise to 0.75 mumol/mL at 24 hr. Of the 38% of the administered glucarate that was recovered, 38% was excreted in the urine, and 30% remained in the gastrointestinal tract at 24 hr. Glucarate was concentrated 3- to 4-fold in the liver and intestinal mucosa, compared to the level in serum. With minor exception, the pharmacokinetics of [14C]13-cis-retinoic acid administered by gavage to rats was similar or not the semipurified diets were supplemented with 64 mmol/kg of calcium glucarate. During the interval between 5 and 10 hr post-administration of [14C]13-cis-retinoid, there was a transient 35-50% rise in the plasma level in rats on the glucarate-supplemented diet. This rise had no observable effect on the level of retinoid in major organs or in the tumor. A glucarate-binding protein was detected in the tumor cytosol. This potential receptor had a Ka of 1.49 x 10(7) M-1.

Animals↗
PubMed1994

Activity of D-glucarate analogues: synergistic antiproliferative effects with retinoid in cultured human mammary tumor cells appear to specifically require the D-glucarate structure.

D-Glucarate has shown modest chemopreventive and synergistic chemopreventive effects with retinoids in a number of tumor models as well as a similar antiproliferative effect in MCF-7 human tumor cells in culture. It has been postulated that D-glucarate exerts some of its effects by equilibrium conversion to D-glucarolactone, a potent beta-glucuronidase inhibitor. In the present study, D-glucarate and a number of its analogues, including D-glucarolactone, were evaluated as antiproliferatives in the MCF-7 model with and without added retinoid. Results suggest that the effects of glucarate are reasonably specific for its structure and may not require conversion to glucarolactone.

Breast Neoplasms↗
PubMed1994

Transient expression of the recombinant chick brain P2y1 purinoceptor and localization of the corresponding mRNA.

Extracellular ATP acts at specific cell surface purinoceptors to elicit a wide range of physiological responses. We have recently isolated a cDNA for a G-protein-coupled P2 purinoceptor (P2y1) from chick brain. It has been defined as a P2Y-like purinoceptor by the rank order of potency of P2 purinoceptor ligands, determined electrophysiologically in the Xenopus oocyte expression system. Here, we examine the ligand selectivity of this recombinant receptor, expressed transiently in COS-7 cells. The regional distribution of the P2y1 purinoceptor transcript within the one-day-post-hatch chick brain was also determined. It is widely expressed in the cerebellum and telencephalon and in specific nuclei of the mesencephalon and diencephalon, suggesting a neuronal localization of the P21 purinoceptor.

Adenosine Triphosphate↗
PubMed1993

Cloning and functional expression of a brain G-protein-coupled ATP receptor.

A cDNA encoding a novel member of the G-protein-coupled receptor (GCR) superfamily, an ATP receptor, has been isolated from an embryonic chick whole brain cDNA library by hybridization screening. The encoded protein has a sequence of 362 amino acids (41 kDa) and shares no more than 27% amino acid identity with any known GCR. When expressed as a complementary RNA (cRNA) in Xenopus oocytes a slowly-developing inward current was observed in response to application of ATP. The pharmacology of this expressed protein defines it as a P2Y purinoceptor.

Adenosine Triphosphate↗