RESUMEN
Our previous work showed that nerve growth factor (NGF) increased the excitability of small-diameter capsaicin-sensitive sensory neurons by activating the p75 neurotrophin receptor and releasing sphingolipid-derived second messengers. Whole cell patch-clamp recordings were used to establish the signaling pathways whereby NGF augments action potential (AP) firing (i.e., sensitization). Inhibition of MEK1/2 (PD-98059), PLC (U-73122, neomycin), or conventional/novel isoforms of PKC (bisindolylmaleimide I) had no effect on the sensitization produced by NGF. Pretreatment with a membrane-permeable, myristoylated pseudosubstrate inhibitor of atypical PKCs (aPKCs: PKMζ, PKCζ, and PKCλ/ι) blocked the NGF-induced increase in AP firing. Inhibitors of phosphatidylinositol 3-kinase (PI3K) also blocked the sensitization produced by NGF. Isolated sensory neurons were also treated with small interfering RNA (siRNA) targeted to PKCζ. Both Western blots and quantitative real-time PCR established that PKMζ, but neither full-length PKCζ nor PKCλ/ι, was significantly reduced after siRNA exposure. Treatment with these labeled siRNA prevented the NGF-induced enhancement of excitability. Furthermore, consistent with the high degree of catalytic homology for aPKCs, internal perfusion with active recombinant PKCζ or PKCι augmented excitability, recapitulating the sensitization produced by NGF. Internal perfusion with recombinant PKCζ suppressed the total potassium current and enhanced the tetrodotoxin-resistant sodium current. Pretreatment with the myristoylated pseudosubstrate inhibitor blocked the increased excitability produced by ceramide or internal perfusion with recombinant PKCζ. These results demonstrate that NGF leads to the activation of PKMζ that ultimately enhances the capacity of small-diameter capsaicin-sensitive sensory neurons to fire APs through a PI3K-dependent signaling cascade.
Asunto(s)
Potenciales de Acción/fisiología , Factor de Crecimiento Nervioso/farmacología , Proteína Quinasa C/metabolismo , Células Receptoras Sensoriales/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Activación Enzimática/efectos de los fármacos , Masculino , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacosRESUMEN
PKC-theta (PKC-θ), a member of the novel protein kinase C family (nPKC), regulates a wide variety of functions in the periphery. However, its presence and role in the CNS has remained largely unknown. Recently, we demonstrated the presence of PKC-θ in the arcuate hypothalamic nucleus (ARC) and knockdown of PKC-θ from the ARC protected mice from developing diet-induced obesity. Another isoform of the nPKC group, PKC-delta (PKC-δ), is expressed in several non-hypothalamic brain sites including the thalamus and hippocampus. Although PKC-δ has been implicated in regulating hypothalamic glucose homeostasis, its distribution in the hypothalamus has not previously been described. In the current study, we used immunohistochemistry to examine the distribution of PKC-θ and -δ immunoreactivity in rat and mouse hypothalamus. We found PKC-θ immunoreactive neurons in several hypothalamic nuclei including the ARC, lateral hypothalamic area, perifornical area and tuberomammillary nucleus. PKC-δ immunoreactive neurons were found in the paraventricular and supraoptic nuclei. Double-label immunohistochemisty in mice expressing green fluorescent protein either with the long form of leptin receptor (LepR-b) or in orexin (ORX) neurons indicated that PKC-θ is highly colocalized in lateral hypothalamic ORX neurons but not in lateral hypothalamic LepR-b neurons. Double-label immunohistochemistry in oxytocin-enhanced yellow fluorescent protein mice or arginine vasopressin-enhanced green fluorescent protein (AVP-EGFP) transgenic rats revealed a high degree of colocalization of PKC-δ within paraventricular and supraoptic oxytocin neurons but not the vasopressinergic neurons. We conclude that PKC-θ and -δ are expressed in different hypothalamic neuronal populations.
Asunto(s)
Hipotálamo/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C-delta/metabolismo , Proteína Quinasa C/metabolismo , Animales , Arginina Vasopresina/metabolismo , Histidina Descarboxilasa/metabolismo , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Oxitocina/metabolismo , Proteína Quinasa C-theta , Ratas , Ratas Long-Evans , Receptores de Leptina/metabolismoRESUMEN
Persistent dephosphorylation has been implicated in the molecular mechanisms of long-term depression (LTD). Dephosphorylation may be due to either a persistent increase in phosphatase activity or a persistent decrease in kinase activity. We have previously found that protein kinase Mzeta (PKMzeta), the autonomously active form of the atypical PKCzeta isozyme that increases in long-term potentiation (LTP), decreases in LTD. This is consistent with the hypothesis that decreased levels of phosphorylation by PKC are important in LTD. Recently, however, increased phosphorylation by PKC has also been implicated in LTD. These contradictory results might be explained, in part, by the multiple isoforms of PKC, which may be independently regulated during the different phases of LTD. We now find that 45 s after low-frequency (3 Hz) stimulation that induces LTD in the CA1 region of hippocampal slices, conventional Ca(2+)/lipid-dependent PKC isoforms translocate from the cytosol to the membrane. This translocation was transient, lasting less than 15 min. In contrast, PKMzeta was persistently decreased through 2 h of LTD maintenance. Therefore, the activation and downregulation of distinct PKC isoforms may participate in the induction and maintenance mechanisms of LTD.
Asunto(s)
Plasticidad Neuronal , Proteína Quinasa C/metabolismo , Animales , Western Blotting , Regulación hacia Abajo , Hipocampo/enzimología , Técnicas In Vitro , Isoenzimas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Protein kinase C (PKC) is a multigene family of at least ten isoforms, nine of which are expressed in brain (alpha, betaI, betaII, gamma, delta, straightepsilon, eta, zeta, iota/lambda). Our previous studies have shown that many of these PKCs participate in synaptic plasticity in the CA1 region of the hippocampus. Multiple isoforms are transiently activated in the induction phase of long-term potentiation (LTP). In contrast, a single species, zeta, is persistently activated during the maintenance phase of LTP through the formation of an independent, constitutively active catalytic domain, protein kinase Mzeta (PKMzeta). In this study, we used immunoblot and immunocytochemical techniques with isoform-specific antisera to examine the distribution of the complete family of PKC isozymes and PKMzeta in rat brain. Each form of PKC showed a widespread distribution in the brain with a distinct regional pattern of high and low levels of expression. PKMzeta, the predominant form of PKM in brain, had high levels in hippocampus, frontal and occipital cortex, striatum, and hypothalamus. In the hippocampus, each isoform was expressed in a characteristic pattern, with zeta prominent in the CA1 stratum radiatum. These results suggest that the compartmentalization of PKC isoforms in neurons may contribute to their function, with the location of PKMzeta prominent in areas notable for long-term synaptic plasticity.
Asunto(s)
Encéfalo/enzimología , Proteína Quinasa C/metabolismo , Ratas/metabolismo , Animales , Cerebelo/enzimología , Hipocampo/enzimología , Immunoblotting , Inmunohistoquímica , Isoenzimas/metabolismo , Ratas Sprague-Dawley , Distribución TisularRESUMEN
Long-term potentiation (LTP) and long-term depression (LTD) are persistent modifications of synaptic strength that have been implicated in learning, memory, and neuronal development. Despite their opposing effects, both forms of plasticity can be triggered by the activation of NMDA receptors. One mechanism proposed for this bidirectional response is that the specific patterns of afferent stimulation producing LTP and LTD activate to different degrees a uniform receptor population. A second possibility is that these patterns activate separate receptor subpopulations composed of different NMDA receptor (NR) subunits. To test this hypothesis we examined the inhibition of LTP and LTD by a series of competitive NMDA receptor antagonists that varied in their affinities for NR2A/B and NR2C/D subunits. The potency for the inhibition of LTP compared with inhibition of LTD varied widely among the agents. Antagonists with higher affinity for NR2A/B subunits relative to NRC/D subunits showed more potent inhibition of LTP than of LTD. D-3-(2-carboxypiperazine-4-yl)-1-propenyl-1-phosphonic acid, which binds to NR2A/B with very high affinity relative to NR2C/D, showed an approximately 1000-fold higher potency for LTP than for LTD. These results show that distinct subpopulations of NMDA receptors characterized by different NR2 subunits contribute to the induction mechanisms of potentiation and depression.
Asunto(s)
Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Inhibición Neural/fisiología , Subunidades de Proteína , Receptores de N-Metil-D-Aspartato/metabolismo , Envejecimiento/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica/métodos , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Regulación del Desarrollo de la Expresión Génica , Hipocampo/citología , Hibridación in Situ , Técnicas In Vitro , Plasticidad Neuronal/fisiología , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/genética , Tiempo , Transfección , XenopusRESUMEN
N-Methyl-D-aspartate (NMDA) receptor activation initiates both homosynaptic long-term depression (LTD) and long-term potentiation (LTP) in the CA1 region of the hippocampus. The mechanism by which two opposing forms of synaptic plasticity can be initiated through the activation of a single receptor is not known. We examined the effects of two competitive antagonists on the induction of LTP and LTD, D-2-amino-5-phosphonovaleric acid (D-AP5), a broad spectrum inhibitor of the NMDA receptor, and 3-((RS)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), an antagonist that binds with high affinity to conventional NMDA receptors subtypes, but not to atypical subtypes that are relatively independent of voltage-dependent Mg2+-blockade. As has previously been reported, LTP, LTD, and depotentiation were all blocked by applications of D-AP5. In contrast, only LTP, but not LTD or depotentiation, was blocked by CPP. These observations suggest that decreases and increases of synaptic strength are mediated by the activation of distinct NMDA receptor subpopulations.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Plasticidad Neuronal/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/fisiología , 2-Amino-5-fosfonovalerato/farmacología , Animales , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Magnesio/farmacología , Plasticidad Neuronal/efectos de los fármacos , Piperazinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Sinapsis/efectos de los fármacos , Factores de TiempoRESUMEN
Spreading depression (SD) is a propagating depolarization of populations of neurons induced by intense electrical, chemical, or mechanical stimulation, which has been proposed to be an important mechanism in the aura of migraine. SD is characterized by a transient loss of synaptic transmission and thus may involve signal transduction mechanisms known to modulate synaptic strength. To examine the underlying pathophysiological molecular mechanisms of SD, we analyzed the regulation of eight protein kinase C (PKC) isoforms by immunoblot during SD induced by a high-intensity stimulus of synaptic afferents in the CA1 region of hippocampal slices. We observed a downregulation of the conventional (alpha, beta I, beta II, gamma) and the novel (delta, epsilon, eta) PKC isoforms in SD, but no change in the atypical isozyme (zeta). The coordinate downregulation of multiple PKC isoforms may be important in the functional depression of neuronal activity in SD. In contrast, the atypical zeta, and its constitutively active fragment PKM zeta, is a specific PKC isozyme that has been implicated in the maintenance of long-term potentiation (LTP) and long-term depression (LTD), widely studied models for the mechanism of memory. The stability of PKC zeta and PKM zeta in SD indicates that a molecular mechanism for the maintenance of LTP/ LTD is relatively resistant to alterations that occur during pathophysiologically large ionic fluxes. This result could help to explain the retention of information stored in the cortex despite the massive release of excitatory neurotransmitter and neuronal depolarization that may occur during the migrainous aura.
Asunto(s)
Depresión de Propagación Cortical/fisiología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Animales , Western Blotting , Estimulación Eléctrica , Electrofisiología , Hipocampo/química , Hipocampo/enzimología , Isoenzimas/análisis , Isoenzimas/química , Potenciación a Largo Plazo/fisiología , Técnicas de Cultivo de Órganos , Proteína Quinasa C/análisis , Proteína Quinasa C/química , Ratas , Ratas Sprague-DawleyRESUMEN
Long-term potentiation (LTP) and long-term depression (LTD) are persistent modifications of synaptic efficacy that may contribute to information storage in the CA1 region of the hippocampus. Persistently enhanced phosphorylation has been implicated in the maintenance phase of LTP. This hypothesis is supported by our previous observation that protein kinase M zeta (PKM zeta), the constitutively active catalytic fragment of a single protein kinase C isoform (PKC zeta), increases in LTP maintenance. In contrast, dephosphorylation may be important in LTD maintenance, because phosphatase inhibitors reverse established LTD, in addition to blocking its induction. Because phosphorylation is determined by a balance of phosphatases and kinases, both increases in phosphatase activity and decreases in kinase activity could contribute to LTD. We now report that the reduction of protein kinase activity by H7, as well as selective inhibition of PKC by chelerythrine, mimics and occludes the maintenance phase of homosynaptic LTD in rat hippocampal slices. Conversely, saturated LTD occludes the synaptic depression caused by chelerythrine. Biochemical analysis demonstrates a decrease of PKM zeta, as well as PKCs gamma and epsilon, in LTD maintenance and a concomitant loss of constitutive PKC activity. LTD and the downregulation of PKM zeta are prevented by NMDA receptor antagonists and Ca(2+)-dependent protease inhibitors. Both LTD and the downregulation of PKM zeta are reversible by high-frequency afferent stimulation. Our findings indicate that the molecular mechanisms of LTP and LTD maintenance are inversely related through the bidirectional regulation of PKC.
Asunto(s)
Potenciación a Largo Plazo , Proteínas Quinasas/metabolismo , Alcaloides , Animales , Benzofenantridinas , Calpaína/metabolismo , Péptido Hidrolasas/metabolismo , Fenantridinas/farmacología , Proteína Quinasa C/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Sinapsis/efectos de los fármacos , Sinapsis/fisiologíaRESUMEN
The maintenance of long-term potentiation (LTP) in the CA1 region of the hippocampus has been reported to require both a persistent increase in phosphorylation and the synthesis of new proteins. The increased activity of protein kinase C (PKC) during the maintenance phase of LTP may result from the formation of PKMzeta, the constitutively active fragment of a specific PKC isozyme. To define the relationship among PKMzeta, long-term EPSP responses, and the requirement for new protein synthesis, we examined the regulation of PKMzeta after sub-threshold stimulation that produced short-term potentiation (STP) and after suprathreshold stimulation by single and multiple tetanic trains that produced LTP. We found that, although no persistent increase in PKMzeta followed STP, the degree of long-term EPSP potentiation was linearly correlated with the increase of PKMzeta. The increase was first observed 10 min after a tetanus that induced LTP and lasted for at least 2 hr, in parallel with the persistence of EPSP enhancement. Both the maintenance of LTP and the long-term increase in PKMzeta++ were blocked by the protein synthesis inhibitors anisomycin and cycloheximide. These results suggest that PKMzeta is a component of a protein synthesis-dependent mechanism for persistent phosphorylation in LTP.
Asunto(s)
Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Proteínas Quinasas/metabolismo , Animales , Anisomicina/farmacología , Hipocampo/fisiología , Inmunohistoquímica , Potenciales de la Membrana/fisiología , Factores de TiempoRESUMEN
We investigated activation of the two major neuronal protein kinase C (PKC) isoforms in Aplysia, Ca(2+)-activated Apl I and Ca(2+)-independent Apl II, during the induction and maintenance of behavioral sensitization of Aplysia defensive reflexes. Activation of PKC occurred during the training stimulus and persisted for at least 2 hr thereafter but was not maintained for 24 hr. The persistent activation required protein synthesis and was blocked by cyproheptidine, an agent that also blocked the initial activation of PKC. Persistent activation involved both an increase in membrane-associated Apl I and an increase in an autonomous kinase activity that may be related to a post-translational modification of Apl II. These results are consistent with the hypothesis that in addition to its role in producing the presynaptic facilitation of mechanosensory-motor neuron synapses that underlie short-term facilitation, PKC is needed for maintaining synaptic changes in an intermediate period that precedes the modifications accompanying consolidation of memory.
Asunto(s)
Aplysia/fisiología , Potenciación a Largo Plazo/fisiología , Proteína Quinasa C/metabolismo , Animales , Western Blotting , Activación Enzimática/fisiología , Ganglios de Invertebrados/fisiología , Técnicas In VitroRESUMEN
Protein kinase C (PKC) is a heterogeneous family of ten or more isoforms which plays an important role in neuronal signal transduction. Isoforms from all subclasses are prominently expressed in the rat hippocampus, as demonstrated by immunoblot with isozyme-specific antisera: Ca(2+)-dependent (alpha, beta I, beta II and gamma), Ca(2+)-independent (delta, epsilon and a newly characterized PKC related to eta) and atypical (zeta). In addition, the zeta isoform is also found as the free, constitutively active catalytic domain, protein kinase M zeta (PKM zeta). Two distinct patterns of expression of PKC isozymes in rat hippocampus are found during development from E18 to P28. PKC zeta, PKM zeta and PKC delta are present at birth and their expression does not increase postnatally. In contrast, the other isoforms are expressed only at low levels at birth and then increase in the first 4 weeks postnatally. These two patterns of expression suggest distinct functions for PKC isozymes during development.
Asunto(s)
Envejecimiento/metabolismo , Hipocampo/enzimología , Isoenzimas/biosíntesis , Proteína Quinasa C/biosíntesis , Secuencia de Aminoácidos , Animales , Anticuerpos , Especificidad de Anticuerpos , Desarrollo Embrionario y Fetal , Edad Gestacional , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Isoenzimas/análisis , Datos de Secuencia Molecular , Proteína Quinasa C/análisis , RatasRESUMEN
We describe a new form of protein kinase C (PKC) with a molecular weight of 97 kDa, higher than the known forms of vertebrate PKC. This putative new high-molecular weight isoform, which we are calling PKC (HMW), is increased in the membrane fraction either upon application of phorbol esters or with afferent synaptic stimulation of Schaffer collaterals in hippocampal slices. The protein cross-reacts on immunoblot with affinity-purified polyclonal antiserum raised against a peptide derived from the carboxy-terminus of PKC eta; it does not cross-react, however, with antiserum against the amino-terminal region of PKC eta. In the tissues examined, PKC(HMW) is localized primarily in brain, in contrast to PKC eta, which is found predominantly in lung and skin.
Asunto(s)
Hipocampo/enzimología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos , Animales , Hipocampo/efectos de los fármacos , Immunoblotting , Isoenzimas/química , Isoenzimas/inmunología , Datos de Secuencia Molecular , Peso Molecular , Neuronas Aferentes/enzimología , Ésteres del Forbol/farmacología , Proteína Quinasa C/química , Proteína Quinasa C/inmunología , RatasRESUMEN
Long-term potentiation in the CA1 region of the hippocampus, a model for memory formation in the brain, is divided into two phases. A transient process (induction) is initiated, which then generates a persistent mechanism (maintenance) for enhancing synaptic strength. Protein kinase C (PKC), a gene family of multiple isozymes, may play a role in both induction and maintenance. In region CA1 from rat hippocampal slices, most of the isozymes of PKC translocated to the particulate fraction 15 sec after a tetanus. The increase of PKC in the particulate fraction did not persist into the maintenance phase of long-term potentiation. In contrast, a constitutively active kinase, PKM, a form specific to a single isozyme (zeta), increased in the cytosol during the maintenance phase. The transition from translocation of PKC to formation of PKM may help to explain the molecular mechanisms of induction and maintenance of long-term potentiation.
Asunto(s)
Hipocampo/fisiología , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos , Animales , Estimulación Eléctrica , Potenciales Evocados , Hipocampo/enzimología , Técnicas In Vitro , Isoenzimas/análisis , Cinética , Modelos Neurológicos , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/inmunología , Fragmentos de Péptidos/análisis , Mapeo Peptídico , Fosforilación , Proteína Quinasa C/análisis , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/fisiología , Factores de TiempoRESUMEN
Binding of cAMP by the five neuronal isoforms (N1-5) of the regulatory (R) subunit of the Aplysia cAMP-dependent protein kinase is diminished in sensory neurons stimulated to produce long-term presynaptic facilitation. To determine how the cAMP-binding activity of the R subunits is lost, we isolated cDNAs encoding N4, which is a homolog of mammalian RI. Immunoblots with antisera raised against the R protein overexpressed in E. coli show that the diminished binding activity, which occurs in long-term facilitation, results from coordinate loss of R protein isoforms. No change was detected in the amount of transcripts for R subunits, suggesting that the down-regulation results from enhanced proteolytic turnover.
Asunto(s)
Aplysia/fisiología , AMP Cíclico/fisiología , Regulación hacia Abajo/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Neuronas Aferentes/enzimología , Proteínas Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Regulación hacia Abajo/genética , Regulación Enzimológica de la Expresión Génica/genética , Sueros Inmunes , Immunoblotting , Isoenzimas/análisis , Isoenzimas/genética , Isoenzimas/fisiología , Sustancias Macromoleculares , Datos de Secuencia Molecular , Neuronas Aferentes/química , Neuronas Aferentes/fisiología , Hibridación de Ácido Nucleico , Proteínas Quinasas/análisis , Proteínas Quinasas/genética , Homología de Secuencia de Ácido Nucleico , Factores de Tiempo , Transcripción Genética/genéticaRESUMEN
We isolated cDNA clones from an Aplysia sensory-cell library encoding two isoforms of protein kinase C (PKC). Several isozyme-specific regions are conserved in the Aplysia kinases, notably the variable regions V5 in the Ca(2+)-dependent PKC (Apl I) and V1 in the Ca(2+)-independent PKC (Apl II). Neuronal proteins with the properties expected of these two isoforms can be identified with antibodies raised against peptides synthesized from the amino acid sequences deduced from the clones. Sacktor and Schwartz (1990) measured the proportion of kinase activity that can be translocated to membrane in Aplysia sensory neurons and ganglia by stimuli that produce the presynaptic facilitation underlying behavioral sensitization. Much less Apl I and Apl II are translocated, suggesting that still other isoforms of PKC exist in these cells.
Asunto(s)
Aplysia/metabolismo , Calcio/fisiología , Clonación Molecular , Neuronas Aferentes/enzimología , Proteína Quinasa C/genética , Animales , Secuencia de Bases , ADN/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Proteína Quinasa C/química , Proteína Quinasa C/metabolismo , Proteína Quinasa C/fisiología , Sinapsis/fisiologíaRESUMEN
We describe some properties on an Mr 30,000 thermolabile and trypsin-sensitive protein that activates phospholipase A2 (PLA2) and which was isolated from nervous tissue of the marine mollusk, Aplysia californica. A similar protein is present in rat cerebral cortex. This protein was partially purified from crude homogenates of nervous tissue by ion exchange chromatography on DEAE-Sephadex followed by size-exclusion high performance liquid chromatography (HPLC). It is loosely associated with membrane fractions, and is extracted by 0.05% Tween 20. Although similar in size to several previously described PLA2-stimulating proteins from non-neural mammalian cells and tissues, it differs from them in some aspects of biological activity. The protein promotes the release of eicosanoids from the membranes of intact Aplysia neurons prelabeled with [3H]arachidonic acid and appears to be an in vitro substrate for protein kinase C (PKC). PLA2-stimulating activity is greatly enhanced after exposing isolated ganglia to phorbol dibutyrate (PDBu) and is reduced by treatment with immobilized E. coli alkaline phosphatase. These observations suggest that phosphorylation of this stimulatory protein by PKC regulates PLA2 in neurons.
Asunto(s)
Ganglios/metabolismo , Neuronas/metabolismo , Fosfolipasas A/metabolismo , Proteína Quinasa C/metabolismo , Proteínas/metabolismo , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico , Animales , Aplysia , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Ácidos Hidroxieicosatetraenoicos/aislamiento & purificación , Ácidos Hidroxieicosatetraenoicos/metabolismo , Cinética , Peso Molecular , Fosfolipasas A2 , Proteínas/aislamiento & purificación , TripsinaRESUMEN
The defensive tail-withdrawal reflex of Aplysia californica, mediated by identified sensory neurons in pleural ganglia that form synapses on motor cells in pedal ganglia, can be sensitized by stimulating the animal with electric shock. The neurophysiological basis of this simple form of learning is thought to be the increased release of transmitter by the sensory neurons. Earlier work has focused on cAMP-dependent protein phosphorylation as the cause of the presynaptic facilitation underlying short-term sensitization. Using physiological concentrations of Mg2+ during fractionation, we now find that, independent from cAMP, protein kinase C is translocated in sensory neurons by sensitizing stimuli. Translocation occurred after behavioral training of the animal and after application to isolated ganglia of serotonin or phorbol esters. Taken together with the neurophysiological evidence presented in the accompanying paper that phorbol esters can produce the facilitation, these biochemical results suggest that protein kinase C plays a role in producing the presynaptic facilitation that underlies short-term sensitization and dishabituation of defensive reflexes.
Asunto(s)
Proteínas de Caenorhabditis elegans , Neuronas Aferentes/fisiología , Proteína Quinasa C/metabolismo , Animales , Aplysia , Proteínas Portadoras , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Ganglios/fisiología , Histonas/metabolismo , Técnicas In Vitro , Magnesio/farmacología , Neuronas Aferentes/efectos de los fármacos , Neuronas Aferentes/enzimología , Forbol 12,13-Dibutirato/metabolismo , Forbol 12,13-Dibutirato/farmacología , Forboles/farmacología , Fosforilación , Receptores de Droga/metabolismo , Serotonina/farmacología , Fracciones Subcelulares/enzimología , Tionucleótidos/farmacologíaRESUMEN
1.) Application of serotonin to Aplysia sensory neurons can result in facilitated synaptic transmission, both short- and long-term. This facilitation is likely to be produced by a complex set of molecular mechanisms: serotonin activates adenylate cyclase, increasing cAMP and protein kinase (Cedar and Schwartz, 1972); serotonin also changes the subcellular distribution of the Ca2+/calmodulin-dependent protein kinase (Saitoh and Schwartz, 1983). Recently, phorbol esters also have been shown to produce facilitation. We have therefore investigated how protein kinase C (PKC) participates in serotonin-mediated synaptic facilitation. 2.) We found that the Aplysia genome encodes PKC, which is expressed in nervous tissue as at least two abundant transcripts (about 0.003% of the total message). Its inferred amino acid sequence is 85% homologous to that of enzymes from mammals and Drosophila, and over 95% homologous if compared to both. The specific activity of the Aplysia kinase is comparable to that found in rat brain, with similar reaction parameters and dependencies on phosphatidylserine (PS), Ca2+, diacylglycerol and phorbol esters. While PKC is found on neuronal membrane in the basal state, the PKC activators, Ca2+ and phorbol esters, further translocate the kinase to membrane in crude extracts of neuronal tissue. The amounts of membrane-bound PKC, as determined by 3H-phorbol-ester binding, are greatest in neuropil and nerve. 3.) Exposure of sensory neurons and their terminals in Aplysia pleural-pedal ganglia to facilitating doses of either phorbol ester or serotonin results in the translocation of PKC from cytosol to membrane, activating the enzyme. cAMP does not produce this translocation.(ABSTRACT TRUNCATED AT 250 WORDS)