Search PubMed⌕ Search

Biomedical subjects

P Warburton

Publications and source records attributed to P Warburton.

14 recordsLinked to original sources
PubMed1997

The conformational change responsible for AT1 receptor activation is dependent upon two juxtaposed asparagine residues on transmembrane helices III and VII.

A model of the angiotensin AT1 receptor and site-directed mutagenesis were used to identify key residues involved in ligand binding. Receptors were stably expressed in human embryonic kidney 293 cells, and their binding properties compared. Wild type receptors exhibited low and high affinity binding sites for peptides. Substitution of Asn111, situated in the third transmembrane helix, resulted in a significant alteration in ligand binding with only high affinity binding of the peptides, angiotensin II, angiotensin III, and [p-amino-Phe6]angiotensin II and a marked loss in the binding affinity of the AT1 receptor selective non-peptide antagonist losartan. From our model it was apparent that Asn111 was in close spatial proximity to Asn295 in the seventh transmembrane helix. Substitution of Asn295, produced identical changes in the receptor's pharmacological profile. Furthermore, the Ser111AT1A and Ser295AT1A mutants did not require the association of a G-protein for high affinity agonist binding. Finally, the Ser295AT1A mutant maintained higher basal generation of inositol trisphosphate than the wild type, indicating constitutive activation. We propose that substitution of these residues causes the loss of an interaction between transmembrane helices III and VII, which allows the AT1 receptor to "relax" into its active conformation.

Alanine↗
PubMed1997

Evidence of an important and direct role for protein kinase C in agonist-induced phosphorylation leading to desensitization of the angiotensin AT1A receptor.

1. The role of protein kinase C (PKC) in the mechanism underlying rapid agonist-induced desensitization of angiotensin AT1 receptors remains unresolved. A major problem has been to isolate these receptors in a sufficiently purified form to allow study of their phosphorylation state. 2. A cleavable (His)6 affinity tag was introduced into the N-terminus of the recombinant AT1A receptor and stably expressed in human embryonic kidney cells. This affinity tag allowed rapid isolation, purification and determination of the phosphorylation state of the AT1A receptor. Using these cells, we determined the role of PKC in both agonist-induced receptor phosphorylation and desensitization under identical conditions. 3. Agonist-induced phosphorylation of the AT1A receptor was observed at both low and high concentrations of angiotensin II (AII). Preincubation of cells with Ro-31-8220 (a PKC specific inhibitor) revealed that at low concentrations of AII (1 nM), PKC appeared to be the main kinase involved in receptor phosphorylation. In contrast, at high concentrations of AII (100 nM), although PKC-mediated phosphorylation of the receptor was observed, this was overshadowed by a second kinase. 4. In preliminary desensitization studies we observed that at a low concentration of AII, preincubation with Ro-31-8220 attenuated desensitization, whilst at high concentrations of AII (100 nM) it had little or no effect on the level of desensitization observed. 5. These data directly demonstrate an association between PKC-induced receptor phosphorylation and desensitization at low concentrations of AII. Since circulating concentrations of AII are in the picomolar range, we propose that PKC is the physiologically relevant mediator of AT1 receptor desensitization.

Angiotensin II↗
PubMed1996

Physical activity levels of 5-11-year-old children in England: cumulative evidence from three direct observation studies.

The aim of this study was to determine the physical activity levels of a sample of young children. Data were collected using direct, continuous observation by trained observers. The observational method utilised a points system to differentiate intensity of physical activity. Observations were carried out during school break times, lunch times and physical education lessons and during free time outside of school. The sample consisted of 93 girls and 86 boys aged between 5 and 11 years. Only 38 children (21%) engaged in a sustained 20-min period of moderate to vigorous physical activity (MVPA), but nearly all children (95%) took part in a 5-min period of MVPA. The highest recordings of MVPA occurred during school break times but MVPA was less prominent during free time outside of school. One hundred and seventy eight children were observed during school physical education lessons. Sustained MVPA was particularly low during physical education lessons with only 13 children (8%) participating in at least one sustained 10-min period. There appeared to be no difference in activity levels between boys and girls or between children of differing ages. During all observation periods the main activity of the child was recorded. Soccer, brisk walking, general play and chasing games were the most common activities. The results are disturbing since preadolescent children appear to be engaging in very little sustained, playful physical activity during their free time outside of school. If childhood is considered to be the most active stage of life there must be concern for the future. There is a need for health professionals to promote active lifestyles from birth.

Child↗
PubMed1995

Functional domains of the C-terminus of the rat angiotensin AT1A receptor.

Previous work has shown that truncating the carboxyl terminus (C-terminus) of the rat angiotensin AT1A receptor to 309 amino acids abolished G-protein coupling and receptor internalization. This suggests that domains responsible for these functions lie beyond amino acid 309 of the C-terminus. The objective of this study was to determine the effect on angiotensin AT1A receptor function and regulation of deleting 41 amino acids from the C-terminus, which include the putative protein kinase C phosphorylation sites. Using site directed mutagenesis, the codon for Tyr319 was converted to a stop codon and the resulting truncated receptor permanently expressed in cultured human kidney cells. The properties of the truncated receptor were compared to those of the full length receptor. Expression of the truncated receptor was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis of photolabelled membrane preparations. Angiotensin II activation of both full length and truncated receptors resulted in mobilization of inositol phosphates. However, whereas this was associated with rapid internalization of the full length receptor, the truncated receptor failed to internalize. Furthermore, pretreatment of cells with phorbol 12-myristate 13-acetate, a direct activator of protein kinase C, markedly attenuated the full length, but no the truncated receptor's ability to mobilise inositol phosphates. Thus, we conclude that the domain between amino acids 309 & 318 is important for G-protein coupling; that amino acids beyond 318 regulate internalization and one or more of the putative protein kinase C phosphorylation sites, present in the C-terminus of the angiotensin At1A receptor, actively regulate the receptor.

Angiotensin II↗
PubMed1995

Glucocorticoids regulate the expression of angiotensin AT1 receptors, in the human hepatoma cell line, PLC-PRF-5.

The effects of different steroids on the expression of angiotensin AT1 receptors by the human hepatoma cell line, PLC-PRF-5 was studied. Dexamethasone and aldosterone decreased the specific binding of [3H]angiotensin II to intact PLC-PRF-5 cells by 57 +/- 4% and 54 +/- 2%, respectively, compared to control, untreated cells. EC50 values for dexamethasone, cortisol and aldosterone were 1.8 +/- 0.6, 40 +/- 6, and 310 +/- 20 nM, respectively, suggesting that these effects were mediated via a glucocorticoid receptor. Scatchard analysis revealed that dexamethasone decreased the number of angiotensin AT1 receptors expressed (50 +/- 4% relative to control) with no change in receptor affinity. Treating cells with dexamethasone in the presence of either an angiotensin converting enzyme inhibitor or an angiotensin II receptor antagonist did not prevent the reduction in angiotensin AT1 receptor expression, ruling out a mechanism involving a dexamethasone induced increase in endogenous angiotensin II production. A ribonuclease protection assay established that the steady state level of angiotensin AT1 receptor mRNA in dexamethasone treated cells was reduced to 34.7 +/- 8.4% of untreated cells. The decrease in the number of angiotensin AT1 receptors expressed on the cell surface after treatment with dexamethasone therefore seems likely to reflect the decreased steady state level of the mRNA coding for this receptor.

Angiotensin II↗
PubMed1994

Comparative pharmacology of recombinant rat AT1A, AT1B and human AT1 receptors expressed by transfected COS-M6 cells.

1. Currently available antagonists and agonists cannot distinguish between angiotensin AT1 receptor subtypes. 2. We synthesized a series of compounds selected on the basis of having the most diverse structural features with respect to losartan (DuP753), the prototype non-peptide AT1 receptor antagonist. Using a radioligand-receptor binding assay and membranes prepared from COS-M6 cells transfected with individual AT1 receptor subtypes, we determined whether any of these compounds could distinguish between the receptor subtypes. 3. The diversity of the structural features of this series of compounds was reflected by the wide range of affinities (pIC50 values) displayed towards competing with [125I]-Sar1Ile8 angiotensin II for binding to the AT1 receptors. 4. Direct comparisons of the pIC50 values of individual compounds for rat AT1A, AT1B and human AT1 receptors revealed only minor differences. 5. It is concluded that compounds based structurally on losartan are unlikely to distinguish between these receptors.

Angiotensin I↗
PubMed1992

Characterization of the angiotensin II receptor expressed by the human hepatoma cell line, PLC-PRF-5.

Radioligand binding studies were undertaken to establish the expression of angiotensin II (AII) receptors on the human hepatoma cell line, PLC-PRF-5. Cell membranes were shown to express a large number of AII receptors with high and low affinity binding sites having Bmax values of 1269 +/- 365 and 4190 +/- 1055 fmol/mg protein and affinities (Kd) of 2.0 +/- 0.3 nM and 8.7 +/- 0.4 nM, respectively. In intact cells a single class of AII binding site was seen with an affinity (Kd) of 6.7 +/- 1 nM and a Bmax value of 315 +/- 32 fmol/mg. In both membranes and intact cells AII, AIII and the selective angiotensin AT1 receptor antagonist, DuP 753, all had a high affinity for the receptor (Ki values in the nanomolar range), but the selective angiotensin AT2 ligands, PD 123177 and p-aminophenylalanine6 AII, had low affinity (Ki values in the micromolar range). These results indicate that the PLC-PRF-5 cells express the angiotensin AT1 receptor subtype. This was further supported by the demonstration of the sensitivity of the receptor to dithiothreitol (DTT). Pretreatment of membranes with DTT reduced [3H]AII binding in a concentration-dependent manner with an IC50 of 4.2 +/- 0.9 mM. The coupling of the AT1 receptor to signal transduction pathways was investigated. In intact cells AII (100 nM) evoked an increase in intracellular calcium ([Ca2+]i). This increase in [Ca2+]i was unaffected by PD 123177 (100 microM) but was abolished by DuP 753 (100 microM). Furthermore, AII (100 nM) did not inhibit forskolin (0.1-10 microM) stimulated cyclic AMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1992

Induction of the angiotensin AT2 receptor subtype expression by differentiation of the neuroblastoma x glioma hybrid, NG-108-15.

In vitro differentiation of the mouse neuroblastoma-rat glioma hybrid cell line, NG-108-15, with dimethyl sulphoxide (1.5%) and low serum (0.5%), produced a marked increase in the number of angiotensin II receptors, from a level at the limit of sensitivity using labelled angiotensin II with a high specific activity ([125I]angiotensin II), in undifferentiated cells, to a Bmax of 1077 (1070-1268) fmol/mg in 5-day-differentiated cells. The affinity (Kd) of radiolabelled angiotensin II for the receptors in differentiated cells was 8.1 (7.5-10) nM. The recently available selective non-peptide antagonists, DuP 753 and PD 123177 and the peptide analogues of angiotensin II, CGP 42112A and p-aminophenylalanine6 angiotensin II, were used to characterize the angiotensin II receptors by competing for 125I-[Sar1-Ile8]angiotensin II binding to membranes prepared from undifferentiated and differentiated cells. The predominant angiotensin II receptor subtype expressed by undifferentiated cells was AT1 and after differentiation AT2. This change in receptor expression was evident 2 days after initiation of differentiation, was maximal at 4-5 days and was stable for at least 8 days. Administration of angiotensin II induced intracellular Ca2+ mobilization in both undifferentiated and differentiated cells. This was antagonised by the selective AT1 antagonist, DuP 753, indicating an action at the AT1 receptor subtype in both undifferentiated and differentiated cells. The selective AT2 antagonist, PD 123177 was without effect on the angiotensin II induced increase in intracellular Ca2+. This effect of DuP 753 on Ca2+ was specific for angiotensin II since the drug had no effect on bradykinin induced increases in intracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1992

Physical activity levels of 5-11-year-old children in England as determined by continuous observation.

Continuous observation of the physical activity of 56 preadolescent children was conducted in four regions of England from October 1988 to July 1989. Observations were undertaken during school break times, lunch times, and physical education lessons, as well as during the children's free time. Of total time observed, the children spent 32.4% of it engaged in moderate to vigorous physical activity (MVPA). Children were shown to be most active during school breaks and least active during free time at home. However, it is thought that cardiorespiratory benefits are best achieved by participation in sustained periods of MVPA. Only 8 (14%) of the 56 children were observed to participate in MVPA for sustained periods of 20 min or longer. A random sample of 55 physical education lessons was observed. Only 6 children engaged in MVPA for sustained periods of 5 min or more, and no children participated in MVPA for 10 min or longer. The implications of these results are that, although the children engaged in a reasonable amount of MVPA, it may not have been sustained for periods of sufficient length to produce a cardiovascular training response. Consideration is given to ways of promoting lifelong involvement in physical activity.

Child↗
PubMed1990

Homologous desensitization of the D1 dopamine receptor.

Preincubation of D384 cells, derived from the human astrocytoma cell line G-CCM, with dopamine resulted in a time-dependent attenuation of cyclic AMP responsiveness to subsequent dopamine stimulation. This effect was agonist specific because the prostaglandin E1 (PGE1) stimulation of cyclic AMP of similarly treated cells remained unchanged. The attenuation by dopamine was concentration dependent with a maximum observed at 100 microM. A comparison of dopamine concentration-response curves of control and dopamine-preincubated cells revealed no change in the Ka apparent value, but a marked attenuation of the maximal response. Preincubation of cells with dopamine in the presence of D1 but not D2 selective antagonists partially prevented the observed attenuation. Attenuations in dopamine responsiveness were also obtained when D384 cells were preincubated with D1 but not D2 receptor agonists. The level of attenuation attained related to agonist efficiency in stimulating cyclic AMP: SKF38393 less than 3,4-dihydroxynomifensine less than fenoldopam less than 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene = dopamine. However, increasing the efficiency of 3,4-dihydroxynomifensine stimulation of cyclic AMP, using the synergistic effect of adding a low concentration of forskolin, produced no further change in the attenuation of the subsequent response to dopamine. Thus, the D1 dopamine receptors expressed by D384 cells undergo homologous desensitization. Uncoupling of the D1 dopamine receptor appears to be independent of cyclic AMP formation, analogous to a mechanism proposed for the beta-adrenergic receptor.

Alprostadil↗
PubMedDate not supplied

Are primary school children gaining heart health benefits from their journeys to school?

Information was collected in relation to 1133 children, aged 4-11 years, regarding methods of travel to and from school, together with distances involved. Ninety-seven per cent of the children walked, travelled by car or alternated between these 2 methods of travel. Some 584 children (51.5%) walked to school every day with an average one-way-distance of less than half a mile. It would seem therefore that few primary school children are walking far enough to gain heart health benefits from their journeys to school. Reasons are given, however, as to why it is important for young children to be encouraged to walk to school providing adequate safety considerations are taken into account.

Child↗
PubMedDate not supplied

Specialty effects.

Explore the source record for details and available documents.

Humans↗