MS Research
The Role of G-proteins in opioid tolerance and µ-opioid receptor downregulation.
A dissertation submitted in partial
fulfillment of the requirements for the degree of
MASTER OF SCIENCE
To the faculty of the
GRADUATE DIVISION,
COLLEGE OF PHARMACY AND ALLIED HEALTH PROFESSIONS
At
St Johns University
New York
By
BENEDICT ASHOKA GOMES
Submitted Approved
Date:__________________ Date:_________________
__________________ _________________
Table of contents
Page
Abstract II
Acknowledgment III
List of abbreviations IV
List of figures V
Sections
I. Introduction 1-15
II. Materials and methods 16-20
III. Results 21-23
IV. Discussion and conclusion 24-28
References 29-38
Figures 39-42
Abstract:
Although opioid receptors are G-protein coupled, the role that G-proteins play in the development of tolerance and the regulation of opioid receptor number is not well-understood. In the present study, we used pertussis toxin (PTX) and a G i a 2 antisense oligodeoxynucleotide (ODN) to examine the contribution of G i/o proteins to µ-opioid receptor downregulation and tolerance in the mouse. Mice were injected intracerebroventricularly (ICV) and intrathecally (IT) for 4 or 5 consecutive days (30µg/site/day), with an antisense ODN or a mismatch ODN directed at mRNA for the G i a 2 subunit of the inhibitory G-protein. Other mice were injected once ICV and IT with PTX (0.1µg/site). Controls were treated with saline. On the 2 nd day of ODN treatment or on the 10 th day following PTX treatment, continuous s.c infusion of etorphine (200µg/kg/day or 150µg/kg/day) or morphine (40mg/kg/day+25mg pellet) was begun. Control mice were implanted with inert placebo pellets. Three days later, pumps and pellets were removed, and mice were tested for morphine analgesia or µ-opioid receptor density was determined in whole brain. Both infusion doses of etorphine produced significant tolerance (ED 50 shift = ~4-6-fold) and downregulation of µ-opioid receptors ( » 15-30%). Morphine treatment produced significant tolerance (ED 50 shift = ~5-8-fold), but no µ-opioid receptor downregulation. PTX and G i a 2 antisense dramatically reduced the acute potency of morphine and reduced (antisense) or blocked (PTX) etorphine induced tolerance. Both PTX and antisense blocked the development of morphine induced tolerance. However, neither PTX nor antisense had any effect on etorphine-induced µ-opioid receptor downregulation. These results suggest that PTX sensitive G-proteins have a minimal role in agonist-induced µ-opioid receptor density regulation in vivo, but are critical in mediating acute and chronic effects of opioids such as analgesia and tolerance.
Acknowledgements : Thanks to Dr. J. Shen, Dr. S. Shah, and Dr. A. Duttaroy for helpful discussions and comments throughout the course of these experiments. Special thanks to Dr. B.C. Yoburn for his invaluable guidance and direction.
Educational Interests:
Chemokines and inflammatory proteins. (Arthritis, Pain etc) .
Role of CCR1 receptor in RA. (Presentation) .
G-proteins and receptors. (molecular basis) .
Tolerance and desensitization a brief introduction (Presentation) .
Cloning and sequencing of a gene....a class project.