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 ( approximately 1 nmole/mg protein) of cytochromes P-450 approximates that of intracellular binding sites (K(d) 1.0-50 microM) 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 (3)H-histamine binding to cytochrome P-450 (K(i) values: testosterone 0.28 microM, progesterone 0.56 microM, flutamide 1.7 microM, tamoxifen 9.0 microM). Furthermore, all the various hormone and drug ligands are mutually inhibitory in their binding to cytochrome P-450; e.g., K(i) values of androstenedione and progesterone, to inhibit imipramine binding to P-450 (determined by spectral analysis), are 11 nM and 26 nM, respectively. The K(i) value of imiprimine to inhibit binding of androstenedione to P-450 is 3.5 microM. 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.

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)phenoxy]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 (K(S) = 4.1 +/- 0.4 microM), CYP3A4 (K(S) = 31 +/- 15 microM) and CYP1A1 (K(S) = 40 +/- 9 microM), 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 K(S) values in the range 80-600 microM. DPPE inhibited HA (600 microM) binding to CYP2D6 (IC50 = 4 microM, 95% CI= 1.8-8.9 microM) and CYP1A1 (IC50 = 135 microM: 95% CI = 100-177 microM), but stimulated HA (500 and 1000 microM) binding to CYP3A4 (EC50 = 155 microM, 95% CI = 104-231 microM). 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 microM, 95% CI = 0.5-25.5 microM), and an additional 70% inhibition occurred at the second site (IC50 = 350 microM, 95% CI = 215-570 microM). 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 K(S) 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.

The site of general anesthesia and cytochrome P450 monooxygenases: occupation of the enzyme heme pocket by xenon and nitrous oxide.

In previous studies, cytochrome P450 monooxygenases were shown to be appropriately sensitive to structurally diverse compounds varying widely in anesthetic potencies and to increasing carbon-number series of straight chain primary and secondary alcohols and rigid cyclic alcohols. We now report that xenon and nitrous oxide, at one atmosphere, occupy the P450 heme cavity and competitively inhibit catalytic activity. The heme enzymes appear to be the most relevant model of the site of general anesthesia, thus far identified.

Occupation of the cytochrome P450 substrate pocket by diverse compounds at general anesthesia concentrations.

Each of a diverse array of compounds, at concentrations reported to effect general anesthesia, when added to liver microsomes, forms a complex with cytochromes P450 to generate, with reference to a cuvette containing microsomes only, a characteristic absorbance-difference spectrum. This spectrum results from a change in the electron-spin state of the heme iron atom induced upon entry by the anesthetic molecule into the enzyme catalytic pocket. The difference spectrum, representing the anesthetic-P450 complex, is characteristic of substances that are substrates for the enzyme. For the group of compounds as a whole, the magnitudes of the absorbance-difference spectra vary only about twofold, although the anesthetic potencies vary by several orders of magnitude. The dissociation constants (Ks), calculated from absorbance data and representing affinities of the anesthetics for P450, agree closely with the respective EC50 (concentration that effects anesthesia in 50% of individuals) values, and with the respective Ki (concentration that inhibits P450 catalytic activities half-maximally) values reported by us previously. The absorbance complex resulting from the occupation of the catalytic pocket by endogenous substrates, androstenedione and arachidonic acid, is inhibited, competitively, by anesthetics. Occupation of and perturbation of the heme catalytic pocket by anesthetic, as monitored by the absorbance-difference spectrum, is rapidly reversible. The presumed in vivo consequences of perturbation by general anesthetics of heme proteins is suppression of the generation of chemical signals that determine cell sensitivity and response.

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 microM; Ks2 = 90 +/- 17 microM) that corresponded to microsomal binding sites (Kd1 = 1.0 +/- 0.9 microM; Kd2 = 57 +/- 13 microM) resolved by 3H-histamine binding; additional low affinity (Kd3 approximately 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 microM; in nuclei, Ki = 13.7 +/- 3.1 microM; 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.

Salutary clinical response of prostate cancer to antiandrogen withdrawal: assessment of flutamide in an in vitro paradigm predictive of tumor growth enhancement.

Salutary clinical responses to withdrawal of flutamide have been widely reported, indicating the potential of this arylalkylamine antiandrogen to stimulate the growth of prostate cancer. Flutamide is known to inhibit cytochrome P450-mediated testosterone synthesis and metabolism. Our laboratory has shown that arylalkylamine potencies in three in vitro assays of P450 binding or function correspond to a propensity of the drugs to enhance tumor growth in vivo. Accordingly, we measured inhibition by flutamide of (a) histamine binding to cytochrome P450 in rat liver microsomes, as determined spectrally, (b) P450-mediated demethylation of aminopyrine, and (c) DNA synthesis in mouse spleen cells stimulated by concanavalin A, and we compared its potencies in these assays with those of other arylalkylamine pharmaceuticals. Flutamide inhibited histamine binding to P450 (Ki = 31 +/- 7 microM), aminopyrine demethylation (Ki = 39 +/- 2 microM), and mitogenesis (IC50 = 12 +/- 1 microM). In overall potency, it ranked with a group of eight drugs, including the antiestrogen tamoxifen, all linked with enhanced tumor growth. In the context of clinical observations that some patients with prostate cancer benefit from flutamide withdrawal, our findings underline concerns that many arylalkylamine drugs have the potential to stimulate the growth or development of malignancies, including prostate cancer. Tumor growth enhancement by flutamide and other arylalkylamines may result from drug perturbation and/or induction of histamine-binding P450 enzymes involved in the synthesis of steroid and eicosanoid mediators that regulate gene function and cell growth.

The site of general anaesthesia and cytochrome P450 oxygenases: similarities defined by straight chain and cyclic alcohols.

1. General anaesthetics disrupt normal cell receptivity and responsiveness while sparing vital respiratory processes. Ultimate elucidation of the molecular basis of general anaesthesia presumes the identification of one or more subcellular components with appropriate sensitivity to the entire array of anaesthetics. 2. Previously, we showed the universal cellular enzymes, cytochrome P450 mono-oxygenases, to be sensitive at relevant concentrations to all anaesthetics tested. The potential significance of P450 inhibition by anaesthetics resides in the contribution of this enzyme family, in conjunction with that of cyclo-oxygenases and lipoxygenases, to the generation from arachidonic acid of lipid second messengers, the eicosanoids. 3. We have shown that P450 enzymes model the site of general anaesthesia in the tadpole with respect to (a) an absolute sensitivity to increasing chain-length series of flexible, straight chain primary and secondary alcohols and straight chain diols, (b) an absolute sensitivity to increasing molecular weight series of rigid cyclic alkanols and cyclic alkanemethanols, (c) the points of abrupt change and of reversal (cut-off) in the linear relationship between increasing anaesthetic potency with increasing carbon chain length, and (d) non-differentiation between secondary alkanol enantiomers. These findings reveal the P450 enzyme family as the most relevant biomolecular counterpart of the site of general anaesthesia, thus far identified.

Subanesthetic concentrations of drugs inhibit cytochrome P450-mediated metabolism of aniline.

We reported previously that 18 compounds varying in general anesthetic potency by up to 66 000-fold inhibited, at anesthetic concentrations, the metabolism of arachidonic acid and aminopyrine by cytochrome P450 monooxygenases in rat liver microsomes. Now, we report that P450-mediated para-hydroxylation of aniline is more sensitive to the anesthetics. The Ki values for enzyme inhibition for seven compounds were close to and for seven compounds 5-40 times less than their respective anesthetic potencies. Endogenous substrates with an aniline-like binding mode to P450 include histamine and related imidazoles. Acetone and each of the halogenated compounds, halothane, enflurane, and chloroform, stimulated aniline hydroxylase activity at concentrations well below and above their EC50 values. These potent actions on the universal P450 isoenzymes may contribute to pharmacological effects of the anesthetics associated with levels of drug well below concentrations that effect general anesthesia.

Enhanced cancer growth in mice administered daily human-equivalent doses of some H1-antihistamines: predictive in vitro correlates.

BACKGROUND: Present studies of drug-induced tumor growth promotion have evolved from earlier investigations into the mechanism of action of N,N-diethyl-2-[4-(phenylmethyl)phenoxy[ethanamine.HCl, a tamoxifen derivative which potently inhibits lymphocyte mitogenesis in vitro and stimulates tumor growth in vivo. It is thought that potency to bind to intracellular histamine receptors (HIC), some of which are on cytochromes P450, may correlate with tumor growth-promoting activity. PURPOSE: We assessed the effectiveness of five in vitro assays in predicting in vivo tumor growth stimulation by the H1-antihistamines loratadine, astemizole, cetirizine, hydroxyzine, and doxylamine. METHODS: Potency of each agent was ranked 1-5 in each of the following in vitro assays: 1) inhibition of [3H]histamine binding to microsomal HIC, 2) inhibition of histamine binding to microsomal P450, 3) inhibition of the P450-catalyzed demethylation of aminopyrine, 4) inhibition of lymphocyte mitogenesis, and 5) stimulation of tumor colony formation. An overall rank score was assigned to each drug and correlated with tumor growth stimulation in vivo. Two laboratories conducted in vivo studies in a blinded fashion. Female C57BL and C3H mice were given a subcutaneous injection on day 1 of syngeneic B16F10 melanoma cells (5 x 10(5)) or C-3 fibrosarcoma cells (1 x 10(5)), respectively. Mice were randomly assigned to treatment groups, then received a single, daily intraperitoneal injection of an estimated human-equivalent dose (or range of doses) of antihistamine or vehicle control for 18-21 days before being killed. Tumors were surgically removed and wet weights compared statistically among groups. RESULTS: The cumulative potency of each drug in affecting tumor growth or growth mechanisms in the five in vitro assays ranked as follows: Loratidine and astemizole ranked highest and were equally potent, followed in decreasing order by hydroxyzine, doxylamine, and cetirizine. A significant correlation (r = .97; P < .02) was observed between the rank order of potency of the antihistamines in all five in vitro assays and the rank order to enhance tumor growth in vivo: Loratidine and astemizole significantly (P < .001) promoted the growth of both melanoma and fibrosarcoma, hydroxyzine significantly (P < .001) promoted the growth of melanoma, while doxylamine and cetirizine did not promote the growth of either tumor. CONCLUSION: Data demonstrate that the in vitro assays predicted the propensity of each H1-antihistamine to stimulate cancer growth in vivo. IMPLICATION: These in vitro tests may prove valuable to screen potential tumor growth promoters.

General anesthetics inhibit cytochrome P450 monooxygenases and arachidonic acid metabolism.

Identification of a specific biomolecular target appropriately sensitive to a wide array of anesthetics has been elusive. At concentrations close to their respective ED50's for anesthesia in man or other species, 18 compounds, differing in potencies up to 66,000 fold, inhibited cytochrome P450 mediated metabolism of aminopyrine, a synthetic substrate, and arachidonic acid (AA), an endogenous substrate, in isolated liver microsomes. There was a highly significant correlation for both substrates between the absolute concentrations required for anesthesia (EC50) and for inhibition of P450 activity (Ki or IC50). The mean Ki/EC50 ratio was 0.97 for inhibition of aminopyrine demethylase. The mean IC50/EC50 ratios were 0.42 and 0.64 for inhibition of two AA-derived products and 2.8 for a third; a mean ratio of 1.4 for inhibition of overall AA metabolism suggests interaction of general anesthetics with a composite of P450 isozymes. The universal cytochrome P450 monooxygenases, in conjunction with other lipid oxygenases (cyclooxygenases and lipoxygenases) participate in the second messenger AA cascade. In nerve cells the sensitivity of these enzymes to hydrophobic neurodepressant drugs may underlie the state of general anesthesia: reversible disruption of intracellular and intercellular signalling without impairment of enzymes vital to cell respiration.

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 microM) 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 microM) was potently inhibited by TP (Ki 0.33 microM). 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 microM and 1.5 mM for histamine, and 0.3 microM and 3.7 microM 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.

Adrenocorticotropin, vasoactive intestinal polypeptide, growth hormone-releasing factor, and dynorphin compete for common receptors in brain and adrenal.

It was previously shown in this laboratory that high affinity binding of [125I]ACTH-(1-24) to membranes from rat brain was inhibited by vasoactive intestinal polypeptide (VIP), GH-releasing factor (GRF), and dynorphin (DYN), but not by other peptides tested. We now show that these peptides compete for [125I]VIP binding in brain and for [125I]ACTH-(1-24) binding in adrenal cortex and promote steroidogenesis. The high affinity sites for [125I]ACTH-(1-24) in the rat brain and bovine adrenal had Kd values of 0.51 +/- 0.41 and 3.9 +/- 1.3 nM, respectively; and the Ki values for VIP were 5.4 +/- 4.2 and 1.4 +/- 0.51 nM, respectively. In rat brain and bovine adrenal the high affinity site for [125I]VIP had Kd values of 2.9 +/- 1.7 and 0.5 +/- 0.8 nM, respectively, and Ki values for ACTH of 23.6 +/- 14.0 and 22.2 +/- 33.0 nM, respectively. In brain, DYN and GRF inhibited binding of [125I]VIP with Ki values of 49 and 30 nM, respectively. Cortisol secretion from isolated bovine adrenal cortical cells was significantly stimulated by 10(-10) M ACTH, VIP, DYN, or GRF, and a maximal response occurred for each at 10(-8) M. However, maximal cortisol production in response to VIP, DYN, or GRF was only about half that by ACTH-(1-24). The combination of ACTH-(1-24) and VIP, each at 10(-10) M, was additive in stimulating cortisol production, whereas each at 10(-8) M caused no greater response than ACTH alone. There was an additive steroidogenic effect of VIP plus ACTH-(1-10), but not VIP plus ACTH-(11-24). Specific binding of [125I]ACTH-(11-24) in adrenal membranes was inhibited by unlabeled ACTH-(11-24), ACTH-(1-24), VIP, GRF, and DYN, but not by ACTH-(1-10), peptide T, TRH, alpha MSH, or beta-endorphin; there was no specific binding of [125I]ACTH-(1-10). Functional studies and binding data, in conjunction with the existence of homologous amino acid sequences, indicate that VIP, GRF, and DYN interact at a subpopulation of ACTH receptors that recognizes a moiety within the 11-24 sequence of the ACTH molecule.

ACTH receptors in nervous tissue. High affinity binding-sequestration of [125I]Phe2,Nle4]ACTH 1-24 in homogenates and slices from rat brain.

We have demonstrated specific, high affinity binding of a biologically active Tyr23-monoiodinated derivative of ACTH, [125I][Phe2,Nle4]ACTH 1-24, in rat brain homogenates. Similarly, in metabolically inhibited and noninhibited rat whole brain slices there is a specific "binding-sequestration" process that is dependent on time, protein concentration, and pH. In homogenates, binding curves were best described by a two-site model and provided the following parameters: Kd1 = 0.65 +/- 0.47 nM, Bmax1 = 21 +/- 41 fmol/mg protein; Kd2 = 97 +/- 48 nM, Bmax2 = 3.5 +/- 1.8 pmol/mg protein. In metabolically viable brain slices, concentration-competition curves of [125I][Phe2,Nle4]ACTH 1-24 binding-sequestration can be described by three components (Kd1 = 14 +/- 24 nM, Bmax1 = 50 +/- 95 fmol/mg protein; Kd2 = 2.4 +/- 1.9 microM, Bmax2 = 44 +/- 49 pmol/mg protein; Kd3 = 0.16 +/- 1.0 mM, Bmax3 = 5.3 +/- 54 nmol/mg protein). Metabolic inhibition, by removal of glucose and addition of 100 microM ouabain, abolishes the lowest affinity, highest capacity binding-sequestrian component only (Kd1 = 7.1 +/- 14 nM, Bmax1 = 8.7 +/- 16 fmol/mg protein; Kd2 = 7.4 +/- 4.49 microM, Bmax2 = 37 +/- 27 pmol/mg protein). The two binding-sequestration parameter estimates obtained from metabolically inhibited tissue slices are not significantly different from those of the two higher affinity components obtained with noninhibited tissue. Thus, metabolic inhibition permits demonstration of ACTH receptor binding only, unconfounded by sequestration or internalization of ligand:receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Surface activity and human blood platelet aggregation-inhibitory potency.

Surface and interfacial activity is correlated with molecular constitution and inhibitory potency of mono- and bis(carbamoylpiperidino)alkanes and aralkanes, and of some corresponding quaternary pyridinium congeners, in ADP-induced human blood platelet aggregation. The measurements of surface and interfacial tension were carried out at concentrations and pH-values approximating those employed in the hemodynamic study. The effect of changes in chemical structure, ranging from relatively minor variations in a specific functional group to the alteration of major components in molecular constitution, was examined and interpreted in terms of contemporary theoretical chemistry.

Ouabain receptor binding of hydroxyprogesterone derivatives.

1 A specific and sensitive radioreceptor assay ahs 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 hydroxyprogesterone having a 17 alpha-acetate substituent: chlormadinone acetate, megestrol acetate, cyproterone acetate and medroxyprogesterone acetate, with IC50s of 2, 7.4, 9 and 21 microM, respectively. Prednisolone-3,20-bisguanyl-hydrazone, reported to have inotropic activity, gave an IC50 of 6.4 microM. Cyproterone-17 alpha-OH was less active (IC50 90 microM) than cyproterone-17 alpha-acetate. 3 A large number of peptide and protein hormones, steroid hormones and their metabolites, amines, and drugs were inactive.

Endorphin activity in anterior, intermediate, and posterior pituitary.

The content of endogenous morphine-like substance in bovine pituitary and brain was determined by an opiate radioreceptor assay. The intermediate lobe was most concentrated in activity and the brain least concentrated. Most of the endorphin is obtained in a 120 000 g-min fraction from pituitary or brain homogenates.

Monolayer cultures of dispersed cells from bovine anterior pituitary: responses to synthetic hypophysiotropic peptides.

Cells were dispersed from bovine anterior pituitary glands, by digestion with collagenase, and cultured. After 4 days the cell monolayers were incubated with fresh medium containing synthetic hypophysiotropic peptides for 2, 6, or 20 h, and hormone released into the medium was estimated by radioimmunoassay. After 2 h, thyroid releasing hormone (TRH) stimulated the release of thyroid-stimulating hormone (TSH) up to eightfold, and of prolactin (PRL) and follicle-stimulating hormone (FSH) about twofold at a minimal effective concentration of 1 ng/ml; enhanced growth hormone (GH) release was not apparent until 20 h, and release of luteinizing hormone (LH) and adrenocorticotrophic hormone (ACTH) was unaffected. Luteinizing hormone releasing hormone (LH-RH) enhanced release of LH maximally (three- to fourfold) during a 2 h incubation and was effective at 0.1 ng/ml; FSH release was significantly enhanced by about 50% above control level. Growth hormone release inhibiting hormone (GH-RIH)(somatostatin) showed significant effects only in the 20 h incubation; GH release was inhibited by 50% and release of PRL was slightly, but significantly, enhanced. Pituitary cell monolayers apparently permit maximal expression of releasing activities inherent in the hypothalamic hormones.