In-vitro metabolism of unconjugated androgens, oestrogens and the sulphate conjugates of androgens and oestrone by accessory sex organs of the mature domestic boar

Minces of the prostate and seminal vesicles of the mature boar were incubated with the following major testicular steroids of the pig: [3H]dehydroepiandrosterone, [3H]5-androstene-3 beta, 17 beta-diol, [3H]oestrone, [3H]oestradiol-17 beta and their respective sulphate conjugates (excluding oestradiol-17 beta). Incubations were also carried out with [3H]testosterone, [3H]5 alpha-dihydrotestosterone and [3H]5 alpha-androstanediols. Minces of the epididymides were incubated with [3H]dehydroepiandrosterone and [3H]oestrone sulphates. The prostate and seminal vesicles converted dehydroepiandrosterone predominantly to weak androgens, whereas 5-androstene-3 beta, 17 beta-diol was primarily converted to testosterone; testosterone and its 5 alpha-reduced metabolites were metabolized in a manner typical of androgen end target organs. Unconjugated oestrone and oestradiol-17 beta were interconverted by the prostate and seminal vesicles. The metabolism of C19 steroid sulphates was less than 1% in all incubations; some oestrone sulphate, however, was converted to unconjugated oestrone and oestradiol, particularly by the caput epididymidis. The significance of these results is discussed in relation to recent studies in vivo.     

Measurement of phospholipase A2 activity in human endometrium during the menstrual cycle

Phospholipase A2 activity was measured in human endometrium throughout the menstrual cycle using an assay based on the liberation of oleic acid from 1-palmitoyl-2-[14C]oleoyl phosphatidylcholine. The enzyme was shown to be calcium dependent, to have an optimum pH of 8-9 and an apparent Michaelis constant of 110 mumol/l. Enzyme activity was low in early proliferative-phase tissue (6.08 +/- 1.42 (S.E.M.) pmol oleic acid released/mg protein per min) but rose significantly (P less than 0.01) during the late proliferative phase (10.86 +/- 2.79 pmol/mg per min). There was a tenfold increase in activity 2-4 days after ovulation (45.6 +/- 13.6 pmol/mg per min) which thereafter declined to reach values which at menstruation were not significantly different from those of the proliferative phase (4.5 +/- 1.76 pmol/mg per min). The results indicate that phospholipase A2 activity in human endometrium is related to the stage of the menstrual cycle and suggest that arachidonic acid release may be influenced by oestradiol and progesterone.     

Renal phospholipase C and diglyceride lipase activity in spontaneously hypertensive rats

Phospholipase C activity and diglyceride lipase activity were studied in the renal cortex and medulla of 10- and 40-week-old stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched normotensive Wistar-Kyoto rats (WKY). Enhanced phospholipase C activity was found in the cortical and medullary cytosol of kidney from SHRSP, and microsomal diglyceride lipase in SHRSP also increased. In SHRSP, phospholipase C and diglyceride lipase activities increased with age, but this increase was not evident in WKY. Phospholipase C had high substrate specificity for phosphatidylinositol in renal cytosol of both WKY and SHRSP. The increased activities were accompanied by prostaglandin E2 synthesis in renal medullary microsomes of 10-week-old SHRSP and were also present in the kidney of 40-week-old SHRSP. Total phospholipid and arachidonic acid contents in kidney were markedly high in the medulla of 10-week-old SHRSP, but these lipids were decreased in 40-week-old SHRSP. These results suggest that phospholipids and arachidonic acid in SHRSP may be genetically high and that the activated phospholipase C and diglyceride lipase hydrolyze phospholipids, providing arachidonic acid for prostaglandin synthesis, which results in a decrease of phospholipids and arachidonic acid in the kidney of 40-week-old SHRSP. These studies demonstrate that a phosphatidylinositol-specific phospholipase C-prostaglandin synthetic system may play an important role in the course of hypertension in SHRSP.     

Prostaglandin endoperoxide analogues stimulate phospholipase C and protein phosphorylation during platelet shape change

We have studied the effects of two stable prostaglandin endoperoxide analogues on platelet lipid metabolism in relation to specific platelet functional changes. During platelet shape change, the endoperoxide analogues induce the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of a phosphoinositide-specific phospholipase C. In parallel, they stimulate the phosphorylation of a 40-kd and a 20-kd protein. During platelet shape change, arachidonic acid is released, but not metabolized by platelet cyclo-oxygenase or lipoxygenase. Phospholipase C activation and platelet shape change are independent of extracellular Ca++ and Mg++, arachidonate metabolism, and release of adenosine diphosphate (ADP). Activation of phospholipase C during platelet aggregation seems, however, to be mediated partly by release of ADP. We conclude that endoperoxide analogues initially stimulate in platelets the formation of products derived from phospholipase C activation, which might serve as intracellular messengers for phosphorylation of specific proteins related to platelet shape change.     

Transport of free and conjugated steroids from the boar testis in lymph, venous blood and rete testis fluid

In 12 anaesthetized boars the concentrations of oestrone sulphate and dehydroepiandrosterone sulphate (DHAS) were 15- to 35-fold higher in lymph collected from a vessel in the spermatic cord than in testicular venous blood plasma from a vein in the spermatic cord. The concentrations of testosterone, total unconjugated oestrogens and dehydroepiandrosterone (DHA) were about twofold higher in lymph. The concentrations of all steroids studied were higher in testicular venous blood plasma than in arterial blood plasma (testosterone about sixfold; total unconjugated oestrogens about fourfold; oestrone sulphate about threefold; DHA and DHAS about twofold), but the concentrations of testosterone, total unconjugated oestrogens and oestrone sulphate in rete testis fluid were comparable to those in arterial blood plasma. Lymph flow from the pig testis was about 7% of plasma flow so that about 80% of the oestrone sulphate and DHAS produced by the testis leaves the organ in the lymph; the comparable values for testosterone, total unconjugated oestrogen and DHA were about 20%. In the 90-min period following an injection of human chorionic gonadotrophin there were substantial increases in the concentration of testosterone and smaller increases in the other steroids in arterial and spermatic venous blood plasma and in testicular lymph, but not in rete testis fluid; there were also small increases in lymph flow, but no change in blood flow.     

ENDOCRINE EFFECTS OF VASECTOMY

In order to provide information on the endocrine effects of vasectomy, unconjugated pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone, oestrone and oestradiol were analysed in the blood plasma of twenty Mexican men on two occasions before and 1, 3, 6 and 12 months after vasectomy. Vasectomy appeared to be associated with a significant decrease in the plasma levels of pregnenolone, dehydroepiandrosterone and androstenedione and a significant increase in the levels of dihydrotestosterone and oestrone. A probably significant increase in oestradiol levels took place 12 months after vasectomy but not before. No consistent changes were found in testosterone (up to 12 months) or in FSH and LH levels (up to 6 months) after vasectomy. The unconjugated steroids indicated above, except oestrone, were also estimated, whenever possible, in seminal plasma specimens obtained from thirty-nine subjects (including the twenty indicated above) on the same occasions. Vasectomy was associated with a highly significant decrease of seminal plasma dihydrotestosterone levels on all occasions and a significant decrease in androstenedione levels after 6 and 12 months. After 12 months there was a decrease in dehydroepiandrosterone and an increase in oestradiol; these changes were both probably significant. In another preliminary study, the levels of pregnenolone sulphate, dehydroepiandrosterone sulphate, testosterone glucuronide, testosterone sulphate and dihydrotestosterone sulphate were estimated before and 1 month after vasectomy in the seminal plasma of fourteen to seventeen subjects. Testosterone glucuronide fell, probably significantly, but other conjugates were unchanged. The data indicate that vasectomy may be associated with significant changes in the circulating and in seminal plasma levels of several steroids. The gradual nature of some of the changes observed suggests the necessity of conducting in several centres large-scale, long-term studies on vasectomized subjects and on a carefully matched control group. During the last decade vasectomy has been widely practised in several parts of the world as a method of fertility control. However, information on the endocrine effects of this intervention appears to be scanty. In most of the human studies reported, a small number of individuals were investigated and the studies have been confined to the assessment of the short-term effects of the operation. Moreover, the hormonal indices assessed by the various investigators have been limited, in most cases, to gonadotrophins and testosterone in blood. The present study was designed to assess in the same subjects the levels of a number of unconjugated steroids, FSH and LH on two occasions before and 1, 3, 6 and 12 months after vasectomy. The studies were extended to include steroid analyses in seminal plasma in the hope that such assays might yield information as to the effects of vasectomy on the distribution of steroids in the fluids of the male reproductive tract.     

ABNORMAL ANDROGEN AND OESTROGEN METABOLISM IN MEN WITH STEROID SULPHATASE DEFICIENCY AND RECESSIVE X-LINKED ICHTHYOSIS

Serum levels of sex hormone binding globulin (SHBG), testosterone, free testosterone, dihydrotestosterone, androstenedione, dehydroepiandrosterone sulphate, oestradiol, oestrone, oestrone sulphate, FSH, and LH were measured in 20 steroid sulphatase-deficient men with recessive X-linked ichthyosis and in normal men. The serum oestrone sulphate level was significantly higher than normal in the patients (P less than 0.0001). In affected men, there was a tendency towards higher dehydroepiandrosterone sulphate levels and no decline with age was seen in the patients as opposed to normal men (interaction: P less than 0.025). Serum androstenedione, and oestradiol levels were lower than normal in the patients (P less than 0.0005 and P = 0.055, respectively), while their LH level was higher than normal (P less than 0.0005). The serum levels of SHBG, total and free testosterone, dihydrotestosterone, oestrone, and FSH were not significantly different from normal in the icthyotic patients. We suggest that the observed abnormalities in these patients are a consequence of the enzyme deficiency which severely impairs the ability of tissues to hydrolyse steroid sulphates.     

PHOSPHOLIPASE C ACTIVITY IN HUMAN ENDOMETRIUM: ITS SIGNIFICANCE IN ENDOMETRIAL PATHOLOGY

Phospholipase C activity was measured in human endometrium using an assay based on the release of total labelled water soluble products (inositol, inositol phosphates) from L-3-phosphatidyl-[2-3H] inositol. The enzyme was shown to be calcium dependent and to have an optimum pH of 5.5. There was no difference between proliferative phase and secretory phase endometrium with respect to phospholipase C activity either in women with normal menstrual blood loss (proliferative phase: 3.7 +/- 0.7 (mean +/- SD), secretory phase: 4.5 +/- 2.0 nmol/mg protein/min) or in those complaining of severe menorrhagia (proliferative phase: 5.8 +/- 2.8, secretory phase: 7.0 +/- 2.8 nmol/mg protein/min). However, women complaining of severe menorrhagia had significantly higher endometrial phospholipase C activity than those in the normal group (P less than 0.01 and P less than 0.02 for proliferative and secretory phases respectively). Endometrial phospholipase C activity was also elevated in the presence of other gynaecological disorders, e.g. dysmenorrhoea, adenocarcinoma of the cervix and endometrial hyperplasia. The results indicate that phospholipase C activity in human endometrium is not related to the stage of the menstrual cycle but that in the presence of menorrhagia and other gynaecological disorders, activity is increased. Phospholipase C could be implicated in the generation of arachidonic acid for prostaglandin synthesis which may in turn be associated with these abnormalities.     

Characterization of human ovarian oestradiol-17 beta oxidoreductase activity.

Oestradiol-17 beta oxidoreductase activity, which catalyzes the interconversion of oestrone and oestradiol, was investigated in preparations of human ovaries. The enzyme activities were localized primarily in the 105,000 X g supernatant fraction; dialyzed supernatant preparations were used in subsequent studies. The pH optima were 6.9 for reduction and 8.1 for 17 beta-dehydrogenation. The apparent Michaelis constants for oestrone and oestradiol were 1 X 10(-7) M and 5 X 10(-7) M, respectively. The enzyme activity was present with either NADP(H) or NAD(H), though (NADP(H) were the preferred cofactors. Non-aromatic steroids androstenedione, dehydroepiandrosterone, testosterone and 5-androstene-3beta,17beta-diol were poor substrates for the enzyme preparation. Methylation of the phenolic hydroxyl of oestrone and oestradiol resulted in slightly enhanced activities. The sulfhydryl reagent, N-ethylmaleimide, inhibited the reduction of oestrone. A dialyzed supernatant preparation retained approximately 79% of the original enzyme activity when stored at -20 degrees C for 6 weeks.     

OESTRONE SULPHATE IN PLASMA FROM POSTMENOPAUSAL WOMEN AND THE EFFECTS OF OESTROGEN AND ANDROGEN THERAPY

The concentration of oestrone sulphate in peripheral plasma from postmenopausal women was investigated using a method which involved extraction of the conjugate, which was then hydrolysed with acid, and determination (by radioimmunoassay) of the purified oestrone fraction obtained. The concentration of unconjugated oestrone in the same plasma samples was also measured. Postmenopausal women had concentrations of oestrone sulphate in plasma (1.1 +/- 0.36 nmol/l, mean +/- SD, n = 39) similar to those found in women in the follicular phase of the menstrual cycle and less than those found in males (3.2 +/- 0.61 nmol/l, n = 21). The mean ratio of the concentration of oestrone sulphate to that of oestrone in plasma from postmenopausal women (7.9 +/- 3.3) was significantly lower (P less than 0.001, t test) than the mean ratio in men (19.8 +/- 3.8). Treatment with conjugated oestrogens, oestradiol in a cream, oestradiol valerate or ethinyl oestradiol, increased the concentration of oestrone sulphate in the peripheral circulation. In contrast, chronic corticosteroid therapy reduced the level of oestrone sulphate (0.5 +/- 0.11 nmol/l, n = 10) but this was partly restored (to 0.7 +/- 0.13 nmol/l) by concomitant oral dehydroepiandrosterone. Ingestion of piperazine oestrone sulphate (Harmogen, 1.5 mg) by three fasting postmenopausal women was followed 4 h later by oestrone sulphate concentrations five to ten times those found at midcycle.     

Postmenopausal serum androgens, oestrogens and breast cancer risk: the European prospective investigation into cancer and nutrition

Considerable experimental and epidemiological evidence suggests that elevated endogenous sex steroids - notably androgens and oestrogens - promote breast tumour development. In spite of this evidence, postmenopausal androgen replacement therapy with dehydroepiandrosterone (DHEA) or testosterone has been advocated for the prevention of osteoporosis and improved sexual well-being. We have conducted a case-control study nested within the European Prospective Investigation into Cancer and Nutrition. Levels of DHEA sulphate (DHEAS), (Delta4-androstenedione), testosterone, oestrone, oestradiol and sex-hormone binding globulin (SHBG) were measured in prediagnostic serum samples of 677 postmenopausal women who subsequently developed breast cancer and 1309 matched control subjects. Levels of free testosterone and free oestradiol were calculated from absolute concentrations of testosterone, oestradiol and SHBG. Logistic regression models were used to estimate relative risks of breast cancer by quintiles of hormone concentrations. For all sex steroids -the androgens as well as the oestrogens - elevated serum levels were positively associated with breast cancer risk, while SHBG levels were inversely related to risk. For the androgens, relative risk estimates (95% confidence intervals) between the top and bottom quintiles of the exposure distribution were: DHEAS 1.69 (1.23-2.33), androstenedione 1.94 (1.40-2.69), testosterone 1.85 (1.33-2.57) and free testosterone 2.50 (1.76-3.55). For the oestrogens, relative risk estimates were: oestrone 2.07 (1.42-3.02), oestradiol 2.28 (1.61-3.23) and free oestradiol (odds ratios 2.13 (1.52-2.98)). Adjustments for body mass index or other potential confounding factors did not substantially alter any of these relative risk estimates. Our results have shown that, among postmenopausal women, not only elevated serum oestrogens but also serum androgens are associated with increased breast cancer risk. Since DHEAS and androstenedione are largely of adrenal origin in postmenopausal women, our results indicated that elevated adrenal androgen synthesis is a risk factor for breast cancer. The results from this study caution against the use of DHEA(S), or other androgens, for postmenopausal androgen replacement therapy.     

Studies on the pattern of circulating steroids in the normal menstrual cycle. I. Simultaneous assays of progesterone, pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone, androstenedione, oestradiol and oestrone.

In an attempt to analyze the multiple changes and interactions in circulating steroid levels in the peri-ovulatory and peri-menstrual periods, the plasma levels of immunoreactive luteinizing hormone (LH), progesterone and unconjugated pregnenolone, dehydroepiandrosterone, testosterone, oestradiol and oestrone were assayed daily during a complete cycle in 17 normally menstruating women. In 14 of the 17 subjects studied androstenedione and unconjugated dihydrotestosterone were also estimated. The day of the LH-peak and the first day of menstruation, respectively, were used to synchronize the peri-ovulatory and peri-menstrual plasma levels of the various steroids. With the exception of dehydroepiandrosterone and dihydrotestosterone, the plasma levels of all steroids exhibited significant, but different changes during the cycle. Testosterone levels showed a slight but significant increase around the LH-peak, whereas the levels of pregnenolone and androstenedione were higher in the post-ovulatory than in the pre-ovulation periods. The levels of oestradiol and oestrone, as well as the ratios of oestradiol to oestrone gradually increased from the low values observed in the early proliferative phase to pre-ovulatory peak values. The relationship between peaks of oestradiol and oestrone and that of LH exhibited great individual variation. The same was true for the individual oestradiol to oestrone ratios. The combination of several steroidal signals did not improve the predictive value of the analyses. However, an increase of individual progesterone values by at least 0.35 ng/ml from the day preceding the LH-peak to the day of the LH-peak was observed in 13 of the 17 subjects. It is suggested that for the early detection of the LH surge and prediction of the subsequent ovulation daily assays of plasma progesterone are of more value than the assay of the other steroids investigated.     

Effect of graded oral doses of oestradiol on circulating hormonal levels

A single oral dose of micronized oestradiol (1, 2 or 4 mg) was administered to 10 normally menstruating women on the second day of 3 consecutive menstrual cycles. The order in which the 3 doses were given was chosen randomly for each subject. Samples of peripheral blood were withdrawn on 3 occasions (10, 5 and 1 min) before and on 9 occasions (0.37, 0.75, 1.5, 30, 6.0, 12.0, 24.0, 48.0 and 72.0 h) after the ingestion of oestradiol. Oestradiol, oesterone, oestradiol sulphate, oestrone sulphate, testosterone and LH were measured by means of radioimmunoassays in blood plasma on all occasions. The levels of all oestrogens increased significantly following the ingestion of oestradiol, although to a greatly different extent. The largest and most prolonged increase was seen in the case of oestrone sulphate, the levels of which--depending on the dose ingested--were 20-50 times higher than the pre-treatment levels 3 after the ingestion of the oestradiol capsule. The increase of the levels of oestrone sulphate was still significant (P less than 0.001) after 24 h, and following the ingestion of 2 and 4 mg of oestradiol even after 72 h (P less than 0.05). The levels of oestrone and oestradiol sulphate exhibited smaller increases 3 h after ingestion, i.e. 4-10 and 2.5-5 times, respectively, and the plasma levels of oestradiol showed a minor increase only (1.4-1.8 times). The concentration of oestradiol metabolites studied increased proportionally with the dose of oestradiol administered. On the other hand, a clear-cut dose--effect relationship could not be observed between the amount of oestradiol administered and the plasma levels of LH and testosterone. The data indicate that the bulk of perorally administered oestradiol is transformed to oestrone sulphate accompanied by smaller quantities of oestrone and oestradiol sulphate and that the ratios of circulating oestrogens seen after the ingestion of oestradiol deviate considerably from those observed in the normal menstrual cycle.     

Enhancement of human amniotic phospholipase A2 activity by steroid-sulphate derived from the foeto-placental unit

To study the control of production of prostaglandins (PG) during pregnancy and parturition, amniotic membranes obtained from normal vaginal delivery were incubated with the substrate of phosphatidylcholine containing [14C]arachidonic acid in the Sn-2 position. Phospholipase A2 activity was calculated as the rate of release of [14C]arachidonic acid from the substrate. Various steroids were added to the incubation medium to elucidate the effect of steroids on the enzymatic activity. The addition of dehydroepiandrosterone-sulphate (DHA-sulphate) to the medium increased by 7-fold the rate of [14C]arachidonic acid release from phosphatidylcholine at a concentration of 1.67 mM. The enhanced rate of arachidonic acid release suggests that DHA-sulphate stimulates phospholipase A2 activity. The same amounts of pregnenolone-sulphate and oestrone-sulphate also enhanced the enzymatic activity, while cholesterol-sulphate, free steroids such as DHA, progesterone, cortisol and oestrogens revealed no effect. These results suggest that steroid sulphates may be involved in the regulatory mechanism for PG synthesis in amniotic membrane.     

Studies on 17 beta-hydroxysteroid dehydrogenase in human endometrium and endometrial carcinoma. III. Partial purification and characterization of the microsomal enzyme.

Microsomal 17 beta-hydroxysteroid dehydrogenase obtained from the human secretory endometrium (17 beta-HSD) was solubilized with triton X-100. A 4-fold purification was achieved by ammonium sulphate precipitation and isoelectric focusing. In the presence of glycerol the partially purified enzyme was stable at 4 degrees C for at least 48 h. Using crude microsomes, the conversion of oestradiol to oestrone was linear with time and with the concentration of protein. The optimum temperature was approximately 40 degrees C and the optimum pH 9.4. For the reduction of oestrone the optimum pH was 6.5. With NAD, oestradiol was oxidized approximately three times more rapidly than with NADP. Km-values for oestradiol were nearly the same in endometrial carcinoma and in proliferative and secretory endometrium (i.e. approximately 3 X 10(-6) M). The maximal velocity was highest in secretory endometrium. Testosterone and androstenedione could also serve as substrates but they were interconverted more slowly than oestradiol and oestrone. Sulphhydryl groups were shown to be essential for catalysis.     

Steroidogenesis in cerebral metastatic chorionepithelioma tissue in vitro.

Homogenates of cerebral metastatic chorionepithelioma tissue were incubated with labelled dehydroepiandrosterone, pregnenolone or 20alpha-hydroxypregn-4-en-3-one. The metabolites of dehydroepiandrosterone which were isolated and identified were androstenedione, testosterone, oestrone, and oestradiol; no oestriol was detected. The only metabolite of pregnenolone and 20alpha-hydroxypregn-4-en-3-one isolated and identified was progesterone. No conversion of C-21 to C-19 steroids occurred in the metastatic chorionepithelioma tissue.     

Dual pathways for carbamylcholine-stimulated arachidonic acid release in rat pancreatic acini

Recent studies suggested the involvement of arachidonic acid in the mediation of pancreatic amylase release. However, an effect of carbamylcholine on arachidonic acid release has not yet been reported in the exocrine pancreas. This study was performed to evaluate the effect of carbamylcholine on arachidonic acid release and determine the underlying intracellular mechanisms. From enzymatic assays, phospholipase A₂ and diacylglycerol lipase were activated by carbamylcholine and these activations were inhibited by the phospholipase A₂ inhibitors, mepacrine and aristolochic acid, and by the diacylglycerol lipase inhibitor RHC 80267. Carbamylcholine also increased arachidonic acid release in a concentration-dependent manner. Both phospholipase A₂ and diacylglycerol inhibitors partially inhibited carbamylcholine-stimulated arachidonic acid release. The phospholipase C inhibitor U73122 and the protein kinase C inhibitor staurosporine also caused partial inhibition. Arachidonic acid release by carbamylcholine was suppressed by the simultaneous addition of RHC 80267 with either phospholipase A₂ inhibitors. Our data demonstrate that phospholipase A₂ and diacylglycerol lipase are activated and arachidonic acid is released in pancreatic acini by carbamylcholine. Dual pathways are responsible for carbamylcholine-induced arachidonic acid release. One such pathway involves the sequential action of phospholipase C, protein kinase C and diacylglycerol lipase, whereas the other involves phospholipase A₂ activation.     

Dissociation between the phospholipases C and A2 activities in stimulated platelets and their involvement in the arachidonic acid liberation

Summary. In previous work we have demonstrated that platelets depleted from secretory phospholipase A2 (sPLA2) produced similar amounts of thromboxane (Tx)B₂ as control platelets upon stimulation by thrombin. However, since depletion of sPLA2 was not total, this sole finding only suggested the non-involvement of sPLA2 in arachidonic acid release.
In the present study we provide further evidence for the non-involvement of sPLA2 in arachidonic acid liberation during platelet activation. Thus, rabbit platelets exposed to thrombin secreted sPLA2, released free arachidonic acid and formed TxB₂ and inositol phosphates. In contrast, U46619, a stable prostaglandin (PG)H₂ analogue, activates phospholipase C (PLC) and induces release of sPLA2 without TXB₂ generation nor arachidonic acid liberation. At each concentration tested of both agonists, stimulation of sPLA2 activity paralleled the production of inositol phosphates. These data suggest that sPLA2 is dependent on phosphoinositide hydrolysis and on the release reaction and that it is not involved in the liberation of arachidonic acid from stimulated platelets. In addition, a dissociation was observed between sPLA2 and the enzyme involved in the arachidonic acid mobilization, suggesting that the liberation of this fatty acid from membrane phospholipids was mediated by cytosolic phospholipase A2 (cPLA2). Finally, PLC does not play a major role in arachidonic acid liberation, since U46619, which induced the breakdown of inositol phospholipids, failed to release arachidonic acid. In confirmation, neomycin, which inhibits PLC activity, failed to inhibit ATP, sPLA2 and arachidonic acid release upon stimulation of platelets by fluoroaluminate. These data demonstrate that sPLA2 is not involved in the arachidonic acid release by stimulated platelets and indicate that the activations of PLC, sPLA2 and cPLA2 are independent events.     

INDICES OF GONADAL FUNCTION IN THE HUMAN MALE

A radioimmunoassay method developed previously for the measurement of unconjugated pregnenolone, dehydroepiandrosterone, androstenedione, testosterone, dihydrotestosterone and oestradiol in peripheral plasma was applied to the assay of these steroids in seminal plasma of normal, oligospermic and azoospermic males. It was not possible to use the plasma assay method for the determination of progesterone and oestrone in seminal plasma, because some of the reliability criteria were not fulfilled. A detailed analysis of these steroids in the peripheral plasma of the same subjects has been described previously. The levels of all steroids in seminal plasma were significantly lower than the corresponding blood levels. The ratios of blood plasma/seminal plasma levels of the various steroids varied from 37 (testosterone) to 1.7 (dihydrotestosterone). There was a positive correlation between the testosterone and dihydrotestosterone levels of the seminal plasma of normal and azoospermic subjects. The concentrations of dihydrotestosterone, pregnenolone and oestradiol were significantly lower in azoospermic subjects than in normals. The only pathological finding in the seminal plasma of oligospermic subjects was a diminished level of dihydrotestosterone. Enzymic hydrolysis of a seminal plasma pool resulted in a 3- to 8-fold increase in the concentration of pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone and oestradiol, indicating that human seminal plasma contains large amounts of steroid conjugates. It is suggested that the analysis of steroids in the seminal plasma in combination with determinations in peripheral plasma may be a valuable aid to the assessment of testicular function.     

Phospholipase C from Clostridium perfringens stimulates phospholipase A2-mediated arachidonic acid release in cultured intestinal epithelial cells (INT 407)

The mechanisms by which phospholipase C from Clostridium perfringens stimulates release of arachidonic acid (AA) in cultured intestinal epithelial cells (INT-407) were investigated. INT-407 cells were first allowed to incorporate 14C-labeled AA into their phospholipids; the labeled cells were then exposed to phospholipase C, and the release of free 14C-AA was determined. Phospholipase C caused a rapid (3 min) intracellular rise of free 14C-AA, followed by a considerable, dose- and time-dependent release of 14C-AA into the extracellular medium. For comparison, the calcium ionophore A23187 also caused a rapid mobilization of free 14C-AA, but a much lower extracellular 14C-AA release than phospholipase C during longer (1 h) incubation. The 14C-AA release was accompanied by a degradation of 14C-myo-inositol-labeled phosphatidylinositols and was reduced by the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). Both phospholipase C- and A23187-stimulated 14C-AA release was associated with degradation of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol and was reduced by nordihydroguaiaretic acid and 4-bromophenacyl bromide, two known phospholipase A2 inhibitors. In addition, the 14C-AA release was reduced by the calmodulin inhibitors trifluoperazine, compound 48/80, and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7). These findings indicate that phospholipase C from C. perfringens stimulates phospholipase A2-mediated AA release from human intestinal epithelial cells and suggest that this stimulation is brought about via processes involving phosphatidylinositol breakdown and activation of calmodulin and protein kinase C. It is possible that this phospholipase C-evoked AA release may contribute to the mucosal pathologic condition in diseases with altered intestinal microbial flora.