Control of steroidogenesis in rat corpus luteum: the rate of access of cholesterol to the active site of the cholesterol side-chain cleavage enzyme.

The pool of cholesterol at the active site of the cholesterol side-chain cleavage enzyme was increased by preincubation with substrate. This process occurred at a linear time-dependent rate over 20 min, whereas uptake of cholesterol into mitochondria was complete by 1 min. Both endogenous mitochondrial cholesterol and added extramitochondrial cholesterol showed similar time-dependent substrate preincubation kinetics. The rate of metabolism of exogenous cholesterol was increased by injection of LH and decreased by treatment with cycloheximide. The rate of entry of cholesterol into the active site in both preparations, however, retained linear time-dependent kinetics. Calcium ions (2 mmol/L) increased the rate of steroidogenesis most in mitochondria preincubated for short periods and least in mitochondria preincubated for longer periods. These studies indicated that the pool of cholesterol at the active site of the enzyme can determine the rate of pregnenolone biosynthesis in the mitochondria of rat corpus luteum. Calcium ions, but not trophic hormones or cycloheximide, appeared to alter the access of cholesterol to the active site of the cholesterol side-chain cleavage enzyme during substrate preincubation.

The biosynthesis of the 3-series prostaglandins in rat uterus after alpha-linolenic acid feeding: mass spectroscopy of prostaglandins E and F produced by rat uteri in tissue culture.

The abnormal uterine activity associated with dietary n-3 fatty acids may result from competitive inhibition of PG2 production. Uterine synthesis of 2- and 3-series prostaglandins F(PGF) and E(PGE) was studied using mass spectrophotometry in rats fed diets containing predominantly n-3 fatty acid, n-6 fatty acid, or control pelleted diet. Mass spectra of PGF (Me, TMS and Me, TBDMS derivatives) synthesised by uteri of n-3 fed rats were characterised by 8 ions containing the n-3 double bond, and m.i.d. of the 651/653 ions of PGF-Me, TBDMS indicated PGF3 alpha synthesis (44 +/- 8% and 13 +/- 2% of PGF release by uteri incubated + or -5 micrograms/ul calcium ionophore A23187 respectively). In uteri from the control diet group incubated with ionophore, PGF3 alpha ions were detected and PGF 3 alpha represented 9.5 +/- 1.0% of PGF alpha release. Similarly, analysis of PGE from uteri of n-3 fed rats indicated that PGE3 (16 +/- 6% of PGE) was released in the presence of ionophore A23187. Synthesis of 3-series PG by rat uteri was detected after only 3 weeks of n-3 diet. The capacity to synthesise 3-series PG increased at intracellular calcium concentrations which mimicked cell calcium during decidual autolysis at parturition. These experiments suggest that uterine synthesis of 3-series PG is regulated by the specifity of enzymes incorporating fatty acids, rather than by the cyclooxygenase enzyme.

Intracellular oxidation by human glioma cell populations: effect of arachidonic acid.

Arachidonic acid (AA) and Gamma linolenic acid have been shown to limit glioma cell growth, stimulate apoptosis and lipid peroxidation. However, brain tumours are characterised by cellular heterogeneity and responding cell populations have not been identified. Brain tumour samples from patients were disaggregated. In cell preparations from 7 gliomas, reactive oxygen species (ROS), morphology and plasma membrane integrity were monitored +/-18-36 microM AA for 15-120 min using flow cytometry. Basal oxidative activity related to cell size/morphology, small granular cells showed lower activity. AA stimulation of ROS formation depended on cell size/morphology. Large, less granular cells showed greater AA stimulation. In 17 gliomas, GFAP immunofluorescence was demonstrated in larger cell populations. The large GFAP positive cell population with low side scatter was the highest responding cell population, suggesting selective tumour cell sensitivity to AA induced ROS formation. ROS may have a role in AA induced cell death and anti-tumour activity of AA in glioma.

Prostaglandin E2 in induction of labour: a pharmacokinetic study of dose and treatment protocols.

The absorption of administered prostaglandin E2 (PGE2) into the term uterus was analysed, by measurement of intrauterine PGE2 and PGF 2 alpha, in 137 women. Amniotic fluid was sampled after elective Caesarean section, or at rupture of the membranes, and fetal membranes were collected after delivery of the placenta. Within 2 h of administration of a PGE2 pessary (500 micrograms), a significant elevation in amniotic fluid PGE2 was detected. Exogenous PGE2 stimulated the production of intrauterine PGE2 and PGF2 alpha, causing an elevated PGE2 concentration in amniotic fluid, and increased PGF2 alpha in fetal membranes. These studies indicate that the administration of as little as 500 micrograms of PGE2 pessary, resulted in elevated intrauterine PGE2. Exogenous PGE2 (2.5 mg) administration resulted in increased concentrations of PGF2 alpha in the fetal membranes. Considerable local release of PGs was observed at the site of membrane rupture, and this influenced the method of amniotic fluid sampling used in this study.

The influence of dietary essential fatty acids on uterine C20 and C22 fatty acid composition.

The effect of dietary fatty acids on uterine fatty acid composition was studied in rats fed control diet or semi-synthetic diet supplemented with 1.5 microliter/g/day evening primrose oil (EPO) or fish oil (FO). Diet-related changes in uterine lipid were detected within 21 days. Changes of 2- to 20-fold were detected in the uterine n-6 and n-3 essential fatty acids (EFA) and in certain saturated and monounsaturated fatty acids. The FO diet was associated with higher uterine C20 and C22 n-3, and the EPO diet, with higher uterine n-6 fatty acid. High uterine C18:2 n-6 was detected in neutral lipid (NL) of rats fed high concentrations of this fatty acid, but there was little evidence of selective incorporation or retention of C18:2 n-6 by uterine NL. The incorporation of EFA into uterine phospholipids (PL) was greater than NL EFA incorporation, and uterine PL n-3/n-6 ratios showed greater diet dependence. Tissue/diet fatty acid ratios in NL and PL also indicated preferential incorporation/synthesis of C16:1 n-9, and C16:0, and there was greater incorporation of C12:0 and C14:0 into uteri of rats fed EPO and FO. Replacement of 50-60% of arachidonate with n-3 EFA in uterine PL may inhibit n-6 EFA metabolism necessary for uterine function at parturition.

Developmental changes in the fatty acids of rat uterus and the influence of dietary essential fatty acids.

The effect of age on uterine fatty acid composition was studied in rats fed diets of differing fatty acid composition. Uteri of newly weaned 23-day rats had a higher fatty acid content and a higher proportion of short-chain (less than or equal to C18) fatty acids. Higher incorporation of C less than or equal to 18 fatty acids into neutral lipid (NL) and phospholipid (PL) of young 42-day rats compared with adult 240-day rats was detected. Uterine NL incorporated predominantly C less than or equal to 18 fatty acids which may be an important metabolic energy store in developing uterine tissue. Incorporation of C less than or equal to 18 fatty acids by uterine PL and NL was relatively unselective. In contrast, there was selective retention of arachidonic acid (AA) and docosahexanoic acid (DHA) throughout uterine development. An effect of dietary EFA on uterine n-3 and n-6 EFA was detected in each age group. There was marked retention of uterine AA when dietary supplies of n-6 EFA were low, but the total AA, eicosapentaenoic acid (EPA) and DHA in uterine PL remained constant in the three diet groups, and a constant content of AA, EPA and DHA was maintained throughout uterine development, regardless of diet. The degree of n-3 substitution achieved in this study inhibited uterine release of PG and parturition in adult rats.

Antitumour and pro-apoptotic actions of highly unsaturated fatty acids in glioma.

The highly unsaturated fatty acids (HUFA) of the n-6 and n-3 series are involved in cell signalling in normal and transformed cells and have recently been associated with pathways leading to tumour cell death. The antitumour activity of three HUFA (arachidonic acid, gamma linolenic acid and eicosapentaenoic acid) were studied in glioma cells and tissue. Using five glioma models, including primary cell suspensions prepared from 46 human glioma samples and an in vivo rat C6 glioma model, we obtained evidence that, following exposure to HUFA, either administered into the medium surrounding human glioma cells or in 16 preparations of multicellular spheroids derived from human and rodent glioma cell lines (C6, MOG, U87, U373) or administered intra-tumourally by infusion using osmotic mini-pumps in 48 rats, glioma regression and apoptosis were detected. Additionally, synergy between gamma irradiation and HUFA administration was observed in 13 experiments analyzing C6 glioma cell apoptosis in vitro. These pro-apoptotic and antiproliferative activities were observed using both C18 and C20 fatty acids of the n-6 and n-3 series, but not when saturated and monounsaturated C18 and C20 fatty acid preparations were used. In the glioma infusion model, in addition to the apoptosis detected in glioma tissue infused with HUFA for 3-7 days, preservation of normal neural tissue and vasculature in adjacent brain was observed. Also, there was little evidence of acute inflammatory infiltration in regressing tumours. Our findings suggest that intraparenchymal infusion of HUFA may be effective in stimulating glioma regression.

The effect of chlamydial infection on the initiation of premature labour: serial measurements of intrauterine prostaglandin E2 in amniotic fluid, allantoic fluid and utero-ovarian vein, using catheterised sheep experimentally infected with an ovine abortion strain of Chlamydia psittaci.

The initiation of premature labour by an ovine abortion strain of Chlamydia psittaci was studied in relation to Prostaglandin E2 (PGE2), which plays a major role in parturition. The local intrauterine concentration of PGE2 was monitored after experimental infection with C. psittaci, during late gestation, using a catheterised sheep model. Indwelling catheters were implanted into the amniotic and allantoic cavities of six control sheep, and into six sheep infected with an ovine abortion of C. psittaci. The release of PGE2 into the utero-ovarian vein of these sheep was also monitored. Infection with C. psittaci was associated with a premature rise in PGE2 in the amniotic fluid between days 122 and 135 of gestation (P less than 0.01). A premature increase in PGE2 was defected between days 127 and 136 of gestation (P less than 0.05) in the allantoic fluid of sheep infected with C. psittaci, but not in the control uninfected sheep. Chlamydial infection significantly decreased the secretion of PGE2 into the utero-ovarian vein. The mean concentration of PGE2 in the utero-ovarian vein of infected sheep was 68.8 +/- 5.2% of the PGE2 concentration of control sheep between days 122 to 141 of gestation (P less than 0.01). The release of PGE2 into the utero-ovarian vein increased between days 126 to 136 of gestation in infected and control sheep (P less than 0.01). The results from the catheterised sheep model indicated that C. psittaci infection is associated with a local intrauterine release of PGE2. The magnitude of this release was similar to the PGE2 release in control sheep prior to parturition.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly unsaturated fatty acid induced tumour regression in glioma pharmacodynamics and bioavailability of gamma linolenic acid in an implantation glioma model: effects on tumour biomass, apoptosis and neuronal tissue histology.

Highly unsaturated fatty acids (HUFAs) are naturally occurring anti-tumour agents. HUFAs act as intracellular signalling molecules in cell proliferation and death. In human glioma, HUFAs may stimulate tumour regression and apoptosis. An implantation glioma model, using the C6 glioma cell line, was used to investigate the bioactivity of locally infused n-6 HUFA gamma linolenic acid (GLA). Rat brains (15 normal and 37 C6 tumour bearing) were infused with vehicle or GLA 200 microM-2 mM. The most active local concentration of GLA for anti-tumour activity was 2 mM, infused at 1 microl/h over 7 days. Tumour regression, increased apoptosis and decreased proliferation were observed in tumours of rats infused with this concentration of GLA. Little effect on normal neuronal tissue was detected. The intraparenchymal route was an effective method of GLA administration in the treatment of glioma. These studies provide further insights into the potential role of HUFAs as anti-glioma agents.

Apoptosis in human primary brain tumours: actions of arachidonic acid.

It has been postulated that loss of proliferative control in tumour cells is a consequence of depletion of cellular arachidonic acid (AA) and that exogenous AA and n-6 fatty acids may restore control of proliferation. To test this hypothesis and to investigate the activity of AA, apoptosis in human primary brain tumour cells was analysed using flow terminal deoxynucleotide transferase uridine nick end-labelling (TUNEL). The effect of exogenous AA (30 microM) was analysed in collagenase-dispersed tissue from seven human primary brain tumours and in the normal brain tissue surrounding one of the tumours. Exogenous AA stimulated apoptosis in tumour tissue. A rapid three-fold increase in endonuclease activity was detected in tumour cells incubated with AA. The increase in apoptosis was significantly greater than the contemporary (< 15%) increase in necrosis detected using propidium iodide permeability and was greater than AA effects on normal brain tissue. These results are consistent with activation of the pathways of apoptosis by AA.

Macrophage prostaglandin E2 and oxidative responses to endotoxin during immunosuppression associated with anaesthesia and transfusion.

The widespread use of blood transfusion in major surgical procedures has led to concern about the immunosuppressive effect of transfusion on patients with underlying malignancy. Transfusion may also suppress the host response to infection. The cellular mechanisms of transfusion-associated immunosuppression may involve macrophage prostaglandin E2 (PGE2) in modulating the host response to cancer and infection. We previously observed that the transfusion of blood increased PGE2 production by unstimulated macrophages. To investigate this PGE2 associated immunosuppression, we studied the effect of transfusion of rats using a physiological stimulus of macrophage PGE2 production, bacterial endotoxin. In the same macrophages, we analysed intracellular oxidative activity. Both allogeneic and syngeneic blood transfusion were associated with increased PGE2 release by macrophages. This stimulation of PGE2 increased with duration of storage of blood. A similar effect of serum indicated that a humoral factor was involved. Endotoxin (50 ng/ml-500 micrograms/ml) stimulated PGE2 production in all transfused subjects. The lowest endotoxin concentration gave proportionately the greatest stimulation. Oxidative activity was down-regulated in macrophages of transfused rats, supporting an immunosuppressive role of PGE2 within the macrophage. An effect of surgery on the oxidative response was also detected.

Effect of blood transfusion on survival after esophagogastrectomy for carcinoma.

BACKGROUND: There is growing evidence that blood transfusion is associated with clinical factors that can lead to transfusion-induced immunosuppression. This effect can be beneficial or deleterious. METHODS: The effect of perioperative allogeneic blood transfusion on survival was studied retrospectively in 524 patients who were discharged from the hospital after esophagogastrectomy for carcinoma performed in a single unit over a 10-year period. RESULTS: The median operative blood loss for the series was 500 mL (range, 50 to 3,750 mL). Three hundred thirty-five patients (64%) received a perioperative allogeneic blood transfusion related to esophagogastrectomy, and 189 (36%) did not. The median perioperative blood transfusion administered was 900 mL (range, 300 to 12,950 mL). Perioperative allogeneic blood transfusion was associated with reduced survival for patients in stage III (p < 0.05) at 1 year, but no significant difference was found in this stage at 3 or 5 years after resection. Stage III disease accounted for 250 (48%) of the 524 patients discharged. CONCLUSIONS: Although perioperative allogeneic blood transfusion does not affect long-term survival after esophagogastrectomy for carcinoma, it does have a significant association with short-term survival in a group whose overall survival is often limited after resection. Attention should be directed toward minimizing operative blood loss and transfusing only for factors known to be clinically important, such as oxygen delivery and hemodynamics, not arbitrary hemoglobin levels.

Effects of N-6 essential fatty acids on glioma invasion and growth: experimental studies with glioma spheroids in collagen gels.

OBJECT: Intracranial infusions of gamma-linolenic acid (GLA), an essential fatty acid, have been used as an adjuvant therapy following malignant glioma resection; however, little is known about the dose response of glioma cells to this therapy. In this in vitro study the authors address this important pharmacological question. METHODS: Glioma spheroids derived from U87, U373, MOG-G-CCM, and C6 cell lines were grown in collagen gel and exposed to a range of GLA concentrations (0-1 mM) for 5 days. The diameter of glioma spheroids was measured, the apoptotic index was assessed using both the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling technique and cell morphological testing, and the levels of proliferating cell nuclear antigen were also measured. CONCLUSIONS: The dose-response patterns were similar for all four glioma spheroids. Low concentrations of GLA (<100 microM) increased both apoptosis and proliferation with a net increase in tumor growth and invasion, whereas high-dose GLA (>100 microM) significantly impaired spheroid cell growth. The proliferative effects of low-dose GLA could be a hazard in the clinical treatment of malignant glioma; however, because of the low toxicity of GLA against normal cells, local delivery of millimolar doses of GLA could significantly reduce tumor size.

Acute phase responses following minimal access and conventional thoracic surgery.

OBJECTIVES: Major thoracic surgery is associated with trauma-related immunological changes. These may impair anti-tumour immunity. We hypothesize that the reduced operative trauma associated with a video-assisted thoracic surgery (VATS) approach may decrease acute phase responses and, consequently, lead to better preservation of immune function. This prospective randomized study compared the effects of conventional open thoracic surgery and VATS on acute phase responses in patients undergoing pulmonary lobectomy. METHODS: Acute phase indicators were analyzed in patients undergoing lobectomy for suspected bronchogenic carcinoma. Surgery was prospectively randomized to pulmonary lobectomy by VATS or limited postero-lateral thoracotomy. Blood was taken pre-operatively and at 4, 24, 48, 72, 120 and 168 h post-operatively for analysis of C-reactive protein (CRP; 41 patients: open, n=22; VATS, n=19) interleukin (IL)-6, tumour necrosis factor (TNF) receptors (TNF-sR55, TNF-sR75) and P-selectin (24 patients: open, n=12; VATS, n=12). Samples taken at 48 and 168 h were also analyzed for phagocyte reactive oxygen species (ROS) production (25 patients: open, n=16; VATS, n=19). RESULTS: Surgery increased acute phase responses. VATS was associated with lower CRP and IL-6 levels. In the open surgery group, significant increases in ROS in neutrophils (up to 36% greater than before surgery, n=12, P<0.02-0.05) were detected at 2 days after surgery, but in the VATS group, the increase after surgery (of up to 17%, n=18) did not reach significance. Similarly, monocyte ROS increases of up to 25% in the mean ROS in the open surgery group and of up to 17% in the VATS group were detected on days 2 and 7 after surgery. CONCLUSIONS: VATS pulmonary lobectomy is associated with reduced peri-operative changes in acute phase responses. This finding may have implications for peri-operative tumour immuno-surveillance in lung cancer patients.

Therapeutic implications of disorders of cell death signalling: membranes, micro-environment, and eicosanoid and docosanoid metabolism.

Disruptions of cell death signalling occur in pathological processes, such as cancer and degenerative disease. Increased knowledge of cell death signalling has opened new areas of therapeutic research, and identifying key mediators of cell death has become increasingly important. Early triggering events in cell death may provide potential therapeutic targets, whereas agents affecting later signals may be more palliative in nature. A group of primary mediators are derivatives of the highly unsaturated fatty acids (HUFAs), particularly oxygenated metabolites such as prostaglandins. HUFAs, esterified in cell membranes, act as critical signalling molecules in many pathological processes. Currently, agents affecting HUFA metabolism are widely prescribed in diseases involving disordered cell death signalling. However, partly due to rapid metabolism, their role in cell death signalling pathways is poorly characterized. Recently, HUFA-derived mediators, the resolvins/protectins and endocannabinoids, have added opportunities to target selective signals and pathways. This review will focus on the control of cell death by HUFA, eicosanoid (C20 fatty acid metabolites) and docosanoid (C22 metabolites), HUFA-derived lipid mediators, signalling elements in the micro-environment and their potential therapeutic applications. Further therapeutic approaches will involve cell and molecular biology, the multiple hit theory of disease progression and analysis of system plasticity. Advances in the cell biology of eicosanoid and docosanoid metabolism, together with structure/function analysis of HUFA-derived mediators, will be useful in developing therapeutic agents in pathologies characterized by alterations in cell death signalling.

Calcium-sensitive mitochondrial membrane potential in human platelets and intrinsic signals of cell death.

The mechanisms involved in storage-induced damage in platelets are not well understood, but membrane signalling via Ca2+ ion flux may affect mitochondrial H+ gradients and metabolism and the intrinsic pathways of cell death, platelet survival and function. In this study, the effects of blood bank storage conditions, including reduced plasma concentration and interrupted agitation, were evaluated in platelets from 136 healthy donors. Mitochondrial membrane potential (DeltaPsim), an indicator of intrinsic cell death, and its sensitivity to Ca2+ ionophore A23187, were monitored using JC-1 by flow cytometry and fluorescence microscopy. Platelet survival was examined using lactate dehydrogenase release, annexin V binding and caspase-3/7 activity. Decreased plasma concentration and interrupted agitation affected DeltaPsim and caspase-3/7. Over 7 days in 30% plasma DeltaPsim showed a significant reduction (86.3 +/- 1.1% platelets with polarised mitochondria day 1; 79.9 +/- 2.1% day 5; 75.1 +/- 3.8% day 7, P = 0.01 day 1 vs. day 7). Whilst DeltaPsim in agitated platelets in 100% plasma was unchanged up to day 7, interruption of agitation was associated with a 44% reduction in the proportion of platelets with polarised mitochondria after 5 days (56 +/- 11%). The Ca2+ sensitivity of DeltaPsim changed earlier: 5 microM A23187 caused a 20-30% change in the fraction of platelets with polarised mitochondria by day 5. Ca2+ sensitivity also increased during interrupted agitation and reduced plasma concentration. DeltaPsim also correlated with indicators of platelet death, caspase-3 activity and annexin V binding (correlation coefficients of 0.8). In conclusion, changes in Ca2+-sensitive DeltaPsim are involved in the initiation of storage-induced cell death signals that influence platelet count and function in vivo.

Arachidonic acid in guinea pig uterus and plasma.

1. The concentration of unesterified arachidonic acid was low in relation to tissue-esterified arachidonic acid. 2. Intracellular esters contain high concentrations of arachidonic acid, and 75% of this ester pool lies in two compounds, phosphatidyl choline and phosphatidyl ethanolamine. 3. The distribution of arachidonic acid among plasma lipids contrasted with the uterine distribution, with microgram/ml quantities in each of the major lipid classes.

Control of endometrial prostaglandin output in vitro during the estrous cycle of the guinea-pig: influence of estradiol 17-beta, progesterone, oxytocin and calcium ionophore A23187.

Agents influencing the output of prostaglandin F2 alpha (PGF2 alpha) from non-pregnant endometrium were investigated by in vitro incubation, for 5 and 19h, using mid-cycle (day 7), end of cycle (day 15), or ovariectomised guinea-pigs. Estradiol 17-beta (10 micrograms/ml) stimulated PGF2 alpha output 24h after incubation with endometrium (p less than 0.05). This stimulation was greater at mid-cycle. Progesterone (50 ng/ml) inhibited output of PGF2 alpha (p less than 0.05) in mid-cycle, end of cycle and ovariectomised guinea-pig cultures. Oxytocin (1 X 10(-5) u/ml) stimulated the output of PGF2 alpha at the end of the cycle, but not at mid-cycle. However, in the presence of estradiol 17-beta (10 micrograms/ml), oxytocin stimulation of mid-cycle PGF2 alpha output was observed. The calcium ionophore A23187 (5 micrograms/ml) stimulated PGF2 alpha synthesis from mid-cycle and end-of-cycle endometrium, and this stimulation resembled that caused by arachidonic acid (100 micrograms/ml), suggesting an action via substrate mobilisation. Co-culture of endometrium and myometrium did not influence endometrial PGF2 alpha or myometrial 6-oxo-PGF2 output. It is suggested that the steroid hormones act as coarse modulators of endometrial PGF2 alpha output, but more rapid changes may be achieved by oxytocin and agents that mobilise substrate supply, possibly via calcium ion fluctuations.