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Biomedical subjects

T M Palmer

Publications and source records attributed to T M Palmer.

At least 19 recordsLinked to original sources
PubMed2005

Inhibition of pro-inflammatory cytokine receptor signalling by cAMP in vascular endothelial cells.

The anti-inflammatory effects of the prototypical second messenger cAMP have been extensively documented in multiple cell types. However, in many instances, the molecular mechanisms by which cAMP elevation disrupts specific pro-inflammatory signalling cascades are unknown. In this review, we will describe the importance of the JAK-STAT (where JAK stands for Janus kinase and STAT for signal transducer and activator of transcription) signalling pathway in vascular endothelial cell function, outline key inhibitory processes that serve to reduce cytokine-stimulated tyrosine phosphorylation and activation of STAT proteins, and discuss possible mechanisms by which intracellular cAMP sensors could interact with these inhibitory processes to diminish cytokine receptor-mediated pro-inflammatory signalling.

Cyclic AMP↗
PubMed2001

The role of pit-and-fissure discoloration in caries assessment.

Occlusal dental caries often is not apparent using traditional diagnostic techniques. The clinical significance of pit-and-fissure discoloration in the absence of dietary and habit substances was examined. The study included 462 extracted teeth (216 with obvious pit-and-fissure discoloration, 216 with subtle discoloration, and 30 with no discoloration); 130 clinical teeth with varying degrees of pit-and-fissure discoloration; and 159 teeth in young adults, which had sealants placed > or = 10 years. All teeth studied were excavated very conservatively using air abrasion and/or the uniquely small H1 004 carbide bur. Presence of caries and its depth and extent were recorded, photographed, and measured with a custom made calibrated probe. Of the 721 teeth with discolored pits and fissures studied (432 extracted teeth and 289 clinical teeth), 660 (92%) had two or more of the four clinical criteria used to define dental caries in this study. Sixty-four percent of the lesions were > 2 mm in depth and 27% were > 3 mm in depth. Of the 159 teeth sealed for > or = 10 years, 47 (92%) were carious, and 26% had large, deep carious lesions penetrating > 3 mm. These data indicate: more effective methods are needed to diagnose pit-and-fissure caries and presence of pit-and-fissure discoloration in the absence of substances causing extrinsic staining should be a strong warning for clinicians to examine carefully for dental caries.

Adult↗
PubMed2000

Identification of threonine residues controlling the agonist-dependent phosphorylation and desensitization of the rat A(3) adenosine receptor.

Activation of the A(3) adenosine receptor (A(3)AR) contributes to the cardioprotective, bronchoconstrictive, and hypotensive effects of adenosine. Agonist occupation of the A(3)AR results in a rapid desensitization of receptor function, which is associated with the phosphorylation of the receptor protein by one or more members of the G protein-coupled receptor kinase family of protein kinases. Although we demonstrated previously that phosphorylation of the C-terminal 14 amino acids of the rat A(3)AR is crucial for rapid desensitization to occur, the identity of the critical phosphorylation sites has remained unknown. Here, we demonstrate that the simultaneous mutation of Thr(307), Thr(318), and Thr(319) to Ala residues dramatically reduces agonist-stimulated phosphorylation and rapid desensitization of the rat A(3)AR. Individual mutation of each residue demonstrated that Thr(318) and Thr(319) are the major sites of phosphorylation. Phosphorylation at Thr(318) appeared to be necessary to observe phosphorylation at Thr(319), but not vice versa. However, the replacement of Thr(318) with a glutamate residue demonstrated that the simple addition of negative charge at position 318 was not sufficient to rescue phosphorylation at position 319. In addition, the mutation of two predicted palmitoylation-site cysteine residues proximal to the regulatory domain resulted in the appearance of an agonist-independent basal phosphorylation. Therefore, G protein-coupled receptor kinase-mediated phosphorylation of the C-terminal tail of the A(3)AR in situ appears to follow a sequential mechanism, perhaps involving receptor depalmitoylation, with phosphorylation at Thr(318) being particularly important.

Alanine↗
PubMed2000

Subtype-specific kinetics of inhibitory adenosine receptor internalization are determined by sensitivity to phosphorylation by G protein-coupled receptor kinases.

Despite coupling to the same class of inhibitory G proteins and binding the same physiological ligand, the human A(1) and rat A(3) adenosine receptors (ARs) desensitize at different rates in response to sustained agonist exposure. This is due to the ability of the A(3)AR, but not the A(1)AR, to serve as a substrate for rapid phosphorylation and desensitization by members of the G protein-coupled receptor kinase (GRK) family. The aim of this study was to investigate whether these differences were also manifested in their abilities to undergo agonist-dependent receptor internalization. For the first time, we report that A(3)ARs internalize profoundly in response to short-term exposure to agonist but not activators of second messenger-regulated kinases. The A(3)AR-selective antagonist MRS1523 blocked both A(3)AR phosphorylation and internalization. Moreover, in contrast to the A(1)AR, which internalized quite slowly (t(1/2) = 90 min), A(3)ARs internalized rapidly (t(1/2) = 10 min) over a time frame that followed the onset of receptor phosphorylation. A nonphosphorylated A(3)AR mutant failed to internalize over a 60-min time course, suggesting that receptor phosphorylation was essential for rapid A(3)AR internalization to occur. In addition, fusion onto the A(1)AR of the A(3)AR C-terminal domain containing the sites for phosphorylation by GRKs conferred rapid agonist-induced internalization kinetics (t(1/2) = 10 min) on the resulting chimeric AR. In conclusion, these data suggest that GRK-stimulated phosphorylation of threonine residues within the C-terminal domain of the A(3)AR is obligatory to observe rapid agonist-mediated internalization of the receptor.

Animals↗
PubMed1999

Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: evidence for regulation by multiple protein kinase C isoforms.

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.

Acetophenones↗
PubMed1999

Resin polymerization problems--are they caused by resin curing lights, resin formulations, or both?

Negative effects of rapid, high-intensity resin curing have been predicted for both argon lasers and plasma-arc curing lights. To address these questions, six different resin restorative materials were cured with 14 different resin curing lights representing differences in intensities ranging from 400 mW/cm2 to 1,900 mW/cm2; delivery modes using constant, ramped, and stepped methods; cure times ranging from 1 second to 40 seconds; and spot sizes of 6.7 mm to 10.9 mm. Two lasers, five plasma-arc lights, and seven halogen lights were used. Shrinkage, modulus, heat generation, strain, and physical changes on the teeth and resins during strain testing were documented. Results showed effects associated with lights were not statistically significant, but resin formulation was highly significant. Microfill resins had the least shrinkage and the lowest modulus. An autocure resin had shrinkage and modulus as high as or higher than the light-cured hybrid resins. Lasers and plasma-arc lights produced the highest heat increases on the surface (up to 21 degrees C) and within the resin restorations (up to 14 degrees C), and the halogen lights produced the most heat within the pulp chamber (up to 2 degrees C). Strain within the tooth was least with Heliomolar and greatest with Z100 Restorative and BISFIL II autocure resin. Clinical effects of strain relief were evident as white lines at the tooth-resin interface and cracks in enamel adjacent to the margins. This work implicates resin formulation, rather than light type or curing mode, as the important factor in polymerization problems. Lower light intensity and use of ramped and stepped curing modes did not provide significant lowering of shrinkage, modulus, or strain, and did not prevent enamel cracking adjacent to margins and formation of "white line" defects at the margins. Until materials with lower shrinkage and modulus are available, use of low-viscosity surface sealants as a final step in resin placement is suggested to seal defects.

Acrylic Resins↗
PubMed1997

Signalling enzymes: bursting with potential.

A newly identified form of phosphoinositide 3-kinase is regulated by G beta gamma subunits and is particularly abundant in phagocytic leukocytes. It is likely to be intimately involved in the process by which inflammatory signals regulate phagocyte activation and is a potential target for new anti-inflammatory drugs.

Amino Acid Sequence↗
PubMed1997

Identification of an A2a adenosine receptor domain specifically responsible for mediating short-term desensitization.

In an attempt to delineate the structural requirements necessary for agonist-induced desensitization, we have stably expressed wild-type and mutant A2a adenosine receptors (A2aARs) in Chinese hamster ovary cells and examined the effects of agonist pretreatment on adenylyl cyclase activity in subsequently isolated membranes. Deletion of 95 amino acids from the carboxyl-terminus of the A2aAR, thereby removing 10 potential phosphorylation sites, failed to alter radioligand binding, adenylyl cyclase activation, or functional desensitization parameters as compared with the wild-type receptor. However, simultaneous mutation of Thr 298 and Ser 305 to Ala residues attenuated the desensitization observed in response to short-term (30 min) agonist treatment while not blocking the ability to desensitize after long-term (24 h) agonist exposure. Individual mutation of these residues revealed that mutation of Thr 298 alone was sufficient to diminish both short-term desensitization and agonist-stimulated receptor phosphorylation. These data suggest that while the majority of the A2aAR carboxyl-terminus is dispensable with regard to observing functional desensitization, the integrity of Thr 298 is essential for observing agonist-stimulated receptor phosphorylation and short-term desensitization but not long-term desensitization of A2aAR function.

Adenosine↗
PubMed1997

Structure-function analysis of inhibitory adenosine receptor regulation.

Pharmacological and molecular cloning studies have revealed the presence of four adenosine receptor (AR) subtypes, termed A1, A2A, A2B and A3. Given that the A1 and A3ARs can both bind adenosine and couple productively to inhibitory G-proteins, the significance of the existence of multiple inhibitory AR subtypes remains obscure, although one possibility is that these receptors are regulated in a subtype-specific manner. In this review, we summarize our investigations into the mechanisms underlying the agonist-induced desensitization of inhibitory AR function. The results of this work demonstrate that while the A1AR desensitizes slowly over a time course of several hours, the A3AR desensitizes within minutes of agonist exposure. Molecular biological studies have begun to delineate the structural requirements responsible for these differences, and will provide a basis for future experiments designed to determine whether the ability of an inhibitory AR receptor subtype to 'turn-off' at a specific rate has implications for the physiological role of that receptor.

Animals↗
PubMed1997

Induction of multiple effects on adenylyl cyclase regulation by chronic activation of the human A3 adenosine receptor.

The A3 adenosine receptor (A3AR) contributes to several cardiovascular effects of adenosine, including antihypertensive and cardioprotective effects. Although several studies have detailed the mechanisms underlying agonist-mediated desensitization of the rat A3AR, the regulation of the human A3AR, which displays only a 70% amino acid identity with the rat homologue, has not been addressed. Using a Chinese hamster ovary cell line stably expressing a recombinant human A3AR, we demonstrated that prolonged treatment with the AR agonist 5'-N-ethylcarboxamidoadenosine induces uncoupling of the A3AR from G proteins and functional desensitization. In addition to A3AR desensitization, a 1.5-2.5-fold increase was noted in the adenylyl cyclase (AC) activity achieved in the presence of GTP with or without forskolin. This sensitization of AC activity was not a consequence of the down-regulation of Gi proteins induced by NECA treatment and was not associated with sustained or transient increases in the expression of Gs. Time course experiments revealed that the onset of sensitization was half-maximal between 2 and 3 hr but was not due to the synthesis of new proteins because cycloheximide treatment failed to inhibit sensitization. The inability of the sensitization process to alter the AC activity obtained in the presence of manganese chloride suggests that prolonged A3AR activation increases the coupling efficiency between Gs and AC catalytic units. This phenomenon has implications for long term cellular adaptation to agonist because in agonist-treated cells, the extent to which a suboptimal concentration of forskolin could increase phosphorylation of the cAMP-responsive element binding protein was elevated compared with vehicle-treated controls.

Adenylyl Cyclases↗
PubMed1996

Molecular basis for subtype-specific desensitization of inhibitory adenosine receptors. Analysis of a chimeric A1-A3 adenosine receptor.

The differing effects of short-term agonist exposure on the two inhibitory adenosine receptor (AR) subtypes have been examined using Chinese hamster ovary cells stably expressing the hemagglutinin epitope-tagged human A1AR and rat A3AR. Under conditions in which exposure of transfected cells to 5 microM (-)-(R)-N6-(phenylisopropyl)adenosine resulted in the functional desensitization and phosphorylation of the A3AR, neither property was exhibited by the A1AR. However, a stably expressed chimeric A1-A3AR, termed A1CT3AR, in which the C-terminal domain of the A1AR distal to its predicted palmitoylation site was replaced by the corresponding region of the A3AR, was able to undergo functional desensitization and agonist-stimulated phosphorylation in a manner similar to that exhibited by the A3AR. Moreover, purified G-protein-coupled receptor kinases 2, 3, and 5 were each capable of enhancing the agonist-dependent phosphorylation of the A3AR and A1CT3AR in vitro. Taken together, these data demonstrate that the C-terminal domain of the A3AR distal to its predicted palmitoylation site is responsible for this receptor's ability to undergo a rapid agonist-dependent desensitization and are consistent with a model in which phosphorylation of the A3AR within this domain by one or more G-protein-coupled receptor kinases initiates the desensitization process.

Adenosine↗
PubMed1995

Agonist-dependent phosphorylation and desensitization of the rat A3 adenosine receptor. Evidence for a G-protein-coupled receptor kinase-mediated mechanism.

A3 adenosine receptor (A3AR) activation contributes to both the cardioprotective and antihypertensive effects of adenosine. To date, no studies have examined the mechanisms by which this receptor undergoes rapid homologous desensitization. Therefore, a functional hemagglutinin epitope-tagged A3AR has been stably expressed in Chinese hamster ovary cells, and its regulation by the AR agonist 5'-N-ethylcarboxamidoadenosine (NECA) has been studied. Cellular exposure to NECA induces rapid (t1/2 = approximately 1 min) A3AR phosphorylation on serine and threonine residues. This is associated with a functional desensitization and a 30-40% reduction in the number of high affinity agonist binding sites as determined by radioligand binding assays. Activation of second messenger-regulated kinases could not mimic the effect of NECA, suggesting a role for G-protein-coupled receptor kinases (GRKs). In vitro phosphorylation assays demonstrate that phosphorylation of agonist-occupied A3ARs is enhanced by GRK2 and that cellular pretreatment with NECA dramatically inhibits subsequent GRK2-mediated phosphorylation in vitro. Therefore, the A3AR is phosphorylated in situ by a kinase similar or identical to GRK2, and this may be involved in rapid functional desensitization of the A3AR.

Adenosine↗
PubMed1995

Differential interaction with and regulation of multiple G-proteins by the rat A3 adenosine receptor.

Interaction of the rat A3 adenosine receptor (A3AR) with G-proteins has been assessed using a stably transfected Chinese hamster ovary cell system. The non-selective AR agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased the labeling of a 41-kDa membrane protein by 4-azidoanilido-[alpha-32P]guanosine 5'-triphosphate (AA-[32P]GTP), a photolabile GTP analogue. Subsequent immunoprecipitation of Gi alpha-subunits indicated that NECA stimulated incorporation of label into both Gi alpha-2 and Gi alpha-3. Additional experiments revealed an A3AR stimulation of label into Gq and/or G11 alpha-subunits, albeit to a lesser degree than that elicited by endogenous P2U purinergic receptors. No interaction with Gs could be detected. Sustained cellular exposure to NECA induced A3AR desensitization and specific down-regulation of Gi alpha-3 and G-protein beta-subunits without changing levels of Gi alpha-2, Gs alpha, or Gq+11 alpha-subunits. Therefore the A3AR can interact with Gi alpha-2, Gi alpha-3, and, to some extent, Gq-like proteins, but sustained agonist exposure down-regulates only one of the G-proteins with which it interacts. This is the first description of the differing specificities of A3AR/G-protein coupling versus down-regulation in situ and provides a potential mechanism by which the A3AR could elicit the heterologous desensitization of signaling events mediated by Gi3.

Animals↗
PubMed1995

125I-4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol, a high affinity antagonist radioligand selective for the A2a adenosine receptor.

The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}-{1,2,4}triazolo{2,3-a}- {1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5'-N-carboxamidoadenosine, 5'-N-ethylcarboxamidoadenosine, and (-)-N6[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM214385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM214385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.

Animals↗
PubMed1994

Desensitization of the canine A2a adenosine receptor: delineation of multiple processes.

Stable cell lines that express the canine-derived A2a adenosine receptor (A2aAR) have been generated. Using a previously characterized anti-A2aAR antibody probe, we have identified the recombinant receptor protein and examined the desensitization process of this G protein-coupled receptor. Agonist exposure induced a rapid desensitization of A2aAR-stimulated adenylyl cyclase activity. This was associated with reduced affinity of the receptor for the A2aAR-selective agonist [3H]CGS21680 and agonist-stimulated phosphorylation of the receptor protein. Agonist-stimulated A2aAR sequestration into a light membrane fraction was also detected over the same time frame but, whereas inhibition of this process did not affect the extent of desensitization, the rapid recovery normally observed after short term agonist exposure was dramatically reduced. Long term agonist treatment resulted in the down-regulation of A2aARs and up-regulation of Gi alpha 2 and Gi alpha 3, as determined by immunoblotting. Recovery of A2aAR function after agonist removal required several hours and was associated with the return of receptor levels to control values. In contrast, inactivation of Gi proteins by pertussis toxin treatment did not alter the extent of agonist-induced desensitization observed. Neither short nor long term desensitization could be mimicked by elevation of intracellular cAMP levels alone. Therefore, these data suggest that A2aAR desensitization is mediated by multiple, temporally distinct, agonist-dependent processes. Agonist-stimulated phosphorylation of the receptor may induce short term desensitization by impairing receptor-Gs coupling, whereas long term down-regulation of receptor number and up-regulation of inhibitory G proteins mediate long term adaptation.

Adenosine↗
PubMed1992

Alterations in G-protein expression, Gi function and stimulatory receptor-mediated regulation of adipocyte adenylyl cyclase in a model of insulin-resistant diabetes with obesity.

The stimulatory effect of Mn2+ (1.5-fold), forskolin (1.6-fold) and low (1 microM) concentrations of GTP (1.9-fold) on the adenylyl cyclase of adipocyte membranes from obese, diabetic CBA/Ca mice was markedly enhanced compared to that seen using membranes prepared from their lean littermates. In contrast, receptor-mediated stimulation, achieved with either isoprenaline or secretin was reduced and that by glucagon abolished in membranes from diabetic animals. The levels of expression of alpha-subunits of Gi-1, Gi-2 and Gi-3 were reduced to some 49, 76 and 54%, respectively, in membranes from diabetic animals compared with those from normal animals. Levels of G-protein beta-subunits and Gs alpha-subunits were similar. Receptor-mediated inhibition of adenylate activity elicited by either nicotinic acid or prostaglandin E1 (PGE1) was of a similar magnitude in membranes from normal and diabetic animals but the inhibitory action of N6-(L-2-phenylisopropyl)adenosine (PIA) was greater in membranes from diabetic animals by about 30%. Gi function was similarly evident in membranes from both lean and diabetic animals, as assessed using low concentrations of guanylyl 5'-imidodiphosphate to inhibit forskolin-stimulated adenylyl cyclase activity. However, assessing Gi function using GTP showed marked dissimilarities in that the elevated GTP concentrations expected to occur physiologically were incapable of reversing the stimulation achieved at low concentrations of GTP in membranes from diabetic but not normal animals. The adipocytes of CBA/Ca mice, as do other animal models of insulin resistance, show lesions in adenylyl cyclase regulation, Gi function and G-protein expression.

Adenylyl Cyclases↗