[3H]5-Hydroxytryptamine ([3H]5-HT) binds to crude brain membrane preparations at two different sites (Kd = 1-3 nM and 10-30 nM). These two sites are present in a limited number as saturable populations and selectively bind 5-HT and related structures. In the same crude membrane preparations, lysergic acid diethylamide (LSD) also binds at two different sites (Kd = 3-4 nM and 20-30 nM). 5-HT binding is found mostly in fractions enriched in synaptosomal and microsomal membranes; fractions rich in mitochondria or in synaptic vesicles have a low binding capacity. The two serotoninergic sites are physically separable; only high-affinity binding sites are found on purified synaptosomal membranes, whereas both types of sites are present in fractions enriched in microsomal membranes. The interaction between LSD and 5-HT shows that high-affinity binding sites for 5-HT are not identical with those for LSD, since the inhibition of binding of one substance by the other is complex.
Low doses of 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), quipazine and cyproheptadine produced facilitation of jumping in mice using the hot plate method. Higher doses produced severe motor disturbances which precluded the assessment of effects on nociception. The observed hyperalgesia might be a consequence of diminution of serotoninergic tone resulting either from triggering of presynaptic serotoninergic receptors in the case of 5-MeODMT and quipazine or from the blockade of postsynaptic serotoninergic receptors in the case of cyproheptadine. The 5-MeODMT-induced hyperalgesia was not attenuated by buprenorphine, which under similar conditions antagonized completely the hyperalgesic effects of naloxone; thus, the hyperalgesic effects of 5-MeODMT do not seemingly involve opioidergic receptors.
Neutral sphingomyelinase-2 (nSMase2) is a key ceramide-producing enzyme in cellular stress responses. While many posttranslational regulators of nSMase2 are known, emerging evidence suggests a more protracted regulation of nSMase2 at the transcriptional level. Previously, we reported that nSMase2 is induced by all-trans retinoic acid (ATRA) in MCF7 cells and implicated nSMase2 in ATRA-induced growth arrest. Here, we further investigated how ATRA regulates nSMase2. We find that ATRA regulates nSMase2 transcriptionally through the retinoic acid receptor-α, but this is independent of previously identified transcriptional regulators of nSMase2 (Sp1, Sp3, Runx2) and is not through increased promoter activity. Epigenetically, the nSMase2 gene is not repressively methylated in MCF7 cells. However, inhibition of histone deacetylases (HDACs) with trichostatin A (TSA) induced nSMase2 comparably to ATRA; furthermore, combined ATRA and TSA treatment was not additive, suggesting ATRA regulates nSMase2 through direct modulation of histone acetylation. Confirming this, the histone acetyltransferases CREB-binding protein and p300 were required for ATRA induction of nSMase2. Finally, use of class-specific HDAC inhibitors suggested that HDAC4 and/or HDAC5 are negative regulators of nSMase2 expression. Collectively, these results identify a novel pathway of nSMase2 regulation and suggest that physiological or pharmacological modulation of histone acetylation can directly affect nSMase2 levels.
