Voditelj:
Prof.dr.sc. Aleksandra Sinđić
Zavod za fiziologiju
Hrvatski institut za istraživanje mozga
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Suradnici:
Dr.sc. Marina Dobrivojević
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Nikola Habek, dr.med.
Naslov projekta: Povoljni učinci natriuretskih peptida u različitim patološkim zbivanjima
Natiuretski peptidi reguliraju krvni tlak i volumena te izlučivanje soli i vode. Članovi ove obitelji su atrijski natriuretski peptid (ANP), natriuretski peptid izoliran iz mozga (BNP), C-tip natriuretskog peptida (CNP) te urodilatin (URO) koji je bubrežna izoforma ANP. Njihova fiziološka uloga se većinom zasniva na aktivaciji receptora vezanih uz enzim guanilat ciklazu te posljedično porast unutarstanične koncentracije cGMP-a. ANP, BNP i URO su agonisti guanilat ciklaze (GC) -A (poznata i kao receptor za natriuretske peptide tip A, NPR-A) dok guanilat ciklaza (GC) - B (receptor za natriuretske peptide tip B, NPR-B) posjeduje veliku specifičnost za CNP. Svi navedeni natriuretski peptidi su agonisti za receptor tip C, poznat i kao receptor koji odstranjuje natriuretske peptide iz cirkulacije (clearance receptor) koji ne posjeduje guanilat ciklaznu aktivnost.Poznato je da natriuretski peptidi imaju povoljan učinak na razvoj edema nakon moždanog inzulta no mehanizam tog učinka je još uvijek nepoznat. Jedan od mogućih mehanizama je interakcija između natriuretskih peptida i bradikininskog (BK) signalnog puta. Da bismo ispitali ovu hipotezu koristili smo HEK293 stanice koje posjeduju sve potrebne dijelove signalnog sustava i za bradikinin i za natriuretske peptide. (rezultat nije prikazan).
Svi prikazani rezultati natriuretskih peptida na B2R-PLC-Ca2+ ovisan signalni sustav BK-a su dodatno potvrđeni mjerenjem unutarstanične koncentracije Ca2+. Iako je cijeli sustav za izvedbu ovih pokusa donirao Prof dr E Schlatter, University of Munster, Germany te je smješten na našoj ustanovi, ova mjerenja su radi nedostatka novca napravljena na PHARIS Biotec GmbH, Hannover, Germany. BK depolarizira HEK293 stanice (tablica 1.) aktivacijom bradikininskog receptora tipa 2 (B2R - Ca2+ signaling put) te kloridnih kanala ovisnih o Ca2+ (rezultati nisu prikazani). Učinak BK se može inhibirati svim natriuretskim peptidima koji su agonisti GC-A receptora te cGMP-om (Sl.1.) (aktivacijom protein kinaze G (sl 2)) dok CNP kao agonist GC-B nema učinak. Vezanje bradikinina za B2R receptor dovodi do aktivacije fosfolipaze C (PLC) (Sl 3.). Poznato je da PKG može inhibirati PLC te kada smo inhibirali PLC specifičnim inhibitorom U-73122 (10 µM), depolarizacija nastala djelovanjem BK je inhibirana na isti način kako i djelovanjem natriuretski peptida, dovodeći do zaključka da je PLC protein od interesa u istraživanju interakcija između signalnog sustava natriuretskih peptida i bradikinina (rezultati nisu prikazani). Ovaj projekt pokazuje da natriuretski peptidi djelovanjem PKG mogu utjecati na PLC ovisan signalni sustav bradikinina te mogu djelovati kao prirodni inhibitori edema uzrokovanog bradikininom u raznim patološkim stanjima kao što su nasljedni angioedem te moždani udar.
Oprema u laboratoriju:
Jedan set up za elektrofiziološka istraživanja (NAPI) (uključuje stol na zračnim jastucima, mikroskop, amplifier, regulator temperature, AD/DA jedinica, kompjuter sa Tida software-om za patch-clamp mjerenja, patch clamp puller za izradu elektroda.
Ostala operma za elektrofiziološka istraživanja (donacija prof dr Eberhard Schlatter, University of Műnster, Germany): WeKa Graph termo pisač, 2x patch-clamp amplifier-a, jedan puls generator s vanjskom jedinicom za upravljanje, pH-metar, spektrofotometar)
Vaga, eppendorf grijači blok te sve potrebno za western blotting, Microforge MF 900 za poliranje pipeta za patch-clamp – nabavljeno povratničkim projektom NZZ-a.
Head:
Prof. Aleksandra Sinđić
Department of Physiology
Croatian Institute for Brain Research
Collaborators:
Dr.sc. Marina Dobrivojević
Nikola Habek, dr.med.
Project title: Effects of natriuretic peptides in physiological and pathological conditions in the brain
Natriuretic peptides regulate blood pressure-volume homeostasis, salt excretion and diuresis. Even they are showing physiological and pathophysiological effects in the brain their function is not well investigated. Members of the human natriuretic peptide family are the atrial natriuretic peptide (ANP), the brain natriuretic peptide (BNP), the C-type natriuretic peptide (CNP), urodilatin (URO - the kidney isoform of ANP), guanylin (GN) and uroguanylin (UGN). They exert their biological functions mainly through activation of guanylate cyclase receptors and subsequently, through an increase in intracellular cGMP. Guanylate cyclase (GC)-A selectively binds ANP, BNP and URO, the GC-B has a high affinity towards CNP while GN and UGN activates GC-C. Additional natriuretic peptide receptor type C (NPR-C) known as clearance receptor binds ANP, URO, BNP and CNP has no guanylate cyclase activity. The function of GC-A and B agonists are more investigated there is almost no research of GN and UGN in the brain. We investigate effects of natriuretic peptides on the primary culture of neurons and astrocytes (table 1.) and their signaling pathways.
Natriuretic peptides depolarized neurons similar to membrane permeable cGMP (8 Br cGMP) which is their well know second messenger. To definitely confirm that GCs are receptors for natriuretic peptides in neurons need further investigation. In astrocytes, ANP and in some cases URO (agonists of GC-A) as well as cGMP lead to cell hyperpolarization. However, natural agonists of GC-B (CNP) and GC-C (GN and UGN) depolarized astrocytes which are opposites of cGMP effects suggesting existence of GC independent signaling pathway.
In neurons all natriuretic peptides depolarized cells (dose dependence curve shown at figure 1). That depolarization was inhibited when BaCl2, an inhibitor of K+- channels is used suggesting that natriuretic peptides as well as cGMP depolarized cells by inhibiting o K+- channels (figure 2.).
Lab equipment:
Set-up for electrophysiological experiments (NAPI) (includes air table, microscope. Amplifier, temperature regulator, AD/DA converter, computer with Tida software for patch-clamp measurements, patch clamp puller for pipette preparation.
Additional equipment for patch-clamp measurements (donation by prof dr Eberhard Schlatter, University of Műnster, Germany): WeKa Graph thermo printer, 2x patch-clamp amplifiers, one pulse generator with control units, pH-meter, spectrophotometer.
scale, Eppendorf thermal block and all equipment for western blotting, Microforge MF 900 for pipette polishing for patch-clamp – financed by the National Foundation for Science, Higher Education and Technological Development of the Republic of Croatia.
- Dobrivojević M, Špiranec K, Sinđić A. Pflügers Archiv. 467:201-12 2015 (4.86)
- Massmann V, Edemir B, Schlatter E, Al-Monajjed R, Harrach S, Klassen P, Holle SK, Sindic A, Dobrivojevic M, Pavenstädt H, Ciarimboli G. Pflugers Arch. 466:517-27 2014. (4,86)
- Schmidt-Lauber C, Harrach S, Pap T, Fischer M, Victor M, Heitzmann M, Hansen U, Fobker M, Brand S-M, Sindic A, Pavenstädt H, Edemir B, Schlatter E, Bertrand J Ciarimboli G. PLOS ONE. 7(12):e52247 2012.
- Dobrivojević M, Sinđić A, Edemir B, Kalweit S, Forssmann W-G, Hirsch JR. Am J Physiol 303(12):C1260-8 2012. (4.2)
- Sinđić A, Dobrivojević M, Hirsch JR. Natriuretic peptides in brain physiology. Translational Neuroscience, 2(3); 246-251, 2011.
- Sinđić A, Sussman CR, Romero MF. Pancreatology, 10:660-3, 2010. (IF 3.0)
