Izražanje endotelijske sintaze dušikovega oksida in kaveolina 1 v normalnem in vnetem sečnem mehurju miši

Fajdiga, Urška (author), Romih, Rok (mentor)

Dušikov oksid ima pomembno vlogo v medceličnem signaliziranju, ki zagotavlja usklajeno delovanje sečnega mehurja med cikli polnjenja in praznjenja. Med vnetjem sečnega mehurja pride do spremenjenega medceličnega signaliziranja, kar povzroči motnje mikcijskega cikla. Lokacija endotelijske sintaze dušikovega oksida (eNOS) in njena regulacija preko kaveol v sečnem mehurju sta slabo raziskani. V nalogi smo proučevali izražanje in razporeditev eNOS in kaveolina 1 v steni normalnega in vnetega sečnega mehurja miši z več primarnimi protitelesi na vzorcih, pripravljenih po različnih postopkih. Normalen sečni mehur je imel dokončno diferenciran urotelij, inducibilna sintaza dušikovega oksida (iNOS) se ni izražala. S protitelesom, ki je s prenosom western potrdilo izražanje eNOS, so bile označene posamezne celice v lamini propriji in endotelijske celice. Z enim protitelesom je bila reakcija pozitivna tudi v citoplazmi posameznih bazalnih celic in pod plazmalemo vmesnih celic urotelija. Kaveolin 1 je bil prisoten v plazmalemi fibroblastov, intersticijskih in endotelijskih celic v lamini propriji ter gladkomišičnih celic detruzorja. Zanesljive kolokalizacije eNOS in kaveolina 1 ni bilo. Prvi in tretji dan po tretiranju s ciklofosfamidom (150 mg/kg in 300 mg/kg) urotelij ni bil dokončno diferenciran, v njem se je močno povečalo izražanje iNOS, kar je dokazovalo prisotnost vnetja. Med vnetjem je prišlo do izražanja eNOS v bazalnih celicah urotelija, kaveolin 1 je bil prisoten v plazmalemi celic kot v normalnem sečnem mehurju, a ga je bilo manj. Naši rezultati kažejo, da se eNOS izraža predvsem v endoteliju in posameznih celicah v lamini propriji v normalnem in vnetem sečnem mehurju. Rezultati imunohistokemije za dokazovanje eNOS pa so odvisni od uporabljenih protiteles in postopkov priprave vzorcev. Kaveolin 1 je prisoten v lamini propriji in detruzorju, ne pa v uroteliju.

• Language: Slovenian
• Keywords: sečni mehur, urotelij, endotelijska sintaza dušikovega oksida, kaveolin 1, dušikov oksid
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-151080
• COBISS.SI-ID: 173256451
• Publication date in RUL: 28.09.2023

Secondary language

• Language: English
• Title: Expression of endothelial nitric oxide synthase and caveolin 1 in normal and inflammed mouse urinary bladder

Abstract

Nitric oxide plays an important role in intercellular signaling, ensuring coordinated functioning of the urinary bladder during filling and emptying cycles. Inflammation of the urinary bladder disrupts intercellular signaling, leading to disturbances in the micturition cycle. The location of endothelial nitric oxide synthase (eNOS) and its regulation through caveolae in the urinary bladder remains poorly understood. In this study, we examined the expression and distribution of eNOS and caveolin-1 in the walls of both normal and inflamed mouse urinary bladders using multiple primary antibodies on samples prepared through various methods. The normal urinary bladder had a fully differentiated urothelium, and inducible nitric oxide synthase (iNOS) was not expressed. Using an antibody that confirmed eNOS expression via Western blot, individual cells in the lamina propria and endothelial cells were labeled. With one antibody, the reaction was also positive in the cytoplasm of individual basal cells and beneath the plasma membrane of intermediate urothelial cells. Caveolin-1 was present on the plasma membrane of fibroblasts, interstitial and endothelial cells in lamina propria, and smooth muscle cells of the detrusor. Reliable colocalization of eNOS and caveolin-1 was not observed. On the first and third days following treatment with cyclophosphamide (150 mg/kg and 300 mg/kg), the urothelium was not fully differentiated, and the expression of iNOS increased significantly, indicating the presence of inflammation. During inflammation, eNOS was expressed in the basal cells of the urothelium, and caveolin-1 was present on the plasma membrane of cells, as in the normal urinary bladder but in lesser amounts. Our results suggest that eNOS is primarily expressed in the endothelium and individual cells in the lamina propria in both normal and inflamed urinary bladders. Immunohistochemistry results for eNOS expression depend on the antibodies used and sample preparation methods. Caveolin-1 is present in the lamina propria and detrusor but not in the urothelium.

• Keywords: urinary bladder, urothelium, endothelial nitric oxide synthase, caveolin-1, nitric oxide