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J F Wootton

Publications and source records attributed to J F Wootton.

15 recordsLinked to original sources
PubMed2003

A comparison between the distinct inward currents activated in rat cultured dorsal root ganglion neurones by intracellular flash photolysis of two forms of caged cyclic guanosine monophosphate.

Whole cell inward currents activated by intracellular photorelease of cyclic guanosine monophosphate (cGMP) were investigated in cultured dorsal root ganglion (DRG) neurones. The actions of two distinct types of caged cGMP (NPE-caged cGMP and a highly water-soluble caged cGMP) were compared. Rapidly activating inward currents were evoked by cGMP in a subpopulation (12.5%) of neurones and these currents may be due to activity of cyclic nucleotide-gated channels. In contrast in 52% of DRG neurones intracellular photorelease of cGMP activated a delayed Ca(2+)-dependent inward current through the generation of cyclic ADPribose and mobilisation of Ca(2+) from ryanodine sensitive intracellular stores. Similar delayed inward currents were activated by both caged compounds but only NPE-caged cGMP evoked rapidly activating currents. Cyclic GMP appears to increase excitability in some DRG neurones by diverse mechanisms.

Animals↗
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↗
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

Kinetics of cytosolic Ca2+ concentration after photolytic release of 1-D-myo-inositol 1,4-bisphosphate 5-phosphorothioate from a caged derivative in guinea pig hepatocytes.

The influence of 1-D-myo-inositol 1,4,5-trisphosphate (InsP3) breakdown by InsP3 5-phosphatase in determining the time course of Ca2+ release from intracellular stores was investigated with flash photolytic release of a stable InsP3 derivative, 5-thio-InsP3, from a photolabile caged precursor. The potency and Ca(2+)-releasing properties of the biologically active D isomers of 5-thio-InsP3 and InsP3 itself were compared by photolytic release in guinea pig hepatocytes. After a light flash, cytosolic free calcium concentration ([Ca2+]i) showed an initial delay before rising quickly to a peak and declining more slowly to resting levels, with time course and amplitude generally similar to those seen with photolytic release of InsP3. Differences were a three- to eightfold lower potency of 5-thio-InsP3 in producing Ca2+ release, much longer delays between photolytic release and Ca2+ efflux with low concentrations of 5-thio-InsP3 than with InsP3, and persistent reactivation of Ca2+ release, producing periodic fluctuations of cytosolic [Ca2+]i with high concentrations of 5-thio-InsP3 but not InsP3 itself. The lower potency of 5-thio-InsP3 may be a result of a lower affinity for closed receptor/channels or a lower open probability of liganded receptor/channels. The longer delays with 5-thio-InsP3 at low concentration suggest that metabolism of InsP3 by 5-phosphatase may reduce the concentration sufficiently to prevent receptor activation and may have a similar effect on InsP3 concentration during hormonal activation. The maximal rate of rise of [Ca2+]i, the duration of the period of high Ca2+ efflux, and the initial decline of [Ca2+]i are similar with5-thio-lnsP3 and lnsP3, indicating that lnsP3 breakdown is not important in terminating Ca2+ release. The second activation ofInsP3 receptors with 5-thio-lnsP3 and particularly the sustained periodic fluctuations of [Ca2+]i indicate persistence of 5-thio-lnsP3,suggesting that InsP3 breakdown prevents reactivation of InsP3 receptors. The photochemical properties of 1-(2-nitrophenyl)-ethyl caged 5-thio-lnsP3 are photolytic quantum yield = 0.57 (cf. 0.65 for caged InsP3) and rate of photolysis = 87 s-I (half-life approximately 8 ms; cf. 3 ms for caged lnsP3; pH7.1; ionic strength, 0.2 M; 21 OC). Caged 5-thio-lnsP3 at concentrations up to 360 pM did not activate lnsP3 receptors to produce Ca2+ release or block Ca2+ release by free 5-thio-lnsP3.

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↗
PubMed1995

Calcium-dependent plateau potentials in a crab stomatogastric ganglion motor neuron. II. Calcium-activated slow inward current.

1. Using intracellular recording and voltage-clamp techniques, we examined the biophysical properties of a Ca(2+)-activated slow inward current and its physiological role in plateau potential generation in the dorsal gastric (DG) motor neuron of the stomatogastric ganglion in the crab, Cancer borealis. 2. As shown in the accompanying paper, a brief puff of serotonin (5-HT) evoked a plateau potential in the DG neuron. Intracellular loading of the Ca2+ chelator ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) prevented 5-HT from evoking a plateau potential. On the contrary, rapid increase of intracellular Ca2+ by photolysis of caged-Ca2+ (bound to DM-nitrophen) evoked a plateau response in DG bathed in normal saline. 3. Extracellular tetrodotoxin (TTX), tetraethylammonium (TEA), 4-aminopyridine (4-AP), and Cs+ and intracellular iontophoresis of Cs+ were used to block voltage-dependent INa, IK, and Ih. Under these conditions we voltage clamped DG using two electrodes and isolated a long-lasting tail current after a short depolarization of the cell. 4. The reversal potential of the slow tail current was extrapolated to be -27 +/- 3.5 (SE) mV. Na+ substitutions with choline+, tris(hydroxymethyl)aminomethane+ (Tris+) or n-methyl-glucamine+ (NMG+) did not significantly affect the reversal potential or the amplitude. 5. The slow tail current was Ca2+ dependent. It was reduced or abolished by the Ca2+ channel blocker Co2+, intracellular injection of EGTA, and by Ba2+ replacement of Ca2+ as the charge carrier. The activation and deactivation of this current do not show an apparent dependence on voltage. 6. When the voltage-dependent Na+, K+, and Ca2+ channels were blocked, a brief puff of caffeine evoked a slow depolarization. In voltage clamp, caffeine evoked a slow inward current with an apparent conductance increase. This current was reduced by intracellular EGTA. The current-voltage (I-V) relationship of the caffeine-evoked current was linear with a reversal potential of -25 +/- 4.8 mV. This was not statistically different from the reversal potential of the depolarization-evoked tail current. 7. 5-HT enhanced the depolarization-evoked slow tail current but had no effect on the caffeine-evoked slow inward current. 8. We conclude that the slow tail current is a Ca(2+)-activated nonselective current, similar to the Ca(2+)-activated nonspecific cation currents described in other preparations. This current appears to play an important role in plateau generation and maintenance in DG. 5-HT has no direct effect on the properties of this current, but it indirectly enhances the current through an increase of voltage-dependent Ca2+ current.

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↗
PubMed1988

Photoactivation of intracellular guanosine triphosphate analogues reduces the amplitude and slows the kinetics of voltage-activated calcium channel currents in sensory neurones.

The influence of guanine nucleotide analogues on calcium channel currents in cultured rat dorsal root ganglion neurones has been studied using a technique in which the rate of diffusion of the analogues to their site of action is by-passed by photochemical release of the analogues within the neurones. The 1(2-nitrophenyl)ethyl P3-ester derivatives of guanosine 5'-0(3-thio)triphosphate (caged GTP-gamma-S) and 5'-guanylylimidodiphosphate (caged GMP-PNP) were synthesised and found to be completely photolysable by light, yielding free GTP-gamma-S and GMP-PNP. Calcium channel currents were recorded using the whole cell patch technique and either caged GTP-gamma-S or caged GMP-PNP (2 mM) were included in the patch pipette. Stable currents were recorded for 5-10 min, and a single pulse of 300-350 nm irradiation was directed using a liquid light guide onto the recording dish. Calcium channel currents were then recorded every 30-120 s following photochemical release of approximately 20 microM GTP-gamma-S. The peak calcium channel current was reduced by about 70% with a slow time course [t1/2 1.5 +/- 0.2 min (mean +/- SEM); n = 5]. The transient component of the peak current was usually completely abolished, whereas the sustained current measured at the end of the 100 ms depolarising pulse was less affected. Qualitatively similar effects were observed on photolysis of caged GMP-PNP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Nitrogen utilization within equine large intestine.

The distribution of nitrogen in ligated segments of the stomach and intestine of two groups of ponies has been examined at sacrifice 2, 4, 8, and 12 h following the final 12-h scheduled feeding of diets designed to provide either 1) ample protein, or 2) limited protein plus supplemental urea as the major nitrogen source. Concentrations and total quantities of total N, NH3, urea plus NH3, and alpha-amino N were determined, and protein N was calculated by difference. Liquid marker (PEG) distribution rate constants and N concentrations were used to calculate rates of entry and exit by digesta flow for the large intestinal compartments. These values, together with measured accumulations of each constituent, allowed assessment of transitory net appearance and net disappearance within each compartment due to processes other than flow along the tract. The data suggested cyclic fluctuations of microbial populations within the large intestine, particularly the dorsal and ventral colon. These segments also were implicated as major sites of microbial protein synthesis and degradation. the former process appeared more evident in animals consuming the urea-supplemented diet.

Amino Acids↗
PubMedDate not supplied

Metabotropic glutamate receptor activation and intracellular cyclic ADP-ribose release Ca2+ from the same store in cultured DRG neurones.

The whole cell patch clamp technique has been used to record Ca(2+)-activated cation and chloride conductances evoked by release of Ca2+ from intracellular stores of cultured neonatal dorsal root ganglion neurones. The aim of this study was to investigate metabotropic glutamate receptor (mGluR) mechanisms and evaluate a possible role for cyclic ADP-ribose as an intracellular signalling molecule. Glutamate and the metabotropic glutamate receptor agonist (1S, 3R)-ACPD-evoked transient depolarizations, Ca(2+)-activated inward currents and rises in intracellular Ca2+. The (1S, 3R)-ACPD-activated currents were insensitive to InsP3 signalling inhibitors, heparin and pentosan polysulphate. Intracellular application of ryanodine alone activated currents in this study and proved a difficult tool to use as a potential inhibitor of cyclic ADP-ribose-mediated responses. However, intracellular dantrolene did attenuate both (1S, 3R)-ACPD and cyclic ADP-ribose responses. Intracellular photo-release of cGMP and cyclic ADP-ribose mimicked the responses to mGluR receptor activation. Intracellular application of nicotinamide and W7 inhibited the responses to photo-released cGMP but did not prevent responses to mGluR activation. The cyclic ADP-ribose receptor antagonist 8-amino cyclic ADP-ribose attenuated responses to (1S, 3R)-ACPD, cGMP and cyclic ADP-ribose, but some Ca(2+)-activated inward currents were still observed in the presence of this antagonist. In conclusion, mGluR receptor activation, cGMP and cyclic ADP-ribose release Ca2+ from intracellular stores. Some evidence suggests that pharmacologically related pathways are involved.

Adenosine Diphosphate Ribose↗