Blockage of 5HT2C serotonin receptors by fluoxetine (Prozac)

Abstract

Fluoxetine (Prozac) inhibited the membrane currents elicited by serotonin (5-hydroxytryptamine; 5HT) in Xenopus oocytes expressing either cloned 5HT2C receptors or 5HT receptors encoded by rat cortex mRNA. Responses of 5HT2C receptors, elicited by nM concentrations of 5HT, were rapidly and reversibly blocked by micromolar concentrations of fluoxetine. For responses elicited by 1 microM 5HT, the IC50 of fluoxetine inhibition was approximately 20 microM. In accord with the electrophysiological results, fluoxetine inhibited the binding of [3H]5HT to 5HT2C receptors expressed in HeLa cells (Ki approximately 65-97 nM), and the binding to 5HT receptors in rat cortex membranes was also inhibited but less efficiently (Ki approximately 56 microM). Our results show that fluoxetine is a competitive and reversible antagonist of 5HT2C receptors and suggest that some therapeutic effects of fluoxetine may involve blockage of 5HT receptors, in addition to its known blockage of 5HT transporters. Similar work may help to design more selective compounds for use in the treatment of brain disorders.

Figure 1

Inhibition of 5HT_2C currents by fluoxetine (100 μM) in an oocyte expressing cloned 5HT_2C receptors (upper trace). The continuing lower trace shows that the response to 10 nM 5HT was still inhibited while that to 10 μM 5HT had recovered substantially after the removal of fluoxetine. For this and following figures, the oocyte’s membrane potential was held at −60 mV. Inward currents are represented by downward deflections, and drug applications are indicated by bars above the traces.

Figure 2

Blocking effects of fluoxetine (100 μM) on 5HT currents in oocytes injected with cloned 5HT_2C receptor mRNA (A) or rat cortex mRNA (B). For these experiments, fluoxetine and 5HT were coapplied for 1–2 min. Results are mean ± SE from 3–10 oocytes. In both A and B, there were no detectable responses to coapplications of 10 nM 5HT and fluoxetine.

Figure 3

Dose-response relation of fluoxetine inhibition of responses to 5HT in oocytes expressing 5HT_2C receptors. (A) Sample traces from one oocyte. (B) Inhibition by different concentrations of fluoxetine coapplied with 1 μM 5HT. Each point represents mean ± SE from four to seven oocytes from one donor frog.

Figure 4

Inhibitory effect of 1 μM fluoxetine on 5HT_2C currents. 5HT was usually applied several times until a stable response was reached before coapplying fluoxetine.

Figure 5

Inhibition of 5HT_2C currents by a low concentration of fluoxetine (1 μM). (A) Sample trace of a nearly complete inhibition of 5HT_2C current. (B) Sample trace of a high concentration of fluoxetine (100 μM) not inhibiting the current elicited by angiotensin III (1 nM) activation of angiotensin II receptors. In this and the following figure, angiotensin II receptors were expressed by injecting 5 ng of bovine AT1 receptor mRNA. (C) Fluoxetine (10 μM) blockage of 5HT_2C currents elicited by 0.3 nM 5HT. Results are mean ± SE from six to seven oocytes.

Figure 6

Effect of fluoxetine on other native or expressed receptors that also utilize the phosphatidylinositol pathway. (A) Effect of fluoxetine on the response elicited by rabbit serum (1/1000 dilution) in noninjected oocytes. (B) Effect of fluoxetine on cloned bovine angiotensin II receptor-mediated responses. Angiotensin III (1 μM) was applied alone or together with fluoxetine (100 μM). Results are mean ± SE from three to four oocytes.

Figure 7

Competition of fluoxetine with [³H]5HT for binding to 5HT_2C receptors expressed in HeLa cells or to native rat cortex 5HT receptors. As indicated, 3.2 or 18 nM of [³H]5HT was used to label the 5HT_2C receptors expressed in HeLa cells (circles), and 3.2 nM [³H]5HT was used to label the rat cortex 5HT receptors. The inhibition by 1 mM 5HT (×) is also included to show that the binding of [³H]5HT to 5HT_2C receptors was completely blocked by fluoxetine. Data points are mean ± SE from three independent experiments in duplicate.