| Sažetak | Gvanilinski peptidi smatraju se unutrašnjim regulatorima funkcija žlijezda slinovnica. Cilj ove disertacije jest odrediti učinak intraperitonealno primijenjenog urogvanilina (UGN) na količinu i ionski sastav stvorene sline nakon stimulacije pilokarpinom.
Istraživanje je provedeno na miševima C57Bl6NCrl (divlji tip, WT) te miševima kojima nedostaje gvanilat-ciklaza C GC-C (GC-C KO) i WT miševima iz istog legla (GC-C WT) starosti 7 mjeseci. Izražaj mRNA za kanale za vodu i ionske transportere je određen u submandibularnoj žlijezdi slinovnici, duodenumu, kori bubrega i plućima. Nakon primjene UGN-a došlo je do smanjenja stvaranja sline stimulirane pilokarpinom te porasta koncentracije Na+ , H+ i Cl-. Učinak UGN-a na stvaranje sline ovisan je o GC-Cu (izvodni kanalići žlijezda slinovnica), dok je učinak na ionski sastav neovisan (Ca2+ signalni put u stanicama acinusa). UGN je povećao izražaj mRNA za Slc26a6 u submandibularnim žlijezdama slinovnicama, a taj izražaj također je bio viši u duodenumu i bubrežnoj kori GC-C KO životinja, što ukazuje na organski nespecifičnu i široko rasprostranjenu ulogu GC-C-a u fiziološkoj regulaciji ovih ionski izmjenjivača. Prikazani rezultati upućuju na mogućnost sistemnog djelovanja UGN-a, a ne kako se do sada smatralo, da je UGN samo unutarnji regulator funkcije žlijezda slinovnica. |
| Sažetak (engleski) | Aim: Guanylin peptides are intrinsic regulators of salivary glands secretion and their existence along with their signalling pathway in salivary glands has been known for more than two decades. Uroguanylin (UGN) is a small peptide and it is a member of the guanylin peptides family. UGN is widely expressed in the intestine. This peptide is secreted in gut lumen and blood after a meal. UGN and its guanylate cyclase C (GC-C) dependent signalling pathway are expressed in many tissues (central nervous, urinary, cardiovascular, respiratory, reproductive, immunological system as well as in salivary glands (parotid and submandibular)). GC-C is located at the apical membrane of the ducts of salivary glands. GC-C agonists are found in intralobular and interlobular ducts. In addition to the GC-C dependent signalling pathway, UGN activates another signalling pathway. This GC-C/cGMP independent signalling pathway exists in the kidneys, intestine, and brain. Its activation leads to an increase in intracellular Ca2+ concentration and a decrease in intracellular cAMP concentration. Since agonists of GC-C are considered to be only intrinsic regulators of salivary glands function, the aim of this study was to determine the effects of systemic UGN of the salivary flow and ion composition. By performing experiments on mice lacking GC-C (GC-C KO mice) we wanted to clarify if the UGN functions via GC-C or other signalling pathway.
Materials and methods: This study was conducted on 7 months old C57Bl6NCrl (wild type, WT) and GC-C knockout (KO) mice and their WT littermates (GC-C WT). The tip of the tail was taken from each animal to determine the genotype and to separate heterozygous animals that was not used in experiments. After induction of anesthesia, the experimental group of WT mice was administered human UGN (30 μg/animal in 250 μl saline) i.p., while the control group of WT mice was administered i.p. 250 μl of saline solution. GC-C WT and GC-C KO mice received the same amount of UGN as the
experimental group of WT mice. Fifteen minutes after application of UGN or saline solution the cholinergic agonist, pilocarpine hydrochloride (0.001 mg/g) was administered i.p. to stimulate salivation. Stimulated saliva collection test lasted 15 minutes. The amount of secreted saliva was determined by the gravimetric method, and the flow rate was expressed as the absolute flow of stimulated saliva in ml over 15 minutes. For the analyte determination, test tubes were centrifuged and concentrations of Na+ , K+ , and Cl- in saliva were measured by indirect potentiometry, while pH was determined by potentiometry on a blood gas analyzer using dedicated pH-sensors. During anesthesia, and after saliva collection, the animals were sacrificed by cervical dislocation in order to isolate submandibular glands, duodenum and kidney cortex. GoScriptTM Reverse Transcription System (ThermoFisher Scientific, Madisnon, Wisconsin, SAD) was used to transcribe RNA into complementary DNA. In order to determine the expression of ion transporters and water channels in the gastrointestinal tract, duodenum and renal cortex, we used polymerase chain reaction in real time (qPCR). For this purpose,
we used the TaqMan Real-Time PCR test (ThermoFisher Scientific) to determine quantitatively gene expression. Used probes used were specific for the genes of interest (ThermoFisher Scientific).
Results: When applied i.p., UGN decreased the pilocarpine stimulated saliva flow rate and increased concentration of Na+, H+ and Cl-. In GC-C KO mice, UGN showed no effect on saliva flow rate, while the concentrations of Na+, H+ and Cl-
were the same in GC-C KO littermates when compared to WT mice. Therefore, effect of UGN on saliva flow rate was GC-C dependent (salivary ducts), while effects on ion concentrations were not dependent (Ca2+ signalling pathway in acinar cells). UGN increased expression of Slc26a6. Slc26a6 expression was found to be higher in GC-C KO mice in comparison to WT suggesting involvement of GC-C independent signalling pathway for UGN. The difference in Slc26a6 in GC-C KO mice was not unique for salivary glands because it was found also in duodenum and kidney cortex.
Conclusions: The effects of systemic UGN via basolateral membrane of salivary glands cells have not been considered so far. In our study, UGN, when applied i.p., decreased salivary flow rate, pH, and changed composition of other ions. Therefore, plasma UGN an hour after a meal could have physiological and pathological importance (development of cavities, inflammations or demineralisations) and inhibition of systemic UGN effects could be considered as a new approach in treatment of those conditions. |
