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A C Dolphin

Publications and source records attributed to A C Dolphin.

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

Facilitation of Ca2+ current in excitable cells.

Voltage-dependent Ca2+ channels are one of the main routes for the entry of Ca2+ into excitable cells. These channels are unique in cell-signalling terms in that they can transduce an electrical signal (membrane depolarization) via Ca2+ entry into a chemical signal, by virtue of the diverse range of intracellular Ca(2+)-dependent enzymes and processes. In a variety of cell types, currents through voltage-dependent Ca2+ channels can be increased in amplitude by a number of means. Although the term facilitation was originally defined as an increase of Ca2+ current resulting from one or a train of prepulses to depolarizing voltages, there is a great deal of overlap between facilitation by this means and enhancement by other routes, such as phosphorylation.

Calcium Channels↗
PubMed1995

Voltage-dependent calcium channel beta-subunits in combination with alpha 1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by G alpha o in rat frontal cortex.

The dihydropyridine-sensitive calcium channel agonist (-)-BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by Go in rat frontal cortex membranes. An anti-calcium channel beta-subunit antiserum abolished the (-)-BayK 8644-stimulated hydrolysis of GTP by Go and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the beta-subunit binding domain of the calcium channel complex, also attenuated (-)-BayK 8644 activation of GTPase. This study suggests that the calcium channel beta-subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Go. This may be a mechanism by which calcium channels can normally act to limit the duration of a G-protein modulatory signal.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Inhibition of the interaction of G protein G(o) with calcium channels by the calcium channel beta-subunit in rat neurones.

1. The beta-subunit has marked effects on the biophysical and pharmacological properties of voltage-dependent calcium channels. In the present study we examined the ability of the GABAB agonist (-) -baclofen to inhibit calcium channel currents in cultured rat dorsal root ganglion neurones following depletion of beta-subunit immunoreactivity, 108-116 h after microinjection of a beta-subunit antisense oligonucleotide. 2.We observed that, although the calcium channel current was markedly reduced in amplitude following beta-subunit depletion, the residual current (comprising both N- and L-type calcium channel currents) showed an enhanced response to application of (-) -baclofen. Therefore, it is possible that there is normally competition between activated G protein G(o) and the calcium channel beta-subunit for binding to the calcium channel alpha 1-subunit; and this competition shifts in favour of the binding of activated G(o) following depletion of the beta-subunit, resulting in increased inhibition. 3. This hypothesis is supported by evidence that an antibody against the calcium channel beta-subunit completely abolishes stimulation of the GTPase activity of G(o) by the dihydropyridine agonist S-(-) -Bay K 8644 in brain membranes. This stimulation of GTPase is thought to result from an interaction of G(o) alpha-subunit (G alpha o) with its calcium channel effector which may operate as a GTPase-activating protein. 4. These data suggest that the calcium channel beta-subunit when complexed with the beta 1-subunit normally inhibits its association with activated G(o). It may function as a GTPase-activating protein to reduce the ability of activated G(o) to associate with the calcium channel, and thus limit the efficacy of agonists such as (-) -baclofen.

Amino Acid Sequence↗
PubMed1995

Use of site-directed antibodies to probe the topography of the alpha 2 subunit of voltage-gated Ca2+ channels.

Polyclonal antibodies were raised against peptides corresponding to residues 1-15, 469-483 and 933-951 of the rabbit skeletal muscle L-type calcium channel alpha 2/delta primary translation product, for use as topological probes. Immunocytochemical comparison of the abilities of the antibodies to bind to the alpha 2 and delta subunits in intact and detergent-permeabilised rat dorsal root ganglion cells enabled the membrane orientation of these regions to be established. The resultant data indicate that the regions containing residues 1-15 and 469-483 of the alpha 2 subunit, and residues 1-17 of the delta subunit, are exposed on the extracellular surface of the membrane, findings consistent with a model that proposes alpha 2 to be entirely extracellular.

Amino Acid Sequence↗
PubMed1995

Antisense depletion of beta-subunits modulates the biophysical and pharmacological properties of neuronal calcium channels.

1. The role of the voltage-dependent calcium channel (VDCC) beta-subunit has been examined in cultured rat dorsal root ganglion neurones (DRGs). An antipeptide antibody was raised and this recognized proteins corresponding to beta-subunits in a number of preparations. Immunoreactivity for the VDCC beta-subunit in DRGs was concentrated on the internal side of the plasma membrane but was also present in the cytoplasm. 2. A twenty-six-mer antisense oligonucleotide with homology to all published VDCC beta-subunit sequences was microinjected into individual cells, and maximal depletion of VDCC beta-subunit immunoreactivity was observed after 108 h suggesting a half-life for the turnover of the beta-subunit greater than 50 h. No depletion was obtained with nonsense oligonucleotide. 3. The effect of depletion of VDCC beta-subunit immunoreactivity on calcium channel currents in these cells was a reduction in amplitude of the maximum current of about 47%, and a shift in the voltage dependence of current activation of about +7 mV. These effects are the converse of those observed following co-expression of cloned beta- with alpha 1-subunits in oocytes and other expression systems. 4. The ability of the 1,4-dihydropyridine (DHP) agonist Bay K 8644 to enhance calcium channel currents was greatly reduced following depletion of beta-subunit immunoreactivity. This result is in agreement with the finding in several systems that co-expression of the beta-subunit with alpha 1-subunits results in an increased number of DHP binding sites. 5. These results show that calcium channel beta-subunits form part of native neuronal calcium channels and modify their biophysical and pharmacological properties.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
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

The involvement of multiple calcium channel sub-types in glutamate release from cerebellar granule cells and its modulation by GABAB receptor activation.

In this study, we have examined both the ability of various Ca2+ channel sub-types to support the release of [3H]glutamate from cerebellar granule neurons and the mechanism of action involved in the modulation of glutamate release by the GABAB receptor agonist, (-)-baclofen. Cerebellar granule neurons were stimulated to release newly synthesized [3H]glutamate by K(+)-evoked depolarization. Stimulated release was entirely calcium-dependent and abolished by the presence of 200 microM cadmium. Release of glutamate was not affected by either tetrodotoxin or 5-aminophosphonovaleric acid but was potentiated by dihydrokainate and inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione. Stimulated glutamate release was partially inhibited by both the L-type calcium channel blocker, nicardipine, and the N-type calcium channel blocker, omega-conotoxin GVIA; however, the P/Q-type calcium channel blocker omega-agatoxin IVA inhibited release of glutamate only after pre-incubation of cells with omega-conotoxin GVIA. K(+)-stimulated release of glutamate was observed when stimulated either in the presence of Ca2+ or of Ba2+ and similar inhibition of release by (-)-baclofen was seen under both conditions. In contrast to these results, ionomycin-evoked glutamate release was greatly reduced as compared to K(+)-evoked release and was not modulated by (-)-baclofen. In the presence of omega-conotoxin GVIA alone, inhibition of release by (-)-baclofen was attenuated but not abolished. Following block of nicardipine-sensitive channels, inhibition of release by (-)-baclofen was still present, and after prior block of omega-conotoxin GVIA-sensitive channels the presence of nicardipine restored the ability of (-)-baclofen to inhibit residual release of glutamate. Modulation of glutamate release by (-)-baclofen was unaffected by the presence of omega-agatoxin IVA alone; however, after block of both omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive channels, inhibition of release by (-)-baclofen was completely abolished. These results indicate that multiple sub-types of voltage-dependent calcium channels are present on the presynaptic terminals of cerebellar granule neurons and support K(+)-stimulated release of [3H]glutamate. Modulation of release by GABAB receptor activation appears to be dependent upon interaction of this receptor with a number of voltage-sensitive calcium channels, including omega-conotoxin GVIA-sensitive and omega-agatoxin IVA-sensitive channels.

Animals↗
PubMed1995

Adenosine A1 agonists and the Ca2+ channel agonist bay K 8644 produce a synergistic stimulation of the GTPase activity of Go in rat frontal cortical membranes.

The identity and role of G proteins in coupling adenosine receptors to effectors have been studied to a limited degree. We have identified the G proteins whose GTPase activity is stimulated by adenosine receptor agonists in neuronal membranes. (R)-Phenylisopropyladenosine, 2-chloroadenosine, and N-ethylcarboxamideadenosine produced a concentration-dependent stimulation of GTPase. At 10(-5) M, the increase above basal GTPase in frontal cortex was 25 +/- 4, 20 +/- 3, and 8 +/- 1%, respectively, and in the cerebellum 55 +/- 2, 41 +/- 4, and 22 +/- 2%, respectively. The effects of (R)-phenylisopropyladenosine and 2-chloroadenosine were inhibited by (1) A1 antagonists (76-96% reduction), (2) pretreatment with pertussis toxin (90-100% reduction), and (3) antibodies raised against the alpha-subunit of Gi and G(o) (55-57% reduction by each), suggesting that A1 receptors interact equally with Gi and G(o). (R)-Phenylisopropyladenosine increased the binding of a nonhydrolyzable analogue of GTP to membranes in a pertussis toxin-sensitive manner, indicative of activation of Gi or G(o). Previously, (+/-)-Bay K 8644 enhanced GTP hydrolysis by G(o) but not Gi. Now we report a profound synergistic stimulation of GTPase in the presence of (R)-phenylisopropyladenosine and (+/-)-Bay K 8644 (10(-7) to 10(-5) M). (+/-)-Bay K 8644 had no effect on nucleotide exchange and, thus, cannot activate G(o). It appears that a positive cooperative stimulation of G(o) occurs when it is first activated by A1 receptors and subsequently interacts with the L-type Ca2+ channel.

2-Chloroadenosine↗
PubMed1994

Cycloheximide abolishes pertussis toxin-induced increase in glutamate release from cerebellar granule neurones.

Release of glutamate from cerebellar granule neurones was stimulated either by adding 50 mM K+ to normal Krebs medium, or by adding 5 mM Ca2+ to neurones continuously depolarised with 50 mM K+ in the absence of Ca2+. Pre-incubation of neurones for 16 h with pertussis toxin (PTX) increased the stimulated glutamate release in both K(+)-stimulated and continuously depolarised neurones. Under both conditions, the PTX-induced increase in release was abolished by cycloheximide. In contrast, in the presence of cycloheximide, PTX still prevented the GABAB agonist (-)-baclofen from inhibiting glutamate release. These results suggest that G-protein ADP-ribosylation by PTX in cerebellar granule neurones may increase synthesis of a protein associated with the L-type calcium channel.

Animals↗
PubMed1994

Low- and high-voltage-activated calcium channel currents and their modulation in the dorsal root ganglion cell line ND7-23.

The dorsal root ganglion-neuroblastoma cell line ND7-23 expresses low-voltage-activated calcium channel currents, and also expresses high-voltage-activated currents in about 50% of differentiated cells. Calcium channel currents were recorded with Ba2+ as the charge carrier. Low-voltage-activated currents were maximally activated at -30 mV and completely inactivated at holding potentials of -60 to -50 mV. omega-Conotoxin GVIA produced a reversible inhibition of low-voltage-activated currents, whereas the inhibition of high-voltage-activated current was largely irreversible. Dihydropyridine antagonists did not inhibit low-voltage-activated currents, whereas they inhibited a sustained, high-voltage-activated current. Low-voltage-activated currents were inhibited to a greater extent than high-voltage-activated currents by Ni2+ (100 microM) and by phenytoin (10 microM). Bradykinin (0.1 microM), baclofen (2 microM) and internal guanosine-5'-O-3-thiotriphosphate (100 microM) inhibited low-voltage-activated currents without affecting their kinetics of activation. Two classes of low-voltage-activated current were distinguished by their kinetics of inactivation. In the majority of cells, currents were slowly inactivating with a time-constant of inactivation of about 50 ms. They also exhibited a sustained component to the current, representing about 20% of the peak current. This component could be distinguished pharmacologically from high-voltage-activated current. The remainder of cells expressed a rapidly and completely inactivating current, with a time-constant of inactivation of about 20 ms. Two distinct single channel currents were observed in these cells, from cell-attached patch measurements, one had a single channel conductance of 7.9 pS, and the ensemble average current showed some inactivation. It is likely that this channel subtype underlies the low-voltage-activated current. The other showed long openings in the presence of a dihydropyridine agonist, had a conductance of 23.1 pS, and was non-inactivating.

Ammonium Chloride↗
PubMed1993

G(o) transduces GABAB-receptor modulation of N-type calcium channels in cultured dorsal root ganglion neurons.

High-voltage-activated (HVA) calcium channel currents (IBa) were recorded from acutely replated cultured dorsal root ganglion (DRG) neurons. IBa was irreversibly inhibited by 56.9 +/- 2.7% by 1 microM omega-conotoxin-GVIA (omega-CTx-GVIA), whereas the 1,4-dihydropyridine antagonist nicardipine was ineffective. The selective gamma-aminobutyric acidB (GABAB) agonist, (-)-baclofen (50 microM), inhibited the HVA IBa by 30.7 +/- 5.4%. Prior application of omega-CTx-GVIA completely occluded inhibition of the HVA IBa by (-)-baclofen, indicating that in this preparation (-)-baclofen inhibits N-type current. To investigate which G protein subtype was involved, cells were replated in the presence of anti-G protein antisera. Under these conditions the antibodies were shown to enter the cells through transient pores created during the replating procedure. Replating DRGs in the presence of anti-G(o) antiserum, raised against the C-terminal decapeptide of the G alpha o subunit, reduced (-)-baclofen inhibition of the HVA IBa, whereas replating DRGs in the presence of the anti-Gi antiserum did not. Using anti-G alpha o antisera (1:2000) and confocal laser microscopy, G alpha o localisation was investigated in both unreplated and replated neurons. G alpha o immunoreactivity was observed at the plasma membrane, neurites, attachment plaques and perinuclear region, and was particularly pronounced at points of cell-to-cell contact. The plasma membrane G alpha o immunoreactivity was completely blocked by preincubation with the immunising G alpha o undecapeptide (1 microgram.ml-1) for 1 h at 37 degrees C. A similar treatment also blocked recognition of G alpha o in brain membranes on immunoblots.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Inhibition of omega-conotoxin-sensitive Ca2+ channel currents by internal Mg2+ in cultured rat cerebellar granule neurones.

The effects of changing the intracellular concentrations of either free Mg2+ ions ([Mg2+]i) or Mg(2+)-bound adenosine triphosphate ([Mg.ATP]i) on Ca2+ channel currents were assessed in cultured rat cerebellar granule neurones using the whole-cell patch-clamp technique. Raising [Mg2+]i from 0.06 mM to 1.0 mM inhibited Ca2+ channel currents by approximately 50%. The action of omega-conotoxin GVIA (omega-CgTX), a selective inhibitor of "N"-type Ca2+ channels was also investigated. With increasing [Mg2+]i, the proportion of current irreversibly blocked by omega-CgTX was reduced, and was negligible (approximately 5 pA of current) in the presence of [Mg2+]i values of 0.5 mM or greater. Block of the omega-CgTX-sensitive current accounted for the reduction in total current by concentrations of [Mg2+]i to 0.5 mM. Raising [Mg2+]i had no effect on the rate of decay of Ca2+ currents, but did produce a negative shift in current activation, possibly due to a non-specific interaction with negative surface charge. Altering [Mg.ATP]i from 0.3 to 5.0 mM caused a twofold increase in the size of currents without affecting the proportion of current sensitive to omega-CgTX. [Mg2+]i was also effective in inhibiting the Ca2+ channel current following potentiation by increasing [Mg.ATP]i. These data suggest that omega-CgTX-sensitive current in these cells is selectively inhibited by internal Mg2+ whereas both omega-CgTX-sensitive and -resistant components of current are potentiated by internal Mg.ATP. The mechanism by which Mg2+ inhibits "N"-type channels is unclear, but may involve an open channel block.

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↗
PubMed1993

Pertussis toxin treatment increases glutamate release and dihydropyridine binding sites in cultured rat cerebellar granule neurons.

This study was designed to examine the ability of pertussis toxin to block various responses due to (-)-baclofen in cultured cerebellar granule neurons of the rat. Treatment with pertussis toxin for 3 h markedly reduced the ability of (-)-baclofen to stimulate GTPase in membranes, and its ability to inhibit forskolin-stimulated adenylyl cyclase in intact cells, whereas the ability of (-)-baclofen to inhibit glutamate release was not affected at 3 h, but was abolished after 16 and 48 h treatment with pertussis toxin. The amount of ADP-ribosylation of Gi/Go due to pertussis toxin in intact cells correlated well with the former two effects, but not with the prevention of the ability of baclofen to inhibit glutamate release. Pertussis toxin treatment for up to 48 h did not significantly affect the levels of Gs, Gi and Go in membranes from granule neurons determined by immunoblotting. Pertussis toxin treatment for 16 or 48 h but not 3 h increased the total amount of stimulated release of glutamate by about 40% under normal conditions, and by 84% under depolarizing conditions. In parallel experiments it was observed that pertussis toxin treatment for 16 h increased the number of dihydropyridine binding sites by about 90% on intact granule neurons. Whole-cell calcium channel currents, recorded under several conditions in the cells, were not increased in amplitude by pertussis toxin treatment for up to 48 h, although the ability of baclofen to inhibit calcium channel currents was blocked by pertussis toxin. These results indicate that the pertussis toxin-induced increase in glutamate release may be due to an increase in dihydropyridine binding sites, possibly localized to the presynaptic terminals.

Adenosine Diphosphate Ribose↗