Prof. Giuliano Meyer, Prof. Maria Novella Orsenigo.

The research lines run by this Unit include the molecular physiology and the physiology of membrane transports. Our scientific interest area in molecular physiology concernes the structure-function correlation and protein-protein interaction study of different membrane proteins. In particular we are interested in the involvement of the ICln protein, an ion channel cloned from a dog renal epithelial cell line, in cell volume regulation. In the field of transport physiology we are studying the function and the molecular identification of membrane carriers and channels.  We are analysing the membrane transport regulations as a function of the cellular homeostasis and of the transepithelial absorptions or secretions. Our laboratory team has a long experience in  electrophysiological, radiochemical, fluorimetric and molecular biology techniques.

Research topics:

Molecular Physiology:

1. The PH domain of ICln and the interaction with phosphatidylinositols

Meyer, Bazzini

In a variety of cell types, swelling coincides with an increase in phosphatidylinositol-3-kinase (PI-3-K) activity. In particular, with patch-clamp experiments, we saw that pre-treatment of the cells with inhibitors of PI-3-K inhibits the volume-sensitive ion efflux. With parallel structural studies (NMR, performed in collaboration with Prof. Konrat, Costanza.) we demonstrated that water soluble ICln is a PH domain (fig 1). This domain is usually involved in protein-protein interactions or phosphatidylinositols (PtdIns) binding, suggesting a possible role of PtdIns in the traslocation of ICln to the membrane and/or the activation of the ICl,swell current.
On these basis we are currently studying the interaction between ICln and different PtdIns, especially 3-phosphoinositols, by in vitro techniques (protein overlay experiments, liposome binding experiments) and in vivo experiments (patch-clamp, FRET).

Fig. 1. NMR-derived structure of water-soluble ICln159.

a, a bundle of 15 final structures and a ribbon plot of ICln with the loop between β6/β7 displayed in magenta, β-sheets are in cyan, and the α-helix is in red. b, T2 versus amino acid sequence of the backbone amide protons. The loop between strand β6 and β7 is disordered and highly mobile and indicated with a bar in magenta. Green columns indicate the β-sheets, and the gray column indicates the  α-helix.

2. ICln interacting proteins

Meyer, Bazzini, Vezzoli

Beside its role in cell volume regulation, additional functions of ICln in other regulatory modules, i.e. the regulation of cell morphology, platelet activation or RNA processing, have been described. The finding that ICln interacts with cytosolic and membrane or membrane associated proteins, suggests that it may function as a connector hub linking various regulatory pathways and compartments within a cell. As a step towards a better understanding of the function of ICln, we are trying to identify the ICln patners in the cytosolic fraction as well as in the membrane fraction and we are mapping the proteome changes in cells during the regulatory volume decrease.
Particularly, we mean to investigate the functional and molecular interaction between ICln and the platelet integrin alpbaIIb chain, as well as a putative zink finger protein of unknown function termed HSPC038. These experiments could help to elucidate the mechanisms leading to the transposition of ICln towards the cell membrane during the regulatory volume decrease, as well as the role of ICln and the interacting proteins in the activation of the anion current elicited by cell swelling (ICl,swell).

3. ICln and apoptosis

Meyer, Bazzini

Cell proliferation and apoptosis are basic biological processes through which new cells are formed, and damaged cells are eliminated to ensure body growth and organ repair. Apoptosis is a complex process, involving several steps that finally lead to cell death. An early key event is the activation of an anion current that closely resembles the swelling activated chloride current (ICl,swell). The activation of the current causes an efflux of ions and consequent cell shrinkage, known as Apoptotic Volume Decrease (AVD, Fig 2). Since ICln is essential for the activation of ICl,_swell, we are investigating the relationship between ICln and the AVD current, apoptosis and cell proliferation. The project involves the use of vitality assays and measures of the activation of key events of the apoptotic pathway, performed on cells over-expressing ICln or after silencing of ICln by RNAi.

4. Transcriptional regulation of the ICln gene

Meyer, Bazzini, Vezzoli

By bioinformatic analysis, we have found in the 5’ flanking region of the ICln gene a sequence element (IRES) common in those genes that are involved in important physiological process like apoptosis and response to the cellular stresses. Consequently we are studying if this element could regulate ICln expression during the regulatory volume decrease.

Fig. 2 Ionic mechanisms of volume regulation and cell death induction

Transport Physiology:

1. Essential hypertension: functional and molecular alteration of distal nephron ion channels

Meyer, Garavaglia

Essential hypertension is closely associated with an increase in  renal Na+ reabsorption. This leads to hypervolemia, which is a crucial step in the development of hypertension.

The distal tracts of the nephron (thick ascending limb, distal convoluted tubule and collecting duct) are the regions where NaCl is actively absorbed, which induces a regulated water reabsorption as a result of the generated osmotic gradient. Since the ion channels both in the apical and basolateral membrane are functionally correlated with the electroneutral transports for NaCl reabsorption in distal nephron, cotransporters modifications could be associated to an altered expression and/or function of ion channels. We are studying  the possible functional alterations of these ion channels in Milano hypertensive rats (MHS), a system model of human essential hypertension. 

2. Effect of S-Carbocysteine-lysine salt monohydrate on glutathione secretion from respiratory epithelium

Meyer, Garavaglia

Glutathione (GSH) is one of the major antioxidative agents in the surface fluids covering the higher airways mucosae. GSH can be secreted in an electrogenic way which is dependent on CFTR (Cystic Fibrosis Trasmembrane Regulator) anion channel. This Research Unit studies, by using electrophysiological techniques, the ability of S-Carbocysteine-lysine salt monohydrate (a promoter of mucolysis) to stimulate both Cl- and GSH secretion in human respiratory epithelium cells. Effects of S-Carbocysteine-lysine salt monohydrate also in the presence of oxidative stress will be analysed. 

3. Functional and molecular characterization of human pendrin

Meyer, Bazzini, Vezzoli

Pendrin is a recently cloned membrane anion exchanger; it is mainly expressed in the thyroid, in the inner ear and in the kidney. It plays a fundamental role for a correct inner ear development and for the thyroid hormones biosynthesis. Mutations of the protein lead to Pendred’s syndrome, an autosomal recessive disease characterized by sensorineural hearing loss and a partial iodide organification defect that could lead to goitre development and hypothyroidism.
We are studying the transcriptional mechanisms leading to Pendrin gene expression, characterizing the promoter region in different cellular lines. Moreover, we are conducting experiments (western blot analysis, immunohistochemistry, transport analysis with fluorescent and radioactive markers, etc) in order to molecularly and functionally characterize the wild type Pendrin and some natural occurring mutants identified in patients with Pendred syndrome. These studies are aimed at individuating  mechanisms (as chaperon proteins, drugs, physical conditions) that could possibly restore the function of the defective proteins.

Fig. 3 Expression of exogenous Pendrin in HEK293-Phoenix cells. The subcellular localization was done by co-expressing Pendrin (red signal) together with a EYFP conjugated plasma membrane label (green signal).

4. Effect of oxidative stress on various functional parameters of small intestine

Orsenigo, Tosco

A number of parameters indicating oxidative stress have been studied in the gut for its peculiarity of being exposed to oxidant agents of both exogenous (diet and lumen) and endogenous (mucosa) origin. In jejunal and ileal tracts of rat intestine we are investigating the effects of different oxidant agents on the activities of (Na+, K+)-ATPase, responsible for transepithelial transport of Na+ ion, and on γ-GT, enzyme involved in glutathione homeostasis. Moreover we are going to study possible direct effects of the same oxidant agents on single mechanisms involved in the transepithelial transports of fluid, glucose, sodium, chloride and lactate, localized either on the apical or on the basolateral membrane of enterocyte.

5. The antioxidant capacity of rat enterocyte in acute and chronic acidosis conditions

Orsenigo, Tosco

It is well known that oxidative stress is involved in the genesis of various gastrointestinal diseases characterized by chronic inflammation; inflammatory state is generally linked with acidosis conditions that, in turn, elicit oxidative stress and/or amplify its effects. Aim of this research is to clarify how acidosis conditions may influence the antioxidant power of epithelial cells and their response to various reactive oxigen species in rat jejunum and ileum. By means of different experimental approaches we are going to evaluate if acute and chronic acidosis may influence lipid peroxidation level, marker of oxidative damage, enzyme activities involved in cellular antioxidant defense (γ-GT, catalase, glutathione peroxidase, glutathione reductase, superoxide dismutase) and expression of heat schock proteins, invoved in cytoprotective mechanisms. To evaluate possible effects of acidosis on intestinal functional features, the expression of (Na+, K+)-ATPase and of the glucose transporters SGLT1 and GLUT2 will be examined.

6. Protective role of creatine on small intestinal mucosa in acidosis and hypoxia conditions

Tosco, Orsenigo

Creatine is an amino acid derivative that has a marked importance in energy metabolism of various cell types, since it belongs to an energy supply system that can provide ATP. Creatine is taken orally by some athletes to improve muscolar mass and performance; moreover it is also used in medicine to treat mitochondrial, neuromuscolar, cardiovascular and brain diseases or also as antitumoral and antiviral agent. Recently creatine was suggested to protect tissues against oxidative stress. Aim of this research is to investigate creatine effects on the main enzymatic activities involved in antioxidant defense of jejunal and ileal rat enterocytes, either in physiological conditions, or in acute and chronic acidosis, or in hypoxia and anoxia. Moreover we will evaluate if, in all the cited conditions, the presence of creatine may influence oxidative stress parameters such as lipid peroxidation level. Finally, to clarify whether creatine may play a cytoprotective role in stress conditions, we will verify if it potentiates heat shock protein expression.

Publications

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