The human 5-HT7 serotonin receptor splice variants: constitutive activity and inverse agonist effects

Abstract

1. Using membranes from stably or transiently transfected HEK293 cells cultured in 5-HT-free medium and expressing the recombinant human 5-HT(7) receptor splice variants (h5-HT(7(a)), h5-HT(7(b)) and h5-HT(7(d))), we compared their abilities to constitutively activate adenylyl cyclase (AC). 2. All h5-HT(7) splice variants elevated basal and forskolin-stimulated AC. The basal AC activity was reduced by the 5-HT(7) antagonist methiothepin and this effect was blocked by mesulergine (neutral 5-HT(7) antagonist) indicating that the inhibitory effect of methiothepin is inverse agonism at the 5-HT(7) receptor. 3. Receptor density correlated poorly with constitutive AC activity in stable clonal cell lines and transiently transfected cells. Mean constitutive AC activity as a percentage of forskolin-stimulated AC was significantly higher for the h5-HT(7(b)) splice variant compared to the h5-HT(7(a)) and h5-HT(7(d)) splice variants but only in stable cell lines. 4. All eight 5-HT antagonists tested inhibited constitutive AC activity of all splice variants in a concentration-dependent manner. No differences in inverse agonist potencies (pIC(50)) were observed between the splice variants. The rank order of potencies was in agreement and highly correlated with antagonist potencies (pK(b)) determined by antagonism of 5-HT-stimulated AC activity (methiothepin >metergoline> mesulergine > or = clozapine > or = spiperone > or = ritanserin > methysergide > ketanserin). 5. The efficacy of inverse agonism was not receptor level dependent and varied for several 5-HT antagonists between membrane preparations of transiently and stably transfected cells. 6. It is concluded that the h5-HT(7) splice variants display similar constitutive activity and inverse agonist properties.

Figure 1

Basal AC activity in membranes of HEK293 cells stably (upper graph) or transiently (lower graph) expressing h5-HT₇ receptor splice variants plotted as a function of receptor density. Membrane AC activity was measured as described in Methods. In the upper graph, each marker represents data from one individual membrane preparation derived from a clonal HEK293 cell line stably expressing either human 5-HT_7(a) (closed circles; n=11, eight clones), 5-HT_7(b) (open squares; n=15, nine clones) or 5-HT_7(d) (grey triangles; n=15, 10 clones) receptors. The dashed line represents the mean basal AC activity of non-transfected HEK293 cells. In the lower graph, each marker represents mean data±s.e.mean from at least three (range 3 – 16) independent membrane preparations from cells with receptor expression densities within the same range.

Figure 2

Effects of 5-HT receptor antagonists on basal AC activity in membranes from HEK293 cells stably expressing h5-HT₇ receptor splice variants at similar densities (∼8 – 10 pmol mg protein⁻¹). Shown are two representative experiments each depicting the effect of four antagonists (A,C,E display methiothepin (solid squares), metergoline (solid circles), methysergide (solid crosses) and clozapine (open diamonds); B,D,F display mesulergine (plus signs), ketanserin (solid triangles), ritanserin (open circles) and spiperone (open squares). Membrane AC activity was measured as described in Methods in the presence of increasing concentrations of antagonist.

Figure 3

Effect of increasing concentrations of methiothepin on basal AC activity in membranes of non-transfected HEK293 cells and two clones stably expressing high densities of h5-HT_7(b) receptors (10 and 15 pmol mg protein⁻¹, respectively). Note that constitutive activity of h5-HT₇ receptors was revealed in clone 1 even though basal AC activity was not elevated above that of non-transfected HEK293 cells. This phenomenon was also observed in clones expressing h5-HT_7(a) and h5-HT_7(d) receptors.

Figure 4

Relationship of inverse agonist and antagonist potencies. Comparison of pIC₅₀ values obtained from antagonist-inhibition of basal AC activity (y-axis) and pK_b values calculated from antagonist-inhibition of 5-HT stimulated AC activity (x-axis) for the h5-HT₇ receptor splice variants. Inverse agonist pIC₅₀ values were taken from Table 2 and antagonist pK_b values are from Krobert et al. (2001).

Figure 5

Effect of increasing receptor density on the efficacy of antagonists (10 μM methiothepin or metergoline; 100 μM methysergide, clozapine, mesulergine or ritanserin; 50 μM ketanserin or 33 μM spiperone) to inhibit basal AC activity in membranes of HEK293 cells transiently expressing 5-HT_7(b) receptors. The reduction of basal was calculated as (1 minus AC activity in the presence of antagonist/AC activity in the absence of antagonist) × 100. The figure shows mean±s.e.mean of 2 – 4 membrane preparations with the average receptor density indicated as fmol mg protein⁻¹ in the legend. Similar data were obtained for the h5-HT_7(a) and h5-HT_7(d) splice variants.

Figure 6

Efficacy of inverse agonism produced by 5-HT antagonists relative to that produced by 10 μM methiothepin (dotted line  –  considered a full inverse agonist) in membranes of HEK293 cells stably (top) or transiently (bottom) expressing the h5-HT₇ receptor splice variants. Membrane AC activity was measured as described in Methods in the presence of either 10 μM metergoline, 100 μM methysergide, clozapine, mesulergine or ritanserin, 50 μM ketanserin or 33 μM spiperone. The data shown for the transiently transfected HEK293 cells are mean±s.e.mean of data as shown in Figure 5 collapsed across receptor density. The values shown for membranes of stable HEK293 clones are the mean±s.e.mean obtained from the asymptote of 3 – 6 concentration response curves.