Role of TRPV1 channel and P2Y1 receptor in Ca2+ signalling in β-cells: A study by single cell microfluorometry
Independent thesis Advanced level (degree of Master (Two Years)), 60 credits / 90 HE creditsStudent thesis
Increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) in the β-cells triggers insulin exocytosis. Among the Ca2+ channels present in the plasma membrane, the transient receptor potential (TRP) channels receptors are currently of great interest. The mechanisms by which the extracellular adenosine diphosphate ribose (ADPr) increases the [Ca2+]i is unknown. Our aims were to study the roles of the TRP channels in the tolbutamide induced [Ca2+]i increase and to identify the surface receptor that is activated by ADPr.
We used S5 cells, a highly differentiated rat insulinoma cell line, as a model for β-cells. Single cell ratiometric microfluorometry was used to measure the [Ca2+]i changes in the Fura-2 loaded cells.
Tolbutamide increased [Ca2+]i in the form of oscillations. After tolbutamide increased [Ca2+]i,capsazepine, a potent blocker of the transient receptor potential vanilloid subtype 1 (TRPV1) channel was added to the β-cells, which reduced the tolbutamide-induced [Ca2+]i increase. capsazepine, N-(p-Amylcinnamoyl) anthranilic acid (ACA), TRPM2 channel blocker, and triphenyl phosphine oxide (TPPO), TRPM5 channel blocker were tested for their effect on potassium chloride (KCl) induced [Ca2+]i response. These blockers did not inhibit the KCl induced [Ca2+]i increase.
Adenosine diphosphate ribose (ADPr) increased [Ca2+]i in the form of initial transient peak followed by an elevated plateau. Application of ADPr shortly after a prior application and washout of Adenosine diphosphate (ADP) elicited only small [Ca2+]i increase indicating desensitization of the receptor involved. 2´deoxy-N6-methyladenosine 3´5´bis-phosphate (MRS2179), and chloro N6-methyl-(N)-methanocarba 2´deoxyadenosine 3´5´ bis-phosphate (MRS2279), two selective inhibitors of P2Y1 receptor, abolished the ADPr-induced [Ca2+]i increase.
Tolbutamide closes ATP sensitive potassium (KATP) channels. Our results demonstrate that besides the closure of the KATP channels, inward cation currents carried by Ca2+through the TRPV1 channel are necessary for depolarization to the threshold for the activation of the voltage gated calcium channels (VGCC) to increase the [Ca2+]i. Our results also show that ADPr increases [Ca2+]i by activating the P2Y1 receptor.
Place, publisher, year, edition, pages
2011. , 21 p.
Insulin exocytosis, P2Y1, tolbutamide, ADPr, microfluorometry, Islets of langerhans, β-cells, calcium signaling, signal transduction.
Cell and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-68955ISRN: LiTH-IFM- A-EX-11-2431OAI: oai:DiVA.org:liu-68955DiVA: diva2:422563
Subject / course
2011-05-27, 13:41 (English)
Islam, Md Shahidul, Associate professor