016P University of Cambridge
Summer Meeting July 2005

 

Characterisation of agonist and inverse-agonist actions on dopamine D3 receptor mediated 35S-GTPγS binding

J. Westaway, C. Langmead, J. Watson, M. D. Wood and C. Scott. Psychiatry CEDD, GlaxoSmithKline Pharmaceuticals, Harlow, Essex, CM19 5AW.

 

The relative lack of functional studies on the human dopamine D3 (hD3) receptor has been suggested to reflect a low efficiency of coupling to the Go subtype of G proteins (Zaworski et al, 1999). In an attempt to overcome this we have transfected the Go protein into a cell line stably expressing the hD3 receptor.

Membranes were prepared from Chinese hamster ovary (CHO) cells stably expressing hD 3 receptors transduced with rat Gαo . Assay buffer contained 20 µM HEPES, 10 mM MgCl 2 and a variety of concentrations of sodium, N-methyl-D-glucamine (NMDG; used to maintain positive charge of the buffer) and GDP (details below). Non-specific binding was defined using 20 m M GTPγS. Following a 30 minute pre-incubation with test compounds, 0.1 nM [35S]-GTPγS was added to membranes and the reaction incubated at 30 oC for 30 min. Assays were terminated by rapid filtration through GF/B filterplates pre-soaked in purified H2O.

(-)-Quinpirole and haloperidol produced a respective sodium dependent increase and decrease in basal [35S]-GTPγS binding (Table 1).

Table 1. Effects of sodium ion concentration on agonist and inverse agonist stimulated 35SGTPγS binding.

 

Sodium/NMDG Buffer Composition

% Change from basal [35S]-GTPγS Binding

pEC50

Quinpirole

Haloperidol

Quinpirole

Haloperidol

0 mM NaCl / 0 mM NMDG

72 ± 5

-21 ± 5

7.57 ± 0.2

8.53 ± 0.8

0 mM NaCl / 100 mM NMDG

74 ± 10

-28 ± 6

8.21 ± 0.4

8.00 ± 0.8

5 mM NaCl / 95 mM NMDG

93 ± 7

-28 ± 3

7.87 ± 0.3

8.75 ± 0.3

25 mM NaCl / 75 mM NMDG

100 ± 14

-39 ± 5

8.31 ± 0.2

8.15 ± 0.4

75 mM NaCl / 25 mM NMDG

103 ± 7

-69 ± 4

7.95 ± 0.1

8.11 ± 0.1

100 mM NaCl / 0 mM NMDG

146 ± 20

-55 ± 4

7.67 ± 0.2

8.44 ± 0.2

 

Data shown are mean ± s.e.m. of 3 independent experiments.

The effects of 1 - 100 µM GDP on [35S]-GTPγS binding were also investigated in buffer containing 100 mM NaCl. Increasing GDP concentration reduced both the levels of basal [35S]-GTPγS binding and the degree to which quinpirole stimulated [35S]-GTPγS binding (from 61 to 35 %). In contrast, haloperidol induced inhibition of [35S]-GTPγS binding decreased with increasing GDP concentrations (from 33 to 56%).

In conclusion, in CHO cells expressing hD 3 receptors, coupling can be increased by transducing the Go protein which increases G protein activation as measured by [35S]-GTPγS binding. Using this approach (-)-quinpirole acted as an agonist and haloperidol as an inverse agonist. The maximal effects of quinpirole and haloperidol were potentiated by increasing sodium ion concentration, whereas increasing the GDP concentration reduced the effects of quinpirole and increased the effects of haloperidol on [35S]-GTPγS binding.

Zaworski, PG, Alberts GL, Pregenzer JF et al (1999) Br. J. Pharmacol. 128, 1181-1188.