Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

The structure of Pavlovian fear conditioning in the amygdala

Do different brains forming a specific memory allocate the same groups of neurons to encode it? One way to test this question is to map neurons encoding the same memory and quantitatively compare their locations across individual brains. In a previous study, we used this strategy to uncover a common topography of neurons in the dorsolateral amygdala (LAd) that expressed a learning-induced and plasticity-related kinase (p42/44 mitogen-activated protein kinase; pMAPK), following auditory Pavlovian fear conditioning. In this series of experiments, we extend our initial findings to ask to what extent this functional topography depends upon intrinsic neuronal structure. We first showed that the majority (87 %) of pMAPK expression in the lateral amygdala was restricted to principal-type neurons. Next, we verified a neuroanatomical reference point for amygdala alignment using in vivo magnetic resonance imaging and in vitro morphometrics. We then determined that the topography of neurons encoding auditory fear conditioning was not exclusively governed by principal neuron cytoarchitecture. These data suggest that functional patterning of neurons undergoing plasticity in the amygdala following Pavlovian fear conditioning is specific to memory formation itself. Further, the spatial allocation of activated neurons in the LAd was specific to cued (auditory), but not contextual, fear conditioning. Spatial analyses conducted at another coronal plane revealed another spatial map unique to fear conditioning, providing additional evidence that the functional topography of fear memory storing cells in the LAd is non-random and stable. Overall, these data provide evidence for a spatial organizing principle governing the functional allocation of fear memory in the amygdala.

Characterization of mouse neuronal Ca2+/calmodulin kinase II inhibitor alpha

We have overexpressed an 8.5-kDa mouse Ca2+/calmodulin kinase II inhibitor a protein (mCaMKIIN alpha) in Escherichia coli and demonstrate that the recombinant protein is a potent inhibitor of Ca2+/calmodulin kinase 11 (CaMKII) in vitro. However, antibodies raised against recombinant mCaMKIIN alpha. react with an similar to 37-kDa protein present in mouse brain. The pattern of expression of the similar to 37-kDa protein is similar to that of mCaMKIIN alpha mRNA as both are expressed in normal but not Japanese encephalitis virus (JEV)-infected mouse brain. Subcellular localization studies indicate that the similar to 37-kDa protein is present in the post-synaptic density (PSD) where mCaMKII alpha is known to perform key regulatory functions. We conclude that the similar to 37-kDa protein identified in this study is mCaMKIIN alpha. and its localization in the PSD indicates a novel role for this protein in the regulation of neuronal CaMKII alpha. (c) 2007 Elsevier B.V. All rights reserved.

Calcium/Calmodulin Dependent Protein Kinase II Bound to NMDA Receptor 2B Subunit Exhibits Increased ATP Affinity and Attenuated Dephosphorylation

Calcium/calmodulin dependent protein kinase II (CaMKII) is implicated to play a key role in learning and memory. NR2B subunit of N-methyl-D-aspartate receptor (NMDAR) is a high affinity binding partner of CaMKII at the postsynaptic membrane. NR2B binds to the T-site of CaMKII and modulates its catalysis. By direct measurement using isothermal titration calorimetry (ITC), we show that NR2B binding causes about 11 fold increase in the affinity of CaMKII for ATP gamma S, an analogue of ATP. ITC data is also consistent with an ordered binding mechanism for CaMKII with ATP binding the catalytic site first followed by peptide substrate. We also show that dephosphorylation of phospho-Thr(286)-alpha-CaMKII is attenuated when NR2B is bound to CaMKII. This favors the persistence of Thr(286) autophosphorylated state of CaMKII in a CaMKII/phosphatase conjugate system in vitro. Overall our data indicate that the NR2B- bound state of CaMKII attains unique biochemical properties which could help in the efficient functioning of the proposed molecular switch supporting synaptic memory.

A influência do metabolismo redox na transmissão da doença de Chagas

As formas epimastigotas de Trypanosoma cruzi proliferam e se diferenciam no interior de diferentes compartimentos do trato digestivo dos triatomíneos. Esses ambientes antagônicos, no que diz respeito à concentração de nutrientes, pH e status redox, constituem um desafio para o protozoário por conterem moléculas e fatores capazes de deflagrar diferentes sinalizações e respostas no parasito. Por isso, testamos a influência de produtos abundantes do metabolismo do vetor e de status redox distintos, frente aos processos de proliferação e diferenciação in vivo e in vitro. Como exemplo temos o heme e a hemozoína, subprodutos da digestão da hemoglobina, e o urato, rico na urina dos insetos. O heme é uma importante molécula em todos os seres vivos. Nosso grupo mostrou seu papel na proliferação in vitro de T. cruzi e que esse sinal é governado pela enzima redox-sensível CaMKII (Lara et al., 2007; Souza et al., 2009). Esse efeito parece depender de uma sinalização redox, onde o heme e não seus análigos induz a formação de EROs, modulando a atividade da CaMKII (Nogueira et al, 2011). Apesar de gerar espécies reativas de oxigênio (EROs) em formas epimastigotas, o heme não alterou a ultraestrutura desses parasitos mostrando uma adaptação a ambientes oxidantes. Além disso, a adição de FCCP inibiu a formação de EROs mitocondrial, diminuindo a proliferação dos parasitos. Em contrapartida, a AA aumentou drasticamente a produção de EROs mitocondrial levando à morte dos epimastigotas. Estes resultados confirmam a hipótese de regulação redox do crescimento de epimastigotas. A formação de β- hematina (hemozoína) constitui uma elegante estratégia para minimizar o efeito tóxico do heme nos insetos hematófagos. Contudo, a β-hematina não influenciou a proliferação ou a metaciclogênese in vitro. Já o urato, e outros antioxidantes clássicos como o GSH e o NAC prejudicaram a proliferação in vitro de epimastigotas. Estes efeitos foram parcialmente revertidos quando os antioxidantes foram incubados juntamente com o heme. Durante a metaciclogênese in vitro, o NAC e o urato induziram um aumento significativo das formas tripomastigotas e levaram a diminuição da porcentagem de formas epimastigotas. Em contrapartida, o heme e a β-hematina apresentaram o efeito oposto, diminuindo a porcentagem de formas tripomastigotas e aumentando a de epimastigotas. No intuito de confirmar a influencia do status redox na biologia do parasito in vivo, nós quantificamos a carga parasitária nas porções anterior e posterior e no reto do triatomíneo alimentado na presença ou na ausência de NAC e urato por qPCR. O tratamento com os antioxidantes aumentou a carga parasitária em todas as partes do intestino analisadas. Posteriormente, para diferenciar as formas evolutivas responsáveis pelo incremento da carga parasitária, foram realizadas contagens diferenciais nas mesmas porções do intestino do inseto vetor. Cinco dias após a infecção foi observado aumento significativo de formas tripomastigotas e diminuição de formas epimastigotas in vivo. Em conjunto, estes dados sugerem que, assim como a concentração de nutrientes e o pH, o status redox também pode influenciar a biologia do T. cruzi no interior do inseto vetor. Neste cenário, moléculas oxidantes agiriam a favor da proliferação, e em contraste, antioxidantes parecem favorecer a metaciclogênese.

Coupling between NMDA receptor and acid-sensing ion channel contributes to ischemic neuronal death

Acid-sensing ion channels (ASICs) composed of ASIC1a subunit exhibit a high Ca2+ permeability and play important roles in synaptic plasticity and acid-induced cell death. Here, we show that ischemia enhances ASIC currents through the phosphorylation at Ser478 and Ser479 of ASIC1a, leading to exacerbated ischemic cell death. The phosphorylation is catalyzed by Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, as a result of activation of NR2B-containing N-methyl-D-aspartate subtype of glutamate receptors (NMDARs) during ischemia. Furthermore, NR2B-specific antagonist, CaMKII inhibitor, or overexpression of mutated form of ASIC1a with Ser478 or Ser479 replaced by alanine (ASICla-S478A, ASIC1a-S479A) in cultured hippocampal neurons prevented ischemia-induced enhancement of ASIC currents, cytoplasmic Ca2+ elevation, as well as neuronal death. Thus, NMDAR-CaMKII cascade is functionally coupled to ASICs and contributes to acidotoxicity during ischemia. Specific blockade of NMDAR/CaMKII-ASIC coupling may reduce neuronal death after ischemia and other pathological conditions involving excessive glutamate release and acidosis.

大鼠海马CA1区神经元高级可塑性（Metaplasticity）的研究

经验（experience）或突触活动依赖的突触信息传递效能的增强（LTP）或抑制（LTD）被称为突触可塑性（synaPticplasticity），是公认的学习和记忆的细胞分子学基础。突触可塑性的方向是突触信息传递效能的增强或降低；突触可塑性的程度是突触信息传递的效能增加量或降低量。突触活动历史可影响随后突触可塑性的方向或程度。这种突触可塑性的可塑性被称为高级可塑性（MetaP1asticity）。最明显的高级可塑性现象是发生在突触效能没有改变的情况下。经验或者突触活动可以同时诱导出突触可塑性和高级可塑性。除非知道突触活动的历史，否则很难预测突触可塑性的大小和方向。高级可塑性简单地讲就是过去的神经信息留下痕迹会对神经信息的加工处理产生持续性影响。目前高级可塑性的机理并不完全清楚。采用全细胞电压钳记录方法，在3周龄wistar大鼠海马脑片中，同时记录schaffer侧枝纤维一以1处兴奋性突触后膜电流（evokedEPsC）和自发性微小兴奋性突触后膜"电流（mEPSC）。结果显示，mEPSC和低频刺激诱导的长时程抑制（Long一TermDePre55ion，LTD）之间呈负相关，mEPsc的频率或幅值越小，诱导出的LTD幅度越大；应激和低钙溶液处理在降低记PsC的频率和幅值的同时并没有明显改变随后LTD的诱导幅，但是这两者负相关性依然存。相关系数在应激条件下有增加，在低钙溶液中有降低。以上结果提示，mPEsc的频率或幅值可能指示了过去的突触活动或经验产牛的高幼可朔件＿田、冲稀恻了喃层的，：。、云导的幅度。C鹉子妙触可黔和高级可黔中起着关键作。作为一种非选择性阳离子通道，van11101drec即tortyPel（VR1或TRPVI）在脑区中广泛表达，尤其在海马高表达。内源性配体可以激活该受体通道。但目前并不清楚vRI在海马突触可塑性当中的作用。我们发现VRI激动剂辣椒素不影响脑片中海马以1区基础突触传递，但是它阻断低频诱导（LFS，3Hz）的LTD，增强高频诱导（HFs，20OHz）的长时程增强（Long一TerlnPotetiantion，LTP）。也就是vRI激活改变了突触可塑性的方向，是一种经典的高级可塑性现象。正常情况下50Hz不能诱导出LTP，而加入辣椒素后易化了LTP的诱导。因此LTP诱导的闽值降低。辣椒素激动剂对突触可塑性或高级可塑性的效应不依赖协。A受体而依赖于H型钙调蛋白激酶（CaMKII）的调控。因为场。A受体电流和它的电流一电压曲线不受此激动剂影响，但CaMKII抑制剂KN-93或者VRI拮抗剂capsazepine却阻断了辣椒素的这种效应。这些发现，显示VRI可以调控海马以1区的高级突触可塑性，可能参与某些类型的学习和记忆。

VEGF-induced retinal angiogenic signalling is critically dependent on Ca2+ signalling via Ca2+/calmodulin-dependent protein kinase II

Purpose. The authors conducted an in vitro investigation of the role of Ca2+-dependent signaling in vascular endothelial growth factor (VEGF)-induced angiogenesis in the retina.

Methods. Bovine retinal endothelial cells (BRECs) were stimulated with VEGF in the presence or absence of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM; intracellular Ca2+ chelator), U73122 (phospholipase C (PLC) inhibitor), xestospongin C (Xe-C), and 2-aminoethoxydiphenyl borate (2APB) (inhibitors of inositol-1,4,5 triphosphate (IP3) signaling). Intracellular Ca2+ concentration ([Ca2+]i) was estimated using fura-2 Ca2+ microfluorometry, Akt phosphorylation quantified by Western blot analysis, and angiogenic responses assessed using cell migration, proliferation, tubulogenesis, and sprout formation assays. The effects of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 were also evaluated on VEGF-induced Akt signaling and angiogenic activity.

Results. Stimulation of BRECs with 25 ng/mL VEGF induced a biphasic increase in [Ca2+]i, with an initial transient peak followed by a sustained plateau phase. VEGF-induced [Ca2+]i increases were almost completely abolished by pretreating the cells with BAPTA-AM, U73122, Xe-C, or 2APB. These agents also inhibited VEGF-induced phosphorylation of Akt, cell migration, proliferation, tubulogenesis, and sprouting angiogenesis. KN93 was similarly effective at blocking the VEGF-induced activation of Akt and angiogenic responses.

Conclusions. VEGF increases [Ca2+]i in BRECs through activation of the PLC-IP3 signal transduction pathway. VEGF-induced phosphorylation of the proangiogenic protein Akt is critically dependent on this increase in [Ca2+]i and the subsequent activation of CaMKII. Pharmacologic inhibition of Ca2+-mediated signaling in retinal endothelial cells blocks VEGF-induced angiogenic responses. These results suggest that the PLC/IP3/Ca2+/CaMKII signaling pathway may be a rational target for the treatment of angiogenesis-related disorders of the eye.

The Gastrointestinal Peptide Obestatin Induces Vascular Relaxation Via Specific Activation of Endothelium-Dependent Nitric Oxide Signalling.

Background and purpose: Obestatin is a recently-discovered gastrointestinal peptide with established metabolic actions, which is linked to diabetes and may exert cardiovascular benefits. Here we aimed to investigate the specific effects of obestatin on vascular relaxation. Experimental approach: Cumulative relaxation responses to obestatin peptides were assessed in isolated rat aorta and mesenteric artery (n=8) in the presence/absence of selective inhibitors. Complementary studies were performed in cultured bovine aortic endothelial cells (BAEC). Key results: Obestatin peptides elicited concentration-dependent relaxation in both aorta and mesenteric artery. Responses to full-length obestatin(1-23) were greater than those to obestatin(1-10) and obestatin(11-23). Obestatin(1-23)-induced relaxation was attenuated by endothelial denudation, L-NAME (NO synthase inhibitor), high extracellular K(+) , GDP-ß-S (G protein inhibitor), MDL-12,330A (adenylate cyclase inhibitor), wortmannin (PI3K inhibitor), KN-93 (CaMKII inhibitor), ODQ (guanylate cyclase inhibitor) and iberiotoxin (BK(Ca) blocker), suggesting that it is mediated by an endothelium-dependent NO signalling cascade involving an adenylate cyclase-linked G protein-coupled receptor, PI3K/Akt, Ca(2+) -dependent eNOS activation, soluble guanylate cyclase and modulation of vascular smooth muscle K(+) . Supporting data from BAEC indicated that nitrite production, intracellular Ca(2+) and Akt phosphorylation were increased after exposure to obestatin(1-23). Relaxations to obestatin(1-23) were unaltered by inhibitors of candidate endothelium-derived hyperpolarising factors (EDHFs) and combined SK(Ca) /IK(Ca) blockade, suggesting that EDHF-mediated pathways were not involved. Conclusions and Implications: Obestatin produces significant vascular relaxation via specific activation of endothelium-dependent NO signalling. These actions may be important in normal regulation of vascular function and are clearly relevant to diabetes, a condition characterised by endothelial dysfunction and cardiovascular complications.

Donepezil attenuates hippocampal neuronal damage and cognitive deficits after global cerebral ischemia in gerbils.

Decreased cerebral blood flow causes cognitive impairments and neuronal injury in vascular dementia. In the present study, we reported that donepezil, a cholinesterase inhibitor, improved transient global cerebral ischemia-induced spatial memory impairment in gerbils. Treatment with 5mg/kg of donepezil for 21 consecutive days following a 10-min period of ischemia significantly inhibited delayed neuronal death in the hippocampal CA1 region. In Morris water maze test, memory impairment was significantly improved by donepezil treatment. Western blot analysis showed that donepezil treatment prevented reductions in p-CaMKII and p-CREB protein levels in the hippocampus. These results suggest that donepezil attenuates the memory deficit induced by transient global cerebral ischemia and this neuroprotection may be associated with the phosphorylation of CaMKII and CERB in the hippocampus.

Abnormal changes in voltage-gated sodium channels NaV1.1, NaV1.2, NaV1.3, NaV1.6 and in Calmodulin/calmodulin-dependent protein kinase II, within the brains of spontaneously epileptic rats and tremor rats

Voltage-gated sodium channels (VGSCs) play a crucial role in epilepsy. The expressions of different VGSCs subtypes are varied in diverse animal models of epilepsy that may reflect their multiple phenotypes or the complexity of the mechanisms of epilepsy. In a previous study, we reported that NaV1.1 and NaV1.3 were up-regulated in the hippocampus of the spontaneously epileptic rat (SER). In this study, we further analyzed both the expression and distribution of the typical VGSC subtypes NaV1.1, NaV1.2, NaV1.3 and NaV1.6 in the hippocampus and in the cortex of the temporal lobe of two genetic epileptic animal models: the SER and the tremor rat (TRM). The expressions of calmodulin (CaM) and calmodulin-dependent protein kinase II (CaMKII) were also analyzed with the purpose of assessing the effect of the CaM/CaMKII pathway in these two models of epilepsy. Increased expression of the four VGSC subtypes and CaM, accompanied by a decrease in CaMKII was observed in the hippocampus of both the SERs and the TRM rats. However, the changes observed in the expression of VGSC subtypes and CaM were decreased with an elevated CaMKII in the cortex of their temporal lobes. Double-labeled immunofluorescence data suggested that in SERs and TRM rats, the four subtypes of the VGSC proteins were present throughout the CA1, CA3 and dentate gyrus regions of the hippocampus and temporal lobe cortex and these were co-localized in neurons with CaM. These data represent the first evidence of abnormal changes in expression of four VGSC subtypes (NaV1.1, NaV1.2, NaV1.3 and NaV1.6) and CaM/CaMKII in the hippocampus and temporal lobe cortex of SERs and TRM rats. These changes may be involved in the generation of epileptiform activity and underlie the observed seizure phenotype in these rat models of genetic epilepsy.

Adult cardiac fibroblast proliferation is modulated by calcium/calmodulin-dependent protein kinase II in normal and hypertrophied hearts

Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts.

Interaction between glucocorticoid and adenosine A2A receptors in the hippocampus : implications for learning and memory

Tese de doutoramento, Ciências Biomédicas (Neurociências), Universidade de Lisboa, Faculdade de Medicina, 2014

Brain-derived neurotrophic factor enhances the hippocampal expression of key postsynaptic proteins in vivo including the monocarboxylate transporter MCT2.

Brain-derived neurotrophic factor (BDNF) promotes synaptic plasticity via an enhancement in expression of specific synaptic proteins. Recent results suggest that the neuronal monocarboxylate transporter MCT2 is a postsynaptic protein critically involved in synaptic plasticity and long-term memory. To investigate in vivo whether BDNF can modulate the expression of MCT2 as well as other proteins involved in synaptic plasticity, acute injection of BDNF was performed in mouse dorsal hippocampal CA1 area. Using immunohistochemistry, it was found that MCT2 expression was enhanced in part of the CA1 area and in the dentate gyrus 6 h after a single intrahippocampal injection of BDNF. Similarly, expression of the immediate early genes Arc and Zif268 was enhanced in the same hippocampal areas, in accordance with their role in synaptic plasticity. Immunoblot analysis confirmed the significant enhancement in MCT2 protein expression. In contrast, no changes were observed for the glial monocarboxylate transporters MCT1 and MCT4. When other synaptic proteins were investigated, it was found that postsynaptic density 95 (PSD95) and glutamate receptor 2 (GluR2) protein levels were significantly enhanced while no effect could be detected for synaptophysin, synaptosomal-associated protein 25 (SNAP25), αCaMKII and GluR1. These results demonstrate that MCT2 expression can be upregulated together with other key postsynaptic proteins in vivo under conditions related to synaptic plasticity, further suggesting the importance of energetics for memory formation.

The role of cyclic nucleotides in modulation of crayfish neuromuscular junctions by a neuropeptide /

DF2, a heptapeptide, is a member of the family of FMRFamide-like peptides and has been shown to increase the amount of transmitter released at neuromuscular junctions of the crayfish, Procambarus clarkit Recent evidence has shown that protein kinase C (PKC), calcium/calmodulin-dependent protein kinase II (CaMKII) and the cAMPdependent protein kinase (PKA) play a role in the neuromodulatory pathway of DF2. The involvement of these kinases led to the prediction that a G-protein-coupled receptor (GPCR) is activated by DF2 due to the role that each kinase plays in traditional GPCR pathways seen in other organisms and in other cells. G-proteins can also act on an enzyme that generates cyclic guanosine monophosphate (cGMP) which mediates its effects through a cGMP-dependent protein kinase (PKG). This thesis addresses the question of whether or not DF2's effects on synaptic transmission in crayfish are mediated by the cyclic nucleotides cAMP and cGMP. The effects of DF2 on synaptic transmission were examined using deep abdominal extensor muscles of the crayfish Procambarus clarkii. An identified motor neuron was stimulated, and excitatory post-synaptic potentials (EPSPs) were recorded in abdominal extensor muscle LI . A number of activators and inhibitors were used to determine whether or not cAMP, PKA, cGMP and PKG mediate the effect of this peptide. Chemicals that are known to activate PKA (Sp-cAMPS) and/or PKG (8-pCPTcGMP) mimic and potentiate DF2's effect by increasing EPSP amplitude. Inhibitors of either PKA (Rp-cAMPS) or PKG (Rp-8-pCPT-cGMPS) block a portion of the increase in EPSP amplitude induced by the peptide. When both kinase inhibitors are applied simultaneously, the entire effect of DF2 on EPSPs is blocked. The PKG inhibitor blocks the effects of a PKG activator but does not alter the effect of a PKA activator on EPSP amplitude. Thus, the PKG inhibitor appears to be relatively specific for PKG. A trend in the data suggests that the PKA inhibitor blocks a portion of the response elicited by the PKG activator. Thus, the PKA inhibitor may be less specific for PKA. Phosphodiesterase inhibitors, which are known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of the peptide. These results support the hypothesis that cAMP and cGMP, acting through their respective protein kinase enzymes, mediate the ability of DFi to increase transmitter output.