Angiotensin II and angiotensin-(1-7) inhibit the inner cortex Na+ -ATPase activity through AT2 receptor

In the present paper, the modulation of the basolateral membrane (BLM) Na⁺-ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) was evaluated. Ang II and Ang-(1–7) inhibit the Na⁺-ATPase activity in a dose-dependent manner (from 10⁻¹¹ to 10⁻⁵ M), with maximal effect obtained at 10⁻⁷ M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1–7) are mediated by AT₂ receptor: The effect of both polypeptides is completely reversed by 10⁻⁸ M PD 123319, a selective AT₂ receptor antagonist, but is not affected by either (10⁻¹²–10⁻⁵ M) losartan or (10⁻¹⁰–10⁻⁷ M) A779, selective antagonists for AT₁ and AT_(1–7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3′,5′-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10⁻⁹ M guanosine 5′-O-(2-thiodiphosphate) (GDPβS; an inhibitor of the G protein activity), and mimicked by 10⁻¹⁰ M guanosine 5′-O-(3-thiotriphosphate) (GTPγS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the G_s protein in the isolated basolateral membrane fraction; (4) (10⁻¹⁰–10⁻⁶ M) cAMP has a similar and non-additive effect to Ang II and Ang-(1–7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1–7); and (6) both angiotensins stimulate PKA activity.     

Bradykinin counteracts the stimulatory effect of angiotensin-(1-7) on the proximal tubule Na+ -ATPase activity through B2 receptor

Recently, we demonstrated that angiotensin-(1–7) (Ang-(1–7)) stimulates the Na⁺-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B₂ receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1–7)-stimulated Na⁺-ATPase activity was evaluated. Preincubation of Na⁺-ATPase with 10⁻⁹ M Ang-(1–7) increases enzyme activity from 7.9±0.9 to 14.1±1.5 nmol Pi mg⁻¹ min⁻¹, corresponding to an increase of 79% (p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10⁻¹⁴–10⁻⁸ M), reaching maximal inhibitory effect at 10⁻⁹ M. Des-Arg⁹ bradykinin (DABK), an agonist of B₁ receptor, at the concentrations of 10⁻⁹–10⁻⁷ M, does not mimic the BK inhibitory effect, and des-Arg⁹-[Leu⁸]-BK (DALBK), a B₁ receptor antagonist, at the concentrations of 10⁻¹⁰–10⁻⁷ M, does not prevent the inhibitory effect of BK on Ang-(1–7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B₂ receptor, abolishes the inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10⁻⁷ M. Taken together, these data indicate that stimulation of B₂ receptors by BK can counteract the stimulatory effect of Ang-(1–7) on the proximal tubule Na⁺-ATPase activity.     

Properties of amino-terminal parathyroid hormone-related peptides modified at positions 11–13

Biological properties of amino-terminal PTHrP analogues modified in the region 11–13 were examined using ROS 17/2.8 cells. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶]PTHrP(1–36)amide had a 17-fold lower binding affinity for the receptor (apparent K_d: 5 × 10⁻⁸ M) than [Tyr³⁶]PTHrP(1–36)amide or [Arg^11,13,Tyr³⁶]PTHrP(1–36)amide (apparent K_d for both: 2 × 10⁻⁹ M). Moreover, it is only a weak partial agonist despite completely inhibiting radioligand binding. [Leu¹¹,D-Trp¹²,Arg¹³,Tyr³⁶,Cys³⁸]PTHrP(7–38) and PTHrP(7–34)amide had similar receptor affinities (apparent K_ds: 5 × 10⁻⁸ M and 8 × 10⁻⁸ M), while that of [Nle^8,18,Tyr³⁴]bPTH(7–34)amide was more than 10-fold lower (apparent K_d: 2 × 10⁻⁶ M). These changes in biological properties suggest that high affinity receptor binding requires both amino- and carboxyl-terminal domains of the PTHrP(1–36) sequence and/or intramolecular interactions which are impaired by the D-Trp substitution for Gly¹².     

Stimulatory effect of vasoactive intestinal peptide (VIP) on the secretory activity of dispersed rat adrenocortical cells. Evidence for the interaction of VIP with ACTH receptors

VIP dose-dependently increased basal, but not submaximally ACTH (10⁻¹⁰ M)-stimulated, aldosterone (ALDO) and corticosterone (B) secretion of dispersed rat capsular and inner adrenocortical cells, respectively. The maximal stimulatory effect (60–70% rise) was obtained with a VIP concentration of 10⁻⁸ M. [4-Cl-D-Phe⁶,Leu¹⁷]-VIP, a VIP-receptor antagonist (VIP-A), and corticotropin inhibiting peptide (CIP), an ACTH receptor antagonist (both 10⁻⁶ M), completely annulled VIP (10⁻⁸M)-evoked rises in basal ALDO and corticosterone secretions. The ACTH (10⁻¹⁰ M)-enhanced (about 5-fold) production of both hormones was completely reversed by CIP (10⁻⁶ M) and only partially reduced (about −30%) by VIP-A (10⁻⁶ M). The hypothesis is advanced that the weak secretagogue effect of VIP on dispersed rat capsular and inner adrenocortical cells may be due to its positive interaction with ACTH receptors.     

Vasopressin and oxytocin modulation of melatonin secretion from rat pineal glands

The rat pineal gland is known to release melatonin in response to noradrenergic stimulation. Since vasopressin (VP)- and oxytocin (OT)-containing fibers innervate the pineal gland, the effects of VP and OT on melatonin release from perifused rat pineal glands were investigated. VP (10⁻⁷ M) and OT (10⁻⁶ M) decreased the basal melatonin secretion. No dose-dependent effect was observed. At high concentrations (10⁻⁵) these peptides potentiated the isoproterenol-induced increase of melatonin secretion. Below 10⁻⁵ M no potentiation was observed. Fragments of VP {[pGlu⁴,Cys⁶]VP(4–9)} and OT {[pGlu⁴,Cys⁶]OT(4–9)} did not display any effect on the isoproterenol-induced melatonin secretion.     

Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

Spectrophotometric and kinetic studies of the binding of Ni, Co, and Mg to poly(dG-dC) · poly(dG-dC). Determination of the stoichiometry of the Ni-induced B → Z transition

Data are reported for the binding of Ni²⁺, Co²⁺, and Mg²⁺ to the B-form of double-stranded poly(dG-dC) at ionic strength conditions I = 0.001 M, 0.01 M, and 0.1 M. The apparent binding constants for Ni²⁺ and Co²⁺ are about the same and are 2- to 3-fold higher than those for Mg²⁺. Kinetic studies indicate that Mg²⁺ binds to the polynucleotide mainly (or solely) as a mobile cloud (electrostatically, outer-sphere), whereas the transition metal ions undergo site binding (inner-sphere coordination) with poly(dG-dC). The kinetic data suggest that an Ni²⁺ ion coordinates to more than one binding site at the polynucleotide, presumably to G-N₇ and a phosphate group.

At low ionic strength conditions the addition of Ni²⁺ induces a B → Z conformational transition in poly(dG-dC). As demonstrated by UV absorption and CD spectroscopy, the transition occurs at I = 0.001 M already when 3 × 10⁻⁵ – 7 × 10⁻⁵ M of Ni²⁺ are added to 8 × 10⁻⁵ M (in monomeric units) of poly(dG-dC), and at I = 0.01 M between 2.5 × 10⁻⁴ and 4.5 × 10⁻⁴ M of Ni²⁺. Using murexide as an indicator of the concentration of free Ni²⁺ ions, the amount of Ni²⁺ which is bound to the polynucleotide could be determined. At I = 0.001 M it was established that the B → Z transition begins when 1 Ni²⁺ is bound coordinatively per four base pairs, and the transition is complete when 1 Ni²⁺ is bound coordinatively per three base pairs. It is this coordinated Ni²⁺ which induces the B → Z transition.     

Effects of neuropeptides on growth of cultivated rat molar pulp fibroblasts

The effect of the neuropeptides substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) on DNA synthesis of dental pulp cells was investigated in cells grown from molar tooth bud explants from 4–6 days old rat pups. A concentration response-assay of the proliferative response of pulpal cells was performed with SP, NPY, NKA, CGRP and VIP (0.01 to 1 nM) in the presence of EGF (10 ng/ml), hydrocortisone (0.4 μg/ml) and 3% FCS, using [³H]thymidine incorporation. The results showed that SP, NKA and CGRP, but not NPY and VIP, increased the cell number in a concentration-dependent manner, with maxima at 10⁻¹⁰ – 10⁻⁹ M (SP, NKA) and 10⁻⁷ M (CGRP). No potentiating effect was noted when cells are simultaneously stimulated with SP and CGRP. The finding that SP, NKA and CGRP have growth regulatory properties on pulpal cells in vitro suggests that sensory neuropeptides may be involved during pulpal development or in wound healing after pulpal injury.     

Effect of nomegestrol acetate on estrone-sulfatase and 17β-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E₂) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17β-hydroxy-steroid dehydrogenase (17β-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E₁S: 5 × 10⁻⁹ M), nomegestrol acetate blocked very significantly the conversion of E₁S to E₂. In the MCF-7 cells, using concentrations of 5 × 10⁻⁶ M and 5 × 10⁻⁵ M of nomegestrol acetate, the decrease of E₁S to E₂ was, respectively, −43% and −77%. The values were, respectively, −60% and −71% for the T-47D cells. Using E₁S at 2 × 10⁻⁶ M and nomegestrol acetate at 10⁻⁵ M, a direct inhibitory effect on the enzyme of −36% and −18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E₁: 5 × 10⁻⁹ M) this estrogen is converted in a great proportion to E₂. Nomegestrol acetate inhibits this transformation by −35% and −85% at 5 × 10⁻⁷ M and 5 × 10⁻⁵ M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, −48%, at 5 × 10⁻⁵ M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17β-HSD activities which converts E₁S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E₂ can open new clinical possibilities in breast cancer therapy.     

Is an endothelin a neurotoxic factor in the brain?

A possible role for endothelin in the pathogenesis of hypoglycemic brain damage in rats was evaluated using an in vitro model with which we could directly monitor the release of dopamine from striatal slices. There was no evidence of impairment in case of non-exposure of the slices to endothelin-3 during 20–40 min of hypoglycemia. The response all but disappeared in striatal slices stimulated twice with 10⁻⁵ M endothelin-3 during 20 min of hypoglycemia. Hypoglycemic damage triggered by endothelin-3 was not observed in the absence of extracellular Ca²⁺. Nifedipine (10⁻⁶ M), but not verapamil (10⁻⁵ M) nor diltiazem (10⁻⁵M), protected striatal tissue from this damage. Our findings provide evidence that endothelins might be etiological factors in the development of hypoglycemic/ischemic brain injury by stimulating dihydropyridine-regulated Ca²⁺ influx.     

Mg2+ ion effect on conformational equilibrium of poly A . 2 poly U and poly A poly U in aqueous solutions

Differential UV spectroscopy and thermal denaturation were used to study the Mg²⁺ ion effect on the conformational equilibrium in poly A · 2 poly U (A2U) and poly A · poly U (AU) solutions at low (0.01 M Na⁺) and high (0.1 M Na⁺) ionic strengths. Four complete phase diagrams were obtained for Mg²⁺–polynucleotide complexes in ranges of temperatures 20–96 °C and concentrations (10⁻⁵–10⁻²) M Mg²⁺. Three of them have a ‘critical’ point at which the type of the conformational transition changes. The value of the ‘critical’ concentration ([Mg_t²⁺]_cr=(4.5±1.0)×10⁻⁵ M) is nearly independent of the initial conformation of polynucleotides (AU, A2U) and of Na⁺ contents in the solution. Such a value is observed for Ni²⁺ ions too. The phase diagram of the (A2U+Mg²⁺) complex with 0.01 M Na⁺ has no ‘critical’ point: temperatures of (3→2) and (2→1) transitions increase in the whole Mg²⁺ range. In (AU+Mg²⁺) phase diagram at 0.01 M Na⁺ the temperature interval in which triple helices are formed and destroyed is several times larger than at 0.1 M Na⁺. Using the ligand theory, a qualitative thermodynamic analysis of the phase diagrams was performed.     

Responsiveness of the 5′-flanking region of the brain type isozyme of creatine kinase to estrogens and antiestrogen

The brain type isozyme of creatine kinase (CKB) has proven to be a useful early marker for the action of steroid and other hormones. An increase in the steady state level of mRNA for CKB was found within 30 min after estrogen stimulation of immature rat uteri. Cycloheximide treatment did not inhibit CKD induction. In order to study the molecular mechanism of this induction, 2.9 kb of the 5′-flanking region of CKB fused with the CAT reporter gene was contransfected into ROS 17/2.8 and HeLa cells along with an expression plasmid for the human estrogen receptor. 17 β-Estradiol at 10⁻⁸ M or greater concentrations and the antiestrogen tamoxifen at 10⁻⁶ M stimulated CAT activity. When given simultaneously with 17 β-estradiol, tamoxifen showed a synergistic effect.     

Redox potentials of the pyridine-haem c system

1. Haem c was synthesized and purified. It was shown unequivocally that the method gives a product with the cysteine residues on the -carbon atoms at the 2 and 4 positions of the haem.

2. Redox potentials of haem c in the presence of 2.5 M pyridine were determined in the pH range 1.5–13; it was found necessary to add cetyl trimethyl ammonium bromide (CTAB) to prevent precipitation in the acid range below about pH 4. The E_m vs pH curve shows three slopes (−dE/dpH) of value, 0.18, 0.01 and 0.06 with points of inflexion at pH 3.8 and 10.6. The potentials are intermediate between those of protohaem and mesohaem obtained under similar conditions.

3. With constant haem c concentration (a) 10⁻⁴ M and (b) 10⁻⁵ M and varying pyridine concentration (0.12–5 M) it was found at pH 9.0 that E_m values increased as the pyridine concentration was increased and there was a tendency to reach a plateau value. The explanation appears to be that pyridine binds more firmly to ferroporphyrin c than to ferriporhyrin c.

4. When the pyridine concentration was kept constant (2.5 M) and the haem c concentration was varied in the range 7 · 10⁻⁴–7 · 10⁻⁶ M, it was found that a decrease in haem c concentration brought about an increase in redox potential. The results are explained as being due to dimerization of the oxidized form.

5. The results are discussed in comparison with a number of related haem systems.     

Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum

The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22–36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 × 10⁻¹¹ M (ΔIsc −2 ± 0.5 μA/cm²). The reduction was maximal at 10⁻⁷ M (Isc −23 ± 2 μA/cm²), and the concentration producing half-maximal inhibition was 10⁻⁹ M. At 10⁻⁷ M, reduction of Isc by PYY reached 90% of response to 5 × 10⁻⁵ M bumetanide. The PYY effect was partly reversed by 10⁻⁵ M forskolin (Isc +13.43 ± 2.91 μA/h·cm², p < 0.05) or 10⁻³ M dibutyryl adenosine 3′,5′ cyclic monophosphate (Isc +12 ± 1.69 μA/cm², p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 μEq/cm² (p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22–36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10⁻⁷ M the biological activity of PYY was lower in fed than fasted rats (p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion.     

Glutathione-proline activation of feeding behavior in the zoanthid Palythoa psammophilia Walsh & Bowers

Palythoa psammophilia Walsh & Bowers has a well coordinated, stereotyped feeding response, the culminating step of which is ingestion; this may be elicited by the synergistic effect of the tripeptide glutathione and the -imino acid, proline. Either activator acting separately causes responses only at high concentrations (above 10⁻⁵ M for glutathione; above 10⁻⁴ M for proline) in a reduced number of animals and at a low rate (5.00 ± 1.73 min in 5 × 10⁻³ M solutions of glutathione; 11.10±3.74 min in 5 × 10⁻³ M solutions of proline). Highest percentages of response were obtained in combinations where glutathione was at a concentration of 5 × 10⁻³ M and proline at 5 × 10⁻⁴ M or in combinations of glutathione at concentrations 5 × 10⁻⁶ M and proline at 5 × 10⁻⁵ M. The speed of ingestion is considerably enhanced when these activators are combined (1.17±1.18 min).     

Molecular changes associated with the acquisition of oestrogen hypersensitivity in MCF-7 breast cancer cells on long-term oestrogen deprivation

The growth dependence of many breast cancers on oestrogen has been exploited therapeutically by oestrogen deprivation, but almost all patients eventually develop resistance largely by unknown mechanisms. Wild-type (WT) MCF-7 cells were cultured in oestrogen-deficient medium for 90 weeks in order to establish a long-term oestrogen-deprived MCF-7 (LTED) which eventually became independent of exogenous oestrogen for growth. After 15 weeks of quiescence (LTED-Q), basal growth rate increased in parallel with increasing oestrogen sensitivity. While 10⁻⁹ M oestradiol (E2) maximally stimulated WT growth, the hypersensitive LTED (LTED-H) were maximally growth stimulated by 10⁻¹³ M E2. By week 50, hypersensitivity was apparently lost and the cells became oestrogen independent (LTED-I), although the pure antioestrogen ICI182780 still inhibited cell growth and reversed the inhibitory effect of 10⁻⁹ M E2 at 10⁻¹² to 10⁻⁷ M. Tamoxifen (10⁻⁷ to 10⁻⁶ M) had a partial agonist effect on WT, but had no stimulatory effect on LTED. Whilst LTED cells have a low progesterone receptor (PgR) expression in all phases, oestrogen receptor (ER) a expression was, on average, elevated five- and seven-fold in LTED-H and LTED-I, respectively, and serine118 was phosphorylated. ERβ expression was up-regulated and the levels of insulin receptor substrate 1 (IRS-1) remained low throughout all phases. The levels of RIP140 mRNA appeared to decrease to approximately 50% of the WT message in LTED-Q and remained constant into the hypersensitive phase. No significant changes were observed in the expression of SUG-1, TIF-1 and SMRT in LTED. The overall changes in nuclear receptor interacting proteins do not appear to be involved in the hypersensitivity. Thus, the resistance of these human breast cancer cells to oestrogen-deprivation appears to be due to acquired hypersensitivity which may be explained in part by increased levels of and phosphorylated ER.     

Fluorescence yield kinetics in the microsecond-range in Chlorella pyrenoidosa and spinach chloroplasts in the presence of hydroxylamine

The kinetics of fluorescence yield inChlorella pyrenoidosa and spinach chloroplasts were studied in the time range of 0.5 μs to several hundreds of microseconds in the presence of hydroxylamine. Fluorescence was excited with a just-saturating xenon flash with a halfwidth of 13 μs (λ = 420 nm). The fast rise of the fluorescence yield which was limited by the rate of light influx, was, in the presence of 10⁻³–10⁻² M hydroxylamine, replaced by a slow component which had a half risetime of 25 μs in essence independent of light intensity. This slow fluorescence yield increase reflects a dark reaction on the watersplitting side of Photosystem II. Simultaneous oxygen evolution measurements suggested that a fast fluorescence component is only present in organisms with intact O₂-evolving system, whereas a slow rise predominantly occurs in organisms with the watersplitting system irreversibly inhibited by hydroxylamine.

The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P⁺, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P⁺ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10⁻² M. (c) A second donor (not transporting electrons from water) transfers electrons to P⁺ with a half time of roughly 25 μs.     

Action of endogenous steroid inhibitors of brain aromatase relative to fadrozole

To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo-(1,5-a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole (K_i < 1 × 10⁻⁹ M) > 4-androstenedione 5-androstanedione > 5-dihydrotestosterone (K_i = 6 × 10⁻⁸ M) > 5β-androstanedione > 5β-dihydrotestosterone (K_i = 3.5 × 10⁻⁷ M) > 5-androstane-3, 17β-diol (K_i = 5 × 10⁻⁶ M) > 5β-androstane-3β,17β-diol. Five other steroids, 5β-androstane-3,17β-diol, 5-androstane-3β,17β-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10⁻⁴ M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole.