Biomedical subjects

R A Challiss

Publications and source records attributed to R A Challiss.

At least 37 records · Page 2Linked to original sources

PubMed1999

Cross talk between m3-muscarinic and beta(2)-adrenergic receptors at the level of receptor phosphorylation and desensitization.

In this study we investigated cross talk between m3-muscarinic and beta(2)-adrenergic receptors coexpressed in Chinese hamster ovary (CHO-m3/beta(2)) cells, focusing on two possible mechanisms of regulation. The first mechanism is based on recent in vitro studies demonstrating that G protein-coupled receptor kinase (GRK) activity, the protein kinase responsible for beta(2)-adrenergic receptor homologous phosphorylation and desensitization, may be regulated by calcium/calmodulin and membrane phosphatidylinositol 4, 5-bisphosphate. Stimulation of the phospholipase C signaling pathway via m3-muscarinic receptors in CHO-m3/beta(2) cells increased intracellular free calcium by approximately 10 fold and membrane phosphatidylinositol 4,5-bisphosphate levels decreased by approximately 74%. However, despite these changes the ability of endogenous kinases, possibly the GRKs, to phosphorylate the beta(2)-adrenergic receptor was not altered. The second mechanism investigated involves a direct heterologous phosphorylation of the beta(2)-adrenergic receptor after muscarinic receptor stimulation. Activation of m3-muscarinic receptors did mediate heterologous phosphorylation of beta(2)-adrenergic receptors in a GRK-independent fashion, via protein kinase C. Heterologous beta(2)-adrenergic receptor phosphorylation correlated with receptor desensitization as measured by a loss in guanine-nucleotide sensitive-high affinity agonist binding and reduction in maximal cAMP response. This receptor cross talk may have a profound physiological importance in a wide variety of cell types, for example smooth muscle, where these two receptors are known to be coexpressed.

Animals↗

PubMed1998

Differential regulation of muscarinic acetylcholine receptor-sensitive polyphosphoinositide pools and consequences for signaling in human neuroblastoma cells.

In this study we have quantitatively assessed the basal turnover of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and M3-muscarinic receptor-mediated changes in phosphoinositides in the human neuroblastoma cell line, SH-SY5Y. We demonstrate that the polyphosphoinositides represent a minor fraction of the total cellular phosphoinositide pool and that in addition to rapid, sustained increases in [3H]inositol phosphates dependent upon the extent of receptor activation by carbachol, there are equally rapid and sustained reductions in the levels of polyphosphoinositides. Compared with phosphatidylinositol 4-phosphate (PtdIns(4)P), PtdIns(4,5)P2 was reduced with less potency by carbachol and recovered faster following agonist removal suggesting protection of PtdIns(4,5)P2 at the expense of PtdIns(4)P and indicating specific regulatory mechanism(s). This does not involve a pertussis toxin-sensitive G-protein regulation of PtdIns(4)P 5-kinase. Using wortmannin to inhibit PtdIns 4-kinase activity, we demonstrate that the immediate consequence of blocking the supply of PtdIns(4)P (and therefore PtdIns(4,5)P2) is a failure of agonist-mediated phosphoinositide and Ca2+ signaling. The use of wortmannin also indicated that PtdIns is not a substrate for receptor-activated phospholipase C and that 15% of the basal level of PtdIns(4,5)P2 is in an agonist-insensitive pool. We estimate that the agonist-sensitive pool of PtdIns(4,5)P2 turns over every 5 s (0.23 fmol/cell/min) during sustained receptor activation by a maximally effective concentration of carbachol. Immediately following agonist addition, PtdIns(4,5)P2 is consumed >3 times faster (0.76 fmol/cell/min) than during sustained receptor activation which represents, therefore, utilization by a partially desensitized receptor. These data indicate that resynthesis of PtdIns(4,5)P2 is required to allow full early and sustained phases of receptor signaling. Despite the critical dependence of phosphoinositide and Ca2+ signaling on PtdIns(4,5)P2 resynthesis, we find no evidence that this rate resynthesis is limiting for agonist-mediated responses.

Androstadienes↗

PubMed1998

A modulatory effect of extracellular Ca2+ on type 1alpha metabotropic glutamate receptor-mediated signalling.

Increasing [Ca2+]e from 1.3 4 mM had little effect on basal phospholipase C activity in baby hamster kidney (BHK) cells stably expressing either type 1alpha metabotropic glutamate (mGlu1alpha) or M3-muscarinic (m3) receptors, but enhanced agonist-stimulated phosphoinositide hydrolysis in BHK-mGlu1alpha, but not BHK-m3 cells, demonstrating that Ca2+(e) selectively modulates phosphoinositide signalling stimulated by this receptor subtype.

Animals↗

PubMed1998

Reversible and non-competitive antagonist profile of CPCCOEt at the human type 1alpha metabotropic glutamate receptor.

In transfected CHO cells expressing the human metabotropic glutamate receptor mGlu1alpha, 7-(hydroxyimino)cyclopropan[b]-chromen-1a-carboxylic acid ethylester (CPCCOEt) was found to antagonize L-quisqualate-induced phosphoinositide hydrolysis in a non-competitive and reversible manner (apparent pKi value, 4.76+/-0.18; n=3). This suggests that CPCCOEt antagonizes type 1alpha metabotropic glutamate receptor activation by interacting with a site distinct from the agonist binding site.

Animals↗

PubMed1998

New developments in the molecular pharmacology of the myo-inositol 1,4,5-trisphosphate receptor.

Receptor-mediated activation of phospholipase C to generate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] is a ubiquitous signalling pathway in mammalian systems. A family of three IP3 receptor subtype monomers form functional tetramers, which act as effectors for Ins(1,4,5)P3, providing a ligand-gated channel that allows Ca2+ ions to move between cellular compartments. As IP3 receptors are located principally, although not exclusively, in the endoplasmic reticular membrane, Ins(1,4,5)P3 is considered to be a second messenger that mobilizes Ca2+ from intracellular stores. Ca2+ store mobilization by Ins(1,4,5)P3 can be shown to contribute to a variety of physiological and pathophysiological phenomena, and therefore the IP3 receptor represents a novel, potential pharmacological target. In this article, Rob Wilcox and colleagues review recent developments in IP3 receptor pharmacology, with particular emphasis on ligand molecular recognition by this receptor-channel complex. The potential for designing non-inositol phosphate-based agonists and antagonists is also discussed.

Animals↗

PubMed1998

Regulation of phosphoinositide turnover in neonatal rat cerebral cortex by group I- and II- selective metabotropic glutamate receptor agonists.

1. The interactive effects of different metabotropic glutamate (mGlu) receptor subtypes to regulate phosphoinositide turnover have been studied in neonatal rat cerebral cortex and hippocampus by use of agonists and antagonists selective between group I and II mGlu receptors. 2, The group II-selective agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC; 100 microM) had no effect on basal total inositol phosphate ([3H]-InsPx) accumulation (in the presence of Li+) in myo-[3H]-inositol pre-labelled slices, but enhanced the maximal [3H]-InsPx response to the group I-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by about 100% in both hippocampus and cerebral cortex. In cerebral cortex the enhancing effect of 2R,4R-APDC occurred with respect to the maximal responsiveness and had no effect on EC50 values for DHPG (-log EC50 (M): control, 5.56+/-0.05; +2R,4R-APDC, 5.51+/-0.08). 2R,4R-APDC also caused a significant enhancement of the DHPG-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass response over an initial 0-300 s time-course. 3. The enhancing effects of 2R,4R-APDC on DHPG-stimulated [3H]-InsPx accumulation were observed in both the presence and nominal absence of extracellular Ca2+, and irrespective of whether 2R,4R-APDC was added before, simultaneous with, or subsequent to DHPG. Furthermore, increasing the tissue cyclic AMP concentration up to 100 fold had no effect on DHPG-stimulated Ins(l,4,5)P3 accumulation in the absence or presence of 2R,4R-APDC. 4. 2R,4R-APDC and (2S, 1'R, 2'R, 3'R)-2-(2,3-dicarboxylcyclopropyl)glycine (DCG-IV), the latter agent in the presence of MK-801 to prevent activation of NMDA-receptors, each inhibited forskolin-stimulated cyclic AMP accumulation by about 50%, with respective EC50 values of 1.3 and 0.04 microM (-log EC 50 (M): 2R,4R-APDC, 5.87+/-0.09; DCG-IV, 7.38+/-0.05). In the presence of DHPG (30 microM), 2R,4R-APDC and DCG-IV also concentration-dependently increased [3H]-InsPx accumulation with respective EC50 values of 4.7 and 0.28 microM (-log EC50 (M): 2R,4R-APDC, 5.33+/-0.04; DCG-IV, 6.55+/-0.09) which were 3-7 fold rightward-shifted relative to the adenylyl cyclase inhibitory responses. 5. The group II-selective mGlu receptor antagonist LY307452 (30 microM) caused parallel rightward shifts in the concentration-effect curves for inhibition of forskolin-stimulated adenylyl cyclase, and enhancement of DHPG-stimulated [3H]-InsPx accumulation, by 2R,4R-APDC yielding similar equilibrium dissociation constants (KdS, 3.7+/-1.1 and 4.1+/-0.4 microM respectively) for each response. 6. The ability of 2R,4R-APDC to enhance receptor-mediated [3H]-InsPx accumulation appeared to be agonist-specific; thus although DHPG (100 microM) and the muscarinic cholinoceptor agonist carbachol (10 microM) stimulated similar [3H]-InsPx accumulations, only the response to the former agonist was enhanced by co-activation of group II mGlu receptors. 7. These data demonstrate that second messenger-generating phosphoinositide responses stimulated by group I mGlu receptors are positively modulated by co-activation of group II mGlu receptors in cerebral cortex and hippocampus. The data presented here are discussed with respect to the possible mechanisms which might mediate the modulatory activity, and the physiological and pathophysiological significance of such crosstalk between mGlu receptors.

Adenylyl Cyclase Inhibitors↗

PubMed1998

Modulation of spasmogen-stimulated Ins(1,4,5)P3 generation and functional responses by selective inhibitors of types 3 and 4 phosphodiesterase in airways smooth muscle.

1. The effects of isoenzyme-selective inhibitors of phosphodiesterases PDE3 and PDE4 on cyclic AMP concentration, two indices of phosphoinositide hydrolysis, and contractile responses to spasmogens have been investigated in bovine tracheal smooth muscle (BTSM). 2. Neither the PDE3-selective inhibitor ORG 9935, nor the PDE4-selective inhibitor rolipram increased cyclic AMP levels in BTSM. However, rolipram addition in the presence of PDE3 inhibition (ORG 9935; 1 microM) concentration-dependently (-log EC50 (M), 6.55+/-0.15; n = 3) increased cyclic AMP levels to about 70% of the maximal response to the beta-adrenoceptor agonist isoprenaline. 3. Rolipram per se inhibited histamine-stimulated [3H]-inositol (poly)phosphate ([3H]-InsP(X)) accumulation by > 80% (-log EC50 (M), 6.92+/-0.11; n = 3). Although ORG 9935 (1 microM) had little effect on histamine-stimulated [3H]-InsP(X) accumulation alone it greatly facilitated the inhibitory action of rolipram (-log EC50 (M), 8.82+/-0.39; n = 3). The effects of PDE3 and/or PDE4 inhibition on [3H]-InsP(X) accumulation stimulated by muscarinic acetylcholine (mACh) receptor activation were less marked. However, combined PDE3/4 inhibition significantly decreased this response at a submaximal concentration of mACh receptor agonist (carbachol; 1 microM). 4. The greater-than-additive effect of combined PDE3/4 inhibition was also observed at the level of contractile responses to histamine and carbachol. In experiments designed to investigate the effects of PDE3 and/or 4 inhibitors on the carbachol-mediated phasic contraction, additions of rolipram (10 microM) or ORG 9935 (1 microM) were without effect, whereas added together the inhibitors caused a significant (P < 0.01) 40% reduction in the peak phasic contractile response. 5. The effect on contraction correlated with a substantial inhibitory effect of PDE3/4 inhibition on the initial increase in inositol 1,4,5-trisphosphate (InsP3) accumulation stimulated by spasmogen. Thus, in the presence of ORG 9935 (1 microM) rolipram concentration-dependently inhibited carbachol-stimulated InsP3 accumulation by > or = 50% (-log EC50 (M), 6.77+/-0.21; n = 4). 6. Carbachol (100 microM) addition caused a rapid decrease (by 67% at 10 s) in BTSM cyclic AMP level in the presence of PDE3/4 inhibition. However, omission of Ca2+ from the incubation medium prevented the carbachol-evoked decrease in cyclic AMP and this coincided with a greater inhibition (> or = 80%) of the carbachol-stimulated InsP3 response. 7. These data indicate that combined PDE3 and PDE4 inhibition has greater-than-additive effects on second messenger and functional responses to spasmogens in BTSM. Furthermore, the ability of PDE3/4 inhibition significantly to attenuate mACh receptor-mediated contractile responses, may be, at least in part, attributed to an effect exerted at the level of InsP3 generation.

Animals↗

PubMed1998

Characterization of an atypical muscarinic cholinoceptor mediating contraction of the guinea-pig isolated uterus.

In many smooth muscle tissues a minor M3-muscarinic acetylcholine (mACh) receptor population mediates contraction, despite the presence of a larger M2-mACh receptor population. However, this is not the case for guinea-pig uterus where radioligand binding and functional studies exclude a dominant role for M3-mACh receptors. Using tissue from animals pre-treated with diethylstilboestrol, estimates of antagonist affinity were made before and after selective alkylation procedures, together with estimates of agonist affinity to characterise the mACh receptor population mediating carbachol-induced contraction of guinea-pig isolated uterus. Antagonist affinity estimates made at 'protected' receptors were not significantly different from those made in untreated tissues. However all estimations were significantly different from those reported in guinea-pig ileum and atria. The rank order of affinities were atropine>zamifenacin=tripitramine> methoctramine. Carbachol-induced contractions were insensitive to the M4-selective muscarinic toxin MTx-3, or PD102807 (0.1 microM) ruling out a role for M4-mACh receptors. The agonist affinity value for L-660,863, a putative 'M2-selective' agonist of 5.44+/-0.30 (n=6) was significantly different from that reported in guinea-pig atria. In contrast, the pKA value for carbachol (4.22+/-0.17 n = 8) agrees with that reported for guinea-pig ileum. Carbachol-induced contractions were insensitive to pertussis toxin although carbachol-induced inhibition of forskolin-stimulated cyclic AMP production was attenuated, ruling out the involvement of Gi-proteins in contraction. Radioligand binding studies revealed a KD for N-[3H]-methylscopolamine of 0.12+/-0.05 nM and a Bmax of 147+/-18 fmol mg protein(-1). Antagonist affinity estimates made using competition binding studies supported previous data suggesting the presence of a homogenous population of M2-mACh receptors. These data suggest a small population of mACh receptors with an atypical operational profile which can not be distinguished using radioligand binding studies may mediate carbachol-induced contraction of guinea-pig isolated uterus.

Adenylyl Cyclases↗

PubMed1998

Effects of human type 1alpha metabotropic glutamate receptor expression level on phosphoinositide and Ca2+ signalling in an inducible cell expression system.

Stable expression of the human type 1alpha metabotropic glutamate (mGlu 1alpha) receptor was achieved in Chinese hamster ovary cells using an isopropyl-beta-D-thiogalactoside (IPTG)-repressible expression system. Treatment of the cells with IPTG resulted in a time- and concentration-dependent induction of receptor expression. Maximal expression was obtained after treatment of the cells with 100 microM IPTG for 20 h, leading to a marked increase in receptor immunoreactivity detected by western blot, >30-fold stimulation of 3H-labelled inositol phosphate (3H-InsP) production, and a robust increase in intracellular calcium concentration in single cells after stimulation with 20 microM quisqualate. The basal level of 3H-InsP accumulation in cells induced with IPTG was increased by two- to threefold as compared with control cells; however, this basal activity was found to be dependent on glutamate released by the cells into the incubation medium. Following IPTG treatment, stable expression of the mGlu 1alpha receptor was maintained for at least 1 week. Taken together, these results clearly indicate the advantages of working with an inducible expression system when studying the biochemical and pharmacological properties of the human mGlu 1alpha receptor in transfected cells.

Animals↗

PubMed1997

Differential expression and regulation of ryanodine receptor and myo-inositol 1,4,5-trisphosphate receptor Ca2+ release channels in mammalian tissues and cell lines.

Ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors (Ins(1,4, 5)P3Rs) represent two multigene families of channel proteins that mediate the release of Ca2+ ions from intracellular stores. In the present study, the expression patterns of these channel proteins in mammalian cell lines and tissues were investigated by using isoform-specific antibodies. All cell lines examined expressed two or more Ins(1,4,5)P3R isoforms, with the type 1 Ins(1,4,5)P3R being ubiquitous. RyR isoforms were detected in only six out of eight cell lines studied. Similarly, of the nine rabbit tissues examined, RyR protein expression was detected only in brain, heart, skeletal muscle and uterus. Specific [3H]ryanodine binding was found in a number of rabbit tissues, although it was not detected in mammalian cell lines. Subcellular fractionation of SH-SY5Y human neuroblastomas revealed that the type 2 RyR and type 1 Ins(1,4,5)P3R co-localize among the fractions of a sucrose-cushion separation of crude microsomal membrane fractions. Manipulation of SH-SY5Y cells by chronic stimulation of muscarinic acetylcholine receptor (mAChR) results in a decrease in their type 1 Ins(1,4,5)P3R levels but not in the abundance of the type 2 RyR. Differentiation of these neuroblastomas by using retinoic acid did not detectably alter their expression of Ca2+-release channel proteins. Finally, differentiation of BC3H1 cells affects the expression of their Ca2+-release channel proteins in an isoform-specific manner. In summary, this study demonstrates that mammalian cell lines display distinct patterns of Ca2+-release channel protein expression. The abundance of these proteins is differentially regulated during phenotypic modifications of a cell, such as differentiation or chronic stimulation of mAChR.

Amino Acid Sequence↗

PubMed1997

Dissociation between beta-adrenoceptor-mediated cyclic AMP accumulation and inhibition of histamine-stimulated phosphoinositide metabolism in airways smooth muscle.

Spasmogen-stimulated phosphoinositide hydrolysis represents one of the major signalling pathways mediating pharmacomechanical coupling in airways smooth muscle (ASM), and cyclic AMP-induced inhibition of phosphoinositidase C has been proposed as an important mechanism underlying the bronchodilator properties of beta2-adrenoceptor agonists. To examine this hypothesis in more detail we have undertaken a direct comparison of the effects of salbutamol and salmeterol, short- and long-acting beta2-adrenoceptor agonists respectively, on cyclic AMP accumulation and histamine-stimulated [3H]-inositol phospholipid hydrolysis in bovine tracheal smooth muscle (BTSM) slices. Although salmeterol displayed a similarly greater potency over salbutamol for both stimulation of cyclic AMP, and inhibition of [3H]-inositol phosphate accumulation, there was a clear disparity between these agents with respect to both their efficacies and the duration of their effects. Hence while salmeterol caused a more protracted, but initially smaller increase in cyclic AMP accumulation compared to salbutamol, the inhibition of histamine-stimulated [3H]-inositol phosphate accumulation observed with salmeterol was of identical duration to salbutamol and was more marked than that of salbutamol at early time points. These data suggest that cyclic AMP accumulation is not the sole mechanism responsible for beta2-adrenoceptor-induced inhibition of phosphoinositide turnover in BTSM, and would support a recent proposal that cyclic AMP-dependent inhibition of agonist-stimulated Ca2+ mobilization in ASM may be mediated by factors independent of inositol phosphate generation.

1-Methyl-3-isobutylxanthine↗

PubMed1997

Pharmacological characterization of type 1alpha metabotropic glutamate receptor-stimulated [35S]-GTPgammaS binding.

1. The activation of G proteins by type 1alpha metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1alpha receptor has been studied by use of a [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding assay. 2. L-Glutamate increased the rate of [35S]-GTPgammaS binding in a concentration-dependent manner (-logEC50 (M) 5.25 +/- 0.07), with an optimal (62.4 +/- 1.6%) increase over basal binding being observed following 60 min incubation at 30 degrees C with 70 pM [35S]-GTPgammaS, 1 microM GDP, 10 mM MgCl2, 100 mM NaCl and 100 microg membrane protein ml(-1). The L-glutamate (100 microM)-stimulated increase in [35S]-GTPgammaS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 microM). 3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K(D)) for [35S]-GTPgammaS binding of 0.76 +/- 0.20 nM and a maximal number of GTPgammaS-liganded G proteins (Bmax) of 361 +/- 30 fmol mg(-1) protein. 4. Metabotropic glutamate receptor agonists, quisqualate (-logEC50 (M) 6.74 +/- 0.06), 1S,3R-ACPD (4.64 +/- 0.08) and (S)-3,5-dihydroxyphenylglycine (5.16 +/- 0.23) also increased [35S]-GTPgammaS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists. 5. (+)-alpha-Methylcarboxyphenylglycine (300 microM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [35S]-GTPgammaS binding, allowing an antagonist equilibrium dissociation constant (K(D)) of 34.0 +/- 7.8 microM to be calculated for this mGluR antagonist. 6. Pretreatment of BHK-mGluR1alpha cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml(-1), 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [35S]-GTPgammaS binding (by 66.0 +/- 0.9%), and an altered concentration-effect relationship for agonist-stimulated [35S]-GTPgammaS binding by the residual PTX-insensitive G-protein population. 7. The modulation of [35S]-GTPgammaS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

Animals↗

PubMed1997

Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen-activated protein kinase.

1. The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2. Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+-response, but resulted in a more rapid decline to basal. There was no response to alpha,beta-MethylATP (alpha,beta MeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3. ATP (log EC50 -5.1+/-0.2) also caused an increase in the total [3H]-inositol (poly)phosphates ([3H]-InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and alpha,beta MeATP giving no detectable response. 4. Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P3. 5. None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPgammaS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 microM forskolin. 6. Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentration-dependency consistent with activation at P2Y2 receptors. 2MeSATP gave a much smaller response with a lower potency than UTP. 7. These results are consistent with brain endothelial regulation by P2Y2 receptors coupled to phospholipase C, Ca2+ and MAPK; and by P2Y1-like (2MeSATP-sensitive) receptors which are linked to Ca2+ mobilization by a mechanism apparently independent of agonist stimulated Ins(1,4,5)P3 levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK coupling of these receptors suggests that they exert fundamentally distinct influences over brain endothelial function.

Adenosine Diphosphate↗

PubMed1997

The effects of insulin on transport and metabolism of glucose in skeletal muscle from hyperthyroid and hypothyroid rats.

The effects of insulin on the rates of glucose disposal were studied in soleus muscles isolated from hyper- or hypothyroid rats. Treatment with triiodothyronine for 5 or 10 days decreased the sensitivity of glycogen synthesis but increased the sensitivity of lactate formation to insulin. The sensitivity of 3-O methylglucose to insulin was increased only after 10 days of treatment and was accompanied by an increase in the sensitivity of 2-deoxyglucose phosphorylation; however, 2-deoxyglucose and glucose 6-phosphate in response to insulin remained unaltered. In hypothyroidism, insulin-stimulated rates of 3-O-methylglucose transport and 2-deoxyglucose phosphorylation were decreased; however, at basal levels of insulin, 3-O-methylglucose transport was increased, while 2-deoxyglucose phosphorylation was normal. In these muscles, the sensitivity of lactate formation to insulin was decreased; this defect was improved after incubation of the muscles with prostaglandin E2. The results suggest: (a) in hyperthyroidism, insulin-stimulated rates of glucose utilization in muscle to form lactate are increased mainly because of a decrease in glycogen synthesis; when hyperthyroidism progresses in severity, increases in the sensitivity of glucose transport to insulin and in the activity of hexokinase may also be involved; (b) in hypothyroidism, the decrease in insulin-stimulated rates of glucose utilization is caused by decreased rates of glycolysis; (c) prostaglandins may be involved in the changes in sensitivity of glucose utilization to insulin observed in muscle in altered thyroid states.

Animals↗

PubMed1997

Enhanced type 1alpha metabotropic glutamate receptor-stimulated phosphoinositide signaling after pertussis toxin treatment.

The regulation of phosphoinositide hydrolysis by the type 1alpha metabotropic glutamate receptor (mGluR1alpha) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S, 3R-dicarboxylic acid resulted in a marked accumulation of [3H]inositol monophosphate (InsP1) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] mass in a time- and concentration-dependent manner. Pretreatment of BHK-mGluR1alpha cells with pertussis toxin [ 100 ng/ml, 24 hr] led to a dramatic 12-16-fold increase in the accumulation of [3H]InsP1 and a 2-fold increase in Ins(1,4,5)P3 in the absence of added agonist. Although only very low levels (</=1 microM) of L-glutamate could be detected in medium taken from control and PTX-treated cell monolayers, the PTX-elicited effect on basal [3H]InsP1 was fully reversed by preincubation of cells in the presence of glutamic-pyruvic transaminase and pyruvate, suggesting that an increased sensitivity to endogenous glutamate was responsible for the apparent agonist-independent activation of phosphoinositidase C (PIC) after PTX treatment. Consistent with this hypothesis, in the presence of glutamic-pyruvic transaminase/pyruvate, the maximal [3H]InsP1 response to quisqualate was increased by >/=75%, and the EC50 shifted leftward by 65-fold [-log EC50 values (molar), 7.26 +/- 0.23 versus 5.45 +/- 0.07; n = 4) in PTX-treated compared with control cells. In contrast, antagonist effects on agonist-stimulated [3H]InsP1 responses were similar in control and PTX-treated BHK-mGluR1alpha cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (Gi/o) and PTX-insensitive stimulatory (Gq/11) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1alpha-mediated PIC activity that could be fundamental in controlling the output of phosphoinositide-derived messengers.

Amino Acid Sequence↗

PubMed1996

Stable overexpression of the type-1 inositol 1,4,5-trisphosphate receptor in L fibroblasts: subcellular distribution and functional consequences.

InsP3 receptor (InsP3R)/Ca(2+)-release channels differ markedly in abundance in different tissues/cell types and InsP3R expression levels may be modulated in response to a variety of external cues. Cell lines overexpressing InsP3Rs will provide useful models for the study of the influence of receptor density and subtype on InsP3-mediated Ca2+ signalling. We have investigated the properties of InsP3Rs in mouse L fibroblast cell lines transfected with either type-1 InsP3R cDNA (L15) or vector control (Lvec). L15 cells express approximately eightfold higher levels of the type-1 InsP3R protein than Lvec cells, as assessed by radioligand binding and immunoblotting. Increased expression was stable since it did not alter over ten cell passages. Both L15 and Lvec cells express predominantly the type-1 InsP3R isoform, indicating that functional differences in the InsP3-mediated Ca2+ signalling in these cell lines are due to alteration in the levels of receptor rather than changes in the isoform expressed. Type-1 InsP3R in L15 cells is largely associated with subcellular membrane fractions bearing the sarco/endoplasmic reticulum Ca2+ ATPase pump, appropriate for rapidly exchanging Ca2+ pools. Functionally, there is an approximately fourfold increase in the sensitivity of permeabilized L15-cell Ca2+ mobilization in response to increasing concentrations of Ins(1,4,5)P3. This study indicates that L15/ Lvec cells provide a suitable model for studying the effects of InsP3R expression level on InsP3-induced Ca2+ mobilization.

Animals↗

PubMed1996

Contrasting effects of phorbol ester and agonist-mediated activation of protein kinase C on phosphoinositide and Ca2+ signalling in a human neuroblastoma.

The effects of protein kinase C (PKC) activation on muscarinic receptor-mediated phosphoinositide and Ca2+ signalling were examined in the human neuroblastoma, SH-SY5Y. Carbachol evoked rapid transient elevations of Ins(1,4,5)P3 and intracellular [Ca2+] followed by lower sustained elevations. Phorbol 12,13-dibutyrate (PDBu) preferentially attenuated transient phases. Removal of the transplasmalemmal Ca2+ gradient coupled with depletion of intracellular Ca2+ stores with thapsigargin also reduced carbachol-mediated Ins(1,4,5)P3 accumulation. Under these conditions, PDBu virtually abolished Ins(1,4,5)P3 responses to carbachol thereby implicating both Ca(2+)- and PKC-sensitive components. PDBu also reduced agonist-mediated accumulation of inositol phosphates and depletion of lipids, thereby eliminating an effect of PKC on Ins(1,4,5)P3 metabolism or phosphoinositide synthesis. In electroporated cells, PDBu inhibited Ins(1,4,5)P3 accumulation mediated by carbachol or guanosine 5'-[gamma-thio]-triphosphate, the latter indicating that some PDBu-sensitive elements were downstream of the receptor. The PKC inhibitor, Ro-318220, protected against PDBu but did not enhance responses to maximal concentrations of carbachol, indicating no feedback inhibition by agonist-activated PKC. Muscarinic antagonist activity of Ro-318220 complicated such assessment at low agonist concentrations. Carbachol or PDBu induced cytosol to membrane translocation of PKC alpha. This was faster and possibly greater with PDBu, which may explain the lack of feedback by agonist-activated PKC. These results indicate that, in SH-SY5Y cells, PDBu activation of PKC preferentially inhibits rapid muscarinic receptor-mediated phosphoinositide and Ca2+ responses via suppression of PtdIns(4,5)P2 hydrolysis. This is at least partially through inhibition of Gq-protein/phosphoinositidase C coupling. However, at least at high agonist concentrations, a major agonist-mediated PKC feedback is not present in these cells.

Animals↗

PubMed1996

Acute regulation of the receptor-mediated phosphoinositide signal transduction pathway.

It is apparent that the phosphoinositide signalling pathway is subject to a variety of regulatory features which will ultimately dictate the magnitude and profile of cellular responses to agonist occupation of PIC-linked receptors. Our understanding of these mechanisms is far from complete but will be crucial in revealing both the specificity of receptor signalling and the integration of signals arising from the potentially wide variety of metabotropic and ionotropic receptors on individual cells.

Animals↗