Interferon-alpha downregulates expression of the oxytocin receptor in cultured human myometrial cells.

Previous studies in the endometrium of ruminants showed that type I interferon (IFN) prevents oxytocin receptor (OTR) formation. We studied the effect of IFN-alpha on human myometrial cells in culture expressing a high density of biologically active OTR. We found that IFN-alpha induced a 35-50% decrease in OTR mRNA and protein and that this inhibition was time and dose dependent. Maximal inhibition of OTR mRNA was obtained after 2-3 days, whereas 1-(beta-mercapto-beta, beta-cyclopentamethyl-enepropionic acid,2-O-Me-Tyr,Thr4,Orn8,Tyr9-amide)-[125I]vasotocin ([125I]OTA) binding reached a nadir after 3-4 days, with half-maximal inhibitory concentration (IC50) = 1,100 U/ml. Mathematical analysis of multiple homologous competition curves for [125I]OTA indicated that IFN-alpha treatment (5,000 U/ml x 3 days) reduced just the binding capacity (Bmax) without changing the binding affinity. Accordingly, the same treatment with IFN-alpha did not affect the half-maximally effective concentration (EC50) for the oxytocin-induced increase in intracellular calcium but significantly decreased maximal responsiveness (Emax) of myometrial cells to OT stimulation. In conclusion, our data demonstrate, for the first time, a negative regulation by IFN-alpha of the steady-state expression of OTR mRNA in cultured human myometrial cells obtained from nonpregnant uteri. This inhibition was followed by a parallel decrease in both the Bmax for [125I]OTA and Emax for oxytocin, suggesting a decreased OTR protein availability.

Platelet-activating factor enhances production of insulin-like growth factor binding proteins in a human adenocarcinoma cell line (HEC-1A)

We have recently demonstrated the existence of an autocrine growth loop driven by platelet-activating factor (PAF) in the human endometrial adenocarcinoma. cell line HEC-1A. To investigate a possible cooperation between PAF and the insulin-like growth factor (IGF) system in this cell line, the effect of PAF on insulin-like growth factor binding protein (IGFBP) production as well as binding and biological activities of IGF-I, IGFv-II, and the analog Des(1-3)IGF-I have been evaluated. Analysis of self- and cross-displacement curves of [125I]IGF-I binding to HEC-1A cells indicates the presence of a single class of binding sites, with affinity constants of 1-4 nM for IGF-I and IGF-II and 70 nM for Des(1- 3)IGF-I, which binds to IGFBPs with lower affinity. Insulin does not apparently bind to this binding site. Moreover, the addition of increasing concentrations of IGF-I leads to a paradoxical increase of binding. These results indicate a similarity of this binding site to IGFBPs. The presence of IGFBPs has been demonstrated by Western ligand blot analysis of HEC-1A conditioned medium which shows the presence of two bands of 32-34 and 40-45 kDa. By Western immunoblotting analysis, the two bands were respectively identified as IGFBP-2 and IGFBP-3. Incubation with PAF (1 microM) highly increases the release of the two IGFBPs from the cells. Such an effect is inhibited by the PAF receptor antagonist L659,989, by the PKC inhibitor sangivamycin, and by the tyrosine kinase inhibitor genistein. PAF also induces a time-dependent increase of mRNA expression for IGFBP-3, suggesting an effect on synthesis of this protein. IGF-I and IGF-II (0.1-100 nM) are almost ineffective in inducing [3H]thymidine incorporation, whereas a slight proliferative effect is observed with Des(1-3)IGF-I which also increases PAF synthesis. These data demonstrate a modulatory action of PAF on IGFBP secretion in HEC-1A cells and indicate that the IGF system plays a minor, if any, modulatory role on proliferation of this cell line.

Endothelin-1 and its receptors in human testis.

We have previously found the presence of endothelin (ET) receptor and ET-like immunoreactivity in rat testis. We now extend our studies from rat to human testis. We found expression of a specific transcript for ET-1 and ET-1-like immunoreactivity in human testis. Positive staining was confined to the Sertoli cells of the tubular compartment, although few peritubular and interstitial cells were also stained. We also identified specific ETA and ETB receptor transcripts in human testis; ETA expression was more abundant than the ETB expression. Mathematical analysis of multiple self- and cross-competition studies among [125I]ET-1, [125I]ET-3, and analogues confirmed the presence of the ETA and ETB isoreceptors. In testicular homogenates, the ETA receptor was sevenfold more concentrated than the ETB receptor. In order to localize the receptors, we performed [125I]ET-1 autoradiography. Binding sites were mostly concentrated into the seminiferous tubules, although interstitial and peritubular myoid cells were also positive. Within the seminiferous tubules, [125I]ET-1 binding sites were confined to primary and secondary spermatocytes and early spermatids, whereas Sertoli cells were negative. We were unable to demonstrate the presence of functional ET receptors in ejaculated spermatozoa. Because ET-like immunoreactivity was present in Sertoli cells, we next asked whether authentic ET-1 is present in human seminal fluid and represents a good index for Sertoli cell function. Reverse-phase high-performance liquid chromatography analysis of ET-like immunoreactivity in seminal fluid indicated that most of the detected peptides correspond to the ET-1 precursor, big-ET-1. The seminal concentration of ET-like immunoreactivity was similar in normospermic, oligospermic, azoospermic, and vasectomized men, indicating that ETs are produced in different parts of the male genital tract and that they do not represent an useful tool for the diagnosis of male reproductive diseases. In conclusion, this study demonstrated, for the first time, the presence of ET-1 and its receptors in human testis.

Identification and characterization of functional angiotensin II receptors in human neuroblastoma cells.

The presence of specific AII receptors in 6 different human neuroblastoma cell lines was investigated using binding, cAMP and [Ca2+]i studies. We found high affinity (0.1 nM), low capacity ((1-2).10(3) sites/cell) binding sites for [125I](Sar-1,Ile-8)AII in one half of the cell lines studied. In the positive cell lines mathematical modeling of multiple competition curves among AII and analogs strongly indicated the presence of a homogenous class of sites, i.e., AT1 receptors. The presence of AT1 receptors was further substantiated by AII-induced inhibition of VIP-stimulated cAMP levels and by AII-evoked [Ca2+]i transient. The density of AT1 receptors in neuroblastoma cells was not affected by treatment with pertussis toxin and retinoic acid but was significantly increased by subacute treatment with VIP. In neuroblastoma cells, AII does not stimulate DNA synthesis, suggesting other roles rather than mitogenesis. Neuroblastoma cells represents an interesting model to investigate the function of AII in neural crest derived tissues.

Platelet-activating factor mediates an autocrine proliferative loop in the endometrial adenocarcinoma cell line HEC-1A.

We investigated the synthesis and biological effects of platelet-activating factor (PAF) in the human endometrial cancer cell line HEC-1A. We found that HEC-1A cells actively synthesize and release PAF, as demonstrated by both [3H]acetate incorporation into PAF and gas chromatography-mass spectrometry studies. HEC-1A cells not only synthesize but also respond to PAF. Indeed, in fura-2-loaded cells, PAF stimulates [Ca2+]i increase with a median effective concentration of 5.6 nM. Furthermore, PAF induces a time-dependent expression increase of the nuclear protooncogene c-fos with a median effective concentration of 130 nM and stimulates DNA synthesis (median effective concentration, 700 nM). All of these effects are inhibited by the PAF receptor antagonist L659,989. Radioligand binding studies indicated the presence of two populations of PAF receptors with affinity constants in the nanomolar and micromolar range. Since the PAF antagonist per se inhibits DNA synthesis and cell proliferation, we suggest that PAF supports an autocrine growth circuit in HEC-1A cells. On the contrary, in the uterine leiomyosarcoma cell line SK-UT-1, which does not express specific binding sites for PAF, neither this phospholipid nor its receptor antagonist affect DNA synthesis. Our results provide evidence for the existence of an autocrine proliferative loop involving PAF in the endometrial cancer cell line HEC-1A.

Platelet-activating factor in human endometrium.

Platelet-activating factor (PAF) is a phospholipid actively produced by human endometrium and deeply involved in the processes of ovoimplantation and labor. We recently found that PAF represents a new autocrine growth factor for a human adenocarcinoma cell line, HEC-1A. Indeed, biologically active PAF is synthesized by HEC-1A cells, under progesterone control. In HEC-1A cells, PAF regulates intracellular calcium concentration ([Ca2+]), DNA synthesis and expression of early oncogenes. All these effects are blocked by the receptor antagonist L659,989. However, while nanomolar concentrations of PAF mobilize [Ca2+], only micromolar concentrations affect cell growth, suggesting heterogeneity of PAF receptors or signaling. Two distinct populations of PAF receptors are present in HEC-1A cells, which bind PAF in nanomolar and micromolar concentrations, respectively. Since HEC-1A cells are producing elevated concentrations of PAF and micromolar concentrations of the PAF antagonist L659,989 inhibit cell proliferation, an autocrine role for PAF is suggested in HEC-1A cells.

Identification, characterization, and biological activity of somatostatin receptors in human neuroblastoma cell lines.

To investigate the presence of biologically active somatostatin (SS) receptors in neural crest-derived tumors, radioligand binding studies, cyclic AMP accumulation, intracellular calcium, and growth assays were performed in eight human neuroblastoma (NB) cell lines. Mathematical modeling of binding experiments strongly indicates the presence of heterogeneity of sites. The first site (SSR1) is present in 40% of the NB cell lines and binds with low capacity (0.5 pmol/mg protein) and high affinity (0.1-1 nM) SS14, SS28, and analogues. The second site (SSR2) is a high capacity site (200 pmol/mg protein), widely distributed in all of the cell lines investigated, that shows relative selectivity yet low affinity (100 nM) for SS14, SS28, and [D-Trp8]SS14 without any apparent biological activity. SSR1 is coupled to a pertussis toxin-sensitive G protein, inhibits forskolin- or VIP-stimulated adenylate cyclase activity, decreases intracellular free calcium, and mediates inhibition (30%) of both DNA synthesis and cell growth. Analysis of cell cycle distribution in aphidicolin-synchronized SSR1-positive NB cells indicated that this inhibitory effect is partially mediated by a transient accumulation in G0-G1. Our data indicate high affinity binding sites for SS14, and analogues are present and biologically active in a subset of NB cells.

Recombinant human basic-fibroblastic growth factor: different medical dressings for clinical application in wound healing.

Recombinant human basic-Fibroblastic Growth Factor (rhb-FGF) is a basic single-chain protein showing high activity as mitogenetic and angiogenetic agent. The application of rhb-FGF in wound healing as stimulator of the tissue repair process is strictly connected with the covering of the wound by means of a proper dressing. A wide number of synthetic occlusive or non-occlusive wound dressings has been developed. Owing to the delicate proteic structure of rhb-FGF, and generally of all the Growth Factors, compatibility with the dressings has to be every time tested, to avoid its inactivation and consequent loss of tissue repair properties.

High-performance liquid chromatographic method for the simultaneous assay of a new synthetic penem molecule and its salt-forming agent in injectable formulations.

Rapid, stability-indicating, reversed-phase high-performance liquid chromatographic method for the direct and simultaneous determination of a new beta-lactam molecule and its salt-forming agent in finished dosage forms was studied for the development of injectable formulations.