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J P Walsh

Publications and source records attributed to J P Walsh.

At least 19 recordsLinked to original sources
PubMed1995

Arachidonoyl-diacylglycerol kinase. Specific in vitro inhibition by polyphosphoinositides suggests a mechanism for regulation of phosphatidylinositol biosynthesis.

We previously described the purification of a membrane-bound diacylglycerol kinase highly selective for sn-1-acyl-2-arachidonoyl diacylglycerols (Walsh, J. P., Suen, R., Lemaitre, R. N., and Glomset, J. A. (1994) J. Biol. Chem. 269, 21155-21164). This enzyme appears to be responsible for the rapid clearance of the arachidonate-rich pool of diacylglycerols generated during stimulus-induced phosphoinositide turnover. We have now shown phosphatidylinositol 4,5-bisphosphate to be a potent and specific inhibitor of arachidonoyl-diacylglycerol kinase. Kinetic analyses indicated a Ki for phosphatidylinositol 4,5-bisphosphate of 0.04 mol %. Phosphatidic acid also was an inhibitor with a Ki of 0.7 mol %. Other phospholipids had only small effects at these concentrations. A series of multiply phosphorylated lipid analogs also inhibited the enzyme, indicating that the head group phosphomonoesters are the primary determinants of the polyphosphoinositide effect. However, these compounds were not as potent as phosphatidylinositol 4,5-bisphosphate, indicating some specificity for the polyphosphoinositide additional to its total charge. Five other diacylglycerol kinases were activated to varying degrees by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, suggesting that inhibition by acidic lipids may be specific for the arachidonoyl-DAG kinase isoform. Given the presumed role of arachidonoyl-diacylglycerol kinase in the phosphoinositide cycle, this inhibition may represent a mechanism for polyphosphoinositides to regulate their own synthesis.

3T3 Cells↗
PubMed1994

Arachidonoyl-diacylglycerol kinase from bovine testis. Purification and properties.

Previous work in our laboratory demonstrated the existence of a membrane-bound diacylglycerol kinase highly selective for diacylglycerols containing arachidonate as the sn-2 fatty acyl moiety (MacDonald, M. L., Mack K. F., Richardson, C. N., and Glomset, J. A. (1988) J. Biol. Chem. 263, 1575-1583). We now report the purification of arachidonoyl-diacylglycerol kinase 34,400-fold to apparent homogeneity from bovine testis. High concentrations of both salt and detergent were required to extract the enzyme from membranes and stabilize its activity, suggesting that in vivo the enzyme is part of a complex with other membrane or cytoskeletal proteins. Arachidonoyl-diacylglycerol kinase had an apparent M(r) of 58,000 both on SDS-polyacrylamide gels and by size exclusion chromatography. The enzyme appeared to be an integral membrane protein. In a mixed micellar assay, arachidonoyl-diacylglycerol kinase followed surface dilution kinetics with respect to diacylglycerol. The purified enzyme retained the arachidonate selectivity observed previously in membranes. Kinetic analyses indicated a Km for sn-1-stearoyl-2-arachidonoylglycerol of 2.4 mol %, as compared to 43 mol % for sn-1-palmitoyl-2-oleoylglycerol. Calcium, an activator of some other diacylglycerol kinases, had no apparent effect on the arachidonate-specific enzyme. Guanosine triphosphate could effectively substitute for ATP as the phosphoryl donor and Mg2+ could be replaced by Mn2+ or Ca2+. Phosphatidylserine and, to a lesser extent, phosphatidylinositol inhibited the purified enzyme. Phosphatidylcholine and phosphatidylethanolamine had only small effects.

3T3 Cells↗
PubMed1994

Loss of paired-pulse facilitation at the corticostriatal synapse of the aged rat.

Changes in calcium (Ca2+) homeostasis have been proposed to contribute to the aging process. Paired-pulse facilitation, a form of synaptic enhancement that relies upon an accumulation of Ca2+ in the presynaptic terminal, was used to examine the effect of aging at the corticostriatal synapse. Intracellular recordings in striatal neurons from young rats demonstrated a consistent enhancement in the second of two synaptic responses evoked by stimulation of the corpus callosum. In contrast, neurons from aged rats showed a consistent depression of the second synaptic response at identical pairing intervals. These differences were not explained by an age-dependent increase in synaptic depression and demonstrate an alteration in the Ca(2+)-mediated process of presynaptic facilitation.

Aging↗
PubMed1993

Evidence for enhanced synaptic excitation in transplanted neostriatal neurons.

Fetal neostriatal tissue was transplanted into either the neostriatum or substantia nigra of adult rats. One to 6 months after transplantation, coronal brain slices were taken through the rostrocaudal extent of the transplants and neurons were characterized electrophysiologically using an in vitro slice preparation. When compared to control neurons taken from intact adult neostriata, transplanted neostriatal neurons (TSNs) had higher input resistances and longer time constants. All other passive and active membrane properties assessed were comparable between transplanted and control neostriatal neurons. Regardless of the transplantation site, local extracellular stimulation outside the graft elicited high-amplitude, long-duration depolarizing synaptic potentials that typically triggered bursts of action potentials. These synaptic potentials contrast with lower amplitude, shorter duration synaptic potentials consistently elicited in control neostriatal neurons. The depolarizing synaptic potentials evoked in the TSNs appeared to be mediated by a combined activation of N-methyl-D-aspartate (NMDA) and non-NMDA excitatory amino acid receptors. Both the broad-spectrum excitatory amino acid antagonist kynurenic acid and the specific non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione significantly reduced postsynaptic potentials elicited in TSNs. The specific NMDA antagonist 2-amino-5-phosphonovalerate had less effect on the amplitude but markedly reduced the duration of the synaptic potentials. The duration and amplitude of the bursts were augmented by the gamma-aminobutyric acid (GABA)A receptor antagonist bicuculline methiodide, indicating that inhibition occurred in TSNs. TSNs were also more sensitive than control neurons to direct application of glutamate or NMDA. These findings demonstrate that TSNs express altered electrophysiological properties. The pharmacological analysis indicates that depolarizing postsynaptic potentials were mediated by activation of excitatory amino acid receptors, suggesting either innervation of the graft by host fibers which contain excitatory amino acids or development of novel local excitatory interactions intrinsic to the graft. Furthermore, the occurrence of high-amplitude, long-duration depolarizing synaptic potentials in TSNs, regardless of the site of transplantation, suggests that grafted neostriatal neurons become hyperexcitable to synaptic input.

2-Amino-5-phosphonovalerate↗
PubMed1993

Synaptic activation of N-methyl-D-aspartate receptors induces short-term potentiation at excitatory synapses in the striatum of the rat.

We examined the effect of augmenting the activation of N-methyl-D-aspartate receptors during tetanic stimulation of the corpus callosum. Excitatory postsynaptic potentials were recorded from striatal neurons in an in vitro brain slice bathed in either normal or Mg(2+)-free artificial cerebrospinal fluid. Post-tetanic potentiation was followed by a long-lasting depression of the excitatory postsynaptic potential in normal artificial cerebral spinal fluid. Mg(2+)-free artificial cerebrospinal fluid increased the duration of the pretetanus excitatory postsynaptic potential and the amplitude and duration of the direct response to the tetanus. Mg(2+)-free artificial cerebrospinal fluid also enabled the expression of a short-term potentiation of the excitatory postsynaptic potential amplitude and duration. N-methyl-D-aspartate receptor antagonists blocked the induction of all tetanus-induced excitatory postsynaptic potential changes specific to the Mg(2+)-free artificial cerebral spinal fluid. These results indicate that maintained activation of N-methyl-D-aspartate receptors by synaptically released glutamate can produce a sustained enhancement of the excitatory postsynaptic potential. This use-dependent increase in synaptic efficacy may contribute to basal ganglia-related motor performance.

Animals↗
PubMed1992

Awareness of heart attack signals and cardiac risk markers amongst the general public in Dublin.

Following myocardial infarction, early access to medical care is essential. In order to assess the ability to recognise and manage heart attack and its risk factors, 302 members of the public were surveyed. A surprisingly comprehensive knowledge base was revealed, together with apparently appropriate ideas about the management of myocardial infarction and its risk factors. The significance of this finding is discussed in the light of the well documented delays by members of the public in summoning help when they suspect heart attack.

Adult↗
PubMed1991

Formation of phosphatidylinositol 3-phosphate by isomerization from phosphatidylinositol 4-phosphate.

We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using diisopropylcarbodiimide to promote migration of the 4-phosphate via a cyclic phosphodiester intermediate. The product was isolated by a thin-layer chromatographic method that depends on the ability of phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate, to form complexes with boric acid. The final yield of the procedure was 8% phosphatidylinositol 3-phosphate, which was approximately 80% pure. The product was shown to be phosphatidylinositol 3-phosphate by the following criteria: (i) cochromatography with an authentic standard on borate thin-layer chromatography, (ii) cochromatography of the deacylated product with glycerophosphoinositol 3-phosphate on high-performance liquid chromatography, (iii) conversion of the product to phosphatidylinositol by homogeneous phosphatidylinositol 3-phosphate 3-phosphatase, and (iv) deacylation and deglyceration of the product to a compound that comigrates with inositol 1,3-bisphosphate on high-performance liquid chromatography. The availability of mass amounts of phosphatidylinositol 3-phosphate will allow further elaboration of reactions in this recently discovered pathway of phosphatidylinositol metabolism.

Autoradiography↗
PubMed1991

Neurophysiological maturation of cat caudate neurons: evidence from in vitro studies.

The membrane properties and synaptic physiology of developing cat caudate (Cd) nucleus neurons were studied in in vitro slice preparations. Recordings were obtained from 98 cells in kittens from fetal day (F) 56 to postnatal day (P) 90. With increasing age, the following maturational changes occurred; resting membrane potentials became more negative, action potential rise times decreased, action potential amplitudes increased, and action potential durations and input resistances decreased. The frequency of occurrence of afterhyperpolarizations and of anomalous rectification increased with age. The primary response to local extracellular stimulation was a depolarization usually accompanied by an action potential. Evoked hyperpolarizing responses were seen after P28 but only occurred if the membrane was depolarized by intracellular current injection. Cells identified by intracellular injection of Lucifer yellow were primarily medium-sized spiny neurons although it was not always possible to determine the cell type in slices from animals less than P5. Somatic diameter, dendritic length, and spine density increased with age. Dye-coupling occurred in slices less than P20. Its frequency decreased with age. These results show that Cd neurons undergo significant maturation during late prenatal and early postnatal periods. In contrast, substantia nigra neurons mature more rapidly and should be capable of influencing the less mature Cd neurons during development.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed1991

Neurophysiological maturation of cat substantia nigra neurons: evidence from in vitro studies.

The membrane properties and synaptic physiology of developing cat substantia nigra (SN) neurons were studied in in vitro slice preparations. Stable intracellular recordings were obtained from 46 neurons in 20 kittens ranging in age from fetal day (F) 51 to postnatal day (P) 120. Only two of these properties changed with development. The percentage of cells displaying inward rectification and the percentage of cells that generated low-threshold Ca++ spikes increased with age. Properties that did not change included resting membrane potentials, action potential amplitudes and durations, and input resistances. At all ages locally evoked synaptic responses consisted of sequences of excitatory postsynaptic potentials followed by inhibitory postsynaptic potentials. Most of the cells recorded had the electrophysiological properties which have been attributed to SN dopamine-containing neurons. To identify neurons morphologically, and verify the recording site, cells were filled with Lucifer yellow at the end of each experiment. Somatic shapes varied widely from oval to fusiform to triangular. Somatic diameters and dendritic length increased with development. Filopodial processes and growth cones were present up to the first postnatal month. Dye-coupling occurred only in the fetal group. These results indicate that cat SN neurons have many mature physiological properties during late fetal and early postnatal development. This contrasts with the significant maturation that occurs in cat caudate neurons during the same developmental period.

Action Potentials↗
PubMed1990

sn-1,2-diacylglycerol kinase of Escherichia coli. Diacylglycerol analogues define specificity and mechanism.

A detailed structure/function analysis of the substrate specificity of Escherichia coli sn-1,2-diacylglycerol kinase was performed with three goals in mind: (a) to define the substrate specificity; (b) to discover inhibitors; and (c) to elucidate the specificity of diacylglycerol-dependent inactivation. Forty-seven structural analogues of sn-1,2-diacylglycerol were prepared and examined as substrates, inhibitors, and irreversible inactivators of the enzyme using mixed micellar assay methods. Modification of the acyl chains or the sn-2 ester affected the apparent Km but had only small effects on Vm; modifications of the sn-1 ester, sn-3 methylene, or sn-3 hydroxyl had large effects on the apparent Vm and smaller effects on Km. Consistent with these observations, diacylglycerol analogues modified only in the acyl chains or sn-2 ester were not diacylglycerol kinase inhibitors, whereas analogues with substitutions of the sn-1 ester or sn-3 hydroxyl frequently caused inhibition. A hydrogen bond-donating group was required for an analogue to be a diacylglycerol kinase inhibitor. Studies of diacylglycerol kinase inactivation by the various analogues were consistent with the previous conclusion that this process involves an interaction of diacylglycerols with an enzyme conformation different from that active in catalysis (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 15062-15069). Studies with a water-soluble diacylglycerol, sn-1,2-dibutyrylglycerol, allowed direct comparison of diacylglycerol kinase activity in mixed micelles with that in native membranes. The results are discussed in relation to the structural requirements of other diacylglycerol-dependent enzymes.

Diacylglycerol Kinase↗
PubMed1990

Synaptic transmission in human neocortex removed for treatment of intractable epilepsy in children.

Synaptic transmission to pyramidal cells was studied in slices of neocortex resected from infants and children (n = 10, age 8 months to 13 years) undergoing surgical treatment for intractable epilepsy. Most specimens were from the least abnormal area of the resection. Stable intracellular recordings could be obtained for up to 8 hours. Most of the recorded neurons had electrophysiological characteristics similar to those of regular-firing pyramidal cells and were in layers III to V, which was confirmed by intracellular staining with Lucifer yellow. Local extracellular stimulation evoked a sequence of excitatory and inhibitory postsynaptic potentials. After application of the gamma-aminobutyric acid antagonist, bicuculline (10-30 microM), extracellular stimulation induced large excitatory postsynaptic potentials and epileptiform bursts. Spontaneous bursts occasionally occurred in bicuculline. This effect of bicuculline was observed in all the tissue samples, even those from infant patients (n = 4, age 8-16 months). Kynurenic acid depressed or abolished both spontaneous and stimulation-induced bursts. The competitive antagonist for N-methyl-D-aspartate receptors, DL-2-amino-5-phosphonopentanoic acid decreased the duration of bicuculline-induced bursts. These data provide evidence that, similar to rat and cat neocortex, excitatory and inhibitory amino acids are important transmitters to pyramidal cells in immature human neocortex.

2-Amino-5-phosphonovalerate↗
PubMed1989

Intracellular neurophysiological analysis reveals alterations in excitation in striatal neurons in aged rats.

Intracellular recordings were used to characterize the physiological changes underlying decreases in excitation observed in striatal neurons during the aging process. Rats were divided into 3 age groups: young (3-5 months), middle-aged (10-12 months) and aged (greater than 24 months). All experiments were performed in urethane-anesthetized rats. Recordings were obtained from 33 neurons in young, 17 in middle-aged and 20 in aged rats. When identified by intracellular injections of Lucifer yellow all recorded neurons were medium-sized spiny cells. Resting membrane potentials were at least -40 mV and action potentials greater than 35 mV. Postsynaptic responses were evoked by stimulation of frontal cortex. In all recorded neurons, regardless of age, excitatory postsynaptic potentials (EPSPs) could be evoked. However, the threshold currents for eliciting both EPSPs and synaptically driven action potentials were significantly higher in neurons obtained from aged rats than those recorded in the other two groups. Other changes in excitation in aged striatal neurons consisted of absence of spontaneously occurring EPSPs, higher current to induce firing by intracellular injections of depolarizing current and an inability of orthodromically induced action potentials to follow paired stimulation pulses to the cortex at short interpulse intervals. These data were interpreted to indicate that a combination of changes in synaptic connectivity and in membrane properties underlie the decreases in excitation. Together with our previous findings obtained from aged cats these results indicate that decreased neuronal excitability is a major effect of aging in the striatum.

Action Potentials↗
PubMed1989

Kynurenic acid antagonizes the excitatory postsynaptic potential elicited in neostriatal neurons in the in vitro slice of the rat.

Stimulation of corpus callosum in rat brain slices evoked an excitatory postsynaptic potential (EPSP) in neostriatal neurons. This EPSP is greatly reduced by exposing the slice to kynurenic acid (KY). The action of KY is reversed when the preparation is bathed in normal Ringer's solution. KY reduces the EPSP amplitude in a dose-dependent fashion, with 1 mM KY attenuating the potential by 86%. The effect is not due to a conductance change, since KY treatment does not alter the neuron's input resistance.

Action Potentials↗
PubMed1989

Dye-coupling in the neostriatum of the rat: I. Modulation by dopamine-depleting lesions.

Evidence from experiments performed in turtle and fish retina suggests that dopamine (DA) modulates the permeability of gap junctions. The present experiment was aimed at determining if DA has a similar role in the mammalian neostriatum. Adults rats received one of four treatments: unilateral electrolytic substantia nigra lesions, unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra, unilateral neocortical aspiration, or no treatment. After 3-5 weeks, neostriata from both sides of the brain were prepared for in vitro intracellular recordings. Recorded neurons (N approximately 150) were filled with Lucifer Yellow (LY), a low molecular weight dye that crosses gap junctions. In animals with electrolytic nigral lesions, dye-coupling in the ipsilateral neostriatum occurred after 38% of the intracellular injections. After 6-OHDA lesions, 19% of the injections produced dye-coupling in the ipsilateral neostriatum. This difference may have been accounted for by the fact that electrolytic lesions produced a greater degree of DA loss than 6-OHDA injections. Both of these percentages contrast with the very small percentage of dye-coupling found in intact animals or in animals with neocortical lesions. Dye-coupling occurred only between medium-sized spiny cells. No morphological differences between dye-coupled and non-dye-coupled cells were observed with light microscopy. Overall, passive and active electrophysiological properties of dye-coupled and single neurons were similar. The results suggest that DA may function in the neostriatum to control permeability of gap junctions.

Action Potentials↗
PubMed1989

Dye-coupling in the neostriatum of the rat: II. Decreased coupling between neurons during development.

Physiological and morphological evidence for coupling between neostriatal neurons was obtained from the developing rat. Intracellular injections of Lucifer Yellow-CH (LY) were made in rat neostriatal slices to study dye transfer (coupling) between neurons. The incidence of interneuronal coupling was 70% in early postnatal (P) periods and declined gradually to 10% in the adult. The number of neurons filled by a single intracellular injection also declined with age. LY injection into single neurons commonly marked aggregates of 4 to 6 cells in neonates. Single injections never produced more than one coupled pair in P20 or older rats. Neurons in which fast prepotentials (FPPs) could be evoked were consistently found to be dye-coupled. FPPs were resistant to collision with action potentials generated by intracellular current injection. When chemical synaptic transmission was blocked Mn2+, short-latency depolarizations (SLDs) could be evoked by extracellular stimulation. The SLDs were distinguished from chemical synaptic potentials by their "all or none" nature and by their insensitivity to changes in membrane potential. No SLDs were observed in adult neurons. FPPs and SLDs may be indicators of electronic transmission between coupled cells. The high incidence of coupling early in development might reflect intercellular communication that contributes to the differentiation and growth of neostriatal neurons.

Action Potentials↗
PubMed1989

Modulation of a steady-state Ca2+-activated, K+ current in tail sensory neurons of Aplysia: role of serotonin and cAMP.

1. The modulation of membrane currents by serotonin (5-HT) was studied in isolated clusters of tail sensory neurons. Serotonin was applied by micropressure ejection onto the somata of sensory neurons voltage-clamped at fixed holding potentials. The range of holding potentials tested in this study was selected to produce a steady-state Ca2+-activated K+ current (IK,Ca). Serotonin induced an inward shift in the holding current associated with a decrease in slope conductance. 2. Intracellular injection of adenosine 3',5'-cyclic monophosphate (cAMP) mimicked the response to 5-HT and induced an inward current associated with a decrease in slope conductance. The responses to 5-HT and cAMP had similar voltage dependencies and both responses were due to an apparent decrease in K+ current. Responses to cAMP were markedly reduced when generated at the peak of a response to 5-HT. The nonsummation of the maximal current responses indicated that 5-HT and cAMP utilize a common, saturable mechanism. 3. In contrast to the consistent decrease in steady-state K+ conductance elicited by cAMP, injection of guanosine 3',5'-cyclic monophosphate (cGMP) produced variable responses. In most cells, cGMP induced outward shifts in holding current that were associated with an increase in slope conductance. 4. Several lines of evidence indicated that IK,Ca contributed to the holding current at the level of membrane potentials that were examined. Inward shifts in holding current associated with a decrease in slope conductance were produced in the presence of agents that block Ca2+ channels, such as Co2+, Cd2+ or Ni2+ and by replacement of extracellular Ca2+ with Ba2+. Reducing the concentration of cytoplasmic Ca2+ through intracellular injection of EGTA had similar effects. Furthermore, inward shifts in holding current were produced by 5 mM tetraethylammonium chloride (TEA), which is known to block IK,Ca in neurons of Aplysia. This concentration of TEA also attenuated the outward current produced in response to direct intracellular injection of Ca2+. 5. Serotonin appears to modulate the IK,Ca that contributes to the steady-state holding current. The same manipulations that block the steady-state IK,Ca (see above) also attenuated the response to 5-HT. Furthermore, K+ currents activated by intracellular injection of Ca2+ were attenuated by 5-HT. 6. These results indicate that the changes in holding current produced by 5-HT are mediated, at least in part, by cAMP. In addition, it appears that 5-HT modulates a steady-state calcium-activated K+ current in addition to the previously described S-current (40, 58) and delayed K+ current (8, 9).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗