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R A Challiss

Publications and source records attributed to R A Challiss.

At least 91 records · Page 5Linked to original sources
PubMed1992

Comparative effects of BRL 38227, nitrendipine and isoprenaline on carbachol- and histamine-stimulated phosphoinositide metabolism in airway smooth muscle.

1. The ability of BRL 38227 and nitrendipine to affect muscarinic agonist and histamine-stimulated [3H]-inositol phosphate accumulation in slices of bovine tracheal smooth muscle has been studied and compared with the established inhibitory effects of isoprenaline on this pathway. 2. Pre-addition of BRL 38227 (5 microM), nitrendipine (1 microM) or isoprenaline (10 microM) significantly inhibited the subsequent inositol phosphate response to histamine at all concentrations studied (10- 1000 microM). BRL 38227 and nitrendipine also significantly inhibited the [3H]-inositol phosphate response to low (1 microM), but not high (100 microM) concentrations of carbachol. Isoprenaline had no effect at any concentration of carbachol studied. 3. Nitrendipine (IC50 = 95 nM) and BRL 38227 (IC50 = 322 nM) caused concentration-related inhibitions of the inositol phosphate response to histamine (100 microM). Similar maximal inhibitions were caused by each agent (55-58%). Inhibitory effect of BRL 38227 was reduced in potency (IC50 = 5.5 microM), but not magnitude, in the presence of glibenclamide (0.5 microM). 4. Time-course studies comparing the effects of BRL 38227 addition 15 min before, and 10 min after histamine challenge showed that for pre-addition a distinct (less than 2 min) lag occurred following histamine addition before the inhibitory effect of BRL 38227 was manifest. In contrast, when BRL 38227 was added 10 min after histamine, an inhibitory effect was immediately apparent. 5. Further evidence for an initial, 'protected' phase of inositol phosphate accumulation was provided by the finding that BRL 38227 pre-addition had no effect on the early (0-300 s) time-course of inositol 1,4,5-trisphosphate mass accumulation. 6. The inhibitory effect of BRL 38227, but not that of nitrendipine or isoprenaline, on histaminestimulated [3H]-inositol phosphate accumulation was completely prevented in the presence of an elevated extracellular K+ (65 mM) concentration. 7. The results demonstrate that membrane hyperpolarization, and/or blockade of voltage-operated Ca2"-channels can regulate agonist-stimulated phosphoinositide metabolism in airway smooth muscle. The possible contribution of this regulatory mechanism to the relaxant properties of these agents is discussed.

Animals↗
PubMed1992

Role of protein kinase C in the regulation of histamine and bradykinin stimulated inositol polyphosphate turnover in adrenal chromaffin cells.

1. The possibility that bradykinin- or histamine-stimulated inositol polyphosphate accumulation may be regulated by protein kinase C (PKC) in bovine adrenal chromaffin cells has been addressed. 2. Initial experiments confirmed that the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate (TPA) dramatically inhibited agonist-stimulated [3H]-inositol phosphate accumulations in [3H]-inositol prelabelled cells. In contrast, the PKC inhibitor, Ro 31-8220, did not affect this response. 3. Histamine (100 microM) or bradykinin (100 nM) evoked rapid increases in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) mass accumulations (maximal accumulations within 10 s and 30 s, respectively) which declined towards basal values over a 10 min incubation period. TPA (1 microM) significantly attenuated the peak Ins(1,4,5)P3 response to bradykinin and histamine by 30% and 70% respectively. In contrast, TPA did not significantly affect agonist-stimulated Ins(1,3,4,5)P4 responses. 4. Ro 31-8220 (10 microM) significantly enhanced the maximal Ins(1,4,5)P3 accumulations elicited by both bradykinin and histamine. 5. The results indicate that the initial Ins(1,4,5)P3 response to either bradykinin or histamine in bovine adrenal chromaffin cells can be attenuated by PKC activation by phorbol ester and enhanced by PKC inhibition by Ro 31-8220. In contrast, agonist-stimulated Ins(1,3,4,5)P4 accumulation does not appear to be affected by these manipulations of PKC activity. Possible bases for differential modulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 are discussed.

Adrenal Medulla↗
PubMed1992

Treatment of experimental NADH ubiquinone reductase deficiency with menadione.

Chronic administration of diphenylene iodonium (DPI) to rats has been shown to model the characteristics of mitochondrial myopathy. Using this model the efficacy of menadione therapy has been assessed. Menadione treatment of rats injected with DPI was associated with improved weight gain and increased survival rate. This was accompanied by an improvement in muscle function as judged by analysis of isometric twitch tension of the gastrocnemius muscle (1 Hz for 20 min). The decline in phosphocreatine (PCr) levels in the gastrocnemius muscle during stimulation and delayed recovery in PCr after stimulation were similar in the menadione treated and untreated models. Menadione treatment of the DPI model resulted in a resting intramuscular pH significantly lower than control or untreated DPI rats, but a similar decline in intramuscular pH to the DPI rats during stimulation. The changes in metabolite levels were broadly similar in both the menadione treated and untreated DPI models following stimulation, although the changes, except for increased lactate concentration, were generally less marked in the menadione-treated DPI model.

Adenosine Diphosphate↗
PubMed1991

Beta-adrenoceptor-agonist and insulin actions on glucose metabolism in rat skeletal muscle in different thyroid states.

1. The actions of the beta-adrenoceptor agonist isoprenaline on glucose and glycogen metabolism, in the presence of various concentrations of insulin, were investigated in isolated soleus muscle preparations taken from eu-, hyper- and hypothyroid rats. 2. Hyperthyroidism, induced by 3,3',5-tri-iodo-D-thyronine (T3) administration for 5 days, increased the rate of lactate formation and suppressed the rate of glycogen synthesis in soleus muscle in response to isoprenaline, even in the presence of physiological or supraphysiological insulin concentrations. 3. Hypothyroidism, induced by administration of 6-n-propyl-2-thiouracil for 4 weeks, decreased the rate of isoprenaline-stimulated lactate formation at all insulin concentrations, but significantly decreased the responsiveness of lactate formation only at low insulin concentrations. In the presence of 100 or 10,000 mu-units of insulin/ml, the ability of isoprenaline to suppress the rate of glycogen synthesis was markedly impaired (inhibition at 100 mu-units of insulin/ml and 1 micro-M-isoprenaline: eu- 72.6 +/- 2.9%; hypo-41.0 +/- 2.1%; P less than 0.001). 4. Hyperthyroidism had no effect on the number or affinity of beta-adrenoceptors, defined by 125I-pindolol binding, or beta-adrenoceptor- or forskolin-stimulated adenylate cyclase activity in membrane preparations of gastrocnemius muscle, whereas hypothyroidism increased the beta-adrenoceptor density and decreased the beta-adrenoceptor-stimulated adenylate cyclase activity, without affecting the receptor affinity or forskolin-stimulated adenylate cyclase activity. 5. It is concluded that there is a complex interplay between insulin, catecholamines and thyroid hormones to regulate skeletal-muscle glucose metabolism. The changes observed in muscles in hypothyroidism may be explained, at least in part, by changes in beta-adrenoceptor-G-protein-adenylate cyclase coupling affecting the generation of cyclic AMP and the regulation of some of the key enzymes of glycogen metabolism; in contrast, the changes observed in muscles in hyperthyroidism do not appear to result from alterations at the level of the receptor-mediated second-messenger generation.

Adrenergic beta-Agonists↗
PubMed1991

Determination of mass changes in phosphatidylinositol 4,5-bisphosphate and evidence for agonist-stimulated metabolism of inositol 1,4,5-trisphosphate in airway smooth muscle.

Stimulation of muscarinic receptors in bovine tracheal smooth muscle (BTSM) causes a sustained increase in muscle tone, but a transient increase in the second messenger Ins(1,4,5)P3. To examine whether this brief increase in Ins(1,4,5)P3 mass results from transient formation or is due to agonist-stimulation of Ins(1,4,5)P3 metabolism, we have studied the relationship between mass changes in PtdIns(4,5)P2 and Ins(1,4,5)P3 accumulation, and changes in [3H]InsP3, [3H]PtdIns, [3H]PtdInsP1 and [3H]PtdInsP2 in carbachol-stimulated myo-[3H]inositol-prelabelled BTSM slices. Carbachol (0.1 mM) caused a rapid transient increase in Ins(1,4,5)P3 concentration (basal, 12.9 +/- 0.8 pmol/mg of protein; 5 s carbachol treatment, 27.1 +/- 1.5 pmol/mg of protein), with values returning to basal levels by 30 s, but a sustained accumulation of total [3H]InsP3s, with [3H]Ins(1,3,4)P3 being the predominant isomer present at later time points. In contrast, PtdIns(4,5)P2 mass, determined by radioreceptor assay of Ins(1,4,5)P3 in desalted alkaline hydrolysates of acidified chloroform/methanol tissue extracts, declined rapidly (basal, 941 +/- 22 pmol/mg of protein; 120 s carbachol, 365 +/- 22 pmol/mg of protein; t1/2 14 s) and remained at this new steady-state level for at least 20 min in the continued presence of carbachol. Addition of 10 microM-atropine 2 min after carbachol caused a prompt return of PtdIns(4,5)P2 concentration to prestimulated values (t1/2 210 s). Ongoing resynthesis of PtdIns(4,5)P2 after carbachol stimulation was demonstrated in [3H]inositol-labelled tissue by observing a persistent increase in the specific radioactivity of [3H]PtdInsP2, shown to be exclusively [3H]PtdIns(4,5)P2, over a 10 min period. These findings strongly suggest the occurrence of persistent receptor-mediated increases in PtdIns(4,5)P2 hydrolysis and Ins(1,4,5)P3 formation which, in conjunction with the transient accumulation of Ins(1,4,5)P3 observed, provide evidence that regulation of the metabolism of Ins(1,4,5)P3 is a major determinant of Ins(1,4,5)P3 concentration in this tissue under agonist-stimulated conditions.

Animals↗
PubMed1991

D-[35S(U)]inositol 1,4,5-trisphosphorothioate, a novel radioligand for the inositol 1,4,5-trisphosphate receptor. Complex binding to rat cerebellar membranes.

D-[35S(U)]myo-inositol 1,4,5-trisphosphorothioate [( 35S]InsPS3), a synthetic, metabolically stable analogue of inositol 1,4,5-trisphosphate (InsP3), binds with high affinity (Kd 58.6 +/- 9.1 nM) to rat cerebellar membranes revealing a high density of specific binding sites (Bmax 21.5 +/- 2.1 pmol/mg of protein). Comparison with [3H]InsP3 binding reveals a higher density of sites labelled by [35S]InsPS3 and complex competition curves for displacement of specific [35S]InsPS3 by InsP3. The results suggest that [35S]InsPS3 labels two sites in rat cerebellar membranes with equal affinity: the InsP3 receptor and a site that displays low affinity for InsP3.

Animals↗
PubMed1991

M3 muscarinic cholinoceptors are linked to phosphoinositide metabolism in rat cerebellar granule cells.

Primary cultures of rat cerebellar granule cells are shown to possess a high density (283 +/- 48 fmol/mg of protein) of muscarinic receptor sites, defined using N-[3H]methylscopolamine [( 3H]NMS), with a KD of 0.18 +/- 0.01 nM measured after culture in vitro for 7 days. Displacement of specific [3H]NMS binding demonstrated a muscarinic receptor with low affinity for pirenzepine (Ki: 240 nM); further investigation using antagonists, AF-DX 116 and 4-DAMP to discriminate between M2 and M3 receptors respectively, revealed low M2 affinity (Ki: 600 nM) and high M3 affinity (Ki: 2.4 nM), indicative of the M3 receptor subtype. The robust muscarinic receptor stimulation of [3H]inositol phosphate formation, previously observed in these cells, was confirmed. Inhibition of this response followed a similar profile to the binding data, exhibiting weak inhibitory effects for pirenzepine (Ki: 710 nM) and AF-DX 116 (Ki: 5000 nM), but a potent action for 4-DAMP (Ki: 2.4 nM). The opposite profile seen for AF-DX 116 and 4-DAMP is indicative of a M3 receptor subtype expressed on these cells and linked to phosphoinositide hydrolysis. Further studies demonstrated that M3 receptor stimulation caused a rapid, transient increase in the second messenger inositol 1,4,5-trisphosphate, suggesting that potential Ca(2+)-homeostatic and neuromodulatory effects may be mediated by this response.

Animals↗
PubMed1991

Characterization of inositol 1,4,5-trisphosphate- and inositol 1,3,4,5-tetrakisphosphate-binding sites in rat cerebellum.

1. The properties of specific Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites have been compared in a crude 'P2' cerebellar membrane fraction. 2. A homogeneous population of [3H]Ins(1,4,5)P3-binding sites was present (KD 23.1 +/- 3.6 nM) at high density (Bmax. 11.9 +/- 1.8 pmol/mg of protein); whereas data obtained for [32P]Ins(1,3,4,5)P4 specific binding were best fitted to a two-site model, the high-affinity binding component (KD 2.6 +/- 0.7 nM) constituted 64.2 +/- 4.3% of the total population and was present at relatively low density (Bmax. 187 +/- 27 fmol/mg of protein). 3. The two high-affinity inositol polyphosphate-binding sites exhibited markedly different pH optima for radioligand binding, allowing the two sites to be independently investigated. At pH 8.0, [3H]Ins(1,4,5)P3 binding was maximal, whereas [32P]Ins(1,3,4,5)P4 specific binding was very low; conversely, at pH 5.0, [32P]Ins(1,3,4,5)P4 binding was maximal, whereas [3H]Ins(1,4,5)P3 binding was undetectably low. 4. Both inositol polyphosphate-binding sites exhibited marked positional and stereo-specificity. Of the analogues studied, only phosphorothioate substitution to form inositol 1,4,5-trisphosphorothioate was tolerated at the Ins(1,4,5)P3-binding site, with only a 2-3-fold loss of binding activity. Addition of a glyceroyl moiety at the 1-phosphate position or addition of further phosphate substituents at the 3- or 6-positions caused dramatic losses in displacing activity. Similarly, complete phosphorothioate substitution of Ins(1,3,4,5)P4 caused an approx. 6-fold loss of binding activity at the [32P]Ins(1,3,4,5)P4-binding site, whereas Ins(1,4,5,6)P4, Ins(1,3,4,6)P4, Ins(1,4,5)P3 and Ins(1,3,4,5,6)P5 were bound at least 100-fold weaker at this site. Therefore, only the phosphorothioate derivatives retained high affinity and selectivity for the two inositol polyphosphate-binding sites. 5. Heparin and pentosan polysulphate were potent but non-selective inhibitors at Ins(1,4,5)P3- and Ins(1,3,4,5)P4-binding sites. N-Desulphation (with or without N-reacetylation) of heparin decreased inhibitory activity at the Ins(1,4,5)P3-, but not at the Ins(1,3,4,5)P4-binding site; however, the selectivity of this effect was only about 4-fold. O- and N-desulphated N-reacetylated heparin was essentially inactive at both sites. 6. The results are discussed with respect to the separate identities of the inositol polyphosphate-binding sites.

Animals↗
PubMed1991

Accumulation and metabolism of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in muscarinic-receptor-stimulated SH-SY5Y neuroblastoma cells.

Stimulation of M3 muscarinic receptors expressed by SH-SY5Y cells induced a dose- and time-related increase in the mass of Ins(1,4,5)P3 (basal 38.3 +/- 5.8 pmol/mg of protein) and Ins(1,3,4,5)P4 (basal 6.1 +/- 1.2 pmol/mg of protein). Comparison of radioreceptor mass assays with [3H]inositol labelling showed higher-fold stimulations with the former protocol. The later accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 mass was dependent upon extracellular Ca2+.

Animals↗
PubMed1991

Platelet-activating factor stimulates a rapid accumulation of inositol (1,4,5)trisphosphate in guinea pig eosinophils: relationship to calcium mobilization and degranulation.

The effect of platelet-activating factor (PAF) on inositol (1,4,5)trisphosphate (Ins[1,4,5]P3) mass, calcium mobilization, and the release of granule enzymes was studied on guinea pig peritoneal eosinophils (EOSs). PAF evoked a concentration-dependent accumulation of Ins(1,4,5)P3 with a drug concentration that elicits 50% of the maximum attainable response (EC50) of 10 nmol/L; the production of this second messenger was maximal at 1 mumol/L of PAF. Kinetic analysis of PAF (1 mumol/L)-induced Ins(1,4,5)P3 accumulation demonstrated it to be transient with a 3.8-fold increase over resting levels observed at 5 seconds. Thereafter, the level of Ins(1,4,5)P3 declined, returning to vehicle-treated levels 60 seconds after PAF challenge. Lyso-PAF, the inactive precursor and metabolite of PAF, was inactive at all concentrations examined. PAF also induced a rapid, concentration-dependent (EC50, 12 nmol/L) rise in the cytosolic-free calcium concentration ([Ca++]i) in fura 2-AM-loaded EOSs that was transient, peaking after the maximum increase in Ins(1,4,5)P3 mass was observed. A highly significant positive correlation was found between the peak increase in Ins(1,4,5)P3 and the peak rise in [Ca++]i. Functionally, PAF evoked a concentration-dependent release of granule constituents from both the small (arylsulfatase B; EC50, 3 nmol/L) and specific (EOS peroxidase; EC50, 2.7 nmol/L) granules that lagged, temporally, behind both Ins(1,4,5)P3 accumulation and the rise in [Ca++]i. Both the biochemical and functional effects of PAF examined in this study were antagonized by WEB 2086 (300 nmol/L), a selective PAF receptor-blocking drug. It is concluded that stimulus (PAF)-response coupling in guinea pig peritoneal EOSs may involve the receptor-mediated formation of Ins(1,4,5)P3 and subsequent release of intracellularly stored Ca++. This sequence of events may link PAF receptor activation to Ca(++)-dependent cellular responses, such as degranulation.

Animals↗
PubMed1991

Differential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes.

Since the discovery of cyclic nucleotide phosphodiesterase 30 years ago, there have been major advances in our knowledge of this group of isoenzymes. Five families, each composed of several isoforms and having differing tissue distributions, have been described. David Nicholson and colleagues compare the tissue distribution of phosphodiesterase isoenzymes and discuss the differential effects of inhibition of particular isoenzymes, with differing subcellular localization, on tissue function. They also review the potential use of isoenzyme selective phosphodiesterase inhibitors in a range of clinical disorders such as heart failure, asthma, depression and dementia.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1991

Lithium and the phosphoinositide cycle: an example of uncompetitive inhibition and its pharmacological consequences.

The ability of lithium to exert profound and selective psychopharmacological effects to ameliorate manic-depressive psychosis has been the focus of considerable research effort. There is increasing evidence that lithium exerts its therapeutic action by interfering with polyphosphoinositide metabolism in brain and prevention of inositol recycling by an uncompetitive inhibition of inositol monophosphatase. Stefan Nahorski, Ian Ragan and John Challiss discuss this unusual stimulus-dependent form of enzyme inhibition, emphasizing that the selectivity exhibited by lithium depends upon the degree of inositol lipid hydrolysis and polyphosphoinositide dephosphorylation.

Animals↗