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R A Challiss

Publications and source records attributed to R A Challiss.

At least 55 records · Page 3Linked to original sources
PubMed1996

Agonist-evoked Ca2+ mobilization from stores expressing inositol 1,4,5-trisphosphate receptors and ryanodine receptors in cerebellar granule neurones.

The mechanisms involved in Ca2+ mobilization evoked by the muscarinic cholinoceptor (mAChR) agonist carbachol (CCh) and N-methyl-D-aspartate (NMDA) in cerebellar granule cells have been investigated. An initial challenge with caffeine greatly reduced the subsequent intracellular Ca2+ concentration ([Ca2+]i) response to CCh (to 45 +/- 19% of the control), and, similarly, a much reduced caffeine response was detectable after prior stimulation with CCh (to 27 +/- 6% of the control). CCh-evoked [Ca2+]i responses were inhibited by preincubation with thapsigargin (10 microM), 2,5-di(tert-butyl)-1,4-benzohydroquinone (BHQ; 25 microM), ryanodine (10 microM), or dantrolene (25 microM). BHQ pretreatment was found to have no effect on the sustained phase of the NMDA-evoked [Ca2+]i response. Both CCh (1 mM) and 1-aminocyclopentane-1S,3R-dicarboxylic acid (ACPD; 200 microM) evoked a much diminished increase in [Ca2+]i in granule cells pretreated with CCh for 24 h compared with vehicle-treated control cells (CCh, 23 +/- 14%; ACPD, 27 +/- 1% of respective control values). In contrast, a 24-h CCh pretreatment decreased the subsequent inositol 1,4,5-trisphosphate (InsP3) response to CCh to a much greater extent compared with responses evoked by metabotropic glutamate receptor (mGluR) agonists; this suggests that the former effect on Ca2+ mobilization represents a heterologous desensitization of the mGluR-mediated response distal to the pathway second messenger. Furthermore, [Ca2+]i responses to caffeine and NMDA were unaffected by a 24-h pretreatment with CCh. This study indicates that ryanodine receptors, as well as InsP3 receptors, appear to be crucial to the mAChR-mediated [Ca2+]i response in granule cells. As BHQ apparently differentiates between the CCh- and NMDA-evoked responses, it is possible that the directly InsP3-sensitive pool is physically different from the ryanodine receptor pool. Also, activation of InsP3 receptors may not contribute significantly to NMDA-evoked elevation of [Ca2+]i in cerebellar granule cells. A model for the topographic organization of cerebellar granule cell Ca2+ stores is proposed.

Animals↗
PubMed1996

Differences in agonist and antagonist activities for two indices of metabotropic glutamate receptor-stimulated phosphoinositide turnover.

1. The abilities of the four diastereoisomers of 1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) to stimulate, and the metabotropic glutamate receptor (mGluR) antagonist (+/-)-alpha-methylcarboxyphenylglycine (MCPG) to inhibit, phosphoinositide turnover in neonatal rat cerebral cortex have been studied. Two indices of phosphoinositide cycle activity were assessed; inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation, and total inositol phosphate [3H]-InsPx accumulation (in the presence of Li+) in myo-[3H]-inositol prelabelled slices. 2. The diastereoisomers of ACPD stimulated each response with a rank order of potency of 1S, 3R > 1R, 3R > 1S, 3S >> 1R, 3S. The response to 1R, 3R-ACPD was largely prevented by pre-addition of the NMDA-receptor antagonist, MK-801, or omission of extracellular Ca2+, suggesting that this isomer acts indirectly on phosphoinositide responses through activation of NMDA-type ionotropic glutamate receptors. In contrast, the responses to 1S, 3R- and 1S, 3S-ACPD were unaffected by prior addition of MK-801, but were blocked by MCPG. 3. The concentration of 1S, 3R-ACPD required to half-maximally stimulate the Ins(1,4,5)P3 response (-log EC50 (M), -4.09 +/- 0.10) was significantly higher than that required to exert a similar effect on [3H]-InsPx accumulation (-log EC50 (M), -4.87 +/- 0.07; P < 0.01; n = 4). A similar marked 8-9 fold discrepancy between these two values was observed for the 1S, 3S isomer, which elicited similar maximal responses to those caused by 1S, 3R-ACPD. 4. Significant differences were also observed with respect to the ability of (+/-)-MCPG (1 mM) to cause a rightward shift in the concentration-response relationships for 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 (5.59 +/- 0.24 fold shift) and [3H]-InsPx (3.04 +/- 0.34 fold shift; P < 0.01; n = 4) responses, giving rise to Kd values of 218 and 490 microM for (+/-)-MCPG antagonism of the respective responses. 5. The potency difference between the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 and [3H]-InsPx responses was reduced when experiments were performed in nominally calcium-free medium ([Ca2+]e = 2 - 5 microM) and EC50 values were almost identical when extracellular calcium was reduced further by EGTA addition ([Ca2+]e < or = 100 nM). Similarly, the Kd value for (+/-)-MCPG antagonism of the 1S, 3R-ACPD-stimulated [3H]-InsPx response decreased under [Ca2+]e-free conditions, approaching those obtained for the 1S, 3R-ACPD-stimulated Ins(1,4,5)P3 response in the presence of normal [Ca2+]e. 6. These data suggest that estimates of the activities of mGluR agonists and antagonists, derived by measuring phosphoinositide turnover, can differ significantly depending on whether Ins(1,4,5)P3 mass or [3H]-InsPx responses are measured. In particular, the possibility that the mGluR-mediated [3H]-InsPx response may not simply reflect direct receptor/G protein/phosphoinositidase C (PIC) activation, but may also be the consequence of stimulation of a facilitatory Ca2+-influx pathway is discussed.

Animals↗
PubMed1996

Stimulatory effects of the putative metabotropic glutamate receptor antagonist L-AP3 on phosphoinositide turnover in neonatal rat cerebral cortex.

1. The effects of the metabotropic glutamate receptor (mGluR) antagonist, L-2-amino-3-phosphonopropionate (L-AP3) on phosphoinositide turnover in neonatal rat cerebral cortex slices has been investigated. 2. At concentrations of < or = 300 microM, L-AP3 inhibited total [3H]-inositol phosphate ([3H]-InsPx) and Ins(1,4,5)P3 mass responses stimulated by the selective mGluR agonist, 1-amino-cyclopentane-1S, 3R-dicarboxylic acid (1S, 3R-ACPD). Comparison with the competitive mGluR antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG) clearly demonstrated that L-AP3 caused inhibition by a mechanism that was not competitive, as L-AP3 decreased the maximal response to 1S, 3R-ACPD (by approximately 40% at 300 microM L-AP3) without significantly affecting the concentration of 1S, 3R-ACPD required to cause half-maximal stimulation of the [3H]-InsPx response. 3. In contrast, at a higher concentration L-AP3 (1 mM) caused a large increase in [3H]-InsPx accumulation which was similar in magnitude in both the absence and presence of 1S, 3R-ACPD (300 microM). D-AP3 (1 mM) had no stimulatory effect alone and did not affect the response evoked by 1S, 3R-ACPD. L-AP3 (1 mM) also caused a large increase in Ins(1,4,5)P3 accumulation. The magnitude of the response (4-5 fold increase over basal) approached that evoked by a maximally effective concentration of 1S, 3R-ACPD, but differed substantially in the time-course of the response. The stimulatory effects of 1S, 3R-ACPD and L-AP3 on Ins(1,4,5)P3 accumulation were also similarly affected by decreases in extracellular calcium concentration. 4. Detailed analysis of the inositol phospholipid labelling pattern and the inositol (poly)phosphate isomeric species generated following addition of L-AP3 was also performed. In the continued presence of myo-[3H]-inositol, L-AP3 (1 mM) stimulated a significant increase in phosphatidylinositol labelling, but not that of the polyphosphoinositides, and the inositol (poly)phosphate profile suggested that substantial Ins(1,4,5)P3 metabolism occurs via both 5-phosphatase and 3-kinase routes. 5. A significant stimulatory effect of L-AP3 (1 mM) on [3H]-InsPx accumulation was also observed in neonatal rat hippocampus, and cerebral cortex and hippocampus slices prepared from adult rat brain. 6. These data demonstrate that whilst L-AP3 antagonizes mGluR-mediated phosphoinositide responses at concentrations of < or = 300 microM, higher concentrations substantially stimulate this response. The ability of (+/-)-MCPG (1 mM) to attenuate significantly L-AP3-stimulated [3H]-InsPx accumulation, suggests that both the inhibitory and stimulatory effects of L-AP3 may be mediated by mGluRs.

Alanine↗
PubMed1995

Divalent cation entry in cultured rat cerebellar granule cells measured using Mn2+ quench of fura 2 fluorescence.

In this study the rate of Mn2+ quench of fura-2 fluorescence evoked by glutamatergic and cholinergic agonists, depolarization and Ca2+ store modulators was measured in cultured cerebellar granule cells, in order to study their effects on Ca2+ entry in isolation from effects on Ca2+ store release. The rate of fluorescence quench by 0.1 mM Mn2+ was markedly increased by 25 mM K(+)-evoked depolarization or by 200 microM N-methyl-D-aspartate (NMDA), with a significantly greater increase occurring during the rapid-onset peak phase compared to the plateau phase of the K(+)- or NMDA-evoked [Ca2+]i response. The stimulatory effect of NMDA on Mn2+ quench was abolished by dizocilpine (10 microM), but nitrendipine (2 microM), while decreasing the rate of basal quench, did not affect NMDA-stimulated Mn2+ entry. This suggests that nitrendipine may not act on NMDA channels in granule cells, at least under these conditions, and that voltage-operated Ca2+ channels are involved in control quench whereas the NMDA-evoked quench is dependent on entry through the receptor channel. The t1/2 of quench was unaffected by alpha-amino-hydroxyisoxazole propionic acid (200 microM) and carbamyl choline (1 mM). Neither thapsigargin (10 microM) nor dantrolene (30 microM) significantly affected the rate of quench under control or NMDA- or K(+)-stimulated conditions, which confirms that the previously reported inhibitory effects on [Ca2+]i elevations evoked by these agents are due to actions on Ca2+ stores. However, thapsigargin elevated [Ca2+]i in the presence of normal [Ca2+]o but not in nominally Ca(2+)-free medium, indicating that it evokes Ca2+ entry in cerebellar granule cells, probably subsequent to store depletion, which appears to be either too small to be detected by Mn2+ quench or to occur via Mn(2+)-impermeant channels.

Animals↗
PubMed1995

Neuronal Ca2+ stores: activation and function.

The intracellular concentration of free Ca2+ ([Ca2+]i) displays complex fluctuations in response to a variety of stimuli, and acts as a pluripotent signal for many neuronal functions. It is well established that various 'metabotropic' neurotransmitter receptors can mediate the mobilization of Ca2+ stores via actions of inositol-polyphosphate second messengers, and more recent evidence suggests that 'ionotropic' receptor-mediated Ca2+ signals in neurones might also involve release of Ca2+ from intracellular stores. These two mechanisms of release of Ca2+ enable considerable temporal and spatial complexity of increases in the [Ca2+]i via multiple interactions at the level of intracellular-receptor activation. The complexity of Ca2+ signalling that is elicited via these interconnecting pathways might underlie mechanisms that are central to information transfer and integration within neuronal compartments.

Animals↗
PubMed1995

Muscarinic cholinoceptor-stimulated synthesis and degradation of inositol 1,4,5-trisphosphate in the rat cerebellar granule cell.

A detailed analysis of the generation and subsequent metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] following muscarinic cholinoceptor stimulation in primary cultures of rat cerebellar granule cells has been undertaken. Following incubation of cerebellar granule cell cultures with [3H]inositol for 48 h, labelling of the inositol phospholipid pool approached equilibrium. Significant basal labelling of inositol pentakisphosphate (InsP5) and inositol hexakisphosphate (InsP6), as well as inositol mono- to tetrakisphosphate, fractions was observed. Addition of carbachol (1 mM) caused an immediate increase in level of Ins(1,4,5)P3 (peak increase two-fold over basal by 60 s), which was well-maintained over the initial 300 s following agonist addition. In contrast, only a modest, more slowly developing, increase in inositol tetrakisphosphate accumulation was observed, whereas labelling of InsP5 and InsP6 was entirely unaffected by carbachol stimulation. Analysis of the products of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate metabolism in broken cell preparations strongly suggested that Ins(1,4,5)P3 metabolism occurs predominantly via the inositol polyphosphate 5-phosphatase route, with metabolism via the Ins(1,4,5)P3 3-kinase being a relatively minor pathway. In view of the pattern of inositol (poly)phosphate metabolites observed on stimulation of the muscarinic receptor, it seems likely that, over the time course studied, the inositol polyphosphates are derived principally from phosphoinositide-specific phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, although some hydrolysis of phosphatidyl-inositol 4-phosphate cannot be excluded.

Animals↗
PubMed1995

Modulation of NMDA effects on agonist-stimulated phosphoinositide turnover by memantine in neonatal rat cerebral cortex.

1. The ability of memantine (1-amino-3,5-dimethyladamantane) to antagonize the modulatory effects of N-methyl-D-aspartate (NMDA) on phosphoinositide turnover stimulated by muscarinic cholinoceptor- and metabotropic glutamate receptor-agonists has been examined in neonatal rat cerebral cortex slices. 2. Memantine antagonized the inhibitory effect of NMDA (100 microM) on both total [3H]-inositol phosphate ([3H]-InsPx) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulations stimulated by carbachol (1 mM) with EC50 values of 21 and 16 microM respectively. 3. Memantine concentration-dependently antagonized (IC50 24 microM) the ability of NMDA (10 microM) to potentiate [3H]-InsPx accumulation in response to a sub-maximal concentration of the metabotropic glutamate receptor agonist, 1S,3R-ACPD (10 microM). 4. The small (approx. 3 fold), concentration-dependent increase in [3H]-InsPx accumulation stimulated by NMDA was completely antagonized by the prototypic NDMA receptor-channel blocker, MK-801 (1 microM) at all concentrations of NDMA studied (1-1000 microM). In contrast, antagonism by memantine (100 microM) was observed only at low concentrations of NMDA (1-10 microM), whilst [3H]-InsPx accumulation stimulated by high concentrations of NMDA (300-1000 microM) was markedly enhanced by memantine. 5. Assessment of the incorporation of [3H]-inositol into inositol phospholipids revealed that memantine (100 microM) caused an approximate 2 fold increase in the labelling of phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. 6H.p.l.c. separation of [3H]-inositol (poly)phosphates demonstrated that whilst memantine (100 microM)alone had no significant effect on the accumulation of any isomer, it substantially altered the profile of accumulation stimulated by NMDA (1 mM), greatly facilitating accumulation of Ins(1,4,5)P3 and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4).7.These data provide evidence that memantine can antagonize the actions of NMDA in neonatal rat cerebral cortex slices in a manner consistent with this agent acting as a NMDA receptor-channel blocker. In addition, at least two further actions of memantine can be proposed. Memantine increases the rate of [3H]-inositol incorporation into the cellular inositol phospholipid fraction, without significantly stimulating phosphoinositide turnover. Furthermore, memantine can substantially alter patterns of inositol (poly)phosphates stimulated by NMDA, promoting the accumulation of the established and putative second messengers Ins(1,4,5)P3 and Ins(1,3,4,5)P4 which are not increased by NMDA in the absence of memantine. It is unknown whether these latter loci of memantine action contribute to known therapeutic actions of this agent.

Animals↗
PubMed1995

Comparative effects of activation of soluble and particulate guanylyl cyclase on cyclic GMP elevation and relaxation of bovine tracheal smooth muscle.

1. The effects of nitric oxide-donating compounds and atrial natriuretic peptide on cyclic GMP accumulation and mechanical tone were compared with the effects of isoprenaline in bovine tracheal smooth muscle. 2. Sodium nitroprusside, glyceryl trinitrate, S-nitroso-N-acetylpenicillamine (SNAP), atrial natriuretic peptide and isoprenaline each caused concentration-dependent inhibitions of histamine-maintained tone (EC50 values 320 +/- 80, 150 +/- 45, 14,000 +/- 4,000, 2.8 +/- 0.8 and 6.6 +/- 4.3 nM respectively). 3. When compared with their effects on histamine-induced tone, sodium nitroprusside was equally potent and effective in causing relaxation of methacholine-supported tone (EC50 290 +/- 90 nM) while isoprenaline was as effective, but less potent (EC50 30 +/- 7 nM). SNAP was more potent and equi-effective as a relaxant of methacholine-supported tone (EC50 340 +/- 140 nM). At the maximum concentrations of glyceryl trinitrate and atrial natriuretic peptide tested against methacholine-supported tone, relaxations of 52% and 14% of the isoprenaline maximum were seen. 4. Sodium nitroprusside, glyceryl trinitrate and atrial natriuretic peptide each induced concentration-dependent increases in cyclic GMP accumulation. The time-courses of accumulation correlated closely with the relaxant actions of these compounds. 5. Pretreatment of tracheal smooth muscle with sodium nitroprusside or SNAP caused a rightward shift of the concentration-effect curve for histamine while reducing the maximum response. 6. LY 83583, a putative guanylyl cyclase inhibitor, caused a concentration-dependent reduction in basal cyclic GMP accumulation in tracheal smooth muscle and inhibited the effects of sodium nitroprusside on cyclic GMP accumulation. 7. LY 83583 also inhibited the relaxation of histamine-supported tone by glyceryl trinitrate, sodium nitroprusside, SNAP and atrial natriuretic peptide, and also sodium nitroprusside- and SNAP-induced relaxation of methacholine-supported tone. However, it had no significant effect on glyceryl trinitrate-induced relaxation of methacholine-supported tone. 8. It is concluded that the relaxant actions of sodium nitroprusside, glyceryl trinitrate, SNAP and atrial natriuretic peptide follow as a result of their ability to activate either soluble or particulate guanylyl cyclase leading to cyclic GMP accumulation. Although there does not seem to be any functional difference in the relaxant response to cyclic GMP generated by the particulate as opposed to soluble form(s) of guanylyl cyclase, atrial natriuretic peptide receptor/guanylyl cyclase activation was much less effective in causing relaxation of methacholine-supported tone when compared to activators of soluble guanylyl cyclase.

Animals↗
PubMed1995

Correlation of cyclic AMP accumulation and relaxant actions of salmeterol and salbutamol in bovine tracheal smooth muscle.

1. The ability of salmeterol to stimulate cyclic AMP accumulation and relaxation has been compared with that of salbutamol in bovine tracheal smooth muscle. In addition, the anti-spasmogenic effects of these agents and their abilities to modulate histamine-stimulated [3H]-inositol phosphate accumulation have also been investigated. 2. In tissue strips, a close temporal correlation was found to exist between salmeterol (0.1 microM)-induced relaxation of methacholine (500 nM)-induced tone and cyclic AMP accumulation, both maximal reversal of induced tone (26.2 +/- 6.0%) and maximal levels of cyclic AMP accumulation being achieved after 30-40 min. In contrast to salmeterol, salbutamol exerted greater and more rapid effects on both parameters. Maximal reversal of methacholine-induced tone (79.3 +/- 14.0%) and maximal levels of cyclic AMP accumulation were produced within 5 min. 3. Salmeterol-induced cyclic AMP accumulation (EC50 = 5.3 [1.8 - 15.2] nM) and inhibition of histamine (0.1 mM)-stimulated [3H]-inositol phosphate accumulation (IC50 = 1.4 [0.3-6.3] nM) were both more potent than those induced by salbutamol (EC50 = 169 [99 - 290] nM; IC50 = 13.8 [7.0 - 27.4] nM). However, maximal effects exerted by each of these agents were similar in magnitude. 4. Anti-spasmogenic effects were examined by beta-adrenoceptor agonist application to tissue strips prior to construction of spasmogen concentration-effect curves. Both salmeterol and salbutamol exerted more marked inhibition of the contractile response induced by histamine than that induced by methacholine, salmeterol being the more potent agent, while salbutamol produced a greater maximal inhibitory effect. 5. The results demonstrate that salmeterol is a more potent agent than salbutamol and have highlighted a close temporal correlation between promotion of cyclic AMP accumulation and tissue relaxation stimulated by each agent when both parameters are measured under identical conditions.

Adrenergic beta-2 Receptor Agonists↗
PubMed1994

Molecular recognition at the myo-inositol 1,4,5-trisphosphate receptor. 3-position substituted myo-inositol 1,4,5-trisphosphate analogues reveal the binding and Ca2+ release requirements for high affinity interaction with the myo-inositol 1,4,5-trisphosphate receptor.

Several novel D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3] analogues equatorially substituted at the 3-position have been synthesized to probe the structure-activity relationship of the Ins(1,4,5)P3-receptor subsite adjacent to the native 3-hydroxy (3-OH) of Ins(1,4,5)P3. This study was prompted, in part, by our observation that myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), the 3-position phosphorylated product of Ins(1,4,5)P3 was a full agonist at the Ca(2+)-mobilizing Ins(1,4,5)P3 receptor of SH-SY5Y cells (Wilcox, R.A., Challiss, R. A. J., Liu, C., Potter, B. V. I., and Nahorski, S. R. (1993) Mol. Pharmacol. 44, 810-817). The 3-position Ins(1,4,5)P3 analogues were equatorially substituted with groups spanning the steric range between the 3-OH of Ins(1,4,5)P3 and the 3-phosphate of Ins(1,3,4,5)P4; in order of increasing 3-position steric bulk these were: 3-fluoro-, 3-chloro-, 3-amino-, 3-bromo-, 3-methoxy-, and 3-phosphorothioate-Ins(1,4,5)P3. The analogues were assessed at the specific Ins(1,4,5)P3 binding-site of bovine adrenal cortex and for Ca2+ mobilizing activity in saponin-permeabilized SH-SY5Y human neuroblastoma cells. A correlation was observed between increasing molecular volume of the 3-position substituent and respective decreases in both affinity and Ca2+ mobilizing efficacy. Further analysis of the data also revealed that Ins(1,4,5)P3 analogues with equatorial 3-OH, 3-phosphate, and 3-phosphorothioate substituents interacted more favorably with Ins(1,4,5)P3 recognition sites than would be predicted by purely steric considerations. In contrast, 3-C-trifluoromethyl-Ins(1,4,5)P3 (which is axially substituted, but retains the native 3-OH of Ins(1,4,5)P3) interacted with Ins(1,4,5)P3 recognition sites with virtually the same potency as Ins(1,4,5)P3, indicating that the binding pocket of the Ins(1,4,5)P3-receptor was not sterically restrictive with respect to axially oriented 3-position substituents. We conclude that the Ins(1,4,5)P3 receptor has favorable non-covalent binding interactions with the equatorial 3-position substituents of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and that these interactions significantly ameliorate the steric constraints of the Ins(1,4,5)P3 receptor binding pocket.

Adrenal Cortex↗
PubMed1994

Effects of membrane depolarization and changes in intra- and extracellular calcium concentration on phosphoinositide hydrolysis in bovine tracheal smooth muscle.

Agonist-stimulated phosphoinositide metabolism plays a central role in pharmacomechanical coupling in airways smooth muscle (ASM). In many other tissues and cells, most noteably excitable cells, membrane depolarization or an increase in intracellular Ca2+ ([Ca2+]i) generated by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release or agonist-mediated Ca2+ influx is able to trigger or augment phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis and/or initiate PtdIns4P/PtdIns hydrolysis by direct stimulation of PIC. To assess the importance of these mechanisms in ASM the effects of KCl-induced membrane depolarization, extracellular Ca2+ ([Ca2+]e) chelation, and addition of ionomycin to elevate [Ca2+]i on basal and agonist-stimulated Ins(1,4,5)P3 concentration and [3H]-InsPx accumulation have been examined. Reducing [Ca2+]e from 1.8 mM to 6 or 0.8 microM caused a progressive inhibition of agonist-stimulated [3H]inositol polyphosphate accumulation over 30 min with the histamine-stimulated response being significantly more sensitive to [Ca2+]e chelation than the response to carbachol. In contrast, the initial accumulation of Ins(1,4,5)P3 was completely unaffected by such reductions in [Ca2+]e. Incubation of [3H]inositol-prelabelled BTSM slices with buffer containing 80 mM KCl failed to stimulate [3H]InsPx accumulation, causing instead a small inhibition of carbachol-stimulated [3H]InsPx accumulation with a similar effect seen with respect to Ins(1,4,5)P3 accumulation. Addition of 5 microM ionomycin to BTSM slices similarly did not stimulate Ins(1,4,5)P3 generation and only increased [3H]InsPx accumulation after prolonged stimulation in the presence of high (mM) [Ca2+]e. These data indicated that in ASM, membrane depolarization or physiological increases in [Ca2+]i did not result in either independent activation of PIC or augmentation of initial agonist-stimulated PtdIns(4,5)P2 hydrolysis. However, while the initial agonist-stimulated generation of Ins(1,4,5)P3 was not dependent on [Ca2+]e, a normal plasmalemmal Ca2+ gradient was required to sustain maximal rates of agonist-stimulated PtdIns(4,5)P2 hydrolysis.

Animals↗
PubMed1994

The novel Ins(1,4,5)P3 analogue 3-amino-3-deoxy-Ins(1,4,5)P3: a pH-dependent Ins(1,4,5)P3 receptor partial agonist in SH-SY5Y neuroblastoma cells.

We have synthesized the first amino-substituted inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogue, D-3-amino-3-deoxy-myo-Ins(1,4,5)P3 (9). Although 9 is a full agonist at the Ca2+ mobilizing Ins(1,4,5)P3 receptor at pH 7.2 and 7.6, it is apparently a high intrinsic activity partial agonist at pH 6.8, releasing only 80% of the Ins(1,4,5)P3-sensitive Ca2+ stores of SH-SY5Y cells. Additionally, 9 was able to fully displace [3H]Ins(1,4,5)P3 from binding sites in rat cerebellum membranes at both pH 6.8 and 7.6, indicating a full interaction with the Ins(1,4,5)P3 receptor. The activity displayed by this amino analogue is unexpected and may be indicative of a pH-dependent conformational change in the amino acid residues comprising the Ins(1,4,5)P3 binding site.

Animals↗
PubMed1994

Potentiation of N-methyl-D-aspartate-evoked elevation of intracellular Ca2+ concentrations by exogenous glycine in cerebellar granule cells.

The effect of glycine on the intracellular free Ca2+ concentration ([Ca2+])i response to N-methyl-D-aspartate (NMDA) was examined in small groups of cerebellar granule cells loaded with fura 2. NMDA alone evoked a long-lasting monophasic [Ca2+]i plateau, which was abolished by removal of extracellular Ca2+, or addition of the NMDA channel antagonist dizocilpine or the glycine site antagonist 5,7-dichlorokynurenic acid, virtually unaffected by the L-type Ca2+ channel antagonist (-)-PN 202 791, and greatly, though variably, potentiated by addition of glycine. In the presence of glycine the response to NMDA was clearly biphasic. However, there was no consistent relationship between the magnitudes of the peak and plateau phases of the response, and their temporal relationship was also highly variable. The potentiation seen with exogenous glycine was highly dependent on plating density, which may be the result of higher levels of endogenous glycine in more dense cultures. Our results provide an explanation of the inconsistent findings previously reported by different groups on the potentiation of the [Ca2+]i response to NMDA by exogenous glycine.

Animals↗
PubMed1994

Effect of temperature on muscarinic cholinoceptor-mediated phosphoinositide metabolism and tension generation in bovine tracheal smooth muscle.

The effect of decreased temperature on phosphoinositide metabolism was studied in flurbiprofen pretreated bovine tracheal smooth muscle (BTSM) by investigating the consequences of cooling on muscarinic-cholinoceptor-mediated [3H]inositol phosphate ([3H]InsP) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) accumulation, basal phosphoinositidase C (PIC) activity and airways smooth muscle (ASM) tone. Cooling of [3H]Ins labelled BTSM slices from 37 degrees C to 27 degrees C for 20 min prior to the addition of agonist caused a substantial (73.0 +/- 2.5%) inhibition of carbachol (100 microM, 30 min)-stimulated [3H]InsP accumulation compared to values measured at 37 degrees C. The degree of inhibition of [3H]InsP accumulation was similar at all agonist time points (2-30 min) studied. In parallel experiments, cooling of unlabelled BTSM slices from 37 degrees C to 27 degrees C resulted in a 34% reduction in basal Ins(1,4,5)P3 mass (37 degrees C, 13.1 +/- 0.6 pmol mg-1 protein; 27 degrees C, 8.9 +/- 0.9 pmol mg-1 protein; P < 0.02) and markedly attenuated carbachol (100 microM)-stimulated increases in Ins(1,4,5)P3 accumulation. Basal PIC activity in the soluble fraction of BTSM homogenates, measured using a [3H]phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) /deoxycholate assay system, was also significantly lower at 27 degrees C compared to 37 degrees C (initial velocities of PtdIns(4,5)P2 hydrolysis of 853 +/- 167 (37 degrees C) and 418 +/- 119 (27 degrees C) pmol min-1 ml-1 (1/400 diluted) BTSM cytosol; p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

(4-(m-Chlorophenylcarbamoyloxy)-2-butynyl)trimethy↗
PubMed1994

Muscarinic acetylcholine receptor subtypes in smooth muscle.

Muscarinic acetylcholine M2 and M3 receptor subtypes are coexpressed in many types of smooth muscle including gastrointestinal smooth muscle, urinary bladder and vascular and airway tissue. Activation of M3 receptors, via the G protein Gq, results in increased polyphosphoinositide hydrolysis, release of Ca2+ ions from the sarcoplasmic reticulum and consequently causes contraction. Quantitation of the relative expression of M2 and M3 receptors has shown that the proportion of M2 receptors often predominates over the M3 receptor population by 4:1 or more. Although it is established that M2 receptors preferentially link, via a pertussis-toxin-sensitive G protein Gi, to inhibition of adenylate cyclase activity, relatively little is known concerning the physiological role of the M2 receptor population. In this review, Richard Eglen and colleagues discuss recent data concerning the possible role(s) of muscarinic receptor subtypes in smooth muscle and appraise the pharmacological methods for dissecting the function of muscarinic receptor subtypes in tissues co-expressing multiple receptors.

Animals↗
PubMed1994

Differential effects of lithium on muscarinic cholinoceptor-stimulated CMP-phosphatidate accumulation in cerebellar granule cells, CHO-M3 cells, and SH-SY5Y neuroblastoma cells.

The ability of lithium to potentiate muscarinic cholinoceptor-stimulated CMP-phosphatidate (CMP.PA) accumulation has been examined in various cells in which muscarinic cholinoceptor agonists evoke a phosphoinositide response. Cell types examined include rat cerebellar granule cells, Chinese hamster ovary cells transfected to express the human muscarinic M3 receptor (CHO-M3 cells), and SH-SY5Y neuroblastoma cells. Neither carbachol (1 mM) nor lithium (10 mM) caused significant increases in CMP.PA accumulation in rat cerebellar granule cells; however, when added together for 20 min a linear 17-fold increase over basal levels was observed. The increase was dependent on the concentration of carbachol and lithium present, and the effect could be reversed by addition of exogenous myo-inositol (10 mM). Addition of carbachol alone to CHO-M3 cells caused a five-fold increase in CMP.PA accumulation. In the presence of lithium, a 70-fold increase was observed at 20 min after carbachol plus lithium addition. This latter response was concentration dependent and could be abolished by preincubation in the presence of 10 mM myo-inositol. In contrast, whereas carbachol elicited a three-fold increase in CMP.PA accumulation in SH-SY5Y neuroblastoma cells, which reached a plateau 10 min after agonist addition, the response could neither be augmented by addition of lithium nor inhibited by addition of myo-inositol. These results emphasise that the ability of lithium to affect agonist-stimulated CMP.PA accumulation is not simply a function of stimulus strength, but is also crucially dependent on the intracellular concentration of inositol.

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