Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors

ATP-gated P2X(2) receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X(2) receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X(2)-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X(2)-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X(2)-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X(2) receptor distribution on the seconds time scale.     

Immunological role of neuronal receptor vanilloid receptor 1 expressed on dendritic cells

Capsaicin (CP), the pungent component of chili pepper, acts on sensory neurons to convey the sensation of pain. The CP receptor, vanilloid receptor 1 (VR1), has been shown to be highly expressed by nociceptive neurons in dorsal root and trigeminal ganglia. We demonstrate here that the dendritic cell (DC), a key cell type of the vertebrate immune system, expresses VR1. Engagement of VR1 on immature DCs such as by treatment with CP leads to maturation of DCs as measured by up-regulation of antigen-presenting and costimulatory molecules. This effect is present in DCs of VR1+/+ but not VR1-/- mice. In VR1+/+ mice, this effect is inhibited by the VR1 antagonist capsazepine. Further, intradermal administration of CP leads to migration of DCs to the draining lymph nodes in VR1+/+ but not VR1-/- mice. These data demonstrate a powerful influence of a neuroactive ligand on a central aspect of immune function and a commonality of mechanistic pathways between neural and immune functions.     

Role for Plk1 phosphorylation of Hbo1 in regulation of replication licensing

In a search for Polo-like kinase 1 (Plk1)-interacting proteins using a yeast two-hybrid system, we have identified histone acetyltransferase binding to the origin recognition complex 1 (Hbo1) as a potential Plk1 target. Here, we show that the interaction between Plk1 and Hbo1 is mitosis-specific and that Plk1 phosphorylates Hbo1 on Ser-57 in vitro and in vivo. During mitosis, Cdk1 phosphorylates Hbo1 on Thr-85/88, creating a docking site for Plk1 to be recruited. Significantly, the overexpression of Hbo1 mutated at the Plk1 phosphorylation site (S57A) leads to cell-cycle arrest in the G₁/S phase, inhibition of chromatin loading of the minichromosome maintenance (Mcm) complex, and a reduced DNA replication rate. Similarly, Hbo1 depletion results in decreased DNA replication and a failure of Mcm complex binding to chromatin, both of which can be partially rescued by the ectopic expression of WT Hbo1 but not Hbo1-S57A. These results suggest that Plk1 phosphorylation of Hbo1 may be required for prereplicative complex (pre-RC) formation and DNA replication licensing.     

Age-dependent variation in the cytosol/membrane distribution of red cell protein kinase-C

An age-dependent increase in membrane association of protein kinase-c and a decrease in the cytosolic enzyme, especially in the densest fraction rich in senescent red cells was observed in Stractan-gradient-separated normal erythrocytes.     

NMDA受体NR1、NR2A/B在丘脑前核-海马CA1、CA3脑区和齿状回的分布与表达

目的 探讨NMDA受体NR1、NR2A/B在丘脑前核-海马CA1、CA3脑区和齿状回的分布与表达,以及丘脑前核-海马神经元的学习记忆功能及作用机制.方法 运用原位杂交检测技术观测丘脑前核及海马CA1、CA3和齿状回内NMDA受体NR1、NR2A 及NR2B mRNA的分布特点.结果 ①原位杂交阳性产物呈棕黄色,主要分布在神经元的胞浆中,胞核基本不着色.②在丘脑前核,阳性神经元分布较密集,细胞形态较一致.③在海马锥体层阳性神经元分布较多,呈带状.在分子层、多形层分布少.④NR1、NR2A/B在丘脑前核和海马CA1、CA3脑区及齿状回均有表达,其中NR1在齿状回表达水平最强,NR2B在丘脑前核、海马CA1、CA3和齿状回表达水平基本相同.结论 在丘脑前核-海马的局部神经元环路中NMDA受体NR1、NR2A及NR2B mRNA分布广泛.其中NR2B mRNA在丘脑前核和海马CA1、CA3脑区及齿状回表达水平基本相同,可能与此环路学习记忆有关.     

Cross-platform validation of neurotransmitter release impairments in schizophrenia patient-derived NRXN1-mutant neurons

Heterozygous NRXN1 deletions constitute the most prevalent currently known single-gene mutation associated with schizophrenia, and additionally predispose to multiple other neurodevelopmental disorders. Engineered heterozygous NRXN1 deletions impaired neurotransmitter release in human neurons, suggesting a synaptic pathophysiological mechanism. Utilizing this observation for drug discovery, however, requires confidence in its robustness and validity. Here, we describe a multicenter effort to test the generality of this pivotal observation, using independent analyses at two laboratories of patient-derived and newly engineered human neurons with heterozygous NRXN1 deletions. Using neurons transdifferentiated from induced pluripotent stem cells that were derived from schizophrenia patients carrying heterozygous NRXN1 deletions, we observed the same synaptic impairment as in engineered NRXN1-deficient neurons. This impairment manifested as a large decrease in spontaneous synaptic events, in evoked synaptic responses, and in synaptic paired-pulse depression. Nrxn1-deficient mouse neurons generated from embryonic stem cells by the same method as human neurons did not exhibit impaired neurotransmitter release, suggesting a human-specific phenotype. Human NRXN1 deletions produced a reproducible increase in the levels of CASK, an intracellular NRXN1-binding protein, and were associated with characteristic gene-expression changes. Thus, heterozygous NRXN1 deletions robustly impair synaptic function in human neurons regardless of genetic background, enabling future drug discovery efforts.

Schizophrenia is a devastating brain disorder that affects millions of people worldwide and exhibits a strong genetic component. In a key discovery, deletions or duplications of larger stretches of chromosomal DNA that lead to copy number variations (CNVs) were identified two decades ago (1, 2). CNVs occur unexpectedly frequently, are often de novo, and usually affect multiple genes depending on the size of the deleted or duplicated stretch of DNA. Strikingly, the biggest genetic risk for schizophrenia was identified in three unrelated CNVs: a duplication of region 16p11.2 and deletions of 22q11.2 and of 2p16.3 (3–9). Of these CNVs, 16p11.2 and 22q11.2 CNVs affect more than 20 genes, whereas 2p16.3 CNVs impact only one or more exons of a single gene, NRXN1, which encodes the presynaptic cell-adhesion molecule neurexin-1 (4, 7, 9–12). NRXN1 CNVs confer an approximately 10-fold increase in risk of schizophrenia, and additionally strongly predispose to other neuropsychiatric disorders, especially autism and Tourette syndrome (13, 14). Moreover, genome-wide association studies using DNA microarrays identified common changes in many other genes that predispose to schizophrenia with smaller effect sizes (15–21). Viewed together, these studies indicate that variations in a large number of genes are linked to schizophrenia. Among these genetic variations, heterozygous exonic CNVs of NRXN1 are rare events, but nevertheless constitute the most prevalent high-risk single-gene association at present.Neurexins are central regulators of neural circuits that control diverse synapse properties, such as the presynaptic release probability, the postsynaptic receptor composition, and synaptic plasticity (22–28). To test whether heterozygous NRXN1 mutations might cause functional impairments in human neurons, we previously generated conditionally mutant human embryonic stem (ES) cells that enabled induction of heterozygous NRXN1 deletions using Cre-recombinase (29). We then analyzed the effects of the deletion on the properties of neurons induced from the conditionally mutant ES cells using forced expression of Ngn2, a method that generates a relatively homogeneous population of excitatory neurons that are also referred to as induced neuronal (iN) cells (30). These experiments thus examined isogenic neurons without or with a heterozygous NRXN1 loss-of-function mutation that mimicked the schizophrenia-associated 2p16.3 CNVs, enabling precise control of the genetic background. The heterozygous NRXN1 deletion produced a robust but discrete impairment in neurotransmitter release without major changes in neuronal development or morphology (29). These results were exciting because they suggested that a discrete impairment in neurotransmitter release could underlie the predisposition to schizophrenia conferred by the 2p16.3 CNV, but these experiments did not reveal whether the NRXN1 mutation induces the same synaptic impairment in schizophrenia patients (31).The present project was initiated to achieve multiple overlapping aims emerging from the initial study on human NRXN1 mutations (29). First, we aimed to validate or refute the results obtained with neurons generated from engineered conditionally mutant ES cells with neurons generated from patient-derived induced pluripotent stem (iPS) cells containing NRXN1 mutations (Fig. 1A). This goal was pursued in order to gain confidence in the disease-relevance of the observed phenotypes. Second, we wanted to test whether the observed phenotype is independent of the laboratory of analysis (i.e., whether it is sufficiently robust to be replicated at multiple sites) (Fig. 1A). This goal was motivated by the observation of limited reproducibility in some studies of the phenotypes of patient-derived neurons. We hypothesized that this lack of reproducibility is due to variations in experimental conditions rather than an experimental failure, and designed our studies to demonstrate robustness of the findings through replication. Third, we aimed to generate reagents that could be broadly used by the scientific community for investigating the cellular basis of neuropsychiatric disorders (32). This goal was prompted by the challenges posed by the finding that many different genes appear to be linked to schizophrenia. Fourth, we aimed to definitively establish or exclude the possibility that human neurons are uniquely sensitive to a heterozygous loss of NRXN1 compared with mouse neurons (Fig. 1B). The goal here was to test whether at least as regards to NRXN1, mouse and human neurons exhibit fundamental differences. Fifth and finally, we hoped to gain further insights into the mechanisms by which NRXN1 mutations predispose to schizophrenia, an obviously needed objective given our lack of understanding of this severe disorder. As described in detail below, our data provide advances toward meeting these goals, establishing unequivocally that heterozygous NRXN1 deletions in human but not in mouse neurons cause a robust impairment in neurotransmitter release that is replicable in multiple laboratories.Open in a separate windowFig. 1.Overall study design illustrating the experimental approach to analyze human heterozygous NRXN1 loss-of-function mutations, to achieve cross-laboratory and cross-platform validation of observed phenotypes, and to perform cross-paradigm evaluations of these phenotypes in human and mouse neurons. (A) Experimental strategy for analyzing the functional effects of heterozygous NRXN1 loss-of-function mutations in human patient-derived neurons and for validating the observed phenotypes in a cross-laboratory and cross-platform comparison. PBMCs from schizophrenia patients with NRXN1 deletions and from control individuals were reprogrammed into iPS cells by Rutgers University (RUCDR Infinite Biologics). iPS cells that passed QC were shipped to Stanford and to FCDI for expansion, banking, and transdifferentiation into induced neurons. The indicated subsequent analyses were carried out at Stanford University and at Rutgers University. FCDI manufactured industry-scale human induced neurons that were shipped to Rutgers for analysis, whereas Stanford generated induced neurons at an academic single-laboratory scale for analysis. (B) Experimental strategy to evaluate the conservation of NRXN1-deletion phenotypes observed in human neurons in mouse neurons (cross-paradigm evaluation). Human and mouse stem cells that carried heterozygous engineered conditional NRXN1/Nrxn1 deletions were transdifferentiated into neurons by Ngn2 expression and analyzed using similar approaches to ensure comparability. In this approach, isogenic human and mouse neurons without or with NRXN1/Nrxn1 deletions were compared to test whether side-by-side analysis of human and mouse neurons prepared by indistinguishable approaches yields similar phenotypes.     

JNK1 controls mast cell degranulation and IL-1{beta} production in inflammatory arthritis

Rheumatoid arthritis (RA) is a chronic inflammatory disease marked by bone and cartilage destruction. Current biologic therapies are beneficial in only a portion of patients; hence small molecules targeting key pathogenic signaling cascades represent alternative therapeutic strategies. Here we show that c-Jun N-terminal kinase (JNK) 1, but not JNK2, is critical for joint swelling and destruction in a serum transfer model of arthritis. The proinflammatory function of JNK1 requires bone marrow-derived cells, particularly mast cells. Without JNK1, mast cells fail to degranulate efficiently and release less IL-1β after stimulation via Fcγ receptors (FcγRs). Pharmacologic JNK inhibition effectively prevents arthritis onset and abrogates joint swelling in established disease. Hence, JNK1 controls mast cell degranulation and FcγR-triggered IL-1β production, in addition to regulating cytokine and matrix metalloproteinase biosynthesis, and is an attractive therapeutic target in inflammatory arthritis.     

Defining roles of PARKIN and ubiquitin phosphorylation by PINK1 in mitochondrial quality control using a ubiquitin replacement strategy

The PTEN-induced putative kinase protein 1 (PINK1) and ubiquitin (UB) ligase PARKIN direct damaged mitochondria for mitophagy. PINK1 promotes PARKIN recruitment to the mitochondrial outer membrane (MOM) for ubiquitylation of MOM proteins with canonical and noncanonical UB chains. PINK1 phosphorylates both Ser65 (S65) in the UB-like domain of PARKIN and the conserved Ser in UB itself, but the temporal sequence and relative importance of these events during PARKIN activation and mitochondria quality control remain poorly understood. Using “UB^S65A-replacement,” we find that PARKIN phosphorylation and activation, and ubiquitylation of Lys residues on a cohort of MOM proteins, occur similarly irrespective of the ability of the UB-replacement to be phosphorylated on S65. In contrast, polyubiquitin (poly-UB) chain synthesis, PARKIN retention on the MOM, and mitophagy are reduced in UB^S65A-replacement cells. Analogous experiments examining roles of individual UB chain linkage types revealed the importance of K6 and K63 chain linkages in mitophagy, but phosphorylation of K63 chains by PINK1 did not enhance binding to candidate mitophagy receptors optineurin (OPTN), sequestosome-1 (p62), and nuclear dot protein 52 (NDP52) in vitro. Parallel reaction monitoring proteomics of total mitochondria revealed the absence of p-S65-UB when PARKIN cannot build UB chains, and <0.16% of the monomeric UB pool underwent S65 phosphorylation upon mitochondrial damage. Combining p-S65-UB and p-S65-PARKIN in vitro showed accelerated transfer of nonphosphorylated UB to PARKIN itself, its substrate mitochondrial Rho GTPase (MIRO), and UB. Our data further define a feed-forward mitochondrial ubiquitylation pathway involving PARKIN activation upon phosphorylation, UB chain synthesis on the MOM, UB chain phosphorylation, and further PARKIN recruitment and enzymatic amplification via binding to phosphorylated UB chains.The RING-Between-RING ubiquitin (UB) ligase PARKIN and the PTEN-induced putative kinase protein 1 (PINK1), both of which are mutated in Parkinson’s disease, promote ubiquitylation of numerous outer membrane proteins on damaged mitochondria, which facilitates mitophagy (1). When mitochondria are healthy, cytoplasmic PARKIN is thought to be in an unphosphorylated and auto-inhibited state (2–6). Upon mitochondrial damage, PINK1 is stabilized on the mitochondrial outer membrane (MOM) and promotes phosphorylation of both Ser65 (S65) on PARKIN’s UB-like (UBL) domain and the conserved S65 residue in UB itself, which is 62% similar to PARKIN’s UBL (1, 7–14). PARKIN is also retained on the mitochondrial surface and ubiquitylates numerous MOM proteins through the formation of both canonical and noncanonical UB chains. The order of events, as well as the precise roles of UB and PARKIN phosphorylation, is poorly defined, and multiple models have been proposed. All models agree that PARKIN activation reverses auto-inhibition but differ in the mechanism and role of p-S65-UB (8, 11–15). In one model, binding of monomeric p-S65-UB to unphosphorylated PARKIN activates its UB ligase activity in vitro and promotes MOM ubiquitylation in vivo (11, 13, 14). Additionally, overexpression of UB^S65E/D mutants as mimetics of p-S65-UB in cells has been reported to activate PARKIN (13–15). However, these studies have differed as to the relative importance of PARKIN and UB phosphorylation on pathway activation. In an alternative model, PINK1 initially phosphorylates a pool of PARKIN on S65 without a requirement for retention of PARKIN on mitochondria, resulting in PARKIN-dependent ubiquitylation of MOM proteins (12, 13, 15). Newly synthesized UB chains are then phosphorylated by PINK1, facilitating recruitment of PARKIN through its p-S65-UB binding ability, resulting in both further PARKIN recruitment and additional MOM ubiquitylation (12). Although PARKIN S65 phosphorylation increases its affinity for p-S65-UB by 20-fold (12), phospho-UB on the MOM could potentially serve to recruit pools of both phosphorylated and unphosphorylated PARKIN.Dissecting roles of UB and PARKIN phosphorylation must take into account the complexity inherent in this system, where UB (i) is transferred between multiple active sites involving the UB-conjugating enzyme UBCH7 and PARKIN, (ii) is linked to primary sites on MOM proteins, (iii) is phosphorylated by PINK1 to generate forms recognized by PARKIN itself, and (iv) is also recognized by UB receptors that promote mitophagy. PARKIN itself has a UBL domain with many similarities to UB, including being phosphorylated on the conserved S65. This system is made more complex by the fact that tools for studying phosphorylation of UB are limited largely to overexpression of phosphomimetics in vivo (13–15). However, as demonstrated here, neither UB nor PARKIN phosphomimetics faithfully mimic the biochemical activities of phosphorylated PARKIN and UB in vitro, raising questions as to the in vivo roles of PINK1-dependent phosphorylation on PARKIN and/or UB deduced using these approaches.Here, we used a UB-replacement strategy (16) to deplete all four mRNAs encoding UB in cultured cells while expressing UB^S65A or UB mutants lacking individual Lys residues involved in chain assembly, thereby allowing an assessment of the role of UB phosphorylation and individual chain linkage types in PARKIN activation, MOM protein ubiquitylation, PARKIN retention on mitochondria, and mitophagy. Moreover, we developed in vitro pulse–chase and multiple-turnover assays to examine the role of p-S65-UB in individual steps in UB transfer by PARKIN. These studies rationalize many previous observations (10–15, 17) and provide further insight into how phosphorylation of both UB and PARKIN promotes a “feed-forward” mechanism controlling mitochondrial ubiquitylation and mitophagy.     

脑通胶囊对VD大鼠海马组织NMDA受体1亚基和2B亚基mRNA表达的影响

目的观察脑通胶囊对血管性痴呆(VD)模型大鼠学习记忆、海马组织N-甲基-D-天冬氨酸(NMDA)受体1亚基和2B亚基mRNA表达的影响。方法采用改良的四血管法(14-VO)制备VD模型,Morris水迷宫测定大鼠学习、记忆能力,实时荧光定量PCR检测NMDA受体1亚基和2B亚基mRNA的表达情况。结果与模型组比较,脑通胶囊大、中剂量组逃避潜伏期缩短,穿越原平台次数增加,NMDA受体1亚基mRNA表达降低,NMDA受体2B亚基mRNA表达升高(P<0.05,P<0.01)。结论脑通胶囊可降低NMDA受体1亚基mRNA的表达,提高NMDA受体2B亚基mRNA的表达,从而改善VD大鼠学习、记忆能力。     

Differential roles of the dopamine 1-class receptors,D1R and D5R,in hippocampal dependent memory

Activation of the hippocampal dopamine 1-class receptors (D1R and D5R) are implicated in contextual fear conditioning (CFC). However, the specific role of the D1R versus D5R in hippocampal dependent CFC has not been investigated. Generation of D1R- and D5R-specific in situ hybridization probes showed that D1R and D5R mRNA expression was greatest in the dentate gyrus (DG) of the hippocampus. To identify the role of each receptor in CFC we generated spatially restricted KO mice that lack either the D1R or D5R in DG granule cells. DG D1R KOs displayed significant fear memory deficits, whereas DG D5R KOs did not. Furthermore, D1R KOs but not D5R KOs, exhibited generalized fear between two similar but different contexts. In the familiar home cage context, c-Fos expression was relatively low in the DG of control mice, and it increased upon exposure to a novel context. This level of c-Fos expression in the DG did not further increase when a footshock was delivered in the novel context. In DG D1R KOs, DG c-Fos levels in the home cage was higher than that of the control mice, but it did not further increase upon exposure to a novel context and remained at the same level upon a shock delivery. In contrast, the levels of DG c-Fos expression was unaffected by the deletion of DG D5R neither in the home cage nor upon a shock delivery. These results suggest that DG D1Rs, but not D5Rs, contribute to the formation of distinct contextual representations of novel environments.The hippocampus is crucial for aversive Pavlovian conditioning, such as contextual fear conditioning (CFC) (1, 2). In CFC, the conditioned stimulus (context) is paired with the unconditioned stimulus (footshock), and after pairing, the context serves as a cue to predict a potential footshock (3, 4). Although the role of dopamine has been studied in the context of reward learning (5), evidence suggests that midbrain dopaminergic neurons are also important for aversive Pavlovian conditioning (6–9). In line with this evidence, hippocampal encoding of novel and contextual information is linked to dopamine release via excitation of dopamine neurons of the midbrain (5, 10, 11). Additionally, delivery of aversive stimuli, such as a footshock, results in increased dopaminergic neuron activity (12). Moreover, inactivation of hippocampal D1Rs and D5Rs attenuates contextual fear memory (13). Thus, it follows that delivery of an aversive stimulus activates midbrain dopamine neurons that project to the hippocampus, which is crucial for encoding novel contextual cues (12, 14, 15). Activation of hippocampal D1Rs and D5Rs may then strengthen the encoding of novel contextual information during CFC.The precise role of subregion-specific D1R or D5R activation in hippocampal-dependent learning and memory is unknown. This is in part due to the inability to discriminate between and spatially restrict D1R from D5R function (16–18), which is an important caveat because each receptor is involved in modulating distinct neuronal processes (19–22). Indeed, there is a lack of consensus of D1R and D5R expression patterns in the rodent hippocampus (23–27). Moreover, pharmacological findings are at odds with D1R and D5R global KO studies, which show that neither D1Rs nor D5Rs are required for fear conditioning (16, 17). Therefore, to reconcile these disparate findings and to test the necessity of D1R and D5R activation for CFC, it is necessary to functionally isolate and spatially restrict hippocampal D1R and D5R activity.In this study, we found that D1Rs and D5Rs exhibit overlapping expression in dentate gyrus (DG) granule cells. DG D1R activation is necessary to increase c-Fos expression in the DG and CA3 to enhance novel contextual encoding. Moreover, DG D1R activation decreases generalization of the conditioned fear response to novel contexts. However, we found no role for DG D5Rs in modulating DG c-Fos expression or contextual fear learning and memory. In using our subregion-specific KO mice, we show that the hippocampal dopamine signal plays a definitive role in CFC.     

HIV-1 gp120 induces autophagy in cardiomyocytes via the NMDA receptor

Background

HIV-1 envelope glycoprotein gp120 (gp120) is considered as one of the major virulent proteins responsible for the involvement of the cardiovascular system. Autophagy as a form of self maintenance plays important roles in cell survival and death. HIV-1 gp120 is reported to induce autophagy in a variety of cells. However, the effect of gp120 on autophagy in cardiomyocytes has not been reported. This study aimed to test our hypothesis that gp120 could induce autophagy in cardiomyocytes.

Methods

Rat cardiomyocyte H9c2 cells were treated with gp120 (100 ng/ml) in vitro for 4 h, 1 day and 7 days. The autophagy related proteins were analyzed by Western blot and the autophagosomes were analyzed by confocal microscopy.

Results

The autophagic proteins and autophagosomes were markedly increased in the H9c2 cells after 4 h of gp120 treatment. Furthermore, gp120 induced autophagic proteins and autophagosomes were significantly inhibited by the N-methyl-d-aspartic acid (NMDA) receptor inhibitor MK801, c-Jun N-terminal kinase (JNK) inhibitor SP600125, and the class III phosphoinositide 3-kinase (PI3K) inhibitor 3-methyladenine (3-MA), while there was no change in the cells pretreated with the CXCR4 antagonist AMD3100. In addition, no apparent cell death was observed in the cardiomyocytes treated with gp120 for up to 7 days.

Conclusions

In summary, our research demonstrated for the first time that HIV-1 gp120 could induce autophagy of cardiomyocytes and the NMDA receptor, JNK and class III PI3K were involved in this process. This observation provides a new insight into the mechanisms of in the cardiovascular involvement during HIV-1 infection.     

Dynamic modulation of Ca2+ sparks by mitochondrial oscillations in isolated guinea pig cardiomyocytes under oxidative stress

Local control of Ca²⁺-induced Ca²⁺ release (CICR) depends on the spatial organization of L-type Ca²⁺ channels and ryanodine receptors (RyR) in the dyad. Analogously, Ca²⁺ uptake by mitochondria is facilitated by their close proximity to the Ca²⁺ release sites, a process required for stimulating oxidative phosphorylation during changes in work. Mitochondrial feedback on CICR is less well understood. Since mitochondria are a primary source of reactive oxygen species (ROS), they could potentially influence the cytosolic redox state, in turn altering RyR open probability. We have shown that self-sustained oscillations in mitochondrial inner membrane potential (ΔΨ_m), NADH, ROS, and reduced glutathione (GSH) can be triggered by a laser flash in cardiomyocytes. Here, we employ this method to directly examine how acute changes in energy state dynamically influence resting Ca²⁺ spark occurrence and properties. Two-photon laser scanning microscopy was used to monitor cytosolic Ca²⁺ (or ROS), ΔΨ_m, and NADH (or GSH) simultaneously in isolated guinea pig cardiomyocytes. Resting Ca²⁺ spark frequency increased with each ΔΨ_m depolarization and decreased with ΔΨ_m repolarization without affecting Ca²⁺ spark amplitude or time-to-peak. Stabilization of mitochondrial energetics by pretreatment with the superoxide scavenger TMPyP, or by acute addition of 4′-chlorodiazepam, a mitochondrial benzodiazepine receptor antagonist that blocks the inner membrane anion channel, prevented or reversed, respectively, the increased spark frequency. Cyclosporine A did not block the ΔΨ_m oscillations or prevent Ca²⁺ spark modulation by ΔΨ_m. The results support the hypothesis that mitochondria exert an influential role on the redox environment of the Ca²⁺ handling subsystem, with mechanistic implications for the pathophysiology of cardiac disease.     

Protein kinase C isoforms α, δ and θ require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells)

Summary Protein kinase C (PKC) isoforms are potentially important as modulators of the insulin signalling chain and could be involved in the pathogenesis of cellular insulin resistance. We have previously shown that phorbol ester stimulated PKC β1 and β2 as well as tumor necrosis factor-α (TNFα) stimulated PKC ɛ inhibit human insulin receptor (HIR) signalling. There is increasing evidence that the insulin receptor substrate-1 (IRS-1) is involved in inhibitory signals in insulin receptor function. The aim of the present study was to elucidate the role of IRS-1 in the inhibitory effects of protein kinase C on human insulin receptor function. HIR, PKC isoforms (α, β1, β2, γ, δ, ɛ, η, θ and ζ) and IRS-1 were coexpressed in human embryonic kidney (HEK) 293 cells. PKCs were activated by preincubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (CTPA) (10^––7 mol/l) following insulin stimulation. While PKCs α, δ and θ were not inhibitory in HEK 293 cells which were transfected only with HIR and PKC, additional transfection of IRS-1 induced a strong inhibitory effect of these PKC isoforms being maximal for PKC θ (99 ± 1.8 % inhibition of insulin stimulated receptor autophosphorylation, n = 7, p < 0.001). No effect was seen with PKC γ, ɛ, ζ and η while the earlier observed insulin receptor kinase inhibition of PKC β2 was further augmented (91 ± 13 %, n = 7, p < 0.001 instead of 45 % without IRS-1). The strong inhibitory effect of PKC θ is accompanied by a molecular weight shift of IRS-1 (183 kDa vs 180 kDa) in the sodium dodecyl sulphate polyacrylamide gel. This can be reversed by alkaline phosphatase treatment of IRS-1 suggesting that this molecular weight shift is due to an increased phosphorylation of IRS-1 on serine or threonine residues. In summary, these data show that IRS-1 is involved in the inhibitory effect of the PKC isoforms α, β2, δ and θ and it is likely that this involves serine/threonine phosphorylation of IRS-1. [Diabetologia (1998) 41: 833–838] Received: 11 February 1998 and in revised form 2 April 1998     

MT1 and MT2 melatonin receptors are expressed in nonoverlapping neuronal populations

Melatonin (MLT) exerts its physiological effects principally through two high‐affinity membrane receptors MT1 and MT2. Understanding the exact mechanism of MLT action necessitates the use of highly selective agonists/antagonists to stimulate/inhibit a given MLT receptor. The respective distribution of MT1 and MT2 within the CNS and elsewhere is controversial, and here we used a “knock‐in” strategy replacing MT1 or MT2 coding sequences with a LacZ reporter. The data show striking differences in the distribution of MT1 and MT2 receptors in the mouse brain: whereas the MT1 subtype was expressed in very few structures (notably including the suprachiasmatic nucleus and pars tuberalis), MT2 subtype receptors were identified within numerous brain regions including the olfactory bulb, forebrain, hippocampus, amygdala and superior colliculus. Co‐expression of the two subtypes was observed in very few structures, and even within these areas they were rarely present in the same individual cell. In conclusion, the expression and distribution of MT2 receptors are much more widespread than previously thought, and there is virtually no correspondence between MT1 and MT2 cellular expression. The precise phenotyping of cells/neurons containing MT1 or MT2 receptor subtypes opens new perspectives for the characterization of links between MLT brain targets, MLT actions and specific MLT receptor subtypes.     

Different responses to EGF in two human carcinoma cell lines, A431 and UCVA-1, possessing high numbers of EGF receptors

EGF binding capacity was examined in 9 different human cell lines which were derived from colon, rectum and pancreas tumors. Among these cell lines, a pancreatic carcinoma cell line, UCVA-1, was found to possess a high number (0.9 X 10(6)/cell) of EGF receptors. This number is comparable to that of EGF receptors in human vulva epidermoid carcinoma A431 cells (2 X 10(6)/cell). However, it was found that, unlike A431 cells, the growth of UCVA-1 cells, in serum-containing and serum-free conditions, was not inhibited by EGF. The UCVA-1 cells have EGF receptor of Mr = 170 K and of two affinity types: Kd1 = 72 X 10(-9) M and Kd2 = 2 X 10(-8) M. The EGF receptors in UCVA-1 cells are less susceptible to proteolytic cleavage than those in A431 cells. In UCVA-1 cells, EGF is apparently processed via a receptor-mediated endocytosis. The UCVA-1 cell membrane contained EGF-stimulated protein kinase as was found in A431 cells. The stimulation of phosphorylation by EGF was only approximately 20% in UCVA-1 while it was over 100% in A431. When angiotensin II was used as a substrate, the relative activity of EGF-dependent tyrosine-specific protein phosphorylation was approximately 8 times less in UCVA-1 cell membrane. The EGF-stimulated phosphorylation was mostly on EGF receptors for both cell lines. However, several other components (Mr = 100 K, 80 K, 72 K and 65 K) were readily detected in A431 cells. These observations indicate that the EGF receptor/protein kinase relation differs in these two cell lines and suggests that it may be related to the growth-inhibitory effect of EGF seen in A431.     

A 973 valine to methionine mutation of the human insulin receptor: interaction with insulin-receptor substrate-1 and Shc in HEK 293 cells

Summary A population-based study in the Netherlands has recently demonstrated that a mutation of the human insulin receptor (HIR-973 valine to methionine) is associated with hyperglycaemia and an increased prevalence of non-insulin-dependent diabetes mellitus (NIDDM). The aim of the present study was to assess whether this mutation leads to a functional alteration of the insulin receptor. We prepared the HIR-973 mutant by in vitro mutagenesis. This mutant was transiently overexpressed in HEK 293 cells either alone or together with insulin-receptor substrate-1 (IRS-1) or Shc. Insulin stimulated autophosphorylation, phosphorylation of the substrates IRS-1 and Shc as well as activation of phosphatidylinositol-3 (PI3)-kinase were studied. Autophosphorylation of HIR-973 and its susceptibility to hyperglycaemia induced inhibition was not different from HIR-wt. Human insulin receptor with a juxtamembrane deletion HIR-ΔJM which is known to impair HIR/IRS-1 interaction was used as control. While the HIR-ΔJM induces a reduced IRS-1 phosphorylation HIR-973 showed even a slightly increased ability to phosphorylate IRS-1 (n = 7, 115 % of control, p < 0.01). Shc phosphorylation was only mediated by HIR-wt and HIR-973 but not by HIR-ΔJM. Again a tendency to higher phosphorylation of Shc was seen with HIR-973 (n = 7, 109 % of control, NS). When PI3-kinase activity was measured in IRS-1 precipitates similar activity was found for HIR-wt and HIR-973 whereas PI3-kinase stimulation was reduced with HIR-ΔJM. In summary, the data suggest that HIR-973 does not impair the first steps of the insulin signalling cascade. It is therefore unlikely that this mutation may cause cellular insulin resistance. The close vicinity of this mutation to insulin receptor domains which are involved in IRS-1 and Shc binding may, however, alter the interaction of the insulin receptor with these substrates. This could explain the slightly increased insulin effect on tyrosine phosphorylation of these docking proteins. These characteristics of HIR-973 might have a compensatory function of impaired signal transduction further downstream of the signalling chain in this specific subgroup of NIDDM patients. [Diabetologia (1997) 40: 1135–1140] Received: 29 January 1997 and in final revised form: 18 June 1997