Gary Queen

Pituitary hormone releasing or inhibiting activity of metal ions present in hypothalamic extracts.

Abstract In vitro trophin-releasing and inhibiting activities of certain purified fractions from bovine hypothalamus were apparently due primarily to the presence of divalent metals. The most abundant metal was copper which at concentrations less than 1 μg/ml markedly stimulated the release of all hormones. Zinc ion which was much less potent than copper, inhibited release of prolactin but stimulated release of the other hormones. Nickel, as low as 1 μg/ml, specifically inhibited prolcatin release; at much higher levels it enhanced release of the other hormones.

H3 receptor antagonist, thioperamide, inhibits adrenal steroidogenesis and histamine binding to adrenocortical microsomes and binds to cytochrome P450.

1 Thioperamide (TP), an imidazole and a highly potent, specific antagonist of the histamine H3 receptor, inhibited the secretion of cortisol from bovine isolated adrenocortical cells (IC50 0.20 μm) and, in the rat (5 mg kg−1) prevented both basal and stress‐induced secretion of corticosterone. 2 In adrenocortical microsomes, low affinity binding of [3H]‐histamine (KD 27.7 μm) was potently inhibited by TP (Ki 0.33 μm). 3 In adrenocortical microsomal membranes, both histamine and TP yielded type II difference absorption spectra, characteristic of the interaction between imidazole and cytochrome P450 enzymes. Dissociation constants for binding to P450, calculated from spectral data, were 15.9 μm and 1.5 mm for histamine, and 0.3 μm and 3.7 μm for TP. 4 In view of previously reported evidence for an intracellular mediator role of histamine in platelets, the present findings suggest a physiological role for histamine in the modulation of adrenal P450 monooxygenases that generate adrenocortical steroids. 5 The results suggest that direct adrenocortical inhibition by thioperamide at a non‐H3 intracellular site must be taken into account in studies designed to elucidate functional roles of H3 receptors.

Lipotropin: Localization by radioimmunoassay of endorphin precursor in pituitary and brain☆

Abstract Beta-Lipotropic Hormone (LPH) was estimated in pituitary and brain by a radioimmunoassay specific for the N-terminal, non-opiate portion of the protein, and endorphin activity by an opiate radioreceptor assay. The intermediate pituitary is most concentrated in LPH and endorphin. Gel filtration indicated the presence in the pituitary of intact LPH and N-terminal fragments but only intact LPH in brain. Endorphin activity in pituitary is associated primarily with a component of 3000 daltons and to a lesser extent with one of about 2000 daltons. Brain endorphin activity is mostly accounted for by a 2000 dalton component, enkephalins representing an apparently minor activity. In pituitary and brain LPH and endorphin are entirely associated with a 12,000 g/10 min fraction.

Prolactin secretion is specifically inhibited by nickel.

PROLACTIN (PRL) secretion from the mammalian anterior pituitary gland is apparently chiefly under hypothalamic tonic inhibition in contrast to the primarily stimulatory influence of the brain on the other trophic hormones1. Extracts of mammalian hypothalamus inhibit the release of PRL in vitro and in vivo1. We report here that PRL-inhibiting-factor (PIF) activity in certain fractions derived from acetic acid extracts of bovine hypothalamus was due to nickel ion (Ni2+) in the extracts. Commercial nickel salts and purified PIF from bovine brain specifically inhibit release of PRL in vivo in the rat and in vitro from bovine pituitary, (a) in basal conditions, (b) in the presence of copper ion which markedly enhances release of all trophic hormones, and (c) following brief exposure of the pituitary tissue to cold, another effective, non-specific stimulant of hormone release.

Subcellular localization of endorphine activity in bovine pituitary and brain.

Summary The endorphine content of bovine anterior (AP), posterior (PP), intermediate (IP) pituitary, brain, and subcellular fractions of these tissues was estimated in an opiate radioreceptor assay. IP was most concentrated in activity, containing 7, 15, 780, and 9,100 times as much as PP, AP, midbrain, and cortex, respectively. More than 90 per cent of total endorphine activity in all lobes of the pituitary sedimented during centrifugation at 12,000 × g for 10 min. Density-gradient centrifugation gave almost identical sedimentation patterns for all pituitary lobes. In brain, endorphine activity sediments similarly but not identically to that of pituitary. Endorphine activity in the pituitary is localized to a secretory granule presumably derived from a cell type common to all lobes. Brain endorphine is localized to a similar granule, but derived at least in part from nerve endings.

N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine (DPPE), a chemopotentiating and cytoprotective agent in clinical trials: interaction with histamine at cytochrome P450 3A4 and other isozymes that metabolize antineoplastic drugs

Purpose: N,N-diethyl-2-[4-(phenylmethyl)phe- noxy]ethanamine · HCl (DPPE), an intracellular histamine (HA) antagonist with chemopotentiating and cytoprotective properties, is currently in phase 2 and 3 clinical trials in breast and prostate cancer. DPPE modulates growth at in vitro concentrations that antagonize HA binding to cytochromes P450 in rat liver microsomes. HA inhibits P450 metabolism of some drugs. Recent in vitro studies in human colon cancer cells have linked DPPE enhancement of paclitaxel, doxorubicin and vinblastine cytotoxicity to inhibition of the P-glycoprotein (P-gp) pump. Many substrates of P-gp are also substrates of CYP3A4, a P450 isozyme that metabolizes a variety of antineoplastic agents and is highly expressed in some malignant tissues. Therefore, we assessed whether (a) DPPE and HA interact at CYP3A4 and other P450 human isozymes, and (b) DPPE inhibits the catalytic activity of CYP3A4. Methods: Using spectral analysis, we measured DPPE and HA binding to insect microsomes that express human P450 isozymes 1A1, 2B6, 2D6 or 3A4. Employing thin-layer chromatography, we assessed the metabolism of DPPE by each isozyme and DPPE inhibition of testosterone metabolism by CYP3A4 and by rat liver microsomes. Results: (1) DPPE evoked “type I” (substrate site binding) absorbance-difference spectra with CYP2D6 (Ks=4.1 ± 0.4 μM), CYP3A4 (Ks= 31 ±15 μM) and CYP1A1 (Ks=40 ± 9 μM), but not with CYP2B6. (2) In correspondence with the binding studies, DPPE was metabolized by CYP2D6, CYP3A4 and CYP1A1; no metabolism occurred with CYP2B6. (3) HA evoked “type II” (heme iron binding) absorbance-difference spectra with all four isozymes, with Ks values in the range 80–600 μM. DPPE inhibited HA (600 μM) binding to CYP2D6 (IC50=4 μM, 95% CI=1.8–8.9 μM) and CYP1A1 (IC50=135 μM: 95% CI=100–177 μM), but stimulated HA (500 and 1000 μM) binding to CYP3A4 (EC50=155 μM, 95% CI=104–231 μM). DPPE did not affect HA binding to CYP2B6. (4) DPPE inhibited the metabolism of testosterone by CYP3A4. The concentration/effect curve was biphasic: DPPE inhibited metabolism by 30% at the first site (IC50=3 μM, 95% CI=0.5–25.5 μM), and an additional 70% inhibition occurred at the second site (IC50=350 μM, 95% CI=215–570 μM). A similar result was observed with rat liver microsomes. Conclusion: DPPE is a substrate for CYP3A4, CYP2D6 and CYP1A1, but not CYP2B6. DPPE inhibits testosterone metabolism by interacting at two sites on CYP3A4, the first correlating with its Ks value to bind the substrate site and the second, with its EC50 value to enhance HA binding to the heme iron. We postulate that (1) the inhibitory effect of DPPE on CYP3A4 activity is mediated directly at the substrate site and indirectly by its enhancement of the binding of HA to the heme moiety; (2) in tumor cells that express high constitutive levels of CYP3A4, potentiation of chemotherapy cytotoxicity by DPPE results, in part, from inhibition of CYP3A4-mediated metabolism and P-gp-mediated efflux of antineoplastic drugs; (3) in normal cells that express low constitutive levels of the isozyme, cytoprotection by DPPE results, in part, from induction of CYP3A4 and P-gp, resulting in an increase both in metabolism and efflux of antineoplastic drugs.

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei, and chromatin from rat hepatocytes

Histamine and polyamines have been implicated in the mediation of cell proliferation. Our previous work linked the growth‐modulatory effects of histamine with its binding to intracellular sites in microsomes and nuclei of various tissues. In this study, we identify cytochrome P450 enzymes as a major component of microsomal intracellular sites in hepatocytes and demonstrate that polyamines compete with high affinity for histamine binding to them. Spectral measurement of histamine binding to P450 in liver microsomes resolved high and intermediate affinity binding sites (Ks1 = 2.4 ± 1.6 μM; Ks2 = 90 ± 17 μM) that corresponded to microsomal binding sites (Kd1 = 1.0 ± 0.9 μM; Kd2 = 57 ± 13 μM) resolved by 3H‐histamine binding; additional low affinity (Kd3 ∼ 3 mM), and probably physiologically irrelevant, sites were resolved only by 3H‐histamine radioligand studies. As determined spectrally, treatment of microsomes with NADPH/carbon monoxide decreased histamine binding to P450 by about 90% and, as determined by 3H‐histamine binding, abolished the high affinity sites and reduced by 85% the number of intermediate sites. Spermine competed potently for 3H‐histamine binding: in microsomes, Ki = 9.8 ± 5.8 μM; in nuclei, Ki = 13.7 ± 3.1 μM; in chromatin, Ki = 46 ± 33 nM. Polyamines inhibited the P450/histamine absorbance complex with the rank order of potency: spermine > spermidine ≫ putrescine. In contrast, histamine did not compete for 3H‐ spermidine binding in nuclei or microsomes, suggesting that polyamines modulate histamine binding allosterically. We propose that certain P450 isozymes that modulate gene function by controlling the level of oxygenated lipids, represent at least one common intracellular target of growth‐regulatory endogenous bioamines and, as shown previously, of exogenous growth‐modulatory drugs including antiestrogens, antiandrogens, and certain antidepressants and antihistamines. J. Cell. Biochem. 69:233–243, 1998.

Ouabain receptor binding of hydroxyprogesterone derivatives.

1 A specific and sensitive radioreceptor assay has been devised which is based on high affinity, saturable binding of 9 nm [3H]‐ouabain to the total particulate fraction isolated from dog heart. Ouabain and other cardiac glycosides, including the aglycones, were about equipotent in their ability to displace [3H]‐ouabain from its receptor, the IC50s ranging from 10 to 30 nm. 2 The only other substances found to compete significantly in the assay were derivatives of hydroxy‐progesterone having a 17α‐acetate substituent: chlormadinone acetate, megestrol acetate, cyproterone acetate and medroxyprogesterone acetate, with IC50s of 2, 7.4, 9 and 21 μm, respectively. Prednisolone‐3,20‐bisguanyl‐hydrazone, reported to have inotropic activity, gave an IC50 of 6.4 μm. Cyproterone‐17α‐OH was less active (IC50 90 μm) than cyproterone‐17α‐acetate. 3 A large number of peptide and protein hormones, steroid hormones and their metabolites, amines, and drugs were inactive.

Displacement of histamine from liver cells and cell components by ligands for cytochromes P450.

Intracellular histamine (HA) and cytochrome P450 monooxygenases (P450) each have been proposed as mediators of cell function, growth, and proliferation. The P450 family of heme enzymes is found in virtually all cells and generates, transforms, or inactivates steroids and other lipids that participate in cell regulation. We previously demonstrated a second messenger role for HA in blood platelets and the formation of a HA‐P450 heme complex when exogenous HA was added to microsomes isolated from rat liver cells or to purified human P450 isozymes. Employing a radioimmunoassay, we now demonstrate that rat liver slices, microsomes derived from the livers of adult male rats and mast cell‐deficient mice, and hepatoma cells, all contain endogenous HA. HA release from microsomes into the incubation medium, as determined by radioimmunoassay, is enhanced in the presence of carbon monoxide, steroids, and certain drugs, all agents that unite either directly with the iron atom or bind elsewhere within the heme cavity. Rat liver slices preincubated with 3H‐HA release labeled amine into the medium in the presence of those same ligands. These findings provide evidence of an in situ HA‐P450 complex and offer further support that the imidazole, HA, is a physiological, intracellular modulator of cytochromes P450 in liver cells, and perhaps of these and other heme proteins in tissues in general. J. Cell. Biochem. 85: 820–824, 2002.

Interactive binding at cytochrome P-450 of cell growth regulatory bioamines, steroid hormones, antihormones, and drugs.

The virtually universal family of P‐450 isozymes contribute to the regulation of cell growth by modulating the levels of steroids and other lipid messengers for cytoplasmic and nuclear processes, including gene expression. In microsomes from rat liver cells, the concentration (∼1 nmole/mg protein) of cytochromes P‐450 approximates that of intracellular binding sites (Kd 1.0–50 μM) for histamine. The potencies of certain therapeutic drugs to inhibit catalytic activity of, and histamine binding to, cytochromes P‐450 in vitro were previously shown by us to be predictive of relative propensities to modulate tumor growth in rodents. Also, we demonstrated that growth‐regulating polyamines potently interact with histamine at P‐450. We now show that several classes of steroid hormones, antiestrogens, and antiandrogens, as well as various arylalkylamine drugs, all potently inhibit 3H‐histamine binding to cytochrome P‐450 (Ki values: testosterone 0.28 μM, progesterone 0.56 μM, flutamide 1.7 μM, tamoxifen 9.0 μM). Furthermore, all the various hormone and drug ligands are mutually inhibitory in their binding to cytochrome P‐450; e.g., Ki values of androstenedione and progesterone, to inhibit imipramine binding to P‐450 (determined by spectral analysis), are 11 nM and 26 nM, respectively. The Ki value of imiprimine to inhibit binding of androstenedione to P‐450 is 3.5 μM. We estimate the total P‐450 content in microsomes to be greater in male than in female rats and correlated with the number of binding sites for histamine, but not for steroids and drugs that appear to be more selective for P‐450 isozymes. Thus, for at least some isozymes, the homeostatic role of the monooxygenases may be governed by histamine, modulated by endogenous ligands, and perturbed by many foreign molecules. J. Cell. Biochem. 76:686–694, 2000.