[(3)H]-SB-269970--A selective antagonist radioligand for 5-HT(7) receptors

Abstract

Binding of the 5-HT(7) receptor antagonist radioligand [(3)H]-SB-269970 to human 5-HT(7(a)) receptors expressed in HEK293 cell membranes (h5-HT(7(a))/293) and to guinea-pig cerebral cortex membranes, was characterized and compared with [(3)H]-5-CT binding. [(3)H]-SB-269970 (1 nM) showed full association with h5-HT(7(a))/293 membranes after 40 min. Specific binding at equilibrium represented >90% of total binding and was fully reversible by methiothepin (10 microM), full dissociation occurring by 100 min. The association (k(+1)) and dissociation (k(-1)) rate constants were 0.05 nM(-1)min(-1) and 0.05 min(-1) respectively, giving a K(D) (k(-1)/k(+1)) of 1.0 nM. [(3)H]-SB-269970 bound saturably and apparently monophasically to both h5-HT(7(a))/293 and guinea-pig cortex membranes, with K(D) values of 1.25+/-0.05 and 1.7+/-0.3 nM respectively. The B(max) for [(3)H]-SB-269970 to both h5-HT(7(a))/293 and guinea-pig cortex membranes (5780+/-380 and 125+/-8.2 fmoles mg protein(-1) respectively) was similar to that for [(3)H]-5-CT (6190+/-940 and 143+/-19 fmoles mg protein(-1) respectively). These data suggest that, in each tissue, both radioligands labelled the same population of receptors, which appear to be present in an agonist high affinity state. The profile of compound inhibition of [(3)H]-SB-269970 binding to h5-HT(7(a))/293 and guineapig cortex membranes correlated well (corr. coeff. 0.98) with those for [(3)H]-5-CT binding and were consistent with the profiles reported previously for the human 5-HT(7(a)) and guinea-pig cortex 5-HT(7) receptors using [(3)H]-5-CT. Hill slopes for inhibition of [(3)H]-SB-269970 and [(3)H]-5-CT binding were close to 1, consistent with binding to a single receptor population in both tissues. [(3)H]-SB-269970 represents the first selective 5-HT(7) antagonist radioligand, which should aid further characterization of 5-HT(7) receptors in recombinant and native tissues and help establish their role in brain function.

Figure 1

Kinetics of association (a) and dissociation (b) of 1 nM [³H]-SB-269970 binding to h5-HT₇/293 membranes. Dissociation was initiated by addition of 10 μM methiothepin. Data points represent the mean±s.e.mean of three separate experiments each performed using triplicate determinations.

Figure 2

(a) Saturation analysis of [³H]-SB-269970 binding to h5-HT₇/293 membranes. Data points show total binding, non-specific binding (defined in the presence of 10 μM 5-HT) and specific binding, calculated by subtracting non-specific binding from total binding. Data are from a typical experiment performed using triplicate determinations and repeated twice (n=3). (b) Scatchard plot (bound in pmoles mg protein⁻¹ versus bound/free (pmoles mg protein⁻¹ nM⁻¹)) of the specific binding data shown in (a).

Figure 3

(a) Saturation analysis of [³H]-SB-269970 binding to guinea-pig cerebral cortex membranes. Data points show total binding, non-specific binding (defined in the presence of 10 μM 5-HT) and specific binding, calculated by subtracting non-specific binding from total binding. Data are from a typical experiment performed using triplicate determinations and repeated twice (n=3). (b) Scatchard plot (bound in pmoles mg protein⁻¹ versus bound/free (pmoles mg protein⁻¹ nM⁻¹)) of the specific binding data shown in (a).

Figure 4

Inhibition of 1 nM [³H]-SB-269970 binding to (a) h5-HT_7(a)/293 membranes and (b) guinea-pig cortex membranes, by 5-CT, SB-269970-A, 5-HT, mesulergine and clozapine. Data points represent the mean±s.e.mean of at least three separate experiments each performed using duplicate determinations. Results are expressed as per cent of specific binding where non-specific binding was defined using 10 μM 5-HT.

Figure 5

Effect of GTPγS (1 nM to 100 μM) on 0.5 nM [³H]-5-CT and 1 nM [³H]-SB-269970 binding to (a) h5-HT_7(a)/293 membranes and (b) guinea-pig cortex membranes. Data points represent the mean±s.e.mean of at least three separate experiments each performed using duplicate determinations. Results are expressed as per cent of specific binding where non-specific binding was defined using 10 μM 5-HT.

Figure 6

Correlation plot of drug potencies (expressed as pK_i; values as shown in Table 1) to inhibit [³H]-5-CT and [³H]-SB-269970 binding to h5-HT₇/293 membranes. Data points shown are for agonists and antagonists. The correlation plot gave a slope of 0.99 and correlation coefficient of 0.98.

Figure 7

Correlation plot of drug potencies (expressed as pK_i; values as shown in Table 1) to inhibit [³H]-5-CT and [³H]-SB-269970 binding to guinea-pig cerebral cortex membranes. Data points shown are for agonists and antagonists. The correlation plot gave a slope of 0.92 and correlation coefficient of 0.98.