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J D Sweatt

Publications and source records attributed to J D Sweatt.

At least 55 records · Page 3Linked to original sources
PubMed1993

Nitric oxide synthase-independent long-term potentiation in area CA1 of hippocampus.

In order to elucidate the role of NO in LTP, we have investigated a biochemical effector of NO action at hippocampal synapses, guanylyl cyclase. We have observed that LTP-inducing stimuli elicit an increase in the activity of guanylyl cyclase, an effect blocked by inhibitors of NO synthase. Extracellular application of hemoglobin, which binds NO and thereby blocks its actions, also attenuated the increase in guanylyl cyclase activity. Taken together, these results provide direct biochemical evidence for an elevation of NO levels with LTP-inducing stimulation, and support the hypothesis that NO can function as a transcellular messenger in the hippocampus. These findings also implicate guanylyl cyclase as a target of NO and demonstrate that while NO is elevated with LTP-inducing stimuli, the activity of NO synthase is not necessary for induction of LTP by all stimulus paradigms.

2-Amino-5-phosphonovalerate↗
PubMed1993

nMDA receptor activation increases cyclic AMP in area CA1 of the hippocampus via calcium/calmodulin stimulation of adenylyl cyclase.

We observed previously that activation of N-methyl-D-aspartate (NMDA) receptors in area CA1 of the hippocampus, through either NMDA application or long-term potentiation (LTP)-inducing high-frequency stimulation (HFS), results in an increase in cyclic AMP. In the present study, we performed experiments to determine the mechanism by which NMDA receptor activation causes this increase in cyclic AMP. As the NMDA receptor-mediated increase in cyclic AMP is dependent upon extracellular calcium, we hypothesized that NMDA receptors are coupled to adenylyl cyclase (AC) via calcium/calmodulin. In membranes prepared from area CA1, AC was stimulated by calcium in the presence of calmodulin, and the effect of calcium/calmodulin on AC in membranes was blocked by the calmodulin antagonists N-(6-aminohexyl)-5-chloro-1- naphthalenesulfonamide (W-7) and trifluoperazine (TFP). In intact hippocampal slices, W-7 and TFP blocked the increase in cyclic AMP levels caused by both NMDA application and HFS of Schaffer collateral fibers. Exposure of hippocampal slices to elevated extracellular potassium to induce calcium influx also caused increased cyclic AMP levels; the increase in cyclic AMP caused by high potassium was also blocked by W-7 and TFP. These data support the hypothesis that NMDA receptor activation is positively coupled to AC via calcium/calmodulin and are consistent with a role for cyclic AMP metabolism in the induction of NMDA receptor-dependent LTP in area CA1 of the hippocampus.

2-Amino-5-phosphonovalerate↗
PubMed1992

Oxidation-induced persistent activation of protein kinase C in hippocampal homogenates.

Incubation of purified protein kinase C with H2O2 results in the generation of a persistently activated form of the enzyme which is no longer dependent on Ca2+ or lipid cofactors. This oxidative activation of purified protein kinase C requires added Fe2+ in the incubation medium. Treatment of the soluble fraction of hippocampal homogenates with H2O2 also leads to persistent activation of protein kinase C; however, oxidative activation of protein kinase C under these conditions does not require the addition of Fe2+. The persistently activated form of protein kinase C appears as a novel peak of activity on DE52 anion exchange columns, suggesting a modification of the charge character of the enzyme. Thus, oxidative modification of protein kinase C can result in its persistent activation, and this mechanism may constitute a pathway for physiological activation of the enzyme in the hippocampus.

Animals↗
PubMed1992

Long-term sensitization training in Aplysia leads to an increase in calreticulin, a major presynaptic calcium-binding protein.

Long-term memory for sensitization in Aplysia requires new protein and RNA synthesis. Here, we identify a late protein as calreticulin, the major Ca(2+)-binding protein of the lumen of the endoplasmic reticulum. An antiserum against Aplysia calreticulin reveals an enrichment of calreticulin immunoreactivity in presynaptic varicosities. Quantitative S1 nuclease analysis indicates that the steady-state level of calreticulin mRNA in Aplysia sensory neurons increases during the maintenance phase of long-term sensitization. The finding that this mRNA increases in expression late, some time after training, is consistent with the idea that long-term neuromodulatory changes underlying sensitization may depend on a cascade of gene expression in which the induction of early regulatory genes leads to the expression of late effector genes.

Amino Acid Sequence↗
PubMed1992

Amygdala kindling alters protein kinase C activity in dentate gyrus.

Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.

Amygdala↗
PubMed1992

Increased phosphorylation of a 17-kDa protein kinase C substrate (P17) in long-term potentiation.

Hippocampal long-term potentiation (LTP) is a persistent increase in the efficacy of synaptic transmission, which is widely thought to be a cellular mechanism that could contribute to learning and memory. Studies on the biochemical mechanisms underlying LTP suggest the involvement of protein kinases in both LTP induction and maintenance. In this report we describe an LTP-associated increase in the phosphorylation in vitro of a 17-kDa protein kinase C (PKC) substrate protein, which we have termed P17, in homogenates from the CA1 region of rat hippocampal slices. This LTP-associated increase in phosphorylation was expressed independent of significant levels of free Ca2+, as phosphorylation reactions were performed in the presence of 500 microM EGTA. The increased phosphorylation of P17 was substantially inhibited by PKC(19-36), a selective inhibitor of PKC. These data support the model that persistent PKC activation contributes to the maintenance of LTP and implicate P17 as a potential target for PKC in the CA1 region of the hippocampus.

Animals↗
PubMed1991

Persistent protein kinase activation in the maintenance phase of long-term potentiation.

Long-term potentiation (LTP) of synaptic transmission in the hippocampus is a robust form of synaptic plasticity that may contribute to mammalian memory formation. A variety of pharmacological evidence suggests that persistent kinase activation contributes to the maintenance of LTP. To determine whether persistent activation of protein kinases was associated with the maintenance phase of LTP, protein kinase activity was measured in control and LTP samples using exogenous protein kinase substrates in an in vitro assay of homogenates of the CA1 region of rat hippocampal slices. After LTP, protein kinase activity was persistently increased, and the induction of this effect was blocked by the N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonovaleric acid. The increased protein kinase activity was found to be significantly attenuated by PKC(19-36), a selective peptide inhibitor of protein kinase C. Thus, LTP is associated with an N-methyl-D-aspartate receptor-mediated generation of a persistently activated form of protein kinase C. These data lend strong support to the model that persistent protein kinase activation contributes to the maintenance of LTP.

2-Amino-5-phosphonovalerate↗
PubMed1991

N-methyl-D-aspartate receptor activation increases cAMP levels and voltage-gated Ca2+ channel activity in area CA1 of hippocampus.

Tetanic stimulation of the Schaffer collateral inputs into area CA1 of the hippocampus causes N-methyl-D-aspartate (NMDA) receptor activation, an effect that contributes to the induction of long-term potentiation (LTP) in this region. The present studies demonstrate that LTP-inducing tetanic stimulation in rat hippocampal area CA1 elicited increased levels of cAMP. The elevation of cAMP was blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV). Bath application of NMDA also resulted in an increase in cAMP in CA1, an effect that was blocked by both APV and removal of extracellular Ca2+. These findings suggest that activation of NMDA receptors elicits a Ca(2+)-dependent increase in cAMP, and taken together with the data from tetanic stimulation, suggest that NMDA-receptor-mediated increases in cAMP could play a role in the induction of LTP in area CA1. One role for cAMP may be to increase Ca2+ influx through voltage-gated Ca2+ channels, as it was observed that application of either 8-bromo-cAMP or NMDA increased the fractional open time of high-threshold Ca2+ channels in CA1 pyramidal cells. Our results raise the possibility that a positive-feedback loop for Ca2+ influx in area CA1 exists. In this model, NMDA receptor-mediated Ca2+ influx leads to an enhancement of further Ca2+ influx via intermediate steps of increased cAMP and subsequent increased voltage-gated Ca2+ channel activity.

2-Amino-5-phosphonovalerate↗
PubMed1990

Protein synthesis during acquisition of long-term facilitation is needed for the persistent loss of regulatory subunits of the Aplysia cAMP-dependent protein kinase.

Depending on the number or the length of exposure, application of serotonin can produce either short-term or long-term presynaptic facilitation of Aplysia sensory-to-motor synapses. The cAMP-dependent protein kinase, a heterodimer of two regulatory and two catalytic subunits, has been shown to become stably activated only during long-term facilitation. Both acquisition of long-term facilitation and persistent activation of the kinase is blocked by anisomycin, an effective, reversible, and specific inhibitor of protein synthesis in Aplysia. We report here that 2-hr exposure of pleural sensory cells to serotonin lowers the concentration of regulatory subunits but does not change the concentration of catalytic subunits, as assayed 24 hr later; 5-min exposure to serotonin has no effect on either type of subunit. Increasing intracellular cAMP with a permeable analog of cAMP together with the phosphodiesterase inhibitor isobutyl methylxanthine also decreased regulatory subunits, suggesting that cAMP is the second messenger mediating serotonin action. Anisomycin blocked the loss of regulatory subunits only when applied with serotonin; application after the 2-hr treatment with serotonin had no effect. In the Aplysia accessory radula contractor muscle, prolonged exposure to serotonin or to the peptide transmitter small cardioactive peptide B, both of which produce large increases in intracellular cAMP, does not decrease regulatory subunits. This mechanism of regulating the cAMP-dependent protein kinase therefore may be specific to the nervous system. We conclude that during long-term facilitation, new protein is synthesized in response to the facilitatory stimulus, which changes the ratio of subunits of the cAMP-dependent protein kinase. This alteration in ratio could persistently activate the kinase and produce the persistent phosphorylation seen in long-term facilitated sensory cells.

1-Methyl-3-isobutylxanthine↗
PubMed1989

FMRFamide reverses protein phosphorylation produced by 5-HT and cAMP in Aplysia sensory neurons.

Neurotransmitter can modulate neuronal activity through a variety of second messengers that act on ion channels and other substrate proteins. The most commonly described effector mechanism for second messengers in neurons depends on protein phosphorylation mediated by one of three sets of kinases: the cyclic AMP-dependent protein kinases, the Ca2+-calmodulin-dependent protein kinases, and the Ca2+-phospholipid-dependent protein kinases. In addition, some neurotransmitters and second messengers can also inhibit protein phosphorylation by lowering cAMP levels (either by inhibiting adenylyl cyclase or activating phosphodiesterases). This raises the question: can neurotransmitters also modulate neuronal activity by decreasing protein phosphorylation that is independent of cAMP? Various biochemical experiments show that a decrease in protein phosphorylation can arise through activation of a phosphatase or inhibition of kinases. In none of these cases, however, is the physiological role for the decrease in protein phosphorylation known. Here we report that in Aplysia sensory neurons, the presynaptic inhibitory transmitter FMRFamide decreases the resting levels of protein phosphorylation without altering the level of cAMP. Furthermore, FMRFamide overrides the cAMP-mediated enhancement of transmitter release produced by 5-hydroxytryptamine (5-HT), and concomitantly reverses the cAMP-dependent increase in protein phosphorylation produced by 5-HT. These findings indicate that a receptor-mediated decrease in protein phosphorylation may play an important part in the modulation of neurotransmitter release.

Animals↗
PubMed1989

Persistent and transcriptionally-dependent increase in protein phosphorylation in long-term facilitation of Aplysia sensory neurons.

From certain perspectives, short- and long-term memory seem to be a single behavioural process whose duration is a graded function of the number of training trials. Yet some clinical conditions can dissociate short- from long-term memory in human beings, and inhibitors of protein or RNA synthesis can selectively block the long-term process in experimental animals. Studies of memory for sensitization in the gill- and siphon-withdrawal reflex in Aplysia indicate that both the behavioural similarities and the differences are reflected in intrinsic cellular mechanisms in the sensory and motor neurons participating in memory storage. Although the long-term change in the synaptic connection between the sensory and motor neurons resembles a graded extension of the short-term change, its induction is selectively blocked by inhibitors of transcription or translation. We have now examined the molecular mechanisms in the sensory neurons that might account for the graded similarity between short- and long-term memory, as well as those that might contribute to the differential sensitivity to inhibitors of macromolecular synthesis. We find that a single exposure to 5-HT (a transmitter released in response to behavioural sensitizing stimuli) or cyclic AMP (a second messenger for 5-HT), which produce short-term facilitation between the sensory and motor neurons lasting minutes, leads to a short-term phosphorylation of 17 substrate proteins that is not dependent on transcription or translation. Repeated or prolonged exposure to serotonin or cAMP, which induce long-term changes in synaptic transmission lasting one or more days, induce long-term changes in phosphorylation of the same 17 proteins that are now dependent for their induction on both translation and transcription. Thus, one of the functions of the genes and proteins required for long-term facilitation may be to maintain actively in the sensory neurons an increased phosphorylation of the same set of substrate proteins involved in eliciting the physiological effects of the short-term process.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Development of a database of amino acid sequences for proteins identified and isolated on two-dimensional polyacrylamide gels.

As part of our continuing studies into the biochemical basis of long-term changes in neuronal function in Aplysia, we have developed a simple method for obtaining amino acid sequence information from proteins isolated on two-dimensional gels. Proteins isolated on preparative two-dimensional gels are digested in situ with Staphylococcus aureus V8 protease, and the resulting peptides electrophoresed, transferred to a polyvinylidene difluoride membrane, and sequenced in a gas-phase sequencer. The method is simple and should be applicable to a variety of other systems where the development of a two-dimensional gel database is underway.

Amino Acid Sequence↗
PubMed1989

5-HT modulates protein synthesis and the expression of specific proteins during long-term facilitation in Aplysia sensory neurons.

Long-term, but not short-term, facilitation induced by 5-HT in Aplysia sensory neurons is blocked by application of inhibitors of protein synthesis or RNA synthesis during the 1.5 hr training period. These findings suggest that genes and proteins not needed for the short-term process are required for long-term facilitation. To identify these proteins, we examined changes in overall and specific protein synthesis in sensory neurons. During the 1.5 hr period of stimulation, 5-HT initiated three temporally distinct changes in total protein synthesis: a small decrease at 0.5 hr, a small increase at 1 hr, and a second and larger increase at 3 hr. 5-HT also produced three temporally distinct changes in the synthesis of individual proteins that could be resolved on 2-D gels. First, there was an increase at 30 min in the rate of synthesis of 10 proteins and a decrease in the synthesis of 5 proteins. These changes in protein expression are rapid and transient, subsiding within 1-3 hr, and are transcription-dependent. Second, at 3 hr, there is a transient increase in 4 different proteins that also is transcription-dependent and lasts approximately 5 hr. Finally, at 24 hr, during the retention phase of facilitation, there was a sustained increase in the expression of two proteins.

Animals↗
PubMed1988

Sequencing proteins from acrylamide gels.

The analytical power of high-resolution two-dimensional gel electrophoresis has been coupled with molecular cloning techniques to allow the sequencing of proteins directly from preparative gels.

Amino Acid Sequence↗
PubMed1988

Sequencing of proteins from two-dimensional gels by using in situ digestion and transfer of peptides to polyvinylidene difluoride membranes: application to proteins associated with sensitization in Aplysia.

We have developed a method for obtaining partial internal amino acid sequence data from proteins isolated directly from preparative two-dimensional polyacrylamide gels. Proteins from a crude cell homogenate are separated using preparative two-dimensional polyacrylamide gel electrophoresis. Then, the gel is stained with Coomassie blue and the protein spots of interest are cut out. The in situ protein is digested with Staphylococcus aureus V8 protease in a second polyacrylamide gel and the peptides are separated by one-dimensional polyacrylamide gel electrophoresis. The peptides are then electroblotted onto a polyvinylidene difluoride membrane, visualized using Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Using this method, we have obtained amino acid sequence data for two proteins that are altered after long-term sensitization: actin and Aplysia protein 407. In addition, we have obtained amino acid sequence data for rat protein 425, a protein that appears to be homologous to Aplysia protein 407.

Actins↗
PubMed1987

Epidermal growth factor (EGF) stimulates inositol trisphosphate formation in cells which overexpress the EGF receptor.

EGF is a low molecular weight polypeptide hormone which acts as a regulator of cell growth and differentiation. The A-431 cell line has been used frequently to examine receptor-mediated biochemical effects of EGF, since this cell line has an increased (20-50 fold) level of EGF receptors. We have utilized A-431 cells to examine the influence of EGF on formation of an intracellular second messenger, inositol, 1,4,5-trisphosphate (Ins-1,4,5-P3), and other inositol phosphates. The results show that EGF induces rapid formation of Ins-1,4,5-P3 as well as Ins-1,3,4-P3 and Ins-1,3,4,5-P4. There is a concurrent decrease in the level of the lipid precursor for Ins-1,4,5-P3, phosphatidylinositol 4,5-biphosphate (PIP2). Furthermore, we have examined five other cell lines that overexpress the EGF receptor and find that EGF treatment induces formation of inositol polyphosphates in those cell lines also.

Calcium↗
PubMed1987

Evidence for a role for Na+-H+ exchange in activation of human platelets by PAF.

We have found previously that inhibitors of Na+-H+ exchange block platelet arachidonic acid release and subsequent secondary aggregation and serotonin release in response to epinephrine, ADP, and thrombin (0.004 U/ml). The present study demonstrates that the addition of ethylisopropylamiloride, an inhibitor of Na+-H+ exchange, leads to an inhibition of platelet activating factor-induced serotonin release and thromboxane B2 production in human platelets in citrated plasma. In addition, platelet activating factor-induced platelet secretion is blocked by the cyclooxygenase inhibitor indomethacin or the thromboxane antagonist SQ 29548, indicating that arachidonic acid mobilization and metabolism is required for platelet activating factor to elicit platelet activation. Our interpretation of the present findings is that platelet activating factor-induced secretion of dense granules from the human platelet requires the production of cyclooxygenase metabolites from arachidonic acid and that Na+-H+ exchange plays an important, albeit not exclusive, role in mobilization of arachidonic acid in response to platelet activating factor.

Amiloride↗