Expression of proinflammatory genes during estrogen-induced inflammation of the rat prostate.

BACKGROUND: Exposure of male Wistar rats to estradiol-17beta (E(2)) in the presence or absence of dihydrotestosterone propionate (DHT) was previously shown to result in prostate inflammation. The present study examines, for the first time, changes in the expression level of several proinflammatory genes during the course of this experimentally induced prostatitis. METHODS: Adult male Wistar rats were given chronic exposure to E(2) + DHT by capsule implantation or were injected with E(2) for short-term exposure. Semiquantitative RT-PCR was employed to measure changes in proinflammatory transcript levels in the separated lobes of the prostate after various times of exposure to estrogen with or without DHT. RESULTS: We observed an upregulation of IL-1beta, IL-6, MIP-2, and inducible nitric oxide synthase (iNOS) after only 4 days treatment with E(2). After 4 weeks of treatment with E(2) + DHT, a significant increase in transcript levels of IL-4, IL-5, IL-6, MIP-2, eotaxin, and iNOS was detected, while IL-1beta and TNF-alpha transcript levels only increased slightly. No increase in transcript levels for cyclooxygenase-2 (cox-2), IFN-gamma, IL-2, or IL-12 was observed. CONCLUSIONS: Upregulation of proinflammatory transcripts occurred shortly after exposure to E(2) and well before any inflammatory cells were observed in the prostate. The pattern of gene expression resembled a T(H)2-type helper-cell response.

Neurotensin stimulation of mast cell secretion is receptor-mediated, pertussis-toxin sensitive and requires activation of phospholipase C.

Pretreatment of isolated rat serosal mast cells with U-73122, an aminosteroid inhibitor of phospholipase C, inhibited histamine secretion in response to neurotensin (NT). This inhibition reached a maximum after 1 h of pretreatment at 37 degrees C and was dependent upon the concentration of U-73122 (IC50 approximately 0.2 microM). The inactive analog, U-73343, had no effect on the secretory response to NT. Pretreatment of mast cells with U-73122 also blocked histamine secretion in response to substance P (SP), mastoparan (MP), compound 48/80, or amidated NT (NT-NH2). Stimulation of mast cells by NT was accompanied by a rise in the level of intracellular free calcium and a rapid (within seconds) increase in the level of inositol trisphosphate (IP3) which was inhibited by pretreatment of the cells with U-73122. Pretreatment of isolated mast cells with pertussis toxin (PTx) blocked histamine release in response to NT as well as to all peptides tested. PTx had no effect on histamine secretion elicited by anti-IgE stimulation of sensitized mast cells. Pretreatment of mast cells with SR 48692, a NT-receptor antagonist, had no effect on histamine release induced by MP. At a high concentration (100 nM) SR 48692 partially inhibited the response to NT-NH2. These results, together with our earlier findings with SR 48692, indicate that the signal transduction pathway in mast cells activated by NT requires a specific NT-receptor, the activation of phospholipase C, and the involvement of a PTx sensitive G protein. The peptides SP and MP, and compound 48/80, while also requiring the activation of PLC and a PTx sensitive G protein, are not inhibited by the NT-R antagonist, SR 48692, suggesting that they exert their actions either via a different mast cell receptor or via a receptor-independent mechanism.

Evidence for a neurotensin receptor in rat serosal mast cells.

OBJECTIVE AND DESIGN: The ability of neurotensin (NT) at nmolar levels to stimulate exocytosis of the mast cell suggested that it could play a role in neuro-immune-endocrine interactions. The inhibition by a specific receptor antagonist of NT's mast cell stimulation suggested the presence of a specific mast cell NT receptor. We have here employed several probes to determine if a specific neurotensin receptor was present on rat serosal mast cells. MATERIAL: Serosal mast cells were isolated from the peritoneal and pleural cavities of male Sprague-Dawley rats. METHODS: Immunocytochemistry with an antibody raised against the C-terminal peptide of the neurotensin receptor was utilized. The same antibody was employed in immunoblotting following SDS gel electrophoresis of mast cell extracts. An RNA probe for ribonuclease protection assays (RPA) was prepared using the rat brain neurotensin receptor cDNA and polymerase chain reaction was carried out using primers based on the rat brain neurotensin receptor sequence. RESULTS: Mast cells showed specific staining with the anti-neurotensin receptor antibody and this same antibody revealed a protein on SDS gels migrating as a 70 kDa species. Ribonuclease protection assays revealed the predicted protected fragment at approximately 450 bp while PCR amplification gave a major product at 843 bp. CONCLUSIONS: These results indicate that a specific neurotensin receptor is present on the rat mast cell.

Inhibition of neurotensin-stimulated mast cell secretion and carboxypeptidase A activity by the peptide inhibitor of carboxypeptidase A and neurotensin-receptor antagonist SR 48692.

BACKGROUND: Neurotensin (NT), a peptide found in brain and several peripheral tissues, is a potent stimulus for mast cell secretion and its actions are blocked by the specific NT receptor antagonist, SR 48692. Subsequent to stimulation, NT is rapidly degraded by mast cell carboxypeptidase A (CPA). In the experiments described here, we tested for the involvement of CPA activity in the activation of mast cell secretion by the peptide, NT. METHODS: Mast cells were isolated from the peritoneal and pleural cavities of rats, purified over metrizamide gradients and incubated at 37 degrees C in Locke solution or Locke containing the appropriate inhibitors. For some experiments, media derived from mast cells stimulated by compound 48/80 were used as a source of mast cell CPA activity. RESULTS: Treatment of mast cells with the highly specific peptide inhibitor of CPA derived from potato (PCI) inhibited histamine release in response to NT and NT8-13 (the biologically active region of NT). This inhibition required some 20 min to develop and was only partially reversed by a 20-min wash period. PCI (10 microM) did not inhibit histamine release in response to NT1-12, bradykinin, compound 48/80, the calcium ionophore, A23187, or anti-IgE serum. PCI also inhibited mast cell CPA activity. SR 48692, a highly selective antagonist of the brain NT receptor and of NT-stimulated mast cell secretion, also inhibited mast cell CPA activity as well as bovine pancreatic CPA activity in a concentration-dependent manner. DISCUSSION: It is suggested that the mast cell binding site for NT and the active site for CPA may share similar characteristics. The results are discussed in terms of NT mechanism of action on the mast cell.

Formation and cell-medium partitioning of autoinhibitory free fatty acids and cyclodextrin's effect in the cultivation of Bordetella pertussis.

Palmitic, palmitoleic and stearic acids were found in the extracted cellular lipids of virulent Bordetella pertussis as unesterified acids in confirmation of earlier taxonomic analyses. The same free fatty acids (FFAs) were found in the spent culture supernatant in concentrations higher than in the uninoculated medium, indicating that they are released into the extracellular medium. These long-chain fatty acids are known to inhibit the growth of B. pertussis at concentrations as low as 1 ppm. Measurement of palmitate cell-medium partitioning demonstrated a strong tendency of FFAs for cellular adsorption. Inhibition kinetics indicated that the cell-bound FFA was responsible for inhibition and that the specific cellular FFA concentrations actually found during growth were similar to those determined to be inhibitory. Autoinhibition by these endogenous FFAs provides an explanation of the low maximum cell concentrations currently attainable in liquid media. Addition of soluble dimethyl-beta-cyclodextrin (MebetaCD) to FFA-inhibited cultures resulted in a rapid reversal of the inhibition. A corresponding shift in the distribution of FFAs from the cells to the extracellular medium demonstrated that MebetaCD sequesters FFAs. Although MebetaCD did not increase final cell concentrations and even had an adverse effect on growth at concentrations above 1 g l-1, it did (at 1 g l-1 extend the initial period of high growth rate leading to shorter cultivation times.

Blockade of mast cell histamine secretion in response to neurotensin by SR 48692, a nonpeptide antagonist of the neurotensin brain receptor.

1. Pretreatment of rat isolated mast cells with SR 48692, a nonpeptide antagonist of the neurotensin (NT) receptor, prevented histamine secretion in response to NT. 2. This inhibition was rapid in onset (approximately 1 min) and dependent upon the concentration of SR 48692 (IC50 approximately 1-10 nM). 3. SR 48692 (1-1000 nM) did not inhibit histamine secretion elicited by substance P, bradykinin or compound 48/80, or by anti-IgE stimulation of sensitized mast cells. 4. When SR 48692 was injected intradermally (5 pmol in 50 microliters) into anaesthetized rats, 15 min before the intradermal injection of NT, it reduced the effect of NT on vascular permeability. 5. When injected intravenously, SR 48692 attenuated the effects of NT on haematocrit and blood stasis. 6. These results demonstrate that SR 48692 selectively antagonizes the actions of NT on rat isolated mast cells as well as mast cells in vivo. Given the demonstrated specific interaction of SR 48692 with receptors for NT in brain, our results suggest the presence of specific NT receptors on mast cells.

Stimulated rat mast cells generate histamine-releasing peptide from albumin.

Media conditioned by compound 48/80-stimulated rat mast cells generated immunoreactive histamine-releasing peptide (HRP) when incubated at physiological pH with bovine serum albumin and the carboxypeptidase inhibitor, O-phenanthroline. The generation of immunoreactive HRP (IR-HRP) was time (after 3 h the concentration of IR-HRP was 20 nM), temperature, and pH dependent and was prevented by omitting albumin, by using media conditioned by nonstimulated mast cells, or by pretreatment of mast cells with disodium cromoglycate, an inhibitor of mast cell secretion. The amount of IR-HRP generated increased linearly with the number of mast cells stimulated and varied directly with the concentration of conditioned media. After removal of the media from stimulated mast cells, the remaining cell pellet retained its ability to generate IR-HRP for up to 8 h. Stimulation of mast cells by either neurotensin or substance P, or of sensitized cells by anti-IgE serum, also produced conditioned media that generated IR-HRP. The amount of IR-HRP formed by various conditioned media or by stimulated cell pellets was dependent upon the concentration of O-phenanthroline used. Including the chymase inhibitor, chymostatin, prevented the formation of IR-HRP in a dose-dependent manner. HPLC analysis showed four peaks of IR-HRP. The major one coeluted with synthetic HRP. These results indicate that the peptide, HRP, can be generated by stimulated mast cells incubated in the presence of albumin. They suggest that a chymase-like enzyme secreted by the mast cell is able to cleave albumin to yield HRP.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of the neurotensin8-13 analogs Asp13-NT8-13 and Asp12-NT8-13 on mast cell secretion.

Pretreatment of isolated mast cells with analogs of neurotensin 8-13 (NT8-13), in which the amino acids Leu13 or Ile12 are replaced with an aspartic acid (Asp13-NT8-13 or Asp12-NT8-13), inhibits the secretion of histamine in response to NT. A 10 min pretreatment with either analog (10 microM) inhibited NT-induced histamine release by 90% (Asp13-NT8-13) or by 98% (Asp12-NT8-13). At concentrations that are inhibitory, Asp13-NT8-13 and Asp12-NT8-13 alone elicit very little release (< 5% at 10 microM). In the continued presence of the analogs, the inhibitory effect lasts for more than 45 min; removal of the analogs resulted in restoration of sensitivity to NT within 10 min. Pretreatment with analog Asp13-NT8-13 resulted in a 39% inhibition of stimulation by substance P and a 52% inhibition of stimulation by histamine-releasing peptide (HRP). In contrast, pretreatment with analog Asp12-NT8-13 gave no inhibition of release by SP or HRP. Neither analog inhibited histamine release in response to bradykinin (BK), NT1-12, compound 48/80 (48/80), the calcium ionophore A23187, or anti-IgE stimulation of passively sensitized mast cells. Although Asp12-NT8-13 and Asp13-NT8-13 differ slightly in regard to the peptides they inhibit, both probably act at a step early in the stimulus-secretion coupling sequence; most likely before the rise in the level of free intracellular calcium that has been shown to accompany secretion in mast cells. It is suggested that these analogs exert their inhibitory effect on NT by competing with NT for a binding site on the mast cell membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid degradation of neurotensin by stimulated rat mast cells.

A RIA towards neurotensin (NT) using C-terminal- and N-terminal-specific antisera was used to study degradation of this tridecapeptide by isolated rat mast cells. Incubation of NT (10 microM) with peritoneal or pleural mast cells resulted in a rapid loss of NT immunoreactivity (iNT), as measured by C-terminal-directed antiserum, with little effect on N-terminal iNT. The rate of the reaction was faster with pleural cells (T1/2, 30 s) than with peritoneal cells (T1/2, 180 s) and was greater than 10-fold slower in the presence of metabolic poisons. The enzyme(s) involved is most likely released from the cells during secretion, as NT was degraded by media conditioned by compound 48/80-stimulated mast cells 40-60 times faster than by media from unstimulated cells. This degradation by conditioned media was concentration dependent, pH dependent, and temperature sensitive. HPLC analyses indicated a near stoichiometric conversion of NT to NT(1-12) (66%) and NT(1-11) (34%) after incubation for 10-30 s with conditioned media. By 30 min only NT(1-11) and NT(1-10) were present. Phenanthroline (1 mM), an inhibitor of carboxypeptidase, prevented the loss of C-terminal iNT and the generation of NT(1-12) and NT(1-11). While NT(1-12) was effective in releasing histamine from mast cells in vitro and increasing vascular permeability in vivo, NT(1-11) was not. These results suggest that carboxypeptidase-like enzyme(s) could modulate the level and form of NT-related peptides in various states involving activation of mast cells.

Effect of inclusion body production on culture turbidity and cell dry weight in growing bacterial cultures.

An approach to the optimization of product yield in an inducible inclusion body-producing system is presented. Following induction by indoleacrylic acid (IAA) of a trpLE-HIVgp41 fusion protein, we found a large increase in culture turbidity and single-cell dry weight. After an initial transition phase, new and constant values for specific growth rate, single-cell light turbidity, and single cell dry weight were achieved, allowing for the determination of optimal conditions for product formation.

In vitro mechanism of inhibition of bacterial cell growth by allicin.

Diallyl thiosulfinate (allicin) is the agent found in garlic which is responsible for the antibacterial and antifungal activity of extracts of this plant. The effect of bacteriostatic concentrations of allicin (0.2 to 0.5 mM) on the growth of Salmonella typhimurium revealed a pattern of inhibition characterized by: (i) a lag of approximately 15 min between addition of allicin and onset of inhibition, (ii) a transitory inhibition phase whose duration was proportional to allicin concentration and inversely proportional to culture density, (iii) a resumed growth phase which showed a lower rate of growth than in uninhibited controls, and (iv) an entry into stationary phase at a lower culture density. Whereas DNA and protein syntheses showed a delayed and partial inhibition by allicin, inhibition of RNA synthesis was immediate and total, suggesting that this is the primary target of allicin action.

Histidine decarboxylase from rat mast cells. Enhanced recovery in cell-free extracts and isotopic labelling.

A procedure for obtaining rat mast-cell histidine decarboxylase in greater than 50% yield in cell-free extracts was developed. The enzyme was found in the supernatant fractions from a 3,500 g and a 105,000 g centrifugation step and was demonstrated to be sensitive to inhibition by alpha-fluoromethylhistidine but not by phenylalanine. Although the enzyme shows a half-life of only 3 h in cell-free extract, the initial high recovery of activity allowed for active-site labelling of the enzyme by [3H]histidine and NaBH4. Labelled protein migrated on non-denaturing polyacrylamide-gradient-gel electrophoresis as a 55,000 Da species.

Viability and recovery from degranulation of isolated rat peritoneal mast cells.

Using a culture system that allows prolonged maintenance of purified populations of peritoneal mast cells, we have examined them following stimulation by non-immunologic or immunologic agents. Employing phase-contrast microscopy of living cells and various pharmacological manipulations, we have noted that the recovery process includes a reduction in cell size, the probable sealing of exocytotic cavities, a pronounced displacement of the cell nucleus and a resynthesis of histamine. During recovery, mast cells can entrap molecules from the extracellular fluid and later release these substances by a Ca-dependent mechanism. Our results suggest that microfilaments, calmodulin, Ca, and metabolic energy are necessary for recovery.

Probing Cu(II)/H2O2 damage in DNA with a damage-specific DNA binding protein.

A human damage-specific DNA binding protein has been employed as a sensitive probe of damage introduction by the combination of Cu(II) and H2O2. Optimal conditions for the introduction of protein-recognizable lesions into DNA in the Cu(II)/H2O2 system were obtained with 10(-5)-M CuCl2 and 0.10-mM H2O2. The absolute requirement for the presence of a metal ion suggests the involvement of a metal catalyzed Fenton reaction. However, damage introduction in the presence of KI and dimethylsulfoxide indicate that hydroxyl radical, while responsible for the introduction of strand breaks, is not the primary species responsible for lesion introduction. Protein-recognizable damage was introduced into DNA and poly d(G-C), but not into poly d(A-T). Loss of label from the five position of cytosine was also observed at high peroxide levels.

Recognition of a cytosine base lesion by a human damage-specific DNA binding protein.

Sodium bisulfite reacts with cytosine and 5-methylcytosine, forming the 5,6-dihydrosulfonate adducts which deaminate to the uracil and thymine adducts, respectively. At alkaline pH, the sulfonate groups are then released, generating uracil and thymine. In DNA, the resulting G:U and G:T base mismatches generated are potential sites of mutagenesis. Using a human damage-specific DNA binding protein as a probe, we have found protein-recognizable lesions in bisulfite-treated DNA and poly d(I-C), but not in treated poly d(A-T) or poly d(A-U). Although this suggests that the lesion recognized is cytosine-derived, there was no correlation between the number of uracils induced and the number of binding sites, suggesting that the protein-bound damage is not a uracil-containing mismatch. Modification of the treatment protocol to reduce elimination of the bisulfite from the base adducts increased the level of binding, suggesting that the protein recognizes a base-sulfonate adduct.

A damage-specific DNA binding protein. Large scale purification from human placenta and characterization.

A new large scale purification procedure for a human damage-specific DNA binding protein is described. Physical characterization suggests that the protein can dissociate into active subunits. A maximum molecular weight of 400,000 was obtained using gel exclusion chromatography, while electrophoresis through a pH 8 polyacrylamide gradient gel suggested a minimum molecular weight for the active protein of 120,000. Binding to UV-irradiated DNA revealed a broad pH optimum, no temperature dependence between 0 and 37 degrees C, and inhibition by intercalating agents. The use of 254 nm irradiation and of 313 nm irradiation in the presence of a triplet state sensitizer with alternating copolymers indicated that the protein was recognizing singlet-state-derived thymine lesions as well as triplet-state-derived adenine lesions. Inhibition of protein binding by pyridoxal 5'-phosphate and NaBH4 suggested a role for lysine at the DNA binding site, while sulfhydryl group involvement was indicated by the sensitivity of the protein to p-hydroxymercuribenzoate inhibition.

Water radiolysis products and nucleotide damage in gamma-irradiated DNA.

The radiation chemistry of nucleotide damage introduction into DNA gamma-irradiated in dilute aqueous solution has been studied with a damage-specific DNA binding protein. Irradiation in air, N2 or N2O in the presence and absence of free radical scavengers revealed that protein-recognizable lesions were introduced both by the hydrated electron and by the combination of the hydroxyl radical and superoxide anion radical, but not by the hydroxyl radical alone. In addition, nucleotide damage was introduced into DNA by an enzymatic superoxide-generating system.

On the substrate specificity of a damage-specific DNA binding protein from human cells.

The nature of the lesion recognized by a damage-specific DNA binding protein from human cells was investigated by examining the substrate specificity of this protein. Protein-recognizable damage was introduced into both T7 DNA and Poly d(A-T) at relatively low UV fluences. In addition, the protein demonstrated binding to both nitrous acid- and bisulfite-treated DNA, but not to DNA crosslinked with trioxsalen plus near-UV nor to non-irradiated uracil-containing DNA. These results suggest that this protein could be recognizing minor helix distortions in DNA rather than any one single lesion.