Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side‐chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7‐dehydrocholesterol (7‐DHC; precursor to cholesterol and vitamin D3) to 7‐dehydropregnenolone (7‐DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7‐DHC into 7‐DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7‐DHC can form 7‐DHP through P450scc side‐chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7‐steroidal dienes (7‐DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB‐induced intramolecular rearrangements to vitamin D3‐like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.

The hypothesis that functionally selective G protein–coupled receptor (GPCR) agonists may have enhanced therapeutic benefits has revitalized interest for many GPCR targets. In particular, although κ-opioid receptor (KOR) agonists are analgesic with a low risk of dependence and abuse, their use is limited by a propensity to induce sedation, motor incoordination, hallucinations, and dysphoria-like states. Several laboratories have produced a body of work suggesting that G protein–biased KOR agonists might be analgesic with fewer side effects. Although that has been an intriguing hypothesis, suitable KOR-selective and G protein–biased agonists have not been available to test this idea. Here we provide data using a G protein–biased agonist, RB-64 (22-thiocyanatosalvinorin A), which suggests that KOR-mediated G protein signaling induces analgesia and aversion, whereas β-arrestin-2 signaling may be associated with motor incoordination. Additionally, unlike unbiased KOR agonists, the G protein–biased ligand RB-64 does not induce sedation and does not have anhedonia-like actions, suggesting that a mechanism other than G protein signaling mediates these effects. Our findings provide the first evidence for a highly selective and G protein–biased tool compound for which many, but not all, of the negative side effects of KOR agonists can be minimized by creating G protein–biased KOR agonists.

The in vitro pharmacological properties of N-(1-Acetyl-2,3-dihydro-1H-indol-6-yl)-3-(3-cyano-phenyl)-N-[1-(2-cyclopentyl-ethyl)-piperidin-4yl]-acrylamide (JNJ-5207787), a novel neuropeptide Y Y₂ receptor (Y₂) antagonist, were evaluated. JNJ-5207787 inhibited the binding of peptide YY (PYY) to human Y₂ receptor in KAN-Ts cells (pIC₅₀ = 7.00 ± 0.10) and to rat Y₂ receptors in rat hippocampus (pIC₅₀ = 7.10 ± 0.20). The compound was >100-fold selective versus human Y₁,Y₄, and Y₅ receptors as evaluated by radioligand binding. In vitro receptor autoradiography data in rat brain tissue sections confirmed the selectivity of JNJ-5207787. [¹²⁵I]PYY binding sites sensitive to JNJ-5207787 were found in rat brain regions known to express Y₂ receptor (septum, hypothalamus, hippocampus, substantia nigra, and cerebellum), whereas insensitive binding sites were observed in regions known to express Y₁ receptor (cortex and thalamus). JNJ-5207787 was demonstrated to be an antagonist via inhibition of PYY-stimulated guanosine 5′-O-(3-[³⁵S]thio)triphosphate binding ([³⁵S]GTPγS) in KAN-Ts cells (pIC₅₀ corrected = 7.20 ± 0.12). This was confirmed auto-radiographically in rat brain sections where PYY-stimulated guanosine 5′-O-(3-[³⁵S]thio)triphosphate binding was inhibited by JNJ-5207787 (10 μM) in hypothalamus, hippocampus, and substantia nigra. After intraperitoneal administration in rats (30 mg/kg), JNJ-5207787 penetrated into the brain (C_max = 1351 ± 153 ng/ml at 30 min) and occupied Y₂ receptor binding sites as revealed by ex vivo receptor autoradiography. Hence, JNJ-5207787 is a potent and selective pharmacological tool available to establish the potential role of central and peripheral Y₂ receptors in vivo.

We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20‐hydroxycholecalciferol and 20,22‐dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)‐hydroxyvitamin D3. P450scc did not metabolize 25‐hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non‐P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized.