Biomedical subjects

J Mazella

Publications and source records attributed to J Mazella.

At least 73 records · Page 4Linked to original sources

PubMed1988

Solubilization and characterization of active neurotensin receptors from mouse brain.

Neurotensin receptors were solubilized from mouse brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). The binding of 125I-labeled [Tyr3]neurotensin to the soluble fraction was time-dependent, saturable, and reversible. Unlabeled neurotensin and its analogues acetylneurotensin (8-13), neurotensin (9-13), and neurotensin (1-12) competitively antagonized the binding of 125I-labeled [Tyr3]neurotensin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane-bound receptors. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of neurotensin binding sites with a Kd of 0.36 nM and a Bm of 63 fmol/mg. As already observed with membrane-bound receptors, the affinity of neurotensin for the soluble binding activity was decreased by Na+ ions. By contrast, soluble receptors were no longer sensitive to GTP and the antihistamine drug levocabastine. A molecular weight of about 100,000 was determined for soluble neurotensin receptors both under native conditions by gel filtration on Ultrogel AcA 34 and under denaturating conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling.

Animals↗

PubMed1987

Distribution of neurotensin binding sites in rat brain: a light microscopic radioautographic study using monoiodo [125I]Tyr3-neurotensin.

The topographic distribution of specifically labeled neurotensin binding sites was examined by light microscopic radioautography in rat brain sections incubated with monoiodo [125I]Tyr3-neurotensin. Preliminary experiments indicated that under the present experimental conditions [125I]neurotensin specifically binds to a single apparent population of sites with a dissociation constant of 7.7 +/- 0.3 nM, and that fixation of the labeled sections with glutaraldehyde ensures regionally proportional retention of more than 70% of bound [125I]neurotensin molecules. High concentrations of [125I]neurotensin binding sites were detected in the olfactory bulb and tubercle, parts of the neocortex, the lateral septum, the diagonal band of Broca, the caudate putamen, the amygdala, the dentate gyrus, the anterior dorsal nucleus of the thalamus, the suprachiasmatic nucleus of the hypothalamus, the medial habenula, the zona incerta, the substantia nigra and the ventral tegmental area. In certain areas, such as in the diagonal band of Broca, the substantia innominata, the nucleus basalis and the pars compacta of the substantia nigra, discrete accumulations of silver grains were apparent over neuronal perikarya and their proximal dendrites. In most areas, however, the label appeared more or less uniformly distributed over nerve cell bodies and surrounding neuropil. In several instances, the labeling conformed with the distribution of cell bodies of origin and terminal aborizations of specific projection systems, suggesting that neurotensin receptors might be distributed both proximally and distally on the plasma membrane of certain neurons. Such putative "neurotensinoceptive" projection systems might involve part of the mesostriatal, mesocortical and mesolimbic dopamine systems, as well as the raphe-prosencephalic serotonin system and the habenulo-interpeduncular and basal forebrain-cortical cholinergic systems. Finally, areas of dense [125I]neurotensin labeling often corresponded to zones previously shown to exhibit intense acetylcholinesterase staining, suggesting the existence of a possible link between the expression of neurotensin binding sites and that of acetylcholinesterase in certain neuronal populations.

Animals↗

PubMed1987

Progestin-dependent effect of forskolin on human endometrial aromatase activity.

We have previously demonstrated that the aromatase activity in human endometrial stromal cells is stimulated by progestin and enhanced by oestrogen. In this study, we have investigated the effect of forskolin (Fk), an agent that stimulates the hormone-sensitive adenylate cyclase in mammalian cells, on the intracellular cAMP content and aromatase activity in endometrial stromal cells in primary culture. Stromal cells were isolated from proliferative and secretory endometria and were individually cultured in nutrient medium or medium supplemented with medroxyprogesterone acetate (MPA), oestradiol (E2) and Fk, separately or in combination. The intracellular cAMP content of stromal cells was increased after incubation with Fk. Stromal cells treated with Fk alone or FK and MPA for 1-3 days sustained the elevated intracellular cAMP content but 90% of this nucleotide was released to the medium. Aromatase activity was either not affected or was increased up to 5-fold over the control by Fk alone. Forskolin exerted a synergistic effect upon aromatase activity in the presence of progestin or progestin and oestradiol. Sequential incubation of the stromal cells with MPA and then Fk indicated that the additional increase in aromatase activity caused by Fk occurred after 24 h of incubation. These results demonstrate that intracellular cAMP exerts a stimulatory effect on aromatase activity in progestin-conditioned stromal cells. We also investigated whether the endometrial aromatase activity changes in vivo during the reproductive cycle by measuring the activities in endometrial specimens obtained from women during their reproductive age.(ABSTRACT TRUNCATED AT 250 WORDS)

4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗

PubMed1987

Effect of relaxin on aromatase activity in human endometrial stromal cells.

Previous studies have shown that the aromatase activity in human endometrial stromal cells was stimulated by progestin and enhanced by estrogen and forskolin (Fk), an agent that stimulates the accumulation of intracellular cAMP. Present study was undertaken to investigate whether any peptide hormone would affect endometrial aromatase activity. Stromal cells were isolated from normal proliferative and secretory endometria and cultured in nutrient medium. Porcine relaxin (RLX) was added to culture medium individually or in combination with medroxyprogesterone acetate (MPA) and estradiol (E2). Cells treated with RLX alone did not affect the aromatase activity. RLX, however, exerted a synergistic effect on aromatase activity in the presence of MPA or MPA plus E2. On the other hand, human CG, epidermal growth factor, human PRL, and insulin did not increase the aromatase activity in the presence or absence of MPA and E2 studied in a limited number of specimens. The progestin-dependent effect of RLX on aromatase activity was dose dependent indicating that the biological effect of RLX is mediated through a saturable mechanism. When RLX was added to MPA-pretreated cells, additional increase of aromatase activity was seen after 24 h incubation indicating that the action of RLX on stromal cells is not an acute effect. Antiprogestin, RU486, inhibited the stimulation of aromatase activity in both MPA and MPA plus RLX treated cells. RLX has either no effect or a moderate increase (up to 2-fold over the control) on intracellular cAMP content. On the other hand, Fk increased the intracellular cAMP level and enhanced the aromatase activity in the presence of progestin. Also RLX did not replace the effect of Fk since additional increase of aromatase activity was noted when stromal cells were incubated with MPA plus RLX plus Fk in comparison to MPA plus RLX or MPA plus Fk. These results suggest that the action of RLX on stromal cells may be mediated through an intracellular messenger independent of cAMP. Present studies provide evidence that RLX exerts a synergistic effect on aromatase activity in the presence of progestin in human endometrial stromal cells. It is evident that human endometrium is a target organ of RLX.

Aromatase↗

PubMed1987

Functional properties and molecular structure of central and peripheral neurotensin receptors.

Membranes prepared from mammalian brain or intestine contain two types of specific binding sites for neurotensin that differ by their affinity and by their sensitivity to sodium ions, GTP, and the antihistamine drug levocabastine. Only the high affinity sites are present in cell cultures and in soluble extracts of CHAPS-treated membranes. These sites represent functional neurotensin receptors coupled to GTP-binding proteins that regulate intracellular levels of cAMP, cGMP and inositol phosphates in neuroblastoma N1E115 cells. The molecular weight of neurotensin receptors in cells and membrane preparations of various origin is about 110,000.

Animals↗

PubMed1986

Photoaffinity labeling of neurotensin binding sites on rat brain sections.

The photoaffinity labeling of neurotensin (NT) binding sites was carried out on rat midbrain sections using a monoiodo analogue of NT (125I-azidobenzoyl [Trp11] NT; 125IAB-NT). Autoradiographic data showed that the 125IAB-NT binding site localization was quite similar to that obtained with 125I-NT, with high densities in both substantia nigra and ventral tegmental area. Covalent specific binding was only observed when sections were irradiated with UV after the incubation, followed by various histological treatments necessary for light and electron microscopy.

Affinity Labels↗

PubMed1986

Endocrine aspects of human uterine sarcoma: a preliminary study.

The biologic effect of estrogen and progesterone in human uterine sarcoma is poorly understood in comparison to that of endometrial adenocarcinoma. In an attempt to elucidate the endocrine status of these tumors, we have investigated the ability of these tumors to synthesize estrogen by measuring the aromatase activity and studied the effect of aromatase inhibitors on the activity. In addition, the effect of estrogen and progesterone on aromatase activity and the growth pattern of these tumors were studied in cell culture and athymic mice systems. Aromatase activities in eight uterine sarcomas ranged from 0.7 to 37 fmol/hr X mg protein, which were within the range or higher than the activity found in normal proliferative endometrium (0.5 to 3 fmol/hr X mg of protein, means = 1.6, n = 10). These results indicate that uterine sarcomas are capable of producing estrogen. However, the enzyme activity showed no correlation with the morphology of tumors or the age of patients. Results from the kinetic studies of aromatase activity in one of the uterine sarcomas indicated that 19-nortestosterone, testolactone, and aminoglutethimide (the most effective one) inhibited aromatase activity. In addition, induction of aromatase activity in two uterine sarcomas was investigated in cell cultures. Progesterone caused an eightfold increase in activity in a sarcoma that was estrogen and progesterone receptor positive but had no effect in a tumor that was estrogen and progesterone receptor negative. The growth rate of two estrogen/progesterone receptor-negative uterine sarcomas was studied in cell culture and in athymic mice. Progestin, but not estrogen, reduced the growth rate in both systems; 30 nmol/L of estrogen had no effect on the growth rate. In summary, we have found that human uterine sarcoma is able to synthesize estrogen. Progesterone is able to induce the aromatase activity in estrogen/progesterone receptor-positive tumors, and progesterone also suppresses the tumor growth rate in estrogen/progesterone receptor-negative tumors. These results suggest that a select group of uterine sarcomas is sensitive to steroid hormone and that progesterone may be potentially beneficial for therapeutic treatment of select uterine sarcomas.

Adult↗

PubMed1986

High-affinity receptor sites and rapid proteolytic inactivation of neurotensin in primary cultured neurons.

The present article describes the interaction of neurotensin with specific receptors in pure primary cultured neurons and the mechanisms by which this peptide is inactivated by these cells. Neurotensin binding sites are not detectable in nondifferentiated neurons and appear during maturation. The binding at 37 degrees C of [monoiodo-Tyr3]neurotensin to monolayers of neurons 96 h after plating is saturable and characterized by a dissociation constant of 300 pM and a maximal binding capacity of 178 fmol/mg of protein. The binding parameters as well as the specificity of these receptors toward neurotensin analogues reveal close similarities between the binding sites present in primary cultured neurons and those described in other membrane preparations or cells. Neurotensin is rapidly degraded by primary cultured neurons. The sites of primary inactivating cleavages are the Pro7-Arg8, Arg8-Arg9, and Pro10-Tyr11 bonds. Proline endopeptidase is totally responsible for the cleavage at the Pro7-Arg8 bond and contributes to the hydrolysis mainly at the Pro10-Tyr11 site. However, the latter breakdown is also generated by a neurotensin-degrading neutral metallopeptidase. The cleavage at the Arg8-Arg9 bond is due to a peptidase that can be specifically inhibited by N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanyl-p- aminobenzoate. The secondary processing occurring on neurotensin degradation products are: a bestatin-sensitive aminopeptidasic conversion of neurotensin11-13 to free Tyr11, and a rapid cleavage of neurotensin8-13 by proline endopeptidase. A model for the inactivation of neurotensin in primary cultured neurons is proposed and compared to that previously described for purified rat brain synaptic membranes.

Animals↗

PubMed1986

Modulation of aromatase activity in human endometrial stromal cells by steroids, tamoxifen and RU 486.

The regulation of aromatase activity (AA) in human endometrial stromal cells by various steroids was studied in primary cell culture. Various progestins, but not androgens or glucocorticoids, stimulated AA. Medroxyprogesterone acetate (MPA) was the most potent progestin. Estrogen (E) alone did not change the activity but it potentiated the stimulation of AA by progestin. Biphasic regulation of AA by progestin was noted in both time- and dose-dependent manners. Endometrial AA was stimulated by MPA and reached the maximum rate between 2-5 days of incubation with subsequent decline of AA in prolonged culture. When stromal cells were treated with MPA (0.03 to 30 microM) for 3 days, AA was increased over the control at all the concentrations tested. The maximum was found at doses between 0.1-1 microM. The activities reduced steadily from the maximum stimulation to less than 50% when the concentration of MPA increased from 1-30 microM. In addition, initial treatment of stroma cells with MPA (1-3 days) resulted in further increase of activity after progestin withdrawal. The enhancement of the induction of AA by E did not alter the biphasic pattern regulated by progestin alone, i.e. E enhanced both the stimulation and the decay of AA. The time study of the effect of E showed that enhancement of AA required at least 10 h of incubation of E with MPA conditioned cells. The effect of E is dose dependent between 0.04-40 nM and shows the greatest effect in the presence of MPA between 0.01-1 microM. The optimal concentrations of E and progestin that stimulate AA in culture are similar to the plasma concentrations after pregnancy, suggesting that the physiological function of the endometrial aromatase is at the time of decidualization. The effects of antiprogestin, Ru 486, and antiestrogen, tamoxifen (TAM), on AA were studied. Ru 486 or TAM alone did not alter AA. Ru 486 inhibited the MPA stimulated AA in a dose-dependent manner suggesting that the effect of progestin may be mediated through a receptor mechanism. Enhancement, but no inhibitory effect, was observed when cells were treated with TAM + MPA and TAM + MPA + E. The effectiveness of Ru 486 to inhibit the induction of AA in endometrial cells may be of primary importance for contraception.

Adult↗

PubMed1985

Regulation of cyclic GMP levels by neurotensin in neuroblastoma clone N1E115.

The binding of 125I-labeled [monoiodo-Tyr3]neurotensin to intact neuroblastoma N1E115 cells and the effect of neurotensin on the intracellular concentration of cyclic nucleotides were studied at 37 degrees C and under physiological conditions of pH and ionic strength. The radiolabeled neurotensin analogue bound specifically to differentiated cells with a dissociation constant of 0.75 nM and a maximal binding capacity of 45 fmol/10(6) cells. Incubation of neuroblastoma cells with neurotensin in the presence of calcium ions resulted in a transient increase of 10 fold over basal level of the intracellular cyclic GMP concentration. Half-maximal stimulation was obtained with 2 nM neurotensin. Under identical conditions the cyclic AMP concentration only decreased by 20-30%. These results suggest that cyclic GMP is a second messenger of neurotensin in neuroblastoma clone N1E115.

Animals↗

PubMed1985

Molecular properties of neurotensin receptors in rat brain. Identification of subunits by covalent labeling.

Neurotensin binding sites in rat brain synaptic membranes were specifically and covalently labeled by two methods. In the first, a photoreactive and highly radioactive analogue of neurotensin, 125I-labeled azidobenzoyl[Trp11]neurotensin, was synthesized and used to photoaffinity label neurotensin receptors. In the second, the reversible association between neurotensin receptors and 125I-labeled[Trp11]neurotensin, a radioactive but nonphotoreactive analogue of neurotensin, was made irreversible by means of disuccinimidyl suberate, a bifunctional cross-linking reagent. Analysis of synaptic membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that using both methods the same two protein bands with apparent molecular weights of 49,000 and 51,000 were specifically labeled. Identical results were obtained with or without reduction of the photolabeled membranes by beta-mercaptoethanol before electrophoresis. Variation of the ligand concentration did not modify the relative labeling intensities of the two bands, indicating that the high- and low-affinity neurotensin binding sites previously detected in rat brain synaptic membranes have similar molecular structures. These results indicate that neurotensin receptors in rat brain may be composed of two different protein subunits with similar molecular weight of about 50,000, that are linked together by noncovalent bonds.

Affinity Labels↗

PubMed1985

Estrogen sulfotransferase in human placenta.

Human placental estrogen sulfotransferase (ESFT) was partially purified from the term placental cytosol by (NH4)2SO4 precipitation and agarose gel chromatography. Additional purifications caused a rapid loss of the enzyme activity. The activity was abolished by isoelectrofocusing but partially retained by chromatofocusing. The value of pI of human placental ESFT is 5.8 and the same value was obtained for bovine adrenal ESFT. The enzyme protein was able to bind to the affinity resin, estradiol-17-hemisuccinyl-1,2-diaminododecane sepharose 4B, but difficult to be extracted by estradiol (E2). The extract of the affinity resin showed one major protein band at 68,000 dalton on SDS-polyacrylamide gel electrophoresis. Kinetic studies using partially purified ESFT revealed that E2 is the best substrate for this enzyme. The relative rate of sulfurylation of E2, estrone, estriol and dehydroepiandrosterone at 4 microM (Km for E2) is 1, 0.3, 0.08 and 0.08, respectively.

Chromatography, Affinity↗

PubMed1984

Preparation of neurotensin selectively iodinated on the tyrosine 3 residue. Biological activity and binding properties on mammalian neurotensin receptors.

[Monoiodo- Tyr3 ]neurotensin, a neurotensin analogue that contains a single iodine atom on the side chain of Tyr 3 was prepared and purified. This analogue can be labeled at any specific radioactivity between 0 and 2000 Ci/mmol; its binding and biological properties on rat and guinea pig neurotensin receptors are identical to those of the parent peptide. These properties make [monoiodo- Tyr3 ]neurotensin the best suitable radioligand for detection and characterization of neurotensin receptors in various tissues and species.

Animals↗

PubMed1984

High-affinity neurotensin binding sites in differentiated neuroblastoma N1E115 cells.

This paper describes the interaction of neurotensin with mouse neuroblastoma N1E115 cells. Neurotensin binding sites are undetectable in nondifferentiated neuroblastoma cells. They appear during cell differentiation in the presence of a low serum concentration and dimethyl sulfoxide, and reach a maximal level after 50-60 h of incubation under these conditions. The binding of monoiodo[Trp11]neurotensin to homogenates of differentiated N1E115 cells is specific, saturable, and reversible. The interaction is characterized by a dissociation constant of 150 pM and a maximal binding capacity of 9 fmol/mg of protein at 0 degrees C, pH 7.5. These binding parameters, as well as the specificity toward a series of neurotensin analogues, are similar for neurotensin receptors in N1E115 cells and for the high-affinity binding sites that had been previously characterized in rat brain synaptic membranes by means of the same radiolabeled ligand. The presence of high-affinity binding sites for neurotensin in the neuroblastoma N1E115 provides a useful model to study the cellular responses that are generated by the association of neurotensin to its receptor in electrically excitable cells.

Animals↗

PubMed1984

Preliminary studies of aromatase in human neoplastic endometrium.

The capacity of aromatization in 15 neoplastic human endometrial specimens was investigated. Viable tissue fragments were incubated with [3H]testosterone (10 nM) at 37C for 20 hours. Various estrogens were isolated from the incubated tissues and medium. The mean value of estrogen synthesized in 12 cases of endometrial adenocarcinoma (16.0 +/- 11, means +/- SD fmol/mg protein), is approximately two and five times higher than that of proliferative and secretory endometria, respectively. The highest capacity was found in three mixed mullerian tumors (means = 145 fmol/mg protein). These findings indicate that the malignant endometria, in particular the mixed mullerian tumors, are able to synthesize estrogen in a capacity higher than that in normal endometria. Endometrial aromatase may play an important role in promoting cell growth in estrogen-sensitive tumors.

Adenocarcinoma↗

PubMed1983

Monoiodo-[Trp11]neurotensin, a highly radioactive ligand of neurotensin receptors. Preparation, biological activity, and binding properties to rat brain synaptic membranes.

Iodination of [Trp11]neurotensin, a neurotensin analogue in which tyrosine 11 has been substituted by a tryptophan, led to the incorporation of one or two iodine atoms on the single tyrosine residue in position 3. Both mono- and diiodinated derivatives were purified by ion exchange chromatography and their biological activity in an in vitro bioassay involving rat ileum was found to be similar to that of native neurotensin. The 125I-labeled monoiodo derivative of [Trp11]neurotensin bound specifically and reversibly to rat brain synaptic membranes. The binding isotherm was biphasic and could be described by postulating the existence of two different classes of independent binding sites with dissociation constants of 0.1 and 4.7 nM. The specificity of a series of neurotensin analogues for both high and low affinity binding sites was the same as that previously observed in other neurotensin radioreceptor assays. The low affinity binding sites appeared to be similar to the single class of sites described in other binding studies. The high affinity binding sites which were not previously detected might represent either a new class of neurotensin receptors or a high affinity state for a fraction of a single population of neurotensin receptors.

Animals↗

PubMed1983

Estrogen sulfatase and estrogen sulfotransferase in human primary mammary carcinoma.

Estrogen sulfatase (ES) and estrogen sulfotransferase (ESFT) activities were measured in a group of primary breast tumors. The mean value of ES activities, measured in 66 breast tumor specimens, was 0.9 nmol of estrone formed from estrone sulfate/mg tissue protein per hr regardless of the hormone receptor status of the specimen. However, the average value of the ESFT activity, expressed in nmol of estradiol-3-sulfate (E2S) formed from estradiol (E2)/mg of cytosol protein per hr, was found to be significantly higher in ER +/PGR + tumors (n = 26, 0.18 +/- 0.15, means +/- SD) than in ER -/PGR - tumors (n = 31, 0.08 +/- 0.06, P less than 0.005). Normal breast tissues also contain ES and ESFT but the activities were lower than those in tumors. When fresh breast tumor tissue fragments were incubated with radioactive E2 (0.4 nM) and E2S (3 nM) separately, E2 was not sulfurylated appreciably while E2S was extensively hydrolyzed to free estrogens indicating that the combined effect of ES and ESFT in breast tumor is favored towards the hydrolysis of estrogen sulfate. These results imply that the circulating estrogen sulfate could be utilized as the precursor of active estrogen to promote the cell growth in hormone sensitive tumors.

Breast Neoplasms↗

PubMed1982

Estrogen synthesis in normal and malignant human endometrium.

The ability of human endometrium to synthesize estrogen from testosterone (T) was investigated. Normal and malignant endometrial specimens were incubated in a complete nutrient medium with 1 muC/ml (approximately 10 pmol/ml) of [3H]T for 20 hrs. Various estrogens: estrone (E1), estradiol (E2), estrone sulfate (E1S), and estradiol-3-sulfate (E2S) were isolated from cultured tissue and medium. The capacity of aromatization, expressed in pmol of estrogen formed/g of tissue, of proliferative endometria was found to be significantly higher than that of secretory endometria (prol. n=12, 0.53 +/- 0.21, vs sec. n=13, 0.15 +/- 0.09, mean +/- s.d., P less than 0.005). In nine cancer endometrial specimens studied, the estrogen produced varied from 0.3 to 15 pmol/g of tissue. These studies represent the first evidence that human endometrium is capable of synthesizing estrogens from delta 4 androgens at a concentration similar to plasma level. The changes of the capacity of aromatization during the two phases of the menstrual cycle indicate that the estrogen synthesis in endometrium is apparently regulated by hormones. The presence of aromatase in cancer endometria may play an important role in promoting the cell growth in estrogen sensitive endometrial cancer.

Aromatase↗