Cholesterol depletion reduces serotonin binding and signaling via human 5-HT(7(a)) receptors.

Lipids, including cholesterol, are critical components of the cell membrane where they are enriched in microdomains, lipid rafts, which organize and concentrate receptors and intracellular proteins involved in signal transduction. The present study examined the effects of cholesterol depletion on serotonin (5-HT) binding and signaling via 5-hydroxytryptamine(7) (5-HT(7)) receptors in stably transfected HeLa cells. Immunohistochemical, ligand-binding and biotinylation experiments demonstrated that the studied cells expressed high levels of 5-HT(7) receptors at their surface with a pharmacological profile resembling 5-HT(7) receptors in native tissue. Depletion of cholesterol, by combined treatment with mevastatin, fumonisin B(1) and mevalonate or methyl-beta-cyclodextrin (MbetaCD), caused highly significant reductions in the B(max) values of [(3)H]5-HT- and [(3)H]-(R)-3-(2-(2-(4-methylpiperidin-1-yl)-ethyl)pyrrolidine-1-sulfonyl)phenol ([(3)H]SB269970)-binding to 5-HT(7) receptors. Cholesterol depletion also reduced the total level of 5-HT(7) receptor protein determined by Western blot analysis. None of the examined treatments affected the affinity of [(3)H]5-HT- or [(3)H]SB269970-binding to 5-HT(7) receptors. Treatment with serotonin caused strong inductions in the phosphorylation states of Ser(63)-ATF-1 and Ser(133)-CREB. These effects of serotonin on signal transduction were significantly counteracted by pre-treatment with cholesterol synthesis inhibitors. Altogether, the present study demonstrates that cholesterol depletion decreases binding of both agonist and antagonist radioligands to 5-HT(7) receptors and counteract 5-HT(7) receptor-mediated intracellular signaling.

Differential agonist-mediated internalization of the human 5-hydroxytryptamine 7 receptor isoforms.

The human 5-hydroxytryptamine 7 (5-HT(7)) serotonin receptor is a class A G-protein coupled receptor that has three isoforms, 5-HT(7(a)), 5-HT(7(b)), and 5-HT(7(d)), which are produced by alternative splicing. The 5-HT(7) receptors are expressed in discrete areas of the brain and in both vascular and gastrointestinal smooth muscle. Central nervous system 5-HT(7) receptors may play a role in mood and sleep disorders. 5-HT(7) receptors show high affinity for a number of antidepressants and typical and atypical antipsychotics. We report here that the human 5-HT(7(d)) isoform expressed in human embryonic kidney (HEK) 293 cells exhibits a pattern of receptor trafficking in response to agonist that differ from 5-HT(7(a)) or 5-HT(7(b)) isoforms. We employed a modification of a live cell-labeling technique to demonstrate that surface 5-HT(7(d)) receptors are constitutively internalized in the absence of agonist. This is in contrast to 5-HT(7(a)) and 5-HT(7(b)) isoforms, which do not show this profound agonist-independent internalization. Indeed, the 5-HT(7(d)) isoform displays this internalization in the presence of a 5-HT(7) -specific antagonist. In addition, the human 5-HT(7) isoform shows a diminished efficacy in stimulation of cAMP-responsive reporter gene activity in transfected cells compared with 5-HT(7(a)) or 5-HT(7(b)) receptors expressed at comparable levels. Thus, the carboxy-terminal tail of 5-HT(7(d)), which is the longest among known human 5-HT(7) isoforms, may contain a motif that interacts with cellular transport mechanisms that is distinct from 5-HT(7(a)) and 5-HT(7(b)).

Serotonergic regulation of membrane potential in developing rat prefrontal cortex: coordinated expression of 5-hydroxytryptamine (5-HT)1A, 5-HT2A, and 5-HT7 receptors.

The developing prefrontal cortex receives a dense serotonergic innervation, yet little is known about the actions of serotonin [5-Hydroxytryptamine (5-HT)] in this region during development. Here, we examined the developmental regulation of 5-HT receptors controlling the excitability of pyramidal neurons of this region. Using whole-cell recordings in in vitro brain slices, we identified a dramatic shift in the effects of 5-HT on membrane potential during the postnatal developmental period. In slices derived from young animals [postnatal day (P) 6 to P19], administration of 5-HT elicits a robust depolarization of layer V pyramidal neurons, which gradually shifts to a hyperpolarization commencing during the third postnatal week. This progression is the result of coordinated changes in the function of 5-HT7 and 5-HT2A receptors, which mediate different aspects of the depolarization, and of 5-HT1A receptors, which signal the late developing hyperpolarization. The loss of the 5-HT7 receptor-mediated depolarization and the appearance of the 5-HT1A receptor-mediated hyperpolarization appears to reflect changes in receptor expression. In contrast, the decline in the 5-HT2A receptor depolarization with increasing age was associated with changes in the effectiveness with which these receptors could elicit a membrane depolarization, rather than loss of the receptors per se. Together, these results outline coordinated changes in the serotonergic regulation of cortical excitability at a time of extensive synaptic development and thus suggest a key role for these receptor subtypes in the postnatal development of the prefrontal cortex.

Congenitally learned helpless rats show abnormalities in intracellular signaling.

BACKGROUND: Affective disorders and the drugs used to treat them lead to changes in intracellular signaling. We used a genetic animal model to investigate to what extent changes in intracellular signal transduction confer a vulnerability to mood or anxiety disorders. METHODS: Levels of gene expression in a selectively bred strain of rats with a high vulnerability to develop congenitally learned helplessness (cLH), a strain highly resistant to the same behavior (cNLH) and outbred Sprague-Dawley (SD) control animals were compared using quantitative reverse transcription polymerase chain reaction. RESULTS: Congenitally learned helpless animals had a 24%-30% reduced expression of the cyclic adenosine monophosphate response element binding protein messenger ribonucleic acid (mRNA) in the hippocampus and a 40%-41% increased level of the antiapoptotic protein bcl-2 mRNA in the prefrontal cortex compared to cNLH and SD rats. Other significant changes included changes in the expression levels of the alpha catalytic subunit of protein kinase A, glycogen synthase kinase 3beta, and protein kinase C epsilon. CONCLUSIONS: Congenitally learned helpless animals show evidence of altered signal transduction and regulation of apoptosis compared to cNLH and SD control animals.