Cellular distribution and bioactivity of the key steroidogenic enzyme, cytochrome P450side chain cleavage, in sensory neural pathways.

Neurosteroids are steroids produced within the nervous system. Based on behavioural responses evoked in animals by synthetic steroid injections, several studies suggested neurosteroid involvement in important neurophysiological processes. These observations should be correlated only to neuroactive effects of the injected steroids. Neurosteroids mostly control the CNS activity through allosteric modulation of neurotransmitter receptors within concentration ranges used by neurotransmitters themselves. Therefore, neurosteroid production within pathways controlling a neurophysiological process is necessary to consider neurosteroid involvement in that process. Because of the increasing speculation about pain modulation by neurosteroids based on pharmacological observations, we decided to clarify the situation by investigating neurosteroidogenesis occurrence in sensory pathways, particularly in nociceptive structures. We studied the presence and activity of cytochrome P450side chain cleavage (P450scc) in rat pain pathways. P450scc-immunoreactive cells were localized in dorsal root ganglia (DRG), spinal cord (SC) dorsal horn, nociceptive supraspinal nuclei (SSN) and somatosensory cortex. Incubation of DRG, SSN or SC tissue homogenates with [3H]cholesterol yielded the formation of radioactive metabolites including [3H]pregnenolone of which the synthesis was reduced in presence of aminogluthetimide, a P450scc inhibitor. These first neuroanatomical and neurochemical results demonstrate the occurrence of neurosteroidogenesis in nociceptive pathways and strongly suggest that neurosteroids may control pain mechanisms.

Neuropeptide Y inhibits the biosynthesis of sulfated neurosteroids in the hypothalamus through activation of Y(1) receptors.

We have recently shown that hydroxysteroid sulfotransferase (HST), the enzyme responsible for the biosynthesis of pregnenolone sulfate (Delta(5)PS) and dehydroepiandrosterone sulfate (DHEAS), is expressed in neurons located in the anterior preoptic area and the dorsal magnocellular nucleus of the frog diencephalon. As these two nuclei are richly innervated by NPY-immunoreactive fibers, we investigated the possible implication of NPY in the control of Delta(5)PS and DHEAS biosynthesis. Double labeling of frog brain sections revealed that 42% of the HST-immunoreactive perikarya in the diencephalon were contacted by NPY-containing fibers. In situ hybridization studies showed that Y(1) and Y(5) receptor mRNAs are expressed in the anterior preoptic area and the dorsal magnocellular nucleus. Pulse-chase experiments with (35)S-labeled 3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor demonstrated that frog NPY (fNPY) inhibited the conversion of [(3)H]Delta(5)P and [(3)H]dehydroepiandrosterone ([(3)H]DHEA) into [(3)H,(35)S]Delta(5)PS and [(3)H,(35)S]DHEAS by diencephalic explants. The inhibitory effect of fNPY on Delta(5)PS and DHEAS formation was mimicked by (pPYY) and [Leu(31),Pro(34)]pNPY, which is an agonist for non-Y(2) receptors in mammals, and was completely suppressed by the Y(1) receptor antagonist BIBP3226. Conversely, the Y(2) receptor agonist pNPY-(13-36) and the Y(5) receptor agonist [D-Trp(32)]pNPY did not significantly modify the biosynthesis of [(3)H,(35)S]Delta(5)PS and [(3)H,(35)S]DHEAS. The present study provides the first evidence for the innervation of neurosteroid-producing neurons by NPY fibers. Our data also demonstrate that NPY, acting via Y(1) receptors, exerts an inhibitory effect on the biosynthesis of sulfated neurosteroids.