Cytosolic Supernatants Research Articles

Protein kinase enzymes in the human vagina-relation to key mediators of the cyclic AMP and cyclic GMP pathways.

Aside from phosphodiesterase (PDE) isoenzymes, protein kinases (cAK=cyclic AMP-binding protein kinase, cGK=cyclic GMP-binding protein kinase) have also been identified as important receptors for cyclic nucleotides. A significance of protein kinases in the control of the function of the male and female reproductive tract has been suggested; however, up until today, only a few approaches have addressed these enzymes in female genital tissues. The present study aimed to investigate by means of biochemical and immunohistochemical methods the expression of cAK and cGK. The distribution of cAK(I) and cGK(I) in relation to the vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and PDE type 4 (PDE4) was also evaluated. Cytosolic supernatants prepared from specimens of vaginal wall smooth muscle or epithelium were subjected to anion exchange chromatography and the activities of cAK and cGK(I) measured. To evaluate the distribution of cAK(I) and cGK(I) in relation to VIP, CGRP and PDE4, immunohistochemistry was conducted in sections of the human vaginal wall (full-wall specimens). Activities representing cGK(I) and cAK(I) were resolved from the chromatography column. Staining specific for cAK(Iα) was identified in both vascular and non-vascular vaginal smooth musculature, immunoreactivity for cGK(Iβ) was observed in the smooth muscle and endothelium of small arteries interspersing the sections. cAK(Iα)-positive vessels were found innervated by slender varicose nerve fibers presenting the expression of VIP and CGRP. These arteries also expressed PDE4. Localization of cAK and cGK in close relation to key mediators of the cyclic AMP (PDE4, VIP) and cyclic GMP (CGRP) pathways indicate that both signaling systems may synergistically work together in human vaginal tissue.

Expression and Distribution of Phosphodiesterase Isoenzymes in the Human Male Urethra

To investigate the expression and distribution of phosphodiesterase (PDE) isoenzymes PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A in human urethral tissue. Specimens of penile urethra were obtained from male subjects who had undergone male-to-female sex reassignment surgery. Using immunohistochemistry (immunofluorescence), the occurrence of PDE1A, PDE2A, PDE4A, PDE4B, and PDE5A, the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide was examined in urethral sections. Cytosolic supernatants prepared from isolated human urethral tissue were subjected to Western blot analysis using specific anti-PDE antibodies. Immunosignals specific for PDE1A, 4A, 4B, and 5A were observed in the urethral smooth musculature. The smooth muscle bundles were seen innervated by slender nerve fibers, characterized by the expression of the neuronal nitric oxide synthase, calcitonin gene-related peptide, and vasoactive intestinal polypeptide. The expression of the PDE isoenzymes mentioned was confirmed by Western blotting. The results provide evidence for a significance of both the cyclic adenosine monophosphate and cyclic guanosine monophosphate signaling in the control of human urethral smooth muscle. The selective inhibition of PDE isoenzymes might represent a pharmacologic option to influence the function of smooth musculature in the human outflow region.

Expression and Distribution of Phosphodiesterase Isoenzymes in the Human Seminal Vesicles

Phosphodiesterase (PDE) isoenzymes have been shown to play a role in the control of human male genital tissues. There are hints from basic research and clinical studies that PDE5 inhibitors may have the ability to retard the male ejaculatory response. While the expression of PDE isoenzymes in the human seminal vesicles (SVs) has been described, the distribution of cyclic adenosine monophosphate (AMP)- and cyclic guanosine monophosphate (GMP)-PDEs has not yet been investigated. The aim of this study was to elucidate the expression and distribution of PDE isoenzymes PDE3A, PDE4 (isoforms A and B), PDE5A, and PDE11A in human SV tissue. Using immunohistochemistry (double-labeling techniques, laser fluorescence microscopy), the occurrence of PDE3A, PDE4A, PDE4B, PDE5A, and PDE11A, the vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), and protein gene product 9.5 (PGP 9.5) was examined in sections of SV. Cytosolic supernatants prepared from isolated human SV tissue were subjected to Western blot analysis using specific anti-PDE antibodies. The expression and distribution by of PDE3A, PDE4A, PDE4B, PDE5A, and PDE11A in the human SV were investigated by means of immunohistochemistry and Western blot analysis. Immunosignals specific for PDE3A were seen in both the smooth muscle and the glandular epithelium, whereas staining for PDE4A, PDE5A, and PDE11A was mainly limited to epithelial cells. Varicose nerve fibers transversing the sections also presented staining for PDE3A. In nerve fibers and nerve endings, PDE4A and PDE4B were found co-localized with VIP; PDE5A-positive nerves also presented immunosignals specific for CGRP. The expression of said PDE isoenzymes was confirmed by Western blotting. The results indicate that cyclic AMP- and cyclic GMP-PDE isoenzymes are involved in the control of secretory activity and efferent neurotransmission in the SV. These findings might be of importance with regard to the identification of new therapeutic avenues to treat premature ejaculation.

Expression and Distribution of Cyclic AMP- and Cyclic GMP-Binding Protein Kinases in the Human Vagina— An Immunohistochemical Study

In contrast to research findings describing the localization of nitric oxide synthases (NOS), guanylyl cyclases, and cyclic adenosine monophosphate (cAMP)- and cyclic guanosine monophosphate (cGMP)-degrading phosphodiesterase isoenzymes in the human vagina, the distribution of proteins known as major targets for cyclic nucleotides has not yet been evaluated. cAMP- and cGMP-dependent protein kinases (cAK, cGKI) have been identified as important receptors for cyclic nucleotides downstream the signaling cascades. To investigate, by means of immunohistochemistry, the expression of cAK and cGKI in relation to endothelial NOS (eNOS), vasoactive intestinal polypeptide (VIP), and protein gene product 9.5 (PGP 9.5) in the human vagina. Expression and distribution of cAK and cGKI(alpha,beta) in relation to eNOS, VIP, and PGP 9.5 in human vaginal tissue. Immunohistochemical techniques were applied to sections of human vaginal full wall specimens in order to evaluate the presence of cAK and cGKI(alpha,beta) in relation to VIP, PGP 9.5, and eNOS, respectively. Western blot analyses were conducted using cytosolic supernatants of homogenized specimens of the vaginal wall and epithelium. Immunostaining specific for cGKIbeta was observed in vascular and nonvascular smooth muscle of the vagina. In the endothelial layer, cGKIbeta was found colocalized with eNOS. In contrast, no signals indicating cGKIalpha were registered. cAK-positive subepithelial vessels were found to be innervated by a dense meshwork of PGP-containing varicose nerve fibers, some of which presented expression of VIP. The expression of cAK and cGKIbeta was confirmed by Western blotting. Our results demonstrate the expression of cAK and cGKIbeta in the human vagina. The colocalization with VIP and eNOS underlines the significance of both the cAMP and GMP pathway in the control of human vaginal vascular and nonvascular smooth muscle.

A Proteasomal ATPase Contributes to Dislocation of Endoplasmic Reticulum-associated Degradation (ERAD) Substrates

Endoplasmic reticulum (ER)-associated degradation (ERAD) eliminates aberrant proteins from the ER by dislocating them to the cytoplasm where they are tagged by ubiquitin and degraded by the proteasome. Six distinct AAA-ATPases (Rpt1-6) at the base of the 19S regulatory particle of the 26S proteasome recognize, unfold, and translocate substrates into the 20S catalytic chamber. Here we show unique contributions of individual Rpts to ERAD by employing equivalent conservative substitutions of the invariant lysine in the ATP-binding motif of each Rpt subunit. ERAD of two substrates, luminal CPY*-HA and membrane 6myc-Hmg2, is inhibited only in rpt4R and rpt2RF mutants. Conversely, in vivo degradation of a cytosolic substrate, DeltassCPY*-GFP, as well as in vitro cleavage of Suc-LLVY-AMC are hardly affected in rpt4R mutant yet are inhibited in rpt2RF mutant. Together, we find that equivalent mutations in RPT4 and RPT2 result in different phenotypes. The Rpt4 mutation is manifested in ERAD defects, whereas the Rpt2 mutation is manifested downstream, in global proteasomal activity. Accordingly, rpt4R strain is particularly sensitive to ER stress and exhibits an activated unfolded protein response, whereas rpt2RF strain is sensitive to general stress. Further characterization of Rpt4 involvement in ERAD reveals that it participates in CPY*-HA dislocation, a function previously attributed to p97/Cdc48, another AAA-ATPase essential for ERAD of CPY*-HA but dispensable for proteasomal degradation of DeltassCPY*-GFP. Pointing to Cdc48 and Rpt4 overlapping functions, excess Cdc48 partially restores impaired ERAD in rpt4R, but not in rpt2RF. We discuss models for Cdc48 and Rpt4 cooperation in ERAD.

Suppression of Apoptosis by Cyclophilin D via Stabilization of Hexokinase II Mitochondrial Binding in Cancer Cells

The permeability transition pore is involved in the mitochondrial pathway of apoptosis. Cyclophilin D, a pore component, has catalytic activity as a peptidyl prolyl cis, trans-isomerase (PPIase), which is essential to the pore opening. It has been reported that cyclophilin D overexpression suppresses apoptosis in cancer cells. To clarify the mechanism of this effect, we generated glioma cells overexpressing wild-type or a PPIase-deficient mutant of cyclophilin D. Interestingly, we found that the PPIase-dependent apoptosis suppression by cyclophilin D correlated with the amounts of mitochondrial-bound hexokinase II, which has anti-apoptotic activity. Inactivation of endogenous cyclophilin D by small interference RNA or a cyclophilin inhibitor was found to release hexokinase II from mitochondria and to enhance Bax-mediated apoptosis. The anti-apoptotic effects of cyclophilin D were canceled out by the detachment of hexokinase II from mitochondria, demonstrating that mitochondrial binding of hexokinase II is essential to the apoptosis suppression by cyclophilin D. Furthermore, cyclophilin D dysfunction appears to abrogate hexokinase II-mediated apoptosis suppression, indicating that cyclophilin D is required for the anti-apoptotic activity of hexokinase II. Based on the above, we propose here that cyclophilin D suppresses apoptotic cell death via a mitochondrial hexokinase II-dependent mechanism in cancer cells.

Protein-protein, protein-RNA and protein-lipid interactions of signal-recognition particle components in the hyperthermoacidophilic archaeon Acidianus ambivalens.

The signal-recognition particle (SRP) of one of the most acidophilic and hyperthermophilic archaeal cells, Acidianus ambivalens, and its putative receptor component, FtsY (prokaryotic SRP receptor), were investigated in detail. A. ambivalens Ffh (fifty-four-homologous protein) was shown to be a soluble protein with strong affinity to membranes. In its membrane-residing form, Ffh was extracted from plasma membranes with chaotropic agents like urea, but not with agents diminishing electrostatic interactions. Using unilamellar tetraether phospholipid vesicles, both Ffh and FtsY associate independently from each other in the absence of other factors, suggesting an equilibrium of soluble and membrane-bound protein forms under in vivo conditions. The Ffh protein precipitated from cytosolic cell supernatants with anti-Ffh antibodies, together with an 7 S-alike SRP-RNA, suggesting a stable core ribonucleoprotein composed of both components under native conditions. The SRP RNA of A. ambivalens depicted a size of about 309 nucleotides like the SRP RNA of the related organism Sulfolobus acidocaldarius. A stable heterodimeric complex composed of Ffh and FtsY was absent in cytosolic supernatants, indicating a transiently formed complex during archaeal SRP targeting. The FtsY protein precipitated in cytosolic supernatants with anti-FtsY antisera as a homomeric protein lacking accessory protein components. However, under in vitro conditions, recombinantly generated Ffh and FtsY associate in a nucleotide-independent manner, supporting a structural receptor model with two interacting apoproteins.

Phosphodiesterase isoenzymes as pharmacological targets in the treatment of male erectile dysfunction

Based on the increasing knowledge of intracellular signal propagation in cavernous smooth muscle tone regulation, which is of major importance to the understanding of both the physiology of erection and the pathophysiology of erectile dysfunction, selective phosphodiesterase (PDE) inhibitors have recently been introduced in the treatment of erectile dysfunction. The first promising clinical data on the use of the orally active PDE5 inhibitor Sildenafil in the treatment of erectile dysfunction were accompanied by boosting research activities on cavernous intracellular signal transduction and phosphodiesterase characterization with the aid of molecular biology and protein chemistry. The presence of mRNA transcripts specific for 14 different human phosphodiesterase isoenzymes and isoforms in human cavernous tissue was shown by RT-PCR: Three isogenes of PDEI, PDE2A and 10A, which hydrolyse cAMP as well as cGMP, the cAMP-specific PDE3A, four isogenes of PDE4, PDE7A and PDE8A, as well as cGMP-specific PDEs PDE5A and PDE9A. Using anion exchange chromatography, the activities of PDE isoenzymes 2, 3, 4, and 5 were detected in cytosolic supernatants of human cavernous smooth muscle. To date, the efficacy and safety of several next generation PDE5 inhibitors for use in the treatment of male erectile dysfunction are under evaluation in vitro and in vivo. Further research will possibly allow identification of diagnostic tools for erectile dysfunction and of even more selective drugs in its therapy.

Relaxation of human ureteral smooth muscle in vitro by modulation of cyclic nucleotide-dependent pathways.

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide turnover and, thus, smooth muscle tension. Recent reports have indicated the presence of PDE isoenzymes 1, 2, 4, and 5 in cytosolic supernatants prepared from human ureteral smooth muscle homogenates and the ability of second-generation inhibitors of PDE 3, 4, and 5 to relax KCl-induced tension of human ureteral muscle in vitro. The aim of the present study was to evaluate the functional effects of recently developed, third-generation isoenzyme-selective PDE inhibitors, the nitric oxide (NO)-donating agents sodium nitroprusside (SNP) and dihydropyridine (DHP), which is also described as an antagonist of L-type calcium channels, and the adenylyl cyclase-stimulating drug forskolin on tissue tension and cyclic nucleotide levels of human ureteral smooth muscle segments in vitro. Relaxant responses of human ureteral smooth muscle were investigated in vitro using the organ bath technique. Cyclic nucleotides cAMP and cGMP were determined by specific radioimmunoassay following time and dose-dependent incubation of the ureteral tissue with the drugs. The most pronounced relaxing effects on KCl-induced tension of ureteral smooth muscle were exerted by nitrovasodilator SNP, PDE4 inhibitor rolipram, and PDE5 inhibitors E 4021 and morpholinosulfonyl-pyrazolopyrimidine (MSPP). Relaxing potency of the drugs was paralleled by their ability to elevate intracellular levels of cGMP and cAMP, respectively. Our data suggest the possibility of using selective inhibitors of PDE isoenzymes 4 and 5 in the treatment of ureteral stones and ureteral colic.

Specific cross-linking of the proline isomerase cyclophilin to a non-proline-containing peptide.

A peptide corresponding to an efficient peroxisomal targeting sequence, the carboxy terminal 12 amino acids of PMP20 from Candida boidinii, was employed as an affinity ligand to search for a peroxisomal targeting receptor. Two proteins from yeast extracts with apparent molecular masses of 20 and 80 kDa were detected by chemical cross-linking to radioiodinated peptide. Both proteins were present in cytosolic supernatants. The 20-kDa species did not cross-link to a control peptide with reversed sequence, whereas the 80-kDa protein cross-linked to both peptides. The cross-linking assay was used to purify the 20-kDa protein from Saccharomyces cerevisiae. Partial protein sequencing identified this protein as cyclophilin, the product of the CYP1 gene. This protein, a peptidyl-prolyl cis-trans isomerase, is the yeast homologue of the protein that mediates the immunosuppressant effects of the drug cyclosporin A (CsA). Cross-linking of peptide to cyclophilin was inhibited by CsA. The cross-linking of cyclophilin to the PMP20-derived peptide was unanticipated because the peptide contains no prolines. The CYP1-encoded protein was not required to target proteins to peroxisomes because this organelle appeared to be assembled normally in a CYP1-disrupted strain. Furthermore, the final three amino acids of the peptide, which are critical for peroxisomal sorting, were not required for cross-linking to cyclophilin. We conclude that either cyclophilin is playing a nonessential facilitating role in peroxisomal targeting or that the interaction of the targeting peptide to cyclophilin is mimicking an interaction with an unidentified substrate or effector of cyclophilin.

Effects of S-carboxymethyl-L-cysteine on pulmonary sialyl transferase activity in vitro, in healthy and in sulphur-dioxide-induced bronchitic rats.

S-carboxymethyl-L-cysteine (carbocysteine) improves the visco-elastic properties of bronchial mucus in vivo, possibly as a result of an increase in the relative proportions of sialomucins in bronchial mucus. Carbocysteine was therefore studied in vitro and ex vivo in both normal and bronchitic rats on pulmonary sialyl transferase, responsible for the addition of sialic acid to mucus glycoproteins. Bronchitis was induced in male Sprague-Dawley rats by repeated exposure to sulphur dioxide for two weeks. During this time they received either 500 mg kg-1 day-1 carbocysteine or its vehicle by the oral route. Rats not being exposed to SO2 received the same treatment. The animals were then killed, and subcellular fractions prepared by differential centrifugation of lung homogenates. Sialyl transferase was assayed using CMP-14C sialic acid as substrate and desialysed fetuin as exogenous acceptor. Enzyme activity was located in both the (Golgi-containing) 10,000 g and 100,000 g pellets with minor activity in the cytosolic supernatants. When tested in vitro between 10(-6) and 10(-3) M, carbocysteine had no effect on sialyl transferase activity in microsomes taken from healthy or bronchitis rats. Repeated administration of carbocysteine was without effect on the sialyl transferase activity in 10,000 g pellets taken from healthy rats. However, in bronchitic rats there was a small but statistically significant (P less than 0.05) increase in enzymic activity in the treated group compared to the animals receiving the vehicle. There was no difference in the activity of the microsomal enzyme compared to vehicle-treated controls in either healthy or bronchitic rats. We conclude that it is possible that an increase in sialyl transferase activity in a Golgi-containing fraction of bronchitic lungs could explain the relative increase in sialomucins in bronchitic subjects.

The lysosomal enzymes of the iris‐ciliary body are retained in their organelles and exhibit increased activities during acute uveal inflammation

Acute uveal inflammation, characterised by conjunctival hyperaemia, limbal vascular injection, iritis and aqueous flare, was induced in the rabbit eye by a single intravitreal injection of 10 micrograms of Shigella endotoxin. Twenty-four, 48 and 72 h after endotoxin administration, lysosomal-rich fractions and cytosolic supernatants were prepared from iris-ciliary bodies and the enzymatic activities of some lysosomal hydrolases in these fractions were assayed. Acid phosphatase, aryl sulphatase and N-acetyl-B-D-glucosaminidase exhibited increased activities in the lysosomal preparations from inflamed iris-ciliary bodies, relative to the activities of the same enzymes in lysosomal fractions from normal iris-ciliary bodies. No significant differences in activities were obtained for these enzymes in cytosolic supernatants from normal and inflamed iris-ciliary bodies. The results suggest that during acute ocular inflammation the lysosomal hydrolases are retained in their organelles at an elevated state of activity.

Intestinal apolipoprotein synthesis and secretion in the suckling pig.

The present studies report characterization of intestinal apolipoprotein (apoLp) synthesis and secretion in the suckling pig. Lipoproteins (d less than 1.006 g/ml) from mesenteric lymph were found to contain both apoB-100 and B-48, in addition to apoA-IV, E, A-I, and Cs. Lymph low density lipoproteins (LDL) and high density lipoproteins (HDL) contained mainly apoB-100 and apoA-I, respectively. Analysis of core cholesteryl ester fatty acid composition suggested filtration from plasma as the major source of lymph LDL and HDL. Dual radioisotope labeling of intestinal and hepatic apoLps in lymph, as well as immunoprecipitation of radiolabeled intestinal mucosa, demonstrated intestinal synthesis of apoB-48, A-IV, and A-I. There was no evidence for apoB-100 synthesis by intestinal mucosa. By contrast, piglet liver synthesized apoB-100, E, A-I, and Cs, but not apoB-48. Newly synthesized intracellular intestinal apoA-I was mainly (basic) isoform 1 (pI 5.58), while lymph and plasma HDL apoA-I were predominantly isoform 3 (pI 5.33), mature apoA-I. Lymph apoB (P less than 0.001) and apoA-I (P less than 0.04) mass output increased significantly during lipid absorption. Studies were subsequently conducted in fasting, fat-fed, bile-diverted, and sham-operated animals to determine the role of both dietary and biliary lipid in regulating intestinal apoLp biosynthesis. Proximal and distal small intestinal loops were pulse-radiolabeled with [3H]leucine, and apoB-48 and A-I were immunoprecipitated from cytosolic supernatants. Although a proximal to distal gradient in intestinal synthesis rates for both apoB and A-I was noted in all groups, the acute absorption of dietary lipid did not significantly increase apoB or A-I synthesis in either location. Complete removal of biliary lipid for 48 hr did not alter synthesis rates in jejunum or ileum. These studies suggest that mesenteric lymph apoLps in the suckling pig are derived both by filtration from plasma and by direct secretion from the intestine. Physiologic regulation of intestinal apoA-I and B-48 synthesis rates appears to be independent of luminal lipid availability.

Sulphasalazine and PhCL28A inhibit the formation of ethanol- and phenylbutazone-induced rat gastric ulcers: lack of involvement of endogenous prostaglandins?

1. The effects of sulphasalazine (SZP) and PhCL28A on macroscopic lesion formation and ex vivo prostaglandin inactivation were studied in the ethanol (ETOH) and phenylbutazone (PBT) models of gastric ulcers in the rat. Prostaglandin 'synthesis' during homogenisation of the stomachs was also studied in the latter model. 2. Both PhCL28A and SZP when injected i.p. prevented the formation of ETOH- and PBT-induced gastric ulcers with ED50 values of 13 and 41 mgkg-1 (vs ETOH) and 3 and 32 mgkg-1 (vs PBT) for PhCL28A and SZP respectively. However, neither compound was active orally in the dose ranges used (up to 30 mg kg-1 for PhCL28A and 100 mg kg-1 for SZP). 3. Irrespective of the route of administration, SZP (100 mg kg-1) and PhCL28A (30 mg kg-1) produced slight but statistically significant decreases in ex vivo prostaglandin inactivation by 100,000 g cytosolic supernatants prepared from stomachs not receiving ulcerogen. When tested in vitro, PhCL28A (IC50 = 230 nM) was approximatively 480 times mor potent than SZP (IC50 = 110 microM) against rat stomach cytosolic prostaglandin inactivation. 4. Both ETOH (50%, 5 ml kg-1, orally) and PBT (200 mg kg-1, orally) significantly decreased ex vivo gastric cytosolic prostaglandin inactivation. PhCL28A (30 mg kg-1, orally or i.p.) decreased prostaglandin inactivation still further after ulcerogen treatment except when given i.p. before ETOH treatment. SZP (100 mg kg-1) had a similar effect when given orally before PBT treatment. 5. When the prostaglandin content of the stomach homogenates was used as a measure of ex vivo prostaglandin synthesis in the PBT experiments, PhCL28A 30 mg kg-' orally (but not i.p.) produced an 88% increase in prostaglandin E2 (PGE2) levels, but had no effect on 6-keto-PGF,, or thromboxane B2 formation during homogenization. SZP (100mg kg' i.p. or orally) was without effect. 6. We conclude from these results that the anti gastric ulcer activity of SZP and PhCL28A is independent of prostaglandin inactivation and endogenous prostaglandin formation is probably not involved.

Identification of 6‐oxo‐prostaglandin E1 as a naturally occurring prostanoid generated by rat lung

The spontaneous release of prostanoids from rat isolated perfused lungs was studied after acid/organic extraction of perfusates by bioassay, radioimmunoassay, thin layer and high performance liquid chromatographic methods and by gas chromatography-negative ion mass spectroscopy (g.c.n.i.m.s.). An acid/organic extractable anti-aggregatory vasodilator prostaglandin which inhibited the twitch response of the field-stimulated guinea-pig vas deferens was released from the Krebs-perfused rat lung in nanogram amounts similar to those of other detected prostanoids. Parallel biological assay suggested that this prostaglandin had very closely similar pharmacological activity to authentic 6-oxo-prostaglandin E1 (6-oxo-PGE1), a metabolite of prostacyclin (PGI2) generated by the action of the enzyme 9-hydroxyprostaglandin dehydrogenase (9-PGDH). 6-oxo-PGE1 was identified conclusively in extracts of rat lung perfusate by thin layer chromatography, high performance liquid chromatography and g.c./m.s. combined with bioassay (inhibition of platelet aggregation), and its covalent structure was defined by g.c. negative ion chemical ionization mass spectroscopy. The rank order of spontaneous release of prostanoids (measured by radioimmunoassay) from the perfused rat lung was 6-oxo-PGF1 alpha greater than thromboxane B2 (TXB2) greater than PGE2 greater than 6-oxo-PGE1 (measured biologically) greater than PGF2 alpha. Release of all five prostanoids was inhibited by indomethacin, but only that of 6-oxo-PGE1 was inhibited by naringenin. Rat lung 100,000 g cytosolic supernatants contained 9-PGDH activity capable of removing 9 beta-tritium from labelled prostacyclin and forming an acid/organic extractable 6-oxo-PGE1-like anti-aggregatory substance. This 9-PGDH activity was inhibited by naringenin (IC50 10.3 microM). 6 The relevance of these findings to the possible physiological role of 6-oxo-PGE1 in the lung is discussed, and we propose that 6-oxo-PGE, should be accorded the status ofa physiologically relevant, naturally occurring metabolite of arachidonic acid.

Effects of diethyl maleate on non-protein sulfhydryl content and cellular functions of mouse thymocytes in vitro.

The electrophilic compound diethyl maleate (DEM, 10(-5)-10(-3) M) decreased the content of non-protein sulfhydryl groups in mouse thymocytes in vitro and affected mitotic activity, spontaneous migration and viability of the cells. The mitotic activity of mouse thymocytes was most sensitive to DEM. It is probable that the decrease of the cell content of reduced glutathione (GSH) led to a failure of GSH-dependent cellular functions, such as mitosis and migration. Cytosolic supernatants of mouse and rat thymocytes exhibited GSH S-transferase activities on DEM and 1-chloro-2,4-dinitrobenzene as electrophilic substrates.

Meso-2,3-dimercaptosuccinic acid increases the inhibition of glutathione S-transferase activity from rat liver cytosol supernatants by di-n-butyltin dichloride.

Di-n-butyltin dichloride (DBTC) and tricyclohexyltin chloride (TCHTC) inhibited the glutathione S-transferase activity of rat liver cytosolic supernatants towards 1-chloro-2,4-dinitrobenzene. Meso-2,3-dimercaptosuccinic acid increased inhibition of glutathione S-transferase by DBTC but lowered inhibition of the enzyme by TCHTC.

Enzymatic inactivation of 6-keto-prostaglandin E 1 in vitro: Comparison with prostaglandin E 1

The inactivation of 6-keto PGE 1, a biologically active and stable metabolite of prostacyclin, was studied in 100,000 g cytosolic supernatants by bioassay on rat stomach strip (contraction) and human platelets (inhibition of ADP-induced aggregation). PGE 1 was used as a reference compound. Both PGs were inactivated in supernatants from colon, kidney and liver of rat, rabbit and guinea-pig. Inactivation was time- and NAD +-dependent and was generally greater for PGE 1 than 6-keto-PGE 1. The enzyme responsible for 6-keto-PGE 1 inactivation in cytosolic supernatants is distinct from prostaglandin 15-hydroxydehydrogenase and 9-keto reductase, is not inhibitable by sulphasalazine-like drugs and its activity is recoverable after precipitation by ammonium sulphate. We conclude that 6-keto-PGE 1 can be inactivated by enzymes with wide tissue distribution, but further studies are needed for identification of these novel enzymes and the products formed as well as to assess their significance in the intact animal.

Inhibition of prostaglandin 15-hydroxydehydrogenase by sulphasalazine and a novel series of potent analogues

The ability of sulphasalazine, its colonic metabolites and various analogues to inhibit prostaglandin inactivation by two purified preparations of type INAD +-dependent prostaglandin 15-hydroxydehydrogenase or in various 100,000 g cytosolic supernatants was investigated using PGF 2a as substrate and radio-TLC. Bovine lung and human placental PGDH were inhibited in a dose-dependent and apparently non-competitive manner by sulphasalazine and most of the 26 salazine/sulphasalazine analogues tested, but the potencies of the analogues varied considerably. In a survey of structure-activity effects testing 30 drugs at a fixed dose (50 μM) in six test systems, it was established that only two aromatic rings are needed and that optimal PGDH inhibition requires -CH 2COOH and -OH at positions 1 and 2 in the salicyl C ring system. Homosalazine was thus established as the type compound of a novel series of powerful PGDH inhibitors. Electronegative substituents meta or para in ring B produce compounds with > 150 × inhibitory potency of sulphasalazine, and a significant linear corrlation ( r = 0.82, P < 0.002) was found between the inhibitory activity and the Hammett σ substituent Constant in this series of ten homosalazine analogues.