Degree Type

Dissertation

Date of Award

2005

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

First Advisor

Walter H. Hsu

Second Advisor

Anumantha Kanthasamy

Abstract

Somatostatin (SS) is a peptide hormone that is known to inhibit insulin secretion in beta-cells. In the presence of araginine vasopressin (AVP), SS increases [Ca2+]i, leading to insulin release from clonal beta-cells HIT-T15 via PLC-beta pathway. Since SS alone fails to increase [Ca2+]i, it is hypothesized that SS increases PIP2 synthesis. In this dissertation, I demonstrate that the betagamma-dimer coupled to SS receptors was involved in phospholipase D1 (PLD1) activation and subsequent increase in PIP2 levels. I also determined that this activation of PLD1 was mediated through Arf6. EFA6A, an Arf6 guanine nucleotide exchange factor, was partially cloned and its involvement in PLD1 activation was determined. Results: (1) SS increased intracellular concentrations of PIP2 and decreased those of PIP in the presence and absence of AVP. (2) Antibody specific to Gbeta inhibited the response to SS, but those vs. Galphai/Galphao failed to do so. (3) zLYCK, a PLD inhibitor, and the antibody vs. PLD antagonized the response to SS. (4) Western blot analysis indicated that only PLD1 was detectable in HIT-T15 beta-cells. (5) SS was able to increase PLD activity, while AVP had no effect on PLD activity. (6) Ct-GRKII completely abolished the ability of SS to activate PLD. (7) Western blot analyses determined the presences of various Arf isoforms in HIT T-15 beta-cells. (8) Dominant negative Arf6 mutant construct Arf6(T27N) completely abolished the ability of SS to activate PLD and wild type Arf6 further enhanced this PLD activation. Furthermore, to determine if the effect of Arf6 on SS-PLD activity was non-specific, over-expression of dominant negative Arf1 mutant construct Arf1(T31N) was used in HIT-T15 beta-cells. (9) Arf1(T31N) had no effect on the SS-inducted PLD activation. (10) EFA6A was cloned from HIT-T15 beta-cells and was 92.8% identical to rat EFA6A sequence at the nucleotide level. (11) Dominant negative EFA6A construct completely abolished the ability of SS to activate PLD. We concluded that in the HIT-T15 cell model activation of SS receptors leads to activation of PLD via Gbetagamma. Gbetagamma signals through EFA6 to activate Arf6-PLD pathway that catalyzes various phospholipids into PA, which is known to increases PIP2 synthesis.

DOI

https://doi.org/10.31274/rtd-180813-16455

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Justin Adam Grodnitzky

Language

en

Proquest ID

AAI3184616

File Format

application/pdf

File Size

139 pages

Included in

Pharmacology Commons

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