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R H Scott

Publications and source records attributed to R H Scott.

At least 19 recordsLinked to original sources
PubMed2001

Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception.

Cannabinoids modulate nociceptive processing in models of acute, inflammatory and neuropathic pain. We have investigated the location and function of cannabinoid receptors on cultured neonatal dorsal root ganglion (DRG) neurones and F-11 cells, a dorsal root ganglionxneuroblastoma hybridoma which displays several of the features of authentic DRG neurones. CB(1) receptor immunolabelling was observed on the cell bodies and as fine puncta on processes of both cultured DRG neurones and F-11 cells. Additionally, fluorescence-activated cell sorting (FACS) analysis provided evidence that both CB(1) and CB(2) receptors are expressed on populations of cells within the cultured DRG and F-11 cells. The cannabinoid receptor agonist (+)-WIN55212 (10 and 100 nM) inhibited the mean voltage-activated Ca(2+) current in DRG neurones by 21% and 30%, respectively. The isomer, (-)-WIN55212 (10 and 100 nM) produced significantly less inhibition of 6% and 10% respectively. The CB(1) selective receptor antagonist SR141716A (100 nM) enhanced the peak high voltage-activated Ca(2+) current by 24% and simultaneous application of SR141716A (100 nM) and (+)-WIN55212 (100 nM) resulted in a significant attenuation of the inhibition obtained with (+)-WIN55212 alone. These data give functional evidence for the hypothesis that the analgesic actions of cannabinoids may be mediated by presynaptic inhibition of transmitter release in sensory neurones.

Animals↗
PubMed2000

Analysis of the structure and electrophysiological actions of halitoxins: 1,3 alkyl-pyridinium salts from Callyspongia ridleyi.

We have chemically characterized a preparation of halitoxins, (1,3 alkyl-pyridinium salts) isolated from the marine sponge Callyspongia ridleyi. At concentrations of 50 and 5 microg/ml the halitoxin preparation caused irreversible membrane potential depolarization, decreased input resistance and inhibited evoked action potentials when applied to cultured dorsal root ganglion neurones. Under whole cell voltage clamp the halitoxins produced an increase in cation conductance that was attenuated by replacing sodium with N-methyl-d-glucamine. Fura-2 fluorescence ratiometric calcium imaging was used to directly measure calcium flux into neurones after exposure to halitoxins. Calcium influx, evoked by the halitoxins, persisted when the neurones were bathed in medium containing the voltage-activated calcium channel antagonists cadmium and nickel. Experiments on undifferentiated F-11 cells showed little or no calcium influx in response to depolarizing concentrations of potassium and indicated that halitoxins evoked massive calcium influx in the absence of voltage-activated calcium channels. The halitoxins also produced transient increases in intracellular calcium when F-11 cells were bathed in calcium-free medium suggesting that the toxins could release calcium from intracellular stores. The pore-forming action of the halitoxins was identified when the toxins were applied to artificial lipid bilayers composed of phosphatidylcholine and cholesterol. Halitoxins evoked channel-like activity in the lipid bilayers, with estimated unitary conductances of between 145pS and 2280pS, possibly indicating that distinct channels could be produced by the different components in the preparation of halitoxins.

Animals↗
PubMed2000

Mobilisation of intracellular Ca2+ by mGluR5 metabotropic glutamate receptor activation in neonatal rat cultured dorsal root ganglia neurones.

The ability of metabotropic glutamate receptor activation to mobilise intracellular calcium was investigated in cultured dorsal root ganglion (DRG) neurones from neonatal rats using the calcium sensitive fluorescent dye Fura-2. L-glutamate (10 microM) caused sustained and oscillatory increases in intracellular calcium concentration ([Ca2+]i) in a subpopulation of cultured DRG neurones. The oscillatory responses were not blocked by combined application of the ionotropic glutamate receptor antagonists MK 801 (2 microM) and CNQX (20 microM). Oscillations in [Ca2+]i were also observed following application of the nonselective metabotropic glutamate receptor (mGluR) agonist, trans-(1S,3R)-1-aminocyclopentane-1S, 3R-dicarboxylic acid (1S,3R)-ACPD, 20 microM) and the mGluR5 agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 500 microM). These responses were blocked by the selective Group I mGluR antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) (100 microM) and Ca2+ release channel inhibitors ryanodine (100 microM) and dantrolene (10 microM). The predominantly Group II agonist (2S,2'R,3'R)-2-(2'3'-dicarboxy-cyclopropyl)glycine (DCG-IV, 100 microM) failed to produce Ca2+ transients alone but suppressed responses to CHPG. Reverse transcriptase PCR techniques, using primers specific to Group I mGluRs, revealed the presence of mGluR5 but not mGluR1 mRNA in these cells. Therefore, glutamate can cause a slowly activating and reversible mobilisation of [Ca2+]i in sensory neurones by activation of ionotropic receptors, and can induce oscillatory calcium transients by selectively activating metabotropic glutamate receptors that are likely to be of the mGluR5 subtype.

Animals↗
PubMed1999

The effects of changing intracellular Ca2+ buffering on the excitability of cultured dorsal root ganglion neurones.

The whole cell patch clamp technique was used to study the effects of changes in [Ca2+]i on the excitability of cultured dorsal root ganglion (DRG) neurones. Increases in [Ca2+]i caused membrane depolarization, altered the characteristics of evoked action potentials and activated potassium, chloride and non-selective cation conductances. Mobilization of Ca2+ from intracellular stores with caffeine (1 mM) or ryanodine (10 microM) or photorelease of Ca2+ from DM-nitrophen gave rise to depolarizations suggesting a dominant inward current under our recording conditions of low [Ca2+]i buffering capacity. The actions of [Ca2+]i could be partially reversed by intracellular flash photolysis of diazo-2 but not by diazo-3. The electrophysiological properties of some DRG neurones were not influenced by changes in [Ca2+]i and we suggest that this is because in this heterogeneous culture some neurones do not express Ca2+-activated ion channels.

Acetates↗
PubMed1999

The actions of ryanodine on Ca2+-activated conductances in rat cultured DRG neurones; evidence for Ca2+-induced Ca2+ release.

The whole-cell recording technique was used to investigate the actions of a calcium release channel ligand, ryanodine, on calcium-activated chloride conductances, and to evaluate ryanodine-sensitive Ca2+-induced Ca2+ release from intracellular stores in cultured neonatal rat DRG neurones. The aim of the project was to use ryanodine as a pharmacological tool to evaluate calcium-induced calcium release in the cell bodies of cultured DRG neurones. Action potential after-depolarizations were attenuated by extracellular application of the chloride channel blocker, niflumic acid (10 microM), and by ryanodine (10 microM); these actions occurred without concurrent changes in evoked action potentials. Ryanodine and caffeine (10 mM) activated calcium-dependent conductances and the responses to ryanodine were attenuated by depletion of caffeine-sensitive Ca2+ stores. The current clamp data were complicated by changes in potassium conductances so studies were carried out under voltage clamp and voltage-activated calcium currents and calcium-activated chloride and non-selective cation currents were isolated pharmacologically. Ryanodine (10 microM) evoked delayed, inward, calcium-activated non-selective cation and chloride currents which reversed close to 0 mV and were attenuated by N-methyl-D-glucamine, niflumic acid and dantrolene. Consistent with actions on action potential after-depolarizations, niflumic acid (10 microM) and ryanodine (10 microM) attenuated calcium-activated chloride currents evoked by calcium entry through voltage-activated calcium channels. Niflumic acid and ryanodine had no effects on voltage-activated calcium currents evoked from a holding potential of -90 mV by voltage step commands to 0 mV. In conclusion calcium-activated chloride conductances appear to be activated in part by calcium released from ryanodine-sensitive stores, and significant calcium-induced calcium release may occur locally in cell bodies of DRG neurones as a result of calcium entry through voltage-activated channels during an action potential.

Action Potentials↗
PubMed1999

Extracellular and intracellular actions of azadirachtin on the electrophysiological properties of cultured rat DRG neurones.

The distinct electrophysiological actions of extracellular and intracellular azadirachtin (type A) were investigated using cultured dorsal root ganglion neurones from neonatal rats and the whole cell variant of the patch clamp technique. The cultured mammalian neurones were relatively insensitive to azadirachtin compared with some invertebrate preparations, such as insect chemosensory systems. Insect preparations have been found to respond to 1-100 nM azadirachtin. However, at concentrations of azadirachtin between 10 and 100 microM significant changes in the electrophysiological properties of cultured DRG neurones were seen. Extracellular application of azadirachtin prolonged the late repolarization phase of evoked action potentials and consistent with this produced modulation of voltage-activated K+ currents. This modulation involved an initial transient increase in K+ current followed by reversible inhibition when azadirachtin was applied at a concentration of 10 microM. Higher concentrations of azadirachtin (50 and 100 microM), had only inhibitory effects on voltage-activated K+ currents. Azadirachtin produced a degree of voltage-dependent inhibition, with a greater level of K+ current inhibition observed when neurones were held at -90 mV compared with -40 mV. Prolonged application of azadirachtin for 24 h reversibly reduced action potential amplitude. In contrast intracellular application of 100 microM azadirachtin via the patch pipette solution had no significant effects on action potentials but produced an increase in conductance. Intracellular azadirachtin activated several conductances including a TEA-sensitive K + current and an inward current that had an estimated reversal potential close to 0 mV. In conclusion, data showed that azadirachtin had different effects when it was applied inside and outside the neurones and that azadirachtin at high concentrations reversibly altered neuronal excitability mainly by modulating potassium conductances. It appears that rat cultured DRG neurones are less affected by azadirachtin than some insect sensory systems.

Action Potentials↗
PubMed1998

Inhibitory actions of synthesised polyamine spider toxins and their analogues on Ca2+-activated Cl- currents recorded from cultured DRG neurones from neonatal rats.

The whole cell variant of the patch clamp technique was used to investigate the actions of polyamine spider toxins and their analogues on high voltage-activated Ca2+ currents and Ca2+-activated Cl- currents (I(Cl(Ca))). The actions of synthesised FTX (putative natural toxin from the American funnel web spider), sFTX-3.3, Orn-FTX-3.3, Lys-FTX-3.3, and argiotoxin-636 on cultured dorsal root ganglion neurones from neonatal rats were investigated. Synthesised FTX (1 microM) inhibited I(Cl(Ca)) but did not inhibit high voltage-activated Ca2+ currents. In contrast, sFTX-3.3 (10 microM) inhibited both high voltage-activated Ca2+ currents and the associated I(Cl(Ca)) in near equal proportions. Argiotoxin-636 (1-10 microM) inhibited I(Cl(Ca)) evoked by Ca2+ entry through voltage-activated channels and by intracellular photorelease of Ca2+ from a caged precursor DM-nitrophen. This data indicates that synthesised FTX and argiotoxin-636 directly inhibit Ca2+-activated Cl- channels. In conclusion, the potency of polyamines as non-selective inhibitors of Ca2+ channels and Ca2+-activated Cl- channels is in part determined by the presence of a terminal arginine and this may involve an interaction between terminal guanidino groups and Ca2+ binding sites.

Animals↗
PubMed1998

Extracellular or intracellular application of argiotoxin-636 has inhibitory actions on membrane excitability and voltage-activated currents in cultured rat sensory neurones.

The whole cell variant of the patch clamp technique was used to investigate the actions of the polyamine amide spider toxin, argiotoxin-636, on the excitability of cultured dorsal root ganglion neurones. Synthesized argiotoxin-636 (0.1-100 microM) reduced neuronal excitability when applied to the extracellular environment by low pressure ejection or to the intracellular environment via the patch pipette solution. The toxin prolonged the duration of evoked action potentials and reduced the peak amplitude of action potentials. Intracellular and extracellular application of argiotoxin-636 also decreased the number of action potentials evoked in response to 800-ms depolarizing current commands. This action of the toxin was mimicked by 100 microM tetraethylammonium. Extracellular application of argiotoxin-636 inhibited voltage-activated K currents in a dose-dependent manner over the complete voltage range. This inhibition occurred without any significant changes in the voltage dependence of activation or inactivation. Intracellular application of argiotoxin-636, during 5-10 min of whole cell recording, also inhibited voltage-activated K+ currents without changing the voltage dependence of activation or steady-state inactivation. Extracellular or intracellular spermidine (250 microM) reversibly attenuated the inhibitory actions of extracellular argiotoxin-636. Argiotoxin-636 also inhibited voltage-activated Na + currents; this effect was dependent on repeated activation of the currents and the period during which the neurones were in culture. We conclude that application of argiotoxin-636 to either the extracellular or intracellular environment reduced excitability of cultured sensory neurones from neonatal rats and that this involved inhibition of both voltage-activated K+ and Na+ currents. The data suggest that the toxin was more effective at attenuating action potentials when neurones were repeatedly excited, and that access to inhibitory sites of action on the voltage-activated ion channels can be achieved from the inside of the neurone.

Action Potentials↗
PubMed1998

Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked responses which were attenuated by photolysis of the caged Ca2+ chelator Diazo-2. Experiments also suggested that extracellular application of dihydrosphingosine can activate membrane conductances. We conclude that dihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine-sensitive intracellular stores in cultured sensory neurones.

Animals↗
PubMed1997

Inhibition of neuronal high voltage-activated calcium channels by insect peptides: a comparison with the actions of omega-conotoxin GVIA.

The whole cell variant of the patch clamp technique was used to investigate the actions of two novel insect peptides on high voltage-activated Ca2+ currents in cultured dorsal root ganglion (DRG) neurones. The insect peptides (PMP-D2 and PMP-C) were isolated originally from insect brains and fat bodies, and have been found to have similar three-dimensional structures to the N-type Ca2+ channel inhibitor omega-conotoxin GVIA (omega-CgTx GVIA). High voltage-activated Ca2+ currents were activated from a holding potential of -90 mV by depolarizing step commands to 0 mV. Extracellular application of synthetic PMP-D2 or PMP-C (1 microM) attenuated high voltage-activated Ca2+ currents. The effects of PMP-C were strongly dependent on the frequency of current activation, but inhibition was apparent and reached a steady state after 20 steps when currents were evoked for 30 msec at 0.1 Hz. The actions of the two insect peptides overlapped both with each other and with omega-CgTx GVIA, suggesting that N-type Ca2+ current was predominantly sensitive to these peptides. Low voltage-activated T-type current and 1,4-dihydropyridine sensitive L-type Ca2+ currents were insensitive to 1 microM PMP-D2 and PMP-C, which indicates a degree of selectivity. The presence of a fucose group on PMP-C abolished the ability of this peptide to attenuate high voltage-activated Ca2+ currents, which may reflect a mechanism by which peptide function could be regulated in insects. The electrophysiological data are supported by studies on 45Ca2+ influx into rat cerebrocortical synaptosomes. Both PMP-D2 (10 microM), PMP-C (10 microM) and omega-CgTx GVIA (1 microM) attenuated a proportion of 45Ca2+ influx into the synaptosomes, but additive effects of these peptides were not observed. We conclude that these naturally occurring peptides obtained from invertebrate preparations have inhibitory effects on N-type Ca2+ channels. Although the peptides have related three-dimensional structures, they have distinct amino acid sequences and appear to have different mechanisms of action to produce inhibition of mammalian neuronal high voltage-activated Ca2+ channels.

Amino Acid Sequence↗
PubMed1997

Activation of Ca2+-dependent currents in dorsal root ganglion neurons by metabotropic glutamate receptors and cyclic ADP-ribose precursors.

Cultured dorsal root ganglion neurons were voltage clamped at -90 mV to study the effects of intracellular application of nicotinamide adenine dinucleotide (betaNAD+), intracellular flash photolysis of caged 3',5'-cyclic guanosine monophosphate (cGMP), and metabotropic glutamate receptor activation. The activation of metabotropic glutamate receptors evoked inward Ca2+-dependent currents in most cells. This was mimicked both by intracellular flash photolysis of the caged axial isomer of cGMP [P-1-(2-nitrophenyl)ethyl cGMP] and intracellular application of betaNAD+. Whole cell Ca2+-activated inward currents were used as a physiological index of raised intracellular Ca2+ levels. Extracellular application of 10 microM glutamate evoked the activation of Ca2+-dependent inward currents, thus reflecting a rise in intracellular Ca2+ levels. Similar inward currents were also activated after isolation of metabotropic glutamate receptor activation by application of 10 microM glutamate in the presence of 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione and 20 microM dizocilpine maleate (MK 801), or by extracellular application of 10 microM trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid. Intracellular photorelease of cGMP, from its caged axial isomer, in the presence of betaNAD+ was also able to evoke similar Ca2+-dependent inward currents. Intracellular application of betaNAD+ alone produced a concentration-dependent effect on inward current activity. Responses to both metabotropic glutamate receptor activation and cGMP were suppressed by intracellular ryanodine, chelation of intracellular Ca2+ by bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid, and depletion of intracellular Ca2+ stores, but were insensitive to the removal of extracellular Ca2+. Therefore both cGMP, possibly via a mechanism that involves betaNAD+ and/or cyclic ADP-ribose, and glutamate can mobilize intracellular Ca2+ from ryanodine-sensitive stores in sensory neurons.

Adenosine Diphosphate Ribose↗
PubMed1996

Electrophysiological and ultrastructural events evoked by methacholine and intracellular photolysis of caged compounds in cultured ovine trachea submucosal gland cells.

Cultured ovine trachea submucosal gland cells release lysozyme in response to extracellular application of secretagogues, including the muscarinic receptor agonist methacholine (20 microM). Investigation of the ultrastructure has shown that these cells contain electron-dense cored granules, which differ from the intact tissue, but appear to be released in response to the application of methacholine and can be arrested during exocytosis by the application of tannic acid. The release process appears to be linked to electrophysiological events activated by methacholine. Extracellular application of methacholine and intracellular photorelease of Ca2+ from DM-nitrophen evoked similar events suggesting that a rise in intracellular Ca2+ may occur following muscarinic receptor activation. Measurements of the reversal potential and the inhibitory action of the chloride channel blocker niflumic acid (10 microM) indicated that Ca(2+)-activated Cl- channel activity underlies these events. Some of the cultured submucosal gland cells also responded similarly to intracellular photorelease of inositol 1,4,5-trisphosphate, suggesting a possible link between muscarinic receptor occupation by agonist, release of calcium from stores, and activation of Ca(2+)-activated Cl- current. Secretion of lysozyme, methacholine-activated currents and currents evoked by intracellular photorelease of Ca2+ were also attenuated by the potent bronchodilator Ro 31-6930 (1 microM). We conclude that Ca(2+)-activated Cl- conductances play an important role in secretory processes in cultured submucosal gland cells. This may have a bearing on both physiological control of secretory events and regulation of the nature of airway surface liquid. Ca(2+)-activated Cl- channels may offer a potential target site for novel therapeutic agents.

Acetates↗
PubMed1995

Characterization of Ca2+ channel currents in cultured rat cerebellar granule neurones.

1. High-threshold voltage-gated calcium channel currents (IBa) were studied in cultured rat cerebellar granule neurones using the whole-cell patch clamp technique with 10 mM Ba2+ as the charge carrier. The putative P-type component of whole-cell current was characterized by utilizing the toxin omega-agatoxin IVA (omega-Aga IVA) in combination with other blockers. 2. omega-Aga IVA (100 nM) inhibited the high voltage-activated (HVA) IBa by 40.9 +/- 3.4% (n = 27), and the dissociation constant Kd was 2.7 nM. Maximal inhibition occurred within a 2-3 min time course, and was irreversible. The isolated omega-Aga IVA-sensitive current was non-inactivating. 3. omega-Aga IVA exhibited overlapping selectivity with both N- and L-channel blockers; omega-conotoxin GVIA (omega-CTX GVIA) (1 microM) and the dihydropyridine (-)-202-709 (1 microM), respectively. Together these toxins reduced the omega-Aga IVA-sensitive component to just 4.5 +/- 1.4% (n = 3). Thus only a small proportion of the current can be unequivocally attributed to P-type current. Inhibition of the HVA IBa by omega-Aga IA also reduced the proportion of omega-Aga IVA-sensitive current to 28.0 +/- 3.2% (n = 3). 4. Application of omega-Aga IVA and a synthetic form of funnel-web toxin, N-(7-amino-4-azaheptyl)-L-argininamide (sFTX-3.3; 10 microM), produced an additive block of the HVA IBa. Consequently these two toxins do not act on the same channel in cerebellar granule neurones. 5. omega-Aga IVA inhibition of low voltage-activated (LVA) IBa was studied in the ND7-23 neuronal cell line. omega-Aga IVA (100 nM) reduced the LVA current by 41.3 +/- 3.2% (n = 17) in a fully reversible manner with no shift in the steady-state inactivation of the channel. 6. A component of current insensitive to N-, L- and P-channel blockers remained unclassified in all our studies. This component, and also that remaining following block by omega-Aga IVA and omega-Aga IA, exhibited relatively rapid, although incomplete, inactivation compared to the other currents isolated in this study. 7. In conclusion, omega-Aga IVA inhibits a component of current in cultured cerebellar granule neurones which overlaps almost completely with that inhibited by L- and N-channel blockers. In addition, a large component of whole-cell current in these neurones still remains unclassified.

Animals↗
PubMed1995

Activation of Ca(2+)-dependent Cl- currents in cultured rat sensory neurones by flash photolysis of DM-nitrophen.

1. Voltage-gated Ca2+ currents (ICa) and Ca(2+)-activated Cl- currents (ICl(Ca)) were recorded from cultured rat dorsal root ganglion (DRG) neurones using the whole-cell configuration of the patch clamp technique. Intracellular photorelease of Ca2+ by flash photolysis of DM-nitrophen elicited transient inward currents only in those cells which possessed Ca(2+)-activated Cl- tail currents following ICa. The reversal potential of the flash responses was hyperpolarized when extracellular Cl- was replaced by SCN-. The flash responses and the Ca(2+)-activated Cl- tail currents were inhibited by the Cl- channel blockers niflumic acid (10-100 microM) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (10 microM). 2. After activation by ICa, the Ca(2+)-activated Cl- current could be reactivated during its decay by photorelease of caged Ca2+. Experiments carried out on neurones held at 0 mV demonstrated that ICl(Ca) could be chronically activated due to residual Ca2+ influx. These data directly demonstrated that the decay of ICl(Ca) is not due to inactivation but rather to deactivation as a result of removal of the Ca2+ load from the cell cytoplasm. 3. Photorelease of caged inositol 1,4,5-trisphosphate (IP3) failed to activate any Ca(2+)-dependent current responses in cultured DRG neurones, although application of caffeine elicited transient inward currents, and responses to photoreleased IP3 could be obtained from freshly dissociated smooth muscle cells. 4. Photorelease of Ca2+ provides a useful method for investigating the properties of ICl(Ca) independently from other physiological parameters. In addition, we have directly demonstrated that ICl(Ca) in DRG neurones does not inactivate, and so may continue to modulate membrane excitability as long as the intracellular Ca2+ concentration ([Ca2+]i) close to the cell membrane is elevated.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗
PubMed1995

Effect of membrane potential on surface Ca2+ receptor activation in rat osteoclasts.

Osteoclasts are known to possess a divalent cation-sensitive receptor, the Ca2+ receptor (CaR). The latter monitors changes in the local Ca2+ concentration generated as a result of hydroxyapatite dissolution. CaR activation elevates cytosolic [Ca2+] and thereby inhibits osteoclastic bone resorption. Recent studies have used Ni2+ as a surrogate CaR agonist to elicit changes in cytosolic [Ca2+]. This article examines the effects of membrane potential changes on the kinetics of the cytosolic [Ca2+] signal resulting from such Ni(2+)-induced CaR activation. Membrane potential was altered through variations in the extracellular [K] in combination with applications of the K+ ionophore, valinomycin. Membrane potential changes were confirmed by independent electrophysiological patch clamp studies of whole osteoclasts. The application of valinomycin produced a distinct, sustained elevation of cytosolic [Ca2+] in single fura 2-loaded cells, a "primary" response. This response was independent of valinomycin concentration (between 5 nM to 5 microM) and persisted in Ca(2+)-free, EGTA-containing solutions. It also persisted both in high (105 mM) and low (5 mM) extracellular [K+]. A gradual "secondary" elevation of cytosolic [Ca2+] then followed with the continued application of valinomycin, but this was eliminated by sequestering the extracellular [Ca2+] or by increasing extracellular [K+] from 5 to 105 mM. In a separate set of experiments, the presence of 5 microM [valinomycin]-([K+] = 5 mM) prolonged the cytosolic [Ca2+] signal elicited by 50 microM-[Ni2+] application. These prolonged kinetics persisted in low extracellular [Ca2+] (zero-added Ca2+), but reverted to a rapid time-course in the presence of 105 mM-[K+] or at higher [Ni2+] (500 microM and 5 mM). The experiments thus indicate that membrane voltage modifies the kinetics of CaR activation by Ni2+ and therefore suggests that the CaR is an integral protein in the osteoclast surface membrane.

Animals↗
PubMed1995

An electrophysiological investigation of the effects of cisplatin and the protective actions of dexamethasone on cultured dorsal root ganglion neurones from neonatal rats.

In this study we have investigated the acute and chronic effects of cisplatin on whole cell currents in cultured dorsal root ganglion neurones. Consistent with effects on action potentials measured under current clamp, acute (5 min) application of cisplatin (5 microM) attenuated voltage-activated potassium, and mixed cation currents by approximately 50% in both cases. Chronic treatment (5-7 days) of cultured neurones with 5 microM cisplatin also resulted in greatly reduced voltage-activated potassium currents (by 50%) and calcium currents (by 60%) compared to events recorded from neurones not treated with cisplatin. In contrast, the amplitude of inward cation current activated by hyperpolarization was doubled by 5-12 days treatment with cisplatin. Studies on action potential after-depolarizations and calcium-activated chloride currents suggest that cisplatin disturbs calcium homeostatic mechanisms. These observations may account for anode break spike excitation and the low efficiency with which cells buffer intracellular calcium following cisplatin treatment. Dexamethasone has been found to enhance the anti-emetic effects of 5-HT3 receptor antagonists in patients treated with cisplatin. For this reason the actions of dexamethasone were studied in combination with cisplatin treatment. Although acute application of dexamethasone (1-10 microM) produced transient depolarizations and bursts of action potentials, after 5 minutes application it had no effect on membrane potential, input resistance, or the properties of action potentials evoked by depolarizing current commands.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗