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A C Dolphin

Publications and source records attributed to A C Dolphin.

At least 73 records · Page 4Linked to original sources
PubMed1993

GABAB receptor modulation of Ca2+ currents in rat sensory neurones by the G protein G(0): antisense oligonucleotide studies.

1. Calcium channel currents (IBa) were recorded in cultured dorsal root ganglion neurones (DRGs), 24-32 h after microinjection with 20-mer phosphorothioate antisense oligonucleotides complementary either to a G alpha o or a G alpha i unique sequence, or with a nonsense sequence. 2. The ability of the GABAB agonist (-)-baclofen (50 microM) to inhibit IBa was examined. The maximum peak current was inhibited by 35.3 +/- 4.0% (n = 11) in control non-injected cells, and by 38.1 +/- 2.6% (n = 11) and 34.8 +/- 4.2% (n = 5) in nonsense- and G alpha i oligonucleotide-injected cells. Following G alpha o oligonucleotide injection, (-)-baclofen inhibited IBa by 21.0 +/- 3.2% (n = 19). 3. Confocal immunocytochemical localization of G alpha o showed prominent staining at the plasma membrane in control DRGs, and this was also present in G alpha i and nonsense oligonucleotide-injected cells. The G alpha o staining at the plasma membrane was reduced by 76% in G alpha o oligonucleotide-injected cells. In contrast, confocal immunocytochemical localization of G alpha i showed immunostaining in the membrane and cytoplasm of control, G alpha o- and nonsense-injected DRGs, whereas this was depleted by 68% in G alpha i oligonucleotide-injected cells. 4. These results indicate that the GABAB receptor couples to voltage-sensitive calcium channels via the G protein G(o) and not Gi, and that antisense oligonucleotides can be used to deplete G proteins in DRGs.

Animals↗
PubMed1993

Changes in protein synthesis accompanying long-term potentiation in the dentate gyrus in vivo.

The possibility that the induction of long-term potentiation (LTP) is followed by changes in protein synthesis has been examined using high-resolution two-dimensional gel electrophoresis. 35S-methionine, infused into the third ventricle of anesthetized rats, was used to label hippocampal proteins. LTP was induced unilaterally in the dentate gyrus by tetanic stimulation of the perforant path, and followed either for 1 hr or for 3 hr. Two-dimensional gel autoradiographs were quantitatively analyzed using the PDQUEST system (Protein Databases Inc.). One hour after the unilateral induction of LTP, only one protein spot was found to be statistically different in intensity from corresponding spots in the contralateral control side. Three hours after LTP, however, 11 spots were found to have altered densities. Examination of basic proteins using the nonequilibrium pH gel electrophoresis system revealed changes in three proteins in the 3 hr group. Reductions as well as increases in spot intensities were observed. The results indicate that LTP is associated with a complex pattern of changes in protein synthesis.

Animals↗
PubMed1992

1,4-Dihydropyridines modulate GTP hydrolysis by Go in neuronal membranes.

Several lines of evidence suggest that L-type Ca2+ channels (1,4-dihydropyridine receptors) are modulated by GTP-binding proteins. We have further examined this interaction by measuring the effect of 1,4-dihydropyridines on GTPase activity in brain membranes. Dihydropyridine agonists significantly increased GTPase, reflected by an increase in the maximal rate of GTP hydrolysis, without affecting the affinity for GTP or the binding of a non-hydrolysable analogue of GTP. The stimulating effect on GTPase was abolished by antisera raised against Go alpha but not Gi alpha. L-type Ca2+ channels may act as endogenous GTPase activating proteins (GAPs) to stimulate GTP hydrolysis by Go.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1992

The effect of phosphatase inhibitors and agents increasing cyclic-AMP-dependent phosphorylation on calcium channel currents in cultured rat dorsal root ganglion neurones: interaction with the effect of G protein activation.

Ca2+ channel currents have been recorded in cultured rat dorsal root ganglion neurones. The amplitude of IBa(GTP gamma S), recorded in the presence of GTP[ gamma S] (200 microM) in the patch pipette solution, is enhanced by external application of forskolin (10 microM), and there is an increase in the proportion of the rapidly activating component of the current. When forskolin (1 microM) is present in the bathing solution at the start of recording, or when 8-bromocyclic AMP (100 microM) is present in the patch pipette solution, the amplitude and rate of activation of IBa(GTP gamma S) are also increased compared to control IBa(GTP gamma S). The effect is mimicked by internal application of a 5 microM solution of a phosphopeptide fragment of inhibitor 1 (I1 PP), which inhibits phosphatase 1. The enhancement of IBa(GTP gamma S) caused by I1PP is not additive with that due to forskolin. Furthermore, the enhancement due to I1PP is reversibly lost when the holding potential is shifted from -80 mV to -30 mV, as was the enhancement due to forskolin and 8-bromocyclic AMP. I1PP also produced a less marked stimulation of the control Ca2+ channel current in the absence of G protein activation. The results suggest that phosphorylation regulates the interaction between calcium channels and G proteins in these neurones, and that phosphatase 1 is tonically active to dephosphorylate the relevant protein(s).

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

G-protein mediation in nociceptive signal transduction: an investigation into the excitatory action of bradykinin in a subpopulation of cultured rat sensory neurons.

Bradykinin is one of several pro-inflammatory, pain-inducing substances produced during inflammation--the body's response to injury. In previous work we have shown that bradykinin and guanosine-5'-O-3-thiotriphosphate increase excitability in a subpopulation of cultured neonatal rat dorsal root ganglion neurons. We now describe experiments in which the mechanism underlying the stimulatory action of these two substances has been examined in more detail. Using the whole-cell voltage-clamp technique, bradykinin-sensitive cells were distinguished by their response to a 1-s depolarizing voltage-pulse which evoked more than one inward current during the step command. The secondary inward currents are likely to represent action potentials generated at the poorly clamped neurites of these cells. Bradykinin- and guanosine-5'-O-3-thiotriphosphate-induced changes in excitability were measured indirectly by a change in the number of inward currents recorded during the 1-s depolarizing voltage-step. The effect of activators and inhibitors of protein kinase C, arachidonic acid metabolism, G-protein activation and release of intracellular Ca2+ were examined on this response. In the presence of extracellular staurosporine (1.0 microM) or nordihydroguaiaretic acid (10 microM), these excitatory effects were reduced but not abolished, whilst indomethacin (20 microM) had no effect. Intracellular application of guanosine-5'-O-2-thiodiphosphate (10 mM) or ryanodine (100 microM) substantially reduced the effect of bradykinin. The excitatory effect of internal guanosine-5'-O-3-thiotriphosphate (500 microM) occurred gradually over time, and this was mimicked by internal application of myo-inositol 1,4,5-trisphosphorothioate (1.0 microM). From the results, it is proposed that G-protein activation is an essential component of the bradykinin response, which may also require a Ca(2+)-activated conductance modulated by protein kinase C and lipoxygenase metabolites of arachidonic acid.

Alkaloids↗
PubMed1992

Intracellular calcium regulates the survival of early sensory neurons before they become dependent on neurotrophic factors.

To investigate the role of intracellular Ca2+ in the survival of developing neurons before they become neurotrophic factor dependent, we have studied chick embryo nodose neurons, which have a particularly protracted period of neuorophic factor independence. Pharmacological reduction of intracellular free Ca2+ or depletion of either Ca(2+)-regulated or inositol trisphosphate-regulated intracellular Ca2+ stores kills early neurotrophic factor-independent neurons, but has a negligible effect on older neurons growing in the presence of brain-derived neutrotrophic factor. Shortly before they become dependent on brain-derived neurotrophic factor, nodose neurons express L-type Ca2+ channels and their survival can be enhanced by depolarization-induced Ca2+ influx. We conclude that intracellular Ca2+ plays a role in regulating neuronal survival both prior to and after the onset of neurotrophic factor dependence, but does not mediate the survival-promoting effects of neurotrophic factors.

Animals↗
PubMed1992

Actions of arginine polyamine on voltage and ligand-activated whole cell currents recorded from cultured neurones.

1. Toxins from invertebrates have proved useful tools for investigation of the properties of ion channels. In this study we describe the actions of arginine polyamine which is believed to be a close analogue of FTX, a polyamine isolated from the American funnel web spider, Agelenopsis aperta. 2. Voltage-activated Ca2+ currents and Ca(2+)-dependent Cl- currents recorded from rat cultured dorsal root ganglion neurones were reversibly inhibited by arginine polyamine (AP; 0.001 to 100 microM). Low voltage-activated T-type Ca2+ currents were significantly more sensitive to AP than high voltage-activated Ca2+ currents. The IC50 values for the actions of AP on low and high voltage-activated Ca2+ currents were 10 nM and 3 microM respectively. AP was equally effective in inhibiting high voltage-activated currents carried by Ba2+, Sr2+ or Ca2+. However, AP-induced inhibition of Ca2+ currents was attenuated by increasing the extracellular Ca2+ concentration from 2 mM to 10 mM. 3. The actions of AP on a Ca(2+)-independent K+ current were more complex, 1 microM AP enhanced this current but 10 microM AP had a dual action, initially enhancing but then inhibiting the K+ current. 4. gamma-Aminobutyric acid-activated Cl- currents were also reversibly inhibited by 1 to 10 microM AP. In contrast N-methyl-D-aspartate currents recorded from rat cultured cerebellar neurones were greatly enhanced by 10 microM AP. 5. We conclude that at a concentration of 10 nM, AP is a selective inhibitor of low threshold T-type voltage-activated Ca2+ currents. However, at higher concentrations 1-10 microM AP interacts with ion channels or other membrane constituents to produce a variety of actions on both voltage and ligand gated ion channels.

Animals↗
PubMed1991

Ca2+ channel currents in rat sensory neurones: interaction between guanine nucleotides, cyclic AMP and Ca2+ channel ligands.

1. The characteristics have been examined of the high threshold calcium channel current in cultured rat dorsal root ganglion (DRG) neurones recorded in the presence of guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S; 200 microM in the patch pipette). This current, termed IBa, GTP gamma S, was slowly activating and showed little inactivation over 100 ms. 2. External application of forskolin (10 microM) to elevate internal cyclic AMP levels increased the amplitude of IBa, GTP gamma S whereas it had no effect on the control IBa. This cyclic AMP-dependent protein kinase (PKI; 25 microM). 3. The cyclic AMP-dependent phosphorylation induced enhancement of IBa, GTP gamma S was voltage dependent and either did not occur or was observed only transiently at a holding potential (VH) of -30 mV. The forskolin-stimulated enhancement seen at VH -80 mV was lost with a t1/2 of about 1 min when VH was depolarized to -30 mV. Cholera toxin pre-treatment also increased the amplitude of IBa, GTP gamma S at VH -80 mV but not at VH -30 mV. 4. The calcium channel antagonist (-)-202-791 (5 microM) increased the amplitude of IBa, GTP gamma S when applied at VH -80 mV, but either not, or only transiently, at VH -30 mV, as previously observed. This 'agonist' effect of (-)-202-791 was prevented by PKI and was occluded by prior enhancement of IBa, GTP gamma S with forskolin. (-)-202-791 did not increase cyclic AMP levels in DRG neurones. 5. The 'agonist' response of IBa, GTP gamma S to D600 (10 microM) was also occluded by application of forskolin (10 microM) in the patch pipette. Forskolin alone, applied in this manner, increased IBa, GTP gamma S to a similar extent to D600 applied alone. 6. The agonist effect of (+)-202-791 (5 microM) on IBa, GTP gamma S was not prevented by prior enhancement with forskolin, nor was it prevented by PKI. 7. In conclusion, internal GTP gamma S activates G proteins which may interact directly with calcium channels to influence the kinetics of activation and to reduce steady-state inactivation of the channels. There is also an indirect effect on the generation of second messengers such as cyclic AMP. It is likely that forskolin enhances IBa, GTP gamma S by increasing activated Gs coupling to adenylyl cyclase and increasing cyclic AMP generation. The mechanism of action of (-)-202-791 to enhance IBa, GTP gamma S also involves cyclic AMP-dependent phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Increased efflux of a haemoglobin-like protein and an 80 kDa protease into push-pull perfusates following the induction of long-term potentiation in the dentate gyrus.

In a previous communication, we reported an increase in protein efflux into perfusates obtained from push-pull cannulation of the dentate gyrus, following induction of long-term potentiation (LTP) in the perforant path. LTP was accompanied by a delayed but general increase in protein efflux. Protein B5 (MW 14 kDa), because of its relatively high concentration in the perfusates and absence from serum, has been chosen for further characterization. Spectrophotometric analysis, in situ proteolysis, two-dimensional electrophoresis and immunoblotting, revealed that protein B5 is indistinguishable from haemoglobin. A persistent increase in an 80 kDa protease, detected by SDS-PAGE zymography, was also seen after the induction of LTP. We consider the possibility that the increased haemoglobin content of perfusates after the induction of LTP may reflect an LTP-associated increase in the release (or activation) of proteases into extracellular space, leading to proteolytic breakdown of blood clots in the vicinity of the cannula. Evidence in favour of this hypothesis is provided.

Animals↗
PubMed1990

Modulation of neuronal T-type calcium channel currents by photoactivation of intracellular guanosine 5'-O(3-thio) triphosphate.

Low voltage-activated T-type Ca2+ channel currents were recorded from cultured rat dorsal root ganglion neurons using the whole-cell clamp technique with Ba2+ as the charge carrier. The T-type Ca2+ channel current was identified by its low threshold of activation (Vc -50 to -20 mV from VH - 90 mV), its kinetics of inactivation and its sensitivity to NiCl2 (100 microM). It was also sensitive to 1-octanol (1 microM). omega-Conotoxin (1 microM) markedly reduced the high threshold voltage-activated Ca2+ channel currents but did not inhibit the T-type Ca2+ channel current. Photorelease of intracellular guanosine 5'-O(3-thio) triphosphate from a photolabile "caged" precursor had dose-dependent effects on the T-type Ca2+ channel current. At a concentration of 6 microM, guanosine 5'-O(3-thio) triphosphate enhanced the current, but further photorelease of guanosine 5'-O(3-thio) triphosphate (up to 20 microM) inhibited the current. Only the inhibitory response was sensitive to pertussis toxin. These data suggest that more than one G-protein is involved in T-type Ca2+ channel current modulation. Inclusion of guanosine 5'-O(2-thio) diphosphate (1 mM) in the patch solution prevented guanosine 5'-O(3-thio) triphosphate from potentiating the current, and greatly attenuated the inhibitory effects observed when larger amounts of guanosine 5'-O(3-thio) triphosphate were photoreleased. Photorelease of guanosine 5'-O(2-thio) diphosphate had no effect on T-type current but did significantly increase the high voltage-activated current. A low concentration of (-)-baclofen (2 microM), potentiated T-type current, while 100 microM(-)-baclofen inhibited T-type current.

Animals↗