Investigaciones de organismos internacionales

The enhancement of the GABA-induced response by benzodiazepines has previously been shown to depend on the composition of the GABA^ receptors, with the benzodiazepine enhancement of GABA-induced responses only occurring in heteromeric constructs containing a y subunit (Pritchett et al.,

1989; cf Malherbe et al., 1990a; for review see Sieghart, 1989; Doble and M artin, 1992). In order to determine the effect of subunit composition on the modulation of the GABA response by benzodiazepines and other drugs acting at the barbiturate and neurosteroid binding site(s) five murine GABA^ receptor subunit constructs were assembled, a l p l, alply2S, aipiy2L, a lp lô , and alply2Sô.

Bath-application of 1 pM flurazepam had no effect on the GABA-induced current and conductance increase in oocytes expressing a l p l GABA^ receptors (Fig. 3.12A). Conversely, application of pentobarbitone (25 pM) to this receptor construct potentiated the GABA-evoked response (Fig. 3.12A). The degree of potentiation of the GABA-induced response produced by different concentrations of pentobarbitone was examined by repeatedly exposing the oocytes to a fixed dose of GABA together with different concentrations of pentobarbitone. The resultant pentobarbitone potentiation curve of the GABA- evoked conductance increase revealed an ECgg value for pentobarbitone of 56 ± 2.4 pM with the maximal increase in the GABA response induced by 500 pM pentobarbitone of 708.7 ± 15% (Fig. 3.12B; Table 3.5). Equilibrium dose-response curves for GABA (0.01 to 500 pM) revealed th a t flurazepam (1 pM) had no effect on the dose-response curve; however pentobarbitone (50 pM) shifted the curve to the left with an increase in the maximum

Chapter three

response (Fig. 3.12B). The EC50 values and Hill coefficients remained relatively unaffected by flurazepam, whilst pentobarbitone decreased the EC50 and increased the Hill coefficient for GAB A (Table 3.6).

As expected, the addition of the t2S subunit to this receptor complex yielding receptors with al(3l72S subunit constructs revealed a sensitivity to the benzodiazepines which appeared to be absent in a l p l GABA^ receptor complexes (Levitan et oZ., 1988; Pritchett et al., 1989; Horne et al., 1993). Application of 1 pM flurazepam potentiated the GABA-induced current and conductance change (Fig. 3.13A). Pentobarbitone (50 pM) and pregnanolone (500 nM) also potentiated the GABA-induced response (Fig. 3.13A). The potentiation produced by diazepam, flurazepam and pentobarbitone on the GABA-induced response was dependent on the concentration of each drug applied. As the concentration of each drug was raised the conductance change evoked by 10 pM GABA increased along a sigmoidal curve with the maximal increases in the GABA response induced by flurazepam, diazepam and pentobarbitone being: 52.2 ± 2.4% (2.5 pM), 57.1 ± 1.6% (0.25 pM) and 190 ± 4.9% (500 pM) respectively (Fig. 3.13B; Table 3.5). The EC50 values for the potentiation induced by diazepam, flurazepam and pentobarbitone of the GABA-induced responses are 0.05 ± 0.003, 0.17 ± 0.02 and 21.97 ± 1.9 pM respectively (Table 3.5). Construction of equilibrium dose-response curves for GABA revealed a shift to the left in the presence of 1 pM flurazepam and 50 pM pentobarbitone with an increase in the maximum only in the presence of the barbiturate (Fig. 3.13B). The Hill coefficient values remained relatively unaffected with a decrease in the EC50 values for GABA in the presence of flurazepam and pentobarbitone (Table 3.6).

A similar picture to the a lp 1^28 GABA^ receptor complex was seen for the alply2L subunit receptor construct. Flurazepam (1 pM) and pentobarbitone (25 pM) potentiated the GABA-induced current and conductance increase (Fig. 3.14A). The potentiation of the response to a fixed dose of GABA (10 pM)

Chapter three

by flurazepam and pentobarbitone was concentration dependent (Fig. 3.14B). The maximal increase of the GABA-induced response for flurazepam and pentobarbitone was 51.4 ± 1.3% (2.5 pM) and 239.8 ± 7.6% (500 pM) with EC50 values for the potentiation of 0.17 ± 0.01 and 28.23 ± 2.8 pM respectively (Table 3.5). Construction of equilibrium dose response curves for GABA again revealed similarities with the a l p l 72S receptors with a shift to the left in the presence of 1 pM flurazepam and 50 pM pentobarbitone and an increase in the maximum in the presence of the barbiturate (Fig. 3.14B). The Hill coefficient values remained relatively unaffected with a decrease in the EC50 values for GABA in the presence of flurazepam and pentobarbitone (Table 3.6).

The addition of a 72 subunit, t2S or 72L, to the a l p l GABA^ receptor was shown to confer a sensitivity of the resulting receptor construct to the benzodiazepines. Therefore, it was interesting to ascertain whether or not the Ô subunit could also modulate the benzodiazepine sensitivity of GABA* receptors. Oocytes were injected with cDNAs encoding murine a ip iô GABA^ subunits. These receptor constructs revealed a similar sensitivity to benzodiazepines and barbiturates to the a l p l GABA^ receptor construct. Application of 1 pM flurazepam and 1 pM diazepam failed to potentiate the GABA-induced response indicating th at the presence of the Ô subunit did not induce the same benzodiazepine sensitivity as the 72 subunits in the GABA& receptor (Fig. 3.15A). Conversely, application of 50 pM pentobarbitone potentiated the GABA response (Fig. 3.15A). Interestingly, murine alpl72S6 GABA^ receptors revealed a similar sensitivity to the positive modulators of the GABA^ receptor as the a l p l 72S receptor constructs. The GABA-induced current and conductance increase was potentiated by the application of flurazepam (1 pM), diazepam (1 pM), pentob£u*bitone (50 pM) and pregnanolone (500 nM). As for the alpl72S receptor construct, 1 pM diazepam appeared to be more potent in potentiating the GABA-induced response than 1 pM flurazepam in this receptor complex. Interestingly, the

Chapter three

addition of the ô subunit to either a l p l or a l p l 72S receptor constructs yielding receptors with the subunit composition a lp lô and aipi72S5 seemingly had little or no effect on the sensitivity of the GABA^ receptor to positive modulators,

3.2.4 COMPARISON OF GABA^ RECEPTORS FROM TWO DIFFERENT