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R H Scott

Publications and source records attributed to R H Scott.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1995

Aspects of calcium-activated chloride currents: a neuronal perspective.

Ca(2+)-activated Cl- channels are expressed in a variety of cell types, including central and peripheral neurones. These channels are activated by a rise in intracellular Ca2+ close to the cell membrane. This can be evoked by cellular events such as Ca2+ entry through voltage- and ligandgated channels or release of Ca2+ from intracellular stores. Additionally, these Ca(2+)-activated Cl currents (ICl(Ca)) can be activated by raising intracellular Ca2+ through artificial experimental procedures such as intracellular photorelease of Ca2+ from "caged" photolabile compounds (e.g. DM-nitrophen) or by treating cells with Ca2+ ionophores. The potential changes that result from activation of Ca(2+)-activated Cl- channels are dependent on resting membrane potential and the equilibrium potential for Cl-. Ca2+ entry during a single action potential is sufficient to produce substantial after potentials, suggesting that the activity of these Cl- channels can have profound effects on cell excitability. The whole cell ICl(Ca) can be identified by sensitivity to increased Ca2+ buffering capacity of the cell, anion substitution studies and reversal potential measurements, as well as by the actions of Cl- channel blockers. In cultured sensory neurones, there is evidence that the ICl(Ca) deactivates as Ca2+ is buffered or removed from the intracellular environment. To date, there is no evidence in mammalian neurones to suggest these Ca(2+)-sensitive Cl- channels undergo a process of inactivation. Therefore, ICl(Ca) can be used as a physiological index of intracellular Ca2+ close to the cell membrane. The ICl(Ca) has been shown to be activated or prolonged as a result of metabolic stress, as well as by drugs that disturb intracellular Ca2+ homeostatic mechanisms or release Ca2+ from intracellular stores. In addition to sensitivity to classic Cl- channel blockers such as niflumic acid, derivatives of stilbene (4,4'diisothiocyanostilbene-2,2'-disulphonic acid, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid) and benzoic acid (5-nitro 2-(3-phenylpropylamino) benzoic acid), ICl(Ca) are also sensitive to polyamine spider toxins and some of their analogues, particularly those containing the amino acid residue arginine. The physiological role of Ca(2+)-activated Cl- channels in neurones remains to be fully determined. The wide distribution of these channels in the nervous system, and their capacity to underlie a variety of events such as sustained or transient depolarization or hyperpolarizations in response to changes in intracellular Ca2+ and variations in intracellular Cl- concentration, suggest the roles may be subtle, but important.

Animals↗
PubMed1995

Modulation of Ca(2+)-dependent currents in metabolically stressed cultured sensory neurones by intracellular photorelease of ATP.

1. The whole cell recording technique was used to study high voltage-activated Ca2+ currents and Ca(2+)-activated Cl- tail currents from cultured neonatal dorsal root ganglion neurones of the rat which were metabolically stressed. The neurones were metabolically stressed with 2-deoxy-D-glucose (5 mM) for 30 min to 3 h. The aim of the project was to examine the actions of intracellular photorelease of ATP on the properties of Ca(2+)-dependent currents and determine if the effects of metabolic stress could be reversed. 2. The mean duration of Ca(2+)-activated Cl- tail currents was significantly increased by metabolic stress and this effect was reversed by intracellular photorelease of approximately 300 microM ATP. Intracellular photolysis of 'caged' photolabile compounds was achieved with a xenon flash lamp. 3. Intracellular photorelease of ATP and adenosine 3':5'-cyclic monophosphate (cyclic AMP) (about 40 microM) also accelerated the inactivation of high voltage-activated Ca2+ currents evoked by 500 ms depolarizing step commands from -90 mV to 0 mV. This effect was prevented by intracellular application of the calcineurin (protein phosphatase-2B) inhibitor cyclosporin A (14 nM) and cyclophilin A (50 nM) either applied together or individually. In contrast the protein phosphatase 1 and 2A inhibitor, calyculin A, increased voltage-activated Ca2+ currents, but failed to prevent enhanced inactivation induced by intracellular photorelease of ATP. Intracellular photorelease of ATP had no effect on Ca2+ currents recorded from control neurones which were not metabolically stressed and supplied with glucose and ATP in the extracellular and patch pipette solutions respectively. 4. In conclusion, intracellular photorelease of ATP increases the decay of Ca2+-activated Cl- tail currents in metabolically stressed neurones suggesting that the efficiency of intracellular Ca2+ buffering was improved. Additionally, an ATP/cyclic AMP-dependent component of high voltage-activated Ca2+current inactivation which is mediated by calcineurin is revealed following photolysis of 'caged' ATP or cyclic AMP in metabolically stressed neurones.

Adenosine Triphosphate↗
PubMed1994

Actions of cisplatin on the electrophysiological properties of cultured dorsal root ganglion neurones from neonatal rats.

In this study we have employed the whole cell patch clamp technique to investigate the effects of an anti-cancer drug cisplatin on basic electrophysiological properties of cultured dorsal root ganglion neurones from neonatal rats. The results show that within the clinical concentration range, cisplatin (0.1 to 10 microM) caused a decrease in input conductance, and complex changes in resting membrane potential in these cultured sensory neurones. The dominant effects of cisplatin on input conductance may be due to inhibition of leak conductances. Transplatin (5 microM) was significantly less effective than cisplatin at reducing input conductance which suggests a degree of stereoselectivity. Cisplatin (1 to 5 microM) transiently increased excitability of the cultured neurones as reflected by a reduction in the threshold for activation of action potentials by 8 mV. The rise time, peak amplitude and duration of action potentials were not changed by acute application of 5 microM cisplatin. Long term treatment of neurones with cisplatin (5 microM), for up to 1 week reduced the viability of the cultures, and attenuated neurone excitability, although input conductance of the cells was significantly increased to 322 +/- 49 M omega (n = 9) compared with controls of 210 +/- 20 M omega (n = 30; P < 0.05). Acute and chronic treatment of cultured neurones with cisplatin therefore produced contrasting actions.

Animals↗
PubMed1994

Properties of K+ currents recorded from cultured ovine trachea submucosal gland cells.

We have used the whole cell recording technique to investigate voltage-activated outward currents in cultured ovine trachea submucosal gland cells. The cultured gland cells secreted lysozyme in response to secretagogues, methacholine (20 microM), phenylephrine (100 microM) and substance P (10 microM). Most cells in culture for 7-21 days expressed a voltage-activated outward current at potentials positive to -30 mV. This outward current inactivated slowly, by 44 +/- 6% during a 3 sec depolarization to +30 mV. The voltage-activated outward current was sensitive to the potassium channel inhibitors tetraethylammonium bromide (5 mM), 4-aminopyridine (500 microM) and glibenclamide (1 microM). These data suggest that the outward voltage-activated currents observed are due to K+ channel activity. In cells with little or no outward current present the potassium channel opener Ro 31-6930 produced an additional voltage-activated net outward current. This effect of Ro 31-6930 was sensitive to glibenclamide (1 microM). Our results suggest that some cultured submucosal gland cells express voltage-activated K+ currents with a mixed pharmacology to antagonists and that a portion of this current is sensitive to modulation by Ro 31-6930.

Adenosine Triphosphate↗
PubMed1994

Effects of metabolic blockers on Ca(2+)-dependent currents in cultured sensory neurones from neonatal rats.

1. The whole cell variant of the patch clamp technique was used to record high voltage-activated Ca2+ currents and Ca(2+)-activated Cl- tail currents from cultured neonatal rat dorsal root ganglion neurones. The aim of the project was to use these currents as physiological indices of intracellular Ca2+ regulation under control conditions and in the presence of metabolic inhibitors. 2. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (5 microM) and sodium cyanide (1 microM) inhibited Ca2+ currents within 20 s, even when ATP was present in the patch pipette solution, suggesting a direct action on Ca2+ channels. These metabolic inhibitors did not affect Ca2+ current 'run down' or inactivation kinetics. 3. Cultured neonatal dorsal root ganglion neurones of the rat were relatively insensitive to the removal of glucose and ATP from the recording solutions for up to 3 h. These data suggest that the Ca2+ homeostatic mechanisms in these cells are highly resistant to metabolic insult. 4. However 2-deoxy-D-glucose (5 mM) in the extracellular recording medium with no ATP or glucose present did prolong the deactivation time of Ca(2+)-activated Cl- tail currents and increase the total charge flow following activation of a 500 ms voltage-activated Ca2+ current. This effect was prevented by inclusion of D-fructose 1,6-diphosphate (500 microM) in the patch pipette solution. 5. We conclude that some agents used to induce chemical hypoxia, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone and sodium cyanide, may interact directly with voltage-activated Ca2+ channels and are therefore not appropriate for use in studying disturbed neuronal Ca2+ homeostasis. However, the use of 2-deoxy-D-glucose in the absence of glucose and ATP does represent a model of disturbed Ca2+ homeostasis in cultured dorsal root ganglion neurones. In this study we have combined the whole cell recording technique with cultured neurones under conditions which produce a degree of metabolic stress as reflected by prolonged Ca(2+)-activated Cl- tail currents. The reduced efficiency of handling of intracellular Ca2+ loads may be an important factor contributing to the onset of neuronal damage during hypoxia and ischaemia.

Adenosine Triphosphate↗
PubMed1993

Ca2+ currents in cerebellar granule neurones: role of internal Mg2+ in altering characteristics and antagonist effects.

Using the whole-cell patch-clamp technique, Ca2+ channel currents were measured in cultured rat cerebellar granule neurones in the presence of 10 mM Ba2+. Two different solutions were used to fill patch pipettes, one containing mainly tetraethylammonium acetate (TEA-Ac solution), and the other mainly caesium and HEPES (Cs-HEPES solution). Under these two different intracellular conditions markedly different Ca2+ channel currents were recorded. When TEA-Ac solution was used intracellularly, small, Cd(2+)-sensitive inward currents (approx. -55 pA) that were inhibited by the dihydropyridine antagonist (-)-202-791 and the GABAB agonist (-)-baclofen were observed. These currents were insensitive to the Ca2+ channel clocking toxins omega-conotoxin GVIA (omega-CgTX) and omega-agatoxin IVA and were enhanced by the dihydropyridine agonist (+)-202-791. In contrast, when the Cs-HEPES solution was used, currents were 2-3 times larger (approx. -130 pA), inhibited by (-)-202-791, omega-CgTX and omega-agatoxin IVA but were unaffected by (-)-baclofen. Furthermore, both (+)-202-791 and Bay K8644 in the presence of Cs-HEPES solution produced only a transient enhancement that was followed by an inhibition. Analysis of steady-state inactivation revealed two components of current in both cases, with similar voltage dependencies. The factor(s) giving rise to these differences were investigated in terms of current amplitude and responses to (-)-baclofen and omega-CgTX and were found to be mainly due to the concentrations of Mg2+ and ATP added to the patch pipette solutions. Furthermore, free internal Mg2+ concentrations of greater than 0.2 mM selectively inhibited omega-CgTX-sensitive Ca2+ channels. Preliminary evidence indicates that the same may be true of omega-Aga IVA-sensitive P-type current. These data suggest that the N-type Ca2+ channels in these cells are preferentially inhibited by intracellular Mg2+ and this may provide an explanation for discrepancies between the results of different groups investigating Ca2+ channel currents in similar cell types.

Adenosine Triphosphate↗
PubMed1993

Interactions of polyamines with neuronal ion channels.

Polyamines, a group of aliphatic amines, exert selective and complex actions on a variety of ion channels. Polyamines are found endogenously, as normal metabolic intermediates, and also in the venoms of several invertebrates, where they act as potent neurotoxins. In addition, evidence suggests that polyamines may mediate or potentiate excitotoxic mechanisms responsible for neuronal damage during ischaemia. Now that the structures and functions of various polyamines are beginning to be deduced, and synthetic analogues become available, these compounds are of importance, not only as pharmacological tools to study specific receptor/ion channel complexes, but also as templates on which to base drugs designed for neuroprotective effects in a number of neurodegenerative disorders.

Animals↗
PubMed1992

Activation of Ca(2+)-dependent currents in cultured rat dorsal root ganglion neurones by a sperm factor and cyclic ADP-ribose.

The effects of intracellular application of two novel Ca2+ releasing agents have been studied in cultured rat dorsal root ganglion (DRG) neurones by monitoring Ca(2+)-dependent currents as a physiological index of raised free cytosolic Ca2+ ([Ca2+]i). A protein based sperm factor (SF) extracted from mammalian sperm, has been found to trigger Ca2+ oscillations and to sensitize unfertilized mammalian eggs to calcium induced calcium release (CICR). In this study intracellular application of SF activated Ca(2+)-dependent currents in approximately two-thirds of DRG neurones. The SF induced activity was abolished by heat treatment, attenuated by increasing the intracellular Ca2+ buffering capacity of the cells and persisted when extracellular Ca2+ was replaced by Ba2+. In addition, activity could be triggered or potentiated by loading the cells with Ca2+ by activating a series of voltage-gated Ca2+ currents. Ca(2+)-activated inward current activity was also generated by intracellular application of cyclic ADP-ribose (cADPR), a metabolite of NAD+, which causes Ca2+ release in sea urchin eggs. This activity could also be enhanced by loading the cells with Ca2+. The cADPR induced activity, but not the SF induced activity, was abolished by depleting the caffeine sensitive Ca2+ store. Ruthenium red markedly attenuated SF induced activity but had little action on cADPR induced activity or caffeine induced activity. Our results indicate that both SF and cADPR release intracellular Ca2+ pools in DRG neurones and that they appear to act on subtly distinct stores or distinct intracellular Ca2+ release mechanisms, possibly by modulating CICR.

Adenosine Diphosphate Ribose↗