Martin Heine

Differential modulation of Ih by 5‐HT receptors in mouse CA1 hippocampal neurons

CA1 pyramidal neurons of the hippocampus express various types of serotonin (5‐HT) receptors, such as 5‐HT1A, 5‐HT4 and 5‐HT7 receptors, which couple to Gαi or Gαs proteins and operate on different intracellular signalling pathways. In the present paper we verify such differential serotonergic modulation for the hyperpolarization‐activated current Ih. Activation of 5‐HT1A receptors induced an augmentation of current‐induced hyperpolarization responses, while the responses declined after 5‐HT4 receptors were activated. The resting potential of neurons hyperpolarized (−2.3u2003±u20030.7u2003mV) after 5‐HT1A receptor activation, activation of 5‐HT4 receptors depolarized neurons (+3.3u2003±u20031.4u2003mV). Direct activation of adenylyl cyclase (AC) by forskolin also produced a depolarization. In voltage clamp, the Ih current was identified by its characteristic voltage‐ and time‐dependency and by blockade with CsCl or ZD7288. Activation of 5‐HT1A receptors reduced Ih and shifted the activation curve to a more negative voltage by −5u2003mV at half‐maximal activation. Activation of 5‐HT4 and 5‐HT7 receptors increased Ih and shifted the activation curve to the right by +5u2003mV. Specific activation of 5‐HT4 receptors by BIMU8 increased membrane conductance and showed an increase in Ih in a subset of cells, but did not induce a significant alteration in the activation curve. In order to verify spatial differences, we applied BIMU8 selectively to the soma and to the dendrites. Only somatic application induced receptor activation. These data are confirmed by immunofluorescence stainings with an antibody against the 5‐HT4A receptor, revealing receptor expression at the somata of the CA1 region. A similar expression pattern was found with a new antibody against 5‐HT7 receptors which reveals immunofluorescence staining on the cell bodies of pyramidal neurons.

The Prostate Specific Membrane Antigen Regulates the Expression of IL-6 and CCL5 in Prostate Tumour Cells by Activating the MAPK Pathways1

The interleukin-6 (IL-6) and the chemokine CCL5 are implicated in the development and progression of several forms of tumours including that of the prostate. The expression of the prostate specific membrane antigen (PSMA) is augmented in high-grade and metastatic tumors. Observations of the clinical behaviour of prostate tumors suggest that the increased secretion of IL-6 and CCL5 and the higher expression of PSMA may be correlated. We hypothesized that PSMA could be endowed with signalling properties and that its stimulation might impact on the regulation of the gene expression of IL-6 and CCL5. We herein demonstrate that the cross-linking of cell surface PSMA with specific antibodies activates the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 in prostate carcinoma LNCaP cells. As downstream effects of the PSMA-fostered RAS-RAC1-MAPK pathway activation we observed a strong induction of NF-κB activation associated with an increased expression of IL-6 and CCL5 genes. Pharmacological blockade with specific inhibitors revealed that both p38 and ERK1/2 participate in the phenomenon, although a major role exerted by p38 was evident. Finally we demonstrate that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically and in a dose-dependent manner and that CCL5 functioned by receptor-mediated activation of the STAT5-Cyclin D1 pro-proliferative pathway. The novel functions attributable to PSMA which are described in the present report may have profound influence on the survival and proliferation of prostate tumor cells, accounting for the observation that PSMA overexpression in prostate cancer patients is related to a worse prognosis.

Distinct Cytoskeletal Tracks Direct Individual Vesicle Populations to the Apical Membrane of Epithelial Cells

A key aspect in the structure of epithelial and neuronal cells is the maintenance of a polarized organization based on highly specific sorting machinery at the exit site of the trans Golgi network (TGN). Epithelial cells sort protein and lipid components into different sets of carriers for the apical or basolateral plasma membrane. The two intestinal proteins lactase-phlorizin hydrolase (LPH) and sucrase-isomaltase (SI) are delivered to the apical plasma membrane of epithelial cells with high fidelity but differ in their affinity to detergent-insoluble, glycolipid-enriched complexes (DIGs). Using a two-color labeling technique, we have recently characterized two post-Golgi vesicle populations that direct LPH and SI separately to the apical cell surface. Here, we investigated the structure and identification of protein components in these vesicle populations and assessed the role of cytoskeletal post-Golgi transport routes for apical cargo. Apart from the central role of microtubules in vesicle transport, we demonstrate that the transport of SI-carrying apical vesicles (SAVs) occurs along actin tracks in the cellular periphery, whereas LPH-carrying apical vesicles (LAVs) are transferred in an actin-independent fashion to the apical membrane. Our data further indicate that myosin 1A is the actin-associated motor protein that drives SAVs along actin filaments to the apical cell surface.

5-Hydroxytryptamine 4(a) receptor expressed in Sf9 cells is palmitoylated in an agonist-dependent manner.

The mouse 5-hydroxytryptamine 4(a) receptor [5-HT(4(a))] was expressed with a baculovirus system in insect cells and analysed for acylation. [(3)H]Palmitic acid was effectively incorporated into 5-HT(4(a)) and label was sensitive to the treatment with reducing agents indicating a thioester-type bond. Analysis of protein-bound fatty acids revealed that 5-HT(4(a)) contains predominantly palmitic acid. Treatment of infected Sf9 (Spodoptera frugiperda) cells with BIMU8 [(endo-N-8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-2,3-dehydro-2-oxo-3-(prop-2-yl)-1H-benzimid-azole-1-carboxamide], a 5-HT(4) receptor-selective agonist, generated a dose-dependent increase in [(3)H]palmitate incorporation into 5-HT(4(a)) with an EC(50) of approx. 10 nM. The change in receptor labelling after stimulation with agonist was receptor-specific and did not result from general metabolic effects. We also used both pulse labelling and pulse-chase labelling to address the dynamics of 5-HT(4(a)) palmitoylation. Incorporation studies revealed that the rate of palmitate incorporation was increased approx. 3-fold after stimulation with agonist. Results of pulse-chase experiments show that activation with BIMU8 promoted the release of radiolabel from 5-HT(4(a)), thereby reducing the levels of receptor-bound palmitate to approximately one-half. Taken together, our results demonstrate that palmitoylation of 5-HT(4(a)) is a reversible process and that stimulation of 5-HT(4(a)) with agonist increases the turnover rate for receptor-bound palmitate. This provides evidence for a regulated cycling of receptor-bound palmitate and suggests a functional role for palmitoylation/depalmitoylation in 5-hydroxytryptamine-mediated signalling.

5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel

Abstract The HvCNG channel from the moth Heliothisvirescens is highly sensitive to cAMP concentrations ranging between 0.1xa0µM and 5xa0µM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0xa0µM and 100xa0µM in the pipette, the half-maximal activation voltage (V1/2) was shifted by +28.5±1.7xa0mV. The activation time constant (τa) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. τa changed from 1106±60xa0ms at 0xa0µM cAMP to 265±7xa0ms at a saturating concentration of 1xa0mM cAMP. A dose–response relationship yielded values of 0.6xa0µM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50xa0µM forskolin induced an elevation of the cAMP level by about 1.6±0.2xa0µM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4(a)- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4(a)-receptors by 0.1xa0µM 5-HT induced an increase of cAMP of about 4.6±1.5xa0µM, whereas cAMP levels decreased from a control value of 1±0.2xa0µM to 0.41±0.1xa0µM after stimulation of the m5-HT1A-receptors.

Impact of glycosylation and detergent-resistant membranes on the function of intestinal sucrase-isomaltase.

Abstract Sucrase-isomaltase (SI) is a highly N- and O-glycosylated intestinal brush border membrane protein. SI is sorted with high fidelity to the apical membrane via O-linked glycans that mediate its association with lipid rafts or detergent-resistant membranes (DRMs). Here, we show that N- and O-glycosylation and DRMs are implicated in the regulation of the function of SI in intestinal Caco-2 cells. The activities of sucrase (SUC) and isomaltase (IM) increase substantially in DRMs when N- and O-glycosylation patterns are intact. Disruption of DRMs by solubilization with Triton X-100 at 37°C substantially reduces the activities of SUC and IM. Furthermore, modulation of O-glycosylation by benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside and N-glycosylation by deoxymannojirimycin is linked to a decreased capacity of SI to associate with DRMs, with a subsequent reduction of the enzymatic activities of SUC and IM. This is the first report that reveals a direct role of N- and O-glycans in association with DRMs in regulating the function of a membrane glycoprotein.

Different glycoforms of prostate-specific membrane antigen are intracellularly transported through their association with distinct detergent-resistant membranes

Hormone-refractory prostate carcinomas as well as the neovasculature of different tumours express high levels of PSMA (prostate-specific membrane antigen). PSMA is a type II-transmembrane glycoprotein and a potential tumour marker for both diagnosis and passive immunotherapy. Here, we report on the association of PSMA with DRMs (detergent-resistant membranes) at different stages of the protein maturation pathway in human prostate carcinoma LNCaP cells. At least three PSMA glycoforms were biochemically identified based on their extractability behaviour in different non-ionic detergents. In particular, one precursor glycoform of PSMA is associated with Tween 20-insoluble DRMs, whereas the complex glycosylated protein segregates into membrane structures that are insoluble in Lubrol WX and display a different lipid composition. Association of PSMA with these membranes occurs in the Golgi compartment together with the acquisition of a native conformation. PSMA homodimers reach the plasma membrane of LNCaP cells in Lubrol WX-insoluble lipid/protein complexes. At the steady state, the majority of PSMA remains within these membrane microdomains at the cell surface. We conclude that the intracellular transport of PSMA occurs through populations of DRMs distinct for each biosynthetic form and cellular compartment.

Functional variants in the sucrase-isomaltase gene associate with increased risk of irritable bowel syndrome

Objective IBS is a common gut disorder of uncertain pathogenesis. Among other factors, genetics and certain foods are proposed to contribute. Congenital sucrase–isomaltase deficiency (CSID) is a rare genetic form of disaccharide malabsorption characterised by diarrhoea, abdominal pain and bloating, which are features common to IBS. We tested sucrase–isomaltase (SI) gene variants for their potential relevance in IBS. Design We sequenced SI exons in seven familial cases, and screened four CSID mutations (p.Val557Gly, p.Gly1073Asp, p.Arg1124Ter and p.Phe1745Cys) and a common SI coding polymorphism (p.Val15Phe) in a multicentre cohort of 1887 cases and controls. We studied the effect of the 15Val to 15Phe substitution on SI function in vitro. We analysed p.Val15Phe genotype in relation to IBS status, stool frequency and faecal microbiota composition in 250 individuals from the general population. Results CSID mutations were more common in patients than asymptomatic controls (p=0.074; OR=1.84) and Exome Aggregation Consortium reference sequenced individuals (p=0.020; OR=1.57). 15Phe was detected in 6/7 sequenced familial cases, and increased IBS risk in case–control and population-based cohorts, with best evidence for diarrhoea phenotypes (combined p=0.00012; OR=1.36). In the population-based sample, 15Phe allele dosage correlated with stool frequency (p=0.026) and Parabacteroides faecal microbiota abundance (p=0.0024). The SI protein with 15Phe exhibited 35% reduced enzymatic activity in vitro compared with 15Val (p<0.05). Conclusions SI gene variants coding for disaccharidases with defective or reduced enzymatic activity predispose to IBS. This may help the identification of individuals at risk, and contribute to personalising treatment options in a subset of patients.

Structure of Excitatory Synapses and GABAA Receptor Localization at Inhibitory Synapses Are Regulated by Neuroplastin-65

Background: Synaptic excitatory to inhibitory ratio is crucial for brain function but its regulation is poorly understood. Results: The cell adhesion molecule neuroplastin-65 regulates contact and stability of excitatory synapses and localization of GABAAR α2 subunits at inhibitory synapses. Conclusion: Neuroplastin-65 mediates specific neuronal connections and regulates the number and function of synapses in hippocampal neurons. Significance: Correct neuronal network activity in specific brain regions may depend on neuroplastin-65. Formation, maintenance, and activity of excitatory and inhibitory synapses are essential for neuronal network function. Cell adhesion molecules (CAMs) are crucially involved in these processes. The CAM neuroplastin-65 (Np65) highly expressed during periods of synapse formation and stabilization is present at the pre- and postsynaptic membranes. Np65 can translocate into synapses in response to electrical stimulation and it interacts with subtypes of GABAA receptors in inhibitory synapses. Here, we report that in the murine hippocampus and in hippocampal primary culture, neurons of the CA1 region and the dentate gyrus (DG) express high Np65 levels, whereas expression in CA3 neurons is lower. In neuroplastin-deficient (Np−/−) mice the number of excitatory synapses in CA1 and DG, but not CA3 regions is reduced. Notably this picture is mirrored in mature Np−/− hippocampal cultures or in mature CA1 and DG wild-type (Np+/+) neurons treated with a function-blocking recombinant Np65-Fc extracellular fragment. Although the number of GABAergic synapses was unchanged in Np−/− neurons or in mature Np65-Fc-treated Np+/+ neurons, the ratio of excitatory to inhibitory synapses was significantly lower in Np−/− cultures. Furthermore, GABAA receptor composition was altered at inhibitory synapses in Np−/− neurons as the α1 to α2 GABAA receptor subunit ratio was increased. Changes of excitatory and inhibitory synaptic function in Np−/− neurons were confirmed evaluating the presynaptic release function and using patch clamp recording. These data demonstrate that Np65 is an important regulator of the number and function of synapses in the hippocampus.

Congenital sucrase-isomaltase deficiency: heterogeneity of inheritance, trafficking, and function of an intestinal enzyme complex.

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